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A woman in her 30s with acquired immunodeficiency syndrome (aids) diagnosed by the fourth - generation hiv testing, with a cd4 count of 3 (1%) cells / mm, presented with weight loss and cachexia . She was started on lamivudine, zidovudine, and nevirapine and then developed hypersensitivity rash and hepatitis . At that time, she also had pancytopenia (white blood cells 2,950/l, neutrophils 79%, lymphocytes 13%, hemoglobin 9.6 g / dl, hematocrit 29%, platelets 325,000 10/l) and intra - abdominal lymphadenopathy . She was treated initially for presumptive disseminated mycobacterial infection with isoniazid, rifampicin, pyrazinamide, ethambutol, and clarithromycin . She improved clinically, and her antiretroviral regimen was restarted after three weeks with lamivudine, tenofovir, and efavirenz . Later on, she had gained weight, and laboratory results showed the resolution of pancytopenia and hepatitis . Two months later, the antibiotic regimen was readjusted to ciprofloxacin, clarithromycin, and streptomycin injection (for one month) according to the susceptibility, and her clinical improvement was remarkable . Two months after the initiation of antiretroviral therapy (art), she had an hiv viral load of <40 copies / ml and a cd4 count of 1 (2%) cell / mm; however, she had recurrent transaminitis . She was given prednisolone 20 mg / day with a tapered dose for five weeks for the treatment of hepatitis possibly due to immune reconstitution inflammatory syndrome . Five months after the initiation of art, her cd4 count was 13 (3%) cells / mm, with an hiv viral load of <40 copies / ml . She developed cytomegalovirus retinitis and received intravitreal ganciclovir injection and oral valganciclovir for six weeks . She then developed abdominal pain, with a palpable large suprapubic mass, for three weeks . A computed tomography (ct) scan of the abdomen showed a large lobulated mass (8.5 10 cm) with the epicenter at the right adnexa and with the invasion of the uterus, right ovary, right lateral wall of the sigmoid colon, appendix, superior wall of the urinary bladder, and right distal ureter, causing right ureter obstruction and a moderate degree of right hydronephrosis . Multiple matted lymph nodes were present along the mesenteric root and in the para - aortic, aortocaval, and retrocaval regions . Focal circumferential wall thickening of the left side of the jejunum caused moderate intraluminal narrowing, with evidence of small bowel obstruction (fig . She underwent exploratory laparotomy; intraoperative findings revealed a large pelvic mass with nodular surface and with dense adhesion to the omentum, the anterolateral wall of the uterus, the bladder, and the sigmoid colon . Only partial resection of the omentum and a biopsy of the mass at the anterior aspect of the uterus were performed . Acid - fast and gomori methenamine stains of the tissue specimen were negative; however, microbial culture was not sent for laboratory analysis . Her hospital course was complicated by wound infection / dehiscence, with the formation of an enterocutaneous fistula . She suffered from abdominal pain as well as malnutrition due to the nature of the unresectable residual mass and the enterocutaneous fistula . She was discharged home on palliative care, with antimicrobial therapy, and was lost to follow - up . Disseminated m. simiae infection presented with pancytopenia, hepatitis, and lymphadenopathy, similar to mycobacterium avium infection in an aids patient.1 effective antimicrobial treatment of m. simiae included rifampicin and ciprofloxacin . Our patient had an abnormal host immune response to infection, malakoplakia, which occurred after m. simiae infection, a condition that has not been previously described in the literature . However, we were unable to demonstrate a direct correlation due to the lack of microbiological data from tissue specimens . Malakoplakia has a gross appearance of round, oval, or mushroom - shaped yellowish structures.2 histologically, there is a proliferation of histiocytes with abundant granular eosinophilic cytoplasm (known as von hansemann histiocytes) that contain the pathognomonic michaelis gutmann bodies.2 these bodies are round to oval in shape and are visible as deeply basophilic structures, which are typically described as target - like and are periodic acid schiff stain - positive and calcium - positive.2 pathogenesis of malakoplakia is associated with the lysosomal dysfunction of macrophages in the intracellular killing process of ingested organisms and/or a defect in elimination, resulting in the accumulation of partially degraded bacteria within the cytoplasm and phagolysosomes of histiocytes, forming michaelis gutmann bodies around the undigested bacteria.2 the most common infectious etiologies described are bacteria, such as escherichia coli, rhodococcus equi, pasteurella multocida, and mycobacterium tuberculosis.211 malakoplakia often occurs in immunocompromised patients, such as those with aids, organ transplant recipients (mostly solid organ transplants), and in one case a patient who had undergone stem cell transplantation.5,1114 malakoplakia affecting a major organ could result in various organ dysfunctions . One of the most serious adverse events reported was irreversible renal allograft dysfunction due to malakoplakia in a renal transplant recipient with e. coli infection, pyelonephritis, and bacteremia.14 in an allogeneic stem cell transplant recipient, pleural malakoplakia caused by r. equi, presenting with a large tumor mass, required a lobectomy.12 in patients with residual disease, successful results have been reported by treating the underlying infection, reducing immunosuppression, improving the bactericidal activity of monocytes with a cholinergic agonist, such as bethanechol, and administering a multivitamin supplement.4,15,16 this is the first report of malakoplakia occurring after m. simiae infection.
Postoperative complications of blepharoplasty range from skin changes to vision - threatening emergencies.1 some of these complications occur early in the postoperative period, such as retrobulbar hemorrhage, infection, and eyelid hematoma . Other complications occur later in the postoperative period, such as eyelid malposition, strabismus, scar and over- and under - resection of skin or orbital fat.1 there has been a case report of persistent lateral hooding after upper lid blepharoplasty that has been treated with lacrimal gland repositioning.2 to the best of our knowledge, we report the first case of lacrimal gland fistula after upper lid blepharoplasty . She had a history of repeated lid swelling for almost 3 years before the age of 10 years . The patient underwent bilateral blepharoptosis repair at 12 years of age, re - operation on the right lid at the age of 18 years, and laser resurfacing of periocular region at the age of 20 . On examination, the patient had bilateral blepharoptosis (margin reflex distance of + 2.5 mm on the right and + 2 mm on the left side), dermatochalasis with mild lateral hooding, and medial and preaponeurotic fat protrusion . Upper blepharoplasty included skin - orbicularis muscle flap excision, medial fat excision, and partially preaponeurotic fat excision through a hole in the center of the septum . She had an uneventful postoperative follow - up with the exception of lateral hooding of the right lid . An elliptical lateral hooding excision was performed 2 months after upper blepharoplasty . At 1 week post - hooding excision, there was wound dehiscence with clear watery drops discharging from the wound [figure 1]. Post - lateral hooding excision right upper eyelid wound dehiscence and watery drops show a fistulous tract from lacrimal gland to the wound the patient was scheduled for repair of the wound and repositioning of the lacrimal gland . There was a tract from the lacrimal gland to the skin of the upper eyelid at the site of wound dehiscence . The lacrimal gland was repositioned into the lacrimal gland fossa using 4 - 0 prolene suture . The postoperative course was uneventful out the last visit at 6 months after repair [figure 2]. Last follow up (6 months) after repair of the wound and repositioning of the right prolapsed lacrimal gland despite the facile nature of the blepharoplasty, high patient expectations can make this procedure quite challenging to the surgeon . In order to attain a good result and avoid patient dissatisfaction, the surgeon must perform a careful history and physical exam and address specific patient complaints and expectations.3 blepharochalasis is a rare eyelid disorder characterized by exacerbation and remission of painless edema, eventually leading to atrophy of the periorbital skin . These episodes of eyelid swelling usually become less frequent with age, and eventually most cases enter a relatively quiescent stage . Ptosis is a common finding in blepharochalasis; however, the levator function is preserved.4 surgical management should be performed during the quiescent phase of blepharochalasis to avoid recurrent bouts of lid swelling leading to further ptosis and lid atrophy . Surgeons advocate that blepharochalasis symptoms should be quiet for 6 - 12 months before surgical treatment is contemplated.4 our case did not report any exacerbation for at least 15 years prior to the recent operation . A prolapsed lacrimal gland occasionally occurs due to atrophic changes in the septum in patients with blepharochalasis . In cases of frank preoperative prolapsed lacrimal gland, a blepharoplasty and suspension of the lacrimal gland should be performed.4 our patient did not present with a prolapsed lacrimal gland preoperatively . She only had post - blepharoplasty lateral hooding on the right side without a palpable lacrimal gland . We assume that blepharoplasty and consequently right - side hooding excision pulled an undetected partially prolapsed lacrimal gland inferiorly into the wound and resulted in the formation of a fistula . A similar case has been reported with hooding after blepharoplasty due to lacrimal gland prolapse; however there was no history of blepharochalasis or postoperative wound dehiscence and fistula.2 repositioning of the lacrimal gland to the right side was successful without postoperative sequelae out to 6 months postoperatively . The procedure can complex especially in the context of chronic inflammatory conditions.5 this case report illustrates an uncommon post - blepharoplasty complication in patients with blepharochalasis . Surgeons who perform blepharoplasty should have a high index of suspicion for lacrimal gland herniation in the presence of lateral hooding specially in patients with a history of blepharochalasis.
The erythrocyte sedimentation rate (esr) is an acute phase reactant (apr). The rate of sedimentation in a period of one hour called esr and also biernacki test . Esr test is a common hematologic nonspecific indicator of inflammation . To perform a test, nonclothing blood is placed in a vertical tube (westergren) and erythrocyte sedimentation rate is measured and is reported in units of mm / h . The best way to test was presented in 1921 by westergren, and it is still the golden standard method for measuring erythrocyte sedimentation rate . This method is considered to be simple and cheap, accessible, and accurate . Esr is a valuable laboratory tool in evaluation of infectious, inflammatory, and malignant diseases [1, 3]. Red blood cells in outside from the body precipitate due to their higher density than the plasma; in normal state these cells reject each other because of their negative surface charges and prevent rolex formation . In order to overcome the negative charge of the red cells it was shown that several factors such as ph levels of plasma other than the size of molecules or rolex formation contribute to erythrocyte sedimentation . Esr levels increase with age and are higher in women [1, 4, 5], anemia, and the black people . Clinical factors that do not influence the esr are [1, 4] obesity, body temperature, recent food, and nsaid [1, 4, 5]. Ivig with the half - life of 3 - 4 weeks was first produced in 1960 [6, 7]; ivig in high doses is used for the treatment of many autoimmune diseases including autoimmune thrombocytopenia, chronic inflammatory polyneuropathy, kawasaki disease, and guillain - barr syndrome [79]. High - dose ivig effects on various proteins, including inflammatory profiles in 63 children with kawasaki disease, were studied . All children had clinical manifestations of kawasaki and received 2 gr / kg ivig and aspirin during 12 hr . Serial testing was carried out before receiving ivig, 24 hours and 7 days later . After ivig infusion, mean esr was 12.7 6.46 mm / h before receiving ivig, 53.3 11.9 mm / h in 24 h, and 48.8 15.2 mm / h in the 7 days after the ivig infusion . Iga and igm immunoglobulin levels did not change 24 hours and 7 days after injection; however, igg is significantly increased . The result is that the high dose of ivig leads to rapid decreased changes of various proteins except for iga and igm and esr . In the same study on patients with myasthenia gravis and guillain - barr syndrome, we try to answer this question too, is esr a valuable apr marker in evaluation of inflammatory response to treatment when ivig is used previously? Total dose of ivig was 2 gr / kg (400 mg / kg in five days or 2 gr / kg in single dose). Esr before infusion of ivig and within 24 hours after administration of the last dose of ivig was checked (1 - 2 gr / kg/12 h or 400 mg / kg dose ivig). The erythrocyte sedimentation rate was measured by westergren method; ivig side effects were not observed in any patient . Results have been shown in different age groups, neonatal, infancy, childhood, and school age . Spss 16 and paired t - test were used to statistical software analysis; significance level of less than 0.05 was considered meaningful . 23 (46%) patients were males and 27 (54%) were females (table 1). The mean and median esr before and after receiving ivig the mean of esr before ivig was 31.8 29.04 and after ivig was 47.2 36.9; this difference was meaningful (p = 0.05). In male group the mean esr before ivig was 34.6 33.5 and after ivig was 49.1 36.2; the difference with (p = 0.003) is statistically significant . Before receiving ivig the mean esr was 29.4 25.2 in females and after receiving ivig it was 45.6 38.04 results of different age groups, 6 patients less than 28 days, 13 patients from 1 month to 1 year, 20 patients from 1 to 6 years old, and 11 patients from 6 to 12 years, have been shown in table 3 . Esr is known as an important factor in the evaluation of infectious and inflammatory processes . Important point for the use of the esr as an apr is its role in the evaluation of response to treatment; decreasing levels of esr are considered as a marker of response to therapy . Plasma fibrinogen and globulins are the major factors affecting esr [1, 2]. Alpha and gamma globulin provide half the ability of fibrinogen and albumin has the lowest ability in the sediment of red cells . However, as mentioned, ivig as an intravenous immune globulin is used in many diseases [6, 7]. Whether the administration of ivig, influences the esr value in monitoring of the response to therapy? In review of the literature generally two studies were found about the effect of ivig on esr . One of them has been done on kawasaki patients and has discussed numerous parameters of protein and also esr affected by ivig and the subsequent study has been limited to patients with myasthenia gravis and guillain - barr syndrome showing numerous parameters and blood esr affected by ivig, but this study has discussed 16 different diseases following the administration of ivig and has focused on the changes in esr . Ivig as an immunoglobulin, mostly igg, has aggregator effect on red cells and prevents their negative discharge forces . Although ivig has many anti - inflammatory effects and it is expected to reduce esr rate, in vivo its biologic effect is more effective than the immune modulation effect . The mean esr was 31.8 29.04 before receiving ivig and after ivig it was 47.2 36.9, with significant differences (p 0.05). These changes were statistically significant in both sexes; it could be interpreted because of lack of physiologic difference in children . In different age groups except the neonatal period, 1 month to 1 year (13 patients), 1 to 6 years (20 patients), 6 to 12 years (11 patients), the mean esr before ivig and after receiving ivig increased with significant differences; p values were, respectively, p = 0.001, p = 0.025, and p = 0.006 . Rapid physiological changes in various neonatal plasma proteins such as albumin and fibrinogen are the variables leading to these results . Different immune regulatory functions of ivig through its interaction with innate and adaptive immune system and immune homeostasis in neonatal period could be another reason . Among the apr, the esr is the most valuable criteria for evaluating and monitoring of response to inflammation; on the basis of this study on patients who are receiving ivig as a therapy, esr increased falsely (noninflammatory rising); therefore use of esr for monitoring of response to treatment may not be reliable . Based on the knowledge of authors and review of the literature this study seems to be the only work about esr and ivig administration effect in children . Although these results do not apply to neonatal group we suggest that, in patients who receive ivig, interpretation of esr should be used cautiously on the followup process.
However, the source of thrombi in these patients is not necessarily from the dilated left ventricle (lv). Several studies have documented that dilated lv is associated with dilated left atrium (la) and left atrial appendage (laa). On the other hand, la size serves as a significant and useful clinical predictor for ischemic stroke or all - cause mortality . Furthermore, laa size, flow velocity, and ejection fraction (ef) have been shown to correlate very well with laa presence of thrombus and subsequent thromboembolic events . The aim of our study was to analyze epidemiological features of our patients with dilated heart of mild to moderate systolic dysfunction, who were at sinus rhythm . Our main aim was to analyze the relationship and prediction of these epidemiological data to la and laa size and function as predisposing features of thromboembolic events . This was a prospective cross - sectional study conducted in university clinical center of kosova from 2009 to 2014 . The study included 101 patients with dilated lv (regardless of its etiology) in sinus rhythm . We excluded patients with swallowing problems, acute myocardial infarction, atrial fibrillation / flutter, severe systolic dysfunction, mechanical valves, oral anticoagulation therapy, and/or patients with a history of stroke / systemic thromboembolic event . The study was approved by our ethical board and written informed consent was taken from every patient that entered the study . Epidemiological data, physical examination, laboratory tests, electrocardiography (ecg), chest x - ray, transthoracic echocardiography (tte), and transesophageal echocardiography (tee) were obtained for every patient that entered the study . Tte (phillips ie 33) examinations and measurements were performed according to the recommendations of the american society of echocardiography . Left ventricular end diastolic diameter (lvedd), left ventricular end systolic diameter, septal wall, and posterior wall thickness were measured from parasternal m - mode view according to standard criteria . Lv ef <50% was considered as systolic dysfunction, whereas lv ef under 30% was considered as severe lv dysfunction . La diameter was measured in two - dimensional projection at end - ventricular systole in parasternal short axis view at the level of the aortic valve . Measurement of la volume was done with area - length (l) method using apical 4-chamber and apical 2-chamber views at ventricular end - systole (maximum la size), whereas l was measured from back wall to line across hinge points of mitral valve . Calculation of la volume was made by the following formula: 8/3 ([a1][a2]/l). Tee (phillips ie 33, agilent image - point hewlett packard or toshiba nemio xg ssa-580a) was performed in all patients that entered this study, with the main aim at analyzing laa . Maximum and minimum laa area was measured by planimetry method by tracing the laa starting from the top of the limbus of the upper pulmonary vein along the entire appendage endocardial border . The maximal area of the laa was measured during laa diastole, while the laa minimal area was measured at systole . The laa ef was calculated from the following equation: laa ef (%) = 100 (laamax laamin)/laamax . Patients with laa peak emptying velocity <40 cm / s and/or spontaneous echo contrast (sec) and/or thrombus in laa were considered as having laa dysfunction . Laa flow velocities were obtained with pulsed - wave doppler interrogation by placing the sample volume at the orifice of the laa . Four waves were identified: e wave, which represents the early diastolic emptying flow; a wave, which corresponds to the laa intrinsic late diastolic contraction; early systolic negative wave, which presents the laa filling; and the systolic reflection waves, which appear if the heart rate is slow enough and if the laa function is normal . Thrombus was diagnosed in case of the presence of a distinct, well contoured echogenic mass, which was identifiable in at least two different views . Correlation of selected variables was estimated using spearman's correlation coefficient as we assumed nonlinear correlation existence . Multiple regression analysis was used as a prediction model, and the data were expressed as odds ratio with 95% confidence intervals (cis) and probability value adjusted for all other measured risk factors . All data analysis was performed using the spss version 13.0 (ibm spss, inc . Tte (phillips ie 33) examinations and measurements were performed according to the recommendations of the american society of echocardiography . Left ventricular end diastolic diameter (lvedd), left ventricular end systolic diameter, septal wall, and posterior wall thickness were measured from parasternal m - mode view according to standard criteria . Lv ef was determined from apical views with modified simpson's rule . Lv ef <50% was considered as systolic dysfunction, whereas lv ef under 30% was considered as severe lv dysfunction . La diameter was measured in two - dimensional projection at end - ventricular systole in parasternal short axis view at the level of the aortic valve . Measurement of la volume was done with area - length (l) method using apical 4-chamber and apical 2-chamber views at ventricular end - systole (maximum la size), whereas l was measured from back wall to line across hinge points of mitral valve . Calculation of la volume was made by the following formula: 8/3 ([a1][a2]/l). Tee (phillips ie 33, agilent image - point hewlett packard or toshiba nemio xg ssa-580a) was performed in all patients that entered this study, with the main aim at analyzing laa . Maximum and minimum laa area was measured by planimetry method by tracing the laa starting from the top of the limbus of the upper pulmonary vein along the entire appendage endocardial border . The maximal area of the laa was measured during laa diastole, while the laa minimal area was measured at systole . The laa ef was calculated from the following equation: laa ef (%) = 100 (laamax laamin)/laamax . Patients with laa peak emptying velocity <40 cm / s and/or spontaneous echo contrast (sec) and/or thrombus in laa were considered as having laa dysfunction . Laa flow velocities were obtained with pulsed - wave doppler interrogation by placing the sample volume at the orifice of the laa . Four waves were identified: e wave, which represents the early diastolic emptying flow; a wave, which corresponds to the laa intrinsic late diastolic contraction; early systolic negative wave, which presents the laa filling; and the systolic reflection waves, which appear if the heart rate is slow enough and if the laa function is normal . Sec was identified if dynamic smoke - like echos with swirling motion in the cavity were seen . Thrombus was diagnosed in case of the presence of a distinct, well contoured echogenic mass, which was identifiable in at least two different views . All data were expressed as mean standard deviation and percentages . Correlation of selected variables was estimated using spearman's correlation coefficient as we assumed nonlinear correlation existence . Multiple regression analysis was used as a prediction model, and the data were expressed as odds ratio with 95% confidence intervals (cis) and probability value adjusted for all other measured risk factors . All data analysis was performed using the spss version 13.0 (ibm spss, inc ., one hundred and one patients (n = 101) were selected to enter our study, and their basic characteristics are presented in table 1 . The main inclusion criterion for our patients was dilated lv, with the mean lvedd being 66.6 6.07 mm, and the mean lv ef 39.02 6.4% . Basic characteristics of all patients in the study (n=101) data are presented as meansd or n (%). Af = atrial fibrillation, bmi = body mass index, bsa = body surface area, cad = coronary artery disease, dm = diabetes mellitus, ht = hypertension, sd = standard deviation they were on an average age of 58.13 12.66 years and predominantly male (69.3%). According to the body mass index (bmi) world health organization classification, our study population was in the preobese group (principal cut - off limits: 25.029.99 kg / m), while taking gender into account, the female patients were classified in obese class i group (principal cutoff limits: 30.034.99 kg / m) and statistically were more obese than males (p = 0.0001). As for risk factors for cardiovascular diseases, hypertension was present in half of our study population (51.5%) without any difference between genders, while smoking (29.7%) and especially diabetes mellitus (29.7%) were presented with similar frequency and rather remarkably . Smoking was significantly more frequent in males (38.6% males vs. 9.7% females; p = 0.002) while the frequency of diabetes mellitus was without any difference between genders . History of coronary artery disease (cad) was rather frequently present in our study population (56.4%) with statistical difference between genders in favor of males (67.1% vs. 32.3%; p = 0.001). As for la dimensions, areas, and volumes, analysis of mean values showed greater than reference values for all parameters [table 2]. La diameter and its normalization for body surface area (bsa) were significantly greater in favor of patients with a history of cad (46.93 4.98 mm vs. 44.70 4.68 mm, p = 0.024; 24.10 3.16 mm / m vs. 22.68 2.76 mm / m, p = 0.02, respectively). Characteristics of left atrium size assessed by transthoracic echocardiography in all patients (n=101) data are presented as meansd . La = left atrium, bsa = body surface area, la a1=left atrial area at 4-chamber view, la a2=left atrial area at 2-chamber view, sd = standard deviation as for characteristics of laa size and function assessed by tee, mean values showed normal laa ef [table 3]. Besides that laa dysfunction was documented in 86.1% of patients, considering the definition of dysfunction: laa peak emptying velocity <40 cm / s and/or presence of sec and/or thrombus in laa . Characteristics of left atrial appendage size and function assessed by transesophageal echocardiography in all patients (n=101) data are presented as meansd or n (%). Laa = left atrial appendage, ef = ejection fraction, sd = standard deviation analysis of relationship among epidemiological parameters and tte assessed la diameter, area, and volume [table 4] showed that male gender, older age, lower body weight, lower bmi, presence of diabetes, and history of cad were related to higher la diameter and la / bsa . Higher lav was related to older age, whereas higher lav / bsa was related to older age, lower weight and bmi, and history of cad . Relevant relationships among epidemiological and echocardiographic parameters of left atrium diameter, area, and volume assessed by transthoracic echocardiography in all patients (n=101) cad = coronary artery disease, bmi = body mass index, bsa = body surface area, dm = diabetes mellitus, la = left atrium, lav = left atrial volume analysis of relationship among clinical parameters and tee assessed laa area and function [table 5] showed that older age, lower weight, lower bmi, and presence of hypertension were significantly related to lower laa ef . Relevant relationships among epidemiological and echocardiographic parameters of left atrial appendage area and function assessed by transesophageal echocardiography in all patients (n=101) bmi = body mass index, ef = ejection fraction, ht = hypertension, laa = left atrial appendage taking into account that la enlargement could be a source of thrombus formation, we analyzed if there were independent predictive epidemiological variables that could affect la size and indirectly lead to thrombus formation . Results of multiple linear regression analysis showed that male gender was responsible for 25.5% la diameter change (r = 0.255), which means that for every male patient the la diameter (on the average) increases by 5.421 mm (95% ci: 1.7659.078) (p = 0.005). When la area average was taken into account, then weight appeared responsible for 20.5% la area average change (r = 0.205), which means that for every decrease of one kilo body weight, the la area average (on the average) increases by 0.197 cm (95% ci: 0.0440.351) (p = 0.014). Weight was responsible for 17.5% la volume change (r = 0.175), which means that for every decrease of one kilo body weight the la volume (on average) increases by 1.113 ml (95% ci: 0.1602.067) (p = 0.024). Binary logistic regression analysis showed a lack of any significant independent predictive clinical variables for laa dysfunction . Our study population was dominated by men (69.3%), and this is similar to most of the studies with heart failure patients . Under - representation of women 1157 patients with heart failure demonstrated an 82% representation of male patients . On the other hand, we found a similar ratio of gender representation to ours in a study by galrinho et al ., where 34% of patients with dilated cardiomyopathy were women . The reasons for this are yet unclear although the incidence of heart failure is higher in men than in women, whereas the prevalence is equal . Mean age (58 13 years) of our heart failure patients was somewhat younger than the mean patient age in most heart failure trials . Thus, the meta - analysis research group in echocardiography heart failure meta - analysis, which included data of 3540 patients from 18 studies, revealed a mean patient age of 62 13 years . Perhaps, the younger patient age in our study population could be explained by the fact that patients with severe lv systolic dysfunction were not included in our study that might have been in more advanced age . The second explanation might be that our sample patient population reflects the overall age demographics in kosovo, where around 50% of population represents those <24 years of age . The framingham heart study has shown that as bmi increases so does the risk for congestive heart failure . As far as our patients habits are concerned, we found that around 30% of our patients were current cigarette smokers, predominantly males . Smoking is not known to be related to dilated cardiomyopathy; however, it is related to cad, which was present in ~56% of our patients and was responsible for ischemic cardiomyopathy . Consumption of alcohol as another habit taken into account was present in ~10% of our patients, all of them being men . Otherwise, it is known that long - term heavy alcohol consumption is the leading cause of a nonischemic, dilated cardiomyopathy, referred to as alcoholic cardiomyopathy . Among the cardiovascular risk factors, high blood pressure was the most prevalent in our study population hypertension is known to be highly prevalent in the general population, and it is the main contributor for the incidence of heart failure as it increases threefold the risk for heart failure . However, the incidence of heart failure in relation to high blood pressure increases with the severity of hypertension . Despite the high prevalence of hypertension in cardiovascular and heart failure patients, after thorough investigation of etiology of heart failure, the role of hypertension decreases as opposed to increase the impact of cad . Furthermore, lv dilation with impaired contractility is the least frequent form of hypertensive heart disease . There are reports that pure dilated cardiomyopathy due to hypertension was present in only 4% of patients with congestive heart failure . Other risk factors for cardiovascular disease, such as diabetes and dyslipidemia, were relatively frequent in our patients (30% and 25%, respectively), which may be justified by a high proportion of patients with cad in our study population (~56%). History of cad was documented by either coronary angiography information or by hospital discharge list confirming experienced myocardial infarction at least 3 months prior to entering the study . Dilated cardiomyopathy of ischemic origin is known to be the most common type of dilated cardiomyopathies . Framingham heart study investigators found that la size remained a significant predictor of stroke in men and death in both genders after adjusting for age, hypertension, diabetes, smoking, ecg lv hypertrophy, prevalent atrial fibrillation, and prevalent congestive heart failure or myocardial infarction . The northern manhattan stroke study also showed that la size was associated with an increased risk of ischemic stroke . Several theories have been proposed to find explanations for the relationship of la enlargement and stroke or all - cause mortality . One of the theories states the possible cause of thrombus formation in patients with dilated la might be blood stasis . Increased la size occurs as a result of raised intra - atrial pressure, which in turn influences a reduction in laa flow velocity, resulting in an increased risk of thrombus formation and potential subsequent embolic stroke . On the other hand, dilation of the la may serve as an indicator for structural heart disease, hypertension, or increased lv mass and by this means, it may be related to augmented risk for stroke and mortality . The laa, on the other hand, is the most common cardiac location of thrombi formation . Three specific variables of the laa have been studied with respect to thrombus formation and stroke, which include: laa size, flow pattern, and flow velocity . As mentioned earlier, laa size, determined at surgery or by tee, has been shown to correlate very well with the laa presence of thrombus and subsequent thromboembolic events . Laa has also been found to be of larger size in patients with laa thrombus . Reduced or absent laa inflow and outflow velocities and that is why we included in the study only patients in sinus rhythm to avoid the influence of atrial fibrillation on thrombus formation and la size . Several studies have demonstrated that obesity is associated with increased la dimension independent of hypertension . Patients with higher bmi have higher fibrinogen levels, which are associated with the presence of la / laa thrombus . Interestingly, our results showed that body mass correlated inversely with both, la and laa size . However, chronic heart failure is known to induce general wasting in heart failure patients . This might be the reason that in our patients with dilated hearts, the relationship of body weight to la and laa size did not correspond to findings by other authors, in contrary it showed a significant inverse relation . Furthermore, reduction of body weight resulted to be an independent predictor of la area and volume enlargement in our study population . We believe to be the first authors to notify that lower body weight in patients with dilated cardiomyopathy, in sinus rhythm, should raise doubts regarding larger la . Our data showed a positive relationship between age and la size (diameter, area, and volume). Likewise, a relation between la dilation and aging, even in healthy patients, has been documented . Male gender is found to be in relationship to la diameter, but it also resulted to be an independent predictor of la size in our study population . As discussed earlier, framingham heart study investigators found that la size remained a significant predictor of stroke in men . Diabetes and coronary disease were related to la size in our patients, and this is comparable to results reported by other authors . Unfortunately, we were unable to find epidemiological predictors that could suggest us for laa dilation or dysfunction . Framingham heart study investigators found that la size remained a significant predictor of stroke in men and death in both genders after adjusting for age, hypertension, diabetes, smoking, ecg lv hypertrophy, prevalent atrial fibrillation, and prevalent congestive heart failure or myocardial infarction . The northern manhattan stroke study also showed that la size was associated with an increased risk of ischemic stroke . Several theories have been proposed to find explanations for the relationship of la enlargement and stroke or all - cause mortality . One of the theories states the possible cause of thrombus formation in patients with dilated la might be blood stasis . Increased la size occurs as a result of raised intra - atrial pressure, which in turn influences a reduction in laa flow velocity, resulting in an increased risk of thrombus formation and potential subsequent embolic stroke . On the other hand, dilation of the la may serve as an indicator for structural heart disease, hypertension, or increased lv mass and by this means, it may be related to augmented risk for stroke and mortality . The laa, on the other hand, is the most common cardiac location of thrombi formation . Three specific variables of the laa have been studied with respect to thrombus formation and stroke, which include: laa size, flow pattern, and flow velocity . As mentioned earlier, laa size, determined at surgery or by tee, has been shown to correlate very well with the laa presence of thrombus and subsequent thromboembolic events . Laa has also been found to be of larger size in patients with laa thrombus . Reduced or absent laa inflow and outflow velocities and that is why we included in the study only patients in sinus rhythm to avoid the influence of atrial fibrillation on thrombus formation and la size . Several studies have demonstrated that obesity is associated with increased la dimension independent of hypertension . Patients with higher bmi have higher fibrinogen levels, which are associated with the presence of la / laa thrombus . Interestingly, our results showed that body mass correlated inversely with both, la and laa size . . This might be the reason that in our patients with dilated hearts, the relationship of body weight to la and laa size did not correspond to findings by other authors, in contrary it showed a significant inverse relation . Furthermore, reduction of body weight resulted to be an independent predictor of la area and volume enlargement in our study population . We believe to be the first authors to notify that lower body weight in patients with dilated cardiomyopathy, in sinus rhythm, should raise doubts regarding larger la . Our data showed a positive relationship between age and la size (diameter, area, and volume). Likewise, a relation between la dilation and aging, even in healthy patients, has been documented . Male gender is found to be in relationship to la diameter, but it also resulted to be an independent predictor of la size in our study population . As discussed earlier, framingham heart study investigators found that la size remained a significant predictor of stroke in men . Diabetes and coronary disease were related to la size in our patients, and this is comparable to results reported by other authors . Unfortunately, we were unable to find epidemiological predictors that could suggest us for laa dilation or dysfunction . Male patients with dilated cardiomyopathy at sinus rhythm with lower body weight tend to have larger la and consequently might be at higher risk of developing atrial thrombus and its subsequent consequences . Therefore, these patients should be watched more carefully and possibly initiate the anticoagulation therapy, despite the fact that they are in sinus rhythm and their lv systolic function is not severely impaired.
A time - motion study is a business efficiency technique combining the time study work of frederick winslow taylor with the motion study work of frank and lillian gilbreth . It is a major part of scientific management (taylorism). (1) a time and motion study is used to determine the amount of time required for a specific activity, work function, or mechanical process . During the last decade, the number of patients seeking outpatient department (opd) services has increased many folds, but the facilities in the opd have not increased at the same rate . The outpatient department thus requires a systematic study of its services for its efficient management and function . It is therefore imperative that a simple time and motion study of an opd system and suitable inexpensive interventions can go a long way to improve the efficiency of a hospital . Time motion study is concerned primarily with increasing performance by measuring and then minimizing the time taken to perform various operations without compromising the quality of services . The fundamental philosophy of this discipline suggests that (a) although there are numerous ways to perform any task, one method will be superior to others, and (b) the superior method can be determined by observing and analyzing the time taken to carry out parts of the activity. (2) few such studies have been reported in the outpatient department of hospitals, and such studies based exclusively on immunization clinic of an institute is a rarity . Hence, this present study was carried out in the immunization clinic of a tertiary care hospital, with the objective of determining the activity time at the service points in the immunization clinic, as such clinics dealt with the most vulnerable and sensitive section of the population, for whom satisfactory preventive / promotive care was essential . Kar medical college, kolkata, over a period of 1 month (september 2010). The total sample was 482 . Before starting the study, ethical clearance for conducting the study the registration records of last 3 years of the immunization clinic were reviewed and average daily registration including both old and new immunization cards was found to be 60 in number . Every third mother / caregiver registering in the clinic on the day of study was selected by systematic random sampling method; as the number working days in the month of september 2010 was 21, the minimum sample fixed to be achieved was 420 . Predesigned and pretested schedules were used to record time and other information, and pre - synchronized stopwatches were used to record total activity time (which included waiting time + service time). As the immunization clinic was open from 10.00 time was recorded at the following points: at the entrance (entry time)immunization table 1 (after completion of initial registration by public health nurse)immunization table 2 (after completion of nutritional assessment of child and health education to mothers by interns)immunization table 1 (after completion of final registration by public health nurse)immunization table 3 (after completion of vaccination and post vaccination advice delivery by public health nurse)exit time at the entrance (entry time) immunization table 1 (after completion of initial registration by public health nurse) immunization table 2 (after completion of nutritional assessment of child and health education to mothers by interns) immunization table 1 (after completion of final registration by public health nurse) immunization table 3 (after completion of vaccination and post vaccination advice delivery by public health nurse) service delivery time at the different activity points (n=483) service delivery time (trimmed mean in seconds) in relation to old//new registrations, day of visit, hour of visit the following activities were carried out in the respective immunization tables . In immunization table 1, initial and final registrations of new and old registration cards were done . In the case of initial registration of new cards, a day - specific serial number was given and in final registration, a new registration number was allotted to the card; it was also checked whether nutritional assessment was done in immunization table 2 and vaccines scheduled for that day were prescribed . In the case of initial registration of old cards, a day - specific serial number was also given and in final registration process it was checked whether nutritional assessment was done in immunization table 2 and vaccines scheduled for that day were prescribed . In addition, in case of old cards, information related to previous vaccination and the already allotted registration number were verified and matched with the records maintained in the clinic during initial and final registration process . In immunization table 2, nutritional assessment of the child was done by clinical and anthropometric methods along with plotting of growth chart and health education was given to mother / caregiver . Also, a comparison with previous records was done in the case of old cards . In immunization table 3, vaccination was given and appropriate post vaccination advices were given to the mothers / caregivers along with information regarding next visit . (the sequence of movement of mother / caregiver was as follows: after entry the participants went to immunization table 1 for initial registration, after which they approached immunization table 2 for nutritional assessment of the child and health education; thereafter the participant returned to immunization table 1 for final registration and was subsequently sent to immunization table 3 for vaccination and post vaccination advices followed by exit from the clinic . The reason for following a two - step registration process, as stated by the clinic health staffs was to ascertain that the mothers / caregivers attend immunization table 2 for nutritional assessment of the child and health education as they had the tendency of bypassing this table to attend immunization table 3 directly for vaccination and post vaccination advices .) Kar medical college, kolkata motion of every mother / caregiver selected for the study was followed from the entry till exit from the immunization clinic in the above sequence, and time spend in the above - mentioned activity points was recorded . Time has been expressed as mean, median, trimmed mean (all in seconds), and interquartile range (iqr). Trimmed mean (5% of the extreme values on either side was trimmed) was calculated as data was very much skewed leading to large standard deviations . Table 1 presents the service delivery time at the different activity points . In the initial registration table the mean time taken was 165.9 167.7 s, while the median value was 120 s (iqr=120). The mean time taken at nutrition and health assessment table was 179.5 176.8 s and median time was 120 s (iqr=120). Vaccination and post vaccination advice table took 46.30% of total activity time, while the mean and median time was 465.25 453.75 and 300 s, respectively . Table 2 shows the service delivery time (trimmed mean in seconds) in relation to old//new registrations, day of visit, time of visit . In the case of new registrations, time taken at vaccination and post vaccination advice table was more, while in all other tables participants with old registration cards took more time . Regarding the day of visit it was found that the maximum time for initial registration was taken on monday (219.2 s), while tuesday (181.3 s) took the maximum time for final registration . Maximum time was also spent on the vaccination and post vaccination advice table on monday (538.1 s) while nutrition and health assessment table took maximum time on friday (217.1 s). Time taken in the first half of immunization session was more for all the tables except for final registration . Historically, time motion studies were used in the manufacturing industry to evolve pay scales with the thought that money was the only motivation for work. (3) today, time motion studies can be effective for performance evaluations, for planning purposes in order to predict the level of output that may be achieved and can be used to unmask problems and create solutions and also can be used for time cost analysis . The outpatient department is the point of contact between the health care facility and the community . The problems of opds of developing countries are long waiting time, long queues, inefficient staffs, absence of staffs, etc . The problem is more grave when the opd deals with pediatric patients like in the immunization clinics . No comparable time motion studies carried out in any other immunization clinic have been found, but other time motion studies have been carried out in relation to surgical interventions, nursing activity monitoring . Few salient features of this study were that the overall time taken at the vaccination and post vaccination table was substantially more than other tables (46.3%). Old registrations cards took more time in immunization tables 1 and 2 probably due to the fact that verifications and comparisons were made with previous records in the clinic in the case of these cards . Similarly, time taken at the different tables during the first half of the clinic (10.00 a.m12.00 noon) was generally more probably due to more number of mother / caregivers attending the clinic in the first half . On mondays, more time was taken in initial registration table (immunization table 1 in the figure 1) and in vaccination and post vaccination table (immunization table 3 in figure 1) which may have been due to more number of participants coming to the immunization clinic on the first day of week (n=136). Such bottlenecks identified may have relations to number of staff present on the particular day of study, unequal efficiency of the members of the staffs, pattern of arrangement of activity tables in the immunization clinic, availability of vaccines uniformly, varying number of participants in relation to different days / hour of visit, etc . Again, factors affecting waiting time and service time in different tables may be studied separately for further future discussions . A time motion study of operation theater time use during laparoscopic cholecystectomy by surgical specialist residents commented regarding the need of change of the current layout of the operating room for time saving. (4) similarly, a time motion study at new delhi and north india showed that cataract surgical output can be increased in the country if operation theatre time is utilized optimally. (5) the quality and efficiency of the immunization services can be improved if the constraints and bottlenecks in the system are identified, analyzed, and attempts are made to overcome them . Perhaps this study will help in the initiation of further in - depth analysis of the bottlenecks related to this particular system of health care delivery . The goal for achieving universal immunization against vaccine preventable diseases requires multifaceted collated response from many stakeholders . Time management at all levels of health care system is the need of the hour which has to be recognized and necessary steps must be taken . This study had the limitation of being observational and would have been more effective if more related variables like number of staff present at each table on different days could also have been included and interventions could have been done leading to comparisons between different time utilizations in different layout settings of the clinic or by changing other contributory factors . At the end, it may be stated that this study aims to initiate an effort to study the utilization of time at a certain health care unit with the invitation of much more in - depth analysis of the method of functioning and subsequent remedial steps for optimal functioning of the system.
Dual antiplatelet therapy consisting of aspirin and an adenine diphosphate (adp) p2y12 receptor antagonist is recommended as the standard antiplatelet regimen for patients with coronary artery disease.1 clopidogrel, an adp p2y12 receptor antagonist, strongly inhibits formation of thrombosis by impeding platelet binding regardless of the degree of degranulation or blood flow status.2,3 aspirin has antiplatelet effects by irreversible inhibition of the formation of thromboxane a2 by acetylation of platelet cyclooxygenase, thereby contributing to decreasing the mortality caused by myocardial infarction, stroke, and cardiovascular diseases.4 in several studies, combined clopidogrel and aspirin treatment increased platelet aggregation, and this effect was superior to administration of aspirin alone in reducing the incidence of cardiovascular death and nonfatal myocardial infarction or stroke.5 a recent meta - analysis concluded that short - term aspirin in combination with clopidogrel is more effective than monotherapy as a secondary preventive treatment for stroke or transient ischemic attack without increasing the risk of hemorrhagic stroke and major bleeding events.6 in addition, approximately 50% of asians, including koreans, have a gene variant that leads to resistance to clopidogrel; therefore, there is a safety issue of thrombus formation.1 however, it is well recognized that low - dose aspirin strongly enhances cyp2c19 enzyme activity and increases drug efficacy in patients having clopidogrel resistance.6 fixed - dose combination (fdc) formulations have an important role in the management of coronary artery disease . The fdc of aspirin and clopidogrel has increased therapeutic compliance, which is a major factor in the poor outcome of stenting patients for acute coronary syndrome, by simplification of the medical regimen.7 in addition, overall costs can be lowered by decreasing the number of prescriptions, bottles, labels, etc.8 clopidogrel is rapidly absorbed from the gastrointestinal tract, with a maximum concentration (cmax) of 2 ng / ml at 1.4 hours following administration of clopidogrel 75 mg, and is eliminated with a half - life (t1/2) of 1.7 hours.9 in contrast, enteric - coated formulations of aspirin 100 mg show continuous absorption from the intestine with a time - to - reach cmax approximately 4 hours, and then the drug is rapidly eliminated with a t1/2 less than 1 hour . Based on these pharmacokinetic properties of the two drugs, there should be little possibility of interaction in the absorption profiles, and their short t1/2 indicates that they are suitable for fdc because both drugs are almost completely eliminated within 12 hours after dosing . In this study, we compared the pharmacokinetics of clopidogrel, aspirin, and its active metabolite, salicylic acid, in healthy men after administration of an fdc formulation and coadministration of clopidogrel and aspirin . The study protocol and written informed consent form were reviewed and approved by the institutional review board of inje university busan paik hospital (irb number 11 - 100). The study was carried out in compliance with the declaration of helsinki (2010), the international conference on harmonization of good clinical practice, and the current korean good clinical practice guidelines.10 healthy men aged between 20 and 55 years within 20% of the ideal body weight according to broca s formula were enrolled in this study . All subjects were judged to be healthy based on the results of a detailed physical examination, 12-lead electrocardiogram, clinical laboratory tests, and vital signs at the screening . The following exclusion criteria were used: excessive consumption of caffeine (> 5 cups / d), cigarettes (> 10 cigarettes / d), or alcohol (> 30 g of alcohol / d), administration of inducers or inhibitors of drug - metabolizing enzymes including proton pump inhibitors (ppis) (lansoprazole, omeprazole, pantoprazole, and rabeprazole), rifampicin, carbamazepine, and barbitals within 28 days; history of disease influence on drug absorption, distribution, metabolism, and excretion (eg, gastrointestinal ulcer, ulcerative colitis, and history of abdominal operation); history of hemorrhagic symptoms and disease; aspartate aminotransferase (ast) or alanine aminotransferase (alt) exceeding 1.25 times the upper limit of the normal range; total bilirubin exceeding 1.5 times the upper limit of the normal range; prothrombin time, activated partial thromboplastin time, and bleeding time extended beyond the normal range; platelet level <15010/l or> 35010/l; and participation in another clinical study within 90 days prior to the start of the study . A randomized, open, two - period, two - treatment, two - sequence crossover study was conducted at the clinical trial center of inje university busan paik hospital, busan, republic of korea (clinicaltrials.gov identifier: nct01496261). The 60 enrolled subjects were divided into two groups and randomized to one of two sequences (rt, tr) at the beginning of the study at a ratio of 1:1 using a randomization schedule generated using the sas software (v 9.3; sas institute inc ., each group of 30 subjects was administered the study drugs in consecutive periods with a gap of 3 days between them . The assigned study drugs were administered orally with 240 ml of water after a 10-hour overnight fast, and subjects continued fasting until 4 hours postdose . Subjects were administered two capsules of clopidogrel / aspirin 75 mg/100 mg fdc (chong kun dang pharm ., subjects were coadministered two tablets of clopidogrel 75 mg (plavix; sanofi - aventis, schiltigheim, france) and two capsules of aspirin 100 mg (astrix; boryung pharm ., seoul, republic of korea) in the other period with a 14-day washout period . The length of the washout period was estimated to be greater than seven times the reported t1/2 of clopidogrel, aspirin, and salicylic acid (7.27.6, 0.4, and 2.1 hours, respectively).9,11 blood samples for clopidogrel measurement were collected at preset time intervals of predose and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 hours after dosing . Samples for aspirin measurement were taken predose and 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hours after dosing . Whole blood samples (5 ml) collected in edta (k2) tubes were centrifuged at 1,800 g for 10 minutes, and plasma samples were transferred to microtubes and stored at 70c . Subjects were asked about the occurrence of any adverse events (aes) between consent and the last poststudy visit . Physical examinations and clinical laboratory tests were performed before and 24 hours after dosing in each period . Vital signs were monitored before and at 12 and 24 hours after dosing in each period . Subjects who took the study drugs at least once during the study were included in the safety assessment . Concentrations of clopidogrel, aspirin, and the active aspirin metabolite salicylic acid were analyzed using validated ultraperformance liquid chromatography tandem mass spectrometry (uplc ms / ms), acquity uplc system (waters, milford, ma, usa). Clopidogrel bisulfate and its internal standard (rac clopidogrel - d4 hydrogen sulfate) and aspirin and salicylic acid and their internal standards (aspirin - d4 and salicylic acid - d4, respectively) were prepared using a 96-well solid - phase extraction plate, bond elut, plexa 10 mg (varian, palo alto, ca, usa). Detection and quantification were performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization interface in positive mode and multiple reaction monitoring mode . Chromatographic separation of the compounds was accomplished using an acquity uplc beh c18 column (1.7 m, 2.150 mm; waters) with acetonitrile in 1,000 ml of water containing 0.1% formic acid as the mobile phase . A full validation of the assay was carried out with respect to selectivity, accuracy, precision, recovery, calibration curve, and stability . The calibration curves for clopidogrel, aspirin, and salicylic acid were linear over ranges of 0.0120.0 ng / ml, 10.02,000 ng / ml, and 10020,000 ng / ml, respectively, with coefficients of determination (r) greater than 0.995 for all . The pharmacokinetic parameters of clopidogrel, aspirin, and salicylic acid were estimated using a noncompartmental method using the winnonlin software (v 6.1; pharsight corp, mountain view, ca, usa). The below the limit of quantitation values occurring prior to the first measurable concentration were set to zero, and those occurring after the first measureable concentration were not included in the pharmacokinetic analysis . The cmax and time to cmax (tmax) were calculated directly from the concentration the area under the plasma concentration time curve (auc) from time 0 to 24 hours (auclast) was calculated according to the linear trapezoidal rule . Auc0 was calculated using the following equation: auc0 = auclast + ct/z, where ct is the last observed concentration and z is the elimination rate constant . The log - transformed cmax and auclast for clopidogrel, aspirin, and salicylic acid were compared using the average bioequivalence approach . This comparison was performed using analysis of variance (anova) with effects for sequence, subject within sequence, treatment, and period . Because, for logistical reasons, the two groups were administered the study drugs at different times, group effect was also included as a factor.12 using anova, the 90% confidence intervals (cis) of the least - squares geometric means were calculated for cmax and auclast . The fdc formulation was considered bioequivalent if the 90% cis of cmax and auclast for clopidogrel, aspirin, and salicylic acid fell within the range of 0.801.25.12 to evaluate the difference of tmax between coadministration and fdc, wilcoxon signed - rank test was used . All statistical calculations were performed using the sas software (v 9.3; sas institute inc . ). Healthy men aged between 20 and 55 years within 20% of the ideal body weight according to broca s formula were enrolled in this study . All subjects were judged to be healthy based on the results of a detailed physical examination, 12-lead electrocardiogram, clinical laboratory tests, and vital signs at the screening . The following exclusion criteria were used: excessive consumption of caffeine (> 5 cups / d), cigarettes (> 10 cigarettes / d), or alcohol (> 30 g of alcohol / d), administration of inducers or inhibitors of drug - metabolizing enzymes including proton pump inhibitors (ppis) (lansoprazole, omeprazole, pantoprazole, and rabeprazole), rifampicin, carbamazepine, and barbitals within 28 days; history of disease influence on drug absorption, distribution, metabolism, and excretion (eg, gastrointestinal ulcer, ulcerative colitis, and history of abdominal operation); history of hemorrhagic symptoms and disease; aspartate aminotransferase (ast) or alanine aminotransferase (alt) exceeding 1.25 times the upper limit of the normal range; total bilirubin exceeding 1.5 times the upper limit of the normal range; prothrombin time, activated partial thromboplastin time, and bleeding time extended beyond the normal range; platelet level <15010/l or> 35010/l; and participation in another clinical study within 90 days prior to the start of the study . A randomized, open, two - period, two - treatment, two - sequence crossover study was conducted at the clinical trial center of inje university busan paik hospital, busan, republic of korea (clinicaltrials.gov identifier: nct01496261). The 60 enrolled subjects were divided into two groups and randomized to one of two sequences (rt, tr) at the beginning of the study at a ratio of 1:1 using a randomization schedule generated using the sas software (v 9.3; sas institute inc ., each group of 30 subjects was administered the study drugs in consecutive periods with a gap of 3 days between them . The assigned study drugs were administered orally with 240 ml of water after a 10-hour overnight fast, and subjects continued fasting until 4 hours postdose . Subjects were administered two capsules of clopidogrel / aspirin 75 mg/100 mg fdc (chong kun dang pharm ., subjects were coadministered two tablets of clopidogrel 75 mg (plavix; sanofi - aventis, schiltigheim, france) and two capsules of aspirin 100 mg (astrix; boryung pharm ., seoul, republic of korea) in the other period with a 14-day washout period . The length of the washout period was estimated to be greater than seven times the reported t1/2 of clopidogrel, aspirin, and salicylic acid (7.27.6, 0.4, and 2.1 hours, respectively).9,11 blood samples for clopidogrel measurement were collected at preset time intervals of predose and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 hours after dosing . Samples for aspirin measurement were taken predose and 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, and 24 hours after dosing . Whole blood samples (5 ml) collected in edta (k2) tubes were centrifuged at 1,800 g for 10 minutes, and plasma samples were transferred to microtubes and stored at 70c . Subjects were asked about the occurrence of any adverse events (aes) between consent and the last poststudy visit . Physical examinations and clinical laboratory tests were performed before and 24 hours after dosing in each period . Vital signs were monitored before and at 12 and 24 hours after dosing in each period . Subjects who took the study drugs at least once during the study were included in the safety assessment . Concentrations of clopidogrel, aspirin, and the active aspirin metabolite salicylic acid were analyzed using validated ultraperformance liquid chromatography tandem mass spectrometry (uplc ms / ms), acquity uplc system (waters, milford, ma, usa). Clopidogrel bisulfate and its internal standard (rac clopidogrel - d4 hydrogen sulfate) and aspirin and salicylic acid and their internal standards (aspirin - d4 and salicylic acid - d4, respectively) were prepared using a 96-well solid - phase extraction plate, bond elut, plexa 10 mg (varian, palo alto, ca, usa). Detection and quantification were performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization interface in positive mode and multiple reaction monitoring mode . Chromatographic separation of the compounds was accomplished using an acquity uplc beh c18 column (1.7 m, 2.150 mm; waters) with acetonitrile in 1,000 ml of water containing 0.1% formic acid as the mobile phase . A full validation of the assay was carried out with respect to selectivity, accuracy, precision, recovery, calibration curve, and stability . The calibration curves for clopidogrel, aspirin, and salicylic acid were linear over ranges of 0.0120.0 ng / ml, 10.02,000 ng / ml, and 10020,000 ng / ml, respectively, with coefficients of determination (r) greater than 0.995 for all . The pharmacokinetic parameters of clopidogrel, aspirin, and salicylic acid were estimated using a noncompartmental method using the winnonlin software (v 6.1; pharsight corp, mountain view, ca, usa). The below the limit of quantitation values occurring prior to the first measurable concentration were set to zero, and those occurring after the first measureable concentration were not included in the pharmacokinetic analysis . The cmax and time to cmax (tmax) were calculated directly from the concentration the area under the plasma concentration time curve (auc) from time 0 to 24 hours (auclast) was calculated according to the linear trapezoidal rule . Auc0 was calculated using the following equation: auc0 = auclast + ct/z, where ct is the last observed concentration and z is the elimination rate constant . The log - transformed cmax and auclast for clopidogrel, aspirin, and salicylic acid were compared using the average bioequivalence approach . This comparison was performed using analysis of variance (anova) with effects for sequence, subject within sequence, treatment, and period . Because, for logistical reasons, the two groups were administered the study drugs at different times, group effect was also included as a factor.12 using anova, the 90% confidence intervals (cis) of the least - squares geometric means were calculated for cmax and auclast . The fdc formulation was considered bioequivalent if the 90% cis of cmax and auclast for clopidogrel, aspirin, and salicylic acid fell within the range of 0.801.25.12 to evaluate the difference of tmax between coadministration and fdc, wilcoxon signed - rank test was used . All statistical calculations were performed using the sas software (v 9.3; sas institute inc . ). Sixty healthy men (30 subjects per group) were enrolled in the study and randomized the mean age of the subjects was 25.53 years (range, 2138 years), and the mean body weight was 69.95 kg (range, 50.387.5 kg). Three subjects withdrew consent during the study and four subjects withdrew because of laboratory abnormalities before dosing . Time profiles of clopidogrel, aspirin, and salicylic acid are shown in figure 1 . Descriptive statistics for the pharmacokinetic parameters of clopidogrel, aspirin, and salicylic acid are provided in table 2 . Similar to the coadministration of clopidogrel and aspirin, the tmax of clopidogrel and aspirin administered as fdc were 0.67 (p=0.85) and 4.00 hours (p=0.88), respectively . Anova indicated a lack of group, period, sequence, and treatment effects for both cmax and auclast . The mean t1/2 of coadministered clopidogrel was 6.31 hours, which was comparable with that of fdc clopidogrel at 5.21 hours . The mean t1/2 of aspirin was also similar for coadministration (0.65 hour) and fdc administration (0.63 hour). The 90% cis for the ratio (fdc / coadministration) of the geometric means for cmax, auclast, and aucinf are presented in table 3 . The mean log - transformed ratios of the primary parameters and their 90% cis were all within the predefined equivalence limit of 0.801.25 . No serious or significant aes occurred during the study . Two aes were reported for one subject (1.67%) after administration of fdc capsules: increases in alt and ast . Four aes were reported for three subjects (5% of study population) after coadministration of clopidogrel 75 mg and aspirin 100 mg: an increase in alt and ast (in the same subject), an increase in blood bilirubin, and catheter site pain . Of these aes, increases in alt and ast were considered possibly related to the study drugs and the others were assessed as unlikely to be or definitely not related to the study drugs . Sixty healthy men (30 subjects per group) were enrolled in the study and randomized the mean age of the subjects was 25.53 years (range, 2138 years), and the mean body weight was 69.95 kg (range, 50.387.5 kg). Three subjects withdrew consent during the study and four subjects withdrew because of laboratory abnormalities before dosing . Mean plasma concentration time profiles of clopidogrel, aspirin, and salicylic acid are shown in figure 1 . Descriptive statistics for the pharmacokinetic parameters of clopidogrel, aspirin, and salicylic acid are provided in table 2 . Similar to the coadministration of clopidogrel and aspirin, the tmax of clopidogrel and aspirin administered as fdc were 0.67 (p=0.85) and 4.00 hours (p=0.88), respectively . Anova indicated a lack of group, period, sequence, and treatment effects for both cmax and auclast . The mean t1/2 of coadministered clopidogrel was 6.31 hours, which was comparable with that of fdc clopidogrel at 5.21 hours . The mean t1/2 of aspirin was also similar for coadministration (0.65 hour) and fdc administration (0.63 hour). The 90% cis for the ratio (fdc / coadministration) of the geometric means for cmax, auclast, and aucinf are presented in table 3 . The mean log - transformed ratios of the primary parameters and their 90% cis were all within the predefined equivalence limit of 0.801.25 . Two aes were reported for one subject (1.67%) after administration of fdc capsules: increases in alt and ast . Four aes were reported for three subjects (5% of study population) after coadministration of clopidogrel 75 mg and aspirin 100 mg: an increase in alt and ast (in the same subject), an increase in blood bilirubin, and catheter site pain . Of these aes, increases in alt and ast were considered possibly related to the study drugs and the others were assessed as unlikely to be or definitely not related to the study drugs . The objective of the current study was to compare the pharmacokinetics of a newly developed fdc of clopidogrel 75 mg / aspirin 100 mg with that of coadministration of corresponding doses of clopidogrel and aspirin . Although exposure to clopidogrel can be highly variable with a coefficient of variance over 30%, we applied conventional bioequivalence criteria using a nonreplicative single - dose crossover design because of the narrow therapeutic window and safety issues with clopidogrel.13 pharmacokinetic analysis showed that the fdc capsule was bioequivalent to the coadministration of the individual drugs . In practice, clopidogrel is prescribed as a loading dose of 300600 mg, with 75 mg given subsequently as a maintenance dose . In this study, a single oral dose of 150 mg, half the loading dose, was administered because of the high intraindividual variability in the degree of absorption . Although the aspirin dose of 200 mg is twice the usual maintenance dose (corresponding to 75100 mg), it was considered acceptable because this was a single - dose study and aspirin shows linear pharmacokinetic characteristics.14 at the time this study was planned, there had been no studies investigating the intraindividual variability of the primary pharmacokinetic parameters of enteric - coated aspirin . Therefore, we calculated the sample size using the ratio of intra- and interindividual variability for the general formulation of aspirin.15 the sample size, based on an estimated intraindividual variability of cmax for aspirin of 38%, was calculated to be 24 subjects per group to detect a 20% difference between the test and reference treatments with a power of 80% at a significance level of 5% . Thus, 60 subjects were considered sufficient to compare the relative pharmacokinetics of fdc and the coadministration of individual formulations . Our extrapolated intraindividual variability is in agreement with the 36.68% for an enteric - coated formulation of aspirin estimated by jung et al.16 when we recruited subjects for this study, strict selection criteria were applied for alcohol uptake, smoking status, and other medications that have an effect on the clopidogrel - metabolizing enzyme cyp2c19, because variability in clopidogrel pharmacokinetics has been attributed to alcohol, smoking, cyp2c19 genotype, and genotype - related drug drug interactions . Clopidogrel is probably converted to ethyl clopidogrel in the presence of ethyl alcohol, the major ingredient of alcoholic beverages, causing a decrease in the total metabolism of clopidogrel . In addition, ethyl alcohol increases the cytotoxic effect of clopidogrel and the pharmacological activity by prolonged presence of clopidogrel by decreasing the total metabolism of clopidogrel (hydrolysis and transesterification) in the presence of ethyl alcohol.17 a potential role of smoking in the metabolism of clopidogrel and high on - treatment platelet reactivity were suggested . Pharmacodynamic studies and post hoc analyses of large clinical trials support a link between smoking status and the efficacy of clopidogrel therapy.18 in addition, clopidogrel is mainly metabolized to its active metabolite by cyp2c19,19 so it is important to consider comedication with drugs having the same metabolic route . The present study has several limitations . In clinical situations, dual therapy with clopidogrel and aspirin however, all participants of this study were healthy men, who may not represent the target patients, and only a single dose of treatment was given . Because the antiplatelet effect of clopidogrel decreases with patient age,20 further multiple dosing studies, preferably in patients with cardiovascular disease, are needed to apply the fdc to clinical practice . The results from the present study indicate that the clopidogrel 75 mg / aspirin 100 mg fdc capsule was bioequivalent to individual formulations of corresponding doses of the two drugs.
Cross sensitivity is defined as sensitivity to one substance that renders an individual sensitive to other substances of similar chemical structure . Cross sensitivity has been reported commonly among various lactam antibiotics and sulfonamides . For the diagnosis of cross sensitivity, various methods like prick test, oral challenge test is considered as the most reliable . Due to broad antimicrobial spectrum, frequently reported adverse reactions with fluroquinolones are nausea, abdominal pain, diarrhea, dizziness, restlessness, headache, depression and somnolence or insomnia . . Immediate type of reactions are more common than delayed type of reactions . Frequently reported immediate hypersensitive reactions are urticaria, anaphylactic shock, rashes and erythema . Ciprofloxacin and norfloxacin are the most common drugs causing hypersensitivity reactions, while levofloxacin is the least common with incidence of 1 per million population . However, we could not find a case of immediate hypersensitivity reaction with levofloxacin or case report of cross sensitivity between ciprofloxacin and levofloxacin . Here with, we report a case of cross sensitivity between ciprofloxacin and levofloxacin for immediate type of hypersensitive reaction . Seven years old male child (24 kg weight) admitted in paediatric ward, sir takhtsinhji general hospital, government medical college, bhavnagar, gujarat, india with complaints of fever, headache, vomiting and abdominal pain for 7 days . Fever was of high grade, intermittent and associated with chills, which was followed by colicky abdominal pain . He had a past history of similar attacks two times, at that time pain was relieved after oral treatment . Various investigations like hemoglobin, total and differential wbc count, platelet count, esr, urine and stool examination were normal . As per surgical opinion conservative treatment was started with ciprofloxacin infusion (20mg / kg / day in two divided doses), inj . Metronidazole (30mg / kg / day, in three divided doses), inj . Five minutes after starting infusion of ciprofloxacin, patient developed itching followed by rashes at the site of infusion . On dermatological examination, reaction started from the site of injection and spread upward involving whole arm up to chest . Infusion was stopped immediately and injection dexamethasone (0.5mg / kg) was given along with injection chlorphenaramine maleate (0.1mg / kg). Levofloxacin (10mg / kg / day) was added in treatment in place of ciprofloxacin from next day under close observation . Immediately after levofloxacin infusion, itching and rashes in arm appeared so, infusion was withdrawn . Ceftriaxone (100mg / kg / day) injection added in the place of fluoroquinolones along with metronidazole . It was moderate in severity according to modified hartwig and siegel's scale and non preventable as per modified schumock and thornton scale . All fluoroquinolones have similar core structure (4-oxo-1, 4-dihydroquinoline ring) with fluorine atom attached at position 6 except, first generation quinolone, nalidixic acid . Norfloxacin, a second generation fluroquinolone, is the result of replacement at c-7 methyl side chain with piperazine group, while replacement of n-1 ethyl group of norfloxacin with cyclopropyl group generates ciprofloxacin . Levofloxacin, third generation flouroquinolone, has modification of piperazine group with methyl group at c-7 . Although changes in position 1, 7 and 8 generated different fluoroquinolones, core structure remains same . 54.5% immediate type of reactions to fluoroquinolones are ige mediated and occurs due to binding of ige to 7 position of core structure of fluoroquinolones . Patient had a past history of two similar attacks, at that time he might be treated with fluoroquinolones, which may resulted in production of ige antibodies . As a result, treatment with fluoroquinolones this time might have caused the reaction . Ciprofloxacin and levofloxacin have similar core structure; this may be the reason for the cross sensitivity in this case . It is advisable to avoid other fluoroquinolones when hypersensitivity reaction detected to one and should be shifted to other class of antimicrobials . Overall risk of adverse effects especially hypersensitivity reactions with fluoroquinolones is low but due to widespread use, such rare hypersensitivity reactions should be kept in mind . Improvement in diagnostic tests like detection of ige antibodies, cellular tests and skin test may help in preventing such adverse drug reactions.
Amblyopia is the leading cause of decreased vision among children, with refractive errors as the most important cause of this condition . Because of the importance of early diagnosis and treatment of amblyopia, however, this method is limited because it requires an experienced examiner and an excessive amount of time . To overcome these limitations, several photorefractors were developed . Among these was the plusoptix s09 photorefractor (plusoptix gmbh, nuernberg, germany), developed for children and the disabled, and is a noninvasive tool for the rapid assessment of both eyes for refractive errors, pupil size, and inter - pupillary distance . In total 77 eyes in 40 children aged 2 to 10 years were evaluated in uijeongbu st . This study was conducted according to the guidelines of the association for research in vision and ophthalmology and the 1975 declaration of helsinki . Children were excluded when they had eccentric fixation, optical media opacity, or exceeded the plusoptix s09 manufacturer's recommendations of a refractive error of a maximum spherical range of 7.00 to + 5.00 d and a pupil size range of 3 to 8 mm . The patients were categorized according to 2 methods: (i) 11 eyes with hyperopia +3.0 d and 66 eyes with hyperopia <+ 3.0 d, emmetropia, and myopia; (ii) 12 eyes with cycloplegic and noncycloplegic refraction of spherical power +2.0 d and 65 eyes with cyclopegic and noncycloplegic refraction of spherical power <+ 2.0 d. all refractive errors (spherical power, cylinder power, and spherical equivalent) were determined in the following order: (1) photorefraction without cycloplegia using the plusoptix s09; (2) autorefraction without cycloplegia (noncycloplegic) using the cannon autorefractometer (rk - f1, canon, tokyo, japan); (3) cycloplegic autorefractometer using 1% cyclopentolate (1% cyclogyl). All cycloplegic autorefractomery was performed within 30 minutes of the use of 1% cyclopentolate 3 times per 5 minutes . Comparisons between the measurements were performed using paired t - tests and pearson's correlation analysis . All statistical analyses were performed using the spss statistical package 19 (spss for windows, chicago, il). A value of p <0.05 was taken to indicate statistical significance . There was no statistical difference between the 11 eyes with hyperopia of +3.0 d and the 66 eyes with hyperopia of <+ 3.0 d, emmetropia and myopia for age and sex . In addition, there was no statistical difference between the 12 eyes with cycloplegic and noncycloplegic refraction of spherical power +2.0 d and the 65 eyes with cyclopegic and noncycloplegic refraction of spherical power <+ 2.0 d (table 1). There was no difference between the plusoptix s09 and cycloplegic autorefractometer for both spherical power and equivalent in all of the children . In contrast, there was a statistical difference between the plusoptix s09 and noncycloplegic autorefractometer (p <0.001) (table 2). Pearson's correlations between the plusoptix s09 and cycloplegic autorefractometer for spherical power, cylinder power, and spherical equivalent were 0.748, 0.893, and 0.782, respectively, whereas between the plusoptix s09 and noncycloplegic autorefractometer these were 0.559, 0.870, and 0.580, respectively . These results indicate that the spherical power and spherical equivalent determined using the plusoptix s09 correlated more closely with those determined using the cycloplegic autorefractometer than using the noncycloplegic autorefractometer (table 3). Although there was no statistical difference between the plusoptix s09 and cycloplegic autorefractometer in the spherical power and spherical equivalent in all of the patients (table 2), the mean spherical power and equivalent recorded using the plusoptix s09 was 2.91 0.90 d and 2.22 0.93 d, respectively, whereas these recorded using the cycloplegic autorefractometer were 4.66 0.93 d and 4.25 0.80 d in hyperopia of +3.0 d, respectively . There was a statistical difference between the plusoptix s09 and cycloplegic autorefractometer for the spherical power and spherical equivalent in hyperopia of +3.0 d (paired t - test, p <0.05). In addition, in patients of cycloplegic and noncycloplegic refraction of spherical power +2.0 d, there was a statistical difference between the plusoptix s09 and cycloplegic autorefractometer for the spherical equivalent (paired t - test, p = 0.015) (table 4). There was significant difference between the plusoptix s09 and cycloplegic autorefractometer according to the scatter plot for spherical power and equivalent in hyperopia of +3.0 d (fig . Cpr = cycloplegic refraction, se = spherical equivalent, sp = spherical power . Amblyopia is the leading cause of decreased vision among children and affects 2% to 5% of the population . Early diagnosis and treatment may lead to better visual outcomes and decrease the severity and prevalence of amblyopia . Because of the importance of early diagnosis, several screening methods for amblyopia are used . However, this method is limited because it requires an experienced examiner, is time consuming, and in general needs sedation of children <3 years old with limited co - operation . In contrast, the photo refractor is easy to use in examining younger children without the need for cycloplegic drops or sedation . Although an ideal screening test would have 100% specificity, 100% sensitivity, and 100% positive predictive value, there is no screening method with this level of accuracy . Many studies have shown the plusoptix to be a rapid, accurate, and noninvasive refractometer in preschool children and in individuals with intellectual or physical disabilities, and it has excellent specificity and sensitivity . The mean spherical power and spherical equivalent recorded using the plusoptix s09 were 1.53 2.01 d and 1.33 1.97 d, respectively, whereas using the cycloplegic autorefractometer they were 0.61 2.02 d and 0.73 2.05 d, respectively, in all the children, which were not significantly different between the 2 devices (paired t - test, p> 0.05). We observed significant pearson's correlation coefficients of 0.748, 0.893, and 0.782 between the plusoptix s09 and cycloplegic autorefractometer for spherical, cylindrical, and spherical equivalent refractive errors, respectively . These values are similar to those of 0.76, 0.86, and 0.76, respectively, reported by rajavi et al . In contrast, the mean spherical power and spherical equivalent were significantly different using the plusoptix s09 (2.91 0.90 d and 2.22 0.93 d, respectively) and the cycloplegic autorefractometer (4.66 0.93 d and 4.25 0.80 d, respectively) in hyperopia of +3.0 d, (paired t - test, p <0.05). Because accommodative capacity leads to a myopic shift in over - moderate hyperopia, a statistical difference between the plusoptix s09 and cycloplegic autorefractometer for the spherical power and equivalent was expected . This shift was reported to be 3.0 d and 1.9 d in some children by erdurmus et al and dahlmann - noor et al, respectively . Schaeffel et al demonstrated a myopic shift of 2.4 d in children wearing + 3.0 d glasses with noncycloplegic photorefraction . In conclusion, the plusoptix s09 can provide a rapid and easy method of refraction for amblyopia screening in young children and detecting risk factors for refractive amblyopia . However, when it is used to estimate the refractive error in a child with hyperopia of +3.0 d, the refractive error should be corrected after a comparison of the refractive errors of the plusoptix s09 with those of a cycloplegic autorefractometer.
Recently, magnetic thin films used in magnetic components, such as inductive devices and magnetic heads, have attracted more and more attention because of the increasing demand for improvement and miniaturization of electromagnetic devices [1 - 3]. The basic requirements for these films operated in the ghz range are high resistivity, high permeability, high saturation magnetization ms and appropriate large anisotropy field hk so as to effectively suppress eddy current loss and to possess high ferromagnetic resonance (fmr) frequency fr (which determines the cut - off frequency for high frequency application). Insulator granular films (migf) consisting of magnetic metal nano - granules uniformly distributed in an insulator matrix are one of the best candidates for satisfying above demands, because these films have the advantages of high ms and high of magnetic metals and high of insulator . Up to now, soft magnetic properties of granular films composed of magnetic metals (and their alloys) and various x-(oxide, nitride and fluoride) where x is nonmagnetic elements such as hf, al, si, zr, rare earth, etc . Have been investigated [4 - 8]. The mechanism of the soft magnetic properties of migfs has been confirmed to be the exchange coupling between granules through the intergranular regions . However, the microscopic origin of exchange coupling has not been totally understood, such as the medium of the exchange coupling for different constitution nano - granular films . By considering the high resistivity of semiconductor oxides, metal semiconductor granular films (msgf) consisting of magnetic metal nano - granules uniformly distributed in semiconductor matrix, such as the feco zno systems, may possess good high frequency properties . As one kind of wide used semiconductor oxides due to the excellent electrical and optical properties, the current carriers of zno may mediate the exchange coupling between magnetic granules and hence favor good soft magnetic properties . So far, there is no work studying systematically the soft magnetic properties of the feco zno granule films reported . In order to test whether zno can mediate the exchange coupling, the structure, magnetic properties and electronic transport properties of feco zno msgfs were systematically studied in this work . (fe65co35)x(zno)1x granular thin films were deposited on water - cooled glass substrates by radio frequency (rf) magnetron sputtering at room temperature with a background pressure lower than 4 10 pa . A three - inch fe65co35 alloy disk covered with zno chips (5 5 1 mm) the sputtering power was 100 w, and sputtering ar gas was kept at 0.40 pa . An external magnetic field obtained by a pair of permanent magnets located on both sides of the substrates was applied during deposition to induce an in - plane uniaxial anisotropy for the samples, and the intensity of external magnetic field is 500 oe . The saturation magnetization 4ms, coercivity hc and in - plane magnetic anisotropy field hk were determined by a vibrating sample magnetometer (vsm, lakeshore 7304 model). The composition of the samples was measured by energy dispersive x - ray spectroscopy (eds). The structure and microstructure were analyzed by x - ray diffraction (xrd) (philips xpert model) with cu - k radiation and high resolution transmission electronic microscopy (hrtem), respectively . The permeability spectra were carried out with a pna e8363b vector network analyzer using the shorted microstrip transmission - line perturbation method, which works from 100 mhz to 5 ghz . All the above measurements were performed for as - deposited samples without any post - heat treatment . Figure 1 shows the bright - field hrtem image (a) and the corresponding electron diffraction (ed) pattern (b), as well as the xrd spectrum (c) for a typical sample of x = 0.67 . It can be seen that the sample consists of bcc fe65co35 particles embedded uniformly in the insulating zno matrix from the image (a). The average size of fe65co35 particles and the distance between them are about 2 and 4.5 nm, respectively . Figure 1b exhibits the diffraction peaks from (110) and (200) planes of bcc fe65co35 alloys, (111) and (103) planes of zno, respectively, which are totally consistent with those of xrd shown in fig . The average diameter of fe65co35 grains calculated by the full width at half maximum (fwhm) of the diffraction peak in xrd spectrum through scherrer s equation is about 2.1 nm, which is close to that of fe65co35 granules . Hrtem image (a) and the corresponding ed pattern (b) as well as the xrd spectrum (c) for a typical sample of x= 0.67 figure 2 expresses the x dependence of hc, hk and . It is seen that increases slowly with decreasing x and then increases rapidly after x <0.64 . Hc is less than 15 oe, and hkis over 43 oe in the range 0.63 <x <0.72, which means good soft magnetic properties are obtained in this range . Especially, for the sample with x = 0.67, its in - plane hysteresis loop shown in fig . The coercivities in hard and easy axes are 1.43 and 7.08 oe, respectively, 4ms = 9.85 kg, and reaches 2.06 m cm, which is three orders higher than that of pure feco alloy . These good soft magnetic properties and the high imply that the films are promising for high frequency applications . H curves for (fe65co35)0.67(zno)0.33 film figure 4 shows the dependence of complex permeability = j on frequency f for the film of x = 0.67, where and represent the real and imaginary parts of complex permeability, respectively . It can be seen that is more than 100 below 1.83 ghz and then gradually decreases with frequency, while the imaginary part gradually increases to a maximum at f = 2.31 ghz, which can be ascribed to the ferromagnetic resonance (fmr). Lifshitz equations taking account of the coherent spin procession, as shown by the solid lines in fig . The fmr frequency obtained by this fitting is around 2.31 ghz, consistent with the experimental result . The high fmr frequency also implies that the granular films are promising for applications in the high frequency range . Permeability spectra (~ f) for the sample of x = 0.67 considering the mechanism of the good soft magnetic properties, we turn to the exchange coupling model . According to the herzer s statement, if the average size d of magnetic particles and the distance s between particles are reduced to be smaller than a characteristic length, namely, the exchange length lex, the exchange interaction between particles may take place, which tends to align the magnetic moments of neighboring particles parallel and so overcome the magneto - crystalline anisotropy and the demagnetization effect of individual particles . As a result, the effective magnetic anisotropy of materials is reduced significantly, which leads to the decrease in coercivity and hence the increase in permeability, and then good soft magnetic properties are obtained . For fe65co35, the characteristic length lex is about 26 nm, while the d and s are 2.3 and 4.5 nm, respectively, based on the results of hrtem spectrum . Thus, (d + s) lex holds, exchange coupling can be realized in this film, which is responsible for the good soft magnetic properties . In order to study the microscopic origin of the exchange coupling between granules, the temperature dependence of resistivity figure 5a depicts thecurve, which exhibits a negative temperature coefficient of resistivity (tcr). 5b, which indicates that the mechanism of electronic transportation of the sample is weak localized electron electron interaction . These weak localized electrons may also be the medium of exchange coupling between magnetic granules . Resistivity as a function of temperature (a) and fitting result (b) for a typical sample of x = 0.67 (fe65co35)x(zno)1x granular films were fabricated by magnetron sputtering, and good soft magnetic properties have been obtained in x range from 0.63 to 0.72 with small hc and appropriate large hk . For the typical sample of x = 0.67, the coercivity of 1.43 oe in the hard axis and 7.08 oe in the easy axis are obtained, and the resistivity reaches 2.06 m cm . At a frequency lower than 1.83 ghz, the real part of the complex permeability of this sample is more than 100, and the fmr frequency reaches 2.31 ghz, which implies that the film is promising for high frequency applications . The good soft magnetic properties origin from the exchange coupling between magnetic granules and the weak localized electrons existing in films may be the medium of this exchange coupling . This work was supported by national natural science foundation of china (grant no.50571041). This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Genomic structures corresponding to crisprs were observed first in 1987 in escherichia coli (1) and were subsequently reported in other organisms under different names [trep (2), srsr (3,4), drvs (5), lctr (6), spidr (7)] until the crispr acronym was proposed by jansen et al . The direct repeat sequences carry in general a low level of palindromic symmetry; they are remarkably well conserved within a species (up to 248 exact copies in verminephrobacter eiseniae ef01 - 2). However, one of the flanking drs is frequently truncated or diverged (see supplementary data). The dr size varies from 24 to 47 bp whereas the spacer sequence is generally within the range of 0.62.5 the dr size . The originality of spacers is that they apparently derive from conjugative plasmids or bacteriophages (2,911). A prokaryotic genome may harbour up to 16 crispr clusters with the same or a different dr . In a genome, a single crispr is generally associated with a family of genes called cas for crispr - associated (8,12), encoding proteins showing functional similarity with components of the eukaryotic rna interference (rnai) systems (13). In addition, it was demonstrated in two archaea, archaeoglobus fulgidus (14) and sulfolobus solfataricus (15), that the crispr locus is transcribed into small rnas (smrna) probably from one of the flanking regions, the leader, acting as a promoter . These observations and the viral origin of spacers have led to the hypothesis that the crispr - associated system (cass) is a prokaryotic defence mechanism against genetic aggressions (10,13,16). Within species, crisprs may be present in a subset of strains, where they sometimes show polymorphism . The dr and the order of the spacers are well conserved, but the number of motifs (dr + spacer) differs from strain to strain . To better understand the mechanisms underlying the crisprs evolutionary scenario, three evolution rules were proposed by pourcel et al . (15): (i) polarized acquisition of spacers near the leader sequence; (ii) random loss of motifs and (iii) shared ancestry when spacers are identical . Crisprs in silico analyses started in 1995 (2) but no specific stand - alone crispr software tool was created . Several software were used by different authors to identify these particular repeats but usually a manual discard of background was necessary, and generally some crispr clusters were missed or neglected, especially the shortest one (less than three motifs). This is the case, for example, of tandem repeat finder (17) when considering a motif (dr + spacer) as a degenerate repeat (10,18), or locating uniform poly - nucleotide areas (luna), a program for finding degenerate repeats in microbial genomes on a desktop computer . The repeats can be filtered using several parameters including length, distance and level of conservation . Another program, patscan (19) a pattern - matching tool that searches sequences fitting the introduced pattern, was applied to identify crisprs containing at least three (20) or four exact direct repeats (8). Pygram (21) is a visualization program browsing all the repeats in the submitted genomic sequence and showing perfectly conserved palindromic repeats as pyramids . The pygram program is mostly efficient in visually displaying large crisprs (crisprs with as many as seven motifs are considered as being very short in this work) since they will be recognized as a concentration of horizontal bars referring to a group of co - occurring repeats that differ by only a few nucleotides . Finally, haft et al . (12) used repfind (http://bibiserv.techfak.uni-bielefeld.de/reputer/), a part of the reputer package (2224) and blastn to identify smaller repeat clusters . These programs are the most used tools in crispr detection, although none of them is especially conceived for this purpose . They require further manual manipulations to eliminate background data (tandem repeats for example) and importantly, do not define accurately the dr consensus (due to errors on the boundaries). Recently, two crispr- dedicated software tools were proposed, crt (http://www.room220.com/crt) and piler - cr (25). Both of them run fast and perform well in finding crisprs . However, crt results in a considerable background since tandem repeats are considered as putative crisprs and in addition, the same crispr is sometimes detected more than once with different consensus drs . Piler - cr has also some drawbacks since it often misidentifies the dr boundaries and omits the truncated dr . A specialized program to automatically identify crisprs seems to be mandatory for their optimum, rapid exploration and in - depth analysis, in order to increase the efficiency of crisprs investigations . Crisprfinder is a web service offering fundamental tools for crispr detection, including the shortest ones, allowing an accurate definition of the dr consensus boundaries and extraction of the related spacers . It offers also additional tools to analyze the crispr loci: (i) obtain the crispr and the flanking sequences according to flexible size; (ii) make a blast of selected spacers or flanking sequences against the genbank database and (iii) check if the dr is found elsewhere in prokaryotic sequenced genomes . The input of the web tool is a genomic query sequence of length up to 67 mb in fasta format . Possible locations of crisprs (consisting of at least one motif) are detected by finding maximal repeats . A maximal repeat (26) is a repeat that cannot be extended in either direction without incurring a mismatch . The total number of maximal repeats in a sequence of size n is linear (less than n) which is interesting since the computation may be done in linear time using a suffix - tree - based algorithm . A crispr pattern of two drs and a spacer may be considered as a maximal repeat where the repeated sequences are separated by a sequence of approximately the same length . The operation of the program can be divided into four main steps summarized in figure 1: (step 1) browsing the maximal repeats of length 2355 bp interspaced by sequences of 2560 bp, (step 2) selecting the dr consensus according to a defined score taking into account the number of occurrences of the candidate dr in the whole genome and privileging internal mismatches between the drs rather than mismatches in the first or the last nucleotides, (step 3) defining candidate crisprs after checking if they fit crispr definition, (step 4) eliminating residual tandem repeats . (step 1) browsing the maximal repeats to get possible crispr localizations using the vmatch program . (step 2) consensus dr selection according to candidate occurrences and a score computation: the score privileges internal mismatches between direct repeats of a cluster rather than boundary mismatches . (step 1) browsing the maximal repeats to get possible crispr localizations using the vmatch program . (step 2) consensus dr selection according to candidate occurrences and a score computation: the score privileges internal mismatches between direct repeats of a cluster rather than boundary mismatches . (step 4) tandem repeats elimination using clustalw for aligning spacers . In the first step, maximal repeats are found by the software vmatch (http://www.vmatch.de/), the upgrade of reputer (2224). Vmatch is based on a comprehensive implementation of enhanced suffix arrays (27) which provides the power of suffix trees with lower space requirements . A one nucleotide mismatch is allowed permitting minimal crisprs with a single nucleotide mutation between drs to be found . Hereafter, the obtained maximal repeats are grouped to define regions of possible crisprs with a display of consensus dr candidates related to each cluster . The difficulty resides especially in the identification of boundaries, which is very important to extract the correct spacers and compare drs . In fact, the consensus dr is selected as the maximal repeat which occurs the most in the whole underlying genome sequence with respect to the forward and the reverse complement directions (since two crisprs having the same dr consensus may be in opposite directions). Thus, ambiguity in the choice of a dr will be eliminated in the case of presence of similar drs in other crisprs of the related genomic sequence . However, if occurrence numbers are equal, more than a single dr consensus candidate are kept and later compared . Given a candidate consensus dr, the pattern search program fuzznuc of the emboss package (28) is applied to get drs positions in the related cluster . As the first or the last dr in a crispr may be diverged / truncated, a mismatch of one - third of the dr length is allowed between the flanking drs and the candidate consensus dr, whereas smaller nucleotide differences are allowed between the other drs to take into account possible single mutations . In case of multiple dr candidates, a score is computed and the best one (minimum) is picked . This score favours candidates which are encountered more frequently, rather than consensus dr showing less internal mismatches . Once the dr consensus is determined, the corresponding spacers (step 3) are extracted according to the dr boundaries determined previously . The spacer length is not allowed to be shorter than 0.6 or longer than 2.5 times the dr length . Therefore, tandem repeats are eliminated by comparing the consensus dr with the spacer if there is only one spacer, or by comparing spacers between each other . The comparison is done with the clustalw program (29) and the percentage of identity between spacers is not allowed to exceed 60% . Finally, candidates having at least three motifs and at least two exactly identical drs are considered as confirmed crisprs . These should be critically investigated by, for example, checking for intraspecies size variation of the locus . Ns characters are accepted, iub / gcg letters (mrwsykvhdbx) will be converted to ns and considered as mismatches but any other characters will be deleted . One can either paste the genomic sequence into the input field or upload it from a file on the local machine . Users may use the default version or click on the advanced version link to set and modify all the program parameters, which may be especially useful for fixing the dr size . After querying a genomic sequence by crisprfinder, results are summarized in a table with the number of confirmed and questionable crisprs (figure 2a). A crispr locus is presented according to a colour code showing drs in yellow and spacers in different colours . The respective positions are displayed, in addition to links to two files: a summary of the displayed properties (number of motifs, dr consensus, positions, etc .) And a fasta file containing the list of spacers . In addition, a png (portable network graphics) figure displays the different candidates location in the analysed sequence . In the case of presence of crispr clusters, further analysis may be done through three hyperlinks in the left menu: (i) blast spacers against the genbank databases with a cutoff of 0.1 for the e - value and a matching length of at least 70% the queried spacer size (figure 2b); (ii) obtain crispr and flanking sequences which are especially useful to define the leader sequence . As the size of the leader sequence depends on the species (it varies from 100 to 500 bp), the retrieved sequence may be manually modified by the user (figure 2c) and (iii) display identical drs in other known crispr loci (figure 2d). Submitted for publication). Figure 2an example of crisprfinder output using the aquifex aeolicus vf5 genomic sequence (refseq: nc_000918). (panel a) (1) home page where the genomic sequence is submitted . (2) table listing the detected crisprs candidates (questionable and confirmed) providing links to each one . (3) crisprs details, the dr is showed in yellow and the spacers in different colours . (4) a fasta file displaying the first crispr spacers . (5) figure showing the aquifex circular chromosome with crisprs positions . (panel b) one or several spacers may be blasted against ncbi databases by clicking on the blast_spacers button . (panel c) the flanking and the crispr sequences may be viewed by clicking on the get sequences button . (panel d) the list of consensus drs for all crisprs is shown with a link to identical drs in the crispr database . An example of crisprfinder output using the aquifex aeolicus vf5 genomic sequence (refseq: nc_000918). (panel a) (1) home page where the genomic sequence is submitted . (2) table listing the detected crisprs candidates (questionable and confirmed) providing links to each one . (3) crisprs details, the dr is showed in yellow and the spacers in different colours . (4) a fasta file displaying the first crispr spacers . (5) figure showing the aquifex circular chromosome with crisprs positions . (panel b) one or several spacers may be blasted against ncbi databases by clicking on the blast_spacers button . (panel c) the flanking and the crispr sequences may be viewed by clicking on the get sequences button . (panel d) the list of consensus drs for all crisprs is shown with a link to identical drs in the crispr database . Crisprfinder is a program that allows the identification of structures with the principal characteristics of crisprs, the smaller being composed of a truncated or diverged dr, a spacer and a complete dr . In their analysis, 20) using patscan had chosen to retain only crisprs with at least three exact repeats (eliminating crisprs constituted of a first truncated repeat plus two exact repeats) thus ignoring most crisprs containing less than three spacers . Similarly in the work by durand et al. (21), the pygram program is mostly efficient in visually displaying large crisprs . Such stringent criteria were appropriate in order to avoid ambiguities in early investigations which were essentially describing these new structures . However, it is now important, in order to better understand the evolution and spreading of crisprs, to provide tools which will not eliminate the smallest crisprs . The major drawback is that when looking for the shortest structures, such as those with a unique spacer, it is clear that the background of spurious candidates can be very high . The output of patscan and crt also contains a large quantity of noised data that needs a manual treatment . We provide several samples on the website as demonstrators . Upon submission of the complete genome of aquifex aeolicus vf5 (sample1), on the contrary, while using the webservice for patscan (http://www-unix.mcs.anl.gov/compbio/patscan/), it is necessary to first define a pattern (which is not straightforward) and it is not possible to seek for crisprs in a single genomic sequence but rather in an entire predefined database . Crt requires either to install jre (java runtime environment) or compile the source files, and piler - cr needs to be compiled before use . A comparison between layouts of available online programs (reputer, patscan, trf) and of crisprfinder is provided in the supplementary data . To check that crisprfinder was efficient in recovering all the crisprs from a genome the data were generally in good agreement, the differences being always in the dr boundaries identification (more accurate with crisprfinder) or in the number of motifs found, as the truncated dr is sometimes neglected or short clusters are not detected with other programs . Interestingly, some strains were claimed to be devoid of crisprs by godde and colleagues but proved to have short crisprs with crisprfinder, such as in different shigella sp . (s. sonnei ss046, s. flexneri 2a str . 301, s. flexneri 2a str), or even long crisprs such as in pseudomonas aeruginosa ucbpp - pa14 . The latter example is shown on the crisprfinder website (sample2), and as can be seen by using the blast spacer function, six spacers out of thirty six at two different crispr loci, correspond to a bacteriophage sequence (bacteriophages f116, b3, d3112, dms3 and phi ctx). Furthermore, the possibility to identify crisprs containing one or two motifs may help understand how new crisprs are created . The very small candidates will need to be typed across different isolates within the same species or very closely related species to search for variations . For instance, as shown with the sample file provided on the website (yp1 yersinia), five yersinia pestis strains possess at the same crispr locus two to eight spacers, some being unique and others shared by two or more strains (10). This strain - dependent polymorphism is especially interesting for epidemiological and phylogenetic studies (30,31). A tool to easily create a dictionary of spacers from different strains is proposed in a crispr - dedicated web database (http://crispr.u-psud.fr/crispr/). The crisprfinder web server is an interface to extract with precision and to further analyse crisprs from genomic sequences . Four main advantages may be cited: (i) short crispr - like structures are detected, they are labelled questionable but may be of great interest if later confirmed; (ii) drs are accurately defined to single base pair resolution; (iii) summary files may be uploaded (crispr properties summary and spacers file in fasta format) and (iv) flanking sequences or spacers can be easily extracted and blasted against different databases.
Every year, approximately 7.7 million individuals develop dementia or alzheimer's disease, a number that translates to one new case every 4 seconds . This figure is expected to increase as assessments and diagnostic criteria improve and as life expectancies continue to increase . Dementia affects more people in latin america (8.5%) than it does in the united states (6.5%) and western europe (6.9%), and since dementia is more prevalent among older adults, the regions expected to be most heavily affected by the disease are the regions with a projected population increase in this age demographic . Countries in latin america and the caribbean are expected to experience huge increases in the population group of individuals aged 60 and older in the next 40 years; the number of older adults is expected to quadruple in this time period, reaching about 186 million, with older adults outnumbering the young population by approximately 30% . Dementia is a syndrome that affects several areas of the brain and leads to a decrease in cognitive functioning usually characterized by progressive deceleration of brain function over time . The primary symptoms of dementia include impairments in memory, language and/or communication difficulties, visual perception problems, a decreased ability to focus or pay attention, difficulties in planning or problem solving, and changes in mood and personality . Short - term memory problems, especially, make it difficult for people with dementia to carry out everyday activities on their own . As a result of the progressive nature of the disease dementia caregiving is usually the responsibility of the spouse of an individual with dementia or an adult child . Caring for aging adults with dementia is associated with increases in burden, distress, and decrements in mental health and well - being . Studies generally suggest that caregiving for individuals with dementia is more stressful than caregiving for individuals with many other diseases . This is because dementia caregiving is characterized by specific problems such as the lack of free time, isolation from others, behavioral problems and personality changes, and fewer positive experiences resulting from the lack of expressed gratitude by the care recipient . Given that the vast majority of individuals with dementia are assisted by family members or other informal caregivers, it is important to consider the role of family dynamics in the caregiving experience . Family communication, adaptability / flexibility, and marital cohesion, for example, have all been connected to the emotional functioning of caregivers . Research utilizing a structural family framework has shown that family functioning plays a significant role in the stress processes of dementia caregivers . Depression and anxiety, for instance, are more likely to occur among caregivers in families with poor functioning, and conflicted family dynamics can intensify caregiver depressive symptoms and caregiver strain . Similarly, the poor functioning of families is also likely to result in a decrease in the time spent on patient care, potentially impacting the quality of care the individual with dementia receives . Conversely, healthier family dynamics, such as family support, are associated with lower levels of caregiver strain . For example, when families give more support to primary caregivers, they often provide more help to the individual with dementia . Caregivers experience less burden and depression when family cohesion is high, and greater family communication also plays an important role in reducing caregiver burden . When caring for a family member with dementia, culture and ethnicity play a role in the caregiver's stress and coping processes . According to the sociocultural stress and coping model, culture influences the perception of social support and the appraisal of stressors, which may impact the well - being of caregivers [2527]. Latino cultures in particular place more emphasis on the family with the cultural value of familismo, defined as strong identification and attachment of individuals to their nuclear and extended families and feelings of loyalty, reciprocity, and solidarity among family members, having an important role . Familismo values encourage the reliance on family members for support, feeling the obligation to care for family in need, and the reliance on relatives to provide guidance on life [28, 29]. Because latinos report stronger commitment to their families, they may have different caregiving experiences compared to caregivers in more individualistic cultures . For example, latino dementia caregivers are expected to avoid placing their family members in nursing homes or adult day care . Since family - centered care is highly favored in latin america, caregivers who consider placing a relative at such facilities often experience feelings of guilt and shame . Subsequently, informal caregivers in latin america spend more time caregiving compared to other racial / ethnic groups and experience poorer mental health outcomes . Additionally, the culture of familismo influences the emotional distress experienced by latino dementia caregivers through dysfunctional thoughts . These dysfunctional thoughts are generally related to depressive symptoms and, among alzheimer's caregivers, to poor health outcomes . Consistent with the sociocultural stress and coping model, caregivers of people with dementia appraise stressors in a variety of ways that may also aid in buffering the negative effects of caregiver burden . For instance, the literature identifies resilience, optimism, and sense of coherence as three of the most important personal strengths exhibited by caregivers that assist in adaptation to caregiver stress [37, 38]. Research on caregiver resilience shows that it is more related to caregiver variables than to situational variables . Similarly, optimism and sense of coherence are caregiver variables related to coping adaptations . As such, all three personal strengths are expected to help buffer the negative effects of caregiver burden . These personal strengths are also of interest to family caregivers as research has shown that family functioning influences the development of coping strategies such as resilience and sense of coherence . And in the context of family dementia caregivers, optimism has been related to caregiver burden, health, and feelings of anger . As such, the impact of family dynamics on a caregiver's adaptation to stress may be especially relevant for latino caregivers as the role of familism is likely to affect their coping style and personal strengths . Resilience is a psychological phenomenon characterized by effective coping and adaptation in the face of loss, hardship, or adversity . In the context of dementia, resilience is believed to be a protective factor for caregiver stress and is related to lower levels of depression and better emotional and physical health outcomes for caregivers . Higher levels of perceived control and the belief that life's challenges are opportunities to increase skills and self - knowledge are some characteristics associated with increased resilience . High levels of social support, especially from within the family, are also associated with higher resilience . Optimism is a personality trait characterized by a disposition to expect positive outcomes in the face of adversity and hardship . Optimism is associated with more positive affect, less negative affect, and better mental health . Caregivers high in trait optimism view their own coping behaviors more positively and are more likely to perceive their coping strategies as effective . Optimism is associated with decreased stress and fewer symptoms of depression, and optimistic caregivers experience more positive affect, less negative affect, and better mental health . Optimism is also shown to be related to greater satisfaction in martial and child - parent relationships and healthier family communication [48, 49]. Sense of coherence is a personality trait that includes a set of positive coping strategies which enable people to use available resources efficiently and has often been used in the research literature interchangeably with the term coping . Sense of coherence is composed of three factors: comprehensibility (the feeling that the world makes sense and that information regarding the environment is ordered, consistent, and explainable), manageability (the feeling that adequate resources are available for meeting internal and external demands), and meaningfulness (the feeling that these internal and external demands deserve investment and engagement) [50, 51]. Higher sense of coherence benefits dementia caregivers in many ways and has been consistently associated with lower burden, anxiety, and depression . Within the context of the family, greater sense of coherence the relation between depressive symptoms and caregiver strain has been found to be mediated by sense of coherence . Higher sense of coherence is thus characterized by a salutogenic (promoting health) orientation toward stressors . Although latin america is expected to experience increases in dementia rates, not enough research has been conducted on dementia caregivers in the region . The existing research has primarily operated from a deficit model examining the negative aspects of care such as burden, depression, and other psychosocial problems in caregivers . Furthermore, very little research has examined particular cultural strengths that may aid in dementia caregiving in latin america, and no studies have been conducted on the relationship between family dynamics and personal strengths in this population despite the research documenting the importance of family values in this population . The purpose of this study is to examine whether healthier family dynamics are associated with higher sense of coherence, resilience, and optimism in dementia caregivers in latin america . It is hypothesized that healthier family dynamics will be related to a higher sense of coherence, greater resilience, and more optimism . From the research on the significance of family functioning in reducing caregiver burden [19, 20, 22, 23], family pathology and cohesion are hypothesized to be uniquely related to greater resilience, sense of coherence, and optimism . Consistent with prior work, greater family communication is also hypothesized to be uniquely associated with resilience, sense of coherence, and optimism . Finally, empathy, a factor associated with social support, is also hypothesized be related to resilience and sense of coherence . Caregivers of individuals with dementia (n = 105) from argentina participated in this study . For a summary of caregiver demographics recruitment of participants was from the instituto de neurosciencias de san lucas in rosario, argentina . Caregivers were identified as individuals caring daily for an individual with dementia, and the inclusion criteria were as follows: being at least 18 years of age, identifying as primary caregiver of the person with dementia, providing care for a minimum of three months, being well - informed about the patient's medical and family history, and not having a history of serious psychiatric or neurological disorders . For all participants, informed consent was provided and the data were collected according to the institutional review board approval at the university of deusto . Many measures had spanish versions readily available including the family adaptability and cohesion evaluation scale fourth edition (faces - iv;), the family communication scale (fcs;), the family satisfaction scale (fss;), and sense of coherence scale (soc-13;). Measures that did not have spanish versions (i.e., resilience scale for adults (rsa;), life orientation test - revised (lot - r;), relationship - focused coping scale (rfcs;), and the family assessment device general functioning (fad - gf;) utilized chapman and carter's methodology for translation . Measures were translated by a bilingual and bicultural researcher in spanish and then translated back into english by a different bilingual and bicultural researcher . Any inconsistencies between the original english version and the spanish - to - english were jointly addressed . Family cohesion and flexibility were assessed using the spanish version of the faces - iv . Balanced and unbalanced domains of flexibility in the family (e.g., it is important to follow the rules in our family) and cohesion (e.g., family members seem to avoid contact with each other when at home) were measured with the current scale . The subscales purport to measure the lower and upper limits of cohesion (e.g., disengagement and enmeshment) and flexibility (e.g., rigid and chaotic) in the family . These constructs are evaluated with two ratio scores that assess the amount of balance versus unbalance within its respective domain with higher ratio scores indicative of healthier or more balanced systems . The spanish version of the faces - iv has demonstrated good internal consistency and adequate convergent, concurrent, and content validity (= 0.87;). The quality of each family's communication patterns was assessed using a spanish version of the fcs . Total scores for this 10-item measure (e.g., family members are very good listeners) range from 10 to 50 with higher scores indicating better communication . Even though no psychometric data are available for the spanish version of the fcs, the english version has shown good internal consistency (= 0.97) and test - retest reliability . The extent to which family members are happy, content with each other, and satisfied with their overall family functioning was assessed with a spanish version of the fss . This 10-item scale (e.g., your family's ability to cope with stress) assesses participants' level of family satisfaction . The minimum score is 10, and the maximum is 50, with higher scores indicating more satisfaction . The fss has demonstrated excellent internal consistency (= 0.93) and good discriminant validity . This measure is composed of 12 items (e.g., planning family activities is difficult because we misunderstand each other) with higher mean scores indicating more problematic functioning (range: 0 to 4). Since a spanish version was not readily available, the fad - gf was translated for the purpose of this study . The english version of the fad - gf has been shown to have good discriminant validity and internal consistency (= 0.83;); however, the scale evidenced good internal consistency within the current sample (= 0.89). Familial empathy was assessed using the rfcs that aims to evaluate the protection and maintenance of familial relationships during stressful periods using empathic responses . This 10-item measure is composed of two facets of empathic responding, behavioral (e.g., tried to help the other person(s) involved by listening to them) and cognitive / affective (e.g., tried to experience what the other person was feeling) along a 4-point scale . Total scores range from 0 to 40 with higher scores indicative of higher empathic responding . The scale has been shown to have excellent internal consistency (= 0.93;). The degree to which an individual's protective resources promote resilience was measured using the rsa . Originally, this scale had 45 items and included five dimensions: social support, personal structure, family coherence, personal competence, and social competence . Per the recommendation of the scale authors, a 33-item version was used for the current study . Adequate reliability has been shown for each subscale with's ranging from 0.67 to 0.90 . Sense of coherence, a global view of life as predictable, meaningful, and viewed as a form of resilience or coping, was assessed by the soc-13 . Items are rated along a 7-point scale with each item requiring different responses, such as very often to very seldom and never happened to always happened, depending on the item . The scale has been validated on eight spanish samples with satisfactory psychometric properties for spanish speakers across various ages, genders, levels of functioning, and disability . Total scores range between 0 and 40, with higher scores indicating greater optimism and a more positive view on life . This scale has shown satisfactory internal consistency (= 0.78), with additional evidence showing appropriate convergent and divergent validity . At the instituto de neurociencias de san lucas in rosario, argentina, participants were recruited through routine visits to the attending neurologist . During the appointment irb approval from the university of deusto was received for the study location, and participants from the site fully consented to participate in the study . The means for the family dynamics variables appear in table 2 . According to criteria established by the authors of the measures, the majority of the caregivers in this sample had connected families (70.5%), some had very connected families (18%), and the others had somewhat connected families (11.5%). The majority of caregivers came from flexible families (88%), some came from somewhat flexible families (10%), and only a few came from very flexible ones (2%). Of the caregivers' families, 6.5% were considered to have very high communication, 26.5% high communication, 26% moderate communication, 13% low communication, and 28% very low communication . On the family satisfaction scale, the majority of the sample exhibited low satisfaction, with 43% of caregivers rating their satisfaction as very low, 20% rating as low, 4.5% as moderate, 25% as high, and only 7.5% as very high . On the relationship - focused coping scale, the sample's mean (sd) of 1.96 0.53 was higher than the original scale's mean (sd) of 1.64 0.64, which indicates that the level of empathy in this sample was high . On the family assessment device, which measured family problems, 51.4% of the sample was classified as having healthy family functioning and 48.6% as having unhealthy family functioning . Skewness values ranged from 0.577 to 0.010, and kurtosis values ranged from 0.873 to 1.470 . As a result, no transformations were needed . Tolerance values (range: 0.23 to 0.96) were greater than 0.2 and vif values (range: 1.04 to 4.47) were less than 10 indicating no significant issues with multicollinearity . A correlation matrix (table 2) was generated to examine the bivariate relationships among all variables in the current study . Empathy, cohesion, flexibility, and communication were found to be negatively correlated with family problems and positively related to all other variables except for optimism . Family satisfaction, resilience, and sense of coherence were also found to be negatively correlated with family problems but were positively correlated with all variables including optimism . Family problems were negatively correlated with all variables at a 0.01 level except for optimism, which was significant at the 0.05 level . Bivariate correlations between caregiver demographics and resilience indicated that only income was significantly related to resilience (r = 0.33 and p = 0.001) and as a result, income was controlled for in analyses . In the first hierarchical multiple regression (table 3) with resilience as the dependent variable, income was entered into the first step, which was significant, with f(1,103) = 12.18, p = 0.001, and r = 0.11 . The second step including all six variables of family dynamics was also significant, with f(7, 97) = 10.11, p <0.001, and r = 0.42, and the amount of variance in resilience that was explained increased significantly, with f(6, 97) = 8.84, p <0.001, and r = 0.32 . In the second model, the family dynamics of empathy and family problems were both uniquely associated with resilience with income remaining statistically significant (table 3). Bivariate correlations between caregiver demographics and sense of coherence indicated that gender was significantly related to sense of coherence (r = 0.23 and p = 0.017), such that women caregivers reported higher sense of coherence than men caregivers . Subsequently, further analyses controlled for gender . In the second hierarchical multiple regression (table 4) with sense of coherence as the dependent variable, gender was entered into the first step, which was significant, with f(1, 103) = 5.88, p <0.05, and r = 0.05 . The second step included all the family dynamics variables, which was also significant, with f(7, 97) = 11.22, p <0.001, and r = 0.45, and the amount of variance in sense of coherence that was explained increased significantly, with f(6, 97) = 11.22, p <0.001, and r = 0.39 . While gender was significant in the first model, it failed to maintain significance in the second model (p = 0.221). Of the family dynamics variables, only family problems and communication were significant, with empathy approaching significance (p = 0.067; table 4). Consequently, no demographics were entered as controls in the third regression with optimism as the dependent variable (table 5). When the six family dynamics were entered as independent variables, the overall model was found to be statistically nonsignificant, with f(6, 98) = 1.20, p = 0.311, and r = 0.07, indicating that family dynamics did not together predict optimism in the current sample . Furthermore, no family dynamics were significant unique predictors in the overall model (all ps 0.231). The purpose of the current study was to examine whether healthier family dynamics were associated with higher resilience, sense of coherence, and optimism among dementia caregivers in argentina . After controlling for significant demographic and caregiver characteristics, family dynamics explained approximately 32% of the variance in resilience and 39% of the variance in sense of coherence . The primary results of the study found that greater empathy was uniquely associated with greater resilience and was trending in significance with increased sense of coherence . Having few family problems was uniquely associated with greater resilience and increased sense of coherence, while greater communication was independently associated with greater sense of coherence . Almost two - thirds of caregivers indicated that their families had moderate to very high levels of communication with a vast majority of caregivers rating their families as connected and flexible . Such broad healthy family dynamics may be due to the importance of familismo imbedded in the latino culture that values having stronger family values . This sample, however, was found to be generally less satisfied with their family dynamics . This could be due to the measure of family satisfaction being originally created without cultural sensitivity for familism . It is also likely that because our sample comes from a culture with a high emphasis on family they held higher standards of family ideals and how their families should function . The overall pattern of findings from the study is consistent with previous literature in which caregivers from families with overall healthier dynamics are more likely to have greater personal strengths through better quality of life . Individuals from families with poor functioning are likely to experience increased caregiver burden, which is associated with decreased coping . Research on the origin of psychological strengths has emphasized the role of sense of coherence and general resistance resources (similar to resilience) in the face of stressors and suggests the importance of social support and expressions of support in increasing sense of coherence and coping abilities . This is consistent with the findings of the current study that sense of coherence and resilience are associated with environmental factors such as family dynamics . One family factor that was identified as particularly important for the development of caregiver personal strength is empathy, which partially supported our hypothesis . These results are consistent with prior research on resilience that has identified increased empathy as a protective factor in dealing with stressors and may thus promote greater adaptive coping and facilitate resilience . By extension, empathy has been derived as a core component for benefit finding among caregivers for individuals with cancer, suggesting that caregivers of people with long - term illnesses who are more empathic may be more likely to derive meaning from the caregiving experience and view life as more meaningful . Family pathology was also identified as being particularly important for resilience and sense of coherence, which generally supports the study hypothesis . Previous research on the salutogenic nature of sense of coherence in the face of stressors is consistent with the current study's finding connecting family problems and sense of coherence . In a family with increased family problems, it may be difficult for an individual to cope with stressors, especially in cultures of familism such as in latin america . Conversely, caregivers who have little family conflict may be likely to perceive stronger family relationships, which is a strong influence on resilience . Since the family is a primary source of social support and identity, an individual from a family high in conflict is likely to have difficulties coping with the daily stressors of caregiving . While sense of coherence may help individuals cope with a stressful family environment, a supportive environment is also likely to help an individual better cope with personal stressors and promote resilience . Though less consistent across the different personal strengths and in partial support of the study hypothesis, family communication was uniquely associated with sense of coherence . Prior work with adolescents has found that those who found it easier to communicate with their parents had greater sense of coherence . Indeed, maintaining satisfactory family communication facilitates adaptive coping within the family system and promotes individual growth and development . This may play a unique role among caregivers as many may view the caregiving role as an opportunity for personal growth, especially as the disease progresses and the individual with dementia becomes increasingly more dependent on the caregiver . The role of familism in this population may additionally direct the caregiver to view their caregiving role as an opportunity to show family loyalty and reciprocity, bolstering the positive effects of healthy family communication and viewing life as meaningful . When examining family dynamics regressed onto optimism, the results were nonsignificant . These findings may be explained by the nature of optimism as a character trait rather than one that is influenced by external factors . Studies on caregivers of children with cancer have failed to find relationships between optimism and environmental factors such as cancer severity, suggesting the trait - like nature of optimism and that environmental factors like family dynamics are not expected to play a major role in increasing optimism . Other studies also have shown that levels of optimism vary by nation, which suggests that culture, rather than family, may have a greater influence on individual optimism . First, this study does not establish causation or temporal inference because of its cross - sectional design . For these reasons, causal and temporal inferences should be established in future studies exploring the association between family dynamics and personal strengths over time using cross - lagged panel designs, latent growth models, or growth mixture models . Secondly, the self - report nature of the measures may have contributed to participant response bias . Participants with heightened caregiver depression or greater optimism, for example, could have over- or underexaggerated their assessments of family dynamics . Future research should include more objective reports of family dynamics by other family members or by trained professionals . Thirdly, the study sample was only comprised of caregivers from argentina indicating that participants may not be representative of the diverse latin american population including noncaregivers . Consequently, future studies should examine the relationship between family dynamics and personal strengths in participants from a diverse range of countries and cities in latin america as well as among noncaregiving samples . Lastly, while the focus of the study was on examining the general pattern of relationships between family dynamics and personal strengths, we did not test any potential moderators or mediators of any of the proposed relationships . It is possible that family dynamics and personal strengths fit within larger and more complex theoretical family system models such as structural family theory applied to caregivers . Thus, future work with sample sizes larger than 200 participants could test these models more directly using different statistical analyses such as structural equation modeling . The findings of the current study can inform future research on the role of key family dynamics in caregiver personal strengths among understudied and collectivist populations . The family environments in which dementia caregivers function may play an important role in the development and maintenance of personal strengths . Additionally, caregiver personal strengths act as protective factors in the face of daily stressors associated with caregiving, and cultivating these strengths could have a ripple effect on the family environment in which these individuals function . Incorporating family systems interventions targeting communication, empathy, and family problems may enhance personal strengths in caregivers and allow them to provide a higher quality of care for individuals with dementia . This study demonstrated that family dynamics were associated with personal strengths in an argentinian sample of dementia caregivers . In general, greater empathy, more communication, and fewer family problems were unique predictors of greater personal strengths suggesting that healthier family dynamics may be important for caregivers with strong family values . While the findings of this study are preliminary, they in part do suggest that they may be important for informing family systems intervention research for dementia caregivers particularly on increasing empathy and communication and decreasing family problems in the family system . Doing so may improve quality of care for individuals with dementia through greater personal strengths in caregivers.
Cognitive dysfunction is known as one of the common complications that occur after stroke and it accounts for 10 - 82% of total patients with stroke24,25). Cognitive impairment after stroke can cause serious social problems, and these include decreased quality of life of patients and severe economic loss with an increase in the medical expense by the community members . It would therefore be mandatory to accurately assess and to perform rehabilitation treatments of patients who present with cognitive impairment, which is essential for achieving successful treatment outcomes in patients with stroke and helping them to return to the daily lives8). A computer - assisted cognitive rehabilitation (cacr) has been frequently used since it was first used by glisky et al.11) in 1986 . Moreover, it is advantageous in providing the standardized task and the immediate feedback for task performance . Furthermore, it is useful in performing a follow - up of patients' clinical course and conducting a clinical study by constantly analyzing and comparing data about the performance results . It has been reported that memory, auditory and visual attention were significantly improved using the korean computer - assisted cognitive rehabilitation program in patients with brain injury14). Recent ongoing studies examine the clinical usefulness of non - invasive cerebral cortex stimulation technique such as transcranial direct current stimulation (tdcs) in the treatment of patients with brain disorder . Andrews et al.1) reported that the working memory was improved by stimulating the dorsolateral prefrontal cortex with tdcs in healthy adults . In addition, a variety of studies have shown that the tdcs had a significant effect in rehabilitating the cognitive functions in patients with brain injury20). Jo et al.12) demonstrated that the tdcs over the left dorsolateral prefrontal cortex was associated with enhanced working memory performance . Ko et al.17) showed that the tdcs was applied to the scalp over the right posterior parietal cortex and it was effective in improving the visual scanning performance in stroke patients with spatial neglect . The tdcs is a non - invasive brain stimulation technique and there is strong evidence that neurons underlying the anode are' excited', with resting membrane potential shifting towards depolarization and an increased rate of spontaneous neuronal firing27). It has already been reported that the effect of either cacr or tdcs on cognitive function . But there are insufficient studies about the effect of both cacr and tdcs on cognitive function . Given the above background, we conducted this study to examine the synergistic effects of both cacr and tdcs on cognitive function in patients with stroke . To do this, we performed cognitive function test with the korean mini - mental state examination (k - mmse) and seoul computerized neuropsychological test (scnt). The current study enrolled a total of 11 patients, comprising five men and six women, whose mean age 65.6 years old . They were newly diagnosed with stroke, whose lesions were confirmed by a magnetic resonance imaging (mri) or a computerized tomography . Of them, six patients had cerebral infarction, four had cerebral hemorrhage and the remaining one had secondary cerebral infarction due to vasospasm after aneurysmal subarachnoid hemorrhage . In addition, there were six right - sided cases and five left - sided one . Moreover, they had no notable history of brain diseases, including dementia, that could cause cognitive dysfunction other than stroke . To rule out significant aphasia, patients underwent bedside language screening test which was consisted of 5 language domains such as comprehension, production, reading, writing, and repetition . The patients were composed of mild - to - moderate cases of cognitive dysfunction, and participated in the scnt by pushing the button according to our instructions . Their k - mmse scores were lower than 25 points, whose mean value was 18.36 points (range, 10 - 24 points) prior to the combination of tdcs with cacr . The patients equipped with metal in the head and the subjects with skin lesions in electrode attachment site were excluded . The institutional review board of our hospital approved the study protocol, and written informed consent was obtained from all subjects before participation . The patients were randomly assigned to two groups: the tdcs group (n=6) and the control group (n=5). Between the two groups, however, there were no significant differences in the age, sex, years of education, the disease duration and the national institutes of health stroke scale score during the hospital stay (table 1). The location of lesions and etiologic causes are summarized in table 2 . As a transcranial direct current stimulator, we used the phoresor ii auto model pm850 (iomed, salt lake city, ut, usa) generating direct current with batteries, where the sponge electrodes of 55 cm in size (area: 25 cm) were attached . The anode and the cathode were attached to the bilateral prefrontal cortex and the non - dominant arm, respectively . The sham stimulus was set identical except that the current was reduced to zero after 30 seconds in such a manner that the patients were blinded to the turning - off of the power . The tdcs and the cognitive function test were performed by two independent personnels . The korean computer - assisted cognitive rehabilitation program (comcog, v1.0, maxmedica, seoul, korea, 2004) consists of ten traning programs related to the attention and another ten related to the memory . In combination with the tdcs, the patients underwent the cognitive rehabilitation program 30 minutes (a 15-minute attention training and a 15-minute memory training) a day five times a week until the discharge since the hospitalization at the rehabilitation center . Cognitive function test was performed by implementing the k - mmse and the scnt (scnt, v2.0, maxmedica, seoul, korea, 2001) prior to and following the concomitant use of tdcs with cacr . The scnt were composed of a total of ten items and it was performed by a single personnel . These ten items include digit span test, verbal learning test for verbal memory function, visual span test, visual learning test for visuospatial memory, auditory continuous performance test (cpt), auditory controlled cpt, visual cpt, visual controlled cpt, word - color test for attention function, and trail making test for visuomotor coordination function15). The post / pre ratio of scores on the k - mmse and scnt item was calculated between prior to and following the concomitant use of tdcs with cacr . The post / pre ratio of scores on the k - mmse and scnt item was compared using the mann - whitney u test between the two groups . Statistical analysis was done using the statistical package for social science (spss) 18.0 (spss inc ., the current study enrolled a total of 11 patients, comprising five men and six women, whose mean age 65.6 years old . They were newly diagnosed with stroke, whose lesions were confirmed by a magnetic resonance imaging (mri) or a computerized tomography . Of them, six patients had cerebral infarction, four had cerebral hemorrhage and the remaining one had secondary cerebral infarction due to vasospasm after aneurysmal subarachnoid hemorrhage . In addition, there were six right - sided cases and five left - sided one . Moreover, they had no notable history of brain diseases, including dementia, that could cause cognitive dysfunction other than stroke . To rule out significant aphasia, patients underwent bedside language screening test which was consisted of 5 language domains such as comprehension, production, reading, writing, and repetition . The patients were composed of mild - to - moderate cases of cognitive dysfunction, and participated in the scnt by pushing the button according to our instructions . Their k - mmse scores were lower than 25 points, whose mean value was 18.36 points (range, 10 - 24 points) prior to the combination of tdcs with cacr . The patients equipped with metal in the head and the subjects with skin lesions in electrode attachment site were excluded . The institutional review board of our hospital approved the study protocol, and written informed consent was obtained from all subjects before participation . The patients were randomly assigned to two groups: the tdcs group (n=6) and the control group (n=5). Between the two groups, however, there were no significant differences in the age, sex, years of education, the disease duration and the national institutes of health stroke scale score during the hospital stay (table 1). As a transcranial direct current stimulator, we used the phoresor ii auto model pm850 (iomed, salt lake city, ut, usa) generating direct current with batteries, where the sponge electrodes of 55 cm in size (area: 25 cm) were attached . The anode and the cathode were attached to the bilateral prefrontal cortex and the non - dominant arm, respectively . The sham stimulus was set identical except that the current was reduced to zero after 30 seconds in such a manner that the patients were blinded to the turning - off of the power . The korean computer - assisted cognitive rehabilitation program (comcog, v1.0, maxmedica, seoul, korea, 2004) consists of ten traning programs related to the attention and another ten related to the memory . In combination with the tdcs, the patients underwent the cognitive rehabilitation program 30 minutes (a 15-minute attention training and a 15-minute memory training) a day five times a week until the discharge since the hospitalization at the rehabilitation center . Cognitive function test was performed by implementing the k - mmse and the scnt (scnt, v2.0, maxmedica, seoul, korea, 2001) prior to and following the concomitant use of tdcs with cacr . The scnt were composed of a total of ten items and it was performed by a single personnel . These ten items include digit span test, verbal learning test for verbal memory function, visual span test, visual learning test for visuospatial memory, auditory continuous performance test (cpt), auditory controlled cpt, visual cpt, visual controlled cpt, word - color test for attention function, and trail making test for visuomotor coordination function15). The post / pre ratio of scores on the k - mmse and scnt item was calculated between prior to and following the concomitant use of tdcs with cacr . The post / pre ratio of scores on the k - mmse and scnt item was compared using the mann - whitney u test between the two groups . Statistical analysis was done using the statistical package for social science (spss) 18.0 (spss inc ., some patients had pricking sensation at the sites of stimulation after the tdcs but there were no notable side effects . The mean period of cacr was 18.5 days in the tdcs group and 17.8 days in the control group . There were no significant differences in the k - mmse scores between the two groups at baseline (table 3). The post / pre ratio of scores on the k - mmse was no significant differences between two groups (table 4). There were no significant differences in the scnt scores between the two groups at baseline except auditory cpt (p=0.032) (table 3). The post / pre ratio of scores on the scnt item was significantly higher in the tdcs group as compared with the control group on the auditory cpt (p=0.017) and visual cpt (p=0.017) for attention function (table 4). There were no significant differences in the k - mmse scores between the two groups at baseline (table 3). The post / pre ratio of scores on the k - mmse was no significant differences between two groups (table 4). There were no significant differences in the scnt scores between the two groups at baseline except auditory cpt (p=0.032) (table 3). The post / pre ratio of scores on the scnt item was significantly higher in the tdcs group as compared with the control group on the auditory cpt (p=0.017) and visual cpt (p=0.017) for attention function (table 4). Of note, the simultaneous application of tdcs during the cacr was more effective than the single use of cacr in improving the cognitive function after stroke . The current intensity of recent conventional tdcs is relatively very weak, therefore the tdcs does not induce the neuronal firing of resting cell . Rather, it is believed to modulate the increased rate of spontaneous neuronal firing depending on the polarity27). According to these technical characteristics, the combination of tdcs with functional training can be a more effective therapeutic approach than the single use of either tdcs or functional training . In the current experimental study, we attached the electrodes to the prefrontal cortex . In the tdcs group, of the scnt items, only those associated with attention had a significant improvement . Many previous reports have also shown that the prefrontal cortex is involved in the detection of external changes and the interaction with the environment16). It has also been reported that patients with prefrontal cortex damage present with apathy16). Yamasaki et al.28) demonstrated a correlation between the attention and prefrontal cortex on functional mri scans . Furthermore, bloch et al.3) reported that there was an improvement in the attention following the application of repetitive transcranial magnetic stimulation to the prefrontal cortex using the non - invasive brain stimulation technique in children with attention deficit hyperactivity disorder . In the current study, of the scnt items, auditory cpt and visual cpt involved in the attention had a significant improvement in the tdcs group as compared with the control group . This suggests that the degree of attention can be significantly improved with the application of the tdcs to the prefrontal cortex in patients with stroke . However, there were no significant differences in the post / pre ratio on the k - mmse score and other scnt items (memory function and visual motor coordination function). The mmse is widely used to assess the cognitive function after stroke; its korean version, k - mmse, is valid for testing patients with dementia7,13). And, many previous studies reported that the prefrontal cortex played a crucial role in regulating the cognitive function19). Prefrontal cortex performs short - term storage of input data, and it is also known to play a role in controlling the working memory that is closely associated with the cognitive processes such as long - term memory, language learning and executive function2,5,10). Cerebral cortexes involved in the working memory include bilateral medial posterior parietal cortex, bilateral premotor cortex, cingulate gyrus, bilateral frontal pole, bilateral dorsolateral prefrontal cortex and bilateral ventrolateral prefrontal cortex22). Previous studies have been conducted in healthy adults and parkinsonian patients, thus reporting that there was a significant improvement in the working memory following the tdcs for prefrontal cortex9,12,21). Boggio et al.4) reported that 18 parkinsonian patients achieved a significant improvement in the accuracy of verbal three - back working memory after receiving the tdcs to left prefrontal cortex at an intensity of 2 ma for 20 minutes . Moreover, jo et al.12) also reported that ten patients with cerebral infarction achieved a significant improvement in the accuracy of verbal two - back working memory after receiving the tdcs to left prefrontal cortex at an intensity of 2 ma for 30 minutes . Furthermore, fregni et al.9) also reported that 15 healthy young adults achieved a significant improvement in the accuracy of verbal three - back working memory after receiving the tdcs to left prefrontal cortex at an intensity of 1 ma for 10 minutes . Our study did not show significant improvement in the k - mmse scores and memory function test and visuomotor coordination function test of scnt in the tdcs group as compared with the control group . Due to a small number of enrolled patients (n=11), the uneven distribution location and size of the lesions and the etiologic causes between the two groups is the limitations of our study . According to desmond et al.7), the incidence of cognitive dysfunction after stroke was reduced by 54% at a long - term follow - up in patients with left - sided lesions and those with major hemispheric syndrome . These authors also noted, however, that the incidence of diabetic complications was reduced by 11.9% at a long - term follow - up . Patel et al.23) reported that the cognitive dysfunction was affected by the smoking, unilateral neglect syndrome, location of lesions and the degree of functional perfomance . In addition, the cognitive dysfunction after stroke was affected by many variables . In the current study, however, we could not match between the two groups becauce of a small number of enrolled patients . We hope further large - scale and case - control studies could show significant improvement in k - mmse score and other scnt items following concomitant use of tdcs with cacr . In the current study, we failed to clarify whether there is a persistent presence of the improvement in the cognitive function following the concomitant use of the tdcs with cacr at a long - term follow - up because the cognitive function was evaluated only twice in the tdcs group (at the time of hospitalization and discharge). According to desmond et al.7), the degree of cognitive function was improved by 35.9% at a 1-year follow - up in patients with stroke . Kotila et al.18) conducted a study in 154 patients, thus reporting that the degree of cognitive function was improved until a year since the onset of stroke . These authors also noted, however, that most of the patients achieved an improvement in the degree of cognitive function between the onset of stroke and three months after it . Wade et al.26) conducted a study in 85 patients with stroke, thus reporting that both the degree of tapping performance and that of non - verbal memory were improved between three and six months after the onset of stroke . Further long - term follow - up studies are therefore warranted to suggest the appropriate period and guidelines for the initiation and termination of the tdcs for the rehabilitation of cognitive function in patients with stroke . Also, all patients who participated in this study had never received objective test for screening their cognitive function before stroke . If some patients would have mild cognitive impairment, themselves and their family can't notice the cognitive impairment . Although we excluded patients had history of brain diseases, including dementia, we can't convince patients' cognitive impairment absolutely induced by stroke . Our results showed that there were no significant differences in the word - color test and trail making test scores on the scnt items between the two groups . This indicates that the concomitant use of the tdcs with cacr was less effective in helping the patients to return to the daily lives . Despite the limitations of the current study, we found that the concomitant use of non - invasive tdcs with cacr had a significant effect in improving attention in stroke patients with mild - to - moderate cognitive dysfunction . But, further studies are warranted to examine the long - term effects of the concomitant use of non - invasive tdcs with cacr on the cognitive function including memory and higher cognitive function in patients with stroke . With the concomitant use of the tdcs with cacr, a relatively safe, convenient, non - invasive treatment modality, there was a significant improvement in the attention in the tdcs group as compared with the control group . However, this deserves further large - scale and case - control studies . In conclusion, our results indicate that the concomitant use of the tdcs with cacr to the prefrontal cortex may provide additional beneficial effects in improving the cognitive dysfunction for patients with stroke.
Female genital tuberculosis (fgtb) is common in developing countries and associated with significant morbidity in the form of menstrual dysfunction, infertility, tubal block, peritubal adhesions, intrauterine adhesions, and perihepatic adhesions . Fallopian tubes are involved in about 90% of the cases with findings such as congested tubes, tubercles, or caseous nodules on them, peritubal adhesions, and tubal blockage at various levels such as cornual block, mid - tubal block, multiple tubal blocks, or fimbrial block, hydrosalpinx, pyosalpinx, and tubo - ovarian masses . A 25-year - old woman with family history of pulmonary tb was presented to the gynecology outpatient department with primary infertility and oligomenorrhea of 3 years . On examination, vaginal examination demonstrated anteverted uterus with fullness and induration in both fornices . On investigations, hysterosalpingogram which was already performed by the referring doctor for infertility before diagnosis of fgtb was made showed bilateral tubal block . Endometrial aspirate performed in premenstrual phase showed positive polymerase chain reaction based on the amplification of the 240 bp region of the mpt 64 gene using primers mpt 1 (59-tccgctgccagtcgtcttcc-39; nt 460479) and mpt 2 (59-gtcctcgcgag tctaggcca-39; nt 700681) with equipment using amplitron thermocycler (barnstead / thermolyne), it also tested positive for culture for mycobacterium tuberculosis using mycobacterium growth indicator tube-960 ., there were findings of tb in the pelvis in the form of multiple tubercles on tubes and uterus, bilateral hydrosalpinx with beading of tubes, and few caseous nodules . When methylene blue dye was injected in the uterus, the fallopian tubes became distended and blue colored with beading looking with alternate constrictions and dilatation looking - like blue pythons diagnosis of genital tb was made, and she was started on anti - tb therapy . Her postoperative period and follow - up were uneventful . Left fallopian tube with distension, multiple ballooning, and blue coloration with fimbrial block showing blue python sign although gold standard in diagnosis of fgtb is demonstration of mycobacterium tuberculosis either on microscopy or culture of endometrial biopsy or presence of epithelioid granuloma on histopathology, they are positive in only few cases leading on to missing of diagnosis in many cases . Polymerase chain reaction on endometrial biopsy is a sensitive and rapid method for detecting mycobacterium dna (mpt 64 gene) but can be false positive and may not be able to differentiate between infection and disease . Gene xpert has recently been introduced in both pulmonary and extra - pulmonary tb as a sensitive and specific method, but its data in fgtb are very sparse . Imaging modalities such as ultrasound, computed tomography scan, magnetic resonance imaging, and positron emission tomography scan have main role in tubo - ovarian masses . Hysterosalpingography is generally avoided in a suspected case of fgtb due to risk of flare up of the disease but may demonstrate tubal block and other tubal patterns, especially in advanced case . Hysteroscopy is useful in endometrial disease and may show pale looking endometrium with endometrial cavity being partially or completely obliterated by adhesions of varying grade (asherman's syndrome) which may involve ostia . However, laparoscopy is the most reliable tool to diagnose fgtb, especially for tubal, ovarian, and peritoneal disease . There can be tubercles on peritoneum or tubes, tubo - ovarian masses, caseous nodules, encysted ascites, various grades of pelvic adhesions, hydrosalpinx, pyosalpinx, beaded tubes, tobacco pouch appearance, and inability to see tubes due to adhesions . Other authors have also found laparoscopy very useful in diagnosis and management of genital and peritoneal tb with the advantage of avoidance of laparoscopy which is more traumatic and hazardous especially in a case of abdominopelvic tb . In a case of fgtb, if there is no cornual block, there is a partial or complete fimbrial block with multiple blocks at various parts of tube as is typical of fgtb, dye enters the tube but remains in various parts of tubes with alternate dilatation and constriction making the tube look like a blue python as happened in the present case . The new sign case is easily diagnosed by gynecologists during routine laparotomy performed for infertility patients with suspected fgtb and can aid in its diagnosis
Periodontitis, a worldwide complication of diabetes (known as diabetic periodontitis), is diagnosed by destruction of periodontal cementum, ligament, and alveolar bone . Both types of diabetes in clinic, type 1 diabetes (t1d) and type 2 diabetes (t2d), exhibit dramatically higher risk and severity of periodontitis [24]. In recent years, researches are focused on the bidirectional communication between diabetes and periodontitis . Because of the complexity of clinical trials, establishing reliable animal models is particularly necessary . In diabetic periodontitis, significantly increased inflammatory cytokines could be found in serum and gingival cervical fluid . Previous studies have confirmed immune and inflammatory responses in the gingival epithelium play an initial role on the onset and development of diabetic periodontitis [7, 8]. Protein tyrosine phosphatase nonreceptor type 2 (ptpn2) is an intracellular tyrosine - specific phosphatase, which is expressed in epithelial cells, fibroblasts, or endothelial cells . The biological function of ptpn2 is believed to vary in response to proinflammatory stimuli such as interferon - gamma (ifn-), tumor necrosis factor (tnf-), hyperosmotic stress, or hyperglycemia [10, 11]. Moreover, proteins on janus family kinase (jak)/signal transducer and activator of transcription (stat) pathway, an important signaling in inflammation, have recently been found to be targets of dephosphorylation by ptpn2 . Inactivation of those substrates by dephosphorylation lead to the negative regulation of signaling pathways involved in inflammatory responses induced by the proinflammatory cytokines like tnf-, which is also increased in serum and gingival cervical fluid of patients with periodontitis . Mice are usually used as the experimental animal models for the research of human disease, because of its unique host response . A large number of researchers have studied the experimental mice models of chronic periodontitis and diabetes disease, respectively [15, 16]. But the research about the establishment and characteristics of the experimental diabetic periodontitis mice models is yet insufficient . In this study, we established three mice models of experimental diabetic periodontitis and compared characteristics in metabolism and periodontal inflammation . The levels of ptpn2 expression and key factors on jak / stat pathway were analyzed to reveal the potential biological reasons behind the differences . Twenty 4-week male db / db mice, 20 male tallyho / jngj (th) mice, and 40 male c57bl/6j (c57) mice were fed commercial mouse food (finely ground autoclaved low - fat diet) and housed under a controlled environment (temperature around 22c and relative humidity 4555% with a 12 - 12-hour light - dark cycle) for the entire research process . At 6 week, 20 c57 mice were received streptozotocin (stz, 55 mg / kg body weight; sigma - aldrich, st . Louis, mo, usa) for 5 days successively by intraperitoneal injection to establish diabetic mice (stz - c57). Animal treatment was approved by the ethics committee of animal welfare (wccsirb-2015 - 133). Porphyromonas gingivalis (p. gingivalis) w50 was acquired from the state key laboratory of oral diseases, sichuan university and cultured anaerobically in blood - agar (oxoid, oxoid ltd ., hampshire, england) with hemin / menadione (sigma - aldrich). At 6 weeks, the mice in the infection groups were inoculated with p. gingivalis as the following methods: 10 colony - forming units of p. gingivalis were diffused in 100 l phosphate buffered saline (pbs) with 2% carboxymethylcellulose and then orally inoculated three times every second day . The mice in shame - infection groups were infected with the equal volume of pbs (100 l). Fasting blood glucose was determined every 2 weeks in tails blood collected from tail veins of the mice after an eight - hour fast using the glucose meter (onetouch; lifescan, milpitas, ca, usa)., the serum tumor necrosis factor - alpha (tnf-) levels were determined in tails blood collected as above in triplicate by elisa kit (cusabio; sino - american biotechnology, wuhan, china) according to manufacturer's instructions . Level of alveolar bone loss was determined using sem (scanning electronic microscope; zeiss evo, craic technologies inc ., the alveolar bone loss level was measured by the average area (mm) bordered as the cementoenamel junction, the mesial and distal line angles, and the alveolar bone crest on the lingual sides of the mandibular second molars . Maxillas of db / db and c57 mice were dissected after sacrifice and were made into section slides and stained with immunohistochemical (ihc) as presented previously . In short, both sides of maxillas were decalcified in 10% edta solution (biorad, biorad laboratories, hercules, ca) for 14 days and embedded in paraffin . The paraffin - embedded tissues were cut into thin sections (4 m) and stained with immunohistochemical . The primary antibodies, anti - ptpn2 (1: 50), anti - jak1 (1: 100), anti - stat1 (1: 100), and anti - stat3 (1: 100), and the secondary antibodies (1: 1000) were all from santa cruz biotechnology (santa cruz, santa cruz biotechnology, inc ., santa cruz, ca). The staining pictures were captured by an optical microscope (nikon 80i, nikon ltd ., pancreas tissues of four types mice were dissected, rinsed in pbs, and fixed in 4% paraformaldehyde (sigma - aldrich) for 24 hours . The paraffin - embedded tissue samples were cut into a series of 4 m section slides and stained with hematoxylin and eosin (sigma - aldrich). The morphological characteristics of pancreas tissues were observed in the images captured by an optical microscope (nikon 80i). Data were shown as mean standard error of the mean (sem) and analyzed with student t - test when comparing two groups or one - way analysis of variance (anova) test followed by snk - q multiple comparisons when comparing three or more groups using spss software (spss 17.0, chicago, il, usa). The fasting blood glucose and body weight levels of all mice were determined every 2 weeks and changes over time are presented (figure 1). Within the mice with p. gingivalis infection, db / db mice exhibited hyperglycemia from the beginning of research and the fasting blood glucose was maintained at a high value as averagely 26 th mice increased spontaneously and exhibited high glucose at 8 weeks old and then elevated continuously to a final value as approximately 25 stz - c57 mice exhibited hyperglycemia 2 weeks after stz injection at 8 weeks old and continued to increase from weeks 810 . The level maintained steady as evenly 25 mmol / l from weeks 1018, while the fasting blood glucose level of c57 mice maintained normal from 6 mmol / l to 7 mmol / l for the entire experimental period . The body weight level of db / db mice increased spontaneously from weeks 610 but decreased gradually after 10 weeks . The body weight level of c57 mice was steady, but the level of stz - c57 mice in the infection groups decreased . As shown in figure 2, comparing infection groups with shame - infection groups, no statistical difference was observed in the fasting glucose levels among the four types of mice (p> 0.05), which indicated that p. gingivalis infection did not affect the metabolic feature in the mice . However, the body weight of the db / db infected mice exhibited a gradual decrease since weeks 10, while the shame - infection group continued to increase . At sacrifice the body weight of infection group was dramatically higher than that of shame - infection group in db / db mice (p <0.01). The level of alveolar bone loss was significantly higher in injection groups compared to shame - injection groups (figure 3). Within the infection groups, db / db mice exhibited more alveolar bone loss than those of th mice, stz - c57 mice, and c57 mice (db / db mice versus th mice, stz - c57 mice, and c57 mice: p <0.01). Similar results could be found in the shame - infection groups (db / db mice versus th mice, stz - c57 mice, and c57 mice: p <0.05). As shown in figure 4, the levels of serum tnf- after sacrifice were higher in the infection groups compared to the shame - infection groups (c57 mice: p <0.01; stz - c57 mice: p <0.05; th mice: p <0.05; db / db mice: p <0.05). Within the infection groups, c57 mice exhibited less serum tnf- levels than those of diabetic mice (stz - c57, th, db / db mice versus c57 mice: p <0.01). Similar results could be observed in the shame - infected groups (stz - c57, th, db / db mice versus c57 mice: p <0.01). No statistical difference was found in the serum tnf- levels among diabetic mice in neither the infection nor the shame - infection groups (p> 0.05). To further illuminate the underlying mechanism of the alveolar bone loss in these mice models, we evaluated the protein expression of ptpn2, jak1, stat1, and stat3 in gingival epithelium at sacrifice (figure 5). C57 mice share the same genetic background with db / db mice and are often used as controls to study the diseases mechanism . The results demonstrated that high glucose seriously reduced ptpn2 expression in gingival epithelia of diabetic periodontitis mice . Jak1, stat1, and stat3 expression levels were dramatically elevated in infection groups of both db / db and c57 mice . Moreover, infected db / db mice exhibited the lowest ptpn2 expression compared to their shame - infection controls and c57 mice . As shown in figure 6, in c57 mice, round or oval islets cells clusters with clear border were located in the central part of islets and contained a large number of islets cells with round nuclei . In db / db mice and th mice, the volume of islets cells clusters shrinked slightly; vessels enlarge mildly, which were the histological appearances of type 2 diabetes, while in stz - c57 mice, the volume of islets clusters decreased dramatically . Vacuolar degeneration, necrosis, or disappearance in islets cells and proliferous fibrotic tissue between islets cells could be observed; thus the islets appeared empty . In this study, three mice models of diabetes periodontitis were established by p. gingivalis oral inoculation and the metabolic and periodontal features were compared . We found no contrary impact could be observed between this periodontal infection and onset and severity of diabetes in both types 1 and 2 diabetic mice . Variations of ptpn2 and jak / stat pathway in gingival epithelia and different amount of serum tnf- may lead to the different inflammation response to periodontal pathogen . Currently, chemical induced and transgenic mice are two main types of diabetic models . Db / db mice had a long history of utilization as t2d spontaneous model . They shared the same genetic background with c57, which often used as the genetic controls to explore disease mechanisms . Th mice are relatively new model for t2d characterized by glucose intolerance and hyperglycemia and show metabolic abnormalities . Chemical induced diabetic mice were generally induced by streptozotocin (stz), which destroy pancreatic cells, resulting in irreversible insulin - dependent diabetes mellitus (t1d). Oral infection of periodontal pathogens has been generally used to establish periodontitis in experimental mice because of limited oral space . P. gingivalis is widely implicated as an crucial etiological agent in the pathogenesis of periodontitis . The selection of bacteria with different virulence is important in making sure of the alveolar bone loss . P. gingivalis w50 is more aggressive in experimental mice for severe periodontal tissue damage because of its high virulence, compared to other strains such as p. gingivalis 381 and w83 [27, 28]. This research implied that p. gingivalis w50 could cause significant periodontal tissue destruction and alveolar bone loss in all infected mice, indicating that oral infection of w50 is an available method to establish periodontitis mice models with both types 1 and 2 diabetes . The alveolar bone loss levels positively correlated with the severity of periodontal inflammation . In our study, we could observe that all the diabetes mice exhibited alveolar bone loss and db / db mice exhibited more compared to other diabetes mice in both the infection and shame - infection groups . It proved that three types of diabetes mice could all exhibited the characteristic of diabetes periodontitis and db / db mice showed more severe periodontal infection than th and stz - c57 mice . On the infection groups of all mice, no effect of periodontal infection on the fasting blood glucose was observed . However, the reverse change was found in the body weight of db / db mice between the infection and shame - infection groups . As db / db mice exhibited more severe periodontal damage, leading to the more difficulty in feeding than other mice with periodontitis, we supposed that the reverse change was elicited by the more periodontal damage, resulting in less food - intake . In all, the results revealed that periodontal infection could not affect the risk and severity of diabetes in both type 1 diabetes and type 2 diabetes mice . Prior research has proved that the serum tnf- levels positively correlate with fasting blood glucose level, which cannot only destroy pancreatic cells and reduce the sensitivity of insulin but also active the signaling pathways like jak / stat [30, 31]. In our study, we observed that the serum tnf- levels of diabetic mice were higher compared to their normal controls, and the diabetic periodontitis mice showed higher serum tnf- levels than those of c57 mice in the infection group . The results indicated that diabetes mice models could all represent the degree of diabetes periodontitis inflammation . Its crosstalk with inflammatory pathways such as jak / stat has been found in immune cells like astrocytes and macrophages . Researches about human macrophages also demonstrated the communication between ptpn2 and jak / stat signaling . A recent research implied that the expression of ptpn2 decreased in gingival epithelial under condition of periodontal disease . Based on this research, we found the ptpn2 expression in the gingival epithelium negatively correlated with the severity of periodontal destruction and hyperglycemia . Moreover, the expression of jak1, stat1, and stat3 increased in the gingival epithelium of diabetic periodontitis . Jak / stat pathway is important in the progress of chronic human inflammatory diseases including diabetes . The main proteins in this pathway (jak1, stat1, and stat3) are proven to be enhanced in periodontal inflammation . Our result demonstrated that the interaction between jak / stat pathway and ptpn2 may contribute to the development of diabetic periodontitis . However, further comprehensive studies are needed to clarify the accurate mechanisms . Taken together, severe periodontitis with t2d could be observed in db / db, while mild t2d periodontitis was found in th mice . The changes of inflammation - related protein expressions in gingival epithelia might lead to the differences in the mouse models.
Porokeratoses are a group of cutaneous entities of unknown etiology, characterized by disordered epidermal keratinization and, at histological examination, by the presence of coronoid lamella, a column of parakeratotic cells extending through the stratum corneum . Disseminated superficial porokeratosis (dsp) is a distinct form of porokeratosis that, clinically, is characterized by numerous, small, superficial keratotic papules with verrucoid aspects, an atrophic center and peripheral keratotic border . Only 7 cases of this association have been described in the literature (table 1). We report a eighth case of this type and an atypical immunohistochemical method used to characterize the amyloid substance . A 76-year - old woman was referred to our department in june 1999 . She reported a 20 years history of an erythematous and squamo - papular eruption located on the extremities, without itching or pain . The patient had noted a progressive extension of these lesions in the last year and an exacerbation of the lesions during the summer . Upon physical examination, numerous papules with a size of less than 15 mm, an atrophic center and hyperkeratotic borders were distributed on the neck, chest and extremities . Palms, soles, mucous membranes and nails were spared, and the patient denied any family history of similar skin lesions . We examined a skin biopsy that revealed atrophy of the epidermis with hyperorthokeratosis and presence of homogeneous and compact cornoid lamella of keratin, typically oriented sideways with respect to the epidermal tissue (fig . 1 a). The superficial dermis showed a slight chronic inflammatory infiltrate with vascular ectasia and edema . Moreover, in the papillary dermis a compact deposit of a cellular eosinophilic material suspicious for amyloid was observed (fig . 1b). To demonstrate the epithelial origin of the amyloid, we performed two immunohistochemical stainings by using specific moabs to pan - cytokeratin ae1-ae3 . Interestingly, there was a striking difference between the two results: in fact, the acellular deposits, mentioned above, were negative for ckae1-ae3, whereas a strong immunoreactivity was demonstrated for ck5, supporting a specific origin of the amyloid substance from the basal cell layer of the epidermis (fig . In 1937, andrews first described dsp and introduced this term to indicate a clinical variant of mibelli's porokeratosis . Later, chernoski and freeman proposed a possible actinic etiology of this dermatosis and coined the term disseminated superficial nowadays, this term is generally accepted in european dermatological literature, and this definition is based on clinical and histological findings . Dermatological manifestations are typically confined to sun exposed areas, with actinic induction and exacerbations . In the literature, two types of localized cutaneous amyloidosis (lca) are described: primary lca (macular amyloidosis and lichen amyloidosis), which is not associated with other dermatoses or systemic involvement, and secondary lca, which is associated with inflammatory, hamartomatous or neoplastic skin disorders . The mechanism by which dsp induces dermal amyloid deposits is not clear, but piamphongstant et al . First suggested that this process can derive from degenerated epidermal keratinocytes . We hypothesize that a mutant keratinocyte clone is responsible for induction of porokeratotic lesions, because these necrotic epidermal keratinocytes (colloid bodies) might be transformed into amyloid by dermal macrophages and fibroblasts . Immunohistochemical staining has shown an overexpression of p53 protein in porokeratotic lesions; this is a tumor suppressor protein, an important gatekeeper and effector of the cell cycle . Mutations of the p53 gene in all forms of porokeratosis, also in dsap, create a permissive state of uncoordinated cell cycling, and predispose cells to death . In our case, the lack of systemic involvement led to our hypothesis of a secondary dermal deposition of amyloid proteins and the existence of a close relationship between these two processes . This hypothesis was confirmed by positive staining with congo red and immunohistochemical staining with the anticytokeratin moabs strongly positive for ck5, just below the epidermal porokeratotic zone in close proximity to the cornoid lamella . This cytokeratin is, in fact, strongly represented in the basal cell layer and these results indicate that the dermal deposits were amyloid originating from the epidermis (type ii keratin). Nowadays, more studies are necessary to clarify the exact mechanism that leads to secondary deposition of dermal amyloid in porokeratotic disease and the frequency of this association . We also suggest utilization of moabs for ck5 as a first - line target in these conditions, encouraged by the immediate and strong positivity of this protein in immunohistochemical studies, whereas pan - cytokeratin at first produced negative results.
Hemangiomas are common lesions of the head and neck area, and they predominantly originate from the lips, tongue and buccal mucosa . The capillary type is lobulated and it lacks a capsule, it is purplish in color and it infiltrates the involved glands . Microscopically, solid masses of cells and multiple anastomosing capillaries that replace the acinar structure of the gland are seen . The cavernous type is formed by dilated blood vessels or sinusoids lined by endothelium (1). Cavernous hemangioma of the external auditory canal (eac) and tympanic membrane is rare, but this lesion is more common in the eac . According to the previously reported cases (2 - 8), cavernous hemangioma seems to most frequently arise in the posterior portion of the eac . However their incidence in the tympanic membrane is very rare and there have been only 4 such case reports in the literature according to a medline search from 1970 to 2008 (9 - 12). We report here the 5th case of isolated cavernous hemangioma that was limited to the tympanic membrane and we review the relevant literature . A 49-year - old man patient was referred to our department for evaluation of right - sided pulsatile tinnitus that he had experienced for the previous 2 years . The patient denied having hearing loss or vertigo and he had no evidence of facial palsy . Endoscopic examination of the eac revealed a red - colored soft non - pulsatile mass that occupied part of the tympanic membrane (fig . The tympanic membrane was partially obscured by the mass and it was not clearly mobile . A pure tone audiogram (pta) showed high frequency mild sensorineural hearing loss in the right ear at 3,000 - 8,000 hz (fig . 2). Temporal bone computerized tomography (ct) showed an isolated soft tissue mass just lateral to the tympanic membrane . A tympanomeatal flap was elevated to reveal a mass with involvement of the tympanic membrane . The mass was spongy on palpation, and it was removed en bloc with little bleeding . The defect of the tympanic membrane was reconstructed by type i tympanoplasty and using a piece of fascia taken from the temporal muscle . Pathologic examination of the specimen showed a well - demarcated nodular lesion composed of dilated vascular spaces lined by a single layer of flat endothelial cells (fig . The postoperative endoscopic examination showed an intact state of the neo - drum with good ventilation . In 1983, andrade et al . (10) reported the first case of cavernous hemangioma limited to the tympanic membrane in a 59-year - old man with a contralateral ear infection . According to the previously reported cases, cavernous hemangioma seems to most frequently arise in the posterior portion of the eac . Conductive hearing loss, tinnitus and aural fullness may be present . In the present case, the differential diagnosis includes aural polyp, glomus tumor, arteriovenous malformation, granulation tissue and carcinoma of the eac . A temporal bone ct scan is the first choice for the evaluating the location and size of the lesion and middle ear involvement . The majority of the previously reported cases showed a normal middle ear . Only 1 case reported by magliudo et al . (8) had cavernous hemangioma that initially involved the middle ear space through a small perforation in the tympanic memrane and the tumor had reached the handle and the short process of the malleus . For an advanced lesion, diagnostic angiography should be obtained to identify the blood supply to preoperatively assess the safety and feasibility of performing embolization (5). Surgical treatment with en bloc excision is generally curative, and preoperative embolization is usually not necessary except for an advanced lesion . The endaural approach is generally used . In this case, we removed the mass by the postauricular approach due to the size of the mass.
A subset of patients with myositis have unique autoantibodies . This has informed and altered our approach to the diagnosis and management of the inflammatory myopathies . The identification of myositis - specific autoantibodies (msa) and myositis - associated autoantibodies (maa) is important because they are associated with specific clinical phenotypes, and may guide the physician in terms of treatment planning . This study reports the experience and clinical utility of the extended myositis antibody (ema) panel across a range of specialties in a large teaching hospital . Cork university hospital (cuh) is the largest university teaching hospital in ireland, and is a multi - specialty tertiary referral centre serving a population of 1.1 million . We conducted a retrospective review of the electronic and paper records of all patients who had serum samples tested for msa and maa from april 2014 -mar 2015 . Euroline autoimmune inflammatory myopathies immunoblot was performed at university hospital galway . This assay uses membrane strip antigen testing to detect anti-: mi2, tif1 gamma, mda3, nxp2, sae1, ku, pm- scl 100, pm - scl 75, oj, ej, jo-1, pl-7, pl-12, scl 70, centromere a, centromere b, rna pol iii, fibrillarin, nor 90, th / to, ku, pdgfr and ro-52 . The use of additional investigations (electromyography, mri, muscle biopsy, ct thorax) and laboratory results, including creatine kinase and autoantibody profile, were documented . We reviewed the utility of the assay in clarifying diagnosis, directing the investigative pathway and selecting the appropriate treatment . Twenty two patients (mean age: 55, sd:15) had an ema panel sent during the study period . Departments across the hospital included respiratory medicine (n=8, 36%), rheumatology (n=5, 23%), neurology (n=4, 18%), and other (n=5, 23%). Additional investigations performed depended on the clinical picture but included cardiac or musculoskeletal mri (n=8, 36%), ct thorax (n=14,64%), muscle biopsy (n=7, 32%) and emg (n=6, 27%). These autoantibodies were ana (n= 10, 45%), ena (n=4, 18%), anti - ro (n=3, 14%), anti - la (n=1, 5%), anti - dsdna (n=1, 5%) and p- anca (n=1, 5%). Of the 17 patients who had a ck recorded, six (27%) were elevated . Investigations and outcomes of patients with a positive ema panel are shown in table 2 . Patient 3 was a 35-year - old woman who presented in acute heart failure, nyha ii . She had an elevated troponin (600s) and ck (1787), yet had a normal cardiac mri and transthoracic echocardiogram . Ema panel was positive for anti pm - scl 75 and anti pm - scl 100 antibodies, providing evidence that her cardiac failure was secondary to an autoimmune process . Her antibody profile resulted in first line treatment with rituximab, avoiding use of cyclophosphamide in a young woman who intended to start a family . One - month post rituximab infusion her dyspnoea had resolved and both her ck and troponin levels had normalised . Patient 4 presented with a parietal stroke, and had a ck of 1539 on admission . Ema panel was positive for anti - pl7, resulting in a diagnosis of anti - synthetase syndrome . Consequently, ct thorax and pulmonary function tests were performed, as well as onward referral to a respiratory physician . Treatment with a combination of rituximab and steroids has halted the progression of her dyspnoea, and has lead to a resolution of her weakness . Patient 6 tested positive for anti - tif1 gamma, and as a result has an annual ct - thorax abdomen and pelvis screening for an occult malignancy . Two were treated with steroids alone, three received steroids in combination with rituximab, and one received steroids followed by azathioprine and then mycophenylate mofetil . Ema panel is entering standard clinical practice but is not yet a routine tool in the investigation of idiopathic inflammatory myopathy (iim) in all centres . Diagnosis of autoimmune myopathy was previously dependent on muscle biopsy, emg and radiological investigations . With the advent of the ema panel use of the panel avoided an invasive procedure (muscle biopsy) in two patients . Antibodies are detectable early in the disease course, and are specific for autoimmune myopathy the ema panel was diagnostic in 27% of patients, when traditional testing had not been definitive . A positive ema panel is of significant clinical utility in facilitating decisions on appropriate investigations . Patient 6 in our study has entered a cancer surveillance programme after testing positive for anti - tif1 gamma; an antibody associated with a significantly increased risk of malignancy . In anti - synthetase patients 4 and 5 in our study had dyspnoea on presentation, and features of ild on imaging . All patients diagnosed with anti - synthetase syndrome should have a high resolution ct thorax and pulmonary function tests performed . Onward referral to a respiratory physician, as was the case for patients 4 and 5 in our study, should be considered . Autoimmune myopathies are important to identify as they often respond to immunosuppression . In our study all patients with a positive ema panel (n=6, 27%) experienced symptomatic improvement on receiving immunosuppressants . A positive panel in patient 3 provided evidence for use of rituximab, as opposed to cyclophosphamide . In addition to myositis, a constellation of clinical features have been described in inflammatory myopathies, including dyspnoea, raynaud s phenomenon, polyarthritis, fever and weight loss . Ild may precede the occurrence of overt myositis in up to 20% of cases, and is estimated to result in an excess mortality of up to 50% . The multisystem nature of autoimmune myopathy means patients need collaborative input from different medical specialities . Ongoing involvement of these physicians is particularly important; all of whom need to be familiar with the diverse clinical presentation of iim . This study illustrates the value of the ema panel in defining a heterogeneous patient population into clinicoserological phenotypes, thus guiding treatment pathways . Furthermore, it highlights the diversity of these presentations, the need for multi - speciality input and serves to heighten awareness among clinicians of the diagnostic use of extended myositis antibody testing in these cases.
Modes of transmission and distribution factors - various scenarios should be considered when trying to understand the transmission dynamics of t. cruzi . The emergence of transmission of t. cruzi and the maintenance cycle of chagas disease involve a diversity of elements . There is diversity in vector species, their behaviours and biotopes (fig . 1) 2012), diversity in its wild and human hosts, in their potential susceptibility to infection and in their response and there is also diversity in the behaviours of the people infected and their social environment, generating a variety of risk or protection scenarios for the various forms of transmission . Fig . This map was provided by dr r salvatella, pan american health organization, uruguay . The conjunction of this diversity creates some specific scenarios that must be taken into account when making a diagnosis of the situation and designing control strategies to prevent transmission or mitigate the existing damage . Regarding the determinants related to the vector and its behaviour, we could consider scenarios where the main vector is exclusively domestic and introduced, as in the case of rhodnius prolixus in costa rica, which could be eliminated (hashimoto & schofield 2012). Scenarios in which the main vector is essentially domestic, but not exclusively (as it can be found in wild niches close to homes), with the consequent risk of the reintroduction of this and other species, as in the case of triatoma infestans in the phytogeographic region of south america s gran chaco (constituted by geographical areas of argentina, bolivia and paraguay) (ceballos et al . Scenarios where the main vector is exclusively peridomestic in peridomestic and wild environments, as in the case of triatoma dimidiata in mexico, with the consequent difficulty of intervening to control its reproduction niche (pacheco - tucuch et al . A different context is that of vector and oral transmission, which is mainly associated with the presence of infected vectors that can contaminate food (shikanai - yasuda & carvalho 2012). The conditions for food contamination with infected vectors in the amazon region to become a risk of oral transmission of t. cruzi are partly the jungle environment where the infected triatomines reproduce and survive and which forms the domestic landscape in which the inhabitants of that region prepare and consume their food (ops 2009b). T. cruzi is genetically diverse and understanding the population structure of this parasite is critical due to its association with transmission and disease cycles . Currently, t. cruzi is divided into six discrete typing units (dtus), tci - tcvi, with different prevalence according to the geographic region . Dtus tci are prevalent in domestic settings, which have a higher risk for vector transmission, in northern south america and central america, while dtus tcii, v and vi are prevalent in southern south america (zingales et al . The authors that made this description concluded that major advances in the understanding of the pathogenicity and epidemiologic characteristics of these parasites can be expected in the next decade from the comparative analysis of the genomes of several dtu strains (zingales et al . 2012). However, potential differences with respect to the performance of diagnostic methods and therapeutic responses must be considered . To all the above, we must add the prominent role played by social factors, which determine the behaviour of the people and their living conditions . Unfavourable geographical and political phenomena that prevent people from living under decent conditions play a fundamental role in determining the risk of vector infestation in human habitats and the consequent risk of becoming infected through these routes . For example, a study conducted in 1984 in south america s gran chaco region (termas de ro hondo, santiago del estero, argentina), in which there was no vector control or recent insecticide treatments at the time of the study, analysed the factors associated with the presence of infection in children . The combination of the presence of hiding places for vectors within the houses (cracked walls and thatched roofs), having more than two dogs or cats, animals sleeping in the bedrooms and peridomestic structures (sheds, pens etc .) Close to the bedrooms were associated with an infection rate in children between five-14 years of age of 36.4%, while families in the same area where these same factors were absent had infection rates in children of the same age of 7.7% . Given that no vector control with insecticides was a factor in both cases, the presence of environmental and behavioural factors posed a significant risk for acquiring the infection through vector transmission (chuit et al . The routes of transmission by blood are not conditioned by environmental factors, as in the case of vector and oral transmission, but almost exclusively by sociodemographic determinants associated with the migration of people . The risk of transmission by blood exists where there are infected persons, the main factor to consider when planning public health interventions being the amount of infected people in a locality, whether or not the area suffers from triatomine infestation and vector transmission . Thus, in areas where no conditions exist for vector transmission (absence of vectors), but where there is intense immigration of people from an area with vector transmission, there may be a risk of transmission by blood that requires the implementation of measures to control transmission through these routes, such as screening of blood and organ donors and monitoring of pregnant women and newborns from women infected with t. cruzi (mallimaci et al . Control strategies must combine two general courses of action including prevention of transmission to prevent the occurrence of new cases (these measures are cost effective) (vazquez - prokopec et al . 2009), as well as opportune diagnosis and treatment of infected individuals in order to allow them to recuperate their health, when possible, to provide comfort to the patient and to their family and community and to break the chain of transmission by blood by reducing the amount of parasites in donors and mothers (sosa - estani et al . It must be noted that reducing the burden of the disease on society would lead to a consequent reduction in health - care spending (lee et al . Evolution of intervention strategies for control - intervention strategies for the control of insect vectors were designed and implemented in the 50s and 60s and for the control of transmission by transfusion in the 60s and 70s, respectively . The pioneers in implementing these strategies were venezuela, brazil and argentina (ops 2002). Strategies for control of congenital transmission began to be implemented in the 80s; while it still cannot be prevented, it can be controlled by early diagnosis and timely treatment of children with congenital infection (blanco et al . 2000). The chronology described above occurred in several countries in the americas, with different times of implementation and different levels of efficacy . There also countries without formally established programs for controlling the transmission of t. cruzi . Since the end of the 90s, other regions such as north america, europe and australia have launched research projects and some countries have implemented programs for controlling a specific route of transmission (the only one in the area), such as universal screening of blood donors in the usa, or, as in spain, of donors coming from regions of endemic vector transmission (who 2002). The practices for taking care of people infected with this disease have evolved from the time when carlos chagas first described it; they currently include health team interventions and care guides (ms 2012a), which have been implemented in very different ways in different endemic countries . Only in the mid-90s some countries started to implement the practice of detecting infected children and providing them with etiological treatment as part of a number of strategic actions for controlling the disease (ops 2002). This is now part of the protocol used by endemic countries, which have institutional control programs through which they certify the interruption of vector transmission by demonstrating the absence of infection in children less than five years of age . A certification of the interruption of transmission is complemented by the absence of the vector, low domestic infestation (<1%) and no reported cases of acute vector chagas, among other indicators . The status reached of interruption is characterised by a sustained surveillance after intensive activities of control of vectors (ops 2002). A review conclude that the combination of spray activities for control of focus of infestation and the community participation should become a strategic component of chagas disease surveillance (abad - franch et al . The activities to prevent transmission of t. cruzi by transfusion are based on the implementation of blood bank control . These strategies started in 70s by 2005 did not reached an adequate rate of implementation in latin america (schmunis & cruz 2005), but it was improved during last decade and more recently these programs were implemented in nonendemic countries (requena - mndez et al . 2 shows the progress of the interruption of vector transmission in the countries of the americas . Only recently has emphasis been placed on the comprehensive care of infected people in any stage of the infection (ms 2012b) as essential to the goal of eliminating chagas disease as a public health problem (sosa - estani 2012, sosa - estani et al . 2012). Source: pan american health organization (paho.org/hq/index.php?option=com_topics&view=article&id=10&itemid=40743). Achieved effect - the sum of the measures for prevention of transmission have had different a different effect depending on the possibility of implementing interventions . The factors on which this depends include: (i) the political and technical decision to implement the strategies with sufficient evidence of efficacy [some examples that demonstrate the feasibility of the various factors described in (i) are the success in interrupting vector and blood transmission, with total or partial geographic coverage in some countries, such as argentina, brazil, chile, uruguay and some others in central america (fig . 2)], (ii) the complexity of the scenario in which the intervention takes place [examples of (ii) could correspond to specific sub - regions, such as the complexity of determinants in the amazon region that cause outbreaks of oral transmission] and (iii) the lack of tools or strategies or insufficient evidence of efficacy [examples of (iii) could be special situations such as the occurrence of pockets of triatomines resistant to insecticides approved for vector control] (germano et al . The measures implemented in recent decades have had an effect, as confirmed by the world health organization (who) when describing that the number of infected people decreased from 30 million in 1990 to 15 million in march 2006 (ops 2006). However, these estimates may have methodological shortcomings in their calculations and may not allow us to make an accurate assessment due to their high potential margin of error . It is also clear that the various control measures that have been systematically carried out since the formal establishment of control programs for chagas in endemic countries at the start of the 60s have served to prevent new cases in many countries in the americas . However, the diversity of socioepidemiological scenarios and implementation status of control programs has meant that while the main routes of transmission of public health interest (vector and blood) have been successfully interrupted in some regions, new cases involving these routes of transmission are still occurring in other regions . The control of vector and blood transmission of t. cruzi clearly improved the transmission profile in latin america . Some specific examples of these progress in different geographical dimension are the control in the state of so paulo, brazil, which started by 70s with continuous surveillance and high impact avoiding new cases by vector transmission for several decades (carvalho et al . 2011). On the other hand in central america (guatemala, el salvador, honduras and nicaragua) an example of interaction between international cooperation by the japan international cooperation agency, japan, official control programs of the countries involved and community organised this model highlighted for the success: the knowledge for what actions should be taken to control the disease, the competence to obtain resources and capacity to implement control actions, the channels for the institutional network and infrastructure and the control of the technical and administrative management (hanada & hashimoto 2014). A very important issue to warranty the success of the chagas disease programs is to implement evaluations programs of each activity, control quality, to monitoring its efficacy . These aspects were recently reviewed by a paho / who group of experts and in addition were discussed that subjects involving sustainability, coverage, financing and quality of the program must be taken into account in each evaluation . Also it is necessary to offer process such as social, administrative and political issues involving an official declaration of disease elimination, considering the possible positive and negative points that can arise from this declaration taking into account gaps and needs for this goal . Some examples of the new scenario and the role of different actors are the recent constitution of the federation of people affected for chagas disease (findechagas.com/), the coalitions of nongovernmental organisations (ngos) (web.mundosano.org/novedades/mundo-sano-hoy/lanzan-una-coalicion-global-para-luchar-contra-la-enfermedad-de-chagas/) in same case with the industry (unitingtocombatntds.org/resource/london-declaration), forum for research (dndial.org/es/doencas-negligenciadas/enfermedad-de-chagas/plataformachagas.html), the constitution or revision of a national plan with political support and international declaration of who assembly . Beyond the measurement of progress, several questions still remain, which can only be answered by further scientific research . At present, there is the need of designing and implementing a quality control system for the supply of domestic insecticides and for making accurate estimates of the effect of control measures on the incidence of new infections . Considering the response of health systems to the reemergence of another infection caused by insects, dengue, it is surprising how fast they reacted, despite the fact that several programs for control of vector - borne diseases have started to be dissolved . In some regions of latin america, this fast response was made possible by the strong influence of the control initiatives carried out by the paho of the who . Although we cannot quantify this influence, it can be said that it is a cultural influence generated by the initiatives . The case of the reemergence of the dengue virus and that of the emergence of the chikungunya virus, both transmitted by the aedes genus, calls into question whether it is not time to strengthen the control programs and the scientific and technical training of the members of those programs in order to make them more effective and less costly in the context of the current challenges . Having achieved the desired effect of interrupting the transmission of a disease, a major challenge is to achieve sustainable control measures to prevent a reestablishment of transmission, as happened with dengue and malaria in the americas (dick et al . Some of the current strategies aimed to achieve sustainability involve empowering local governments and promoting community mobilisation and participation through organised communities and ngos . The establishment of integrated programs constructed collectively allows proposing strategies that address as many determinants as possible for the control of chagas disease . As an example, the recently revised national program for control of chagas disease in argentina, implemented between 2010 - 2016, consists of five strategic lines: (i) strategies for the prevention and control of vector transmission (vector control and surveillance, resistance monitoring, improvement of the environment - domestic and peridomestic), (ii) strategies for the prevention and control of nonvector transmission, particularly of transmission by blood, (iii) strategies for the control of congenital infection, (iv) strategies for the control of acute and chronic infection in carriers of t. cruzi and (v) general and transversal strategies for the whole program including strengthening of the political dimension, strengthening of the resources of the national and local programs for chagas, social communication and community participation, assessment and monitoring of the program (ms 2012b). These nationwide programs should consider particular situations, mainly related to vulnerable communities, such as care in indigenous communities where the perception and acceptability of the strategies should be approached with particular caution . This based on the evidence of the failure of interventions in these communities due to several factors, one of them being the use of standard procedures which in these populations may cause a kind of culture shock (sosa - estani et al . 2009a, dellarciprete et al . 2014). In 2010, the who estimated the existence of seven - eight million people infected with t. cruzi distributed throughout the world . The region of the americas, particularly latin america, where those with the highest proportion of infected people, being endemic areas for vector transmission the most efficient type of transmission . However, the phenomenon of human migration has created scenarios in need of attention in other regions of the world such as europe, japan and australia, mainly with respect to the diagnosis and treatment of infected people to prevent and control congenital transmission and transmission by contaminated blood (who 2013). There is limited information about the effect of health care activities in infected people on the overall activities of prevention and control . However, two fundamental concepts must be taken into account, either with evidence or when suggesting a hypothesis . The first is the direct effect that timely cares with early interventions have on the quality of life of the patients, with the consequent reduction of the burden of the disease by preventing or stopping its progression (secondary and tertiary prevention) (sosa - estani et al . The second concept is that indicating a trypanocidal treatment, which has proven effective in eliminating or reducing the parasitic infection, modifies the population parasitaemia, with consequential benefits . These benefits may include changing the natural history of the disease by producing less severe clinical chronic forms and/or decreasing the influence of parasitaemia (sosa - estani et al . 2009b) as a determining factor in the occurrence of congenital transmission (bua et al . There are guidelines advising regarding management and care of people infected with t. cruzi . A precocious and adequate clinical management is required to all acute or chronic cases . A timely and adequate clinical, pharmacological and chirurgical management if necessary is essential . The specific treatment has to be considered currently among other therapies (who 2002). For instance, timely management of initial arrhythmia, heart failure or megaesophagus is fundamental to avoid or delay the appearance of severe and irreversible situations, according several longitudinal studies have demonstrated (viotti et al . A challenge to each health system is how to implement the activities to provide an adequate care of people infected . There is a diversity of system, but all of them must to warrant a care with quality . There is a consensus that most of patients can to be care in the primary health care level and to be included into the schedule of national control programs . The role of etiological treatment for t. cruzi infection on several levels of prevention in public health - the etiological treatment allows for actions on several levels of public health prevention . Previous studies provide evidences useful for applying health care strategies in control programs in several countries . When a high number of people can get diagnosis, treatment and cure, it generates a new scenario for the future reduction of the burden of the disease . Primary prevention level - if the goal is to prevent the occurrence of new infections, etiological treatment could have an indirect effect when applied to children and young people . Curing children and women in reproductive age would prevent future events of congenital t. cruzi transmission (sosa - estani et al . In addition, blood and organ donors would increase if infected people are treated . Unfortunately, the effectiveness of the etiological treatment with respect to these primary prevention indications remains unknown, although it can be assumed to be at least equal to the seronegativisation rates observed in some studies . Another strategy would be to obtain a feasible treatment for pregnant women, such as that used for human immunodeficiency virus (hiv) infection, to prevent congenital transmission during pregnancy . However, safe drugs are necessary for this strategy . Etiological treatment in cases of accidents with material contaminated with parasites or with blood samples of patients infected with t. cruzi could also be considered as an indication for primary prevention . In fact, the treatment is not strictly prophylactic, as it is not possible to prevent infections, but the infection can be aborted immediately after the accident with a timely treatment and an appropriate concentration of specific drugs (herwaldt 2001). Recent results show the benefit, in terms of primary prevention, of treating girls and women of childbearing age before pregnancy, preventing the congenital transmission of t. cruzi to children born afterwards (sosa - estani et al . Secondary prevention level - if prevention activities cannot prevent infection in children, curing infected children is still possible by prescribing etiological treatment (cerisola 1977, moya et al . In this regard, etiological treatment should be indicated when damages from cardiac or digestive disease are not strongly present in children . This is the best opportunity to get seronegativisation and avoid disease, thus preserving social, mental and physical health into adulthood (de oliveira jr et al . National control programs have been implemented in more and more latin - american countries since 1994 . They consist of the screening of children populations as a regular strategy providing early diagnosis and treatment (silveira 2002). The positive effect of curing infected children detected by serological screening must be assessed by taking into account the number of infected people, the transmission, evolution and burden of chagas disease, so that it becomes possible to analyse the usefulness of serology as an indicator of action against the vector . Another indication for etiological treatment in secondary prevention is to prevent the reactivation of a chronic infection . 2007) or hiv / acquired immune deficiency syndrome (cordova et al . Even though the effectiveness of etiological treatment for the clinical control of reactivation episodes has been demonstrated, it is necessary to gather evidence as to whether preventive treatment is effective in patients with no signs of clinical reactivation and with abnormal immunological parameters (nishioka 2000). In this regard, some protocols recommend treating organ donors infected with t. cruzi to reduce the risk of transmission by transplant (altclas et al . Tertiary prevention level - the use of etiological treatment against t. cruzi infection in order to reduce the negative effect of established disease is being evaluated using two randomised clinical trials for assessing its efficacy in patients with ongoing cardiac disease (reyes & vallejo 2005, riarte et al . These trials are appraising the efficacy of benznidazole for preventing the progression of cardiac disease . Several observational studies have been published showing the effects of etiological treatment on patients infected with t. cruzi with respect to preventing the progression of chronic chagasic cardiomyopathy (sosa - estani et al . These studies achieved quality of evidence type ii and strength of recommendation b and c. the prognosis of patients with heart failure or advanced stages of chagasic cardiomyopathy is poor (rassi jr et al . 2006), but similar to that of other patients who develop heart failure . Since the disease is chronic and heart damage develops over decades, it is very important to recognise the factors that determine the progression of the disease in the early stages (viotti et al . The evolution of knowledges addressed to the current scenario with a new paradigm regarding benefit of trypanocide treatment (viotti et al . 2014). The etiological treatment should be considered as a protective factor in the model of the physiopathology of chagasic cardiomyopathy . As mentioned above, the effectiveness of etiological treatment in the control of reactivation episodes has been proved, showing recovery from severe manifestations of reactivation such as meningoencephalitis, myocarditis, panniculitis etc . (diez et al . 2007, cordova et al . 2008). Based on our current understanding of the disease, there is a consensus that every patient infected with t. cruzi must (children) or should be treated (adults). Treatment can cure infection and reduce or prevent the progression of chagas - related heart disease / cardiomyopathy . The current evidence of the benefits and limitations of etiological treatment based on clinical and implementation research help prioritise primary health care strategies, focusing on completing the course of treatment, rather than demonstrating serological negativisation . Incorporating etiological treatment as a public health strategy useful at the primary, secondary and tertiary prevention levels is essential to reduce the burden of the disease and to eliminate chagas disease as a public health issue . There are guidelines advising regarding management and care of people infected with t. cruzi . A precocious and adequate clinical management is required to all acute or chronic cases . A timely and adequate clinical, pharmacological and chirurgical management if necessary is essential . The specific treatment has to be considered currently among other therapies (who 2002). For instance, timely management of initial arrhythmia, heart failure or megaesophagus is fundamental to avoid or delay the appearance of severe and irreversible situations, according several longitudinal studies have demonstrated (viotti et al . The medical care all over latin america have different systems with subsystems and it must to have into account to resolve access to diagnosis and treatment, coverage, referential system, medical expertise, among others considerations . This is a new goal for an integrated chagas disease control programs, including the care of people infected as formal objective and activities . Control models and their effect - based on the above, we can graph three intervention models and their expected effect on the evolution of the number of infected people . 3 defines three scenarios according to the intervention models and three times (baseline, medium and long term regarding time lapsed). These three scenarios are described just as a concept and not defined through a mathematical model . The first scenario where no intervention against the vector is carried out and vector transmission continues unabated generates an increase in the number of infected people . The second scenario is where only primary prevention actions are implemented, such as vector control and control of blood and organ donors . These actions achieve a progressive reduction of the number of infected people, but without care for infected people they remain infected for the rest of their lives; due to the phenomenon of population parasitaemia and migration, the risk of transmission by blood would increase . Finally, the third scenario considers primary prevention actions in conjunction with care of infected people, including etiological treatment . This combination can reduce the number of people infected with t. cruzi in a shorter time . Fig . 3:combined effect of prevention and care for people in the control of chagas disease . The implementation of actions with sufficient evidence of efficacy carried out in an integrated manner throughout the country is a challenge of political and technical decision - making . The proof of success in some communities should be a stimulus / incitation for communities with similar scenarios where these actions have not been implemented . The field of research is oriented towards finding alternatives when the tools / strategies used stopped working or are unavailable . Thus, the research priorities for the present situation have been defined different in discussion forums, trying to find the best way of achieving an efficient implementation of control actions for the prevention of new cases and for taking care of those already infected (dujardin et al . Chagas disease cannot be eradicated due to the multiple scenarios that contribute to the occurrence of new cases and mainly because of the demonstrated existence of infected wild triatomines in permanent contact with domestic cycles . However, it is possible to interrupt the transmission of t. cruzi in a large territory, we have discussed before the complexity of dynamic of vector transmission to avoid new cases and having expectance to eradicate or eliminate this disease . Measures available of prevention of chagas disease can reach high level of control due to total interruption of transmission by transfusion, total interruption of vector transmission in large areas and partial interruption or control in areas like amazon region and adequate level of coverage of timely diagnoses and treatment of people infected . The hypothesis is that in the future always will remain just few new cases, but the conjunction of the previous goal described will allow to eliminate chagas disease as a public health problem . In this sense a cut - off point of magnitude of prevalence or incidences of people infected and its burden disease to decide when chagas disease is not a public health problem has to be developed.
The data were collected from the records of patients presenting to outpatient department for piol surgery for myopia . The inclusion criteria for the icl implantation were preoperative best - corrected distance visual acuity (bdva) of 6/24 (snellen equivalent) or better, stable refraction for at least 1 year and a clear central cornea with myopia or myopic astigmatism . The exclusion criteria included the age younger than 18 years, anterior chamber depth <2.8 mm (as measured on pentacam), endothelial cell density <2000 cells / mm, white to white measurement <10 mm (as measured on orbscan ii), presence of cataract, glaucoma, unlasered thinning, breaks or retinal detachment, macular degeneration or retinopathy, a past history of any episode of ocular inflammation, patients with severe dry eyes, or those corneal opacity or irregularity . Staar visian icl (walker avenue, california, usa) was implanted using the prescribed protocol by the same surgeon . Pre- and post - operative visual acuity (snellen equivalent converted to logarithm of the minimum angle of resolution), stereoacuity, refractive error (spherical, cylindrical, and spherical equivalent), and degree of anisometropia (algebraic difference of the spherical equivalent between the two eyes) were analyzed . Visual acuity was recorded postoperatively at day 1, day 8, and 4 weeks . Stereoacuity was tested with best - corrected visual acuity (bcva) and was recorded at 4 weeks of follow - up . Distance randot stereotest (stereo optical co., inc ., chicago, il, usa) was administered while wearing polarizing glasses by trained masked optometrists at 3 m for distance stereopsis (range 40060 arcsec). Near stereoacuity was measured using the tno test (lameris instrumenten b.v ., utrecht, the netherlands) and was administered at 40 cm using red, green anaglyphic filters . Chicago, il). For the purpose of analysis in the absence of actual quantitative value of stereoacuity tests for gross and null stereopsis, categorization of stereopsis was done into three categories as fine stereopsis (<480 on tno and <400 arcsec on distance randot stereotest), gross stereopsis (> 500 arcsec on tno and 7002000 arcsec on distance randot), and null stereopsis (nonmeasurable stereopsis on test). Mcnemar's test was applied for calculating differences in frequency between the paired nominal data of stereoacuity categories pre- and post - operatively . Wilcoxon signed rank test was used to calculate significance levels for the difference before and after surgery . Two sample paired t - test was applied to calculate the relationship among change in stereoacuity with change in visual acuity, spherical equivalent, and anisometropia . All patients were operated bilaterally, except one who had unilateral myopia (total of 95 eyes). The surgery and postsurgical course were uneventful in all eyes except in one eye, which showed raised intraocular pressure postoperatively, for that augmentation of yag iridotomy had to be performed . The mean spherical error was 9.9 5.5 d (range: 1 to 19), and the mean cylindrical error was 1.11 1.7 d (range: 6 to 0 d). The average spherical equivalent of the population was 10.49 5.69 d (range: 2.75 to 21 d). Pre- and post - operative demographic data of patients undergoing icl surgery there was a significant improvement in visual acuity (both uncorrected distance visual acuity and bdva) as compared to preoperative levels (p <0.001; wilcoxon signed rank test). There was a significant improvement in stereoacuity for both near (p = 0.001) and distance (p = 0.0005) (mcnemar's test). The difference (two - sample paired t - test) was not found to be statistically significant for change in near stereoacuity with change in ucva (p = 0.3), bcva (p = 0.8), spherical equivalent (p = 0.8), and anisometropia (p = 0.5). The change in numbers of distance stereoacuity postoperatively did not allow a similar analysis for distance stereopsis . 1a and b. (a) graph showing change in near stereoacuity pre- and post - operatively . (b) graph showing change in distance stereoacuity pre- and post - operatively (values in sec represents arc seconds) the overall improvement in stereopsis was observed in 15/48 (31.25%) and 13/48 (27.10%) subjects for near and distance, respectively, with no significant difference between the two (p = 0.82; fisher's exact test) [table 2]. Improvement of stereoacuity among the stereoblind subjects was observed in 10/12 (83.33%) and 13/36 (36.11%) for near and distance, respectively [fig . There was a significantly greater improvement of near stereopsis in stereoblind subjects when compared with distance stereopsis (p = 0.007; fisher's exact test). The odd's ratio for near stereoacuity to improve in comparison to distance stereoacuity among the stereoblinds was 8.85 (95% confidence interval: 1.6846.70; p = 0.01). The change in stereoacuity pre- and post - operatively when divided into the categories nonrecordable, gross, and fine stereoacuity in our series, the patients registered a significant improvement in both near and distance stereopsis postsurgery (p <0.001). There are only anecdotal reports in literature describing the improvement in stereopsis following posterior chamber piols implantation . However, these studies were limited by reporting outcome for change in stereopsis in a small number of patients that were treated for varied refractive errors, at differing age and with different types of piol . This may suggest that stereopsis is susceptible to recovery even later in adulthood if appropriate visual rehabilitation is offered . Enhanced stereoacuity postsurgery could be due to the greater retinal magnification of images (1147%) at intraocular plane versus that at the spectacle plane, corresponding to an improved bdva of 0.4 and 1.8 lines . Other factors include the elimination of prismatic effect of spectacles, enhanced quality of vision in both eyes, correction of anisometropia, and infrequent use of glasses preoperatively . The absence of any relationship of stereoacuity with anisometropia could be due to low degree of anisometropia in our patients (median 0.87 d) as anisometropia> 3d has the maximum potential to create blur and affect stereoacuity . About 84% and 37% patients who were stereoblind for near and distance, respectively, improved following surgery . The greater probability (8.84) of near stereoacuity to improve than distance may be because myopes have a good near vision despite having a poor distant vision . Further, stereoscopic disparity was measured dichoptically using two different principles, that is, polaroid vectograph - based and anaglyph - based principles for distance and near stereoacuity, respectively . It could have resulted in slight differences in accuracy while comparing the distance and near stereo acuities obtained in this study . Moreover, patients with low, pre- and post - operative bcva could also have confounded the results in not producing improvement in stereoacuity . Nevertheless, this is the only study that sought the impact of icl implantation on the change in stereoacuity in myopia . To conclude, icl implantation aids in improving stereoacuity postoperatively . It may be utilized as a therapeutic procedure for poor stereopsis preoperatively in certain sportspersons, e.g. In athletes, shooters, car racers . Near stereopsis
Two great changes in developed countries are taking place: populations are ageing and becoming increasingly overweight . The average weight of both men and women at the age of 45 years is 20% higher than it was 20 years age [24]; on the other hand, ageing workers can be found in almost all types of occupation . The international labor organization (ilo) has estimated that by the year 2025, the proportion of individuals over the age of 55 years will be 32% in europe, 30% in north america, 21% in asia, and 17% in latin america . The health and safety laboratory used the term older workers to refer to people 50 years of age [7, 8]. This is due to the following: with advancing age, decrements can be expected in aerobic capacity, general health, grip strength, lifting strength, balance, eyesight, hearing, reaction time, limb motility, and tolerance for paced work [810]. The work ability concept is a dynamic process that changes greatly for several reasons throughout an individual's work life . One of the main factors inducing change is aging and its effect on human resources . Work ability is defined as the ability of a worker to perform his / her job, taking into account work demand and physical and mental conditions . Work ability is a tool to identify workers at risk for imbalance between health, capabilities, and demands at work . Improving work ability is one of the most effective ways to enhance the ability and to prevent disability and early retirement [13, 14]. Another factor that influenced work ability and consequence in early retirement is body mass index (bmi) in study by lund et al ., and van den berg et al . Underweight as well as obesity compared to normal weight decreases the wai score [12, 15]. Night shift work, as compared with day work, is a high risk for poor work ability, when combined with aging (44.5 years old) is significantly higher than the risk associated with their additive effect . In a study by capanni et al . In relation to working hours, wai proved to be worse in shift workers than in day workers, and, particularly, in continuous 3-shift workers . Measures of subjective workload show that what may appear to be a simple assignment is in fact quite demanding; on the other hand, jobs without specific demands will not require additional ergonomics attention or actions to keep older workers fit in the job [1820]. These specific demands can be classified into (or combinations of) physical, mental, or psychosocial job characteristics . Mental work load is the amount of effort that the mind can do in duty and requires input from such cognitive domains as concentration, memory, decision making, or attention [20, 21]. There are three principal methods for measuring workload: physiological (heart rate and blood pressure as responses to stress such as that induced by physical activities), procedural (measuring time spent on secondary tasks), and perceptual or subjective (workload measurement that uses rating scales to evaluate participants' perceived workloads). Although physiological and procedural measurements may appear to be more accurate and objective, subjective measurement of workload has been reported to be less invasive, easier, and less expensive to obtain more easily reproduced, and of higher face validity . A study by bridger and bennett among seafarers indicate that mental workload had a moderately high level of satisfaction with work performance, and all other scores were generally clustered around the midpoints of the rating scales . The aim of the current investigation was to quantify the mental demands placed on textile workers during their normal daily work and shift activities in relation to their self - assessed work ability and mental workload . A second aim was to identify factors that affected mental workload and work ability index in textile workers . This cross - sectional study was carried out in a textile industry in isfahan, between september and november 2012 . Employees were selected from each job based on their job groups and work conditions, and they have been divided into 6 categories (table 1). Finally, one hundred and ninety - four employees completed questionnaires; then employees were invited for anthropometric measurements . At the conclusion of the shift, participants returned to a briefing room where they completed two questionnaires . Perceived work ability was measured by a questionnaire - based index composed of the following seven items: (i) current work ability compared with lifetime best (010 point); (ii) work ability in relation to both physical and mental demands of work (210 point); (iii) number of current diseases (17 points); (iv) estimated work impairment due to diseases (16 points); (v) sick leave during the past year (12 months) (15 points); (iv) own prognosis of work ability 2 years from now (1, 4, or 7 points); (vii) mental resources (enjoying daily task, activity and life spirit, and optimistic about the future) (14 points). Translating the wai questionnaire into persian and identifying its reliability and validity in iran have been done by abdolalizadeh et al . . It is divided into the following categories: poor (727 points), moderate (2836 points), good (3743 points), and excellent work ability (4449 points). Subjects at or above 37 points were classified as having satisfying work ability . Perceived mental workload was measured by nasa - tlx (the national aeronautics and space administration task load index) questionnaire . Nasa - tlx has six subscales, mental demand (md), physical demand (pd), temporal demand (td), performance (pf), effort (ef), and frustration (fr), which can be divided into three groups: characteristics of the task: mental, physical, and time demands; behavioral characteristics: performance and effort; individual characteristic: frustration . Each of the bipolar subscales of nasa - tlx consists of 20 five - point steps from 0100 . Each subject makes a paired comparison, deciding with all 15 possible pair combinations of the 6 dimensions which pair element is more important with regard to workload in the rated task . From the results, a rank order of the dimensions from 05 is derived by which the individual subscale scores of the rated task are weighted . By summing up the weighted subscale scores and dividing them by the sum of the weights (= 15), the mean weighted workload score is obtained that indicates workload in percent . Participants were asked to report their shift schedules and these were categorized into, the following: morning shift from (7 am15 pm), evening shift from (15 pm23 pm), and night shift from (23 pm7 am). Anthropometric measurements were made at the end of the shift, using the following equipment and procedures according to standardized guidelines.stature: volunteers removed their shoes before standing on a stadiometer (invicta, leicester, uk) with the feet together . Heels, buttocks, and scapulae were in contact with the stadiometer, and participants were instructed to look straight ahead to inhale steadily, and the measurement was taken to the nearest 0.1 cm.mass: volunteers were barefoot, in general working clothes, with all items removed from their pockets . Weight was measured to the nearest 0.1 kg (seca, hamburg, germany).body mass index was calculated using the formula weight / height and divided into four bmi categories: underweight was bmi 19 kg / m, ideal weight was 19 <bmi 25 kg / m, overweight was 25 <bmi 30, and severe overweight was bmi> 30 kg / m . Stature: volunteers removed their shoes before standing on a stadiometer (invicta, leicester, uk) with the feet together . Heels, buttocks, and scapulae were in contact with the stadiometer, and participants were instructed to look straight ahead to inhale steadily, and the measurement was taken to the nearest 0.1 cm . Mass: volunteers were barefoot, in general working clothes, with all items removed from their pockets . Weight was measured to the nearest 0.1 kg (seca, hamburg, germany). Finally, the information was analyzed using spss 20 and statistic tests, namely; multiple multivariate regression test has been used for relationships between individual factors such as age and bmi with wai and nasa - tlx . Differences between three work shifts with mean wai and nasa - tlx were analyzed by anova test . Differences in wai score between workplace groups were analyzed by anova test . A p value less than 0.05 was considered to be statistically significant . The workers were between 24 and 62 years old, and their mean age was 40.22 6.99 years . Differences in age between workplace groups were significant (one - way analysis of anova, p <0.001). All subjects who responded were men . The average period during which participants had been working at their current workplace was 15.17 years with range of 127 years . Table 2 summarizes participant anthropometric characteristics; mean body mass index (bmi) was 25.5 kg / m (overweight). The percentage of the participants with underweight (bmi 19 kg / m) was 11.3%, at a healthy weight (19 <bmi 25 kg / m) was 45.9%, and severe overweight (bmi> 30 kg / m) was 7.2% (figure 1). Multiple multivariate regression test showed that relationships between age and wai were statistically significant (p = 0.007), but these relationships were not statistically significant with mental workload (p> 0.05). Relationships between bmi with wai and mental workload were not statistically significant (multiple multivariate regression test, p> 0.05). Anova test revealed that relationships between work shifts and wai were statistically significant (p <0.05), and these relationships were not significant with mental workload (p> 0.05). The lowest work ability was recorded among the doubling workers, followed by spinning workers, while the highest work ability was recorded among the supervisor workers . Anova test showed that after adjusting for age, there was a significant difference in the wai scores between the workplace groups (p <0.001) (table 3). The nasa - tlx mean ratings of the demands fell towards the high point except for time pressure and frustration that were placed at the midpoint of the scales, indicative of high demands as a percentage of maximum: mental demands 66.4%, sd 25.2%; physical demands 71.3%, sd 28%; time pressure 58.9%, sd 25.8%; effort 75.5%, sd 24.2%; frustration 44.5%, sd 30%; satisfaction with daily performance 73.5%, sd 23% . The average nasa - tlx scores for job group indicative high mental workload . The highest mental workload was recorded among supervisors and the lowest was recorded among office workers (table 3). The result of one - way analysis of anova shown between wai and nasa - tlx in job groups was statistically significant (p = 0.01). The perceived work ability of textile workers, as assessed by the wai, decreased at an early age . The first significant turning point was observed after 30 years of age, as shown in figure 2; at age 2030 years mean wai was 38.2 that placed in good work ability and after 30 years of age mean wai was decreased that placed in poor work ability, and the second, steeper decrease occurred after 40 years of age, and this can because changes in physical work capacity that the maximal oxygen consumption (vo2max) shows linear decline with age . Generally, the changes in physical capacity in relation to aging are often difficult to distinguish because work and living habits can accelerate or slow down such changes . Changes in musculoskeletal capacity can also be pronounced after the age of 4550 years . In a follow - up study, physically demanding jobs decreased by 4050% during a 10-year period . Multiple multivariate regression test showed that age relationships were not statistically significant with mental workload and this is maybe result in nasa - tlx scores are significantly affected by both mental and physical workload and among six subscale only one subscale related to physical demand and this subscale may affect by age but determining the total score related to six subscale . Until now, extensive research has been performed on the separate assessment of physical and mental workloads, but there are no validated techniques available for measuring overall workload in multitask situations that involve substantial levels of both physical and mental workloads . As shown in table 4, weight gain in adult men is normal up to the age of 50 years due to the presence of a small, positive energy balance . It tails off at about the age of 50 years . At the age of 45 years, the average body mass of men and women is 20% higher than it was 20 years before . The best predictor of wai score was the interaction between bmi and age, in the expected direction . Shift work implies that working hour differs from the traditional diurnal work period (7 pm and 6 am) [32, 33]. Shift work may cause several problems in physical and mental health and work ability in workers . In relation to shift work, wai proved to be worse in shift workers than in day workers . In morning shift, mean wai score was 35.1 (4.9); in evening shift, it was 32.8 (6.2); in night shift, it was 33 (4.9). Similar findings have been reported in the study by costa et al . Among health care workers; the result revealed that shift workers showed a more pronounced decrease of wai over the years compared to their colleagues day workers . On the other hand, nasa - tlx score among night shift workers had a bigger mark than day shift workers . Studies of shift work have demonstrated that working shifts may lead to health complaints and social problems . Alternating the working hours has decreased the amount of time available for rest and recovery within the context of current working hours, and this factor can affect mental workload during shift work . The average wai score among textile industry workers less than 37 points indicated unsatisfying work ability . In the present study, the average wai score for all workers showed unsatisfying work ability (wai <37). These results are lower than the finnish reference data in mentally demanding work (mean 39). This may result in textile industry due to the nature of the work in which the physical conditions of the workplace consist of stressors such as harmful physical agents (noise, lighting), harmful chemical agents (air borne cotton dust, ventilation condition of saloons), ergonomic risks (lifting and handling equipment, bad posture during working), and another occupational hazard that can affect wai . The result is similar to the study by aittomki et al . Which was conducted among municipal employees . And comparative with studies in an oil company in croatia, among fire fighter in belgium, and in constructer worker in netherland that wai was satisfying for all workers . The lowest mental workload was recorded among office workers and the highest was recorded among the supervisor workers with mean 70.8 (17.5) and 89.5 (7.8), respectively, and consist with study by jin et al . Among drivers in japan that result indicated high mental demand such as this study . The result of one - way analysis of anova showed that relationships between wai and nasa - tlx in job group were statistically significant (p = 0.01). As shown in table 3, mean wai in all job groups was low, and mean nasa - tlx score was high; these two variables have direct inverse effect that was revealed in a study by sjgren - rnk among office workers (2002), and the result showed that low mental demand at work was directly related to higher work ability . The results of the current study showed that the most important factor that influenced work ability among textile workers was age . Unlike the previous study, a decrease point in wai score started in early age that may be due to life - style work and another psychological factor; on the other hand, nasa - tlx revealed high score in six subscales that can be another reason for low wai . The result indicated that nasa - tlx score was more affected by working conditions in contrast to individual factors such as age and bmi.
In 2001, we enrolled 603 children (116 years of age) attending the sickle cell clinic at mulago hospital, kampala, in our study . By design, plasma specimens were tested by using an elisa for hcv antibodies to recombinant antigens c22, c200, and ns5b (elisa-3.0, ortho - clinical diagnostics, raritan, nj, usa) according to the manufacturer's instructions . To conserve samples we did not confirm positive results by riba but instead tested for virus in plasma by using a real - time reverse transcription (rt)pcr assay for hcv rna developed in our laboratory . This quantitative assay amplified a conserved 155-nucleotide target sequence within the hcv 5 untranslated region and had an absolute sensitivity of 9 iu / ml of viral load (43 copies / ml). Total rna was extracted from 140 l plasma by using the qiagen viral rna mini kit (qiagen, valencia, ca, usa). Rt - pcr was performed in a thermo hybaid mbs 0.2s (fisher scientific international, hampton, nh, usa) in triplicate reactions that used 10 l rna per reaction . After cdna synthesis, quantitative pcr was performed by using an abi prism 7700 or 7900 sequence detection system (applied biosystems, foster city, ca, usa). Hcv rna positive specimens were further characterized by sequencing parts of the core / e1 and ns5b regions . Nested pcr was performed with sets of published pcr primers to amplify dna from core / e1 or ns5b regions (6,7). The amplification products were separated in an agarose gel and purified by using the promega dna purification kit (promega, madison, wi, usa). The bigdye terminator kit (applied biosystems) separate alignments were generated for the ns5b and core / e1 regions, each including sequences from our samples and genotype 1 to 6 reference sequences obtained from genbank (7). These sequences were aligned in clustalx (version 1.81, plate - forme de bio - informatique, illkirch, france) by using the clustalw (version 1.6, plate - forme de bio - informatique) matrix and edited in genedoc version 2.6 (http://www.psc.edu/biomed/genedoc). In mega version 2.1 (http://www.megasoftware.net), the clustal alignments for core / e1 and ns5b were used to generate neighbor - joining trees by using the kimura 2-parameter plus g distribution (k80+g) distance model . Free parameters were reduced to the k80 model, and values were determined by using a maximum likelihood approach in paup*4.0 (sinauer associates, inc . Estimated phylogenies of hepatitis c virus genotype 4; ns5b phylogenetic analysis based on 350 bp of ns5b nucleotide sequence . Numerical values (presented when> 60%) represent the statistical support for the tree topology as determined by 1,000 bootstrap replicates . Reference sequences for genotypes 13, 5, and 6 (7) were included in both analyses and retained as the outgroup . Of 603 children (median age 6.8 years, interquartile range 3.910.9 years), 27 (4%) were hcv antibody positive . No significant trends were found between hcv status and demographic (age, sex, tribe) or household (urban / rural location, water supply, room density) variables . Three (5%) of 61 children born to hcv - seropositive mothers were hcv seropositive, compared with 16 (3%) of 456 children born to seronegative mothers (p = 0.58). Eleven (3%) of 322 children with a history of transfusion (mean 1.5 transfusions, range 110) were hcv seropositive, compared with 12 (5%) of 245 children with no history of transfusion (p = 0.38). Prevalences in children with (2%) and without (4%) history of scarification were also similar (p = 0.38). Using rt - pcr to test for hcv rna, we examined 58 samples from children: all 27 samples with antibody - positive results, 11 with high - negative (near the positive cutoff) results, 10 with negative results from children with a history of frequent transfusions, and 10 with low - negative results from children with no history of transfusion . Only 3 samples, all from seropositive children (ages 6, 7, and 13 years), had positive results by rt - pcr, and viral rna levels in these samples were 2.310, 2.810, and 3.810 hcv iu / ml plasma . Of 525 serum samples available from mothers, 62 (12%) were antibody positive . We sought hcv rna in all 62 mothers with positive elisa results and in 20 mothers with high - negative results . Five (8%) antibody - positive mothers (ages 28, 30, 38, 39, and 42 years) had rna - positive samples . Viral levels varied from 6.610 to 5.710 hcv iu / ml (median 8.110). All hcv - seronegative mothers (median age 32.3 years, mean 33.2 years, standard deviation 7.6) were rna negative . We amplified samples for sequencing from 5 mothers and 1 child and obtained core / e1 sequences for all 6 samples and ns5b sequences for 3 samples . None of the sequences was identical to any other or to any sequence in genbank . Phylogenetic analysis of both the ns5b and core / e1 regions showed that all viruses amplified clustered within genotype 4 (figure 1 and figure 2). In the core / e1 phylogeny (figure 2), 2 clusters of hcv strains and 1 outlier (ug1) appeared . Each of the ns5b sequences obtained (figure 1) was from a different core / e1 group, and they did not cluster . Estimated phylogenies of hepatitis c virus genotype 4; core / e1 phylogenetic analysis based on 340 bp spanning the junction between the core and e1 regions . Numerical values (presented when> 60%) represent the statistical support for the tree topology as determined by 1,000 bootstrap replicates . Reference sequences for genotypes 13, 5, and 6 (7) were included in both analyses and retained as the outgroup . We found hcv infection to be uncommon in uganda, consistent with reports from other ugandan studies . This cohort of patients with sickle cell disease had frequent blood transfusions, but only 4% of the children were hcv positive by elisa . Of the seropositive children, only 11% had hcv rna detectable by rt - pcr . Seropositivity in their mothers was higher, 12%, but only 8% of the seropositive women had positive rt - pcr results . If we assume that untested seronegative patients were not viremic, the overall prevalence of hcv rna was 0.5% in children and 1% in mothers . False - positive results could be due to nonspecific antibody binding or to cross - reactivity with other tropical pathogens, e.g., flaviviruses found in africa (2,3). Alternatively, perhaps true antibody - positive participants did not have positive rt - pcr results because they had cleared hcv viremia; however, in the industrialized world, most antibody - positive persons have persistent viremia . We did not confirm elisa positivity with riba because, in africa, few hcv elisa - positive samples are confirmed by riba (3,8). Our rt - pcr results confirm that hcv is present in uganda, albeit at a low prevalence . We did not find correlations between infection in children and either seropositivity or viremia in their mothers . We expected to see iatrogenic transmission (1,8) because the children in this study all had sickle cell disease and more than half had had blood transfusions, and many had had multiple transfusions . Most children would have also had potential exposure to hcv from contaminated needles used during the course of their medical care or blades used in scarification by traditional healers (9). However, despite these potential exposures, documented hcv infections were uncommon . Most hcv isolates can be classified into 6 major genotypes on the basis of sequence data from the ns5b, core, and e1 genomic regions . In this study, as in others (8), multiple sets of primers were needed to amplify these regions because different primer sets worked for different samples, which reflects the diversity of hcv . Neither the core / e1 nor ns5b sequences in uganda clustered closely with genotype 4 sequences reported from other areas (figure 1 and figure 2). Because the number of sequences was limited, we have not assigned subtypes to our newly described variants . In sub - saharan africa, genotype 4 has been found in the west - central nations of africa (nigeria, cameroon, gabon, and central african republic), but it also occurs in tanzania (11). Phylogenetic analysis shows that types 1 and 4 share a common ancestor (12). (13) noted that genotypes 1 and 4 in cameroon have considerable genetic diversity and suggested that these types may have arisen there . Our detection of hcv genotype 4 in uganda is consistent with their hypothesis, but we do not know the direction of spread . Type 4 is also found in egypt, where its epidemic distribution is attributed to widespread iatrogenic transmission during schistosomiasis eradication campaigns from 1960 to 1980 (in which needles for injecting potassium antimony tartrate were reused after rudimentary cleaning) (7,14), and in the arabian peninsula (15). For many centuries, arab traders have had extensive contact with african populations of the nile watershed region, including uganda, which provides routes for genotype 4 to spread between east africa and the middle east.
For the better part of the past 20 years, dentistry has seen the development of many new all - ceramic restorative systems . The need to develop such materials and associated technology has been driven by the patient expectations for excellent esthetics and by biocompatibility concerns of metals intraorally . The combination of advancements in dental materials as well as in computer technology has made computer - assisted design / computer - assisted manufacturing (cad / cam) fabricated restorations possible and plentiful in dental clinics . It is interesting to note that the term cad / cam does not provide any information on the method of fabrication . All cad / cam systems consist of three components: (1) a digitization tool / scanner that transforms the geometry into digital data that can be processed by the computer . (2) software that processes data and depending on the application, produces a data set for the product to be fabricated . (3) a production technology that transforms the data set into the desired product . Since its introduction in the early 1980s, it has evolved in three directions depending on the type of the production line, (a) chair - side production, e.g. Cerec system (sirona dental gmbh; salzburg, sterreich), (b) laboratory production, e.g., ineos x5 scanner and inlab mc xl milling unit (sirona dental gmbh; salzburg, sterreich), and (c) centralized fabrication in a production center, e.g. Nobel procera (nobel biocare, zrich switzerland). The list of various materials that may be processed by cad / cam devices depends on the respective production system . Some milling units are specifically designed for the production of zro2 frames; other more versatile ones accommodate a selection of materials from metals, resins, glass ceramics, and high - performance ceramics . Glass ceramics are grindable silica bases blocks and due to their higher stability values, lithium disilicate ceramic blocks are particularly important in this group; they can be used for full anatomical crowns and copings in the anterior and posterior regions, and for three - unit fpd frameworks in the anterior region due to their high mechanical stability of 360 mpa . High - performance oxide ceramics are mainly of two types: (a) aluminum oxide (al2o3), and (b) yttrium stabilized zirconium oxide (zro2, y - tzp) a high - performance oxide ceramic which when compared to other all - ceramic systems, exhibits superior mechanical properties, high flexural strength (900 mpa to 1200 mpa), and higher fracture toughness (6 mpa to 10 mpa m1/2). A 19-year - old female of excellent health was referred to the department of prosthodontics for the esthetic restoration of peg - shaped right and left maxillary lateral incisors (lis). During the examination, it was noted that the patient had congenitally missing maxillary canines on both sides and a midline diastema [figure 1]. The treatment plan involved fabrication of full coverage crowns for the right and left maxillary li's and ceramic veneers for both the maxillary central incisors . However, the patient had made it clear at the onset that she was seeking treatment only for restoring the shape of her li's and wanted the midline diastema to be left as it was . The patient was given the option of a porcelain - fused - to metal or an all - ceramic restoration . For obvious reasons, the patient chose to have the li's restored with the all - ceramic option . This clinical report describes the procedure for restoring the esthetic appearance of both the maxillary peg - shaped li's with a metal - free sintered finely structured feldspar ceramic material using the latest laboratory cad / cam system . During the first visit, diagnostic impressions are taken and the study models were articulated in a mean value articulator (e200 simplex, cori dent, daegu, south korea). The entire procedure, including the tooth preparation, restoration fabrication, and cementation, was scheduled for the second visitat the second visit, preparations were carried out with both the maxillary li's with a resultant shoulder finish line and vinyl polysiloxane impressions (express, 3 m espe dental products; st . Paul, mn, usa) were made following gingival retraction using impregnated retraction cords (ultrapak e cord #00, ultradent products inc ., st . The models were poured in type iv gypsum (gladstone 3000, dentsply gac, ny, usa)the models were taken to our on - site laboratory which housed the inlab mc xl cad / cam station (sirona dental gmbh; salzburg, sterreich) and a new digital file was created using the inlab 4.2 software . After entering the dentist and patient information, the system prompted the operator to select various parameters that included the tooth number, type of restoration, type of bite, block selection among others, and the scan was initiated using the ineos blue scannerindividual scans of both the maxillary and mandibular models along with a buccal scan of the articulated models were performed and the system then generated a digital model using the composite imagesthe digital models were then aligned in the orientation mode and the margins were traced using the margin tools from the tool pane on the right side of the screen and the restorations were designed from the tooth library which contains thousands of biogeneric tooth morphologies [figure 2]. The restorations were moved, resized, and then the interproximal and occlusal contacts were evaluated [figure 3] using the model contact feature of the analyzing tool pane . The occlusion with the antagonist teeth was on the palatal middle third of the crowns and manually adjusted until the occlusal contacts were in the light blue color zone indicative of light contact (050 m) in maximum intercuspationonce satisfied, the location of the sprue and the position of the restorations in a multiblock data was adjusted [figure 4], and the data were sent to the inlab mc xl milling unit . The selected block (cerec s2 pc, 14, sirona dental gmbh; salzburg, sterreich) was fitted in the mill and the milling was initiated which lasted for approximately 8 min for each individual crown [figure 5] after completion of the milling, the crowns were retrieved and the sprue areas were ground smooth . The restorations were then sintered and luted with a dual - cure resin cement (calibra esthetic resin cement, dentsply intl, ny, usa) [figure 6]. During the first visit, diagnostic impressions are taken and the study models were articulated in a mean value articulator (e200 simplex, cori dent, daegu, south korea). The entire procedure, including the tooth preparation, restoration fabrication, and cementation, was scheduled for the second visit at the second visit, preparations were carried out with both the maxillary li's with a resultant shoulder finish line and vinyl polysiloxane impressions (express, 3 m espe dental products; st . Paul, mn, usa) were made following gingival retraction using impregnated retraction cords (ultrapak e cord #00, ultradent products inc ., st . The models were poured in type iv gypsum (gladstone 3000, dentsply gac, ny, usa) the models were taken to our on - site laboratory which housed the inlab mc xl cad / cam station (sirona dental gmbh; salzburg, sterreich) and a new digital file was created using the inlab 4.2 software . After entering the dentist and patient information, the system prompted the operator to select various parameters that included the tooth number, type of restoration, type of bite, block selection among others, and the scan was initiated using the ineos blue scanner individual scans of both the maxillary and mandibular models along with a buccal scan of the articulated models were performed and the system then generated a digital model using the composite images the digital models were then aligned in the orientation mode and the margins were traced using the margin tools from the tool pane on the right side of the screen and the restorations were designed from the tooth library which contains thousands of biogeneric tooth morphologies [figure 2]. The restorations were moved, resized, and then the interproximal and occlusal contacts were evaluated [figure 3] using the model contact feature of the analyzing tool pane . The occlusion with the antagonist teeth was on the palatal middle third of the crowns and manually adjusted until the occlusal contacts were in the light blue color zone indicative of light contact (050 m) in maximum intercuspation once satisfied, the location of the sprue and the position of the restorations in a multiblock data was adjusted [figure 4], and the data were sent to the inlab mc xl milling unit . The selected block (cerec s2 pc, 14, sirona dental gmbh; salzburg, sterreich) was fitted in the mill and the milling was initiated which lasted for approximately 8 min for each individual crown [figure 5] after completion of the milling, the crowns were retrieved and the sprue areas were ground smooth . A bisque try - in was done and the occlusion was adjusted minimally . The restorations were then sintered and luted with a dual - cure resin cement (calibra esthetic resin cement, dentsply intl, ny, usa) [figure 6]. Final restoration design on the articulated digital models occlusal contacts being evaluated the location of the sprue and position of the crown was adjusted in a polychromatic block the selected block (cerec s2 pc, 14/14) being fitted in the mill pre and post- operative view finally, the patient received postoperative care instructions, and a recall appointment was scheduled . During the first visit, diagnostic impressions are taken and the study models were articulated in a mean value articulator (e200 simplex, cori dent, daegu, south korea). The entire procedure, including the tooth preparation, restoration fabrication, and cementation, was scheduled for the second visitat the second visit, preparations were carried out with both the maxillary li's with a resultant shoulder finish line and vinyl polysiloxane impressions (express, 3 m espe dental products; st . Paul, mn, usa) were made following gingival retraction using impregnated retraction cords (ultrapak e cord #00, ultradent products inc ., st . The models were poured in type iv gypsum (gladstone 3000, dentsply gac, ny, usa)the models were taken to our on - site laboratory which housed the inlab mc xl cad / cam station (sirona dental gmbh; salzburg, sterreich) and a new digital file was created using the inlab 4.2 software . After entering the dentist and patient information, the system prompted the operator to select various parameters that included the tooth number, type of restoration, type of bite, block selection among others, and the scan was initiated using the ineos blue scannerindividual scans of both the maxillary and mandibular models along with a buccal scan of the articulated models were performed and the system then generated a digital model using the composite imagesthe digital models were then aligned in the orientation mode and the margins were traced using the margin tools from the tool pane on the right side of the screen and the restorations were designed from the tooth library which contains thousands of biogeneric tooth morphologies [figure 2]. The restorations were moved, resized, and then the interproximal and occlusal contacts were evaluated [figure 3] using the model contact feature of the analyzing tool pane . The occlusion with the antagonist teeth was on the palatal middle third of the crowns and manually adjusted until the occlusal contacts were in the light blue color zone indicative of light contact (050 m) in maximum intercuspationonce satisfied, the location of the sprue and the position of the restorations in a multiblock data was adjusted [figure 4], and the data were sent to the inlab mc xl milling unit . The selected block (cerec s2 pc, 14, sirona dental gmbh; salzburg, sterreich) was fitted in the mill and the milling was initiated which lasted for approximately 8 min for each individual crown [figure 5] after completion of the milling, the crowns were retrieved and the sprue areas were ground smooth . The restorations were then sintered and luted with a dual - cure resin cement (calibra esthetic resin cement, dentsply intl, ny, usa) [figure 6]. During the first visit, diagnostic impressions are taken and the study models were articulated in a mean value articulator (e200 simplex, cori dent, daegu, south korea). The entire procedure, including the tooth preparation, restoration fabrication, and cementation, was scheduled for the second visit at the second visit, preparations were carried out with both the maxillary li's with a resultant shoulder finish line and vinyl polysiloxane impressions (express, 3 m espe dental products; st . Paul, mn, usa) were made following gingival retraction using impregnated retraction cords (ultrapak e cord #00, ultradent products inc ., st . The models were poured in type iv gypsum (gladstone 3000, dentsply gac, ny, usa) the models were taken to our on - site laboratory which housed the inlab mc xl cad / cam station (sirona dental gmbh; salzburg, sterreich) and a new digital file was created using the inlab 4.2 software . After entering the dentist and patient information, the system prompted the operator to select various parameters that included the tooth number, type of restoration, type of bite, block selection among others, and the scan was initiated using the ineos blue scanner individual scans of both the maxillary and mandibular models along with a buccal scan of the articulated models were performed and the system then generated a digital model using the composite images the digital models were then aligned in the orientation mode and the margins were traced using the margin tools from the tool pane on the right side of the screen and the restorations were designed from the tooth library which contains thousands of biogeneric tooth morphologies [figure 2]. The restorations were moved, resized, and then the interproximal and occlusal contacts were evaluated [figure 3] using the model contact feature of the analyzing tool pane . The occlusion with the antagonist teeth was on the palatal middle third of the crowns and manually adjusted until the occlusal contacts were in the light blue color zone indicative of light contact (050 m) in maximum intercuspation once satisfied, the location of the sprue and the position of the restorations in a multiblock data was adjusted [figure 4], and the data were sent to the inlab mc xl milling unit . The selected block (cerec s2 pc, 14, sirona dental gmbh; salzburg, sterreich) was fitted in the mill and the milling was initiated which lasted for approximately 8 min for each individual crown [figure 5] after completion of the milling, the crowns were retrieved and the sprue areas were ground smooth . A bisque try - in the restorations were then sintered and luted with a dual - cure resin cement (calibra esthetic resin cement, dentsply intl, final restoration design on the articulated digital models occlusal contacts being evaluated the location of the sprue and position of the crown was adjusted in a polychromatic block the selected block (cerec s2 pc, 14/14) being fitted in the mill pre and post- operative view finally, the patient received postoperative care instructions, and a recall appointment was scheduled . Irregularities in tooth morphology resulting from late disturbances during the differentiation process most commonly result in peg - shaped li's . The esthetic defect in patients with peg li's consists of both the malformed teeth and the presence of diastema between teeth . The treatment includes two primary objectives: to restore the hypoplastic dental crowns and to close the diastema, but can vary according to patient demand as in this case wherein the patient requested the diastema to be left untouched . Treatment options for diastema closure include procedures such as fixed orthodontic therapy, porcelain laminate veneers as well as minimally invasive procedures such as direct resin composite bonding and treatment options for the correction of peg - shaped li's include metal - ceramic restorations and cad / cam all - ceramic crowns . The incorporation of the cad / cam technology in dentistry and the advances in recent years which include faster production, precision - fit, excellent esthetics, and the possibility of chair - side production made it the preferred choice . At present, the expanded palette of materials for definitive prostheses production using cad / cam technology provides access to both newer and conventional materials such as zirconium, titanium, titanium alloys, and chrome cobalt alloys . The stability values of zirconium oxide ceramics permit the use of this material as an alternative to metal frames for permanent prostheses . On the one hand, resin material blocks are being used for a full anatomical long - term temporary prosthesis and on the other hand, they can also be used for the milling of lost wax frames for casting technology . The uniqueness of this report does not lay in the case selection itself, but the use of cad / cam technology which makes it possible to produce restorations faster with precision- fit and good esthetics overcoming the errors associated with traditional ceramo - metal technology . Cad / cam technologies have started a new era of procedures and restorations in dentistry . However, a major drawback of this technology is the high investment cost for the milling equipment which might hinder its growth in the lower wage third world countries . In spite of all the benefits of these new methods, the dentist working procedures will have to be adapted in the methods of cad / cam milling technology . These include appropriate tooth preparations with rounded contours and the creation of a continuous preparation margin, which is clearly recognizable to the scanner . Last but not least, cad / cam technology has made it possible to machine newer materials such as the high - performance oxide ceramics and titanium with high accuracy that meet industrial standards.
Parry romberg syndrome (prs) is an orphan or rare disease and is a poorly understood degenerative condition, seen most commonly as asymmetry of the face affecting dermatomes of one or multiple branches of the trigeminal nerve and rarely affects the limbs causing hemiatrophy . Some authors refer to it as parry romberg syndrome / en coupe de sabre (prs / ecds) since both conditions can occur separately or together . It most commonly presents in the first two decades of life and is commoner in females with an incidence of 0.3 - 2.5 cases per 100,000 population per year . Prs / ecds can also be associated with neurologic, ophthalmologic, cardiac, rheumatologic, endocrinologic, infectious, orthodontic and maxillofacial manifestations and autoimmune findings . Whilst trophic malfunction of the sympathetic system has been proposed as the etiological basis, other reports suggest an autoimmune pathogenesis . Serological abnormalities in support of this putative mechanism have included the presence of antibodies to anticentromere, extractable nuclear antigen, rheumatoid factor, histone, anticardiolipin, anti scl 70 and hla b 27 antigens . Recent evidence has suggested that there are diverse neurological presentations and late progression can occur with a fundamental immunologically mediated vascular pathogenesis with subcortical white matter changes on magnetic resonance imaging (mri) suggestive of a chronic encephalopathic process . We report a case with a 3 decade history which highlights intracerebaral calcification and the bilateral white matter changes noted on t2-weighted and fluid -attenuated inversion recovery (flair) mri views and suggest this is typical of the periventricular hyperintensities seen in intracranial vascular disease of other etiologies inclusive of collagen vascular disease and referred to as leucoariaosis but not traditionally used in descriptions of this illness . The landmark study on the international epidemiology of prs / edcs by stone reported no cases from the west indies . A 39-year - old woman of indo - trinidaian descent was referred for investigation of possible multiple sclerosis after periventricular white matter changes were noted on mri of the brain following assessment for intermittent blurred vision of 8 weeks duration . At age 8, she had noticed bald spots on her head, which were followed at age 11 by three dark lines that appeared on her forehead and right side of her face . At 18 years of age, she had her first partial seizure and experienced occasional headaches and intermittent visual loss, which lasted for a few seconds followed by generalized tonic - clonic seizures, which was managed with carbamazepine 400 mg orally twice daily . She had approximately two to three seizures per year, which occurred only when she missed meals . At age 21, she noted upward deviation of her cheek - bones . After she had her first child at age 25, her chin deviated down and dark marks appeared at the middle of her chin and at the right side of her mouth . She had no episodes of status epilepticus . Despite seeing many doctors of diverse specialties over the intervening years the prs / ecds was not recognized due to unfamiliarity with the entity . At age 39, when she presented to us, she complained of blurred vision in the 2 preceding months, whenever she focused on obycts . Her ophthalmologist assessment reported anisocoria with the right pupil larger than the left (figure 1a). In particular, there was no hypertension, diabetes, dyslipidemia, smoking, family history of intracranial, collagen vascular disease or stroke . She went on to complete her secondary school education and then completed an accounting degree . There was asymmetry of the right half of the face with atrophy of the muscles and a demarcation line between normal and abnormal skin . The right eye was mildly depressed in the socket, with asymmetry of the pupils and less prominent eyebrows . In addition, there was loss of subcutaneous fat with prominent bony ridges on the affected side and increased pigmentation on the ipsilateral upper forehead . There was atrophy of the right ala region of the nose (figure 1a). Other aspects of her clinical examination were normal . Based on this constellation of clinical features investigations showed that hemogram, international normalized ratio, prothrombin time, partial thromboplastin time, renal, thyroid and liver function and urine analysis were normal . The antinuclear antibodies showed a fine speckled and homogenous pattern at 1/10 dilution: double stranded dna antibody, rheumatoid factor, c3, c4, p and c antinuclear cytoplasmic antibodies, autoantibodies to extractable nuclear antigens, homocysteine and erythrocyte sedimentation rate were within normal limits . Ct brain imaging showed areas of right parietal lobe hyperdensities consistent with calcification and mild atrophy of the right cerebral hemisphere as evidenced by prominence of the cerebral fissures and sylvian fissure (figure 2). T1 flair mri axial views showed periventricular hyperintensities consistent with leukoariaosis more prominent in the right cerebral hemisphere with right frontal, temporal and parietal cortical atrophy (figure 3a, b). Sagittal t2 flair mri view showed similar hyperintensities involving the corpus callosum (figure 3c). Skin biopsy of an area of alopecia showed sclerosis of the dermis with normal adnexal structures and minimal inflammatory infiltrates of the mid - dermis . Prs has overlapping features of scleroderma en coup de sabre and the pathogenesis for both entities is unknown with no effective therapy available . While trophic malfunction of the sympathetic system has been proposed as a cause of prs other reports suggest an autoimmune pathogenesis . Whilst conflict exists between the differentiation of prs from localized scleroderma, it is considered as a clinical subtype of linear scleroderma based on the recent literature and the classification of mayo clinic . In en coup de sabre atrophy of underlying muscle or bone is not classically seen . Furthermore, there is prolonged nerve conduction in areas affected by scleroderma, which do not exist in prs . Additionally, anti - nuclear anti - body titers are often raised with active linear scleroderma, but rarely so with prs . While testing for immunological serologies has been done commonly in this condition, it is important to continue to do so as the presence of a positive rheumatoid factor or ana is associated with increased risk of developing connective tissue diseases . A positive ana as in our case is the most common laboratory abnormality in prs / ecds, with approximately 25 - 52% of patients having an elevated titer . A theory supports that progressive hemi - facial atrophy (pha) is an autoimmune condition . This is supported by the presence of other autoimmune conditions such as systemic lupus erythematosus (sle), generalized myopathy, and rheumatoid arthritis (ra) in some patients with pha . In one study, 17 and 10% of surveyed respondents with pha described a medical history of vitiligo and thyroid dysfunction, respectively . Other autoimmune conditions seen in these responders were inflammatory bowel disease (5%), ra (4%), ankylosing spondylitis (2%), and sle (2%). On mri, t2 hyperintensities is one of the most common finding in these patients suffering from prs / ecds, even with mri evidence of microbleeds or microcalcifications . Ct findings of calcification though rare have also been noted as in our case . In one report, it was noted that seizures were a common finding, however neurologic abnormalities were not correlated with the mri imaging abnormalities . Furthermore the severity of superficial disease did not correlate with neurological symptoms or findings on neuroimaging . Whether the mri abnormalities have any correlation to serum immunological titers has not been previously documented . While the pathogenesis of neurological involvement is unclear, a vasculitic process has been suspected . In this report by chiu et al ., perivascular lymphocytic infiltrate with features of vasculitis was observed with ectatic blood vessels seen on both biopsies and angiographic studies, which supported the theory that vasculopathy or vasculitis are at the root of the cerebral changes . The presence of neurological involvement, hyperintensities on mri and positive serologies of immunological parameters in our case, suggest that the underlying etiology for her condition is vasculitic . Other reports supporting this immunologic phenomenon have advocated research regarding the efficacy of immunosuppressants in these patients suffering from prs . Brain histological studies in prs / ecds have been few and may be non - specific, consistent with changes of vasculitis . Whilst the periventricular white matter changes in figure 3b in our case is radiologically similar to leucoariaosis described in other vascular disease, leucoariaosis is not a term used in descriptions of this illness and may be another observation for further examination . Definitive postmortem studies of the brain were not found and this is also an area of further research . Despite the numerous neurological manifestations as summarized by vix and colleagues in their recent review and late deterioration with a hemiparesis as noted by dibaj and colleagues, in our patient apart from seizures, the illness has run a benign course . In conclusion, we report the first case of prs / ecds from the west indies and highlight the typical clinical, serological and the rare neuroradiologic findings observed in larger case series . Stone recorded diagnostic delay up to 41 years in his global survey, 10 years longer than our patient.
Flat foot is a disease that is either congenital or acquired, having characteristics such as talus medial rotation, decreased medial arch height, and forefoot supination and abduction1 . Flat foot also causes excessive movement to be pronated and shock absorption to be decreased . In general, a normal foot experiences a pressure 1.5 times body weight when in contact with the ground, whereas people with flat feet feel more fatigue because of the problem of shock absorption . Flat foot is a dysfunction of the posterior tibial tendon, one of the important supporters of the medial arch, a dysfunction of the spring ligament2,or an injury to the plantar fascia3 . It can also result from obesity increasing the load on the feet during the stance phase, causing abnormal foot movement4 . Most previous studies have been causal analyses of flatfoot and studies of treatment effectiveness through sugery . Few studies have investigated the extent to which dynamic activities, such as gait, affect the lower extremities of flat - footed people . Gait is a natural action in daily life, and there is great diversity in individual gait patterns, especially at different gait velocities5, 6 . Because the human foot has evolved for standing and upright movements, such as gait, the alignment of the foot and ankle joints plays an important role in supporting weight during gait . Thus, the human foot uniquely contacts the ground, supplying the momentum for movement via the ground reaction force and playing an important role in the weight - bearing function of subtalar movement7 . Ultimately, gait is possible through interactions that link the human calcaneus, the sole of the foot, and the tips of the toes, and this is why we have conducted this study, to determine the difference between flat feet and the normal feet at different gait velocities using emg and foot pressure analysis . People with normal feet (n = 15) and people with flat feet (n = 15), all of whom have no neurological history and were between the ages of 21 and 30, participated in this study . Age, weight, and height were measured to determine the body characteristics of the subjects . Flat foot was confirmed by posture analysis (gps400, redbalance, italy). As described by clarke8, strake strake s line passes from the forefoot s medial line to the rearfoot s medial line, and marie s line passes from the center of the 3 metatarsal bone to the center of the rearfoot . If the line of the medial sole falls outside marie s line, it is confirmed as a normal foot . If the line of the medial sole falls inside marie s line, it is confirmed as a flat foot . All the subjects received explanation of the research and provided their consent to participation . A treadmill (ac5000 m, scifit, uk) was used to analyze kinematic features during gait, using a slope of 0% with gait velocities of 1.101.25 m / s9 . The average gait velocity of the average man at slow, normal, and fast rates are 3, 4, and 5 km / h, respectively, and those of the average woman are 2.7, 3.7, 4.7 km / h, respectively10 . Subjects walked for about one minute to determine their natural gait velocity before the experiment began . Then all subjects walked barefoot for five minutes on the treadmill, looking forward . Muscle activity data were collected and analyzed using a wireless surface electromyograph (telemyo 2400 t, noraxon co., usa). The electrode diameter was 11.4 mm, and the distance between the electrodes was 20 mm . The sampling rate for the emg signal was set at 1000 hz, the bandwidth was set between 20450 hz, and the notch filter was set at 60 hz . Emg was performed after depilating the electrode - attachment areas with a razor, removing the horny layer with sand paper, and cleansing the areas with an alcohol swab . To measure muscle activations in the lower extremities during gait, electrodes were attached to the abductor hallucis, tibialis anterior, peroneus longus, medial gastrocnemius, lateral gastrocnemius, vastus medialis, vastus lateralis, and biceps femoris muscles . The frequency range of the emg signal was band - pass filtered between 20 and 500 hz, and the sampling frequency was 1024 hz . We normalized the signal of each muscle to the maximal voluntary isometric contraction (mvic). Data on the change of foot pressure was collected using a foot pressure analyzer (fsa, vista medical, canada). The pressure measurement pad is 0.88 mm thick, and it has 128 numbered resistance sensors (916 mm each) arranged in a 816 grid . We used a sampling rate of 3,072 hz, and measurement range of 030 psi . The sizes of the pressure measurement mats were 230100 mm, 250100 mm, and 270100 mm, and they were fitted to subjects insoles according to their foot size . In this study, the foot was divided into eight areas for analysis according to foot regions: two toe regions (1st toe region and 2nd5th toe region), three forefoot regions (1st metatarsal region, 2nd3rd metatarsal region, and 4th5th metatarsal region), the midfoot region, and two heel regions (medial heel region and lateral heel region)11 . The average pressure of each region was measured during the experiment . The general subject characteristics (age, height, and weight) data were analyzed by repeated anova in spss for windows (version 17.0), and the differences between groups at the different gait velocities were examined with the independent t test . Eg: experimental group; cc: control group * p<0 .05, eg: experimental group; cg: control group rf: rectus femoris; vm: vastus medialis; vl: vastus lateralis; ta: tibialis anterior; pl: peroneus longus; mg: medial gastrocnemius; lg: lateral gastrocnemius; ah: abductor hallucis * p <0.05; eg: experimental group; cg: control group t1: toe1; t2: toe25; f1: 1st metatarsal; f2: 2~3rd metatarsal; f3: 4~5th metatarsal; m: midfoot; h1: medial heel; h2: lateral heel the general characteristics of the subjects are shown in table 1 . Muscle activities (excepting that of the rectus femoris) of the flat - footed subjects were significantly different from those of the normal - footed subjects at all of the different gait velocities (p <0.05), especially those of the vastus medialis and abductor hallucis muscles (p <0.05) (table 2). Significant differences in pressure were also detected on the forefoot, midfoot, and the medial area of the hindfoot (p <0.05). Table 3 shows a significant difference in the 2nd3rd metatarsal area (p <0.05). Nakajima et al.12 studied the relationship between the foot s arch height and the shock effect on the knee . They concluded that a correlation exists between the height of the foot s arch and the shock on the knee . Thus, we know that the adduction moment of the knee joint of flat - footed subjects is higher than the adduction moment of sunjects with normal feet . This is due to higher muscle activation of the vastus medialis muscle in flat - footed people . Furthermore, muscle activation of the abductor hallucis muscle in flat - footed people decreases relative to the changes in velocity compared to people with normal feet is relatively lower according to the changes in velocity . Thus, we can conclude that, compared to subjects with normal feet, the medial longitudinal arch of flat - footed subjects does not work well as a dynamic stabilizer . Fiolkowski et al.13 confirmed that the abductor hallucis muscle affects the height of the navicular bone in a tibial nerve block study, and that foot pressure changed with gait velocity . We confirmed the weight of flat foot was not moved onto the toe until the terminal stance phase, and that the weight of flat footed subjects was focused on the 2:3rd metatarsal area . We also confirmed that hindfoot eversion of a flat foot is higher with increasing gait velocity, and the foot pressure of a flat foot is higher on the medial side of foot in the terminal stance phase . We suppose the load of moving the lower extremity increases with velocity and ability of medial longitudinal arch of flat foot is less than normal foot resulting in pressure being focused on 2:3rd metatarsal area in the stance phase.
Acute mesenteric ischemia (ami) is an emergency situation that is caused by circulatory disturbances of mesenteric vessels and is frequently associated with atherosclerosis and cardiovascular disorders . Ami is a complex disease entity including acute mesenteric arterial embolism and thrombosis, mesenteric venous thrombosis, and non - occlusive mesenteric ischemia (nomi). Nomi results from low - velocity blood flow states or from mesenteric vasoconstrictions caused by some drugs . The abdominal pain caused by intestinal ischemia is not proportional to the physical examination findings, especially in elderly persons . Although computed tomography (ct), magnetic resonance imaging and angiography are valuable diagnostic steps, definite diagnosis of intestinal ischemia frequently depends upon the clinical history because the representation would be subtle and obscure . The majority of patients with ischemic colitis can be treated medically, but approximately 20% of the patients clinically deteriorate to develop peritonitis and sepsis, necessitating surgical intervention with an associated 60% mortality rate [1, 2]. A laparotomy is helpful for rapid confirmation of the diagnosis and for optimal treatment, which often consists of revascularization and resection of the affected bowel . Alcohol is well recognized as a systemic toxin that causes serious health problems, including psychosocial problems, myocardial infarction, stroke and atherosclerosis . We reported a case of sub - acute ileal and colonic ischemia in an elderly patient, not having a history of cardiovascular disorders, after heavy alcohol intake, and we present a review of the literature . An 80-year - old male patient was admitted due to uncontrolled diarrhea and abdominal pain accompanied by fever . He became unconscious after drinking 1,800 ml of soju (korean traditional drink, 25% distilled alcohol) in one sitting and was then admitted to a local clinic . After conservative management, he woke up coherent, but he was referred to ewha womans university mokdong hospital because his diarrhea, fever and abdominal pain had not subsided after 10 days . He had no history of chronic diseases such as diabetes, cardiovascular disease, hypertension, hepatitis or smoking . He had had a drinking habit of half a bottle of soju (90 ml) daily for forty years . On admission, his blood pressure was 110/60 mmhg, his pulse rate was 84 beats / min, his respiratory rate was 24 breaths / min, his body temperature was 36, and his mental status was clear . He had continuously suffered from diarrhea from the onset of the illness, 6 to 7 times daily, including night - time defecation . Upon physical examination, there was mild generalized tenderness with abdominal distension on the whole abdomen and hyperactive bowel sound . Laboratory data included the following: leukocyte count, 14,100/l with no leftward shift (neutrophil count, 61.8%); hemoglobin level, 12.1 g / dl; platelet count, 263,000/dl; total protein, 4.8 g / dl; albumin, 2.2 g / dl; aspartate transaminase, 78 iu / l; alanine transaminase, 103 iu / l; blood urea nitrogen 8 mg / dl; serum creatinine, 0.7 mg / dl; amylase, 82 u / l; creatine kinase, 48 upright and supine roentgenograms of the abdomen showed the paralytic ileus with bowel distension (fig . Abdominal ct scan showed circumferential wall thickening of the bowel from the distal ileum to the ascending colon (fig . Frequently the clinician finds no evidence of alcohol - induced hepatitis or pancreatitis that could be the cause of abdominal pain in patients suffering from alcohol abuse . Upon the suspicion of ischemic colitis, the patient was treated with intravenous hydration, bowel rest, and broad - spectrum antibiotics . After 5 days of treatment, he still had intermittent fever above 39 and severe cramping abdominal pain . On day 7 diffuse wall thickening of the terminal ileum to the transverse colon and a large amount of ascites in the pelvic cavity were noted in the ct scan (fig . Mucosal edema and multiple phlegmons were found on the ascending colon with colonoscopy (fig ., symptoms such as diffuse abdominal distension, abdominal tenderness and rebound tenderness were aggravated; hence, a laparotomy was performed . When the abdomen was opened, a small amount serous colored ascites was noted in the pelvic cavity . The outer surface of the ileum (from the ileocecal valve to the proximal 30 cm) looked dark brown to blackish with severe edema and a weakening of the tissues of the bowel wall was observed . Also, the findings of the cecum and the ascending colon appeared to be similar to those of the affected ileum . The gross appearances of the transverse and the descending colon and the sigmoid colon were almost normal . Pathologically, a diffuse acute infarction was present in the ileum and cecum, with acute vasoconstrictions of the mesenteric vessels . A polyp of 0.4 cm was found on the ascending colon, 13 cm distal to the ileocecal valve, and was revealed to be a mucosal adenocarcinoma . The postoperative course was uncomplicated, and the patient was discharge in good condition on the 15th postoperative day . Nomi, the dominant non - thrombotic cause of acute mesenteric ischemia, results from low - flow states (cardiogenic shock, sepsis, and hypovolemia), vasoconstriction, or occasionally drugs . Nomi is defined as " intestinal infarction without mesenteric vascular occlusion, " and is the most lethal form of ami, with mortality rates of up to 70% [3, 4]. The condition commonly occurs in elderly patients, but a subset of young patients with mesenteric ischemia has been reported in the literature . In this young population, etiologies were reported as hypovolemic shock, vasculitis, and drug use . Medication and addictive drugs, such as digitalis, oral contraceptives, phenobarbital, ergotamine, furosemide and cocaine, have been implicated in the development of ischemic intestinal disease . For a long time, alcohol has been known to be a precipitating factor of vasodilatation; thus, after alcohol intake, the face becomes red, the blood pressure falls and the blood flow increases in the splanchnic vasculature . Unlike such physiologic changes, pathological binge drinking is known to act as a vasocontricting situation and to be the cause of cardiovascular events . During the past ten years, alcohol consumption has come to be known as a risk factor for developing cerebral vascular diseases, such as stroke . Several studies suggest that ethanol produces prolonged constrictions in cerebral blood vessels, and the vasoconstrictive actions of ethanol are involved in the hypoxic, ischemic and hemorrhagic actions on the brain [5, 6]. Although several studies have investigated alcohol's effect on the vascular system, there are no reports that alcohol - caused mesenteric vasoconstriction directly causes intestinal ischemia . The mechanism of alcohol - induced splanchnic vasoconstriction is unclear and is thought to be multifactorial . Several mechanisms want for consideration to explain how alcohol causes bowel ischemia in spite of the difficulty in proving a direct causal relationship between the abuse of alcohol and intestinal ischemia . At first, alcohol may have a direct toxic effect on the gut mucosa and increase bacterial overgrowth in the small intestine [7, 8]. This persistent mucosal irritation is also likely to impair the susceptibility to abundant normal bacterial floras inside the bowel . Another indirect effect of alcohol on the mucosal microcirculation is an enhanced trans - capillary fluid filtration, increasing the permeability of the mucosa to macromolecules, such as endotoxin and/or other bacterial toxin that cause sepsis . In such a state, the intestinal infection has already started and may result in an irreversible clinical course . Alcohol intake induces abnormalities in the plasma proteins that are required for blood clotting, leading to the formation of blood clots and the thrombotic occlusion of arterioles . Alcohol may induce mesenteric vasoconstriction directly by suppressing the endothelium - dependent vasorelaxation, leading to reduced blood flow and ischemia . In our case, the patient had no history of any specific infection, had no predisposing factors for systemic vascular problems, and was healthy for his age . We tried conservative treatment under the clinical diagnosis of acute gastroenteritis because there was no evidence of thrombus or constricted vessels in the first series of ct scans, but, in spite of bowel rest and conservative management, the symptoms continued for 15 days (including the treatment period at another hospital) without improvement . There were some differences between the operation findings and the ct findings . During surgery, however, the distal ileum to the mid - transverse colon showed diffuse ischemic change, and there were much ascites . Pathological examination of the mesenteric vessels harvested revealed neither thrombosis or embolus within arteries or veins nor evidence of inflammatory bowel disease, such as systemic vasculitis and chronic granulomatous inflammation . Extensive mucosal necrosis, which was associated with inflammation of the mucosa and vascular congestion, was consistent with nomi . It is doubtful that the clinical course of this patient would have been positive if prompt surgery had not been performed . Recently, with increasing average life span, the elderly population has been increasing rapidly . Many studies on systemic vascular disorders in the elderly have been reported . In the case of emergency situations such as cerebrovascular disease or cardiovascular disease, the best procedure has been established relatively well . However, there are frequent occasions where even doctors cannot diagnose an intestinal infarction when a patient presents with an acute abdomen . Fortunately, with the improvement of high resolution ct, the accuracy of the diagnosis of an acute abdomen in the elderly is much improved . Ct may be helpful in determining the primary cause of bowel ischemia and in allowing direct evaluation of the bowel wall, the adjacent mesentery, and vascular structures . Ct is fundamental for early diagnosis and management when evaluating elderly patients with an acute abdomen, and we should have intestinal infarction in mind when evaluating patients with a history of binge drinking . Drug abuse, including excessive alcohol consumption in particular, has become a major health problem in korea . This report established that, for successful treatment, mesenteric ischemia should be considered in the differential diagnosis of an acute abdomen in patients with a history of alcohol abuse.
Pica actually refers to the latin word for magpie, a bird thought to have odd feeding habits . Incidence of pica has also been linked to iron and zinc deficiency . Whether the pica is an eating disorder or obsessive compulsive disorder is still controversial . The patient was a 27 year old young indian lady living in the baruipur with no previous psychiatric or medical history . Patient complained of an unusual sensation in her abdomen for the last 3 months while her parents gave a history of habitual consumption of nails for the same duration . The patient was a single child to her parents and she was living in the suburbs since her birth . She had attended public schools and had completed her graduation on arts from a college in kolkata . Soon after her marriage, she expressed sexual aversion with history of dyspareunia . Within a month of her marriage, she found it extremely difficult to live in her in - laws house and preferred to stay at her parental home, although her parents repeatedly insisted her to go back to her in - laws residence . Depressed and also admitted that for the last 3 months, she was having a sense of hopelessness and worthlessness over her future days . Vanished. She temporarily enjoyed the gritty and spiky sensation of nails along her throat, but at night she used to have a vague abdominal sensation which she described to be funny. According to her mother, she did not want to go back to live with her husband . On close interviewing of the patient, initially her response to same- sex relationship was guarded but later it was found that she was actually having such a relationship with another lady of her age for the past 3 years . She also accepted that she used to consume glass piece occasionally since last 3 years but never disclosed them to anyone . She had once had an x - ray of her abdomen done 2 years back due to complaints of low back pain which was inconclusive; perhaps glass beads which are radiolucent were missed . No further investigation was done . The patient was decently dressed in a pastel shade salwar with a boyish cut hairstyle . On mental state examination on interviewing the husband, it was known they had never had any sexual intercourse since marriage . On routine examination, possibility of iron deficiency anemia, serum ferritin abnormality and zinc deficiencies were ruled out . X ray of abdomen showed multiple radio opaque pointed objects in her gut (ingested iron nails). The patient had been nurtured in an overprotected environment by her parents in her early childhood days . However immediately following her high school, she had to take many responsibilities of the family all of a sudden because of loss of job of her father . She was more bonded to her mother and in the elderly, often avoided her father because he was alcoholic . The cultural, psychodynamic, and physiological aspect of this case is a unique one . The patient's initial experimentation with ingestion of glass beads, though in a very remote sense, may be culture based considering the fact that the practice of ingesting such beads and sharp metallic objects has been reported . Considering the fact that the patient was initially nurtured in an overprotected environment by her parents and suddenly being exposed to a comparatively hostile situation where she found a discouraging picture of the members of the opposite sex from that of her alcoholic father, the patient's inclination toward same - sex relationship can be explained . Psychologically, the inclination of the patient toward same sex relationship and her unfamiliarity and uneasiness with the members of the opposite sex led to marital dissatisfaction . Also, since the patient had to take many burdens of her family at an age which may be considered quite an early one considering the indian standards and her socioeconomic profile, this probably has caused her to resort to some, harsh robust means like ingestion of nails which may have been an act to depict her her intelligent quotient as measured by wechsler's adult intelligence scale had been found to be 98 which fall in normal age . As in most cases of pica, she initially did not come with a complaint of pica . Rather, she had reported to the hospital with complaints of unusual sensation in her abdomen . The present case presented a unique conglomeration of cultural, psychodynamic, and physiological determinants which together contributed to the expression of pica in comparison with previous reports and reviews . However, an interesting fact in this case is the age of the patient which is quite unusual for pica to occur . This perhaps necessitates further research in dealing with the epidemiology and other aspects of this quite rare but interesting disease entity.
Recently, global nursing experts have been aggressively encouraging nurses to pursue creativity and innovation in nursing in order to improve nursing outcomes (1, 2). The world of healthcare is changing (3). Nurses worldwide are engaged in innovative activities on a daily basis; activities motivated by the need to improve care outcomes and reduce costs to the health system (4). Failure to acknowledge and encourage creativity in nurses may hinder future development and innovations in nursing practice and in nursing science (5). Nurses often encounters unexpected situations and involves taking care of patients with different backgrounds and health conditions; hence, they need to go beyond nursing routine and acquire creative thinking to make useful decisions (6). In most health systems, nurses are the main professional component of front line staff providing up to 80% of primary health care . As such, they are critically positioned to provide the creative and innovative solutions for current and future global health challenges (4). Moreover, being effective, health care organizations must facilitate creative responses to increasingly complex health problems . These organizations and their staff must innovate and make change as health care needs and demands shift . Meeting these challenges requires the complete use of entire available human and mental resources (4). Among the most important of these resources is creativity, which is the capacity to think of old problems in new ways to change the perspectives, or to create novel and useful approaches to construct our organizations work better and to meet the needs of patients (7). Creativity is a major issue in nursing profession and the most important characteristic of nurses and in the health care, it is a complex issue (8). The term creativity and innovation are often used interchangeably; however, there is a clear distinction between them, creativity is the generation of novel and useful ideas, while innovation is the implementation of the ideas (9). Recent nursing literature has repeatedly proclaimed the need for creativity in nursing (10). Studies have shown that creativity is not highly rewarded in practice; however, it is supposed to be in the theory (11) and although creativity has been featured in the nursing literature for many years, it has been the subject of only a small amount of research in nursing . Therefore, an edified understanding of creativity and its dynamics and place in nursing has never been attained (12). Also, the most studies on creativity have been conducted in nursing education (13), but creativity in the clinical settings and its outcomes for patients, nurses and health care organizations have not been extensively studied and its consequences is still not clear . Hence, the area of creativity within the medicine and nursing appears underdeveloped and the outcomes of creativity in these fields are unknown (14). In addition, most researches about the nursing creativity in iran have been conducted with quantitative approach (15 - 17). Therefore, considering the existing evidence in iran and lack of comprehensive study on the nurses creativity, this study explores the perception and experiences of creative nurses about outcomes of their creativity in clinical settings in the cultural context of iran . Considering the importance of creativity in nursing and the role of nurses in health care organizations, this study aimed to determine the iranian nurses perceptions and experiences of creativity in clinical settings and the outcomes of their creativity for health care organizations . A qualitative approach using content analysis was adopted for this study to facilitate a rich description of the understanding and experiences of creative clinical nurses about outcomes or consequences of creativity in health care settings in an iranian context . It was composed of two open - ended questions (interview guide) as follows: have had any experience regarding creativity in your workplace? And, what were the outcomes of creativity for you, patients, and hospitals? Also, some probing questions were asked for additional clarification to answers given by the participants . No prejudices or personal opinions were involved in the interview process, and semistructured guidelines were adopted to guide interviewees to express their experiences as far as possible . Interviews lasted between 30 - 90 minutes and were performed in a quiet place in participants working units . All interviews were performed by one female interviewer and were audiotaped with the nurses consent . The interviewer received formal qualitative curriculum training and had clinical practicum teaching experience of 21 years, which would help the participant enter the interview situation and build a trustworthy relationship . The data collection, data analysis and participants selection was continued until data saturation occurred and a rich description of nurses experiences was obtained . The data collection was interrupted after 14 interviews, because it was clear that no new concepts had emerged after 13 interviews . In this study, 14 nurses (5 females and 9 males) who had worked in different units of educational hospitals affiliated to jahrom and tehran medical universities, iran, were recruited by purposeful sampling with the maximum variation sampling to achieve variation in nurse's gender and work experiences as well as educational levels (table 1). Two nurses refused to participate in the study after initial agreement (both because of problems in setting time for interview). Inclusion criteria, including the nurses who had offered novel and useful ideas, products, services or procedures in clinical settings and had willingness to participate in the study . In this study, matrons, supervisors, and head nurses were asked to nominate nurses who provided creativity and innovation at workplace . Abbreviations: msn, master of science in nursing; bsn, bachelor of science in nursing; adn, associate degree in nursing; lpn, licensed practical nurse . The ethical and research committee of university of social welfare and rehabilitation sciences in tehran approved this study (2id11767). Before starting each interview, all participants were informed about the objectives and method of the study . They were also informed that participation in the study is voluntary; therefore, they could refuse to participate or withdraw from the study at any time . Moreover, the participants were reassured that their responses would be kept confidential and their identities would not be revealed in research reports and publications of the study . Finally, the participants who agreed to participate in the study were asked to sign a written consent . . Framework is an analytical process, which involves a number of distinct though highly interconnected stages (18). It has five key stages . In the first stage or familiarization stage, we transcribed the data verbatim and read each interview several times to gain a sense of content . The second stage or identifying a thematic framework, involved dividing the text into meaning units . The condensed meaning units were abstracted and labeled with a code, which constitute the manifest content . In the third stage or indexing, we compared the various codes based on differences and similarities and sorted them into subcategories and categories and collated all the relevant coded into data extracts within the identified categories . In the fourth stage or charting, we read all the collated extracts for each category and considered whether they appeared to form a coherent pattern . Then, we considered the validity of individual categories in relation to the dataset and whether our candidate categories accurately reflected the meaning evident in the dataset as a whole . Two researchers independently examined the data for categories . In the fifth stage or mapping and interpretation, we defined and further refined the categories (18). Similar to all qualitative approaches, content analysis entails discovering and exploring the processes in the findings (19). To ensure credibility, member checking was used in which the first author performed a member check for 9 participants through providing a summary of identified themes for clarification . Data were coded by the authors after they were compared with each another . In cases of disagreement, explanation and clarification it was composed of two open - ended questions (interview guide) as follows: have had any experience regarding creativity in your workplace? And, what were the outcomes of creativity for you, patients, and hospitals? Also, some probing questions were asked for additional clarification to answers given by the participants . No prejudices or personal opinions were involved in the interview process, and semistructured guidelines were adopted to guide interviewees to express their experiences as far as possible . Interviews lasted between 30 - 90 minutes and were performed in a quiet place in participants working units . After recording the interviews, they were transcribed verbatim immediately and analyzed . All interviews were performed by one female interviewer and were audiotaped with the nurses consent . The interviewer received formal qualitative curriculum training and had clinical practicum teaching experience of 21 years, which would help the participant enter the interview situation and build a trustworthy relationship . The data collection, data analysis and participants selection was continued until data saturation occurred and a rich description of nurses experiences was obtained . The data collection was interrupted after 14 interviews, because it was clear that no new concepts had emerged after 13 interviews . In this study, 14 nurses (5 females and 9 males) who had worked in different units of educational hospitals affiliated to jahrom and tehran medical universities, iran, were recruited by purposeful sampling with the maximum variation sampling to achieve variation in nurse's gender and work experiences as well as educational levels (table 1). Two nurses refused to participate in the study after initial agreement (both because of problems in setting time for interview). Inclusion criteria, including the nurses who had offered novel and useful ideas, products, services or procedures in clinical settings and had willingness to participate in the study . In this study, matrons, supervisors, and head nurses were asked to nominate nurses who provided creativity and innovation at workplace . Abbreviations: msn, master of science in nursing; bsn, bachelor of science in nursing; adn, associate degree in nursing; lpn, licensed practical nurse . The ethical and research committee of university of social welfare and rehabilitation sciences in tehran approved this study (2id11767). Before starting each interview, they were also informed that participation in the study is voluntary; therefore, they could refuse to participate or withdraw from the study at any time . Moreover, the participants were reassured that their responses would be kept confidential and their identities would not be revealed in research reports and publications of the study . Finally, the participants who agreed to participate in the study were asked to sign a written consent . Data were analyzed using a conventional content analysis approach . To analyze the data, . Framework is an analytical process, which involves a number of distinct though highly interconnected stages (18)., we transcribed the data verbatim and read each interview several times to gain a sense of content . The second stage or identifying a thematic framework, involved dividing the text into meaning units . The condensed meaning units were abstracted and labeled with a code, which constitute the manifest content . In the third stage or indexing, we compared the various codes based on differences and similarities and sorted them into subcategories and categories and collated all the relevant coded into data extracts within the identified categories . In the fourth stage or charting, we read all the collated extracts for each category and considered whether they appeared to form a coherent pattern . Then, we considered the validity of individual categories in relation to the dataset and whether our candidate categories accurately reflected the meaning evident in the dataset as a whole . Two researchers independently examined the data for categories . In the fifth stage or mapping and interpretation similar to all qualitative approaches, content analysis entails discovering and exploring the processes in the findings (19). To ensure credibility, member checking was used in which the first author performed a member check for 9 participants through providing a summary of identified themes for clarification . Data were coded by the authors after they were compared with each another . In cases of disagreement, explanation and clarification in total, 14 nurses (9 males and 5 females) aged 27 - 57 years old participated in this study . Their work experiences were between 7 - 30 years and most of them had worked in different units of the hospital (table 1). Data analysis resulted in identifying four main themes: a) improvement in patients quality of care, b) improvement in nurses quality of work, personal and social life, c) promotion of organization and d) unpleasant outcomes . The rest of the text discusses the meaning of each theme, with quotations from participants . When nurses were asked their experiences about outcomes of creativity in clinical settings, they pointed to the more cooperation of patients, doing the work more convenient and achieving better therapeutic results . A nurse with 15 years of work experience stated that: " i made little crafts such as sofas and chairs or little fishes with waste papers, and next morning when i wanted to give medication, first i gave it to the child . Therefore, i communicate with the kids in this way that it caused my work to be more convenient . For example, the child used the drug much better and was more cooperative during change dressing . Furthermore, participants mentioned that creativity caused facilitation of patient care and the patients received better care and had higher satisfaction . For example a nurse with 7 years of work experience who had made a stretcher that equipped with a cassette for taking x - ray and especially for patients with multiple trauma stated: there, they put the radiology cassette in the drawer under the stretcher to take an x - ray; so, there is no need to move the patient from the stretcher to the radiology bed . I used odorless whitex (a commercial disinfectant solution in iran) in surgical wards for cleaning surfaces and this leads to high patients satisfaction because smelling whitex bothered them. Some participants believed that expressing creative ideas in the workplace had had positive effects on their physical and mental health and they achieved self - belief and felt happiness . Also, pleasure of discovering new creative ideas and doing a larger work had given them intrinsic satisfaction and feeling of pride . One nurse with 14 years of work experience cited that: this means that you're satisfied and happy of doing the right thing, it will make you happy. Also, a nurse with 30 years of work experience mentioned that: i thought if i want to go short, do not look for a bigger work or a larger service, i really do not enjoy, and maybe even have a negative impact on my health. A nurse with 17 years of work experience cited that: moreover, participants pointed out to the gaining social respect in the community and they believed that creativity in nursing causes acceptance of friends and the community, being known, being seen with a positive outlook and being a model for other nurses or students . One nurse with 20 years of work experience stated: when friends praise that you offered a new job, had an invention, or an innovation, maybe i am satisfied with these aspects, well it's good in the community . For example, when they say you are creative, one feels that he is difference with the other people and this is due to the feedbacks that community gives to you . In addition, participants mentioned that creativity is the main engine for the development of health care workers and organizations . And nurses creativity leads to innovation, entrepreneurship, and is a source of income . A creative and entrepreneur nurse with 13 years of work experience: if there were problems in the operating room, i designed and made them . Then, i established a company in industrial park and i just make some of the medical devices for production in the company and i have about 8 - 9 workers . According to the nurses who participated in the study, creativity, innovation, and entrepreneurship in nursing has led to growth and promotion of the agency and nurses have had more desire to work . One nurse with 7 years of work experience stated: my creativity and appliances that i had made has led the university to promote a lot . For example, the university announced that it was supposed to have 3 patent annually based on their strategic plan . Vice chancellor of research and technology in the ministry of health wrote that this university has grown by 200 percent . Well, they were grown . Also, they had a positive view and said if we encourage someone, others are also encouraged . Also, participants believed that creativity has led to reduction of hospital costs, building devices similar to foreign samples with better quality, using the supplies in the hospital and greater comfort of patients and coworkers (health workers). I designed and built a bed special for doing percutaneous nephrolitotomy in the operating room, at that time it was about 240 million rials (or around 240,000 dollars). I designed it and put in the hospital, the entire cost for building it, was 8000,000 rials (or around 1000 dollars). In addition to positive or pleasant outcomes, nurses implied to unpleasant or negative outcomes including: lack of support by some managers and authorities including little encouragement, providing low - cost financing, paying out - of - pocket personally, disruption of family roles, hard- working, cutting out a lot of things in life, and long - time warranty of devices that were made by creative nurses . One nurse with 25 years of work experience stated: " well according to the current situation, i can say that what i expected, outcome is not pretty for me both from incentives, (i mean the system), and financial aspects . " And a nurse with 27 years of work experience cited that: " do you think a creative person as a father in the home could be a positive and effective parent for the children . Unfortunately it is not supported, the creative nurse must work very hard, and cutting out a lot of things in the life, and this is a fact . " When nurses were asked their experiences about outcomes of creativity in clinical settings, they pointed to the more cooperation of patients, doing the work more convenient and achieving better therapeutic results . A nurse with 15 years of work experience stated that: " i made little crafts such as sofas and chairs or little fishes with waste papers, and next morning when i wanted to give medication, first i gave it to the child . Therefore, i communicate with the kids in this way that it caused my work to be more convenient . For example, the child used the drug much better and was more cooperative during change dressing . Furthermore, participants mentioned that creativity caused facilitation of patient care and the patients received better care and had higher satisfaction . For example a nurse with 7 years of work experience who had made a stretcher that equipped with a cassette for taking x - ray and especially for patients with multiple trauma stated: there, they put the radiology cassette in the drawer under the stretcher to take an x - ray; so, there is no need to move the patient from the stretcher to the radiology bed . I used odorless whitex (a commercial disinfectant solution in iran) in surgical wards for cleaning surfaces and this leads to high patients satisfaction because smelling whitex bothered them. Some participants believed that expressing creative ideas in the workplace had had positive effects on their physical and mental health and they achieved self - belief and felt happiness . Also, pleasure of discovering new creative ideas and doing a larger work had given them intrinsic satisfaction and feeling of pride . One nurse with 14 years of work experience cited that: this means that you're satisfied and happy of doing the right thing, it will make you happy. Also, a nurse with 30 years of work experience mentioned that: i thought if i want to go short, do not look for a bigger work or a larger service, i really do not enjoy, and maybe even have a negative impact on my health. A nurse with 17 years of work experience cited that: moreover, participants pointed out to the gaining social respect in the community and they believed that creativity in nursing causes acceptance of friends and the community, being known, being seen with a positive outlook and being a model for other nurses or students . One nurse with 20 years of work experience stated: when friends praise that you offered a new job, had an invention, or an innovation, maybe i am satisfied with these aspects, well it's good in the community . For example, when they say you are creative, one feels that he is difference with the other people and this is due to the feedbacks that community gives to you . In addition, participants mentioned that creativity is the main engine for the development of health care workers and organizations . And nurses creativity leads to innovation, entrepreneurship, and is a source of income . A creative and entrepreneur nurse with 13 years of work experience: if there were problems in the operating room, i designed and made them . Then, i established a company in industrial park and i just make some of the medical devices for production in the company and i have about 8 - 9 workers . According to the nurses who participated in the study, creativity, innovation, and entrepreneurship in nursing has led to growth and promotion of the agency and nurses have had more desire to work . One nurse with 7 years of work experience stated: my creativity and appliances that i had made has led the university to promote a lot . For example, the university announced that it was supposed to have 3 patent annually based on their strategic plan . Vice chancellor of research and technology in the ministry of health wrote that this university has grown by 200 percent . Well, they were grown . Also, they had a positive view and said if we encourage someone, others are also encouraged . Also, participants believed that creativity has led to reduction of hospital costs, building devices similar to foreign samples with better quality, using the supplies in the hospital and greater comfort of patients and coworkers (health workers). I designed and built a bed special for doing percutaneous nephrolitotomy in the operating room, at that time it was about 240 million rials (or around 240,000 dollars). I designed it and put in the hospital, the entire cost for building it, was 8000,000 rials (or around 1000 dollars). In addition to positive or pleasant outcomes, nurses implied to unpleasant or negative outcomes including: lack of support by some managers and authorities including little encouragement, providing low - cost financing, paying out - of - pocket personally, disruption of family roles, hard- working, cutting out a lot of things in life, and long - time warranty of devices that were made by creative nurses . One nurse with 25 years of work experience stated: " well according to the current situation, i can say that what i expected, outcome is not pretty for me both from incentives, (i mean the system), and financial aspects . " And a nurse with 27 years of work experience cited that: " do you think a creative person as a father in the home could be a positive and effective parent for the children . Unfortunately it is not supported, the creative nurse must work very hard, and cutting out a lot of things in the life, and this is a fact . " In the present research, nurses were asked about outcomes of their creativity in clinical settings . Findings indicated good information about the unique experiences and perspectives of creative nurses and showed that creativity has many benefits for the patients, nurses, their colleagues, as well as the hospitals and the universities . Also, it had some negative effects on nurses that sometimes led to discouragement and displeasure . However, nurses experiences indicated that positive aspects outweighed the negative ones . Therefore, we can say that nurses creativity for health care system is a privilege, not a disadvantage . As the complexity of health care and nursing escalates, health care providers are challenged to think more creatively and develop innovations that advance the knowledge, learning, and service contributions of their discipline to the health care enterprise . Nursing requires creative thought and innovative action in service of the greater good (20). It prevents more invasive procedures, more cooperation and satisfaction of the patients, and facilitation of patients care . Achieving high quality in patient care is important because of the pivotal role of delivering direct care in nursing as well as the growing emphasis on improving hospitals credibility and clinical status . In addition, patient satisfaction is considered as an indicator of quality of health care services (21). Empowerment energizes the people who are closest to the patients and the technology to continuously look for ways to provide high quality patient care and to improve processes . The accumulation of ideas, both great and little by many people, will result in better patient care and practical efficiencies (22). In addition, creative modalities offer nurses a new perspective on how to care for patients . The link between creativity and healing is well - documented (23). Ultimately, fostering the ethics of change, innovation and creativity led to the development of professional nursing practices and advanced patient care (24). The second theme indicated that creativity causes improvements in nurse s quality of work, personal and social life . Nurses stated that creativity leads to innovation, entrepreneurship, establishment of a company, manufacturing of medical equipment s and this makes financial benefits for them . Nurses innovation is the key to the change and transformation in health care systems (25). In iran, creativity, innovation and entrepreneurial skills has not been considered as part of the " formal education " and socialization of nurses in the workplace . Creativity and innovation in nursing is outspread, accidental issue and is not as organized and managed, and not being seen as part of the work to be done by the health care workers, but is seen as something extra to the " real work " of nurses . Therefore, it is essential to manage creativity and innovation in nursing education and practice since it is a developing process and does not happen quickly . Also, internal climate of health care organizations must provide and support the nurses innovation and entrepreneurship . Nurses experiences indicated that creativity enhanced self - esteem, self - belief and feeling of happiness . Successfully, dealing with a difficult situation increases belief in the ability to handle other similar situations . Creatively resolving a present problem when an individual faces with a worrisome problem and uses his or her ingenuity to solve it, an increased sense of satisfaction will be resulted in (26). Also, satisfaction with creativity is supported, at organizational level, by teamwork, autonomy, domain - relevant competences, and at the individual level, nurses experienced social respect and feeling of pride and they stated that creativity had positive effect on their physical and mental health . Pride is produced when an outcome is perceived as the result of an individual s own actions, particularly if he or she receives accolades from others (26). Also, the creative process is seen as a process of discovery, which stimulates and guides expression by the creator, eliciting positive feelings and emotions, thereby promoting their health and well - being (14). In addition, the results of studies indicated that participating in creative activities can have a positive effect on behavioral changes, self - confidence, self - esteem, levels of knowledge and physical activity (28). Creativity and innovation are keys to the survival and growth of both health care system and nursing . The nurse gains personal satisfaction, rewards, and recognition, whereas the organization survives, thrives, and prospers (29). Therefore, creativity and innovation are key elements for improvement of the organization, and, more specifically, for nursing to find alternatives for solving the problems related to the occupation as a whole (30). Creative nurses experienced some negative outcomes, such as disturbances in family role, high workload and time pressure that caused them being fatigue and also impediment to creativity . Creativity takes time, a great deal of hard work, and strenuous mental energy (31). Therefore, providing sufficient time is a factor for promoting creativity, time is necessary to think creatively, to explore different perspectives, and to play with ideas (32). In addition, when workload pressure goes beyond a certain threshold in the work environment, represent an impediment to creativity (27). The nurses pointed to the lack of financial support and attention of managers, so in order to establish a promoting climate for creativity to prosper, the organization should provide a warm intellectual environment that gives employees recognition, prestige, and an opportunity to participate . Nurse managers should promote creativity through sensitivity that gives nurses the attention they want and treats them as distinct individuals (33). Consequently, creativity is crucial in all institutions where nurses are trained . The critical importance of identifying, nurturing and protecting innovators, as well as the role of managers and mentors, is emphasized . While this study was performed with only 14 nurses, it provided a good description about the phenomenon of nurses creativity in clinical settings . The flexibility and other strengths of the qualitative methodology made it possible to explore nurses experiences . Results from this study can be used to establish guidelines on how to foster creativity in health care professionals, especially practical nurses and nursing students as future nurses . Moreover, as this is a qualitative study, the study group is in no way a representative sample . Hence, the results of this study are predominantly representative of the group of creative nurses who participated in this study and cannot be generalized to all nurses . However, generalizability was neither the aim nor the claim of this qualitative research study . While this study was performed with only 14 nurses, it provided a good description about the phenomenon of nurses creativity in clinical settings . The flexibility and other strengths of the qualitative methodology made it possible to explore nurses experiences . Results from this study can be used to establish guidelines on how to foster creativity in health care professionals, especially practical nurses and nursing students as future nurses . Moreover, as this is a qualitative study, the study group is in no way a representative sample . Hence, the results of this study are predominantly representative of the group of creative nurses who participated in this study and cannot be generalized to all nurses . However, generalizability was neither the aim nor the claim of this qualitative research study.
Cirrhosis secondary to hepatitis c virus (hcv) is the most common indication for orthotopic liver transplantation . Acute recurrence can occur within 6 months after transplantation, is often more severe than the primary hcv disease, and leads to fairly rapid progression to cirrhosis . Recurrent viral infection with progression to cirrhosis and graft failure is the most frequent cause of morbidity in the posttransplant setting [24]. Factors possibly contributing to the recurrence of hcv include viral hcv - related factors (viral load and genotype [5, 6]), coinfection with other viruses, donor - related factors, type and amount of immunosuppression, and steroid pulses for treatment of acute rejection [7, 8]. The choice of calcineurin inhibitor does not seem to influence hcv recurrence . In a prospective randomized study, no significant difference in hcv recurrence or hcv progression was found between tacrolimus- and cyclosporine - based treatments . A relationship between steroids and the severity of hcv recurrence has, however, been observed . Posttransplant tapering of steroids has been found to reduce the progression of recurrent hcv [11, 12] while pulse administration of steroids for treatment of acute rejection has been associated with development of cirrhosis a primary cause of graft loss in liver transplantation . In this study we explored the impact of steroid - free immunosuppression on hcv viral load at 12 months in patients transplanted for hcv cirrhosis . The onset of hcv - related liver disease is difficult to determine as the clinical signs and symptoms of hcv are similar to those of acute rejection and the two conditions can coexist . We therefore used hcv viral load as a surrogate indicator of hcv recurrence because of the potential difficulty in differentiating between acute rejection and hcv disease after transplantation . To test the effect of a steroid - free regimen, we compared two tacrolimus - based protocols: one with steroid administration for 3 months (the reference treatment) and the other with daclizumab in which steroids were avoided for both prophylactic immunosuppression, and wherever possible, antirejection treatment (experimental treatment): we assumed that hcv recurrence would be lower with the steroid avoidance regimen . Safety and efficacy evidence for omitting steroids [15, 16] and for replacing steroids with daclizumab in immunosuppression protocols in liver transplantation has been demonstrated in randomized multicenter clinical trials . This was a prospective, randomized, open - label, parallel arm study that was conducted between june 2005 and june 2008 at 17 centers in 8 european countries . Patients were followed up to 12 months unless they withdrew consent or withdrew from treatment for reasons other than death or graft loss . Inclusion criteria included age above 18 years, hepatitis c virus positive, and first orthotopic (whole or split) liver transplant . Exclusion criteria were on - going steroid administration, hiv positivity, abo incompatibility, and a previous history of malignancy other than treated nonmelanoma skin cancer . Patients with hepatocellular carcinoma were included unless they had> 3 nodules, the nodules were> 5 cm in diameter, and there was evidence of vascular invasion, metastases, or local invasion . The study was conducted in compliance with the declaration of helsinki and good clinical practice guidelines and in accordance with local and national regulatory requirements and laws . All relevant study documents were approved by the institutional review board responsible for the study center . All patients provided signed informed consent and could withdraw from the study at any time . The initial daily dose was 0.100.15 mg / kg . Recommended trough levels from day 0 today 42 were 1015 ng / ml, and from day 43 to day 365 levels were <10 ng / ml . Two doses of daclizumab 2.0 mg / kg were administered; the first dose was given during the anhepatic period and the second dose between days 7 and 10 . Biopsy confirmed that acute rejection was treated by increasing tacrolimus dose to attain trough levels of 15 ng / ml . If rejection persisted, thymoglobulin (ratg) was administered at 1.5 mg / kg for up to 3 days; acetaminophen and intravenous h1-receptor antagonists could be administered for premedication against anaphylactic reaction . Steroids were given at a bolus dose of 500 mg in the perioperative period followed by tapered doses of 1520 mg / day during month 1, 1015 mg / day during month 2, 510 mg / day during month 3, then discontinued . Biopsy confirmed that acute rejection was treated by increasing tacrolimus dose to attain trough levels of 15 ng / ml . If there was no response, pulses of corticosteroids up to 1000 mg / day for 3 consecutive days could be administered . Prophylactic antiviral treatment was required for cytomegalovirus (cmv) in cases where a cmv - positive donor graft was transplanted in a cmv - negative recipient . Treatment with the following drugs was prohibited during the study: any other immunosuppressive agents except for mycophenolate mofetil (mmf) used at the discretion of the investigator for rejection treatment and polyclonal antibodies and okt3 that were allowed for the treatment of intractable rejection . Quantitative and as necessary qualitative serum hcv - rna evaluations were performed using branched dna technology . In cases where a viremia reading was <3200 copies / ml, the tma method was used as a sensitive analysis to measure copies 50/ml . Serum hcv rna has been demonstrated to be a reliable indicator of hepatitis c liver disease . Hcv recurrence was diagnosed by liver biopsy and was performed as clinically indicated (in patients with elevated liver enzymes) or at protocol - defined biopsy control . Recurrence was defined as acute hepatitis with lobular necroinflammatory activity and/or as chronic hepatitis with lymphoplasmacytic portal inflammation with interphase hepatitis, lobular necroinflammatory activity, and with portal fibrosis . The histological grading and staging of biopsies for hcv evaluation were based on the modified hai score by ishak et al . . Other outcomes measured were: the incidence of and time to first biopsy - proven acute rejection (bpar); patient and graft survival at 12 months; renal function that was assessed by calculated creatinine clearance (cockcroft - gault formula); the incidence of adverse events that were classified using meddra before database closure . Samples for the evaluation of qualitative and quantitative serum hcv - rna were collected at baseline and at subsequent regular assessments . Routine hematology and blood chemistry were assessed at 9 regular assessment visits (baseline (day 0, defined as the day of liver transplantation), days 1, 7, and 14, and months 2, 3, 6, 9, and 12) or at the time of premature study discontinuation . Tacrolimus trough levels were measured after the first dose then 2 - 3 times per week during the period of hospitalization and at each scheduled follow - up out - patient visit or more frequently if clinically indicated . Protocol biopsies were performed at the end of months 6 and 12; central assessment of biopsy specimens was conducted in a blinded manner . There were no historical data on hcv viral load available at the time of the design of this exploratory study for the planned patient population . Therefore, sample size was based on a compromise between the considerations of feasibility and reasonable statistical power to detect a signal for a positive effect of the avoidance of steroids on hcv viral load . Based on a (1-sided) mann - whitney u test and assuming a standard deviation of 0.6 log10 (copies / ml) the sample size of 50 evaluable patients per treatment arm was considered large enough to achieve a power> 80% . Assuming a rate of 15% nonevaluable patients, 120 patients (60 per treatment arm) were to be randomized . Allocation to treatment arms was performed using sealed sequentially numbered randomization envelopes provided by the study sponsor . Hcv viral load at 12 months was analyzed after a transformation using log10, which resulted in the unit log10 (iu / ml). The 1-sided wilcoxon rank sum test was used to test for the superiority of the steroid - free arm (tac / dac) over the reference arm (tac / str). Kaplan - meier analysis was used to estimate rates of patient and graft survival and survival over time to recurrence of hcv . The chi - square test was used to compare differences in adverse events between treatment arms . The last - value - carried - forward method was used for replacement of missing laboratory values . At investigator review of the results it became apparent that some patients had received antiviral treatment for hcv during the study . It was decided to perform post hoc analyses of hcv viral load at month 12 excluding patients who had taken antiviral medications for hcv . It was assumed that the elimination of these patients from an analysis of the primary endpoint would provide more information on the effect of the two protocols on hcv recurrence . The full analysis set (fas) population included all randomized patients who received at least one dose of any immunosuppressive medication according to the assigned study arm . The primary analysis set (pas) comprised patients included in the fas and with an hcv viral load above the limit of quantification at baseline (615 iu / m / l) to provide a population for analysis with active hepatitis c. primary endpoint results are presented for the pas and fas populations, and the fas is used for the presentation of all other results . Of 138 patients randomized to receive treatment, 135 were included in the fas population . The rate of study completion was lower in the tac / dac arm at 45% (30 of 67 patients) than the rate of 82% (56 of 68 patients) in the tac / str arm . The main reason for premature withdrawal in both arms was an adverse event (figure 1). Study discontinuation during week 1 was more common in the tac / dac arm (12 of 37 withdrawn patients) than in the tac / str arm (4 of 12 withdrawn patients). An erroneously administered steroid bolus at transplantation was the reason for 5 of the 6 protocol violations in the tac / dac group and a patient in the tac / str arm violated the protocol for receiving basiliximab . A baseline hcv viral load below the limit of quantification was documented in 17 patients in the tac / dac arm and in 16 patients in the tac / str arm and led to exclusion of these patients from the pas . The pas, therefore, comprised 50 patients in the tac / dac and 52 patients in the tac / str arm . Of these, 19 patients (38%) in the tac / dac arm and 35 patients (67%) in the tac / str arm completed the study and were used in the analysis of the primary endpoint . Baseline patient demographics were broadly similar between arms including the model for end - stage liver disease (meld) mean scores (table 1). Hepatocellular carcinoma was present in 29 of 67 patients (43%) in the tac / dac arm and 34 of 68 patients (50%) in the tac / str arm at randomization . As would be expected in this european sample, the most common hcv genotype in both arms was 1b . All allografts were recovered from deceased donors . A greater number of organs were from male donors in the tac / dac arm than in the tac / str: this difference was not considered to affect study results . Patients in both arms received antiviral treatment during the study in contradiction to the study protocol . This was 10 patients in the tac / dac arm and 21 in the tac / str arm included in the fas and 8 patients in the tac / dac arm and 20 in the tac / str arm included in the pas . Antiviral treatment was administered to patients with biopsy - proven recurrence to reduce the risk of early severe hcv recurrence, which is associated with increased risk for graft loss . Mean tacrolimus trough levels in both arms were well within the targeted range of 1015 ng / ml up to day 42 . Mean trough levels at month 12 were 7.6 ng / ml in the tac / dac arm and 8.6 ng / ml in the tac / str arm, which were within the targeted range of <10 ng / ml . The mean (sd) daily dose of tacrolimus at month 12 was 0.1 (0.06) mg / kg in the tac / dac arm and 0.05 (0.04) mg / kg in the tac / str arm . All patients in the tac / dac arm received one dose of daclizumab and 52 of 67 (78%) received a second dose . In violation of the protocol, 6 of 67 patients (9%) in the tac / dac arm erroneously received steroid boluses perioperatively; the median (range) dose was 7.7 (1.99.8) mg / kg . A few patients (13 patients at each scheduled assessment visit) were taking maintenance steroids . In the tac / str arm, 56 of 68 patients were maintained on steroids at 3 months, which decreased to 45 patients at 6 months and to 17 patients at 12 months . The median daily dose of maintenance steroids at 12 months was 0.04 (0.010.15) mg / kg . Pulse steroid treatment for acute rejection was administered to 3 patients in the tac / dac arm and to 10 patients in the tac / str arm . There was no significant difference between the treatment arms in hcv viral load at month 12 (table 2). A post hoc analysis of hcv viral load that was performed on patients who had not received antiviral medications during the study showed comparable median values for hcv viral load for the pas (table 2). The difference in median values between the treatment arms reached statistical significance for the fas . In the interpretation of these results it is important to note that analyses were performed post hoc on patients completing the study and the number of study completers was lower in the tac / dac arm . The rate of patients free of hcv recurrence at 12 months was 19.1% with the tac / dac steroid - free protocol and 13.8% with the tac / str protocol (kaplan - meier method) with a significant difference in survival curves between treatments (95% ci, 0.105 to 0.211%; p = 0.020) (figure 2). Early hcv recurrence at month 3 was less common with the steroid - free protocol at 55% compared with 70% in the steroid arm . Results of the post hoc analysis, in which liver biopsies were centrally reviewed and hcv recurrence was censored for antiviral treatment, favored the tac / dac immunosuppression protocol . The estimated rate of patients free of hcv recurrence in this analysis was significantly higher in the tac / dac arm at 20.2% compared with 13.1% in tac / str (95% ci, 0.091 to 0.234%; p = 0.022) (table 3). Protocol biopsies (performed at months 6 and 12) and nonprotocol biopsies were centrally reviewed to assess fibrosis score . As depicted in table 3, there were no differences between the treatment arms in the total mean modified fibrosis staging score; mean scores were <2 in both arms . More cases of severe fibrosis, as evidenced by a score of 4, were assessed in the tac / dac arm (4 of 35 biopsies) compared with the tac / str arm (0 of 54 biopsies). There was no clinically relevant difference between the treatment arms in mean total modified hai grading score at 12 months (table 3). The overall estimated rate of patient survival (kaplan - meier method) was significantly lower in the tac / dac arm (95% ci, 0.227 to 0.019%; p = 0.025) (table 3). The estimated rate of graft survival was numerically lower in the tac / dac arm (table 3). The rate of graft loss was 19.4% in the tac / dac arm (13 of 67 patients) and 8.8% in the tac / str arm (6 of 68 patients). Patient death accounted for 11 of the 13 grafts lost in the tac / dac arm and for 3 of the 6 grafts lost in the tac / str arm . There were 11 patient deaths in the tac / dac arm; 8 deaths occurred during the study and 3 after premature withdrawal . The causes of death were hcv recurrence (3 patients), cardiovascular complications (5 patients), surgical complications (2 patients), and infection (1 patient). Specifically, cardiac complications were myocardial ischemia, cardiac tamponade, cerebral hemorrhage, cardiac failure (identified as not or unlikely related to study drug), and cerebrovascular accident (identified as possibly related to study drug). There were 3 patient deaths in the tac / str arm, 1 death during the study and 2 after study withdrawal . The causes of death were hcv recurrence, surgical complications, and infection (each 1 patient). The overall frequency of bpar was significantly lower in the tac / dac than in the tac / str arm (p = 0.048, chi - square test) (table 3). Estimated freedom from bpar (kaplan - meier method) was numerically but not significantly higher with tac / dac (78.4%) than with tac / str (66.1%) (95% ci, 0.042 to 0.287%) (table 3). Histological findings showed that the majority of rejections were classified as mild (banff i) in both treatment arms: 8 of 11 (11.9%) in the tac / dac arm and 14 of 21 (20.6%) in the tac / str arm . One rejection in the tac / dac arm was classified as severe (banff iii). Renal function at 12 months, assessed using calculated creatinine clearance (cockroft - gault method) and serum creatinine, was comparable between the two arms . Mean (sd) creatinine clearance was 72.2 (27.3) ml / min in the tac / dac arm and 74.7 (30.6) ml / min in the tac / str arm . Mean serum creatinine in the tac / dac arm was 128.4 (68.8) mol / l and 110.8 (59.7) a summary of the adverse event profile for the two treatment arms is presented in table 4 . An adverse event occurred in 91% of tac / dac and 97% of tac / str patients . There was a significantly lower incidence of hepatitis c reported as an adverse event in the tac / dac arm than in the tac / str arm and a significantly higher incidence of thrombocytopenia in the tac / dac arm than in the tac / str arm . The number of patients discontinuing the study due to a treatment emergent adverse event was threefold higher in the tac / dac arm than in the tac / str arm . In the tac / dac arm, 16 of 67 patients (24%) discontinued the study compared with 5 of 68 patients (7.4%) in the tac / str arm . Malignancies occurred with an incidence of 4.5% (3 of 67 patients) in the tac / dac arm and 3% (2 of 68 patients) in the tac / str arm . These were 2 cases of hepatocellular carcinoma and 1 case of uterine cancer in the tac / dac arm and 1 basal cell carcinoma and 1 peritoneal carcinoma in the tac / str arm . The incidence of new onset diabetes mellitus after transplantation was 1.5% (1 patient) in the tac / dac arm and 6% (4 patients) in the tac / str arm . The results of this study showed that the steroid - free protocol in comparison to the protocol with steroids had a negligible impact on viral hcv rna values at 12 months . Results of the post hoc analysis, which excluded patients who had taken antiviral medications for treatment of hcv, indicated an even smaller between - treatment difference in viral load in the primary analysis set but a significant difference in the full analysis set . This study, despite its limitations, is one of a few randomized controlled multicenter studies to prospectively analyze hcv viral load and disease recurrence using a steroid - free protocol . While we did not observe an impact of this experimental treatment protocol on viral load, we did find that the estimated rate of patients remaining free of hcv recurrence was slightly better with the steroid - free protocol . The exclusion of patients who had received antiviral treatment during the study did not affect rates of hcv recurrence in either arm or the between - treatment arm difference in rates . As reported in other comparative trials in hcv - positive patients [10, 15, 21], we observed a tendency for earlier recurrence in the steroid arm . The higher number of patients in that arm who received steroid pulse treatments for acute rejection might have contributed to this result . Conversely, we observed a tendency for more advanced fibrosis occurring with tac / dac . A longer - time span to register fibrotic changes and a more detailed analysis of fibrosis progression might have yielded more clinically relevant comparative data of longer term outcomes with the two treatment protocols . Using daclizumab in place of steroids had no compromising effect on efficacy as shown by the lower incidence of bpar with tac / dac than tac / str . Adding mmf to the steroid - free protocol may have provided higher immunocoverage with lower bpar incidence as reported in a similar study using a triple - drug steroid - free protocol . We acknowledge that many features of mild rejection and hcv recurrence are shared and that mild rejection in the presence of hcv might be difficult to distinguish from hcv recurrence alone . There were unexpected differences between the treatment arms in patient tolerability of treatment and the incidence of premature withdrawal . Although adverse events occurred with a similar frequency in the two treatment arms, an adverse event was more commonly the cause of premature study withdrawal in the steroid - free arm . No single type of adverse event accounted for the higher number of dropouts . In a parallel arm randomized trial with treatment interventions similar to those we applied, premature study discontinuations were also higher in the tacrolimus + daclizumab than in the tacrolimus + steroids arm . The higher incidence of thrombocytopenia in the tac / dac arm is difficult to explain as other consequences of bone marrow suppression (pancytopenia and leukopenia) were higher in the tac / str arm and incidences of anemia were similar between treatment arms . Lower rates of hepatitis c reported as an adverse event in the tac / dac arm may indicate a potential safety benefit of this protocol over one with steroids . Patient survival was lower, albeit not significantly, in the steroid - free than in the steroid arm but is comparable to results reported from another steroid - free clinical trial . We could find no demographic or comorbidity factors, or common events leading to or causing death or serious adverse events to account for this difference . The higher number of patient deaths caused by cardiac events in the steroid - free arm could be attributed to chance . Contrastingly, patient survival in the steroid arm was 96%, which is exceptionally good considering that the study population was hcv positive . This survival rate is higher than the rate of 88.4% for unadjusted 1-year patient survival (deceased donor) reported in the 2009 optn / srtr annual report on the status of liver transplantation . The first is the low number of patients providing data for the primary endpoint; this affected the statistical power of the study generating inconclusive results . Secondly, only the post hoc analysis, which is biased due to the elimination of patients treated with antiviral agents and limited because of the post hoc method, revealed superiority of the steroid - free protocol over the reference protocol in impacting on hcv viral load in hcv - positive patients . It is difficult to recommend a steroid - free protocol for hcv - positive patients based on study results . The impact of this protocol on hcv viral load at one year was insignificant, and although there was a tendency for later hcv recurrence and lower incidences of rejection, we also observed a higher dropout rate and a lower patient survival rate with tacrolimus and daclizumab compared to tacrolimus and steroids . A study of longer duration could provide important clinical information on the relationship between type of treatment protocol and viral load, hcv recurrence, and fibrosis progression.
Human primary cell cultures and cell lines have become fundamental tools for basic research in numerous life science faculties as well as for the production of bioactive reagents in biomedicine and biotechnology . They are already used for several decades and frozen cell cultures or blood and tissue samples obtained many years ago can be found in numerous laboratories . As known from experience in the transfusion and transplantation medicine, human cells can harbor a number of different human pathogens and conveyed a potential risk for the recipients to become infected before extensive screenings of the material were accomplished . In particular, human pathogenic viruses like human immunodeficiency virus type 1 (hiv-1), human t - cell leukemia / lymphoma virus type i and ii (htlv - i and -ii), and hepatitis viruses, for example, hepatitis b virus (hbv) and hepatitis c virus (hcv), are found in human donor and patient material . Cell lines were usually established from patient material which might similarly be infected with those viruses or perhaps with viruses linked to specific tumors, for example, human herpes virus type 8 (hhv-8) or novel types of papilloma viruses . A considerable percentage of cell lines was established before viral contaminations had been routinely assayed or even before those viruses had been discovered . Indeed, some cell lines are known to harbor human pathogenic viruses, among them the well - known and widely distributed hela cell line which contains the human papilloma virus integrated into its genome . Besides the infection of the primary material which may be traced back to the donor, contaminations of cell cultures can also be introduced secondarily by laboratory personnel or from other infected cells when handled simultaneously . Such means of infection are much more likely, as similar problems were shown for mycoplasma contaminations (an incidence of ca . This type of infection with transmissible viruses might be true for the contamination with squirrel monkey retrovirus (smrv), which was detected in some human and animal cell lines; sequences of the virus were shown to be present in interferon- preparations produced by the human burkitt lymphoma cell line namalwa [6, 7]. An infection of the cells with human pathogenic viruses or bacteria increases the potential risk of a cell culture . Although the probability of the unintentional establishment of a cell line which is infected with a high - risk virus is extremely low, primary cells and cell cultures of unknown origin should be regarded as potentially harmful and are categorized as risk group 2, at least until the infection status of the donor or the cells is clearly determined . Whereas some viruses can be easily propagated in continuous cell lines (e.g., human retroviruses), propagation of other viruses additionally, some viruses exhibit a latent or cryptic infection cycle, during which no active viruses are produced (e.g., epstein - barr virus (ebv), proviruses of retroviruses). However, the latent status can be switched to the productive lytic cycle by certain inducers, or constantly low replication rates can be found . In this report, we describe the use of polymerase chain reaction (pcr) assays, enzyme - linked immunosorbent assay (elisa), southern and western blotting for the detection of latent and active forms of the human pathogenic viruses (ebv, hbv, hcv, hiv-1, htlv - i and -ii), smrv, and retroviruses in general in a large panel of continuous primate cell lines to determine the potential risk for handling these cell lines . Regarding ebv, the inducibility of the lytic cycle was investigated by treatment of the latently infected cell lines with the phorbol ester 12-o - tetradecanoylphorbol-13-acetate (tpa) and sodium butyrate (na - butyrate). The continuous cell lines were provided for accessioning to the cell lines bank by the original or secondary investigators . Cell lines were grown at 37c in a humidified atmosphere of air containing 5% co2 . The basic growth media (invitrogen, karlsruhe, germany) were supplemented with 10%20% fetal bovine serum (fbs) (sigma, deisenhofen, germany). For growth factor - dependent cell lines, specific growth factors or conditioned media containing growth factors were added . All cell lines were free of mycoplasma and other bacterial, yeast, and fungi contaminations as tested by pcr and microbiological growth assays . The authenticity of the cell lines was determined by dna typing . For the induction of the lytic cycle of ebv infection, pcr - positive cell lines were treated for 3 days with 10 m tpa (sigma) and 3 mm na - butyrate (sigma). A total of 1 10 cells were pelleted and washed with ice - cold phosphate buffered saline (pbs), resuspended in 30 l ice - cold protease inhibitor buffer containing 0.1 m nacl, 10 mm tris - hcl, ph 7.5, 1 mm edta, 1 g / ml aprotinin, and 100 g / ml phenylmethylsulfonyl fluoride and boiled for 10 minutes after addition of 30 l 2 sds gel - loading buffer containing 125 mm tris - hcl, ph 6.8, 4% sds, 20% glycerol 2% -mercaptoethanol, and 0.1% bromophenol blue . Proteins were separated on 10% sds - page . Blotting and staining conditions were as described previously . Bzlf1 protein was detected using the mouse anti - zebra antibody (dakocytomation, hamburg, germany) diluted 1: 100 in pbs and 5% fbs . Most of the cell lines were analyzed for general infections with retroviruses applying an elisa . Briefly, using 5 ml cell free culture supernatant, retrovirus particles were concentrated by ultracentrifugation at 100,000 g or by polyethylene glycol precipitation and subsequent centrifugation at 800 g. the pellets were analyzed applying the reverse transcriptase assay, nonradioactive, from roche (mannheim, germany). In this assay, digoxigenin- and biotin - labeled desoxyuridine - triphosphates are incorporated in the presence of viral reverse transcriptase during the synthesis of a dna strand along a synthetic target of single - stranded dna molecules . The newly synthesized dna is trapped on streptavidin - coated microtiter plates and subsequently detected immunologically by binding of peroxidase - labeled antidigoxigenin antibodies and visualized by color development and finally quantified by measuring the absorbance with an elisa reader . Two different buffers were used to detect mg - dependent rt as well as mn - dependent rt of mammalian c - type retroviruses . Episomal and linear forms of ebv dna were distinguished by horizontal gel electrophoresis according to gardella et al . . After electrophoresis, the separated dna was blotted onto nylon filters (see southern blot analysis) and the dna was hybridized with a p labeled ca . 37 kb dna fragment of the 5-region of ebv (cm - sal a). Genomic dna of cell lines or dna from ebv was extracted and purified using the pcr high pure dna kit from roche . Fifteen g of genomic dna or ebv dna from 24 ml supernatant of the b95 - 8 cell line were digested with 150 u xhoi restriction enzyme over night . After electrophoresis, the separated dna was blotted onto a nylon membrane and hybridized with various radioactively labeled cosmid probes from the ebv genome as described earlier . Probes for the 5- and 3-region of the linear ebv genome were prepared by pcr . An 894 bp fragment of the 5-region was amplified using the forward primer f - ebv1 (5-agg cat tta cgg tta gtg tg-3) and the reverse primer r - ebv1 (5-cgg tca gga tag caa gaa t-3). Pcr was carried out with 35 cycles of 30 seconds at 95c, 30 seconds at 52c, and 1 minute at 72c with hotstart taq (takara, lonza, verviers, belgium). A 619 bp fragment of the 3-region of the linear ebv genome was amplified using the forward primer f - ebv2 (5-atc ctc agg gca gtg tgt cag-3) and the reverse primer r - ebv2 (5-caa gcc gca gcg act ttc-3). Pcr was carried out with 35 cycles of 30 seconds at 95c, 30 seconds at 59c, and 1 minute at 72c with hotstart taq . The pcr fragments were isolated from the gels and cloned into pgem - t - easy vectors (invitrogen). Aliquots of the clear lysates were labeled with p by nick translation and hybridized to the southern blots . Signals were detected with a phosphor imager (molecular dynamics, sunnyvale, ca, usa). Pcr or rt - pcr was applied for the detection of specific dna or rna sequences of the human pathogenic viruses ebv, hbv, hcv, hhv-8, hiv-1, htlv - i and -ii, and smrv . Dna sequences integrated into the cellular genome (proviruses) and episomal dna of ebv and hhv-8 were detected using the pcr amplification method . Dna from cell lines was isolated applying the high pure pcr template preparation kit from roche . Specific oligonucleotide pairs from the gag region of hiv-1 (sk 38 and sk 39), the pol region of hltv - i/-ii (sk 110 and sk 111), the gag and env region of smrv (f - smrv - env, r - smrv - env and f - smrv - gag, r - smrv - gag), from the eb2 (bmlf1) gene of ebv (tc 70 and tc 72), and the putative minor capsid protein gene of hhv-8 (f- and r - hhv-8) were used with the appropriate annealing temperatures in the pcr reaction (table 1). The hot start pcr was either carried out by denaturing the reaction - mix which contains no taq - polymerase for 7 minutes at 94c, subsequently adding the taq - polymerase mix (1 u / reaction) during the following cycle step at 72c for 3 minutes, annealing for 2 minutes at the appropriate temperature, and amplification at 72c for 10 minutes, or by using hotstart taq - polymerase with initial cycles according to the manufacturer's recommendations . After the initial cycle, 35 cycles were run at 95c for 30 seconds, template specific annealing temperature for 30 seconds, and 72c for 1 minute plus 2 seconds extension for each cycle . The amplified products were identified by agarose gel electrophoresis and visualized by ethidium bromide intercalation . The active form of hbv was tested by organic extraction of dna from 5 ml supernatant of the cell lines after enrichment of particles by ultracentrifugation (15 minutes at 100,000 g). Pcr was carried out using forward and reverse primers from the conserved region of the precore / core protein gene to detect the different subtypes of hbv (table 1). For the detection of the hepatitis b surface antigen coding region in cell line hep-3b, a second primer pair was applied, consisting of the forward primer f - hbv-3 (5-caa gat tcc tat ggg agt ggg cct-3) and r - hbv-4 (5-ctc tgc cga tcc ata ctg cgg aac-3). For hcv detection, total rna was isolated from cells by organic extraction with trizol reagent (invitrogen) as described by the manufacturer . Five g were reverse transcribed using random primers and superscript reverse transcriptase according to the recommendations of the manufacturer (invitrogen). The amplification was carried applying primers from the 5 untranslated region (5-utr) of hcv (table 1). In all pcr runs appropriate positive, negative, and internal controls were integrated in parallel reactions . As positive control for hiv, a plasmid containing the gag and pol region of hiv-1 was used . As positive control for htlv, we applied genomic dna from the human t leukemia cell line mt-1 . Regarding ebv and hhv-8, genomic dna of the cell lines raji and cro - ap2 were used as positive control dna, respectively . Active hbv reference plasma (subtypes ad and ay) were applied to establish the pcr procedure and used as positive controls . The plasma samples were obtained from the german national reference center for virus hepatitis (gttingen, germany). M. roggendorf from the national reference center for hepatitis c (essen, germany). Except for hhv-8 and smrv, internal control dna for each of the pcr assays was constructed starting from the pcr products of the positive control dna . In general, dna fragments were cloned into the pcr products to retain the target sequences of the respective primer pairs, but to obtain a pcr product which is significantly longer than the original pcr product of the wild type sequence . For hiv and htlv, a 250 bp hcv positive control pcr fragment was inserted into the hiv and htlv fragments, according to the gene splicing by overlap extension for hbv, the pcr product of the positive control reaction was ligated into the pgem - t vector and a 445 bp fragment was excised from the insert employing the restriction enzyme bglii . A 788 bp dna fragment was isolated after digestion of the pgem vector applying mboi . Thus, the shortened and linearized vector containing the hbv pcr product ends can be ligated with the pgem restriction fragment, transformed into e. coli, and the resulting plasmid can be used as internal control dna for hbv . The pcr product from the positive control dna was ligated into the pgem - t vector and a 187 bp fragment was excised using the restriction enzyme smai . Phsse-1 was digested with rsai and a 379 bp fragment was extracted from the gel . Blunt - end ligation of the two dna sequences and transformation into e. coli produced a plasmid containing a dna sequence which can be amplified with the hcv primer and is 192 bp longer than the wild type dna . The pellet was washed with 5 ml pbs, centrifuged again at 200 g for 5 minutes, and the cells were resuspended in 0.5 ml pbs . One hundred l of the cell suspension were centrifuged onto acid washed slides using a cytospin centrifuge . The slides were air - dried and the cells were fixed in a mixture of methanol and acetic acid (3: 1) for 30 minutes in the cold . The samples were dehydrated with an ethanol series (70%, 70%, 90%, 90%, and 100%, each for 2 minutes). The slides were again air - dried for 10 minutes and processed further or stored at 20c with desiccant . The ebv cosmid dna was labeled with cy3 or spectrum green (vysis, stuttgart, germany) by nick - translation (roche) according to the manufacturer's protocols . 40 ng of the labeled probe was added to 1 g of human cot dna (roche) and ethanol precipitated . The pellet was resuspended in 10 l hybrizol vii (qbiogene, heidelberg, germany), placed on the slide, covered with a cover slip, and sealed with rubber cement . The preparations were denatured at 80c on a heating plate and the subsequent hybridization was carried out at 37c over night . Slides were washed in 2 ssc at room temperature for 1 minute, 3 times in 50% formamide/50% 2 ssc at 40c for 3 minutes, 3 times in 2 ssc at 40c for 2 minutes, and 5 minutes at room temperature in 4 ssc . Slides were mounted with vecta - shield containing 1 g / ml counterstain 1 (hoechst, frankfurt, germany) and evaluated with a fluorescence microscope . Ebv became the prime example of a human tumor virus that is etiologically linked to a diverse range of malignancies . Ebv has been associated with a variety of lymphoid and epithelial malignancies, such as burkitt lymphoma, hodgkin lymphoma, t - cell lymphomas, aids - related lymphomas, nasopharyngeal carcinoma, and lymphoepithelioma - like carcinomas of several organs including stomach, salivary glands, thymus, and lung . Additionally, ebv has the unique capability to transform and immortalize resting b cells into permanently growing b - lymphoblastoid cell lines (b - lcl). As ebv is ubiquitously distributed in all human populations and more than 90% of the individuals are infected, the detection of ebv is a priori not a matter of safety . However, for quality control reasons and moreover due to the potential of ebv to transform b cells, cell banks should routinely determine the ebv infection status of cell lines . Not every cell line derived from a tumor patient is necessarily a tumor cell line as nonmalignant cells which are independent of the tumor cells may sometimes be immortalized as well . In a leukemic context, such cell lines are usually normal b - cells which become immortalized through incorporation of the ebv genome . There are several hematopoietic diseases from which it is notoriously difficult to establish cell lines, in particular the mature b - cell malignancies such as chronic lymphocytic leukemia, hairy cell leukemia, plasma cell leukemia (pcl), multiple myeloma, and hodgkin lymphoma . While there are number of bona fide myeloma-, pcl-, and hodgkin lymphoma - derived cell lines, various cell lines which are in reality ebv b - lcls have been described and are still being used as model systems for these diseases . Experimental results from such misclassified cell lines might easily lead to false conclusions regarding the investigated tumor type . Although the contamination with ebv is not detrimental to the cell culture, the effect of the contamination can be manifold . For example, the gene regulation of the cells can be influenced by transcription factors or altered methylation patterns, and central signal transduction pathways might be activated or inhibited by viral products (e.g., nf-b, interferons) and might alter the physiology of the cells significantly . We also investigated the presence of ebv infections in nonhematopoietic cell lines to determine whether some of the carcinoma cell lines of the above mentioned tissues might also be linked to ebv infections . In order to inhibit spread of the infections among cell cultures and for the classification of the cell lines into risk groups, it is important to know whether the infection is latent and no active viruses are produced or whether ebv particles are produced during the lytic phase of infection . In some cell lines the lytic phase of ebv infection can be induced, depending on the culture conditions . The possibility of induction of the release of active viruses was investigated by the treatment of the cells with tpa and na - butyrate, because it is also relevant for the risk group classification of the cell lines . This method detects linear genomes of the active ebv particles as well as ebv genomes integrated into the eukaryotic chromosomes and nonintegrated ebv episomes . The analyses revealed that 39 out of 465 primate cell lines contain sequences of the ebv genome (see tables 2 and 3). All pcr positive human cell lines were established from b - lineage leukemia / lymphoma cells, natural killer cells, or are ebv transformed b - lymphoblastoid cells . No cell lines originating from other tissues were found to be ebv . To demonstrate the integrity and the sensitivity of the pcr runs each sample was assayed in duplicate, one reaction without an internal control, and one reaction was spiked with the internal control dna in a dilution that was close to the detection limit of the pcr assay (figure 1). A number of cell lines were examined for ebv infection by the originators of the cell lines or later on by other investigators . In the majority of cases the ebv status of the cell lines was determined immunologically by the detection of ebv nuclear antigen (ebna), early antigen (ea), and/or viral capsid antigen (vca). Concordance of our pcr results with data for protein expression given in the literature was found in nine cell lines (arh-77, bonna-1, hc-1, jiyoye, l-591, mec-1, mec-2, raji, and yt) [9, 25, 26]. The dohh-2 cell line was originally described as ebv negative but was later on found to be ebv positive in ebna immunofluorescence staining and pcr [27, 28]. In three cell lines (cro - ap2, cro - ap5, and hc-1) ebv sequences were demonstrated by southern blotting [2931] and four cell lines were described to be established by immortalization with ebv particles from supernatant of the b95 - 8 cell line (eheb, jvm-13, jvm-2, and jvm-3) [32, 33]. In contrast to our pcr results, the cell lines ci-1, nk-92, and tmm were specified as ebv negative as examined by ebna and pcr assays [3537]. Our pcr results were verified by fish, in situ lysing gel analysis, southern and western blotting as described below and showed in at least two additional assays that all those cell lines are clearly ebv - positive (see table 2). The ci-1, dohh-2, and tmm cell lines were positive in our pcr assay but showed weak or few signals in in situ lysing gel analysis and fish (see below). This indicates that the ebv load of the cells is very low and that the expression of ebna protein in these three cell lines was below the detection limit of the immunological assays . Thus, pcr is a convenient and reliable assay to detect ebv infections in cell cultures, provided that the appropriate control reactions including internal dna controls are performed to avoid false positive and false negative results . The activation of the lytic phase of the virus is usually closely connected with the production of infectious virus particles in the cells . The permissivity of a cell line relating to ebv is important to determine legally the risk group of the cell line as only cell cultures producing active viruses should represent an elevated risk . To identify the lytic phase of the ebv infections, we analyzed the expression of the zebra protein (bam hi z epstein - barr replication activator) by western blotting applying an anti - zebra monoclonal antibody . Zebra is the product of the bzlf1 gene and is a transcriptional activator that mediates a genetic switch between the latent and lytic states of ebv . Zebra binds to the promoters of genes involved in lytic dna replication activating their transcription . Thus, phorbol esters and na - butyrate are among the most reproducible and most broadly applicable inducers of the lytic cycle of ebv . However, not all latently infected lymphoblast cell lines can be induced by tpa and only a minority of the cells within a culture is inducible at all . In some human b - lcl about 10% of the cells can be induced to permit lytic virus replication, whereas in a few others 20%50% induced cells can be found . Marmoset lymphoblasts seem to be more inducible, for example, the b95 - 8 cell line . For this reason we used the b95 - 8 cell line as positive control to determine the efficiency of the induction with tpa / na - butyrate . All ebv pcr+ cell lines were incubated with and without 10 m tpa for 3 days and analyzed for zebra expression . As shown in table 2, five unstimulated cell lines exhibited a zebra specific band of approximately 40 kda (b95 - 8, bd-215, l-591, nc - nc, and yt). The expression of the protein was weak in all five cell lines but increased after tpa / na - butyrate stimulation . B95 - 8 and bd-215 increased strongly, whereas l-591 and yt increased only moderately . Six additional cell lines which showed no expression in the absence of tpa / na - butyrate produced the zebra protein in various amounts after induction of the lytic phase with tpa (eb-1, granta-519, hc-1, im-9, jiyoye, and val). All other cell lines tested (n = 28) did not express the bzlf1 gene product even after stimulation with tpa / na - butyrate . Figure 2 shows a representative western blot with positive and negative cell lines . Lieberman et al . Showed that the zebra protein is induced by tpa in raji cells . Raji is a nonproducer cell line with at least two deletions in the ebv genome and is defective for ebv dna replication and late gene expression . On the other hand, di francesco et al . Showed a weak induction of zebra in western blots after treatment with tpa in combination with n - butyrate, whereas further addition of cocain caused a high increase in zebra expression . However, importantly all studies demonstrated that zebra was not expressed during normal cell culture, and thus, a production of viruses is very unlikely at nonstimulating conditions . The ebv genome can be present in the host cells as covalently closed circles (episomes), as linear dna of active viruses, or integrated into the host genome . The ebv infected cells can harbor 110 episomes in low load cells or up to several hundred episomes in high load cells . Ebv producer cell lines contain also linear double stranded dna which is packaged into the virions . Ebv genome integration was described only for a small number of cell lines . For example, the namalwa cell line and its subclones are described to contain an integrated ebv genome and no episomes, whereas the raji cell line was reported to contain both, chromosomally integrated ebv genomes as well as episomes . To distinguish between the linear dna of active viruses, episomal dna of ebv - infected cell cultures, and solely integrated ebv genomes, we performed a variation of a southern blot analysis as described by gardella et al . . The autoradiograms showed the episomal dna as clear - cut bands whereas the linear dna usually formed distinct bands within a smear of dna (figure 3). The episomal as well as the linear dna of the cell lines examined showed different migrations indicating different ebv clones . Except for the nawalwa cell line and its subclones, dohh-2, and oci - ly19, all ebv pcr positive cell lines showed at least one band of episomal ebv genomes (table 2). As no denaturing conditions were applied in the in situ lysing gels, different sequences of the ebv strains can result in different conformations and mobilities of the episomes . The cell lines colo-720l, im-9, jvm-13, lcl - ho, tmm, and val displayed two episomal dna bands . The intensities of the episomal bands were almost identical only in the cell line jvm-13 . The existence of two ebv clones in one cell line can be explained by three assumptions: (i) two different ebv clones simultaneously infected the cell which led to the establishment of the cell line, (ii) the cell line was established from two or more different cells of the patient harboring different ebv clones (oligoclonality), or (iii) single and duplicated episomes are present in the nuclei of the cell lines . The nk-92 and tmm cell lines were described to be ebv negative according to ebna detection assay and pcr, respectively [35, 37]. This confirmed the results of the pcr assay as discussed above . Concerning the namalwa cell lines, no episomal dna bands were expected, because the ebv dna was described to be integrated into the eukaryotic genome . Since no linear dna was detected, the integrated genome is not transcribed in those cell lines . Dohh-2 and oci - ly-19 might also only contain integrated ebv genomes, but fluorescence in situ hybridization (fish) indicates that the number of ebv episomes in a cell culture is too low to be detected by in situ lysing gels (see below). The intensities of the episomal bands in the in situ lysing gels were highly variable among the cell lines but were constant for a given cell line, indicating that the ebv loads are different in the cell lines examined . The highest episomal loads were found in the cell lines daudi, im-9, l-591, raji, and sd-1 . Of these, only the l-591 cell line expressed bzlf1 constitutively and im-9 expressed the protein after stimulation with tpa and na - butyrate . This indicates that the lytic cycle induction is completely indepentent from the number of episomes present in the nuclei . High episomal loads were described for the cell line daudi and raji, which carry 100200 and 5060 viral copies per cell, respectively [23, 46]. Based on the intensities of the ebv - specific bands in the autoradiograms of the in situ lysing gel assays, the numbers of episomes in im-9 and sd-1 were comparable to those of daudi or raji, respectively . All other cell lines were low load cell lines with 110 ebv copies per cell compared to the high load cell lines mentioned above (figure 3). In order to obtain more quantitative data on the ebv load of the ebv cell lines, we performed single cell analyses by interphase fish . The analysis showed that the viral load of the nonproducing cells within a culture was heterogeneous . Some cells had high ebv loads, whereas other cells of the same cell culture showed low or even no viral load . For example, the cell line dohh-2 contains only a few cells with several ebv episomes, whereas in most of the cells ebv dna was completely absent (figure 4(b)). Any cross - contamination of the cell line was excluded by dna typing (data not shown). The heterogeneity of ebv load within a cell culture population and the finding that only a small fraction of cells can be stimulated to enter the lytic phase implies that specific cellular factors regulate the propagation of episomes in the nuclei and the permissivity of the cells related to ebv . Phosphorylation or methylation of ebv specific proteins or epigenetic events like dna methylation or histone deacetylation might contribute to the cell specific regulation . Linear ebv dna was detected in a number of cell lines (table 2). The expression of linear ebv genome dna did not correspond to the expression of bzlf1 protein . Although all bzlf1 positive cell lines produced high levels of linear ebv dna, many more bzlf1 negative cell lines also expressed significant levels of linear ebv genomes . This indicates that the lytic status of the ebv infection is not essential for the production of linear ebv genomes . The intensities of the linear ebv dna bands did not correlate to the intensities of the episomes . For example, the cell line im-9 displayed a very strong episomal dna signal, whereas no linear dna was detectable in untreated cells and very low amounts were seen in tpa / na - butyrate treated cells (figure 5). On the other hand, cell line yt showed a strong band for linear dna, but a weak band for the episomal dna . The number of episomes per cell was not affected by the stimulation with tpa / na - butyrate, whereas the amount of linear dna increased with the addition of tpa in those cell lines which can be stimulated with the phorbol ester . Cross - contamination is a serious problem in cell culture and causes mycoplasma infection and cell line cross - contamination . To find out whether a transfer of ebv from one cell culture to another can be demonstrated, we investigated the clonality of the ebv genomes . We performed southern blot analysis with genomic dna of the ebv - pcr positive cell lines digested with the restriction enzyme xhoi . Applying ebv cosmids covering almost the whole ebv genome each cell line displayed a unique ebv genotype the most striking differences occurred in the lengths of the terminal repeat (tr) regions which reflect the numbers of repetitive exons . Several different patterns can be identified per cosmid probe and the combination of several cosmid probes revealed a high number of different genotypes (figure 6). Even the cell lines which were described to be established with ebv particles of the b95 - 8 cell line displayed differences in the lenght of the tr regions of the ebv genotypes . Nevertheless, the cell lines display different patterns concerning the tr region and a few additional bands within the ebv genome . The eheb cell line was also described to be transformed by b95 - 8 ebv viruses . The eheb cell line displays distinct bands in the xhoi - digested ebv dna different from those found in the jvm cell lines . Nevertheless, the bands of the b95 - 8 ebv - transformed cell lines detected in the southern blots are all more or less prominently represented within the pattern of the b95 - 8 cell line . From these results, we conclude that the b95 - 8 cell line harbors different genotypes of ebv with the ability to transform other cells . Taken together, the various patterns of digested ebv indicate that the infections with eb viruses originated from the patient or from the transforming viruses, but not from dissemination during cell culturing . Although mycoplasma and cross contaminants are mainly introduced into cell cultures by inadequate cell culture techniques, this does not seem to be the case with regard to the distribution of ebv in cell cultures . The low number of unintended intercell line contamination by ebv might be based on the low number of cell lines which produce actively ebv . Additionally, only a limited number of cell lines are susceptible to the infection with ebv at all . Hbv can be grown in primary cell cultures of normal adult or fetal human hepatocytes, but propagation was not yet shown in untreated continuous cell lines . Production of hbv particles can be achieved to date only by transfection of hepatocyte cell lines with plasmids containing hbv genomes . In cell lines, the hbv genome is integrated into the eukaryotic chromosomes with variable copy numbers as shown for several cell lines, for example, hccm, hep-3b, huh-1, huh-4, and plc / prf/5 . Except for hccm, all these cell lines express the hepatitis b surface antigen hbsag . The hbv pcr assay used here amplified conserved sequences of the core region of hbv and was evaluated using hbv reference plasma no 1 (subtype ad) and no 2 (subtype ay) which were established in cooperation with the european study group on viral hepatitis (eurohep). In order to determine the detection limit, we performed a dilution series of the hbv particles in conditioned cell culture supernatant of an hbv negative cell line . Subsequently, the hbv particles were recollected by ultracentrifugation, dna was extracted, and the pcr reaction was performed as described in the methods section . We screened 465 primate cell lines (table 3) by performing pcr assays for the detection of active hbv with dna extractions of the cell culture supernatants and found no hbv cell lines . The hep-3b cell line was described to carry the hbv genome integrated into the eukaryotic chromosomes, but the pcr method we applied detected the contamination neither in the supernatant nor using genomic dna of the cell line . 2.3 kb fragment originating from hbv in the hep-3b cell line and the integration was associated with a chromosomal translocation of the chromosomes 4 and 13 . The hbv core region is deleted in the hep-3b cell line and was thus not detected by the pcr analysis . We confirmed the presence of the hbv fragment in the hep-3b cell line by southern blot analysis (figure 7) on genomic dna and by pcr amplification of the coding region of the hbsag . In hep - g2 and sk - hep-1, a hepatocellular carcinoma, and a liver adenocarcinoma cell line, respectively, we did not detect hbv sequences by pcr or southern blot analysis . Hcv infections are widely distributed all over the world and are associated with chronic hepatitis . Until its identification in 1988, the virus was the predominant causative of transfusion - associated hepatitis . Similarly to hbv, until now hcv cannot be propagated in unmanipulated continuous human cell lines . Hcv is a 9.6 kb linear positive - stranded rna virus and can be detected by reverse transcriptase pcr (rt - pcr). The 5-utr is the most conserved part of the virus genome and commonly used for the amplification by rt - pcr . We extracted total rna from the cell lines and the integrity of the rna was evaluated by demonstration of the rrna bands on an ethidium bromide stained gel . The subsequent rt reaction was verified by a pcr reaction amplifying the ubiquitously expressed abl transcript . This pcr allows (i) to demonstrate the sensitivity of the rt - pcr, because abl is only moderately transcribed in human cells, and (ii) to distinguish between the cdna and residual genomic dna, because the primers are located in two different exons of the abl gene . Due to the lack of availability of hcv positive cell lines, we used a plasmid containing the 5-utr of hcv as positive control and produced an internal control dna to verify each pcr reaction . We investigated 465 cell lines (table 3) applying rt - pcr for the conserved 5-utr of the hcv genome . This result supports the observation that hcv cannot be propagated in human or other primate cell lines . The retroviruses hiv-1 and htlv - i and ii infect human t cells and are involved in the development of aids and the adult t - cell leukemia, respectively . The retroviruses contain two rna molecules which are reverse transcribed into dna and integrated as proviruses into the host genome . Hiv and htlv can be propagated in continuous cell lines and a number of cell lines were described as being latently infected or actively producing viruses . Pcr assays were established to detect latent infections in terms of proviruses of hiv-1 and htlv - i and -ii in the cell lines . Dna of the mt-1 cell line was used as positive control for the htlv - pcr and a plasmid containing the hiv-1 gag and pol regions as positive control for hiv-1 . In total 465 primate cell lines of the cell bank (table 3) were analyzed by pcr for the presence of those retroviruses and none of the cell lines were found to be infected with hiv or htlv sequences . Furthermore, 293 of these cell lines were tested for the activity of rt . The activity of 5 ml conditioned kasumi-1 cell culture supernatant showed an rt activity that was equivalent to approximately 2 ng of hiv-1 rt (specific activity> 5 u/g). For comparison, 5 ml supernatant of the ovine cell line flk - blv, which is infected with the bovine leukemia virus, shows an activity equivalent to ca . 10 ng of hiv-1 rt . The cause of the rt activity could not be determined . But pcr analyses excluded the presence of hiv-1, htlv - i/-ii, and smrv . Smrv was first isolated from the lung cell culture of a squirrel monkey after cocultivation with other cell cultures . The endogenous virus belongs to the beta - retroviruses and exhibits mg - dependent rt . Smrv is not pathogenic to its natural host and pathogenicity was not described for human or other species . The xenotropic virus was shown to be expressed in different cell lines of canine, man, chimpanze, rhesus monkey, and mink origin . Natural or alternate hosts other than squirrel monkey were not yet identified . Already in 1974 retroviral particles were detected in the human hematopoietic cell line rpmi-8226 which were later identified as smrv [6, 51]. Oda et al . Reported the detection of retroviruses in the human lymphoblastoid cell line hlb which turned out to be smrv [52, 53]. Reported the insertion of smrv sequences in the genome of the human burkitt lymphoma cell line namalwa . The subclone namalwa.ipn/45 contains two partial smrv copies integrated into the cmyc gene locus and 10 further copies integrated at different sites of the genome of the cell line . They also found smrv sequences in the ebv producing monkey cell line b95 - 8 and demonstrated that smrv was transmitted when ebv particles from the b95 - 8 cell line were used to establish a b lymphoblastoid cell line . Smrv sequences were also detected in interferon- preparations which were produced using the namalwa cell line . As seen in the cell line namalwa, the insertion can affect not only the transcription activity of cellular oncogenes but also tumor suppressor genes or other regulatory genes . Beside the possibility of altering the cellular characteristics, this leads also to a certain, at least theoretically, possible pathogenicity of retrovirus infections per se; hence the release of those viruses especially with a very broad host cell spectrum such as xenotropic smrv should be minimized . Moreover, a potential risk occurs when cells infected with retroviruses are subjected to retroviral - mediated gene transfer . A recombination of the replication deficient retrovirus and the wild type retroviral genetic information might lead to a complementation of the replication defect and to replication - competent retroviruses . We investigated 430 human and 4 monkey cell lines (table 3) for the occurrence of smrv dna sequences in the genome . Six cell lines were found to be positive for smrv: five namalwa subclones (namalwa, namalwa.csn/70, namalwa.ipn/45, namalwa.kn2, namalwa.pnt) and the human lymphoblastoid cell line lcl - ho . Investigated namalwa cell lines from different laboratories and found no smrv - free nalmalwa cell cultures . Presumably, all namalwa cell lines are contaminated with smrv and no smrv - free cell line is available . The lcl - ho cell line is a lymphoblastoid cell line which was established by transformation of a patients primary b cells with ebv . We suspect that smrv was transmitted by the use of ebv from an smrv infected b95 - 8 cell line . Other lcls which were transformed with ebv from b95 - 8 showed no smrv contamination . The low infection rate of the cell lines indicates that smrv is not transmitted to other cell cultures comparable to the distribution of mycoplasma or cell line cross contaminations, although many different cell types were shown to be susceptible to an infection . Nevertheless, the transmission of these xenotropic viruses during cultivation and handling of infected cell cultures to other cell lines cannot be excluded . In summary, we showed that human and other primate cell lines directly obtained from a biological resource center do not carry high - risk viral contaminants and thus do not represent safety risks for cell culturists . The pcr assays provide a robust, fast, inexpensive, sensitive, and reliable method for routine screening . The additional feature of incorporating an internal control in each assay reduces the possibility of false - negative data . Importantly, unlike mycoplasma and other bacterial infections and cell line cross contamination, viruses are not disseminated among cell lines due to poor cell culture technique . Only a minor part of the ebv cell lines produces active viruses or can be stimulated to produce them . Detection of bzlf1 protein by western blot, tr sequences by southern blot, and interphase fish can be applied to recognize cell lines producing active ebv particles and to assign a cell culture to a legal risk group . Newly established cell lines should be tested for ebv - infections to detect ebv - transformed b - cells giving rise to b - lcls . On the other hand, the use of ebv from an smrv b95 - 8 cell line to establish b - lcls might carry the risk of coinfecting the reccipient cells with smrv.
Chikungunya (chikv) and dengue (denv which is divided into 4 serotypes, 1 to 4) viruses are both arthropod - borne viruses belonging respectively to the genus alphavirus, togaviridae family and flavivirus, flaviviridae family, and transmitted to humans via a bite of infected aedes mosquitoes (aedes aegypti, aedes albopictus). Over the past two decades, they have caused multiple worldwide outbreaks, particularly in tropical and sub tropical areas . Recent chikv outbreaks started in kenya in 2004, spread over the indian ocean region, reached india causing in all more than 1.5 million cases . Then, numerous imported cases were reported in different parts of the world, an outbreak occurred in italy, while autochthonous cases were reported in france . In central africa, outbreaks occurred in democratic republic of congo, cameroon, gabon and republic of congo . As for dengue fever (df), its incidence has grown dramatically around the world, rising more than 30-fold in recent decades . Two fifths of the world's population are at risk of infection, and who currently estimates there are 50 million dengue infections each year . The disease is now endemic in more than 100 countries in africa, the americas, the eastern mediterranean, south - east asia and the western pacific . During the last decade, denv epidemics have occurred in some east and west african countries, including cape verde where more than 17,000 df cases due to serotype 3 were notified . In practice, the global picture of both chikungunya and df is similar in their classical forms, so they can be confused particularly in areas where they cocirculate . After variable incubation period, both diseases start abruptly with acute fever, arthralgias, myalgias, headaches, rash and fatigue . Arthralgias are often incapacitating in chikv infection and can persist for several years in some patients . For denv infection, after the initial febrile phase, hemorrhagic manifestations can occur, leading to dengue hemorrhagic fever (dhf) indeed dengue with shock syndrome (dss). And finally, in addition to denv, reports of deaths in la reunion island were directly attributed to chikv . Furthermore, there is no comparative study of the two infections, still lesser in a concurrent outbreak . Here, we compare the clinical features and the laboratory findings for 343 laboratory - confirmed patients from the concurrent chikv / denv outbreak which occurred in gabon in 2010 . Franceville, the main town of the haut ogooue province, 512 km south - east from libreville (capital of gabon), is located at altitude 333 meters, at the end of the transgabon railway and the national 3 road, in a savannah ecosystem constituted by forest gallery (figure 1). There are 5 health care capacities (hcc) including 2 public hospitals with 170 beds in all and the medical unit of the centre international de recherches medicales de franceville (cirmf). A concomitant chikv/ denv outbreak raged 11 towns and one village of the southeast, from february to july 2010, and recorded 1127 laboratory confirmed cases in franceville . The cirmf was included as a partner of the moh, in the response to this outbreak, and implicated in active surveillance, case management and diagnostic . As part of these activities, a prospective study was initiated, and the investigations were considered as a component of the public health response and authorized by the regional health director . Map of gabon with location of the gabonese chikungunya outbreak in 2010 . At the time of the outbreak, the moh provided a case definition including 2 categories: a suspected case was a patient presenting with at least one of the following symptoms: fever, arthralgias, myalgias, headaches, rash, fatigue, nausea, vomiting, diarrhea, bleeding, and jaundice; a confirmed case was a suspected case with laboratory confirmation . All suspected cases consulting in the cirmf medical unit were examined and demographic and clinical data collected in standardized questionnaire . Blood samples were collected in two 7-ml edta vacutainer tubes and one 7-ml dry tube (vwr international, france). The tubes were stored in the dark at + 4c until arrival at the laboratory . Thick and thin blood films were stained with 20% giemsa and examined for malaria parasites . Hematological (hematology analyser act 10, beckman coulter) and biochemical (creatinine, ast, alt) tests were performed (automatic analyser hitachi model 902, roche diagnostics). Samples were tested for chikv and denv rna genomes by using taqman qrt - pcr technology and specific primers and probes . When used during the acute phase (17 days after onset of symptoms), the taqman qrt - pcr technology has a sensitivity of up to 80% and specificity of 100% . Results were expressed as averages (with their sd) and percentages (with their 95% confidence interval, ic). The student's t test was used to compare laboratory parameters and continuous clinical variables . For qualitative variables, the chi square test or fisher's exact test were used as appropriate . Franceville, the main town of the haut ogooue province, 512 km south - east from libreville (capital of gabon), is located at altitude 333 meters, at the end of the transgabon railway and the national 3 road, in a savannah ecosystem constituted by forest gallery (figure 1). There are 5 health care capacities (hcc) including 2 public hospitals with 170 beds in all and the medical unit of the centre international de recherches medicales de franceville (cirmf). A concomitant chikv/ denv outbreak raged 11 towns and one village of the southeast, from february to july 2010, and recorded 1127 laboratory confirmed cases in franceville . The cirmf was included as a partner of the moh, in the response to this outbreak, and implicated in active surveillance, case management and diagnostic . As part of these activities, a prospective study was initiated, and the investigations were considered as a component of the public health response and authorized by the regional health director . At the time of the outbreak, the moh provided a case definition including 2 categories: a suspected case was a patient presenting with at least one of the following symptoms: fever, arthralgias, myalgias, headaches, rash, fatigue, nausea, vomiting, diarrhea, bleeding, and jaundice; a confirmed case was a suspected case with laboratory confirmation . All suspected cases consulting in the cirmf medical unit were examined and demographic and clinical data collected in standardized questionnaire . Blood samples were collected in two 7-ml edta vacutainer tubes and one 7-ml dry tube (vwr international, france). The tubes were stored in the dark at + 4c until arrival at the laboratory . Thick and thin blood films were stained with 20% giemsa and examined for malaria parasites . Hematological (hematology analyser act 10, beckman coulter) and biochemical (creatinine, ast, alt) tests were performed (automatic analyser hitachi model 902, roche diagnostics). Samples were tested for chikv and denv rna genomes by using taqman qrt - pcr technology and specific primers and probes . When used during the acute phase (17 days after onset of symptoms), the taqman qrt - pcr technology has a sensitivity of up to 80% and specificity of 100% . Results were expressed as averages (with their sd) and percentages (with their 95% confidence interval, ic). The student's t test was used to compare laboratory parameters and continuous clinical variables . For qualitative variables, the chi square test or fisher's exact test were used as appropriate . The sex ratio m / f was 0.99 and the mean age was 3015 (range, 177 years). Among these 433 patients, 343 (79.2%) were laboratory confirmed with 270 (62.3%) chikv+, 53 (12.2%) denv+ . The sex ratio m / f was 0.85 for the chikv group, 0.55 for the denv group and 1 for the chikv / denv group, and the mean age was respectively 3016 (range 1 77 years), 3114 (range, 576 years), and 3612 (range, 174). Hospitalization was required for 39 (14.4%) chikv+, 2 (3.7%) denv+ (p=0.02), and 6 chikv / denv+ patients . Headaches and hemorrhage were more frequent (p respectively 0.01 and 0.001) in the denv+ group than in the chikv+ group (table 1), and myalgias were more frequent in the chikv and denv groups than in the chikv / denv group (p = 0.005 and 0.02 respectively). Similarly, leukopenia and lymphopenia were more frequent (p respectively 0.02 and 0.001) in the denv+ group than in the chikv+ group (table 2). There was no difference in biological parameters between the co - infected group and the other groups . Table 1comparison of clinical signs between chikungunya and dengue - infected patients during the 2010 outbreak in gabon.chikv+denv+pn%n%hospitalization3914.323.60.02clinical symptomsfever234865292.90.16arthralgia22783.54783.90.93myalgia19571.74071.40.96headache19069.94885.70.01asthenia21478.74478.60.72skin rash11140.81526.80.06pruritus7326.81119.60.26digestive signs87321934.50.86hemorragy51.8712.50.001chikv, chikungunya virus; denv, dengue virus . Table 2comparison of biological parameters between chikungunya and dengue - infected patients during the 2010 outbreak in gabon.chikv+denv+pmeansdmeansdbiological parametershaemoglobin12.31.711.91.140.49leucocytes52431676339015170.02lymphocytes222821614012430.001platelets23308981750225600608240.77asat171118100.38alat453550340.58cratinine963288320.14chikv, chikungunya virus; denv, dengue virus . We conducted a comparative study of clinical and biological data of patients with chikungunya and dengue viruses infections during a concurrent outbreak which occurred in south east gabon in 2010 . Of the 343 individuals included in the study, 270 were chik+, 53 were denv+, and 20 were co - infected (chik+/den+). Headaches, hemorrhage, leukopenia and lymphopenia were significantly more frequent in denv+ patients . In our opinion, thus, the circulation during this epidemic of other arboviruses such as west nile, rift valley fever and yellow fever viruses, cannot be ruled out . To our knowledge, this is the first comparative study of these two diseases ., chikungunya is described in the literature as a dengue - like disease, due to their common symptoms . Nevertheless, the existence of incapaciting joints pain located at the extremities seems specific of chikungunya . This distinction is difficult in tropical areas where many other diseases, such as malaria, typhoid fever, influenza, share the same symptoms . So, in our field experience, these diseases are clinically indistinguishable, and the problem of differential diagnosis may be compounded during concomitant chikv / denv outbreaks in a malaria endemic area . In their classical form, the hemorrhage constitutes an inconstant sign . Bleeding is generally minimal, non life - threathning, and cannot assist for the differential diagnosis . This entity is more recognizable, leading, with the shock syndrome, to the severity of the disease . These hematological disorders are non specific and cannot also assist for the differential diagnosis . Patients with mixed infection did not seem to have more severe symptoms or biological disorders, suggesting no additive effect of the two viruses . The clinical and biological differences detected in our analysis are not useful in the field . So, our study confirms that the two diseases are clinically similar and need for laboratory confirmation for their recognition . Since their appearance in 2007 in the north of the country, these viruses have spread in the south east, together with the vector aedes albopitus.
A newly diagnosed acromegaly patient aged 45-year - old woman was referred to our clinic for an ophthalmologic examination from the endocrinology department of our hospital . Serum igf-1 level was 750 ng / ml (reference ranges of igf-1 in ages 4685: 94166 ng / ml). Complete ophthalmological examination including cct with ultrasonic pachymetry (tomey corporation, nagoya, japan), and standard threshold perimetry with the humphrey visual field analyzer 24 - 2 program (carl zeiss meditec, inc ., dublin, ca, usa) were performed . After informed consent was obtained from the patient, basal tear samples were collected atraumatically from the inferior tear meniscus of both eyes using glass capillary tubes or micropipettes . Tear samples were diluted in phosphate - buffered saline, placed in microtubes, and stored at 70c until further examination . Tear igf-1 concentrations were measured using a commercial enzyme - linked immunosorbent assay kit (quantikine, r and d systems, minneapolis, mn, usa) according to the manufacturer's instructions . Tear igf-1 level was 0.63 ng / ml . Best - corrected visual acuities were 1.0 in both eyes . Optic disc examination revealed normal in both eyes (cup - to - disc ratio was 0.3 in both eyes). Cct was 615 m in the right eye and 612 m in the left eye . Iop levels were 24 mm hg in the right eye and 23 mm hg in the left eye . After the patient was referred to a neurosurgery by the endocrinology clinic for an operation, he underwent transsphenoidal adenectomy for the pituitary adenoma . The patient's serum igf-1 values had fallen to 125 ng / ml . Besides tear igf-1 values were 0.32 ng / ml . Cct was 585 m in the right eye and 575 m in the left eye . Iop levels were 20 mm hg in the right eye and 19 mm hg in the left eye . Another newly diagnosed acromegaly patient aged 55-year - old woman was referred from endocrinology clinic for an ophthalmologic examination . Serum igf-1 level of the patient was 1340 ng / ml . Tear igf-1 level was 0.98 ng / ml . Cct was 595 m in the right eye and 590 m in the left eye . Iop levels were 22 mm hg in the right eye and 21 mm hg in the left eye . Optic disc examination were also normal in both eyes (cup - to - disc ratio was 0.4 in both eyes). The patient also was referred to the neurosurgery department for an operation, and she also underwent pituitary surgery . Three months after surgery the patient's complete ophthalmological and visual field examination revealed normal . Serum igf-1 value had fallen to 389 ng / ml . Besides tear igf-1 value was 0,25 ng / ml . Cct values were 565 m in the right eye and 560 m in the left eye . Iop levels were 18 mm hg in the right eye and 17 mm hg in the left eye . A newly diagnosed acromegaly patient aged 45-year - old woman was referred to our clinic for an ophthalmologic examination from the endocrinology department of our hospital . Serum igf-1 level was 750 ng / ml (reference ranges of igf-1 in ages 4685: 94166 ng / ml). Complete ophthalmological examination including cct with ultrasonic pachymetry (tomey corporation, nagoya, japan), and standard threshold perimetry with the humphrey visual field analyzer 24 - 2 program (carl zeiss meditec, inc ., dublin, ca, usa) were performed . After informed consent was obtained from the patient, basal tear samples were collected atraumatically from the inferior tear meniscus of both eyes using glass capillary tubes or micropipettes . Tear samples were diluted in phosphate - buffered saline, placed in microtubes, and stored at 70c until further examination . Tear igf-1 concentrations were measured using a commercial enzyme - linked immunosorbent assay kit (quantikine, r and d systems, minneapolis, mn, usa) according to the manufacturer's instructions . Tear igf-1 level was 0.63 ng / ml . Best - corrected visual acuities were 1.0 in both eyes . Optic disc examination revealed normal in both eyes (cup - to - disc ratio was 0.3 in both eyes). Cct was 615 m in the right eye and 612 m in the left eye . Iop levels were 24 mm hg in the right eye and 23 mm hg in the left eye . After the patient was referred to a neurosurgery by the endocrinology clinic for an operation, he underwent transsphenoidal adenectomy for the pituitary adenoma . The patient's serum igf-1 values had fallen to 125 ng / ml . Besides tear igf-1 values were 0.32 ng / ml . Cct was 585 m in the right eye and 575 m in the left eye . Iop levels were 20 mm hg in the right eye and 19 mm hg in the left eye . Another newly diagnosed acromegaly patient aged 55-year - old woman was referred from endocrinology clinic for an ophthalmologic examination . Serum igf-1 level of the patient was 1340 ng / ml . Tear igf-1 level was 0.98 ng / ml . Best - corrected visual acuities were 1.0 in both eyes . Cct was 595 m in the right eye and 590 m in the left eye . Iop levels were 22 mm hg in the right eye and 21 mm hg in the left eye . Optic disc examination were also normal in both eyes (cup - to - disc ratio was 0.4 in both eyes). The patient also was referred to the neurosurgery department for an operation, and she also underwent pituitary surgery . Three months after surgery the patient's complete ophthalmological and visual field examination revealed normal . Serum igf-1 value had fallen to 389 ng / ml . Besides tear igf-1 value was 0,25 ng / ml . Cct values were 565 m in the right eye and 560 m in the left eye . Iop levels were 18 mm hg in the right eye and 17 mm hg in the left eye . In acromegaly, normalization of igf - i in serum has been used to follow - up the success of surgical and medical therapies . Van setten et al . Reported that the igf-1 level was elevated not only in the serum, but also in the subretinal fluid and aqueous humor in a patient with acromegaly . This study inspired us to hypothesize that the eyes might be another target organ for the effects of excess gh and igf-1, hence in our study we measured tear igf-1 levels and evaluated the relationship with cct and iop before and after treatment in two acromegaly patients . Ciresi et al . Emphasized that high gh may have stimulatory effects on the cornea, as well as on other target organs . Bramsen et al . Divided 27 pituitary adenoma patients into two groups as those with and without acromegaly and reported higher cct values in the group with acromegaly . In contrast, polat et al . Reported no statistically significant difference regarding median right and left corneal thicknesses and mean cct values between acromegaly and control groups . They divided 30 patients with acromegaly into two groups as those with active and inactive . We think that possible growth effect of tear igf-1 may have an impact on cct . We found a higher cct, iop, and tear igf-1 values in the active phase of the disease . The activity of the disease may have an impact on ocular parameters such as cct, iop, and tear igf-1 values . However, longitudinal studies including larger populations and controls are needed to confirm this relationship.
Osteoporosis is a worldwide disease characterized by low bone mass and poor bone strength caused by an imbalance between bone forming osteoblasts and bone resorbing osteoclasts.1 an estimated 200 million people are affected worldwide with the great majority being women.2 reports demonstrate that within the lifetime of a patient suffering from the disease, 30%50% of women and 15%30% of men will suffer at minimum one osteoporotic - related fracture.3 a recent systematic review found that not only was life quality for these patients severely decreased, but the incidence of death for patients following hip fracture is at least doubled for an age - matched control without such fractures, and is continuously rising.4 currently the two major pharmacological approaches are anabolic agents such as parathyroid hormone which stimulates bone formation or anti - resorptive agents including bisphosphonates, calcitonin, raloxifene, and estrogen which act by inhibiting bone resorption.5 although much research to date focuses on the prevention of bone loss by using the abovementioned agents, less emphasis has been placed on the repair of defects following fracture while more than 1.5 million fractures occur annually in the united states alone.6 to date the replacement and repair of damaged or diseased tissues is largely driven by the field of tissue engineering which is an interdisciplinary field that draws on a multitude of disciplines including material science, cell biology, biotechnology, and biomaterials.7,8 mesoporous bioactive glass (mbg) has been widely used as a bone replacement material due to its mesoporous structure which increases surface area and allows for preferential osteoblast adhesion, proliferation, and differentiation.9,10 previously we have demonstrated that the incorporation of the trace element boron plays an important role in bone growth.9,11 boron has the ability to stimulate wound healing, facilitate the release of osteo - inductive growth factors and cytokines, and increase the extracellular matrix (ecm) turnover.9,11,12 boron - containing mbg (b - mbg) is an excellent scaffold with significantly enhanced osteogenic properties and drug delivery capability for tissue engineering compared with mbg alone.9 however, mbg as a scaffold for bone repair also has drawbacks such as inherent brittleness.13,14 in contrast, p(n - isopropylacrylamide - co - butyl methylacrylate) (pib) nanogels is a temperature - sensitive cross - linking polymer and its dispersion exhibits three phase states (swollen gel, flowable sol, and shrunken gel) in accordance with temperature changes . It can be used to treat defects of irregular shapes and deformities as an injectable, thermoresponsive, biocompatible hydrogel for tissue engineering applications which undergoes rapid thermal gelation once the sol form reaches body temperature.15,16 recently, pib nanogels have been applied in interventional therapy for liver tumors and vascular occlusion of renal artery as a novel blood - vessel - embolic material.15,17 however to date, no study has applied this new technology for the repair of bone defects . Therefore, the aim of the present study was to investigate the role of pib nanogels as a bone substitute material for the repair of femur defects in rats . Furthermore, a new composite scaffold combining pib nanogels with b - mbg scaffolds was fabricated and evaluated for material characterization, mechanical properties, and in vivo early bone formation in comparison to pib nanogels alone . Pib nanogels were fabricated according to the method of precipitation polymerization, as previously described.17 b - mbg was fabricated according to the method of our previous study.9 then, b - mbg powder was put into the pib nanogels solution, and stirred for 48 hours at 4c . The mass ratios of b - mbg powder and pib nanogels in the mixture were 1:1 respectively . The size of pib nanogels were determined by dynamic light scattering (zetasizer nano - zs 90; malvern instruments, malvern, uk) using a helium neon laser source (=633 nm) with scattering angle 90. all samples were diluted with ultrapure water to 0.5 mg / ml . The size was measured at the various temperatures from 25c to 40c with pre - equilibration for 5 minutes at each point . The dynamic viscoelastic properties of nanogel dispersions were obtained using a stress - controlled rheometer (kinexus; malvern instruments) with a parallel plate (=40 mm, gap set at 1.0 mm) in the range of 20c50c with the following parameters: shear stress = 1.0 pa, heating rate = 5c / min, and frequency = 1.0 hz . Pib nanogels / b - mbg and pib nanogels were respectively implanted into the right and left critical - sized femur defect of mature osteoporotic female wistar rats . The osteogenic properties were then evaluated by histological staining as described in the following section . Animal feeding and surgical procedures were conducted in accordance with the guidelines of animal care and use committee of wuhan university, people s republic of china, and authorized by the ethics committee at the school of dentistry, wuhan university . Ten week old mature female wistar rats weighing between 200300 g were used for this experiment . Ten percent chloralic hydras (4 ml / kg body weight) was injected inside the abdomen for anesthetization . An osteoporosis model was induced by means of bilateral ovariectomy as previously described by our group.10,18,19 briefly, a lumbar bilateral incision was made where the ovaries were located . Femur defect drilling was performed under general anesthesia by intraperitoneal injection of sodium pentobarbital (40 mg / kg body weight). A linear skin incision of approximately 1 cm in the distal femoral epiphysis was made bilaterally and blunt dissection of the muscles was performed to expose the femoral condyle.20,21 then, a 3 mm diameter anteroposterior bicortical channel was created perpendicular to the shaft axis to remove cancellous bone, by using a trephine burr at a slow speed irrigated with saline solution to avoid thermal necrosis . The drilled holes were rinsed by injection with saline solution in order to remove bone fragments from the cavity . Pib nanogels and b - mbg / nanogels scaffolds were gently placed in the right and left defects of each femur in experimental animals . Nine animals were used to confirm the osteoporotic model as well as for the control blank defects (data not shown). Following equal implantation of each scaffold gentamycin was given by intramuscular injection to prevent postoperative infection after the incisions were closed . At each time point of 2, 4, and 8 weeks after femur surgery, six rats were sacrificed by cervical dislocation . The femur samples were fixed in 4% formaldehyde for 24 hours at room temperature and then flushed in water for 8 hours . Femoral condyles were decalcified in 10% ethylenediaminetetraacetic acid (edta) for 2 weeks, changed twice per week, and then dehydrated in a series of graded concentrations of ethanol from 70% to 100% . To get a distinct view of the defect, the orientation and alignment of femurs were carefully considered during paraffin embedding . A series of slices starting at a distance of 1 mm proximal from the end of the growth plate with a length of 2 mm were chosen for evaluation . For analysis of the bone regeneration process within the defect, the central region of the 2.5 mm diameter defect was defined by analyzing a circular contour as area of measurement per slice, thus obtaining a consistent volume of interest and to avoid including the native bone margins . Longitudinal serial sections, 4 m thick, were cut and mounted on polylysine - coated microscope slides . For descriptive histology, hematoxylin and eosin (h&e) staining, safranin o staining (sigma s2255; sigma - aldrich co., st louis, mo, usa) and tartrate - resistant acid phosphatase staining (sigma 387a; sigma aldrich co.) were performed according to manufacturer s protocol . Specimens were examined under microscopic light by using an olympus dp72 microscope (olympus corporation, tokyo, japan). Cells containing more than three nuclei were defined as osteoclasts as previously described.10,22 to validate the results, each experiment was repeated at least three times . Qualification of the regenerated bone was done according to h&e staining by using image pro plus 6.0 software (media cybernetics, bethesda, md, usa). Areas of newly formed bone which acquired a bluish - green stain were delineated manually and then calculated as the percentage of new bone area in total cross - sectional area ([bone area / total area] 100%) as previously described.21,2325 the number of osteoclasts was measured by osteomeasure software (osteometrics, decatur, ga, usa) following nomenclature defined by the american society for bone and mineral research . Nine randomly selected representative fields (2,048x1,536pix) from each section were identified (original magnification 10) and averaged . Pib nanogels were fabricated according to the method of precipitation polymerization, as previously described.17 b - mbg was fabricated according to the method of our previous study.9 then, b - mbg powder was put into the pib nanogels solution, and stirred for 48 hours at 4c . The mass ratios of b - mbg powder and pib nanogels in the mixture were 1:1 respectively . The size of pib nanogels were determined by dynamic light scattering (zetasizer nano - zs 90; malvern instruments, malvern, uk) using a helium neon laser source (=633 nm) with scattering angle 90. all samples were diluted with ultrapure water to 0.5 mg / ml . The size was measured at the various temperatures from 25c to 40c with pre - equilibration for 5 minutes at each point . The dynamic viscoelastic properties of nanogel dispersions were obtained using a stress - controlled rheometer (kinexus; malvern instruments) with a parallel plate (=40 mm, gap set at 1.0 mm) in the range of 20c50c with the following parameters: shear stress = 1.0 pa, heating rate = 5c / min, and frequency = 1.0 hz . Pib nanogels / b - mbg and pib nanogels were respectively implanted into the right and left critical - sized femur defect of mature osteoporotic female wistar rats . The osteogenic properties were then evaluated by histological staining as described in the following section . Animal feeding and surgical procedures were conducted in accordance with the guidelines of animal care and use committee of wuhan university, people s republic of china, and authorized by the ethics committee at the school of dentistry, wuhan university . Ten week old mature female wistar rats weighing between 200300 g were used for this experiment . Ten percent chloralic hydras (4 ml / kg body weight) was injected inside the abdomen for anesthetization . An osteoporosis model was induced by means of bilateral ovariectomy as previously described by our group.10,18,19 briefly, a lumbar bilateral incision was made where the ovaries were located . Femur defect drilling was performed under general anesthesia by intraperitoneal injection of sodium pentobarbital (40 mg / kg body weight). A linear skin incision of approximately 1 cm in the distal femoral epiphysis was made bilaterally and blunt dissection of the muscles was performed to expose the femoral condyle.20,21 then, a 3 mm diameter anteroposterior bicortical channel was created perpendicular to the shaft axis to remove cancellous bone, by using a trephine burr at a slow speed irrigated with saline solution to avoid thermal necrosis . The drilled holes were rinsed by injection with saline solution in order to remove bone fragments from the cavity . Pib nanogels and b - mbg / nanogels scaffolds were gently placed in the right and left defects of each femur in experimental animals . Nine animals were used to confirm the osteoporotic model as well as for the control blank defects (data not shown). Following equal implantation of each scaffold gentamycin was given by intramuscular injection to prevent postoperative infection after the incisions were closed . At each time point of 2, 4, and 8 weeks after femur surgery, six rats were sacrificed by cervical dislocation . The femur samples were fixed in 4% formaldehyde for 24 hours at room temperature and then flushed in water for 8 hours . Femoral condyles were decalcified in 10% ethylenediaminetetraacetic acid (edta) for 2 weeks, changed twice per week, and then dehydrated in a series of graded concentrations of ethanol from 70% to 100% . To get a distinct view of the defect, the orientation and alignment of femurs were carefully considered during paraffin embedding . A series of slices starting at a distance of 1 mm proximal from the end of the growth plate with a length of 2 mm were chosen for evaluation . For analysis of the bone regeneration process within the defect, the central region of the 2.5 mm diameter defect was defined by analyzing a circular contour as area of measurement per slice, thus obtaining a consistent volume of interest and to avoid including the native bone margins . Longitudinal serial sections, 4 m thick, were cut and mounted on polylysine - coated microscope slides . For descriptive histology, hematoxylin and eosin (h&e) staining, safranin o staining (sigma s2255; sigma - aldrich co., st louis, mo, usa) and tartrate - resistant acid phosphatase staining (sigma 387a; sigma aldrich co.) were performed according to manufacturer s protocol . Specimens were examined under microscopic light by using an olympus dp72 microscope (olympus corporation, tokyo, japan). Cells containing more than three nuclei were defined as osteoclasts as previously described.10,22 to validate the results, each experiment was repeated at least three times . Qualification of the regenerated bone was done according to h&e staining by using image pro plus 6.0 software (media cybernetics, bethesda, md, usa). Areas of newly formed bone which acquired a bluish - green stain were delineated manually and then calculated as the percentage of new bone area in total cross - sectional area ([bone area / total area] 100%) as previously described.21,2325 the number of osteoclasts was measured by osteomeasure software (osteometrics, decatur, ga, usa) following nomenclature defined by the american society for bone and mineral research . Nine randomly selected representative fields (2,048x1,536pix) from each section were identified (original magnification 10) and averaged . All statistical analysis was performed by using spss version 17.0 software (spss inc ., data (percentage of new bone formation and osteoclast number) were expressed as mean standard deviation and were analyzed using one - way analysis of variance and post hoc student s t - test . A 5% (p<0.05) level of significance was adopted . As shown in figure 1, pib nanogels exhibited temperature - sensitive volume phase transition . The particle size of nanogels decreased from about 180 nm at 25c to 80 nm at 37c . Most notably, a sudden change of nanogels size occurred at 32.5c, that is, the volume phase transition temperature . The temperature sensitive volume phase transition was attributed to the increase of the hydrophobic force between nanogels particles in response to the increasing temperature . Therefore, concentrated pib nanogels dispersion exhibited different phase states depending on the temperature . At lower temperature (<25c), hydrophilic pib nanogels swelled, and formed a swollen gel phase due to the crowded volume effect . Pib nanogels then shrank with the increasing temperature, and turned from a swollen gel phase into flowable sol phase owing to the volume reduction . When nanogels concentration is below 8.0 wt%, the storage modulus (g) and the loss modulus (g) of pib nanogels always decreased with the increasing temperature in rheological determination (figure 2). Pib nanogels exhibited two phase states: swollen gel phase and flowable sol phase; the shrunken gel phase was not observed . The mixture of 5.0 wt% pib nanogels dispersion and 6.0 wt% mbg, however, exhibited three phase states: swollen gel phase, flowable sol phase, and shrunken gel phase . Moreover, the addition of mbg improved the mechanical strength of pib nanogels dispersion (figure 2). Histological analysis by h&e and safranin o staining revealed more new bone formation in the defects filled with pib nanogels / b - mbg scaffolds compared to pib nanogels scaffold alone (figures 3, 4 and 5). In both the pib nanogels and pib nanogels / b - mbg composite scaffolds, ecm has been formed and cells migrated inside the defects as observed by h&e staining at 2 weeks (figure 3a and d). Defects treated with pib nanogels were however sparsely populated with fibrous tissue along with a clear border of native bone (figure 3a). On the other hand, signs of newly formed irregular bone trabeculae can be seen in the pib nanogels / b - mbg groups (figure 3d). At 4 weeks, the density of the ecm had increased and extensive newly formed bone was observed (figures 3b, e). Furthermore, the formation of new blood vessels randomly dispersed throughout the defect area was also observed (figures 3b, e). The new bone of the pib nanogels / b - mbg groups demonstrated significantly higher levels of new bone formation at 4 weeks (figure 4). At 8 weeks, bone islands were observed in the center of defects with abundant bone formation peripherally . As compared with pib nanogels scaffolds, significantly higher new bone tissue was observed in the critical sized defects of pib nanogels / b - mbg composite scaffold at 8 weeks (figure 4)., shuttle shaped osteoclasts were not observed in the defects either peripherally or centrally indicating that bone remodeling was not initiated (figure 6a and d). At 4 weeks, osteoclasts were found lining the surface of the osteoporotic trabeculae . No significant difference was observed between both groups at 2 or 4 weeks (figure 7). At 8 weeks, a gradual increase in osteoclast number was observed in both groups and a significantly lower number of osteoclasts was found on b - mbg / nanogels scaffolds when compared to control nanogels (figure 7). As shown in figure 1, pib nanogels exhibited temperature - sensitive volume phase transition . The particle size of nanogels decreased from about 180 nm at 25c to 80 nm at 37c . Most notably, a sudden change of nanogels size occurred at 32.5c, that is, the volume phase transition temperature . The temperature sensitive volume phase transition was attributed to the increase of the hydrophobic force between nanogels particles in response to the increasing temperature . Therefore, concentrated pib nanogels dispersion exhibited different phase states depending on the temperature . At lower temperature (<25c), hydrophilic pib nanogels swelled, and formed a swollen gel phase due to the crowded volume effect . Pib nanogels then shrank with the increasing temperature, and turned from a swollen gel phase into flowable sol phase owing to the volume reduction . When nanogels concentration is below 8.0 wt%, the storage modulus (g) and the loss modulus (g) of pib nanogels always decreased with the increasing temperature in rheological determination (figure 2). Pib nanogels exhibited two phase states: swollen gel phase and flowable sol phase; the shrunken gel phase was not observed . The mixture of 5.0 wt% pib nanogels dispersion and 6.0 wt% mbg, however, exhibited three phase states: swollen gel phase, flowable sol phase, and shrunken gel phase . Moreover, the addition of mbg improved the mechanical strength of pib nanogels dispersion (figure 2). Histological analysis by h&e and safranin o staining revealed more new bone formation in the defects filled with pib nanogels / b - mbg scaffolds compared to pib nanogels scaffold alone (figures 3, 4 and 5). In both the pib nanogels and pib nanogels / b - mbg composite scaffolds, ecm has been formed and cells migrated inside the defects as observed by h&e staining at 2 weeks (figure 3a and d). Defects treated with pib nanogels were however sparsely populated with fibrous tissue along with a clear border of native bone (figure 3a). On the other hand, signs of newly formed irregular bone trabeculae can be seen in the pib nanogels / b - mbg groups (figure 3d). At 4 weeks, the density of the ecm had increased and extensive newly formed bone was observed (figures 3b, e). Furthermore, the formation of new blood vessels randomly dispersed throughout the defect area was also observed (figures 3b, e). The new bone of the pib nanogels / b - mbg groups demonstrated significantly higher levels of new bone formation at 4 weeks (figure 4). At 8 weeks, bone islands were observed in the center of defects with abundant bone formation peripherally . As compared with pib nanogels scaffolds, significantly higher new bone tissue was observed in the critical sized defects of pib nanogels / b - mbg composite scaffold at 8 weeks (figure 4)., shuttle shaped osteoclasts were not observed in the defects either peripherally or centrally indicating that bone remodeling was not initiated (figure 6a and d). At 4 weeks, osteoclasts were found lining the surface of the osteoporotic trabeculae . No significant difference was observed between both groups at 2 or 4 weeks (figure 7). At 8 weeks, a gradual increase in osteoclast number was observed in both groups and a significantly lower number of osteoclasts was found on b - mbg / nanogels scaffolds when compared to control nanogels (figure 7). Although a broad range of bone grafting materials has been used for tissue repair application, to date no ideal bone substitute material exists . Natural polymer - based materials are excellent scaffolds for cell attachment and growth due to their tissue - like water content and structure stability, however their weak mechanical properties make them susceptible to pressure when implanted into bone defects.2628 biodegradable synthetic polymer - based materials can readily be manufactured into specific shapes with relatively high mechanical strength, but they are usually hydrophobic and lack biocompatibility.28,29 ceramic - based materials mimic the natural bone mineral,28,30,31 have revealed good bone - bonding ability32 and osteoconductivity,30,31 however their fabrication procedures are more complex and have the inherent brittleness which limits their use in bone replacement procedures.28 in order to overcome the disadvantages of either of these materials, many composite scaffolds have been fabricated . The ideal composite should combine optimum properties such as biocompatibility, bioactivity, biodegradability, mesopore structure, and mechanical strength . This study focuses on a novel biomaterial, pib nanogels, and its ability to be used as a bone replacement graft . Unlike conventional biomaterials, it presents as liquid state at room temperature and solidifies at body temperature.15,17 hydrogels are quite similar to natural living tissues, due to their ability to retain a significant amount of water or biological fluids . They are three - dimensional, hydrophilic, polymeric networks which can be used as bone substitutes and drug delivery systems.33 there are three principles for an ideal hydrogel biomaterial: 1) upon injection, it can be solidified quickly without excessive heat generation; 2) gelation temperature in vivo should be near body temperature; and 3) the gelation products should be biocompatible or bioactive to form a tight bone bonding interface.34 pib nanogels possess all three of these properties but lack some of the mechanical properties offered by ceramic scaffolds.15,17 furthermore, the material itself undergoes material shrinkage when passing from the liquid phase at room temperature to the hardened phase at body temperature . Thus their use alone for bone tissue repair comes with certain disadvantages . As a bioceramic material, mbg has excellent bone - like apatite - formation properties and well - ordered mesoporous channels.35 mesoporous structures of biomaterials are of great importance for the loading and delivery of cells and drugs and can improve angiogenesis for tissue ingrowths and initiate osteoblast differentiation and bone regeneration.9 when in contact with biological fluids, bioactive glass can mechanically and chemically bond to bone by forming a carbonated hydroxyl - apatite layer on the glass surface . This bioactive bone - bonding property ensures the implant osteo - integration36 and makes nanogel one of the leading new and promising scaffold materials.37 the main disadvantage of mbg scaffolds is their brittleness and high rate of degradation.35 in the present study we explored the physical and biological properties of the pib nanogels / b - mbg composite scaffolds . When compared to pib nanogels alone, the incorporation of b - mbg greatly improved the mechanical strength of the pib nanogels as well as the bone regeneration abilities of pib nanogels . H&e and safranin o staining revealed significant new bone formation within the defects both peripherally and centrally in the pib nanogels / b - mbg scaffold groups . The results demonstrate that b - mbg plays an important role in enhancing bone regeneration . Our previous study showed that b - mbg scaffolds have the ability to control the release of boron ions which promotes bone growth.9 we have previously demonstrated that osteoblasts seeded in the presence of boron demonstrate significantly higher cell proliferation and increase the bone - inducing properties by expressing higher levels of collagen 1 and runx2.9 thus it is reasonable to assume that under the present study parameters, the osteoporotic healing of defects is largely driven by the release of boron which is able to stimulate osteoblast bone - forming behavior . It remains to be investigated what role boron may have on osteoclast bone - resorbing activity . In summary, it has been demonstrated that by combining the advantages of the two biomaterials, novel pib nanogels with b - mbg were able to efficiently generate high quantities of early bone formation in ovariectomized rats . The results from the present study indicate that pib nanogels / b - mbg composite scaffolds have the potential to create a new class of bone repair biomaterials with consummate properties suited for bone tissue engineering . The fabrication of pib nanogels / b - mbg enhanced the mechanical properties and bone regeneration abilities compared to pib nanogels alone . The pib nanogels / b - mbg composite has potential future use in bone tissue engineering especially for the repair of bone defects with irregular shapes because of its ability to act as a gel at injection temperatures and solidify at body temperatures . The ordered mesoporous structure, temperature - sensitive phase transition, enhanced mechanical strength, and biocompatibility make pib nanogels / b - mbg a desired biomaterial for bone tissue engineering.
During july december 2012 and 2013, sick raccoon dogs and arctic foxes, which were farmed for fur products on 6 farms (farms a f) in jilin and liaoning provinces, china, were received for quarantine inspection at the fur animal disease laboratory, institute of special animal and plant sciences, chinese academy of agricultural sciences . Several infant raccoon dogs from 1 litter became ill 40 days after birth, and the numbers of sick animals increased by the time they were 3 months of age . Clinical signs included anorexia, emaciation, growth retardation, thirst, chronic diarrhea, and unkempt fur; necropsy often revealed cyanosed splenomegaly, enlargement of mesenteric lymph nodes, and renal cortex congestion and brittleness . For the raccoon dogs showing similar clinical signs, rate of illness was 4%8%; death rate was 60% before the age of 4 months; and rate of illness increased by years on the farms that initially had sick animals . Among arctic foxes, signs varied: emaciation and growth retardation in 3-month - old cubs with pale and swelling kidneys in dead foxes; and severe diarrhea or intermittent tar - like feces in 37-month - old cubs . Because signs in the sick animals sent for quarantine inspection were similar to those in aleutian mink disease, we first used amdv - specific counter - immunoelectrophoresis (ciep) (5) to test serum samples of six 3-month - old sick raccoon dogs from farm a. all 6 were positive . Next, we designed conserved amdoparvovirus primers (av7; table 1) for pcr detection . Viral nucleic acids were extracted by using a minibest viral rna / dna extraction kit (takara, dalian, china). Dna extracted from spleen, kidney, mesenteric lymphonodus, and mucosal tissue and blood of the 4 sick raccoon dogs was all rfav dna positive . After 10 days, the 4 raccoon dogs remained rfav dna pcr positive in blood and ciep positive in serum . Dna extracted from blood of two 37-month - old sick raccoon dogs from farm c was rfav dna pcr positive, and serum samples from these animals were ciep positive . Two 7-month - old raccoon dogs from farm d appeared healthy but on necropsy showed cyanosed splenomegaly . The overall positive rates of rfav dna and ceip antibody in sick raccoon dogs were 90% and 100%, respectively . Serum iodine agglutination test (iat) (6) was positive or strongly positive in 20 sick raccoon dogs that were 7 and 19 months old but was negative in healthy animals . Of the 29 sick raccoon dogs, two 37-month - old animals were both canine parvovirus 2 and rfav dna positive by pcr in blood or spleen samples . However, canine distemper virus was not detected in spleen samples from any sick raccoon dogs . In arctic foxes raised together with sick raccoon dogs on farm b, intestinal mucosa samples from 3 of 7 foxes that died of diarrhea - associated dehydration were rfav dna pcr positive . One kidney sample from a 3-month - old fox of 3 tested on farm f showed renal enlargement and was rfav dna pcr positive . Blood, urine, and feces of two 7-month - old arctic foxes on farm c, which had tar - like feces, were rfav dna pcr positive . Results of all samples tested by pcr, ciep, and iat are summarized in table 2 . * ciep, counter - immunoelectrophoresis; iat, serum iodine agglutination test; na, samples not available; nd, assays not done; +, positive . Farm: a, heishan county; b, qianan county; c, jilin county; d, panjin county; e, haicheng county; f, nongan county . Qianan, jilin, and nongan are located in jilin province; heishan, panjin and haicheng are located in liaoning province . For raccoon dogs, pcr+ for all the tested tissues (blood, spleen, kidney and mesenteric lymphonodus). For arctic foxes, pcr+ results only for 1 of the tested tissues (blood, kidney, or intestinal mucosae). Blood and spleen samples were tested separately by pcr from healthy animals except for the blood samples from the 3-month - old healthy raccoon dogs . We next applied a semiquantitative pcr to quantify the level of rfav dna in the blood and spleens of four 7-month - old sick raccoon dogs (3 from farm d, 1 from farm c). Briefly, dna samples were diluted from 10 to 10 viral genomic copies (vgc)/l by using a quantified rfav dna template and were amplified by using av3 primers with the detection threshold of 100 vgc in a volume of 15 l . Virus titer was determined on the basis of the maximum dilution at which viral dna was detected by agarose gel electrophoresis . Rfav dna ranged from 2 10 to 5 10 vgc / ml in blood and 7 10 vgc / g in spleen . In spleen and kidney tissues of 2 raccoon dogs euthanized at 3 months of age (from farm a) and four 7-month - old sick raccoon dogs (from farm d), bacterial infections, performed by standard methods, were not found . Collectively, these results demonstrate that blood or tissues from the 2 sick animals contained rfav dna, and the dna levels were high in some samples . We used indirect immunofluorescence assay (ifa) to probe rfav antigens in spleen and kidney of the 2 rfav pcr positive 3-month - old sick raccoon dogs from farm a. tissues of the sick raccoon dogs were amdv - g antigen ifa positive, but the healthy animal tissues were not (figure 1, panel a). This result indicates that the spleen and kidney of the sick raccoon dogs contained viral antigens that share immunogenicity with amdv - g . More importantly, we isolated an rfav strain, named xq - jlr, by infecting crfk cells . Ifa using anti - amdv serum showed positive green cells in rfav - infected crfk cells but not in mock - infected cells (figure 1, panel b). Under a transmission electron microscope, virus particles at 23 nm in diameter were visualized in a concentrated supernatant of infected crfk cells (figure 1, panel c). Tissue smears, as described below, were prepared from spleen and kidney samples of sick raccoon dogs and detected with a control mouse serum or anti - amdv - g serum by using an ifa (8). (a) mock smear of a spleen tissue from a sick raccoon dog, detected with normal mouse serum as a primary antibody; (b) control smear of a spleen tissue from a healthy raccoon dog; (c, d) amdoparvovirus antigen - positive smears of spleen tissue (c) and kidney tissue (d) from a sick raccoon dog, detected with anti - amdv - g serum . Original magnification 200 . B) one milliliter filtered (0.22 m) pathological spleen samples collected from a sick raccoon dog that had a virus titer of 7 10 vgc / ml, was inoculated to confluent crfk cells in a t25 flask . Infected cells of the fourth passage were fixed and analyzed with ifa by using anti - amdv serum (mock- or rfav - infected crfk cells). C) twenty - five milliliter supernatant (2 10 vgc / ml) of rfav(xq - jlr)infected crfk cell cultures were concentrated . Virions were agglutinated with an anti - rfav raccoon dog serum and were visualized under a transmission electron microscope (tem). Amdv, aleutian mink disease virus; rfav, raccoon dog and fox amdoparvovirus; ifa, indirect immunofluorescence assay; vgc, viral genome copies . We further proved that rfav is the predominant virus in the lesion tissues of sick animals . A modified sispa (7) was performed for high - throughput sequencing with mixed lesion tissues of 3 spleens and 3 kidneys from 3 sick raccoon dogs . By using illumina miseq sequencing (illumina, san diego, ca, usa), we obtained 478,813 high - quality reads, and 668 contigs were assembled, including 17 contigs for new amdoparvovirus . Cytosine nucleotide content of 62-bp gap, were recovered, which are consistent with the sequences acquired by sanger sequencing of viral dna amplified from tissues . There were 865 reads, by blastn (http://www.blast.ncbi.nlm.nih.gov/blast.cgi), in alignment with amdoparvovirus sequences but only 16 reads with sequences of other non - mammal viruses, such as phage and baculovirus . The identities were <91% of the reads aligning with amdv sequences in genbank by blastn (e value <10). We sequenced av7 primer - amplified pcr products of rfav dna from the tissues of 32 animals (genbank accession nos . Kj396347kj396350) only share a similarity of 82% and 76.7% in the nonstructural protein (ns) 1encoding sequence and ns1 aa sequence, respectively, with amdv . Phylogenetic analyses of 2 neighbor - joining trees of either ns1 or major structural protein (vp2) strongly suggest that rfav strains cluster into a unique clade between amdv and gray fox amdovirus species (figure 2). Rfav and other published amdoparvovirus sequences were aligned by using the muscle program in mega5.2 (9), which used a p - distance model with 1,000 bootstrap replicates to generate phylogenetic trees of ns1 and vp2 aa sequences . Amdv, aleutian mink disease virus; gfav, gray fox amdoparvovirus; rfav, raccoon dog and fox amdoparvovirus; ns, nonstructural protein; vp, viral structural protein . We identified a new virus species, rfav, from farmed raccoon dogs and arctic foxes in jilin and liaoning provinces, china . Raccoon dogs are naturally susceptible to rfav infection, and rfav is most likely the etiologic agent responsible for the disease manifestations of the sick raccoon dogs.
The development of reasonablecost power sources for largescale electric transportation is becoming increasingly important for economic and ecological reasons . Liion batteries (libs) are the most promising energystorage systems for electric vehicles (evs) and hybrid plugin electric vehicles (hpevs).1, 2 however, further improvements to the currently available lib technology are needed to meet the requirements for automotive applications . Enhanced energy density, increased safety and reduced cost are key requirements . In stateoftheart libs, both the cathode operating voltage (v) and capacity (a h kg) determine the energy density (w h kg) of the cell . Standard cathodes [lini1xymnxcoyo2 (nmc), lini1xycoxalyo2 (nca), lifepo4 (lfp)] operate at potentials below 4.2 v versus li / li with limited capacity . Moreover, at the full liion cell level, the cathode capacity is further reduced due to the formation of the solid electrolyte interface (sei) on standard graphitebased anodes . Therefore, cathode materials with either increased specific capacity or increased working potential with respect to those of the currently used active materials are necessary to improve energy density.3, 4, 5 additionally, the cathode materials currently proposed for highenergydensity applications contain cobalt, which is highly toxic and expensive . The substitution of co with more accessible elements is the key to the cost reduction required for largescale applications . Here, we describe a series of cofree, lirich li1+xni0.5mn1.5o4 cathode materials with high operating voltage and high capacity for lib applications . To date, such structures have been obtained by discharging lini0.5mn1.5o4 versus metallic li at potentials below 3 v.6, 7, 8, 9, 10, 11 the nisubstituted spinel lini0.5mn1.5o4 (lnmo) has been investigated widely as a cathode material for highvoltage applications and showed very promising performance in the potential range 3.55 v. intensive research efforts have been devoted to the study of the influence of the morphology and transitionmetal order on the electrochemical performance.12, 13, 14, 15, 16 the lnmo cathode shows a potential plateau at 4.7 v versus li / li, which corresponds to the ni / ni redox reaction, whereas mn remains in the original 4 + oxidation state . The reversible extraction of one li ion per formula unit leads to a theoretical specific capacity of 147 ma h g. theoretically, further li insertion is possible through the reduction of the mn oxidation state from 4 + to 3 + . The reduction of all mn ions into the trivalent state would lead to a final composition of li2.5ni 0.5mn 1.5o4 with a theoretical specific capacity of 347 ma h g and a specific energy higher than 1100 w h kg versus li / li . This value is far beyond those reported to date for layered oxides and li mnrich materials . One of the main limitations arises from the poor cycling stability observed if extra li is inserted electrochemically in the lnmo structure at potentials below 3 v.6, 7, 8, 9, 10, 11 only a few reports deal with the electrochemical behavior of li1+xni0.5mn1.5o4 through the exploitation of both ni and mn redox activity . From a structural point of view, phase transitions from a cubic to tetragonal structure during lithiation to 2 v are commonly reported . The reversibility of those phase transitions depends on different factors including the final li content, the cation ordering of the pristine spinel structure, and its morphology.6, 7, 8, 9, 10, 11 generally, the specific capacity can be increased significantly by expanding the voltage operative window to low potentials but this is associated with rapid capacity fading . Furthermore, to make li1+xni0.5mn1.5o4 structures of technological interest, it is necessary to develop a suitable synthesis method . So far, the electrochemical insertion of li into lnmo remains the only route to obtain li1+xni0.5mn1.5o4 . In this case, the anode provides the li ions that are inserted into the original cathode structure during the reduction process . Although the electrochemical method allows relatively easy control of the amount of li in li1+xni0.5mn1.5o4, this approach is clearly not useful for practical applications in full liion cells . A full cell is assembled with a licontaining cathode and a lifree anode, and extra li sources, such as metallic li, are not available . The cathode provides all of the li for reversible li insertion / extraction reactions and irreversible sei formation and should contain the maximum amount of li in the pristine state for full capacity utilization . Therefore, the high specific capacity of lirich li1+xni0.5mn1.5o4 phases would be very attractive from a practical point of view if the active material could be prepared chemically . To date, synthesis routes for materials with li1+xni0.5mn1.5o4 composition and suitable properties for largescale processing have not been reported . Obtained li2ni0.5mn1.5o4 through the chemical intercalation of a second li ion into lini0.5mn1.5o4 by using lii in acetonitrile.17 however, the cycling behavior and stability involving the ni and mn redox reactions were not provided . To the best of our knowledge, the cycling of a chemically synthesized li1+xni0.5mn1.5o4 over a wide capacity range has not been reported . In the present work, we describe a series of lirich phases with li1+xni0.5mn1.5o4 (0<x<1) composition, which were obtained through a scalable, lowtemperature synthetic method . The material characteristics such as tap density, particle architecture, and size distribution are tailored to meet the requirements for largescale applications and allow easy processability during standard electrode manufacturing processes . The li content is optimized to allow very stable cycling behavior in halfcells with highvoltage and highcapacity performance . In the potential range 2.4 to 4.9 v, the cathode material with li1.5ni0.5mn1.5o4 operates at two different potential plateaus with a specific capacity of 200 ma h g. additionally, we demonstrate the first practical application of a lirich li1+xni0.5mn1.5o4 (0<x<1) cathode in full cells versus graphite anodes, and the cells show very promising stability for the development of nextgeneration highenergy liion batteries . Li1+xni0.5mn1.5o4 samples, with x ranging between 0 and 1, were obtained successfully by the chemical method described in the experimental section . The chemical lithiation method reported here allows the synthesis of the limit composition of li2ni0.5mn1.5o4, in which the average mn oxidation state is 3.3 + . For all of the obtained samples, the experimental li / mn / ni ratios obtained from chemical analysis are in good agreement with the nominal compositions . The particle architecture of the synthetized materials was optimized to reach a tap density of 2.4 g cm . High tap densities are beneficial for obtaining close packing of the active material in the electrodes . Sem analysis of all samples was performed to investigate the influence of the lithiation process on the particle morphology . The insertion of li is expected to produce volume expansion and lattice distortion, because the formation of mn ions and consequent jahn teller distortion may lead to the formation of cracks or rupture the particles . Sem images of selected samples with different li content, namely, lini0.5mn1.5o4, li1.44ni0.5mn1.5o4, li1.88ni0.5mn1.5o4, and li2ni0.5mn1.5o4 are shown in figure 1 . All of the materials present similar morphologies with spherical particles made of densely packed nanosized primary crystallites . These images also reveal that the lithiation process does not affect the integrity of the particles . This suggests that the network of grain boundaries in the particles can buffer the volume expansion that occurs during li insertion and prevent the formation of cracks . In this tailored particle architecture, the random orientation of the crystallites in the spheres allows anisotropic expansion to occur at the crystal level without affecting the grains . Therefore, such features are beneficial for high particle density and, at the same time, provide material integrity upon mechanical stress owing the high amount of li ions inserted . Sem images of samples of composition (a, b) lini0.5mn1.5o4, (c, d) li1.44ni0.5mn1.5o4, (e, f) li1.88ni0.5mn1.5o4, and (g, h) li2ni0.5mn1.5o4 at 3000 and 10 000 magnification . The structures of the two endmembers of the series, lini0.5mn1.5o4 and li2ni0.5mn1.5o4, were investigated by selectedarea diffraction . The selectedarea electron diffraction patterns obtained from the lini0.5mn1.5o4 and li2ni0.5mn1.5o4 samples are shown in figure 2 a and b, respectively . The diffraction patterns were indexed to the [1 0 0] orientation of the ordered cubic p4332 space group and the [0 1 0] zone axis of the tetragonal i41/amd, respectively . The superstructure of the ordered p4332 space group is apparent from the satellite spots in the diffraction pattern in figure 2 a. the simulated electron diffraction patterns are displayed in figure 2 c and d for the [1 0 0] ordered cubic and [0 1 0] tetragonal phases, respectively . (a) selectedarea electron diffraction pattern of lini0.5mn1.5o4 in the [1 0 0] orientation as indexed to ordered cubic p4332 . The pattern is indexed to the [0 1 0] zone axis of the tetragonal i41/amd . (c) calculated electron diffraction patterns for the [1 0 0] orientation of the ordered cubic p4332 . (d) calculated electron diffraction pattern for the [0 1 0] zone axis of the tetragonal i41/amd . The xrd patterns of the samples described above with different li contents are shown in figure 3 . In good agreement with the tem results, the sample with one li ion per formula unit crystallizes in the cubic spinel structure . Two types of lini0.5mn1.5o4 structures are possible: (1) an ordered structure with cubic primitive crystal symmetry (space group p4332), in which the ni and mn ions occupy 4b and 12d sites, respectively, and (2) a cationdisordered structure with facecentered cubic symmetry (space group fd3 m), in which the ni and mn ions occupy 16d positions randomly.18, 19 (a) xrd rietveld refinement results and (b) phase identification of lini0.5mn1.5o4, li1.44ni0.5mn1.5o4, li1.88ni0.5mn1.5o4, and li2ni0.5mn1.5o4 samples . The xrd analysis of the lini0.5mn1.5o4 sample reported in figure 3 shows superstructure reflections (the most pronounced at 2=15.3, see inset in figure 3 a) typical for highly ordered materials (space group p4332). The refinement indicates the presence of a single phase with no crystalline impurities, and the lattice parameter a=8.168 is in good agreement with the results reported previously.9, 18, 20 the xrd peaks of the li2ni0.5mn1.5o4 sample were indexed to a tetragonal phase with i41/amd space group, and no evidence of the original cubic phase was observed . The reflections of the cubic phase become weaker with increasing li content, and the relative intensity of the tetragonal phase increases (figure 3 b). Therefore, the xrd analysis suggests that li insertion by the chemical method leads to a structural conversion from the original cubicphase lini0.5mn1.5o4 to tetragonalphase li2ni0.5mn1.5o4 upon lithiation . The rietveld refinement of the xrd data was performed to determine the formed phases, c / a ratios, and lattice parameters . The results, reported in table 1, are consistent with the tem results described above . The domain sizes, as calculated from xrd analysis, are (10515) and (8525) nm for lini0.5mn1.5o4 and li2ni0.5mn1.5o4, respectively . The values were obtained through integralbreadthbased lvol calculations (volumeaveraged column height from integral breadth; lvolib) assuming intermediate crystallite size broadening modeled by a voigt function . Reported li2ni0.5mn1.5o4 obtained by chemical methods.17 however, they observed no structural differences between the pristine lnmo and the lithiated phase, both of which were identified as pure cubic spinel from xrd analysis . This result disagrees with the commonly reported evidence of structural changes upon electrochemical li insertion, which is expected to produce lattice strain owing to the accommodation of the li ions and the consequent structural expansions associated with the formation of the larger mn ions and the jahn teller effect . Our findings on the structural characterization of the lirich phases li1+xni0.5mn1.5o4 (0<x<1) are in good agreement with the phase evolution from cubic to tetragonal (i4 1/amd) reported in for the electrochemical lithiation of lnmo at low potentials.6, 7, 8, 9 to design li1+xni0.5mn1.5o4 (0<x<1) compounds with optimized li content for practical applications and to define the best operative conditions, the electrochemical behavior at low potentials was first studied through galvanostatic li insertion into lini0.5mn1.5o4 . The discharge profile from 4.9 to 1.5 v versus li the amount of inserted li is calculated on the basis of the specific capacity of lini0.5mn1.5o4 . The lowvoltage region of the galvanostatic discharge curve exhibits two different voltage plateaus at approximately 2.7 and 2.1 v. this indicates that two electrochemical processes may occur during galvanostatic lithiation, the mechanism for which could be explained if it is assumed that two different phase transitions occur . However, the structural analysis of the lirich phases li1+xni0.5mn1.5o4 (0<x<1) did not reveal the formation of any intermediate phase . The xrd results show a single phase transition from cubic to tetragonal, and intermediate compositions are indexed as mixtures of the two phases, the relative amounts of which depend on the li content . The two voltage plateaus at 2.7 and 2.1 v have been reported by several authors, although the electrochemical mechanism is not fully understood.8, 9 further studies on the li insertion / extraction mechanism at low potentials and on the appearance of the two lowvoltage plateaus will be the subject of a later communication . In addition to the highvoltage capacity of 135 ma h g associated with the plateau at 4.7 v, the previously described plateaus at 2.7 and 2.1 v deliver 65 and 70 ma h g, respectively . A further 50 ma h g can be obtained between 1.9 and 1.5 v. the total capacity gained between 1.5 and 4.9 v reaches a very high value of 320 ma h g. nevertheless, the accommodation of high amounts of li is expected to produce strain in the lattice and, thus, decrease the cycling stability upon continuous charge / discharge.8 the use of the first of the lowvoltage plateaus at 2.7 v with the lowerpotential cutoff limited to 2.4 v would lead to a final capacity of approximately 200 ma h g. this in turn would minimize lattice distortions due to the li insertion . Unexpectedly, the impact of the li1+xni0.5mn1.5o4 cathode capacity on the overall energy density at a fullcell level is comparably low, as shown in table 2 . Theoretical energy density of li1+xni0.5mn1.5o4 in halfcells versus metallic li and in full cells versus a graphite anode . [a] a reversible capacity of 350 ma h g and a working potential of 0.2 v were assumed for the graphite anode . The calculations of the theoretical energy density for halfcells and full cells of henmc are also reported for comparison . [b] percentage of the specific energy assuming the value obtained in the full potential range cycling (1.54.9 v vs. li / li) as 100% . The capacity of li1+xni0.5mn1.5o4 was calculated on the basis of the results reported in figure 4, and the values obtained after each of the lowvoltage plateaus are also listed in table 2 . The average voltage was calculated as the ratio between the overall specific energy (w h kg) and the overall specific capacity (a h kg). It is worth noting that the lirich li1+xni0.5mn1.5o4 cathode material can reach energy densities as high as those of highenergy nmc (henmc). However, unlike henmc, it does not require an activation cycle and is cofree . This translates into lower cost, higher safety, and easier application in libs . From the capacity point of view, our calculations indicate that li1+xni0.5mn1.5o4 cathode capacities beyond 200 ma h g, accessible by increasing the potential window, would not contribute significantly to increasing the overall energy density of the full cell . Therefore, the working potential window used to investigate the electrochemical performance of the synthetized lirich samples was set as 2.44.9 v. as shown in figure 4, the amount of li inserted in this potential window is approximatively 1.5 ions per formula unit . Accordingly, we selected and tested the sample with a composition close to this value, namely, li1.44ni0.5mn1.5o4 . However, further li can be provided in the pristine cathode structure as a charge reservoir to compensate the irreversible capacity loss (icl) at the graphite anode for use in full cells . For this purpose the cycling stability of electrodes made with these two materials was tested at a charge / discharge rate of 30 ma g in the potential range 2.44.9 v. the recorded galvanostatic curves are reported in figure 5 . The capacities obtained during the first charges were 220 and 262 ma h g for li1.44ni0.5mn1.5o4 and li1.88ni0.5mn1.5o4, respectively, on the basis of the weights of the nominal compositions . For both samples, the amount of li extracted during the first oxidation corresponds to the value determined through chemical analysis . In good agreement with the previous results, li extraction / insertion occurs at two different potential plateaus . The first one is the highvoltage plateau at 4.7 v related to the ni / ni redox reaction, which provides 130 ma h g for all samples . The second voltage plateau occurs at 2.9 and 2.7 v in charge and discharge, respectively . The associated capacity depends on the amount of mn ions and, thus, of li ions present in the structure . Indeed, the lowvoltage capacities are 43 and 98 ma h g for li1.44ni0.5mn1.5o4 and li1.88ni0.5mn1.5o4, respectively . The sample of composition li1.88ni0.5mn1.5o4 shows an irreversible capacity loss of 50 ma h g during the first cycle . This excess of li, which is not reinserted in the next discharge, can be utilized to compensate the icl of the graphite . In the second cycle, both samples reached a reversible specific capacity of 220 ma h g and high coulombic efficiency close to 100% . The higher stability can be ascribed mainly to the particle morphology, which is beneficial in the highvoltage region by limiting the side reactions with the electrolyte.12, 13 furthermore, the network of grain boundaries in the particles can buffer the volume changes in the lowpotential region and allow good contact to be maintained among the electrode components during cycling . The cathode material presented here offers a unique combination of characteristics in terms of morphology, particle size, and density as well as highcapacity and highvoltage performance . Galvanostatic charge / discharge profiles of electrodes based on lirich compounds (a) li1.44ni0.5mn1.5o4 and (b) li1.88ni0.5mn1.5o4 (b). Charge / discharge current density 30 ma g. charge / discharge specific capacity versus number of cycles for electrodes based on li1.44ni0.5mn1.5o4 cycled between 2.4 and 4.9 v at 30 ma g. the feasibility of the lirich li1+xni0.5mn1.5o4 structures as cathode materials versus graphite in full cells was also investigated . For this purpose, electrodes based on the lirich material with composition li1.5ni0.5mn1.5o4 were assembled versus a standard graphitebased anode . It is worth noting that the electrode mass loadings are comparable to those of commercial electrodes for libs . The full cell was tested by continuous charge / discharge at a constant current density of 30 ma gcathode in the voltage range 1.504.78 v. the evolution of the cathode capacity versus the number of cycles is shown in figure 7 . The sei formation at the graphite anode consumes part of the initial charge available in the cathode . After sei formation, a stable reversible specific capacity of 160 ma h gcathode was obtained . (a) cycling performance and (b) charge / discharge profiles of a full cell with cathode composition li1.5ni0.5mn1.5o4 . (c) cycling performance and charge / discharge profiles (inset) of a full cell with a cathode composition of li1.88ni0.5mn1.5o4 versus graphite . As already mentioned, the chemical method proposed here allows the amount of li in the cathode structure to be tuned to compensate for the icl of the graphite and increase the reversible capacity of the full cell . The obtained results are reported in figure 7 c. the potential curves versus a li reference for the first cycle clearly show the sei formation of the graphite . After the first three cycles, the cathode delivers 170 ma h g. this indicates that it is possible to increase the cell capacity by optimizing the cell balance, that is, by estimating the optimum cathode / anode ratio and li content in the cathode to fully compensate the icl of the graphite . However, the optimization of the cell balance is beyond the scope of this work, and further experiments are needed to achieve the best performance in full cells . Regardless of possible further cellbalance optimization, the cathode materials presented here offer a unique combination of characteristics in terms of morphology, particle size, and density and show highcapacity and highvoltage performance . The very promising cyclability versus graphite in full cells has not been reported previously and is highly encouraging in view of practical applications for liion batteries . We successfully synthetized lirich phases with composition li1+xni0.5mn1.5o4 (0<x<1), which showed highcapacity and highvoltage performance as cathode active materials for liion batteries (libs). The structural and morphological parameters were optimized to obtain the maximum energy density in cell applications . The optimized architecture in combination with the high density leads to fewer surface side reactions at high potentials . Moreover, the polycrystalline architecture of the grains with random orientation of the primary crystallites may help to accommodate the changes to the anisotropic lattice parameters that occur at low potentials . Our findings confirm that the chemical lithiation leads to the same lirich phases as those obtained through electrochemical lithiation . Furthermore, we demonstrated the first application of lirich li1+xni0.5mn1.5o4 (0<x<1) cathodes in full cells versus graphite anodes, and the cells had a capacity of 160 ma h g and a coulombic efficiency of nearly 100% . The high capacity and high potential make the chemical lithiation approach feasible for practical applications . The li1+xni0.5mn1.5o4 materials were synthesized by a coprecipitation process followed by two thermal treatments: the first one in an oxidative atmosphere and the second one under reductive conditions to obtain the final lirich stoichiometry . The precursors were obtained by combining aqueous solutions of ammonia, nickel, and manganese nitrates in an alkaline environment . The relative amount of nickel and manganese in the solution was adjusted to obtain a ratio of 1:3 in the final precipitate . The thermal treatment was performed under air with a final annealing step at 700 c . The obtained product was then mixed with a li source under reductive conditions without any solvent to obtain the desired lirich phase . The temperature of the second thermal step was kept below 600 c for a dwell time of 4 h. a series of products with different compositions were synthesized, and the li content was varied by adjusting the ratio of the reactants . The chemical analysis was performed by inductively coupled plasma optical emission spectrometry (icpoes) with an arcos sop instrument . The structural properties were investigated by powder xrd (cuk radiation, =0.154 nm) with a siemens d5000 diffractometer in the 2 range 10 to 120. the xrd patterns were analyzed by the topas 2.1 program from bruker . The structures of lini0.5mn1.5o4 and li2ni0.5mn1.5o4 were analyzed by tem, for which powder samples were dispersed in dmc and transferred onto holey carbon grids without crushing to reduce artefacts that may be formed from the crushing process . Electron diffraction experiments were conducted at 80 kv with a titan 80300 kv transmission electron microscope to reduce electronbeaminduced damage . The electrodes for electrochemical measurements were manufactured by preparing slurries of active material / carbon superp (timcal)/polyvinylidene fluoride in the ratio 88:8:4 wt% . The slurries were coated on al foil by the doctor blade technique and then dried at 40 c . The electrodes were punched from the obtained coatings, pressed, and dried under vacuum . The halfcell tests were performed with twoelectrode coin cells (cr2032, mti alclad) with metallic li as the counter electrode or threeelectrode cells (elcell germany) with metallic li as the counter and reference electrodes . The anode composition was 90 wt% graphite (hitachi smg) and 10 wt% polyvinylidene fluoride . Anode and cathode pairs were first assembled in a threeelectrode configuration to follow the individual potential profiles versus li . Subsequently, the same anode and cathode were reassembled in a twoelectrode coin cell and cycled for longterm stability tests . Cathode / anode activematerial mass ratios in the range 1.041.98 were used for fullcell balancing . The electrolyte was a solution containing 0.98 m lipf6 (basf) and 0.02 m lithium bis(oxalato)borate (libob, rockwood lithium) in ec / dmc (1:1 w / w, ube industry, japan; ec = ethylene carbonate). All tested electrodes had the same composition and activematerial loading of approximately 15 mg cm . All measurements were performed at room temperature with a vmp2/z electrochemical workstation by biologic science instruments.
Triggered by initial reports on lasers with mirrorless feedback, random lasers with noncoherent (which essentially is amplified spontaneous emission, ase) and coherent feedback moved closer in the focus of extensive research activities . Coherent light amplification using high refractive index dielectric or metallic nanostructures inside a gain medium can be seen as a promising alternative to conventional lasers . While a conventional laser requires accurate fabrication and extremely precise alignment of the resonator, the resonator in a coherent random laser is realized by randomly distributed scattering nanoparticles forming closed - loops for photons . This permits a cheap and easy fabrication method, making coherent random lasers an attractive alternative to conventional lasers in a manifold of potential applications, ranging from smart sensors and medical diagnostics, to lighting devices and displays . Noble metal nanoparticles exhibiting localized plasmon resonances provide two beneficial features for coherent random lasers: much higher scattering cross sections than dielectric nanoparticles and high field enhancements close to their surface, associated with localized plasmons . It turns out that star - shaped gold nanoparticles, so - called gold nanostars, outperform conventionally shaped gold nanoparticles such as spheres and rods in terms of coherent random lasing . Gold nanostars exhibit multiple plasmon resonances in the red and near - infrared spectral region . These plasmon resonances associated with the tips of the gold nanostars lead to large and spectrally broad - band scattering cross sections as well as extremely strong field enhancements localized at the tips, key features for coherent random lasers . However, despite all of the efforts on plasmon - mediated coherent random lasing, there are no reports on hybrid multilayered plasmonic nanostars - based coherent random lasing . Here, we report on the development of hybrid multilayered plasmonic nanostars, which can be mixed with gain materials dissolved in either polar or nonpolar solvents . Two different, but in terms of optical properties similar, gain materials, a dye molecule and a light - emitting polymer, were dispersed in polar or nonpolar solvents, respectively . For extremely low pumping thresholds, the solutions containing gain material and silver - enhanced and silica - coated nanostars exhibit spectrally narrow lasing modes, typical for coherent random lasing . Further, we show that silver - enhancement of the gold nanostars reduces the pumping threshold for nanostar - based random lasing . Additionally, we reveal that the silica coating nearly does not influence the operation of the silver - enhanced gold nanostar - based random lasers in terms of lasing intensity and pumping threshold, in contrast to gold nanostar - based random lasers, where only ase and no coherent random lasing was observed for the silica - coated gold nanostars . Silver nitrate (agno3, 99.9999%), gold(iii) chloride trihydrate (haucl43h2o), l - ascorbic acid (aa), tetraethyl orthosilicate (teos), trisodium citrate dihydrate, 1 n hydrochloric acid solution (hcl), sodium borohydride (nabh4, 99%), hexadecyltrimethylammonium bromide (ctab), o-[2-(3-mercaptopropionylamino)ethyl]-o-methylpoly(ethylene glycol) (mpeg - sh, mw 5k), ammonium hydroxide (nh4oh, 30%), and rhodamine 6 g (r6 g) were purchased from sigma - aldrich at the highest purity grade available . Commercial poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene - vinylene] end - capped with polysilsesquioxane (ads200re, purchased from american dye source) was used without further purification . Citrate - capped spherical gold nanoparticles (125 nm in diameter) were purchased from nanopartz . All glassware and stir bars were thoroughly cleaned with aqua regia and dried prior to use . Ultrapure distilled water (millipore, 18 m cm) was used in all steps of nanoparticle preparation . The hybrid multilayered plasmonic nanostars were synthesized according to the synthesis route in figure 1a . Subsequently, aliquots from the same solution containing freshly synthesized aunsts were branched off for further coating of the aunsts, either for coating with a silver shell for plain silver - enhancement (auagnsts), for silver - enhancement and subsequent coating with a thin silica shell (auagnsts@sio2), or for coating with a thin silica shell only (aunsts@sio2). To avoid aggregation and to improve the stability of the nanostars without silica shell dispersed in ethanol, the ctab was replaced with thiolated mpeg (mpeg - sh) in case of the aunsts and auagnsts via a ligand exchange reaction . Sem images of a representative (b) aunst and of representative (c) auagnsts . The scale bars are 100 nm . Insets: schematics of the corresponding nanostars illustrating the layered composition . For the synthesis of the gold nanostars (aunsts) sodium citrate - functionalized gold nanoparticles of 35 nm in diameter were used as seeds for the growth of the aunsts . The gold seed particles were prepared by adding 0.6 ml of ice cold 0.1 m nabh4 to a 20 ml aqueous solution containing 0.25 mm haucl4 and 0.25 mm sodium citrate under vigorous stirring . For the growth of aunsts, 100 l of the aged seed solution was added to 10 ml of an aqueous solution of 0.25 mm gold chloride (haucl4), hereafter called growth solution . The ph was adjusted by adding 10 l of 1 n hydrochloric acid (hcl). Subsequently, 100 l of 2 mm silver nitrate (agno3) and 50 l of 0.1 m ascorbic acid (aa) were simultaneously added to the growth solution under vigorous stirring . The nanostars were functionalized with hexadecyltrimethylammonium bromide (ctab); that is, 100 l of 0.1 m ctab was added to the growth solution, which was left stirring for 5 min . The solutions containing aunsts were washed by centrifugation (2000 rcf for 20 min), and redispersed in a 10 ml of 1 mm aqueous ctab solution . Such aunsts with long and sharp tips, pointing in arbitrary directions, exhibit multiple plasmon resonances, strong field enhancements located at their tips, as well as large scattering cross sections . For the silver - enhanced gold nanostars (auagnsts), we followed a method that was inspired by the method reported by fales et al . 5 l of 0.1 m agno3 and an equivalent volume of 0.1 m aa were added as a precursor and as a reducing agent, respectively, under vigorous stirring to 1 ml of ctab - functionalized aunsts in solution . After 15 min, the silver enhanced gold nanostars were purified by centrifugation at 2000 rcf for 10 min, discarding the supernatant, and redispersed in 1 ml of distilled water . . The auagnsts still exhibit sharp tips, but the branch length of the previously long gold nanostar tips protruding from the core of the nanostars is reduced while the core diameter is increased due to the silver - enhancement . The exact silver shell thickness of the auagnsts can hardly be measured precisely with a tem, due to the limited material contrast . However, we can distinguish between the gold nanostar and the silver shell in high angle annular dark field tem images, so that we estimate the silver shell thickness to be in the range of 20 nm around the core of the gold nanostar (an example is shown in figure s3). Further, from elemental line and mapping analysis of representative auagnsts using energy dispersive x - ray spectroscopy (edx), we assume that the apexes of the tips are also covered by a very thin (<8 nm) silver shell (figure s3). The presented synthesis methods for the aunsts and auagnsts allow a high reproducibility with negligible batch - to - batch variations in the optical ensemble spectra (figure s4). To obtain a homogeneous and thin (<10 nm) silica shell that follows the morphology of the nanostars, the original recipe, which provides substantially thicker shells, had to be modified . The freshly synthesized and washed aunsts and auagnsts were functionalized with thiolated mpeg (mpeg - sh) via a ligand exchange reaction . Mpeg - sh was mixed with 1 ml of aqueous solution containing nanostars to a final concentration of 5 m under gentle stirring for 1 h. the pegylated nanostars were washed once more by centrifugation (2500 rcf for 10 min) to remove the residual ligands and were redispersed in a mixed solution of ethanol (0.9 ml) and water (0.2 ml). Under gentle stirring, silica (teos) coating of the nanostars was initiated by adding 10 l of nh4oh followed by 3 l of 10% teos in ethanol . The reaction was allowed to proceed for 12 h. after that, the nanostars were washed three times by centrifugation at 3000 rcf for 5 min and redispersed in ethanol or chlorobenzene . In figure 1c and d, tem images of representative silica - coated nanostars, aunst@sio2 and auagnst@sio2, are shown (see figures s5 and s6 for more examples of aunst@sio2 and auagnst@sio2, respectively). The average silica shell thickness was about 10 nm based on tem image analysis of several nanoparticles (figure s7). In both cases, the shell was uniformly and completely formed around the nanostars, maintaining their morphology . As a major benefit, this thin silica shell allows mixing of both nanostar types, aunsts@sio2 and auagnsts@sio2, with polar (ethanol) as well as nonpolar organic solvents (chlorobenzene) without causing aggregation . In addition, the silica shell of around 10 nm is thick enough to prevent chemical degradation such as oxidation of silver in the case of auagnsts@sio2 . It is worth rementioning that all of the displayed nanostars stem from the same original batch of gold nanostars . For a period of 10 h without stirring, figure s8 shows the corresponding extinction spectra of auagnsts with and without silica shell dispersed in ethanol recorded every hour for 10 h. all nanostars have similar tip - to - tip diameters of about 100150 nm (the corresponding histograms can be found in figure s9). For reference, citrate - functionalized spherical nanoparticles (aunps) with diameters of about 120 nm were also coated with silica using the same procedure as for the nanostars (examples of sem and tem images of aunps and aunps@sio2 are displayed in figure s10). Extinction spectra were measured by a varian cary 500 scan uv vis nir spectrophotometer . For the characterization of the morphology, solutions containing aunsts and auagnsts were drop casted on cleaned ito substrates, dried, and analyzed with a zeiss smart sem supra 55 vp scanning electron microscope (sem). For the transmission electron microscope (tem) characterization, solutions containing silica - coated auagnsts and aunsts were drop casted on carbon - coated copper tem grids, dried, and analyzed using a jeol jem-2010 tem . Scanning tem (stem) was performed with bright field (bf) and high angle annular dark field (haadf) detectors . Elemental line and mapping analyses were carried out using energy dispersive x - ray spectroscopy (edx). The samples were investigated with a jeol jem-2200fs transmission electron microscope in stem mode operated at 200 kv, equipped with an oxford sdd x - maxn (80 mm) edx - system . For preparation of the randomly lasing systems, rhodamine 6 g (r6 g) and ads200re (a meh - ppv derivate) were used without further treatment . R6 g (2 mg ml) or meh - ppv (7 mg ml) dissolved in ethanol or chlorobenzene, respectively, were used as stock solutions . In the following, we refer to the r6 g dissolved in ethanol as to the r6 g solution and to the ads200re dissolved in chlorobenzene as to the meh - ppv solution . The nanoparticle concentrations were controlled by gentle centrifugation and redispersion in ethanol or chlorobenzene . Aunsts and aunsts@sio2 in ethanol exhibit the same optical density (od) at 405 nm (d - band absorption of gold). The auagnsts and auagnsts@sio2 were directly synthesized out of a part of the aunsts, so that the concentrations are considered to be similar . Because all four types of the nanostars can be dispersed in ethanol without aggregation, 0.3 ml of each type of the nanostars dispersed in ethanol was mixed with 1 ml of the r6 g solution . As a reference without any nanoparticles, 0.3 ml of ethanol was mixed with 1 ml of r6 g solution . In the case of the meh - ppv solutions, either 0.3 ml of aunsts@sio2, or 0.3 ml of auagnsts@sio2 or silica - coated spherical nanoparticles (aunps@sio2) dispersed in chlorobenzene the concentration of aunp@sio2 dispersion was adjusted in such a way that its od at 405 nm (d - band absorption of gold) was the same as the od of the aunst@sio2 dispersion (figure s11c). As reference mixture of meh - ppv without any nanoparticles, 0.3 ml of chlorobenzene was mixed with 1 ml of meh - ppv solution . The final mixtures contain similar concentrations of nanostars / nanoparticles and either r6 g (1.5 mg ml) or meh - ppv (5.4 mg ml). For more clarity, we denote each suspension as nanostar type / gain material, for example, aunst / r6 g . For random lasing experiments, quartz cuvettes with four transparent windows (inner dimensions 10 mm 4 mm) containing 0.5 ml of the solutions we used a setup, which basically resembles the variable stripe length method for cuvettes, that is, a stripe - like excitation illumination of the cuvette through the 10 mm window . The detection through the 4 mm side window was almost perpendicular to the excitation . The cuvette was slightly tilted with respect to the direction of the excitation stripe . Thus, any signals stemming from multiple reflections are not fed into the detector . Figure 2 shows the excitation and detection scheme for the random lasing experiments . A diode - pumped, passively q - switched solid state laser (crylas gmbh, pulse length shorter than 1.3 ns, maximal pulse energy 20 j) was used to excite the suspensions at a wavelength of 532 nm . The excitation beam was shaped into a thin stripe by two crossed cylindrical lenses and focused in the plane of two razor blades . To establish a 3 mm long, 55 m wide excitation stripe on the suspensions in the cuvette, the emission from the pumped excitation volume was coupled out of the side window and focused onto the entrance slit of the spectrometer (newport ms260i), which was equipped with a peltier - cooled charge - coupled device camera (andor ivac). A 532 nm notch filter was used to suppress detection of scattered excitation light . To quantitatively compare the random lasing from the different gain - nanostar solutions, all measurements were performed under exactly the same experimental conditions . In all random lasing experiments, the size of the excitation stripe, the position of the cuvette in the setup, the position of the excitation stripe on the cuvette s window, and the integration time (1 s) for the detection were fixed . (a) illustration of the excitation stripe and the detection of the emission . (b) the nanostars can randomly form resonant cavities in the gain medium and hence the necessary feedback mechanism for random lasing . Figure 3a displays the absorption (abs) and photoluminescence (pl) spectra of r6 g dispersed in ethanol . The dashed orange curve in figure 3b zooms into the pl spectrum of r6 g from 545 to 580 nm, taken at a pumping fluence of 0.24 mj cm . For this fluence, the pl intensity was magnified 30 times to be visible on the scale of the ordinate in figure 3b . For fluences above 0.30 mj cm, the emission spectrum of the reference r6 g solution shows amplified spontaneous emission (ase) with a substantially reduced spectral bandwidth as compared to pl (see figure s12). The curves shaded from black to red in figure 3b show the ase spectra of the reference r6 g solution without nanostars excited by single pulses at different excitations fluences, ranging from 0.33 to 0.66 mj cm, increasing with equal steps of 0.03 mj cm . (a) normalized absorption (green) and photoluminescence (pl) (orange) spectra of r6 g; (b) ase spectra of the reference r6 g solution without nanostars, excited by single pulses at different excitation fluences from 0.33 to 0.66 mj cm in steps of 0.03 mj cm . The intensity of the pl spectra of r6 g at a pumping fluence of 0.24 mj cm (dashed orange curve) was magnified 30 times for comparison . (c, e, g, i) spectral overlap of the r6 g absorption and emission spectra (dotted curves) with the extinction spectra of respective nanostars in ethanol (solid lines) and (d, f, h, j) the emission (random lasing or ase) spectra at different excitations fluences for nanostars / r6 g mixtures . (c, d) aunsts, (e, f) aunsts@sio2, (g, h) auagnsts, and (i, j) auagnsts@sio2, respectively . Aunsts dispersed in ethanol exhibit a spectrally broad extinction spectrum with a maximum around 630 nm, due to the multiple plasmon resonances of each individual nanostar (figure 3c, blue curve). The plasmon resonances of the aunsts spectrally overlap partially with the pl emission of r6 g (dashed orange curve in figure 3c), but they hardly overlap with the r6 g absorption . At lower pumping fluences, ase is observed from the aunst / r6 g sample (figure 3d), similar to the ase of the r6 g reference (figure 3b). For higher fluences, sharper features are emerging out of the ase background . This behavior is typical for random lasers: ordinary pl is observed for low pumping fluences, ase is observed for a fairly broad range of pumping fluences, followed by coherent laser peaks for even higher pumping fluences . Upon silica - coating, the plasmon resonances of the aunsts red - shift by about 20 nm due to the introduction of a higher refractive index medium around the aunsts as compared to the solvent (see figure 3e). To underline our findings, we performed three - dimensional finite difference time domain (fdtd) calculations on the optical properties of the hybrid multilayered nanostars . The calculated plasmon resonance of a single aunst with silica shell is red - shifted as compared to the plasmon resonance of the same single aunst without the silica shell (figure s14). For a detailed description of the numerical calculation on the plasmonic properties of hybrid multilayered plasmonic nanostars, refer to the corresponding section in the supporting information . Surprisingly, the aunst@sio2/r6 g sample exhibits only pronounced ase even at the highest pumping fluence (figure 3f). The silica shell affects the gold nanostar - based random lasers in several ways . First, the local field enhancements at the surface of the aunsts@sio2 are slightly reduced by the silica coating . The maximal field intensity is localized close to the gold tip in the silica shell (see figure s15), so that outside the silica shell the efficient excitation of the nearby gain material might also be reduced . Second, besides the spectral red - shift, the silica shell induces a spectral broadening of the plasmon resonances, typically for chemical damping of the plasmon resonances, leading to a small reduction in the spectral overlap between the pl of r6 g and the plasmon resonances of the aunsts@sio2 . Third, the silica shell acts as a spacer between the gain material and the gold reducing quenching . While the latter is beneficial for random lasing, the small reduction in the spectral overlap between aunsts@sio2 scattering and the pl of r6 g as well as slight reduction of the field enhancements the silver - enhancement of the aunsts leads to a blue - shift of the plasmon resonances from around 630 nm (aunsts, figure 3b) down to 530 nm (auagnsts, figure 3 g). Hence, the extinction spectra of the auagnsts in ethanol spectrally overlap with both the absorption and the pl of the r6 g . The blue - shift of the plasmon resonances can be attributed to a mixed gold silver dielectric function, as well as to the reduced length of the nanostars tips protruding from the core and to an increased diameter of the core induced by the silver - enhancement (see figures s14 and s16). Additionally, due to the high scattering efficiency of silver, the auagnsts in ethanol exhibit 4 times higher extinction as compared to the aunsts (note the different scales of the ordinate in figure 3b and g), although the concentrations of the nanostars were similar in both dispersions . Spectrally very sharp and intense random lasing peaks, characteristic for coherent random lasing, are observed for the auagnst / r6 g sample . Surprisingly, no pronounced ase background is detected even at low pumping fluences (figure 3h and figure s13), in contrast to the previously mentioned samples . The investigation of the underlying physical mechanisms for this experimental observation is beyond the scope of this article . The maximally observed random lasing intensities are more than 3-fold higher than the random lasing intensities detected from the aunst / r6 g . Silica coating of the auagnsts leads to a spectral red - shift (20 nm) of the plasmon resonances, so that the extinction spectra of the auagnsts@sio2 in ethanol perfectly overlap with the absorption and the pl of the r6 g (figure 3i). No sign of a possible detrimental effect of the silica coating on the random lasing characteristics was observed in the case of the auagnst@sio2/r6 g sample (figure 3j), in contrast to the aunst@sio2/r6 g case (figure 3f). Moreover, the random lasing intensities are comparable for both samples . As for the auagnst / r6 g sample, coherent random lasing is observed also for the auagnst@sio2/r6 g sample even at low pumping fluences (figure 3j). For the sake of better visualization, the random lasing spectra of the auagnst / r6 g and auagnst@sio2/r6 g from figure 3h and j are redisplayed in figure 4a and b, respectively . For both samples, the measured spectral width of the coherent random lasing peaks is as small as 0.3 nm, corresponding to the resolution of the spectrometer . Hence, the values represent an upper limit, while the actual line widths of the lasing modes might be well below . The spectral position, number, and relative intensities of the random lasing peaks vary from pulse to pulse . The closed loop resonator cavities of the random laser, formed by the nanostars, change from shot to shot, due to brownian motion . There are several effects that can contribute to the fact that the auagnsts as well as the auagnsts@sio2 could facilitate the buildup of coherent lasing modes . First, the plasmon resonances of the auagnsts and auagnsts@sio2 spectrally overlap with both the absorption and the emission of r6 g in ethanol, in contrast to aunsts . Second, the stronger hot spots at the tips of the auagnsts or auagnsts@sio2 as compared to hot spots at the tips of the aunsts (see figure s15) might provide a more efficient pumping of the gain material, promoting inversion . Third, the auagnsts could provide coherent feedback for random lasing at 560 nm, due to their high scattering cross sections in the spectral region coinciding to the spectral position of the main pl maximum of r6 g (560 nm) (see figure s14). Random lasing spectra of the (a) auagnst / r6 g and (b) auagnst@sio2/r6 g samples excited by single pulses at different excitation fluences . Next, we show that silica - overcoated nanostars cannot only be used as scatterers for random lasing in the polar solvent ethanol, but also in the nonpolar solvent chlorobenzene . The normalized absorption and pl spectra of meh - ppv dissolved in chlorobenzene are displayed in figure 5a . Figure 5b shows the ase spectra of the reference meh - ppv solution without nanostars excited by single pulses at different excitation fluences, ranging from 0.18 to 0.39 mj cm increasing with equal steps of 0.03 mj cm . In the case of the meh - ppv reference sample and for small excitations fluences (<0.18 mj cm) the dashed orange curve in figure 5b shows the pl spectra of meh - ppv at a pumping fluence of 0.12 mj cm . The pl intensity was magnified 45 times for comparison with ase . For excitation fluences above 0.18 mj cm, the meh - ppv reference sample shows ase (see figure s17). (a) normalized absorption (blue) and photoluminescence (pl) (orange) spectra of the meh - ppv in chlorobenzene solution; (b) ase spectra of the reference meh - ppv solution without nanostars, excited by single pulses at different excitation fluences . The intensity of the pl spectra of meh - ppv at a pumping fluence of 0.12 mj cm (dashed orange curve) was magnified 45 times for comparison . (c h) spectral overlap of the meh - ppv absorption and emission spectra (dotted curves) with the extinction spectra of respective silica - coated nanoparticles in chlorobenzene (solid lines) and the emission (ase or random lasing) spectra at different excitations fluences for nanoparticles / meh - ppv mixtures: (c, d) aunps@sio2, (e, f) aunsts@sio2, (g, h) auagnsts@sio2, respectively . The inset in (d) shows the tem image of a representative aunp@sio2 nanoparticle . The scale bar is 100 nm . As a further reference, spherical nanoparticles with dimension similar to that of the nanostars (125 nm in diameter) and coated with a 10 nm silica shell (aunps@sio2) the plasmon resonances of the aunps@sio2 in chlorobenzene spectrally overlap with the pl spectra of the meh - ppv (figure 5c). The inset in figure 5d displays the tem image of a representative aunp@sio2 nanoparticle . As shown in figure 5d, coherent random lasing arising from the ase background could be observed for the aunp@sio2/meh - ppv sample only for the highest fluences . The random lasing intensity for a pumping fluence of 0.39 mj cm is increased by a factor of 3 with respect to ase intensities detected for the other pumping fluences, which are comparable to the ase intensities observed for the reference meh - ppv sample (figure 5d). The extinction spectrum of the aunsts@sio2 dispersed in chlorobenzene (black curve) is shown in figure 5e . The aunst@sio2/meh - ppv sample exhibits prominent ase (figure 5f), as was previously observed also for the aunst@sio2/r6 g sample (figure 3f). For fluences above 0.30 mj cm, coherent random lasing peaks can be observed on top of the pronounced ase background . It is worth mentioning that, although the plasmon resonances of the aunsts@sio2 dispersed in chlorobenzene spectrally overlap with the second pl emission maximum of meh - ppv (at 630 nm), no ase or random lasing was observed from the aunst@sio2/meh - ppv sample in this spectral region . The plasmon resonances of the auagnsts@sio2 in chlorobenzene spectrally overlap with both the absorption and the pl spectra of meh - ppv . For the auagnst@sio2/meh - ppv sample, coherent random lasing was observed starting with pumping fluences above 0.24 mj cm (figure 5h). In figure s18, the random lasing spectra of the auagnst@sio2/meh - ppv sample excited by single pulses at different excitations fluences are displayed in a fashion similar to those in figure 4 . For a more quantitative comparison of the different samples, we analyze the maximal emission intensity, that is, the maximal intensity of the ase or coherent random lasing in figure 3 and figure 5, as a function of the applied pumping fluence for the samples containing r6 g (figure 6a) or meh - ppv (figure 6b) as a gain material, respectively . (a) maximal emission intensities of the ase or random lasing spectra (figure 3) as a function of the corresponding pumping fluence for r6 g reference sample without nanoparticles (orange), for aunst / r6 g (blue), aunst@sio2/r6 g (black), auagnst / r6 g (purple), and auagnst@sio2/r6 g samples (red). (b) maximum emission intensities of the ase or random lasing spectra (figure 5) as a function of the corresponding pumping fluence for meh - ppv reference solution (orange), for aunp@sio2/meh - ppv (dark cyan), aunst@sio2/meh - ppv (black), and auagnst@sio2/meh - ppv (red) samples, respectively . For the r6 g reference sample without nanoparticles, the ase intensity increases with increasing pumping fluences (the orange curve in figure 6a). As expected from figure 3b, there is no hint for a coherent random lasing threshold . The blue curve in figure 6a refers to the spectra of aunst / r6 g sample in figure 3d . One is able to identify two distinctively different slopes of the blue curve . For low fluences (<0.45 mj cm), the aunst / r6 g sample exhibits ase similar to that of the r6 g reference sample . At around 0.45 mj cm, the slope of the blue curve changes, indicating the coherent random lasing threshold of the aunst / r6 g sample . The black curve in figure 6a, depicting the maximal intensities of the spectra displayed in figure 3h for aunst@sio2/r6 g samples, shows a trend with increasing pumping fluences similar to that of the r6 g reference sample . As one can deduct from the steeper slope of the black curve as compared to the orange curve, the red and purple curves in figure 6a correspond to the maximal random lasing intensities from the auagnst / r6 g (red) and auagnst@sio2/r6 g (purple) samples, respectively . The threshold fluences for coherent lasing are at 0.39 mj cm (red curve) and at 0.42 mj cm (purple curve), respectively . Saturation of peak intensities is observed for high fluences . In the case of auagnst@sio2/meh - ppv, the threshold fluence for coherent lasing is at 0.24 mj cm, while the onset for coherent random lasing for the aunp@sio2/meh - ppv sample (dark cyan curve in figure 6b) is at much higher pumping fluences (0.36 mj cm). At a fluence of 0.39 mj cm, the coherent random lasing emission intensity from the auagnst@sio2/meh - ppv sample is 8.3 times higher than the emission intensities observed from the samples containing aunps@sio2 (dark cyan curve) or aunsts@sio2 (black curve). Moreover, the maximal detected random lasing intensity for the auagnst@sio2/meh - ppv sample is boosted by a factor of 25 as compared to maximal ase intensity from the reference meh - ppv sample (orange curve). Our observations can be explained by the unique properties of the auagnsts as compared to other particles . The auagnsts and the auagnsts@sio2 exhibit higher scattering cross sections than the aunps@sio2 and the aunsts with and without silica shell, due to the silver - enhancement . The higher scattering cross section might be one of the main reasons for the low pumping thresholds at which the solutions containing auagnsts or auagnsts@sio2 show coherent random lasing . Another reason could be the spectral overlap of the plasmon resonances with the absorption spectra of the gain material and the excitation wavelength . The hot - spots at the tips of the auagnsts could facilitate the creation of inversion by providing more efficient pumping of the gain material . Similar correlations between the lasing thresholds and the field enhancement were reported also by others . Finally, we turn our discussion to the comparison of pumping thresholds for the auagnst@sio2/r6 g and auagnst@sio2/meh - ppv - based random lasers . Because the concentrations of auagnsts@sio2 in the r6 g and meh - ppv solutions were the same, we analyze the lasing thresholds with respect to the extinction a = cd of the gain materials in the stock solutions . Is the extinction coefficient of r6 g (219 ml mg cm taken from ref (41)) or meh - ppv (106 ml mg cm from ref (42) assuming that of meh - ppv in chlorobenzene is in the same order of magnitude as in chloroform), c is the concentration of the gain materials, and d is the optical path of 4 mm . The extinction of the auagnst@sio2/r6 g solutions (c = 1.5 mg ml) is ar6 g 132, while the auagnst@sio2/meh - ppv solutions (5.4 mg ml) have an extinction ameh ppv 229 . For the auagnst@sio2/meh - ppv sample, coherent random lasing without pronounced ase background was observed starting with pumping fluences above 0.24 mj cm . Considering that ameh ppv 1.73 ar6 g, the lasing threshold for the auagnst@sio2/r6 g should be 1.73 higher than the lasing threshold for auagnst@sio2/meh - ppv . Indeed, the experimental threshold for coherent random lasing of auagnst@sio2/r6 g (0.42 mj cm) is 1.75 times higher than the threshold for auagnst@sio2/meh - ppv . In summary, we synthesized hybrid multilayered plasmonic nanostars, comprised of gold, silver, and silica in various compositions . The silver - enhanced and silica - coated gold nanostars can be mixed with polar or nonpolar solutions containing gain material . We find that auagnsts and auagnsts@sio2 provide sufficient feedback for coherent random lasing . In single pulse experiments, we show that the silver - enhancement of the gold nanostars reduces the pumping threshold for nanostar - based coherent random lasers substantially in different gain media . We attribute this to the plasmon resonances of auagnsts and auagnsts@sio2, which spectrally overlap perfectly with both the absorption and pl emission of the gain materials as well as the excitation wavelength . First, hot - spots at the tips of the auagnsts and of auagnsts@sio2 can be resonantly excited by the pumping laser, and hence the gain material in the vicinity of the tips can be excited with a higher effective intensity . Therefore, the auagnsts and auagnsts@sio2 can facilitate inversion of the gain material more easily . Second, the auagnsts and the auagnsts@sio2 can provide the necessary feedback for coherent . Considering the perfect spectral overlap of the plasmon resonances with the pl of the gain material and the high scattering cross sections of the auagnsts and auagnsts@sio2, the emitted photons can be scattered efficiently . Further, we reveal that the lasing intensity and pumping threshold of a silver - enhanced gold nanostar - based random laser are basically not influenced by the silica coating, in contrast to the gold nanostar - based random laser, where for the silica - coated gold nanostars only mediate ase but no coherent random lasing was observed . The large scattering cross sections and easily accessible hot - spots provided by auagnsts and the simultaneous compatibility of the auagnsts@sio2 with polar and nonpolar solvents render hybrid multilayered plasmonic nanostars one of the most promising candidates for an effective photon - management in organic and inorganic optoelectronic devices as well as for biosensing and analytical applications.
My lab is fascinated by understanding how tissues renew themselves throughout an organism s lifetime . Specifically, we are interested in understanding how cells orchestrate tissue growth and make cell - fate choices that result in balanced tissue regeneration . Tissue regeneration is often looked at as a process happening in a vacuum: each individual cell is an actor in a play, enacting its role according to an unchanging script . Yet continuous insults, such as tissue tear and somatic mutations, among others, can create continuous variations to the script . Thus, since these variations may call for improvisation, cells must adapt their roles to keep a tissue (play) functional (entertaining). As i started my lab, i felt that the biggest challenge in understanding mammalian tissue regeneration was that the field largely used static analyses that prevent the ability to capture cells in action in the context of an intact organism in which variations occur . During my doctoral thesis, i experienced firsthand how live imaging provided me not only a better understanding of tissue patterning but also allowed us to discover new biology that we had not anticipated, whereby epithelial cells secrete vesicles (called argosomes) that carry morphogens and disperse them throughout the tissue (greco et al ., 2001). Thus, as i set up my lab, we studied tissue regeneration by investing in a high - risk / high - reward approach to establish skin stem cell imaging in live mice . After more than one year of troubleshooting and several discouraging roadblocks, we were finally able to visualize, track, and manipulate stem cells and their niches within the skin epithelium of an intact living mouse (rompolas et al ., 2012; pineda, park, et al ., 2015). These novel approaches have allowed us to get a fresh look at processes that have been investigated for decades, leading to the capture of novel principles of stem cell biology and tissue regeneration . In retrospect, what i had accomplished was combining my passion for visualizing biological processes in vivo with my knowledge of stem cells gained during my postdoc (greco, chen, et al ., 2009). The ability to directly observe a biological phenomenon is the reason why i fell in love with science, and i was able to bring this angle to bear on our research, which uniquely poised my lab to address previously inaccessible questions and distinguished my lab from the laboratories of my previous mentors (park et al ., 2016). With these tools, my lab has contributed to the understanding of fundamental principles of the equilibrium of cell choices reached during tissue regeneration and has explored the edges of this equilibrium, which we describe in more detail below . Questions that fueled our science include, what are the rules that sustain robust daily tissue regeneration? How does this equilibrium evolve when the normal tissue is in the presence of cancerous proliferative clones? Tissue regeneration is achieved through a balance of cell production (growth) and elimination (regression). Yet we still fail to understand how stem cells and their environment balance tissue growth and regression during regeneration in a live mammal . To address these questions, we used the mouse hair follicle, which cycles between these phases while maintaining a pool of stem cells to sustain tissue regeneration . By visualizing stem cell behavior and manipulating stem cells niche during growth, we have shown that 1) stem cell fate depends on the position (surrounding niche) that the cells inhabit, and 2) while the niche is required for tissue regeneration, dedicated stem cells are dispensable . Specifically, we showed that cells can switch their fate to adopt new stem cell functions in the face of loss of a specific stem cell pool . These discoveries have provided a new understanding of the role of stem cell location and how fundamentally important the native / local niche is with respect to stem cell decisions and overall tissue regeneration, which could not have been observed without our ability to track the same cells over time in a live mammal . Our results also reveal a robust mechanism of compensation in which cells from other epithelial compartments can adopt new stem cell functions and fuel tissue regeneration (rompolas et al ., 2012, 2013). We have shown that hair follicle epithelial stem cells are eliminated during regression through a spatial gradient of apoptosis along the same axis utilized for growth . Furthermore, we have demonstrated that hair follicle stem cells collectively act as phagocytes to clear dying epithelial neighbors . Through cellular and genetic ablation, we have shown that epithelial cell death is extrinsically regulated by the local niche through transforming growth factor (tgf)- activation . Strikingly, our data show that regression acts to reduce the stem cell pool, and the inhibition of the regression phase results in excess basal epithelial cells with regenerative abilities (mesa et al ., 2015). These findings are surprising, because the field previously thought of stem cells as having a finite lifetime / capacity of divisions that eventually leads to their elimination by exhaustion . Indeed, this work shifts the understanding to the niche environment, which, if altered, can lead to the aberrant coopting of the system toward deregulated growth . It also demonstrates that reinstalling a proper niche can correct stem cell driven aberrancy . This principle is key to tgf-driven cancer models and, importantly, also elevates the significance of the niche in the broader study of mechanisms of cancer initiation . Currently, we are addressing how these cellular interactions are regulated by surrounding niche populations such as mesenchymal cells and immune cells and structural elements such as the extracellular matrix . Together, these approaches will allow us to determine whether specific niche populations that are interspersed or adjacent to the epithelium serve as regional checkpoints to locally control the regeneration process . Additionally, this work has allowed us to branch out and begin to study how cells ensure rapid tissue repair after injury . We have been able to study how the interplay between repair behaviors, such as migration and proliferation, lead to effective reconstruction of the epithelial tissue and the extent to which homeostatic processes such as differentiation are affected during the repair process . We are particularly excited to study how these interconnected cellular behaviors contribute to the tissue - scale changes observed in the repair process . Yet we lack an understanding of how these individual cellular behaviors are regulated and how mutations may influence them . To answer these questions, we have demonstrated that stem cells do not appear to be intrinsically biased toward either self - renewal or differentiation but instead seem to be influenced by the behaviors of their neighboring sister cells . Additionally, as basal stem cells stochastically commit to differentiation, they reuse existing structural organizations (rompolas, mesa, et al ., 2016; xin et al ., we therefore began to interrogate the interface between normal tissue and cancerous clones by using the evolutionarily conserved pathway, wnt/-catenin . Our efforts uncovered a novel mechanism of action for -catenin that acts non cell autonomously within the hair follicle stem cells by recruiting wild - type cells to induce de novo hair growth that ultimately results in tumors . Additionally, we show that -catenin driven growth is triggered and expands independent of the local niche s influence (deschene, myung, et al ., 2014). This work changes our understanding of how cells that carry mutations can interact with neighboring cells, providing insights into how cancer initiation and progression may be fueled . To understand how to counterbalance cancerous growth, we took advantage of two contrasting mouse cancer models: a unique, benign skin tumor that regresses spontaneously, keratoacanthoma; and a malignant skin tumor, squamous cell carcinoma (scc). We demonstrated that self - regressing keratoacanthoma tumors counterbalance excessive proliferation by employing a homeostatic mechanism of terminal differentiation to regress . When this differentiation cue, retinoic acid, is used on scc it could also induce the regression of these malignant tumors (zito et al ., 2014). Taken together, this body of work modifies prevailing views in the field regarding how cells that carry mutations can interact with neighboring cells, expanding our understanding of how tumor progression and regression is regulated . Currently, we are interested in mutations associated with scc, such as clones bearing hras mutations in combination with a loss of tgf function . Thus, we are using live imaging to study the dynamic behaviors and interactions between mutant clones (double and single mutants) and wild - type neighboring tissues . Functional investigation of both oncogenic signaling pathways and different cellular interactions will help us elucidate the critical set of decisions that lead to cancer . Tissue regeneration is achieved through a balance of cell production (growth) and elimination (regression). Yet we still fail to understand how stem cells and their environment balance tissue growth and regression during regeneration in a live mammal . To address these questions, we used the mouse hair follicle, which cycles between these phases while maintaining a pool of stem cells to sustain tissue regeneration . By visualizing stem cell behavior and manipulating stem cells niche during growth, we have shown that 1) stem cell fate depends on the position (surrounding niche) that the cells inhabit, and 2) while the niche is required for tissue regeneration, dedicated stem cells are dispensable . Specifically, we showed that cells can switch their fate to adopt new stem cell functions in the face of loss of a specific stem cell pool . These discoveries have provided a new understanding of the role of stem cell location and how fundamentally important the native / local niche is with respect to stem cell decisions and overall tissue regeneration, which could not have been observed without our ability to track the same cells over time in a live mammal . Our results also reveal a robust mechanism of compensation in which cells from other epithelial compartments can adopt new stem cell functions and fuel tissue regeneration (rompolas et al ., 2012, 2013). We have shown that hair follicle epithelial stem cells are eliminated during regression through a spatial gradient of apoptosis along the same axis utilized for growth . Furthermore, we have demonstrated that hair follicle stem cells collectively act as phagocytes to clear dying epithelial neighbors . Through cellular and genetic ablation, we have shown that epithelial cell death is extrinsically regulated by the local niche through transforming growth factor (tgf)- activation . Strikingly, our data show that regression acts to reduce the stem cell pool, and the inhibition of the regression phase results in excess basal epithelial cells with regenerative abilities (mesa et al ., 2015). These findings are surprising, because the field previously thought of stem cells as having a finite lifetime / capacity of divisions that eventually leads to their elimination by exhaustion . Indeed, this work shifts the understanding to the niche environment, which, if altered, can lead to the aberrant coopting of the system toward deregulated growth . It also demonstrates that reinstalling a proper niche can correct stem cell driven aberrancy . This principle is key to tgf-driven cancer models and, importantly, also elevates the significance of the niche in the broader study of mechanisms of cancer initiation . Currently, we are addressing how these cellular interactions are regulated by surrounding niche populations such as mesenchymal cells and immune cells and structural elements such as the extracellular matrix . Together, these approaches will allow us to determine whether specific niche populations that are interspersed or adjacent to the epithelium serve as regional checkpoints to locally control the regeneration process . Additionally, this work has allowed us to branch out and begin to study how cells ensure rapid tissue repair after injury . We have been able to study how the interplay between repair behaviors, such as migration and proliferation, lead to effective reconstruction of the epithelial tissue and the extent to which homeostatic processes such as differentiation are affected during the repair process . We are particularly excited to study how these interconnected cellular behaviors contribute to the tissue - scale changes observed in the repair process . Yet we lack an understanding of how these individual cellular behaviors are regulated and how mutations may influence them . To answer these questions, we have demonstrated that stem cells do not appear to be intrinsically biased toward either self - renewal or differentiation but instead seem to be influenced by the behaviors of their neighboring sister cells . Additionally, as basal stem cells stochastically commit to differentiation, they reuse existing structural organizations (rompolas, mesa, et al ., 2016; xin et al ., we therefore began to interrogate the interface between normal tissue and cancerous clones by using the evolutionarily conserved pathway, wnt/-catenin . Our efforts uncovered a novel mechanism of action for -catenin that acts non cell autonomously within the hair follicle stem cells by recruiting wild - type cells to induce de novo hair growth that ultimately results in tumors . Additionally, we show that -catenin driven growth is triggered and expands independent of the local niche s influence (deschene, myung, et al ., 2014). This work changes our understanding of how cells that carry mutations can interact with neighboring cells, providing insights into how cancer initiation and progression may be fueled . To understand how to counterbalance cancerous growth, we took advantage of two contrasting mouse cancer models: a unique, benign skin tumor that regresses spontaneously, keratoacanthoma; and a malignant skin tumor, squamous cell carcinoma (scc). We demonstrated that self - regressing keratoacanthoma tumors counterbalance excessive proliferation by employing a homeostatic mechanism of terminal differentiation to regress . When this differentiation cue, retinoic acid, is used on scc it could also induce the regression of these malignant tumors (zito et al ., this body of work modifies prevailing views in the field regarding how cells that carry mutations can interact with neighboring cells, expanding our understanding of how tumor progression and regression is regulated . Currently, we are interested in mutations associated with scc, such as clones bearing hras mutations in combination with a loss of tgf function . Thus, we are using live imaging to study the dynamic behaviors and interactions between mutant clones (double and single mutants) and wild - type neighboring tissues . Functional investigation of both oncogenic signaling pathways and different cellular interactions will help us elucidate the critical set of decisions that lead to cancer . During my journey, i have often reflected on the features of a successful leader and will explain below the thought process that brought me to evolve a model of leadership that is based on the complementary strengths and weaknesses of all who work in the lab . The search for leadership features revolved around the principal investigator (pi) as the leader . While the pi is fundamental in the initial phases of a lab the founder who needs to begin the lab with a great idea as the group expands in size over time, the lab s effectiveness increasingly depends the vision of all of its constituents, not just the pi . Yet models of leadership i have been exposed to, from grant preparation to promotion to credit in papers, seemed to single out one element in this collective project, the pi, but did nt take into account that the members of the group are also a critical part of the leadership equation (berg, 1998). As a new pi, i faced a number of challenges implementing a model of leadership in my lab . One such challenge was that i did nt recognize in myself what society seems to value as innate successful features of a leader, in that i felt at a disadvantage being a woman and an immigrant . Historically, these characteristics are (consciously or unconsciously) not often associated with people in positions of leadership in the united states . But hard work as well as support from my lab, my peers, and a handful of senior colleagues has made scientific and personal success possible and rewarding . Over the years, in speaking with others in positions of leadership, i have found that, encouragingly, several models were available as alternatives to the trait theory of leadership . These alternatives point to a view of leadership as not something we are born with but rather a diverse set of behaviors and acquired skills . I was then faced with identifying what successful behaviors a person in a leadership role should adopt . While our society seems drawn to behaviors such as self - confidence, assertiveness, and strength, these struck me also as individual behaviors not necessarily connected to the groups with which we work . In fact, leadership is about the effective work of a collective group, and those behaviors seem to have little to do with the group itself . I finally came to realize that, in my view, the most successful behaviors that people in positions of leadership would need to adopt would evolve around the demands of the group . Thus, in my view, this requires people in positions of leadership to acquire at least two essential behaviors: first, knowing one s own weaknesses, and second, effectively adapting to the group by listening to feedback and developing effective solutions to the problems raised . I believe that our society promotes a stereotype that people in positions of leadership are infallible and superior to most of the people around them (is nt that how they got to the top?). In my opinion, this myth reinforces the distance between the pi and the lab members, therefore diminishing the effectiveness of the group s productivity . However, i have also found that recognizing one s limitations is not always simple . While we may be able to get as far as diagnosing our limitations, i felt resistance in myself and in my colleagues when it came to admitting to those limitations and reaching out for help from the group . One hypothesis may be that we fear our disclosure of weakness or limitation will result in our lab members losing trust in us . After growing comfortable with admitting my limitations and feeling vulnerable in front of my lab, i now work with my group and the individual lab members to compensate for my weaknesses (these range from being a procrastinator on specific tasks that do nt come naturally to me to merely admitting that, as a single individual, i have a biased and narrower view on any subject under discussion, despite many years of education and the title of associate professor). As i share my weaknesses, i also engage my lab members in compensating for them (and complementing my strengths) by working together on tasks and creating a better outcome on all fronts . Thus, my weaknesses turn into a positive tool that empowers my lab members and allows them to grow better and faster, preparing them for future roles in positions of leadership while building a more cohesive group in which everyone is valued . Greco lab annual retreat august 2016 . From left to right, top row: kai mesa, katie cockburn, jonathan boucher, cristiana pineda, david gonzalez, tianchi xin, eduard marsh, and samara brown . I would not be where i am today were it not for the insightful and invaluable contributions of each person in my lab (figure 1). I have always felt that we use more reductionist approaches in viewing people, ranking the first and seeing the rest as less valuable . I have found that it takes courage, time, but most importantly, genuine belief to view everyone we have hired within an organization, some of whom fall below in a formal hierarchical structure, as significant contributors . I have come to realize that this belief in the meaningful contribution of all members of the lab is a fundamental component of a successful group . Therefore, i work hard to maintain a culture in my lab in which feedback from lab members is not only encouraged but listened to carefully and very often leads to changes that deeply affect the course of our lab decisions . While it has not always been easy and requires a significant investment of time and a willingness to accept that the pi s ideas are not always welcomed with open arms by the lab members, it has created an evolving entity that empowers each individual within our small organization . I believe an obstacle to establishing this model comes from the fear we carry of not feeling adequate . I believe this feeling is shared within any profession in which present and future performance is what determines our own and our peers appreciation independent of previous accomplishments . This thought was posed to me with the assumption that such a lab member is a potential threat . I started to think that people feel a need to establish a hierarchical scale to rank people from best to worst (similar to the ranking of the traits i was discussing earlier). In a separate conversation with a colleague, i was told that i needed, once in a while, to establish the superiority of the pi over the lab members . My hypothesis is that both conversations were stemming from this fear we carry of becoming dispensable, which in turn may trigger a dominant, repressing behavior in us . Regardless, these behaviors that our group will silently watch and learn from will inhibit a transparent critical dialogue, which in turn limits the power of the lab for discovery . If we instead foster a system in which 1) each individual is valued for his or her strengths and weaknesses (one person s weaknesses leave room for the contributions of others) and 2) the pi is committed to soliciting and responding to lab members feedback on important lab decisions, i believe we create the opportunity for significant scientific achievement . I believe that these reflections on leadership could be applied to any group setting and therefore to any person working in a position of leadership . Thus, our scientific community at large could profit from a similar model, wherein more engagement of our members, seeing them as accountable and driving forces of the group itself, may accelerate both individual and collective growth . I believe that our society promotes a stereotype that people in positions of leadership are infallible and superior to most of the people around them (is nt that how they got to the top?). In my opinion, this myth reinforces the distance between the pi and the lab members, therefore diminishing the effectiveness of the group s productivity . However, i have also found that recognizing one s limitations is not always simple . While we may be able to get as far as diagnosing our limitations, i felt resistance in myself and in my colleagues when it came to admitting to those limitations and reaching out for help from the group . One hypothesis may be that we fear our disclosure of weakness or limitation will result in our lab members losing trust in us . After growing comfortable with admitting my limitations and feeling vulnerable in front of my lab, i now work with my group and the individual lab members to compensate for my weaknesses (these range from being a procrastinator on specific tasks that do nt come naturally to me to merely admitting that, as a single individual, i have a biased and narrower view on any subject under discussion, despite many years of education and the title of associate professor). As i share my weaknesses, i also engage my lab members in compensating for them (and complementing my strengths) by working together on tasks and creating a better outcome on all fronts . Thus, my weaknesses turn into a positive tool that empowers my lab members and allows them to grow better and faster, preparing them for future roles in positions of leadership while building a more cohesive group in which everyone is valued . Greco lab annual retreat august 2016 . From left to right, top row: kai mesa, katie cockburn, jonathan boucher, cristiana pineda, david gonzalez, tianchi xin, eduard marsh, and samara brown . I would not be where i am today were it not for the insightful and invaluable contributions of each person in my lab (figure 1). Paradoxically i have always felt that we use more reductionist approaches in viewing people, ranking the first and seeing the rest as less valuable . I have found that it takes courage, time, but most importantly, genuine belief to view everyone we have hired within an organization, some of whom fall below in a formal hierarchical structure, as significant contributors . I have come to realize that this belief in the meaningful contribution of all members of the lab is a fundamental component of a successful group . Therefore, i work hard to maintain a culture in my lab in which feedback from lab members is not only encouraged but listened to carefully and very often leads to changes that deeply affect the course of our lab decisions . While it has not always been easy and requires a significant investment of time and a willingness to accept that the pi s ideas are not always welcomed with open arms by the lab members, it has created an evolving entity that empowers each individual within our small organization . I believe an obstacle to establishing this model comes from the fear we carry of not feeling adequate . I believe this feeling is shared within any profession in which present and future performance is what determines our own and our peers appreciation independent of previous accomplishments . This thought was posed to me with the assumption that such a lab member is a potential threat . I started to think that people feel a need to establish a hierarchical scale to rank people from best to worst (similar to the ranking of the traits i was discussing earlier). In a separate conversation with a colleague, i was told that i needed, once in a while, to establish the superiority of the pi over the lab members . My hypothesis is that both conversations were stemming from this fear we carry of becoming dispensable, which in turn may trigger a dominant, repressing behavior in us . Regardless, these behaviors that our group will silently watch and learn from will inhibit a transparent critical dialogue, which in turn limits the power of the lab for discovery . If we instead foster a system in which 1) each individual is valued for his or her strengths and weaknesses (one person s weaknesses leave room for the contributions of others) and 2) the pi is committed to soliciting and responding to lab members feedback on important lab decisions, i believe we create the opportunity for significant scientific achievement . I believe that these reflections on leadership could be applied to any group setting and therefore to any person working in a position of leadership . Thus, our scientific community at large could profit from a similar model, wherein more engagement of our members, seeing them as accountable and driving forces of the group itself, may accelerate both individual and collective growth.
Obstructive sleep apnea syndrome (osas), which is caused by a complete or partial obstruction of the upper airway producing apnea or hypopnea, is a sleep disorder characterized by repetitive cessation of breathing occurring during sleep . According to the american academy of sleep medicine, a diagnosis of osas can be made in the presence of five or more episodes of apnea / hypopnea per hour of sleep, accompanied by clinical symptoms (such as daytime sleepiness, mood disorder, insomnia, hypertension), or at least 15 events per hour without additional symptoms . Osas is defined as mild when characterized by 5 - 15 events per hour, moderate in the presence of up to 30 events per hour, and severe when these number more than 30 . The pathogenesis of osas is multifactorial, however two main categories are described in the literature that, in some cases, can occur in combination . The first, and more common, form is due to obstruction of the respiratory lumen, for example at the level of the retropalatal and retroglossal spaces, involving the soft palate and the base of the tongue . This obstruction, which can be partial or total, is linked to tissue factors such as tonsillar hypertrophy, but also to obesity, which can determine fatty infiltration of the pharyngeal wall . The second, less frequent form is central osas, which is due to dysfunction of the cerebral mechanisms that control the pharyngeal muscles that keep the airways open during sleep . In this case, suppression of nerve impulses results in a narrower airspace that is more prone to collapsing on inspiration during sleep . Certain skeletal abnormalities, such as reduced sagittal linear dimensions of the cranial base, of the maxilla, and also of the bony naso - oropharynx, can also predispose to osas . Retrognathia of both the maxilla and the mandible and increased lower face height frequently show a bold relationship with osas . In addition, in osas subjects the hyoid bone is located more inferiorly, between the fourth and the sixth cervical vertebra, and the intermaxillary space length (the distance between the posterior pharyngeal wall and the tip of the lower incisor) is reduced . These alterations decrease the tongue space, causing it to lie in a lower and a more posterior position, where it causes partial or total obstruction of the pharyngeal lumen during sleep . Due to repeated apnea / hypopnea episodes, osas patients have disturbed and less restorative sleep, and thus present several characteristic symptoms, such as daytime sleepiness associated with a higher risk of motor vehicle accidents, morning headache, mood disorder, depression and lack of libido . They also have an increased incidence of medical comorbidities (e.g. Hypertension, stroke, death). In the initial therapeutic approach, osas patients are encouraged to make lifestyle changes, for instance, to lose weight (in the case of obese patients), but also to stop using alcohol or other agents that can facilitate collapse of the upper airway . However, the main therapeutic options also include continuous positive airway pressure (c - pap) therapy, oral appliances and surgical procedures . C - pap therapy is based on the maintenance of constant air pressure, preventing the collapse of the upper airway during sleep, and it is recommended for subjects with an upper airway obstruction . It is currently the gold standard non - invasive treatment for osas, but it is not indicated in patients with nasal obstruction or claustrophobia . The cpap machine is quite voluminous and it must be used every night; this treatment has a high therapeutic efficacy but it is poorly tolerated and refused by 50% of patients . Other therapeutic strategies are the use of oral mandibular repositioning appliances, usually better tolerated by patients, but successful only in the treatment of mild - to - moderate cases of sleep apnea . These appliances cover the upper and lower teeth and hold the mandible in an advanced position, thus widening the upper airway . Surgical modifications of the soft tissue of the upper airway - e.g. Uvulopalatopharyngoplasty (uppp), partial midline glossectomy and radiofrequency volumetric tissue reduction - were, in the past, commonly used to increase the posterior airway space in severe osas . Orthognathic surgical procedures, on the other hand, consist of: anterior inferior mandibular sagittal osteotomy (genioglossal advancement), which, together with hyoid suspension, constitutes phase i surgery, genioplasty, and maxillomandibular advancement osteotomies . Maxillomandibular advancement (mma) is used for osas patients with dentofacial deformities requiring orthognathic surgery and/or for those with extremely severe obstructive disease, in whom all applicable conservative therapies have failed or proved intolerable . It consists of le fort i osteotomy of the maxilla and a bilateral sagittal split osteotomy of the mandible producing simultaneous advancement of both the maxilla and the mandible, and of the soft tissue overlying these structures . This procedure, by modifying the pharyngeal structures and the tongue space, increases the dimension of the upper airway and thus reduces the risk of pharyngeal collapse during negative pressure inspiration . This method is sometimes associated with anticlockwise rotation of the occlusal plane that advances the soft palate . Recently li reported a success rate of 89%, and prinsell, too, demonstrated that mma is a highly effective treatment with a success rate of between 86.5% and 86.6% . For this reason it can be considered the surgical treatment of choice that is able, especially when performed in young patients, to significantly improve the quality of life of individuals with osas . This study set out to evaluate the effects of mma on osas patients treated with orthognathic surgery; specifically, its aim was to establish which cephalometric variables can influence changes in respiratory symptoms . This retrospective study analyzed 15 patients (11 males and 4 females) from an initial pool of 27 patients (table 1, who had clinical and polysomnographic evidence of moderate (1 subject) or severe (14 subjects) osas and were submitted to orthognathic surgery as detailed in table 2 . The mean age of the 15 patients who underwent mma was 42.3 9.5 years (range from 26.3 to 62.5 years). On the basis of anb, nine patients were deemed to have presurgical skeletal class ii malocclusion (anb> 4), five patients class i malocclusion (anb = 2 2), and only one patient class iii (anb <0) malocclusion . All the patients had already undergone other non - invasive treatments that had been unsuccessful (oral appliances) or had been rejected in the long term (c - pap). All the subjects included in this study were adults referred from the department of maxillofacial surgery of the sant'anna hospital in como, italy . Each patient was evaluated with nocturnal polysomnoraphy (psg) and lateral cephalometric radiographs preoperatively (t1) and after the surgery (t2). The variables taken into account were: age, gender, apnea / hypopnea index (ahi), lowest oxygen saturation (lsat) level, and cephalometric data . Selection and exclusion criteria used to recruit osas patients c - pap = continuous positive airway pressure therapy . Psg was performed at the sleep center of the san raffaele hospital in milan and at the department of sleep medicine of the felice villa hospital in mariano comense (co) using a somnocheck effort polygraph (weinmann, hamburg, germany). The following variables were measured for each patient: ahi, as measure of therapeutic efficacy, and lsat . These variables were used to determine the presence and severity of osas before surgery and to establish (on the basis of changes in the psg variables after surgery) the clinical effectiveness of the therapy . Cephalometric analysis pre- and postoperative lateral cephalometric radiographs for each patient were taken with the teeth in occlusion and following a standardized procedure . The cephalometric examination was carried out with a standardization of the magnification factor, and each cephalogram was double hand - traced by two different investigators who were unaware of the clinical results . The following linear and angular variables were analyzed: sna (angle between the sella, nasion and subspinale point a), snb (angle between the sella, nasion and supramentale point b), anb (angle between the maxilla and the mandible), pas (the narrowest distance between the base of the tongue and the posterior pharyngeal wall a measurement used to detect pharyngeal obstruction), mp - h (distance from the mandibular plane to the most anterior point of the hyoid bone), co - a (the amount of maxillary advancement) and co - pog (the amount of mandibular advancement). Cephalometric variables measured: 1 = sna (angle between sella, nasion and subspinale point a); 2 = snb (angle between sella, nasion and supramentale point b); 3 = anb (angle between the maxilla and the mandible); 4 = pas (the narrowest distance between the base of the tongue and the posterior pharyngeal wall); 5 = mp - h (distance from the mandibular plane to the most anterior point of the hyoid bone); 6 = co - a (amount of the maxillary advancement); 7 = co - pog (amount of mandibular advancement). The surgical procedure consisted of le fort i advancement and bilateral mandibular sagittal split osteotomies (figure 2, figure 3a, b). Rigid fixations were used in all patients to hold the bones in their new position: microplates at the maxilla and miniplates with monocortical screws at the mandible (synthes cmf, missisauga, ontario, canada). It consisted of a clinical follow - up, a radiological evaluation with orthopantomography and lateral cephalometric radiographs, and a psg assessment . Mma, whose amount depended on the initial craniofacial characteristics, was evaluated in each patient . Statistical analysis descriptive statistical analysis was performed for all demographic variables (age and gender) and outcome variables at t1 and t2 . The wilcoxon signed rank test was used to determine the correlation between the psg variables and the cephalometric variables for each outcome measure, and p <0.05 was considered statistically significant for all analyses . In addition, pre- versus postoperative reduction in the ahi was calculated to establish the treatment effectiveness . Application of the paired t - test revealed no significant mean differences between the two series of records . Errors were normally less than one unit, ranging from 0.2 to 0.8 mm for linear measurements and from 0.4 to 1 for angular measurements, corresponding of a coefficient of reliability of 96% . Effect of mma on osas table 2 shows preoperative and postoperative ahi and lsat values with corresponding percentage changes . Postoperatively, the overall mean ahi dropped from 58.7 16 to 8.1 7.8 events per hour (p <0.001). The mean preoperative lsat increased from 71% preoperatively to 90% after surgery (p <0.001). Pre- and postoperative polysomnographic recordings ahi = apnea / hypopnea index; lsat = lowest oxygen saturation; m = male; f = female . Cephalometric analysis lateral cephalometric radiographic evaluation (table 3) showed mean preoperative sna and snb values of 79.9 5.8 and 74.3 4.9 respectively; the mean postoperative values were 85.9 5.8 and 78.5 5.0, respectively . The mean overall amount of maxillary advancement measured on x - ray was 5.2 mm (4.5 mm), ranging from 0 mm to 14 mm . The mean amount of mandibular advancement measured on x - ray was 9.5 mm (8.7 mm), ranging from - 2 to 29 mm . Postoperative cephalometric analysis (table 4) showed a statistically significant average variation (p = 0.01) of the sna which correlated with the decrease in the ahi . The mean sna variation was 6 (falling from 79.9 5.8 to 85.9 5.8). In addition, the increase in lsat was found to show a statistically significant correlation both with the amount of maxillary advancement (p = 0.03) and with the amount of mandibular advancement (p = 0.029). In particular, the results show that for each mm of co - a and co - pog increase there was a 1% increase in lsat . No statistically significant correlations were found between the psg parameter ahi and the snb (p = 0.07), anb (p = 0.33), pas (p = 0.74) and mp - h (p = 0.16). The position of the hyoid was recorded in relation to the mandible, and the mean distance from the hyoid to the mandible plane was increased by 1.3 mm (from 28.2 mm to 29.5 mm) on the post - treatment x - ray . Differences between preoperative and postoperative cephalometric values and means of differences sna = angle between sella, nasion and subspinale point a; snb = angle between sella, nasion and supramentale point b; anb = angle between the maxilla and the mandible; pas = the narrowest distance between the base of the tongue and the posterior pharyngeal wall; mp - h = distance from the mandibular plane to the most anterior point of the hyoid bone); co - a = amount of the maxillary advancement; co - pog = amount of the mandibular advancement; sd = standard deviation . Correlation between polysomnographic and cephalometric variables p <0.05 was considered statistically significant for all analyses . Wilcoxon signed rank test was used to determine the correlation between the psg variables and the cephalometric variables for each of the outcome measures . Ahi = apnea / hypopnea index; lsat = lowest oxygen saturation; sna = angle between sella, nasion and subspinale point a; snb = angle between sella, nasion and supramentale point b; anb = angle between the maxilla and the mandible; pas = narrowest distance between the base of the tongue and the posterior pharyngeal wall; mp - h = distance from the mandibular plane to the most anterior point of the hyoid bone; co - a = amount of maxillary advancement; co - pog = amount of mandibular advancement . This study set out to identify what pre- and postoperative cephalometric characteristics might help predict an osas patient's response to mma and also which outcome variables might predict a higher success rate . As shown in the previous osa surgical literature, success corresponds to an ahi of less than 20 and/or an at least 50% reduction of ahi from the preoperative level . On this basis, the therapy could be deemed successful in all the 15 patients undergoing mma in our study (13 had an ahi <20 after surgery and the other two recorded a 50% reduction). Comparison of the pre- and postoperative psg results showed a clear improvement in all patients after mma surgery . Albeit in a small sample, we therefore recorded a 100% success rate of mma, which is similar to the findings of previous studies which reported success rates ranging from 95% to 98% . In accordance with the findings of a recent meta - analysis led by holty and guilleminaul, the mean overall lsat level increased from 71% to 90% . The results of the present study also showed a statistically significant increase in the sna in all the successfully treated cases (p = 0.01). This implies that the improvement in a patient's respiratory symptomatology is affected by the amount of sna increase . As reported in several publications, bimaxillary advancement surgery has been successfully used to correct craniofacial skeletal anomalies in osas patients, inducing a regression of the syndrome . Nevertheless, some authors concluded that mma can be considered a treatment option in osas patients without cephalometrically defined maxillomandibular deficiency; it has been shown to be a successful therapeutic option for moderate or severe osas patients even in absence of skeletal and\or occlusal anomalies . In this study, nine patients had presurgical skeletal class ii malocclusion (anb> 4), five patients class i malocclusion (anb = 2 2), and only one patient class iii (anb <0) malocclusion . A comparable study of a population of 82 osas patients found that only one - third were diagnosed as skeletal class ii, 60% as class i and 10% as class iii . The surgical correction of our class iii patient resulted in less anterior movement of the mandible than is typical in mma surgery . Although the mandibular advancement in this patient was only 3 mm, the ahi decreased from 44 to 7 events per hour, thus confirming that mma surgery is indicated not only in class ii, but also in class i and in class iii malocclusion, as previously demonstrated by others . Other recent studies, moreover, have pinpointed characteristics useful for identifying which patients might benefit from mma . One of these characteristics is the presence of pharyngeal narrowing in the basal lingual area with retrognathia, especially when the length of the pas at the level of the mandibular base is less than 11 mm or less than 9 mm and the snb is less than 77. hochban and brandenburg suggest other traits based on cephalometric measurements, such as pharyngeal narrowing in combination with a retrognathic or dolicofacial profile . Holty and guilleminault, through a systematic review and a meta - analysis including 627 osas adults, found that no pre- or postoperative cephalometric measurements, with the exception of a wider pas, were predictive of successful mma surgery . The only predictive factors noted were the degree of maxillary advancement, similar to our own findings, in association with a lower preoperative body mass index, young age and female gender . Instead, it is the change in sna that determines the improvement in the respiratory symptoms . In our study the mandibular plane to hyoid bone distance did not show a significant change, thus supporting the findings of gale at al . . The width of the pas was measured as the narrowest distance between the base of the tongue and the posterior pharyngeal wall, which is not related to any skeletal structures . This value increased from 5 2.2 mm to 9.5 3.3 mm, replicating the findings of other studies and thus demonstrating that the dimension of the airway increased after surgery . Several published data support bimaxillary advancement as the most effective available surgical option for treating osa patients; therefore mma may be considered, in certain cases, the first - line approach in these subjects . The patients treated in our study, regardless of their skeletal anomalies, experienced a remarkable regression of their symptoms . Our findings suggest that the improvement in the respiratory symptoms is correlated to the increase in the sna after surgery . This factor could help surgeons to plan surgical interventions on osas patients in advance, allowing them to define the amount of maxillary advancement required . Further research in a larger sample is needed to increase the predictive precision and significance of the results with a view to clinical application . Sandro veronesi (department of statistics - university of insubria) for his help in processing the statistical data.
Anterior cruciate ligament (acl) injuries in the skeletally immature athletes have been reported with increasing frequency, accounting for 31% of the total knee injuries (shea et al . Non operative management is an appealing option in reason of the risk of physeal damage, however the recent literature uniformly indicates that conservative treatment of acl tears in children results in a higher rate of instability that may progress to intra - articular damage, including meniscal tears (fabricant et al . For this reason, there has been an increasing interest in the early operative management to restore stability in skeletally immature knees (falciglia et al . Several aspects of the biology and biomechanics of the reconstruction of the anterior cruciate ligament have been underlined, but only few studies have been conducted on the process of ligamentization in childhood . Although tendons have different both biological and histological features compared to ligaments, numerous studies have reported the use of tendons for ligamentous repair . One of the still debatable questions is to clarify how the so - called neo - ligament really progressively loses its tendon specific biological characteristics exhibiting ligamentous histological properties . While there has been a wide number of studies exploring the various aspects of acl reconstruction in adults only limited data are available regarding the human or animal tendon graft healing process in skeletally immature patients . In the early twentieth century roux, bertocchi - bianchetti, and vallone published the first experimental studies on the histological characteristics of tendon grafts (amiel et al . Later, carnevali indicated that the graft preserved its original features on its intra - articular tract (claes et al ., claes examined several animal studies and showed that the implanted tendons indeed seemed to remodel into a ligamentous acl - like structure (claes et al . Surgery should respect the biology and biomechanics of reconstruction of the anterior cruciate ligament in order to minimize the surgeons fear regarding the possibility of consequences on the growth of the lower limbs . Different tendons have been proposed for surgical acl reconstruction in skeletally immature patients but the semitendinosus is certainly the most commonly used . The purpose of this study was to evaluate the morphological and histological changes that occur in the intra - articular tract of a semitendinosus tendon, when used to reconstruct the acl in growing rabbits, preserving its distal insertion . The study was performed on growing animals to determine the type and the extent of the lesions that develop in the intrarticular tract . The hypothesis was that the so - called graft ligamentization process never completes beyond what was already stated during the maturation phase in other experimental studies in mature animal models . This experimental study is an histological qualitative description of the ultrastructural changes of semitendinosus autograft used for the anterior cruciate ligament reconstruction in an immature rabbit model . The experiment was approved by the local ethics committee and followed the guidelines for the care and use of animals at our institution . The principles of laboratory animal care were followed, as well as specific national laws where applied . All rabbits used for the study were provided by the animal enclosure of the catholic university of the sacred heart of rome (rome, italy). The animals received antibiotic prophylaxis with 10 mg / kg amoxicillin (amoxysol - bayer spa) (preoperative administration and two additional doses at intervals of 72 h) and analgesic therapy with 30 mg ketorolac (toradol - recordati spa) for 2 days postoperatively . They have been observed for 1 week following surgery in adequate cages and then sent back to the farm under veterinarian care . In 1934 galeazzi proposed the anterior cruciate ligament reconstruction in humans using the semitendinosus tendon (galeazzi 1934). Carnevali used this technique in animal models in 1938 (carnevali 1938). For the study twenty - one 8-week - old new zeland white rabbits, weighing about 1500 200 g, underwent surgery on the right knee using this last technique (fig . 1). The animals were anaesthetized with diazepam (ziapam laboratoire tvm) (3 to 5 mg / kg) and with intramuscular ketamine (inoketam virbac srl) (500 mg / kg).fig . The st and g tendons are divided from the musculo - tendinous junction, preserving the distal insertion site . Once the acl was accurately removed the oblique 2-mm - diameter tunnels were drilled, mediolaterally in the tibia and anteroposteriorly in the femur (arrows), passing through the original attachments of the acl and through the growth plates . Then, the semitendinosus tendon () was threaded through the tibial and femoral tunnels and then sutured to the periosteum of the lateral part of the femoral metaphysis with 2.0 silk thread carnevali s technique: intraoperative field . The st and g tendons are divided from the musculo - tendinous junction, preserving the distal insertion site . Once the acl was accurately removed the oblique 2-mm - diameter tunnels were drilled, mediolaterally in the tibia and anteroposteriorly in the femur (arrows), passing through the original attachments of the acl and through the growth plates . Then, the semitendinosus tendon () was threaded through the tibial and femoral tunnels and then sutured to the periosteum of the lateral part of the femoral metaphysis with 2.0 silk thread the right knees joints were exposed through a medial parapatellar incision . The acl was accurately removed and the original footprints were marked using a sterile pencil . Oblique 2-mm - diameter tunnels were drilled, mediolaterally in the tibia and anteroposteriorly in the femur, passing through the original attachments of the ligament and through the growth plates . Then, the semitendinosus tendon was cut at its proximal muscle - tendon junction, but left uninjured at its distal attachment site . The tendon was then threaded through the tibial and femoral tunnels and then sutured to the periosteum of the lateral part of the femoral metaphysis with 2.0 silk thread . The operated knees were immobilized in a cast for seven days, with all efforts made to minimize suffering and to avoid weight bearing . After surgery, the animals returned to their cages and were feeded with water and standard rabbit diet the animals were then randomly divided into seven groups of three and sacrificed at 1, 4, 6, 8, 12, 24 and 48 weeks after surgery . The right limb of each animal was dissected, measured with a caliper (din 862, mauser - messzeug - gmbh, oberndorf / neckar, germany) and radiographed to detect deformity and leg - length discrepancy . In two cases the left knee was used to compare the normal ultrastructure of the acl in rabbits (fig . H&e staining (a) original magnification x20; (b) original magnification x60; (c) original magnification x100 normal structure of the anterior cruciate ligament in the growing rabbit . H&e staining (a) original magnification x20; (b) original magnification x60; (c) original magnification x100 for the experimental and control limbs, longitudinal 5 m - thick sections were stained with hematoxylin and eosin (h&e), masson s trichrome, alcian - pas and alcian blue with different mgcl2 concentrations (quintarelli and dellovo 1965). The specimens were evaluated under light microscopy (leica dm rb) to analyze changes in the intra - articular tract of the graft . Quantitative variables were presented as means and sd or proportion where appropriate . The variables that the study had taken in account and qualitatively described the variables that the study had taken in account and qualitatively described were the following: necrosis, neovascularization and organization of the collagen fibers . Any complication with the animals, throughout the entire period of the study, was observed . All grafts were intact at the time of evaluation and they did not have the pearly appearance of the normal acl . No failure of the semitendinosus graft was observed at the sacrificed time . At 1 week after surgery, the graft tissue showed focal cell - free areas with poor necrosis . Tissue necrosis was better evaluable at 34 weeks, as the intense inflammatory response, but it was no longer detectable after 40 days from surgery . 1 month after surgery, many fibroblasts were present, and numerous disorganized collagenous fibers were observed . The fibrillar collagen, such as type i and iii, are the essential building blocks that provide tendon and ligaments with their elasticity and strength . The fibroblastic cells had a central position in the tendon with an initial longitudinal arrangement . The vascularization was still poor and vessels were located on the grafts periphery (fig . 3a).fig . 3h&e staining (a) (original magnification x40) one month after surgery the graft tissue showed regions of cell - free areas and necrosis . * vessel located in the periphery of the graft and runned parallel to the collagen fiber bundles in the septa at forty days; b (original magnification x100) fusiform fibroblast (arrow) in the central portion of the tendon . C masson trichrome staining (original magnification x40) disorganized collagenous fibers at 4 weeks h&e staining (a) (original magnification x40) one month after surgery the graft tissue showed regions of cell - free areas and necrosis . * vessel located in the periphery of the graft and runned parallel to the collagen fiber bundles in the septa at forty days; b (original magnification x100) fusiform fibroblast (arrow) in the central portion of the tendon . C masson trichrome staining (original magnification x40) disorganized collagenous fibers at 4 weeks in the semitendinosus tendon, the fibroblastic cells appeared fusiform (fig . However, after 4 weeks, these fusiform - like cells became more rounded . The collagen fibers became more organized after the first month with areas of disorganized collagen matrix (fig . 3c). From the fourth to the eighth week after surgery the typical inflammatory cell population gradually disappeared and the small necrosis areas were not detectable . At 8 weeks after surgery, the collagen fibers aligned in parallel with the new capillaries and with the fibroblastic cells that had become highly differentiated (fig . 4). The semitendinosus tendon, which initially had no vessels after grafting, gradually became vascularized, expecially after 40 days after surgery . The grafts, however, did not appear to resemble the acl at this timepoint, with the mean graft cross - sectional area significantly higher in the operated knee as compared with the normal left knee ligament . At the end of the healing process of the neo - ligament (24 weeks) the loss of the regular collagen orientation and crimp pattern was observed with the shift of the diameter of the collagen fibrils, from large to small, the reduction of collagen type i fibrils and an increase of collagen type iii synthesis . The histological features observed in the sacrificed animals at 24 weeks (fig . 5) were not subject to further changes at 48 weeks (fig . Weeks the graft had not yet acquired the characteristics of the normal rabbit acl in the intra - articular tract of the sections . C masson trichrome staining (original magnification x60) collagenous fibers of the semitendinosus tendon at 8 weeks after surgeryfig . 5peripheral and core vessels () in the semitendinosus tendon 24 weeks after surgery . B masson trichrome staining (original magnification x40) (c) alcian blue (original magnification x20)fig . 6 a h&e staining (original magnification x60). Despite the histological changes the neoligament at 48 weeks after surgery does not assume the characteristics of the normal rabbits acl . B masson trichrome staining (original magnification x20). Semitendinosus tendon 48 weeks after surgery . An increased longitudinal and parallel orientation of the collagen bundles was observed; c masson trichrome staining (original magnification x40). At 48 weeks the neoligament was viable . Red cells were present in the peripheric portion of the grafttable 1major histological characteristics at each time - point in st graft1 week4 weeks6 weeks8 weeks12 weeks24 weeks48 weeksnecrosisfocal cell - free areas with small necrosiscell - free areas with poor necrosisgradually disappearesno necrosis signneovascularizationno vesselpoor and located on the grafts peripheryvessel located in the periphery of the graft and runned parallel to the collagen fiber bundles in the septavessel located from periphery to central areanormal vascularityorganization of the collagen fibersst tendon characteristicsdisorganized collagenous fibersinitial longitudinal arrangementcollagen fibers aligned in parallel with the new capillaries and with the fibroblastic cellsloss of regular collagen orientation and crimp patternslight separation with increased waviness of fibersshift of the diameter of the collagen fibrils, from large to smallunimodal appearance of collagen fibrilsreduction of collagen type i fibrilsfailure restauration of collagen orientation to normalityincrease of collagen type iii synthesiscellularityinflammatory cell populationaligned longitudinally, more rounded fibroblastic cells with large nuclei and abundant cytoplasmhighly differentiated fibroblastic cellsnormal cellularitydisorganized and randomly arranged fusiform - like fibroblastic cells with plump nucleigradual disappearance of the typical inflammatory cell populationfibrocyte have different characteristics from fibroblastic cells h&e staining (a) (original magnification x40). C masson trichrome staining (original magnification x60) collagenous fibers of the semitendinosus tendon at 8 weeks after surgery peripheral and core vessels () in the semitendinosus tendon 24 weeks after surgery . A h&e staining (original magnification x40). B masson trichrome staining (original magnification x40) (c) alcian blue (original magnification x20) a h&e staining (original magnification x60). Despite the histological changes the neoligament at 48 weeks after surgery does not assume the characteristics of the normal rabbits acl . B masson trichrome staining (original magnification x20). Semitendinosus tendon 48 weeks after surgery . An increased longitudinal and parallel orientation of the collagen bundles was observed; c masson trichrome staining (original magnification x40). At 48 weeks the neoligament was viable . Red cells were present in the peripheric portion of the graft major histological characteristics at each time - point in st graft the most noteworthy findings in the current study were: 1) the semitendinosus graft did not undergo full ligamentization and, after 48 weeks, its original appearance at the microstructural level was different; 2) a marked reduction of cellular necrosis was observed in the early phase of the neo - ligament healing process . The acl reconstruction in pediatric age has become more frequent in these past recent years . In skeletally immature patients, physeal - sparing procedures the use of patellar tendon should be avoided for the potential risk of premature physeal closure and subsequent tibial recurvatum (meller et al . 2008), moreover it may interfere with the delicate changes of the extensor mechanism during growth (progressive reduction of laxity, reduction of q angle, lower limb torsional changes and gradual patella lowering). For these reasons there were only a few papers on growing animal models, but no studies on growing rabbits has ever been performed before . This last aspect makes the study on skeletally immature patients original . In particular in other studies, the experiments were performed on mature rabbits (amiel et al 1986; blickenstaff et al . 2009), mature sheep (blickenstaff et al . 1997; mayr et al . Besides, while we used the semitendinosus tendon, other studies used the achilleous tendon (li et al . 2009) the patellar tendon (amiel et al . 1986; falconiero et al . 1998), the superficial flexor digitorum tendon and the gastrocnemius tendon (li et al . Few studies considered the remodeling of autografts after anterior cruciate ligament reconstruction in mature human patients (claes et al . 2011; scheffler et al . Amiel (1986) proposed the concept of graft ligamentization . In his study, he used a rabbit model for acl reconstruction and transplanted the autogenous patellar tendon in 37 adult rabbits of approximately 6 to 8 months old . He showed that patellar autograft underwent a process of ligamentization when placed in the acl environment and that the cells responsible for this change were of an extragraft origin . He concluded that the ligamentization process was a continuous development of the tendon into a substance similar to a normal ligament . Other studies supported these findings (falconiero et al . 1998; marumo et al . 2005; sanchez et al . 2010). On the opposite side, bosch demonstrated that the autograft tissue was different from the original tendon both structurally and mechanically, and that it would never approach the characteristics of a normal ligament (bosch and kasperczyk 1992). He also stressed the importance of the four different phases of the autograft healing process: necrosis, revitalization, collagen formation and remodeling . This was supported by blickenstaff, who concluded that, at the end of the healing period of 52 weeks, the graft was different from the original ligament, but also had different characteristics from the original tendon (blickenstaff et al . Kennedy analyzed the evolution of intra - articular semitendinosus (st) transfer in rabbits concluding that the surgical acl reconstruction was a failure (kennedy et al ., tohyama published a study showing that the patellar tendon graft underwent important changes in its structure that could be divided into the upper mentioned four stages (tohyama et al . He concluded that the matrix changes were caused by the adaptation of the patellar tendon to the acl environment . In literature only one experimental study on rabbit models used the semitendinosus tendon for acl reconstruction with preservation of the distal insertion . In this study the specimens were taken up to 12 weeks after surgery, instead in our study they were extended beyond 24 weeks after surgery reaching the rabbits skeletal maturity, which verifies approximately at about 28 weeks . Papachristou immobilized rabbits for 2 weeks after surgery, instead in our study we immobilized the animals for only one week to avoid the potential risk of osteonecrosis . Despite the time points of the samples explanted were not completely overlapped, in the first month after surgery the results were similar (absence of avascular necrosis, disorganized collagen fibers). This last finding, in our study, was observed between the 4 and the 6 week . The phase of cell differentiation was more markedly observed between the 6 and the 12 week . After this time point we evidenced the maturation phase of the graft which maintained vital up to 48 weeks after surgery . Our study analyzed the trend and the graft patterns across different timepoints to verify the microscopic changes that occurred during the healing process . After surgery, the space between the graft and the original bone was filled with a fibrovascular tissue . The graft showed a fibroblasts number increase during the early postoperative period (about 1 month). It was believed that the cells were metabolically active because of the presence of plump nuclei . After this timepoint, the fibroblasts aligned longitudinally became rounded and their nuclei less ovoidal (fig . The study confirmed, even in growing rabbits, that the findings reported by other authors such as the graft vascularization began only 3040 days after surgery (amiel et al . 1986) and the initial focal necrosis areas were no more detectable after 46 weeks (ekdahl et al . 2008; stener et al . 2012). The new vessels initially appeared on the grafts periphery and later were present within the central zone (fig . Collagen orientation did not return to normality during the study period (48 weeks). Moreover, at the electron microscopic analysis, the diameter of the collagen fibrils lost their normal bimodality appearance changing in unimodal as described by abe et al . (1993). As a collateral finding, although it was not the aim of this study, any necrosis sign in the physis was observed . Therefore, drilling a tunnel through the region of the growth plate did not alter bone growth; although, there was transient hypertrophy in the portion, which disappeared within the first month after surgery (guzzanti et al . The remodeling phase of the healing process of the tendon graft is the most vulnerable phase because of the increased necrosis, revascularization and extra - cellular infiltration (claes et al . Other animal studies have shown that the patellar tendon, similar to the semitendinosus tendon, underwent more extended avascular necrosis and revascularization after transplantation (amiel et al . These histological observations, compared with human studies, showed that especially in the early phase there was a marked necrosis in the central portion of the graft (amiel et al . 1986; bosch and kasperczyk 1992). Although the preservation of the distal insertion of the semitendinosus tendon did not improve the final graft histological feature, and even the time of the different phases of the remodeling process were not modified, however, this study noted a marked reduction in cellular necrosis, only in focal areas observed . This aspect was due to the increased vascularization of the neoligament directly given by the preservation of the distal attachment of the semitendinosus that improved both revascularization and growth of fibroblasts, as observed by papachristou . This finding is very important because it reduces the vulnerability of the graft especially during the first weeks after acl reconstruction . Even though this study showed a lower percentage of cellular necrosis, it is better to be more cautious and avoid an early and intensive rehabilitation treatment in adolescents, this not for the histological weakness but for the uncertainty on their prudent post - surgical conduct . A thorough knowledge of the graft healing process is necessary to improve aspects of the surgical technique and in particular for the rehabilitation program . However one thing is clear: the neoligament does nt assume the histological characteristics of the normal anterior cruciate ligament, expressing always an intermediate structure between the tendon and the ligament . However, clinical experience in the use of the semitendinosus for the acl injured reconstruction shows that the ultrastructural appearance of the graft, viable at any given time point of the studies, at the end of the healing process provides sufficient guarantees such as to justify its use . The major limitation of our study was the lack of biomechanical analysis and the ability to transfer the findings of this animal model to human tendon transplantation surgeries . However, the minor surgical cases and the rarity of degenerative disease in this population make second - look arthroscopy extremely rare and conditioned by small percentage of biopsies taken from the center of the graft . Findings showed in experimental animal models, in their general concept about the progressive biologic changes that develop in the transplanted semitendinosus tendon, could be useful to lead acl reconstruction in pediatric and adolescent population . The ligamentization process is not present up to 48 weeks after surgery in growing rabbit models . The grafted st tendon changes into a new entity during the healing process, assuming a micro - architecture somehow between the tendon and the ligament ones . Semitendinosus graft undergoes a process of adaptation rather than full restoration of the intact acl s biology properties . The use of semitendinosus for acl reconstruction with preservation of its distal insertion should be the gold standard in skeletally immature patients.
Chronic cough is a frequent presenting complaint to not only the general practitioner but also the otolaryngologist and respiratory specialist . Diagnosis can be difficult, especially in primary care, but empirical treatment for common causes such as asthma, chronic obstructive pulmonary disease, gastro - oesophageal reflux disease, postnasal drip or drug - induced cough (most commonly secondary to angiotensin converting enzyme inhibition) is recommended when there are no associated worrying symptoms, and is successful in the majority of cases . Symptoms that may signify a rarer but more serious cause for chronic cough include the copious production of sputum, fever, sweats, unintentional weight loss, haemoptysis, clubbing, hoarseness and marked dyspnoea . Here we highlight a unique cause of chronic cough: the initial symptom of lymphoma compressing the brainstem . A 69year - old gentleman presented to his general practitioner with a 3-month history of progressive unsteadiness . On further questioning he also volunteered that these symptoms had been preceded by a chronic cough, which had no obvious precipitant, and which had failed to resolve . On examination he had truncal ataxia, to the point where he had difficultly remaining upright when walking, and exhibited gaze - evoked first - degree nystagmus in his left eye on looking to the right . The patient had suffered an episode of labyrinthitis 10 years earlier, and so he was prescribed pharmacological therapy aimed at a presumptive diagnosis of a second episode . Successive trials of prochlorperazine, betahistine, cyclizine and arlevert were not noted to have any positive effect on the patient's dizziness and he was referred for outpatient neurological opinion . By the time of specialist review, the patient's symptoms had worsened and he now described periods of severe nausea, with progression of his ataxia to the point where he was unable to stand unaided . He had also developed an area of hypoesthesia on the left side of his face . Eye movements were adjudged to be markedly abnormal with dysmetric horizontal saccadic movements in addition to his ongoing nystagmus . His right vestibular ocular reflex was reduced and his left upper limb movements lacked coordination . Gadolinium - enhanced magnetic resonance imaging (mri) of the brain revealed a homogenously enhancing, sharply demarcated 13 mm diameter lesion in the left posterior and lateral midbrain extending into the inferior cerebellar peduncle, with some adjacent oedema, suggestive of primary lymphoma (see fig . 1). Stereotactic brain biopsy confirmed diffuse large b - cell lymphoma (dlbcl) and subsequent bone marrow biopsy and computer tomography (ct) imaging showed no evidence of lymphoma at any additional anatomical site . Figure 1:gadolinium - enhanced t1-weighted mri images (panes lettered with coronal (a), axial (b) and sagittal (c) sections) showing a homogenous enhancement of a clearly demarcated lesion in the left posterior and lateral midbrain with extension into the inferior cerebellar peduncle . Gadolinium - enhanced t1-weighted mri images (panes lettered with coronal (a), axial (b) and sagittal (c) sections) showing a homogenous enhancement of a clearly demarcated lesion in the left posterior and lateral midbrain with extension into the inferior cerebellar peduncle . The patient embarked on idaram (idarubicin, cytarabine, methotrexate, dexamethasone) chemotherapy followed by two rounds of high - dose methotrexate mono - therapy . Imaging following treatment showed radiological remission and this is also seen on recent scans, now 2 years later (see fig . 2). While the symptoms of cerebellar dysfunction have largely abated since discharge, the patient has, however, been continually troubled by his cough . Figure 2:compares initial t1-weighted gadolinium - enhanced mri images (seen in panes on left hand side: a, c and e) with those taken following completion of treatment (panes on right: b, d and f). Radiological remission is shown in the post - treatment images . Compares initial t1-weighted gadolinium - enhanced mri images (seen in panes on left hand side: a, c and e) with those taken following completion of treatment (panes on right: b, d and f). Radiological remission is shown in the post - treatment images . Otolaryngology assessment, including symptom review, medication history, endoscopic evaluation and chest x - ray, was unable to elucidate any cause for cough other than the presumed effects of the medullary lesion . Empirical trials of nasal steroids, nasal saline washes, high - dose proton pump inhibitor and inhaled bronchodilators were all unsuccessful . Coughing is a protective motor reflex under both voluntary and involuntary regulation, integrating physiological, psychological and social inputs . Studies in experimental animals have shown that vagal afferents signal peripheral sensory stimulation from the airways and lungs, and project centrally to the nucleus tractus solitarius (nts) with further projections between the nts and the medulla and pons . That such projections constitute a regulatory brainstem cough centre is a concept that has been extensively pursued; attempting to determine the specific central pathways involved in the initiation and regulation of the efferent arc of the cough reflex . The aetiology of chronic cough has been considered in a similar manner to the aetiology of chronic pain, with elements of both peripheral and central sensitization . Most of the common causes of chronic cough, and notably the classically described diagnostic triad of asthma, gastro - oesophageal reflux disease and postnasal drip, arise primarily through the activation of peripheral vagal afferents, but with probable central maladaptation contributing to hyper - sensitization to peripheral stimuli . Pathological impingement of the dorsal medullary region of the brainstem is recognized as a neurogenic cause of chronic cough in cases of patients with chiari i malformations and cough may be the initial symptom . However, to our knowledge, this is the first reported case of a lymphoma around the brainstem with chronic cough as one of the first symptoms . It is difficult to be certain as to the mechanism of chronic cough induced by this lymphoma . It may be that the expanding mass of the dlbcl compressed nearby nerve efferents serving the cough reflex, leading to aberrant hypersentization . However, dlbcl can be an aggressive lesion and has been reported to present with focal neurology from local invasion into nerves . It is possible, therefore, that the chronic cough in this case was caused by tumour infiltration into nerves rather than compression . Although clearly cerebral lymphoma is a very rare cause of chronic cough, we believe that clinicians in primary and secondary care should consider rare central causes of chronic cough in cases where neurological symptoms or signs are present, or in cases where empirical treatment for the more common causes of cough fails to provide symptomatic relief.
Although rare, tumor involvement of the inferior vena cava (ivc) by hepatobiliary, pancreatic or duodenal malignancies can be difficult to assess preoperatively, and can compromise an adequate oncological resection, especially when found incidentally at laparotomy . However, radical resection with negative histological margins remains the only chance of cure, especially for duodenal tumors which have better prognosis . Lessons learned from liver transplantation have improved technical considerations and popularized partial or complete resection of the ivc, with acceptable morbidity . Various techniques are used for venous reconstruction, using a prosthetic graft interposition in most of the cases . However, in case of associated digestive resections, postoperative complications can be responsible for prosthesis infection and related vascular complications . In this setting we present an original technique of a venous autoplasty in a woman who underwent pancreaticoduodenectomy for a duodenal adenocarcinoma involving the anterior part of the infrarenal ivc . A 74-year - old woman was referred in our tertiary center for the exploration of an anemia . She had no previous remarkable medical history . Gastroduodenal endoscopy with biopsies identified an adenocarcinoma of the second part of the duodenum . Abdominal computed tomography (ct) scan showed a huge duodenal mass of 7 cm in size developed from the second part of the duodenum, close to the anterior wall of the ivc, without extension to other major vessels (e.g. Coeliac, hepatic or superior mesenteric artery) or metastases (fig . Figure 1:preoperative ct - scan showing a huge duodenal mass () of 7 cm in size developed from the second part of the duodenum, close to the anterior wall of the infrarenal ivc ( ). Preoperative ct - scan showing a huge duodenal mass () of 7 cm in size developed from the second part of the duodenum, close to the anterior wall of the infrarenal ivc (* ). A kocher maneuver was not possible because of strong adhesions between the tumor and the anterior wall of the ivc . In order to ensure adequate resection margins, we performed a pancreaticoduodenectomy with en - bloc resection of a 3 cm area of the anterior wall of the ivc (fig . 2a), after total clamping of the right and the left renal veins, and of the ivc from either side of the involved area . Direct suture of the ivc was not possible . We decided not to use a prosthetic graft for the venous reconstruction because of the risk of infection regarding the high rate of infectious complications after pancreaticoduodenectomy, particularly in case of soft pancreas texture . Thus, since the posterior wall of the ivc is usually more floppy than the anterior one because of its higher distance from the renal veins, we harvested a transversal patch of 20 30 mm in size from the posterior wall of the ivc, below the confluence with the renal veins (fig . Reconstruction of the posterior wall of the ivc was achieved by a direct hand - sewn hemi - circumferential anastomosis using a single - layer non - absorbable 4/0 polypropylene (fig . Thereafter, the venous patch was disposed longitudinally in place of the resected area at the anterior wall of the ivc, and was sutured using a single - layer non - absorbable 4/0 polypropylene (figs . 2c, d and f). The digestive reconstruction was then performed according to the child procedure with a pancreaticogastrostomy . Figure 2:(a) we performed a pancreaticoduodenectomy with en - bloc resection of a 3 cm area of the anterior wall of the ivc (). Direct suture of the ivc was not possible and venous reconstruction required the interposition of a graft . (b) since the posterior wall of the ivc is usually more floppy than the anterior one because of its longer distance from the renal veins, we harvested a transversal patch of 20 30 mm in size from the posterior wall of the ivc, below the confluence with the renal veins . (c) reconstruction of the posterior wall of the ivc was achieved by a direct hand - sewn hemi - circumferential anastomosis using a single - layer non - absorbable 4/0 polypropylene . (d and e) the transversal venous patch was placed longitudinally on the resected area at the anterior wall of the ivc (* ), and was sutured using a single - layer non - absorbable 4/0 polypropylene . (f) intraoperative view showing the venous autoplasty of the ivc . Srivc, suprarenal inferior vena cava; irivc, infrarenal inferior vena cava; rrv, right renal vein; lrv, left renal vein . (a) we performed a pancreaticoduodenectomy with en - bloc resection of a 3 cm area of the anterior wall of the ivc (). Direct suture of the ivc was not possible and venous reconstruction required the interposition of a graft . (b) since the posterior wall of the ivc is usually more floppy than the anterior one because of its longer distance from the renal veins, we harvested a transversal patch of 20 30 mm in size from the posterior wall of the ivc, below the confluence with the renal veins . (c) reconstruction of the posterior wall of the ivc was achieved by a direct hand - sewn hemi - circumferential anastomosis using a single - layer non - absorbable 4/0 polypropylene . (d and e) the transversal venous patch was placed longitudinally on the resected area at the anterior wall of the ivc (* *), and was sutured using a single - layer non - absorbable 4/0 polypropylene . Srivc, suprarenal inferior vena cava; irivc, infrarenal inferior vena cava; rrv, right renal vein; lrv, left renal vein . Postoperative course was marked by a bile leakage on postoperative day 2 which required surgery to repair the hepaticojejunal anastomosis . Postoperative ct - scan showed good permeability of the ivc with no symptoms related to the reconstruction . Pathological examination confirmed the r0 resection of a t4 n1 m0 duodenal adenocarcinoma involving the ivc . She received a 6 months adjuvant folfox chemotherapy regimen and was followed up every 3 months with no recurrence at time . Resection of the ivc is widely performed during surgery for various abdominal and retroperitoneal malignancies, since a radical resection is the only chance of cure . Suprarenal ivc resection without venous replacement has been proposed because the venous collateral pathways can supply the caval flow through the lumbar and epigastric pathways, the renal veins, and the vertebral venous system, which are often well developed in case of a ivc chronic occlusion . However, large dissection of a retroperitoneal tumor and/or thoracoabdominal incisions can both interrupt the ascending collateral venous pathways, resulting in a high risk of major venous insufficiency . In these cases, reconstruction of the ivc is mandatory and can be performed through a direct end - to - end anastomosis, or after the interposition of a venous / prosthetic patch . Reported that resection 2 cm of the ivc could be reconstructed by a direct suture while resection> 2 cm required the interposition of a graft . In the same way, tsuji et al . Performed a direct suture when the wall defect was less than one - third of the circumference of the ivc . In our case, the venous resection was too large to perform a direct suture of the ivc, either longitudinal or transversal . Therefore, the technique of choice regarding this large venous defect appeared to be a prosthetic reconstruction . However, considering the high risk of infectious complications after duodenopancreatectomy, especially when performed in the settings of a duodenal tumor with a soft pancreatic gland texture and without dilatation of both the pancreatic and the main bile duct, we chose a reconstruction technique that required a large venous patch . Thus, we decided to harvest the posterior wall of the ivc because it was very floppy, that allowed its direct suture . Indeed, these elastic properties of the ivc have already been investigated . Reported the possibility to easily harvest a venous patch from the anterior wall of the ivc, above the confluence of the renal veins, with a low risk to narrow the ivc because its diameter is larger at this point . Our original technique of venous autoplasty could be thus extrapolated to these veins, in order to prevent the use of prosthetic patches and to facilitate reconstruction.
In 1996, deleuze et al . Initially suspected the role of defects in the multidrug resistance 3 p - glycoprotein (mdr3) in a subtype of progressive familial intrahepatic cholestasis (pfic). This refers to a heterogeneous group of familial cholestatic conditions caused by defects in biliary epithelial transporters . The disease usually occurs first in childhood with progressive cholestasis leading to death from liver failure at ages ranging from infancy to adolescence . The underlying genetic and molecular abnormalities related to pfic have led to the identification of three subtypes and the description of several mutations in hepatocellular transport system genes involved in bile formation . Only pfic 3 phosphatidylcholine solubilises cholesterol in mixed micelles and prevents damage to the biliary epithelium from unbound bile acids . Unchaperoned bile acids in the bile of patients with mdr3 deficiency may cause chronic cholangitis . Several other biliary disorders have been associated with abcb4/mdr3 mutations: low phospholipid - associated cholelithiasis (lpac) syndrome, intrahepatic cholestasis of pregnancy (icp), drug - induced liver injury, transient neonatal cholestasis [tnc], adult biliary fibrosis or cirrhosis, and very recently intrahepatic cholangiocarcinoma (ihcc) [49]. In young adults, abcb4/mdr3 alterations are mostly associated with lpac syndrome and icp . In the latter case, patients typically present with pruritus that can lead to complications for both mother and fetus . In the former case, the diagnosis of lpac syndrome is often delayed and relies on clinical and biological elements . This review will focus on imaging features of hepatobiliary anomalies associated with abcb4/mdr3 deficiency in adults, with special attention to mr characteristics . Intrahepatic lithiasis is considered to be very uncommon in europe and much more frequent in asia . Since the description of mdr3 deficiencies, an increasing number of patients are diagnosed with lpac syndrome - related intrahepatic lithiasis . The diagnosis is frequently performed several years after the beginning of the symptoms due to their lack of specificity . The three most important are: (1) biliary symptoms in a young adult (<40 years old); (2) symptom recurrence after cholecystectomy; (3) presence of hyperechoic material in the biliary ducts . Other minor criteria have been described, such as mild chronic cholestasis, at least one episode of cholangitis, acute pancreatitis or biliary colic, efficiency of ursodesoxycholic acid (udca) and similar symptoms in first - degree relatives . Gene mutations have been described in patients with lpac syndrome in 2556% of cases [1113]. At imaging, lpac syndrome is associated with various mr presentations: normal mr cholangiography, isolated intrahepatic lithiasis and, rarely, bile duct dilatations . No imaging differences can be found between patients with or without abcb4 mutation and no specific mutation can be associated with the different presentations [12, 14]. The most common presentation of lpac syndrome is isolated intrahepatic lithiasis . In lpac patients, most stones cholesterol stones may vary in colour from light - yellow to dark - green or brown . To be classified as such, they must be at least 50% cholesterol by weight (or 70%, according to the japanese classification system) [16, 17] brown stones (pigment stones). Those are traditionally favoured by bile infection, frequent in the asiatic population but much rarer in western countries . Ultrasound examination is very accurate in detecting intrahepatic stones, since they appear as heterogeneous and echoic foci centred on the intrahepatic ducts, or as a comet - tail artefact due to ultrasound reverberation (fig 1) [19, 20]. The comet - tail artefact may be due to intrahepatic lithiasis or to the associated cholangiopathy.fig . 1ultrasound / mrcp discrepancy in a 44-year - old man with lpac syndrome . A transverse ultrasound showing typical comet - tail artefacts in the left lobe . B the mrcp shows no sign of biliary stone ultrasound / mrcp discrepancy in a 44-year - old man with lpac syndrome . A transverse ultrasound showing typical comet - tail artefacts in the left lobe . B the mrcp shows no sign of biliary stone if endoscopic retrograde cholangiopancreatography (ercp) has been considered as the gold standard for diagnosing bile duct stone, magnetic resonance cholangiopancreatography (mrcp) is a non - invasive alternative technique that has been shown to be equivalent to ercp in choledocholithiasis diagnosis and superior to ercp in intrahepatic lithiasis diagnosis [2123]. The diagnosis of biliary stone is based on the presence of round or oval shape signal voids in the lumen of the bile ducts on heavily t2-weighted sequences (figs . 2, 3, 4, 5, 6 and 7). However, it is occasionally difficult to diagnose stones when the surrounding liquid is not present . On t1-weighted sequences, stones present with a spontaneous hyperintensity and, recently, studies have shown the superiority of these sequences over the t2-weighted sequences for the detection of biliary stones . Discrepancies between mr and ultrasound have been reported as mr is not able to detect very small stones (fig 1), while ultrasound may be suboptimal in case of massive intraluminal stones (fig 7). 2unifocal mild biliary dilatation with biliary stone in a 40-year - old man with lpac syndrome . The dilatation is located in the segment v on t2-weighted acquisitions (white arrow in a) and 3d mrcp (b) and contains a small signal void corresponding to an endoluminal stone (white arrow in b). The gallbladder and the common bile duct show no abnormalitiesfig . 3severe unisegmental dilatation with endoluminal stones in a 44-year - old woman with lpac syndrome . Several dilated bile ducts in segment viii containing macroscopic signal voids corresponding to biliary stones (white arrows). A transverse t2-weighted acquisition with fat saturation and (b) 3d mcrpfig . Transverse t1-weighted acquisition with fat saturation after injection of a gadolinium chelate at portal phase (a and b), and 3d mcrp (c) show a mild biliary dilatation of both segment iii (white arrow in a) and vi (white arrow in b). The mrcp shows a round signal void in the dilated bile ducts of the segment iii (white arrow in c) corresponding to a biliary stonefig . . Transverse t2-weighted acquisition (a), transverse t1-weighted acquisition with fat saturation (b), and coronal maximum intensity projection mcrp (c) show a biliary dilatation in both right and left lobes containing biliary stones depicted as t2 hypointense and t1 hyperintense endoluminal formations (white arrow in a and b). The mrcp shows large oval shape signal voids in the dilated bile ducts (white arrow in c) corresponding to biliary stonesfig . 6segmental dilatation of segment vi bile duct filled with several stones in a 69-year - old man with lpac syndrome . Coronal maximum intensity projection mcrp (a), transverse t2-weighted acquisition (b), and transverse in - phase t1-weighted acquisition (c) show a dilatation of segment vi bile ducts filled with several stones (white arrow in a). The stones appear as endoluminal signal voids on t2-weighted acquisition (white arrow in b) and hypointensities on t1-weighted acquisitions (white arrow in c)fig . Transverse t2-weighted acquisition (a), and coronal maximum intensity projection mcrp (b) show a rare presentation of severe lpac syndrome consisting in diffuse biliary dilatation containing multiple biliary stones unifocal mild biliary dilatation with biliary stone in a 40-year - old man with lpac syndrome . The dilatation is located in the segment v on t2-weighted acquisitions (white arrow in a) and 3d mrcp (b) and contains a small signal void corresponding to an endoluminal stone (white arrow in b). The gallbladder and the common bile duct show no abnormalities severe unisegmental dilatation with endoluminal stones in a 44-year - old woman with lpac syndrome . Several dilated bile ducts in segment viii containing macroscopic signal voids corresponding to biliary stones (white arrows). A transverse t2-weighted acquisition with fat saturation and (b) 3d mcrp bisegmental dilatation in a 36-year - old woman with lpac syndrome . Transverse t1-weighted acquisition with fat saturation after injection of a gadolinium chelate at portal phase (a and b), and 3d mcrp (c) show a mild biliary dilatation of both segment iii (white arrow in a) and vi (white arrow in b). The mrcp shows a round signal void in the dilated bile ducts of the segment iii (white arrow in c) corresponding to a biliary stone diffuse global bile duct abnormalities in a 48-year - old man with lpac syndrome . Transverse t2-weighted acquisition (a), transverse t1-weighted acquisition with fat saturation (b), and coronal maximum intensity projection mcrp (c) show a biliary dilatation in both right and left lobes containing biliary stones depicted as t2 hypointense and t1 hyperintense endoluminal formations (white arrow in a and b). The mrcp shows large oval shape signal voids in the dilated bile ducts (white arrow in c) corresponding to biliary stones segmental dilatation of segment vi bile duct filled with several stones in a 69-year - old man with lpac syndrome . Coronal maximum intensity projection mcrp (a), transverse t2-weighted acquisition (b), and transverse in - phase t1-weighted acquisition (c) show a dilatation of segment vi bile ducts filled with several stones (white arrow in a). The stones appear as endoluminal signal voids on t2-weighted acquisition (white arrow in b) and hypointensities on t1-weighted acquisitions (white arrow in c) severe lpac syndrome in a 55-year - old man . Transverse t2-weighted acquisition (a), and coronal maximum intensity projection mcrp (b) show a rare presentation of severe lpac syndrome consisting in diffuse biliary dilatation containing multiple biliary stones computed tomography (ct) performance for the diagnosis of intrahepatic lithiasis depends on the calcium content of bile stones . Cholesterol stones present with a typical hypodense aspect and are poorly visible . Rarely, patients may present with more severe dilatations (<10%). Such severe bile duct dilatation may only involve one or two liver segments or may be diffuse (figs . 3, 4, 5, 6 and 7). In such cases, and as opposed to ductal plate malformations (caroli disease, congenital hepatic fibrosis, congenital cystic anomalies of the common bile duct, etc . ), which are directly related to abnormal embryological development of the bile ducts, abnormalities are related to a biliogenesis disorder developed on initially normal ducts . Anomalies are a consequence of the chronic alteration of the bile composition, which results in damage to the biliary epithelium . Bile duct abnormalities are demonstrated as unifocal or multifocal non - cystic large spindle - shaped bile duct dilatations (figs . 2, 3, 4, 5, 6 and 7). However, ductal plate malformations, particularly the caroli disease, have to be ruled out by the absence of specific features, such as the central dot sign, dilated bile ducts associated with focal area of cystic ectasia, or the fact that no biliary dilatation can be found in lpac syndrome without underlying biliary stones [14, 25]. Other possible differential diagnosis is bile duct dilatations related to focal obstacle on the biliary ducts . Traditionally, downstream - acquired stenosis (iatrogenic, biliodigestive anastomosis, sclerosing cholangitis, cholangiocarcinoma) may lead to obstructive dilatations that appear more central without intrahepatic lithiasis . In mice, the multidrug resistance (mdr) glycoproteins that mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte are called mdr2 . Whereas the main feature in mdr2 knock - out mice, which corresponds to the equivalent animal model of human mdr3 deficiency [26, 27], is sclerosing cholangitis, controversies exist whether a genetically determined dysfunction of mdr3 plays a pathogenic role in primary biliary cirrhosis and primary sclerosing cholangitis (psc) in humans . . Found no genetic argument supporting the role of mdr3 in psc . Since then, concepts in psc understanding have evolved and many authors consider that psc may represent a mixed bag of diseases of different aetiologies in which several genes such as abcb4/mdr3 may play a disease modifier role . To support this conceptual view, our group recently reported for the first time, in a series of 13 patients with mdr3 deficiency, imaging presentations mimicking sclerosing cholangitis in two patients at mr imaging (figs . They corresponded to small duct fibro - obliterative lesions at pathology, and may be due to the direct toxic effect of biliary acids on epithelium . To our knowledge, this is the only report of such association of mdr3 deficiency and secondary sclerosing cholangitis but the two patients presented with recurrent cholangitis and not lpac syndrome per se.fig . Three - dimensional mrcp (a) and sagittal ultrasound of the right lobe (b) show right biliary abnormalities (a). These mild irregular calibre intrahepatic bile ducts were not demonstrated with ultrasound; on the other hand, small bile stones were easily depicted as hyperechoic formations with posterior attenuationfig . 9diffuse and severe cholangitis in a 64-year - old woman . Maximum intensity projection coronal mrcp (a), t1-weighted transverse acquisitions with fat saturation after gadolinium chelate injection obtained at arterial (b), portal (c) and delayed phase (d) show biliary irregularities and stenoses (a) associated with intense biliary contrast uptake of the thickened biliary walls at the arterial (white arrows in b) and portal phase (white arrow in c). The common bile duct presents with the same abnormalities (white arrow in d). Note the segment i hypertrophy (white star in b and c) biliary irregularities in a 54-year - old man . Three - dimensional mrcp (a) and sagittal ultrasound of the right lobe (b) show right biliary abnormalities (a). These mild irregular calibre intrahepatic bile ducts were not demonstrated with ultrasound; on the other hand, small bile stones were easily depicted as hyperechoic formations with posterior attenuation diffuse and severe cholangitis in a 64-year - old woman . Maximum intensity projection coronal mrcp (a), t1-weighted transverse acquisitions with fat saturation after gadolinium chelate injection obtained at arterial (b), portal (c) and delayed phase (d) show biliary irregularities and stenoses (a) associated with intense biliary contrast uptake of the thickened biliary walls at the arterial (white arrows in b) and portal phase (white arrow in c). The common bile duct presents with the same abnormalities (white arrow in d). All complications associated with chronic cholangitis and/or cholelithiasis have been described in patients with lpac syndrome: intrahepatic cholangiocarcinoma (ihcc) (fig . 10), portal hypertension, cholangitis and abscess formation (fig 11), hepatic fibrosis or cirrhosis . Ihcc is a rare primary liver tumour (10 - 20%) [31, 32]. Several risk factors have been identified and differ in western and asian populations: primary sclerosing cholangitis, congenital biliary abnormalities and hepatolithiasis . In most cases, no underlying risk factor is found . Recently, tougeron et al . Reported two cases of ihcc in different and unrelated families with mdr3 deficiency . In both cases, no argument supporting the direct relation between abcb4 mutations and tumorigenesis was found and ihcc may be considered as a consequence of the chronic biliary abnormalities . Genetic polymorphisms in biliary transporters genes have been studied but, to date, no relation has been established between ihcc and abcb4 mutations .fig . Maximum intensity projection coronal mrcp (a), and transverse t2-weighted acquisition show right biliary irregularities and dilated left bile ducts filled with several small intrahepatic stones (white arrow in b). Two years later, t1-weighted transverse acquisitions with fat saturation after gadolinium chelate injection obtained at portal phase (c) and transverse t2-weighted acquisition (d) show an intrahepatic large mass with irregular contrast enhancement (white star). . Transverse t1-weighted contrast enhanced acquisitions (a and b) show small round lesions with peripheral enhancement (arrows) corresponding to abscesses in segment ii (a) and iv (b). The patient previously underwent right hepatectomy for multiple and diffuse bile duct stones severe lpac syndrome with secondary intrahepatic cholangiocarcinoma formation in a 55-year - old woman . Maximum intensity projection coronal mrcp (a), and transverse t2-weighted acquisition show right biliary irregularities and dilated left bile ducts filled with several small intrahepatic stones (white arrow in b). Two years later, t1-weighted transverse acquisitions with fat saturation after gadolinium chelate injection obtained at portal phase (c) and transverse t2-weighted acquisition (d) show an intrahepatic large mass with irregular contrast enhancement (white star). Liver biopsy confirmed the diagnosis of intrahepatic cholangiocarcioma biliary abscess formation in a 30-year - old woman . Transverse t1-weighted contrast enhanced acquisitions (a and b) show small round lesions with peripheral enhancement (arrows) corresponding to abscesses in segment ii (a) and iv (b). It is mainly characterised by intrahepatic lithiasis and, in severe forms, by bile duct dilatations and rarely, secondary cholangitis.
Mast cells, like blood cells, are derived from pluripotent bone marrow hematopoietic stem cells but, unlike blood cells, they leave the bone marrow as progenitors and migrate into virtually all vascularized tissues to complete their differentiation under the influence of factors present at each tissue site . It is the microenvironment surrounding the mast cells that determines their mature phenotype [16]. Mast cells are effector cells of allergic and anaphylactic reactions and play a role in many physiological and pathological processes [7, 8]. Recently, they have gained new importance as immunoregulatory cells with the recognition that they are a major source of cytokines and chemokines and play roles in both innate and adaptive immunities [7, 9, 10]. Although mast cells may be activated by a number of stimuli and pathways [11, 12], the major mechanism for their activation and subsequent degranulation is through the high - affinity receptor for immunoglobulin e (fcri), present in the plasma membrane of mast cells, epidermal langerhans cells, eosinophils, and basophils . Fcri is expressed as a heterotetrameric structure composed of one subunit with an extracellular domain that binds ige, a four - transmembrane - spanning subunit, and two identical disulphide linked subunits [1417]. The subunit serves as an important amplifier of ige and antigen - induced signaling events . Furthermore, the subunits are essential for initiating signaling events downstream of fcri [17, 18]. The carboxyl terminal cytoplasmic domains of both the and subunits contain an immunoreceptor tyrosine - based activation motif (itam), common to all multisubunit immune recognition receptors, that is critical for cell activation . Because the receptor subunits lack any known enzymatic activity, fcri must rely on associated molecules for transducing intracellular signals [16, 19, 20]. Mast cell activation is initiated by the binding of oligomeric antigens to receptor - bound ige, which crosslinks fcri and results in its aggregation . The first recognized biochemical event of the cytoplasmic signal transduction cascade involves phosphorylation, presumably by lyn, of two conserved tyrosine residues within the itams of both and subunits of the receptor . The tyrosine - phosphorylated itams create a novel binding surface that is recognized by additional cytoplasmic signaling molecules, such as the protein tyrosine kinase syk which binds mainly to the subunit, via its tandem src homology 2 (sh2) domains . This interaction results in a conformational change in syk, followed by its activation and autophosphorylation . This results in an increased kinase activity that rapidly shifts the equilibrium of the cell from a resting state (where phosphorylation and dephosphorylation activities are approximately equal) to an activated state (where phosphorylation activity increases exponentially and cannot be counteracted by dephosphorylation). This syk - mediated signal amplification results in a direct or indirect activation of several proteins, including linker for activation of t cells (lat), vav, phospholipase c-1 (plc-1), and plc-2 . Finally, downstream activation results in an increase in intracellular calcium levels, activation of other enzymes and adaptors, and rearrangement of the cytoskeleton that culminates in the release of three classes of mediators: (1) preformed mediators (stored in secretory granules), such as histamine, heparin, -hexosaminidase, neutral proteases, acid hydrolases, major basic protein, carboxypeptidases, and some cytokines and growth factors, (2) newly formed lipid mediators, such as prostaglandins and leukotrienes, and (3) newly synthesized mediators, that include growth factors, cytokines, and chemokines [14, 21, 22]. Accumulating evidence suggests that lipid rafts or raft components play a pivotal role in signal transduction via fcri in mast cells and that the organization of various molecules in lipid rafts could modulate many biological processes in these cells . Lipid rafts, present in all eukaryotic cells, are currently defined as dynamic - ordered nanoscale assemblies of proteins and lipids of the plasma membrane and other intracellular membranes, such as golgi membranes, that associate and dissociate on a subsecond timescale [4, 23, 24]. They contain high levels of cholesterol, sphingolipids (such as sphingomyelin), and gangliosides . Lipid rafts selectively concentrate glycosylphosphatidylinositol- (gpi-) anchored proteins on their outer side and proteins anchored by saturated palmitoyl or myristoyl groups and cholesterol - binding proteins on the cytoplasmic side [2529]. Their lipid composition (figure 1), with a preponderance of longer saturated hydrocarbon chains that potentiate interdigitation between leaflets and favors interaction with cholesterol, allows cholesterol to be tightly intercalated . Lipid rafts are highly organized and probably exist in a liquid - ordered (lo) phase, different from the rest of the plasma membrane which consists mainly of phospholipids (with unsaturated tails) in a liquid - disordered (ld) phase . The cis double bond present on unsaturated lipids introduces a rigid bend in the hydrocarbon tail which interferes with the tight packing and results in less stable aggregates . Lipid rafts are characterized by high melting temperature and a resistance to solubilization in nonionic detergents such as triton x-100, at low temperature . They are dynamic in that both proteins and lipids can move in and out of raft domains with different partitioning kinetics, as well as by coalescing or by breaking up into smaller units . Lipid rafts can also form stabile platforms that are important in signaling, viral infection, and membrane trafficking . Despite a body of evidence supporting the existence of raft domains, the raft concept is still being debated because the mechanisms that govern the associations among sphingolipid, cholesterol, and specific membrane proteins in live cell membranes remain unclear . The controversy is largely due to the lack of standardized methodology for lipid raft studies and the difficulty in proving definitively that rafts exist in living cells without causing significant nonphysiological perturbations by using low temperatures or by extensive cross - linking . The majority of the studies involving lipid rafts begin with detergent solubilization of whole cells followed by sucrose density gradient centrifugation and the recovery of detergent - resistant membranes from the light fractions of the gradient [19, 20]. However, the analysis of density gradient centrifugation experiments remains controversial because there is an indication that detergents may force associations between components that are not colocalized in intact cells . Fractionation results are also known to be severely altered by varying the concentration of triton x-100 [39, 40], by the use of different detergents, [41, 42], or by omission of detergents in general [4345]. Another difficulty has been demonstrating the coexistence of lo and ld phases in live cells . However, technological advances have produced compelling data that self - organization of lipids and proteins can induce subcompartmentalization that organizes the bioactivity of cell membranes . Recently, the lipid - based phase separation into liquid - ordered - like and liquid - disordered - like phases has been seen in giant plasma membrane vesicles (gpmvs) obtained by chemically induced blebbing from cultured cells [46, 47] or by using cell swelling to generate plasma membrane spheres (pms). In 2010, johnson et al . Using gpmvs showed that peripheral protein binding may be a regulator for lateral heterogeneity in vivo . These new approaches are very promising, allowing studies of the lipid domains in the absence of detergents and other perturbations of membrane structure . Advances in imaging and studies with improved integrated methodologies, such as flotation of detergent - resistant membranes, antibody patching and immunofluorescence microscopy, immunoelectron microscopy, chemical crosslinking, single fluorophore tracking microscopy, photonic force microscopy, spectrofluorimetry, mass spectrometry, and fluorescence resonance energy transfer (fret) are now providing insights into the existence and behavior of lipid rafts [2, 16, 24, 5056]. The lipid microdomains are variable in stability, size, shape, lifetime, and molecular composition [29, 37]. Due to differing molecular composition, studies of lipid rafts have also been complicated by imprecise nomenclature . For example, caveolae was synonymous with lipid rafts for many years . In 1998, harder et al ., using a cell system lacking caveolae, demonstrated that raft and nonraft markers segregated in the same cholesterol - dependent way in the absence of caveolae . These results showed that clustered raft markers segregate away from nonraft proteins in a cholesterol - dependent, but caveolin independent manner . Today caveolae membrane rafts in most cell types are enriched with signaling molecules by virtue of the affinity of signaling proteins including transmembrane receptors, gpi anchored proteins, g proteins, rhoa and src kinases for rafts [1, 35]. The number of proteins reported to be regulated by specific lipid interaction is steadily increasing, but the precise structural mechanisms behind specific binding and receptor regulation in membranes remain uncharacterized . A wealth of biochemical and genetic data have lent credence to the notion that raft function as a specialized signaling platform in cell membranes [5965]. Most likely, the function of rafts is aided by stimulation - induced association and recruitment of various molecules with raft affinity, as well as varying degrees of raft engagement with the cytoskeleton [3, 4, 29]. Lipid rafts are also thought to be important sites for protein tyrosine kinase - mediated protein - protein interactions that are involved in the initiation of receptor signaling pathways [5, 6, 16]. It is well known that, in the case of tyrosine kinase receptors, adaptors, scaffolding proteins, and enzymes are recruited to the cytoplasmic side of the plasma membrane as a result of ligand binding to form a signaling complex . If receptor activation takes place in an ordered lipid raft, the signaling complex is protected from other proteins, such as membrane phosphatases, localized in the disordered region of the plasma membrane, that otherwise could affect the signaling process [35, 51, 67]. Lipid rafts are implicated in the function of diverse signaling pathways such as those mediated by growth factors, morphogens, integrins and antigen receptors on immune cells, including mast cells [6871]. The structural basis for the association of fcri with lipid rafts is partially understood and appears to involve the transmembrane segments of fcri and/or subunits . However, the structural features of fcri that mediate the detergent - sensitive interaction with lipid rafts occur selectively but not uniquely with this receptor . Both and subunits are palmitoylated, which could facilitate their association with lipid rafts . Studies have shown that establishing and maintaining lipid rafts is important for many biological processes besides cell signaling [73, 74]. These membrane microdomains have been implicated in such processes as exocytosis, endocytosis, membrane trafficking, and cell adhesion . The structure - function relationship of lipid rafts or rafts constitutes are important in various aspects of mast cell biology . The ability to form lipid rafts appears to be important for maintaining the typical morphology of mast cells . Gangliosides (figure 2), lipid raft components, are complex glycosphingolipids that are ubiquitous membrane constituents [5, 7577] and seem to be structurally important for lipid raft assembly and function . The rigid structural nature of the ceramide anchor in gangliosides, coupled with the ability of sphingolipids to associate with cholesterol, is thought to drive the assembly of lipid rafts [16, 78]. The influence of gangliosides and/or lipid rafts on cell structure and organization was examined using a ganglioside - deficient cell line, d1, and the parent cell line, rbl-2h3, a cell line with homology to mucosal mast cells [8084]. The d1 cell line is deficient in gm1 gangliosides and in mast cell specific -galactosyl derivatives of the ganglioside gd1b . The -galactosyl derivatives of the ganglioside gd1b, antigens i and ii, contain, respectively, one and two additional -galactosyl residues when compared with gd1b . These unique gangliosides are present on the surface of rodent mast cells and are specifically recognized by the monoclonal antibody (mab) aa4 . These gangliosides derived from gd1b have been identified as components of lipid rafts in the plasma membrane of rbl-2h3 cells [86, 87]. The mutant cell line d1 showed a cellular morphology which is distinct from rbl-2h3 cells (figure 3), suggesting that the gangliosides are important in the maintenance of normal cell morphology . The morphological changes observed in d1 cells could be related to the lipid composition of these cells . This cell line presents a large decrease in glycosphingolipids, such as gm1 and the -galactosyl derivatives of the ganglioside gd1b, which may affect many physicochemical properties of the plasma membrane . According to kato et al ., the lipid composition could influence membrane stability, membrane fluidity, lipid packing, bilayer curvature, and hydration elasticity, as well as anchorage of the cytoskeleton to the plasma membrane . Silveira e souza et al . Also observed that the d1 cells showed an abnormal distribution of actin filaments and microtubules . A growing body of evidence indicates that lipid rafts are essential for membrane - cytoskeleton coupling, and the association of lyn and other raft markers with crosslinked fcri is regulated by interactions with f - actin [8991]. It is possible that in the d1 mutant cells, the disorganization of both lipid rafts and actin filaments (figure 4) leads to impaired degranulation after fcri stimulation [79, 87]. Furthermore, the actin cytoskeleton is known to participate in regulating and activating raft - associated signaling events [9294]. The factors that govern the formation of lipid rafts continue to be elucidated, but lipid raft formation often requires actin filaments . The connection between lipid raft proteins and actin filaments can affect the lateral distribution and mobility of these membrane proteins [59, 95]. The extent to which the actin cytoskeleton participates in the formation of membrane rafts is not yet established . Observed that perturbations in the actin filaments (with cytochalasin d and latrunculin a) affect the organization of lipid rafts in rbl-2h3 cells . Importantly, the actin cytoskeleton is a dynamic structure that changes in response to extracellular signals, and it may therefore represent one mechanism for governing the establishment and distribution of lipid rafts in the plasma membrane . Chichili and rodgers showed that lipid rafts may be structured by a synergistic interaction between the cortical actin filaments and the lipid rafts themselves, and that many of the structural and functional properties of rafts require an intact actin cytoskeleton . An important regulator of membrane - cytoskeleton interactions is the phosphoinositide pip2, which is a minor lipid component of the plasma membrane that is known to regulate the organization of the actin cytoskeleton and in particular the formation of actin - membrane linkages . Pip2 also serves as a cofactor for many of the proteins that anchor actin filaments to the plasma membrane [99, 100]. Protein binding to pip2 often occurs through a pip2-specific recognition sequence, in many cases represented by a pip2-specific pleckstrin homology (ph) domain [101103]. Some actin binding proteins (abps) are thought to link actin filaments and pip2-enriched rafts . Microtubules are one of the major determinants of cell shape and polarity [105, 106]. In the ganglioside - deficient d1 cells, the results from this study have demonstrated that the abnormal morphology observed in the mutant cell line could be related to the decrease in gangliosides that leads to lipid raft disorganization . When the concept of lipid rafts and the mobility of proteins in the plasma membrane originated, it was observed that plasma membrane associated proteins could suffer a selective reorganization followed by internalization of these proteins [107109]. Receptor - mediated endocytosis, including endocytosis of fcri, is a temporally and spatially organized process [22, 110]. After activation, crosslinked fcri is endocytosed through clathrin - coated vesicles and transported by the endosomal system for eventual degradation in lysosomes [111113]. In unstimulated mast cells, fcri is dispersed throughout the plasma membrane but upon activation the receptors rapidly aggregate and can be found on the cell surface in lipid rafts in association with gm1 [114, 115], gangliosides derived from gd1b, protein tyrosine kinase lyn and lat, [22, 39, 86]. However, only when the mast cells are activated via fcri does a significant internalization of the gd1b derivatives occur [22, 116]. The endocytosis process itself may play an important role in signal transduction [110, 117]. Oliver et al . Showed that upon activation of fcri, the gangliosides derived from gd1b are internalized together with the receptor, following the same pathway to lysosomes (figure 5). This may facilitate the structural preservation of signaling complexes and the prolongation of the signal since these gangliosides and the fcri are associated in lipid rafts . In view of the importance of lipid raft integrity for efficient receptor endocytosis, it has been observed that the fcri ubiquitination is a key mechanism for the regulation and control of antigen - dependent endocytosis of receptor complexes . Moreover, it has been demonstrated that ubiquitin ligases cbl and nedd4 are recruited into lipid rafts upon ige triggered cell signaling . The ubiquitin cbl is a good candidate to mediate fcri ubiquitination since it participates in various functions such as cis - and - trans - ubiquitination . [124, 125] suggested that the recruitment of engaged fcri subunits into lipid rafts precedes their ubiquitination, and that integrity of lipid rafts is required for receptor ubiquitination and endocytosis, contributing to the down - regulation of fcri - mediated signaling . In mast cells, the first signaling complex convincingly shown to involve lipid rafts was immunoglobulin e (ige). Ige signaling was initially thought to be based on protein - protein interactions alone, but several observations indicated that lipid rafts are involved in this process [37, 68, 126131]. The first hint came from the finding that fcri is soluble in triton x-100 at steady state but becomes insoluble in low concentrations of this detergent after crosslinking . Moreover, in unstimulated cells, fcri is dispersed throughout the plasma membrane, but upon activation rapidly aggregates [115, 132] and can then be found on the cell surface in association with the ganglioside gm1 [57, 114, 133] and gpi - anchored proteins [89, 134]. Despite numerous studies on mast cell activation through fcri, the detailed mechanism by which cross - linking promotes the initial phosphorylation by lyn and the molecular mechanisms for lyn activation are still unclear [67, 77, 135, 136]. Davey et al . Suggested that protein - protein interaction (ige - fcri cross - linking) recruits essential signaling proteins and lipid molecules into more ordered domains that serve as a platform for signaling . An approach intensively used to better understand the role of lipid rafts in fcri - mediated signaling has been the study and/or the manipulation of the lipid constituents of rafts, such as cholesterol and gangliosides [16, 87, 136]. Methyl--cyclodextrin (mcd), a carbohydrate molecule with a pocket for binding cholesterol, is extensively used to deplete the surface cholesterol and subsequently disrupt lipid rafts . Mcd has been used to study the role of lipid rafts in fcri - mediated signaling, particularly in early events of signal transduction such as tyrosine phosphorylation of fcri by lyn . Sheets et al . Have demonstrated that phosphorylation of fcri proceeds in a cholesterol - dependent manner and that cholesterol depletion reduces stimulated tyrosine phosphorylation of fcri . In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated fcri and lyn in intact cells . Cholesterol repletion restores receptor phosphorylation together with the structural interactions, providing strong evidence that lipid raft structure, maintained by cholesterol, plays a critical role in the initiation of fcri signaling . Cholesterol depletion by mcd in rbl-2h3 cells also reduced the release of -hexosaminidase activity in cells stimulated via fcri [87, 88, 137, 138]. These data suggest that the cholesterol depletion by mcd affects the ige signaling due to the disruption of lipid rafts and consequently results in a failure to form a signaling complex . Moreover, young et al . Showed evidence that lyn isolated in lipid rafts has substantially higher lyn kinase activity than lyn outside of these membrane microdomains . These data suggest that some unknown components in lipid rafts may influence the kinase activity of lyn and subsequently fcri signal transduction . It was initially identified as a caveolae - associated membrane protein and is a marker protein of lipid rafts, but its physiological role is still not clear . Knockdown rbl-2h3 cells showed that flotillin-1 regulates the kinase activity of lyn in mast cells . In the flotillin-1 knockdown cells, there was a significant decrease in ca mobilization, the phosphorylation of erks, tyrosine phosphorylation of the -subunit of fcri, and ige - mediated degranulation . This study also showed that flotillin-1 is constitutively associated with lyn in lipid rafts in rbl-2h3 cells, and that antigen stimulation induced an increase in flotillin-1 binding to lyn, resulting in enhancement of the kinase activity of lyn . These data suggest that this raft protein is an important component of fcri - mediated mast cell activation and regulates the kinase activity of lyn in lipid rafts . The -galactosyl derivatives of the gangliosides gd1b also seem to be intimately involved with signaling through fcri . Although the functional role of these gangliosides is not clear, previous studies have shown that when the -galactosyl derivatives of ganglioside gd1b are bound by mab aa4, histamine release was inhibited in a time- and concentration - dependent manner . Binding of mab aa4 to rbl-2h3 cells resulted in an increase in intracellular calcium, phosphatidylinositol hydrolysis, and a redistribution of pkc . However, the magnitude of these changes was less than those after fcri aggregation, and unlike fcri activation, these changes were not accompanied by histamine release . The derivatives of the ganglioside gd1b coprecipitated with the src family tyrosine kinase lyn and that in spite of the fact that mab aa4 binds to sites close to fcri the association between lyn and these gangliosides was not mediated by fcri . The association of lyn with these gangliosides is much stronger than the association of lyn with fcri . These associations suggest that a complex of molecules that includes gangliosides, fcri, and lyn is essential for modulation of signal transduction in mast cells [81, 141144]. Furthermore, analysis of the subcellular distribution of the gangliosides recognized by mab aa4 and of fcri on sucrose gradients showed that, following fcri activation, there was a shift in the distribution of the gangliosides to the lipid raft fractions [22, 87]. The movement of these gangliosides into the lipid rafts may be another mechanism that regulates signal transduction in mast cells . As previously stated, using a cell line deficient in the -galactosyl derivatives of ganglioside gd1b, as well as the parent cell line, rbl-2h3, silveira e souza et al . Demonstrated and confirmed the importance of these gangliosides for lipid raft organization and consequently for fcri - mediated degranulation in rodent mast cells . In this study, the authors observed a decreased release of -hexosaminidase activity in the mutant cell line after fcri stimulation, but not after exposure to calcium ionophore . These results show that release of -hexosaminidase activity is calcium - dependent and furthermore indicated that the mutant cell line possesses the capacity to degranulate . Moreover, reduced release of -hexosaminidase activity in rbl-2h3 cells treated with compounds that inhibit ganglioside synthesis was also observed . In addition to lipid raft assembly, another possible role for the mast cell - specific gangliosides in signal transduction could be to facilitate the association of lyn with fcri . Because fcri itself has no intrinsic kinase activity, the tyrosine phosphorylations induced by receptor cross - linking could be a secondary event that occurs after aggregation of fcri and its movement into lipid rafts . Therefore, these lipid raft complexes that include gangliosides, associated proteins, such as lyn, lat, flotillin-1 and fcri, have an important role in receptor - mediated signal transduction . Recently, fifadara et al . Reported that mast cells produce structures such as cytonemes or tunneling nanotubes used for intercellular communication and that intercellular communication may be important during allergic and inflammatory responses following costimulation of fcri and ccr1 . Albeit the process of cytoneme formation remains poorly understood, the fact that cholesterol depletion reduced the formation of cytonemes suggests that lipid rafts may participate in cytoneme formation in mast cells, either by promoting membrane integrity or by participating in cell signaling . The expression of the -galactosyl derivatives of the ganglioside gd1b on the mast cell surface also appears to be related to mast cell development and recruitment . Previous studies using mab aa4 showed that the -galactosyl derivatives of the ganglioside gd1b were present only in mast cells and not in any other cell type in all 23 rat tissues examined [81, 85]. However, in bone marrow, a population of large, poorly differentiated cells, presumably immature mast cells were also stained with mab aa4 . Later these cells were indeed shown to be very immature and immature mast cells [145, 146]. Since the heterogeneity of the maturing mast cells makes them impossible to separate from other cells on the basis of their density and mab aa4 binds only to cells which can be identified as mast cells [146, 147], the gangliosides recognized by mab aa4 may be considered a powerful marker for rodent mast cells . The ability to characterize the maturation of bone marrow - derived and peritoneal mast cells has been impaired both by the lack of mast cell - specific markers and by the inability to rapidly and efficiently separate mast cells in all stages of maturation from a mixed population of cells . They then went on to isolate and characterize bone marrow mast cells at various stages of maturation . In this study, the very immature mast cells, which had not been previously described, were identified by the presence of the derivatives of the ganglioside gd1b on their surface . These cells which could not be recognized as mast cells by standard cytological methods contained only a few small cytoplasmic granules . On the other hand, undifferentiated mast cell precursors in the bone marrow these gangliosides begin to be expressed on the cell surface jointly with fcri and at the same time as the initiation of the formation of cytoplasmic granules in very immature mast cells . The gangliosides derived from gd1b continue to be expressed by mast cells in all stages of maturation . These data suggest that mast cell lipid rafts or raft constitutes are related to mast cell maturation and function . Several aspects of raft structure and function in mast cell biology still need to be elucidated . Undoubtedly, lipid rafts and their constitutes play a role in many aspects of mast cell biology, such as activation through fcri, morphology, endocytosis, and maturation . Further research to better define the role of lipid rafts in mast cells could offer novel targets for immunotherapies and treatment of diseases in which mast cells and/or their mediators are involved.
Http://www.ncbi.nlm.nih.gov/sra/erx162128 http://www.ncbi.nlm.nih.gov/sra/erx162129 http://www.ncbi.nlm.nih.gov/sra/erx162130 http://www.ncbi.nlm.nih.gov/sra/erx162131 http://www.ncbi.nlm.nih.gov/sra/erx162139 http://www.ncbi.nlm.nih.gov/sra/erx162134 http://www.ncbi.nlm.nih.gov/sra/erx162140 http://www.ncbi.nlm.nih.gov/sra/erx162141 http://www.ncbi.nlm.nih.gov/sra/erx197218 http://www.ncbi.nlm.nih.gov/sra/erx197219 http://www.ncbi.nlm.nih.gov/sra/erx197220 http://www.ncbi.nlm.nih.gov/sra/erx197221 http://www.ncbi.nlm.nih.gov/sra/erx162158 http://www.ncbi.nlm.nih.gov/sra/erx162159 http://www.ncbi.nlm.nih.gov/sra/erx162160 http://www.ncbi.nlm.nih.gov/sra/erx162161 http://www.ncbi.nlm.nih.gov/sra/erx162176 http://www.ncbi.nlm.nih.gov/sra/erx162164 http://www.ncbi.nlm.nih.gov/sra/erx162165 http://www.ncbi.nlm.nih.gov/sra/erx162166 http://www.ncbi.nlm.nih.gov/sra/erx162167 http://www.ncbi.nlm.nih.gov/sra/erx162168 http://www.ncbi.nlm.nih.gov/sra/erx162169 http://www.ncbi.nlm.nih.gov/sra/erx162177 ruminants contribute to a significant proportion of the domesticated animal species worldwide, and are the best adapted to the utilization and digestion of the plant cell wall . Nutrition to an animal offers a means of making rapid change in milk composition i.e., concentration of milk fat, where the amount of roughage, forage: concentrate ratio, carbohydrate composition are the key factors to be taken cared of . Improvement in the ability of the rumen microbiota to degrade plant cell wall is generally highly desirable and usually leads to improved animal performance . The understanding of the carbohydrate utilization in ruminants is now expanding with the advent in molecular technologies . The culture dependent methods have remain pitfall in explaining microbial metabolism carried out in the rumen . After the foundation of the term metagenomics, the recent developments in sequencing technologies have allowed the researchers to reach the deeper layer of the microbial community . Here in the present study, four animals were given three different treatments where the dry roughage to concentrate ratio was changed (50%:50%, 75%:25% and 100%). In the first treatment (m1), 50% roughage and 50% concentrate was given as diet . The second treatment (m2) consists of 75% roughage and 25% concentrate . Whereas third treatment (m3) consisted 100% roughage . After the end of the six weeks of each treatment, the rumen sample was collected using stomach tube and then filtered using muslin cloth to separate liquid and solid fractions . In total 24 samples (4 animals 3 treatments 2 fractions = 24 samples) were collected and stored at 20 c . 300 ng of total dna was used for library preparation of ion torrent pgm platform . A total of 24 barcoded libraries were prepared and sequencing run was carried out on 316 chips of ion torrent pgm platform . The sequencing data are available at ncbi with sra experiment ids (erx162128, erx162129, erx162130, erx162131, erx162139, erx162134, erx162140, erx162141, erx197218, erx197219, erx197220, erx197221, erx162158, erx162159, erx162160, erx162161, erx162176, erx162164, erx162165, erx162166, erx162167, erx162168, erx162169, erx162177).the sequencing data were also uploaded to mg - rast web server . The functional classification of the sequences was done by using seed database at 80% identity cutoff . The subsystem classification was carried out at level 1, the major category from which was further sub - classified at level 2 for each metagenome groups . In the first treatment (m1), 50% roughage and 50% concentrate was given as diet . The second treatment (m2) consists of 75% roughage and 25% concentrate . Whereas third treatment (m3) consisted 100% roughage . After the end of the six weeks of each treatment, the rumen sample was collected using stomach tube and then filtered using muslin cloth to separate liquid and solid fractions . In total 24 samples (4 animals 3 treatments 2 fractions = 24 samples) were collected and stored at 20 c . 300 ng of total dna was used for library preparation of ion torrent pgm platform . A total of 24 barcoded libraries were prepared and sequencing run was carried out on 316 chips of ion torrent pgm platform . The sequencing data are available at ncbi with sra experiment ids (erx162128, erx162129, erx162130, erx162131, erx162139, erx162134, erx162140, erx162141, erx197218, erx197219, erx197220, erx197221, erx162158, erx162159, erx162160, erx162161, erx162176, erx162164, erx162165, erx162166, erx162167, erx162168, erx162169, erx162177).the sequencing data were also uploaded to mg - rast web server . The functional classification of the sequences was done by using seed database at 80% identity cutoff . The subsystem classification was carried out at level 1, the major category from which was further sub - classified at level 2 for each metagenome groups . The assignment of the sequences to seed database revealed that at subsystem level, out of the 24 subsystems identified, the genes associated with carbohydrate metabolism were found to be most abundant in each metagenome groups (fig . So, that particular category of subsystem was sub - classified at further level . By sub - classifying, we found that the genes associated with sugar utilization in thermogales were more abundant for both liquid and solid fractions (table 1, table 2). This particular category possesses the enzymes involved in formation of the system for sugar transport and its utilization . The second most dominant category found in our dataset was one carbon metabolism which included the genes involved in serine - glyoxylate cycle . It was found to be increased with the increment in the roughage for liquid fraction (m1dl = 8.11%, m2dl = 8.59%, m3dl = 8.74%) whereas, for solid fraction it was found to be in similar abundance for m2ds and m3ds (7.92%) as compared to m1ds group (7.65%). The category that belonged to enzymes for di and oligo saccharide utilization was found which is composed by the genes related to maltose / maltodextrin utilization, xylose utilization, l - rhamnose utilization, l - arabinose utilization, lactose utilization, mannose utilization etc . The genes linked to maltose utilization decreased in both the fractions with the increment in the roughage . Whereas, the genes associated with the mannose and xylose utilization increased with the increment in the roughage in both the fractions . The genes predicted for carbohydrate hydrolases decreased with the increment in the roughage for liquid fraction and increased in case of the solid fraction . Moreover, the genes related to dehydrogenase complexes increased with the increment in the roughage for both the fractions . As observed by brulc et al ., the bovine rumen harboured the functional genes associated with carbohydrate utilization . A study by wang et al . Revealed that majority of the genes were associated with the carbohydrate metabolism (11%). In the present study, the genes involved in the sugar utilization were observed to be predominated . This finding shows that the ruminal microbiota starts acting on the ingested food within 2 h of the feeding and start materializing that food into sugars to be taken up by the host . Moreover, we found the glycolysis and gluconeogenesis related enzymes both for bacteria and archaea that increased with the increment in the roughages . Glycolysis is the most common pathway for the conversion of glucose-6-p into pyruvate which generates atp and metabolites for essential cellular processes . Whereas, essential glycolytic intermediates are synthesized via gluconeogenesis, the process which is reversion of glycolysis . However, this particular pathway is found to be well conserved in the bacteria and eukaryotes, the archaea have developed unique variants for this pathway which includes zero or very low atp yields; reduction of ferredoxin rather than nadh; many unusual glycolytic enzymes, including adp - dependent gluco- and phosphofructo - kinases, non - orthologous pgms, fbas, non - phosphorylating gap dehydrogenases, etc . . In the present study, the enzymes involved in this pathway were found to be more or less similar in their abundance in both bacteria and archaea . In the present study, the genes involved in maltose utilization decreased with the increment in the roughage, which correlates with the fact that with the increment in roughage and decrease in starch rich concentrate, the maltose and maltodextrin utilization decreases, as they are produced after partial hydrolysis of starch . It has been found that prevotella ruminicola is a major amylolytic organism in the rumen and produces high amylases, which cleaves starch into maltose and maltodextrins . A previous study, done by, also showed that the abundance of genus prevotella decreases with the increment in the dry roughage proportion . On the other hand, the abundance of the enzymes involved in pentose sugar utilization like xylose and arabinoses increases with the increment in the roughages . It has been found that ruminococcus albus is an important fibrolytic ruminal bacteria which degrades hemicelluloses and ferments them to pentose sugars . As per our previous study in the same breed, we have found that the abundance of ruminococcus genus increases with the increment in the roughage . Thus, this study provides the deep insights into the metabolism carried out by ruminal bacteria and its functional relevance . The information gained by this study can be helpful in developing the diet formulation for the better nutrition of livestock.
Culicoides spp . Latreille (diptera: ceratopogonidae) or biting midges are small (0.5 - 2.0 mm in length) bloodsucking insects that have a broad distribution and impact on human and veterinary health as vectors of viruses (bluetongue, african horse sickness, epizootic hemorrhagic disease of deer, akabane, aino, chuzan, and bovine ephemeral fever viruses), protozoan parasites (haemoproteus and leucocytozoon spp . ), and filarial worms (onchocerca and dipetalonema spp .) Throughout their range [1 - 15]. The first nationwide biting midge surveys in the republic of korea (rok) were reported in 1974, with seasonal abundance and host blood meal analysis of culicoides spp . Collected from cattle and poultry farms in gyeonggi - do (province) more recently, a brief summary of the history of research into the culicoides fauna and seasonal prevalence of culicoides spp . Collected at 9 cowsheds in the southern part of the rok were reported by kim et al . Along with new records of 3 culicoides spp . (c. nasuensis kitaoka, c. pallidulus yu, and c. jacobsoni macfie), bringing the total number of culicoides spp . Vector - borne disease surveillance is conducted by the 65th medical brigade in collaboration with the public health command region - pacific (phcr - pac) to determine the species composition and seasonal and geographical distributions for mosquitoes and other biting flies, e.g., culicoides spp ., and pathogens they harbor using mosquito magnet traps in / near the demilitarized zone (dmz) that separates north and south korea in northern gyeonggi - do, rok ., lititz, pennsylvania, usa) use counter - flow technology and propane gas to produce heat and co2 as attractants to capture mosquitoes and other biting flies, such as culicoides . Collections were made at 4 sites, including the neutral nations supervisory commission (nnsc) camp (37 57' 16.39 " n, 26 40' 50.03 " e), daeseongdong village (37 56' 26.92 " n, 126 40' 37.42 " e) inside the 2-km wide demilitarized zone (dmz) and adjacent to the military demarcation line (mdl) that separates north and south korea and at warrior base, a us army training site (37 55' 17.01 " n, 126 44' 30.22 " e), and a beef farm at tongilchon (37 54' 32.18 " n, 126 44' 01.88 " e) located north of the imjin river and approximately 3 km from the dmz, gyeonggi - do, rok, from may - october 2010 - 2012 (fig . Daeseongdong is a village of approximately 200 residents that is under the protection of south korea and is approximately 100 - 200 m from the mdl . Rice farming is the principal activity, with an enclosed (not pastured) beef farm of <50 cattle . The mosquito magnet was placed at the northern most edge of daeseongdong and approximately 100 m north of the beef farm . Warrior base is a us military training site approximately 3 km south of the dmz . Tongilchon is a small village of approximately 200 residents with 2 enclosed (not pastured) beef farms of approximately 50 cattle each . It is located approximately 2 km west of warrior base and 3 km from the dmz . The mosquito magnet was placed outside one of the beef farms adjacent to a rice paddy and dry land vegetable farming . Mosquito magnet traps were operated continuously, with specimens collected twice weekly from may - october in 2010 - 2012 . Specimens were transported to the 5th medical detachment, us army garrison, yongsan, seoul, rok, where mosquitoes and other biting flies were separated . Specimens of culicoides were transferred into 70% ethanol and identified using the keys of arnaud and the checklist of bellis et al . . Voucher specimens were submitted to the northern territory quarantine insect collection (ntqic) in darwin, northern territory, australia, for future reference and genetic studies . A total of 18,677 culicoides females (18,399; 98.7%) and males (248; 1.3%) comprising 16 species were collected from may - october over a 3-year period from 2010 - 2012 . Culicoides nipponensis tokunaga was the most commonly collected species (42.9%), followed by culicoides erairai kono and takahasi (29.2%), culicoides punctatus (meigen) (20.3%), culicoides arakawae (arakawa) (3.3%), c. pallidulus (1.8%), and culicoides circumscriptus kieffer (1.4%), while the remaining 10 species accounted for only 1.1% of all culicoides spp . Collected (table 1). C. nipponensis was the most frequently collected species (females and males) at daeseongdong village (67.1%), and was commonly collected from the nnsc camp (35.6%), tongilchon (33.9%), and warrior base (19.5%). C. erairai was the most frequently collected species at nnsc camp (53.6%) and accounted for 85.1% of c. erairai collected at all 4 sites (table 1), while c. punctatus was the most frequently collected species at both tongilchon (54.4%) and warrior base (28.7%). C. nipponensis females demonstrated bimodal capture rates, with large numbers collected during may (1,754, 82.4%), june (1,479, 29.5%), and then again during september (3,332, 81.7%) (table 2). C. erairai was more frequently collected during june (2,962, 59.2%) and july (2,154, 76.0%) followed by a sharp decline in numbers collected from august - october . C. punctatus was collected infrequently from may - september (range, 84 - 372) with large numbers only collected during october (2,756, 88.0%). Overall, low numbers of c. nipponensis were collected during 2011, while higher numbers were observed during may - june and again during september 2012 at the nnsc camp and daeseongdong (fig . 2). C. erairai was collected more frequently at the nnsc camp during the months of june - july 2011 and 2012, with few collected from august - october for either year . Low numbers of c. punctatus were collected from may - september, with a sharp increases in numbers observed primarily at the beef farm at tongilchon during october for both 2011 and 2012 . This is the first report for collections of culicoides using mosquito magnet traps that use only heat and co2 as attractants . The addition of other chemical attractants, e.g., octenol, has been shown to repel some species of culicine mosquitoes, while enhancing the collection of others, but the relative attractiveness of this attractant has not been investigated for most species of culicoides . A major difference between mosquito magnet and light traps is that mosquito magnet traps operate continuously without bias toward nocturnal and diurnal active species . This difference, however, indicates that comparative analysis between data obtained from mosquito magnet and light traps should be viewed with caution . Biting midges are both nuisance biters and vectors of medical and veterinary important pathogens that impact the economy of the animal industry in the rok . C. nipponensis, and c. punctatus were the most commonly collected species by mosquito magnets during this study and have been associated with the transmission of important livestock pathogens, e. g., fukuoka, aino, and ibaraki viruses, or as biting pests of humans (c. erairai). Consequently, surveillance of these and other species of culicoides and associated pathogens are an important part of the veterinary health service to identify relative vector populations, their distributions throughout the rok, and the presence and prevalence of pathogens affecting livestock and poultry . The dominance of c. nipponensis and c. punctatus in the mosquito magnet collections is similar to findings by cho and chong and kang and yu using new jersey light traps . Similarly, lee found that c. punctatus, c. arakawae, and c. tainanus kieffer were the most commonly collected species at inland sites, whereas c. nipponensis and c. sinanoensis tokunaga were the predominant species collected from the south and eastern coastal areas in the rok . Recently kim et al . Found that c. punctatus and c. arakawae were the dominant species collected from light traps set in 9 cowsheds in the southern part of the rok . The unprecedented predominance of c. erairai and relatively low numbers of c. arakawae in the mosquito magnet traps compared to these and other previous reports may reflect the relative attraction of these species to light and the attractants associated with the mosquito magnet traps . The dominance of c. nipponensis and c. punctatus at a cowshed at tongilchon agrees with the host preference of these species for cattle . Although c. arakawae appears to have a preference for feeding on birds, it also readily feeds on cattle, which would explain its abundance around cowsheds in other reports but not the low numbers collected at the cowshed at tongilchon in this survey . The variable distribution of culicoides spp . Over a limited area (2 km radius) demonstrates the focal species abundance due to little understood environmental and host attraction factors . For example, high numbers of c. erairai were observed at the nnsc camp where humans are believed to be the primary host, while c. nipponensis was the primary species collected at daeseongdong and tongilchon where a variety of hosts (e.g., cattle, dogs, large water birds, and humans) serve as potential hosts . Thus, further studies comparing light and mosquito magnet traps and range of available hosts may reveal differential attraction for different species and assist in surveys targeting particular species . Additionally, studies on the geographical and seasonal distributions, host attraction (e.g., placement of traps near human habitation and poultry, cattle, and swine farms), temporal biting activity, pathogen infection rates, and their role as potential vectors of zoonotic pathogens that impact on human and animal health are warranted . Although limited, the data presented herein provide a better understanding of the biology, ecology, and environmental parameters that affect relative population abundance of culicoides spp . That can be used to predict potential human and animal health risks and develop and implement mitigation strategies.
The esthetic demand for restorative treatments that reestablish natural teeth s aspects is a major concern in dentistry.1 among the available options, ceramic restorations are an excellent alternative because of advantages, such as aesthetic, biocompatibility, chemical durability, fluorescence, compression and wear resistance, and thermal expansion coefficient similar to the dental structure.25 advances in resin - based cements and adhesive systems technology allowed the increase of clinical applications of all - ceramic restorations.6,7 replicating the appearance of natural tooth structure requires careful control of the form, surface texture, translucency, and color of the restoration.8 laboratory procedures that involve ceramic brand and batches,810 ceramic firing temperature and number of firings, and the condensation technique1113 also could affect the final shade of the porcelain . Therefore, the color of the ceramic chosen may not correspond to the exact color of the tooth.14 laboratory procedures involved in the construction of porcelain inlays, onlays, all - ceramic crowns, and veneer restorations demand the use of refractory dies of dental phosphate - bonded investment materials that are used to build, sinter, cast, and press these types of restorations.7,15,16 when fabricating metal - free feldspathic ceramic restorations with the refractory die technique, the application and high burning temperature of the ceramic are accomplished in direct contact with dental phosphate - bonded refractory die products . 7,15 therefore, the ceramic is subjected to the influence of the investment according to its composition, surface, and physical - chemical properties.17,18 although several studies have analyzed the mechanical properties of refractory phosphate - bonded investment materials,16,2025 investment influence on the final color of ceramics remains unclear17,18,26 and deserves further investigation . Because color is considered a sensation obtained through proprioceptive mechanisms, and thus considered subjective, standardized colorimetric techniques were developed with the objective of transforming colors in numeric values.8 for this aim, the cie lab system of colors was created,19 eliminating inconsistencies inherent in color perception and specification among observers.8 this type of evaluation becomes important as the investment / ceramic relationship constitutes the base of the process of manufacturing ceramic restorations and is directly related to the final aesthetic quality of this type of restoration.7,15 the aim of this study was to evaluate the influence of investment type on the color of various conventional powder - slurry feldspathic ceramics . The null hypothesis tested assumed that there would be no color alteration in the ceramics for all investments tested . An auto - polymerized acrylic resin mold was machined as a master die for the fabrication of multiple specimens and duplicated with an elastic jelly (duplicator vipi, pirassununga, sao paulo, brazil). Each investment was placed into the jelly moulds, obtaining cylindrical refractory dies, with a central depression with a diameter of 11 mm and thickness of 1.5 mm, where the ceramics were applied and fired, simulating a refractory die for fabrication of ceramic restorations . The investment was removed from the elastic jelly moulds after 1 hour to acquire appropriate resistance . The investment materials were mixed in a vacuum investor (model a 300; polidental, pelotas, rs, brazil) with speed time, liquid, and powder proportions recommended by the manufacturers (table 1). After setting, the refractory dies were placed into a dental laboratory burnout furnace (model edgcon 5p; edg equipments, so paulo, brazil) at room temperature and heated at different rates (c / min) according to ceramics manufacturers instructions (table 1). Five feldspathic ceramics shade a3 (n=20; table 2) were applied on the refractory dies of the four investment materials according to the manufacturers recommendations, resulting in 25 refractory dies for each investment, with 5 refractory dies for each ceramic evaluated . The discs were fired in a ceramic furnace (titan 99, edg equipments ltda, so paulo, brazil). Refractory dies were cooled and manually divested, and the internal surfaces were airborne - particle abraded (microjet iii; edg equipments ltda, so paulo, brazil) with 50-m aluminum oxide abrasive (aluminum oxide 50; almet gmbh, fellbach, germany). Ernst vetter gmbh, pforzheim, germany) and glazed according to manufacturers recommendations . For the control specimens, five ceramic specimens were fabricated with each ceramic using a circular stainless steel matrix, with a diameter of 11 mm and thickness of 1.5 mm without contact with any investment material . Specimens were removed from the matrix and placed in the porcelain - firing oven, fired, polished with silicone polishers, and glazed, as previously described . The evaluation of the color parameters was accomplished through the cie lab system of colors using a colorimeter (colour - guide, byk - gardner, columbia, md, usa). To simulate the color of an underlying dental structure, a background disc with 30-mm diameter was made with resin composite (filtek z-250, colour a3; 3m - espe, st paul, mn, usa). In the cie lab system, the place of a color in the space is defined through 3 coordinates: l , a , and b. The l coordinate is a measure of the lightness - darkness of the specimen; therefore, the greater the l , the lighter the specimen . The a coordinate is a measure of the chroma along the red - green axis . A positive a * relates to the amount of redness, and a negative a * relates to the amount of green of the specimen . The b * coordinate is a measure of the chroma along the yellow - blue axis, where a positive b * relates to the amount of yellowness, and a negative b * relates to the amount of blue of the specimen . The measure of the total difference of color among 2 objects is described by e . The formula used to calculate the e was: eab * = [(l ) 2 + (a ) 2 + (b ) 2]1/2 . The e values are graded as follows: e <1 = not appreciable, e <2 = clinically acceptable, e> 2 = clinically unacceptable, and e> 3.7 = clinically unacceptable with very poor match.2729 the lab color notation of each specimen was measured thrice consecutively, and an average was calculated to give the initial color of all substrates . The formula used to calculate the lightness is: l * = lf li, with f representing the final value of l * and i the initial value of l * (control group). Statistical analyses were done, with the significance level fixed at 5%, and analyzed by two - way anova, followed by tukey post hoc test . The influence of the different investments was analyzed on the final color of the ceramic specimens . An auto - polymerized acrylic resin mold was machined as a master die for the fabrication of multiple specimens and duplicated with an elastic jelly (duplicator vipi, pirassununga, sao paulo, brazil). Each investment was placed into the jelly moulds, obtaining cylindrical refractory dies, with a central depression with a diameter of 11 mm and thickness of 1.5 mm, where the ceramics were applied and fired, simulating a refractory die for fabrication of ceramic restorations . The investment was removed from the elastic jelly moulds after 1 hour to acquire appropriate resistance . The investment materials were mixed in a vacuum investor (model a 300; polidental, pelotas, rs, brazil) with speed time, liquid, and powder proportions recommended by the manufacturers (table 1). After setting, the refractory dies were placed into a dental laboratory burnout furnace (model edgcon 5p; edg equipments, so paulo, brazil) at room temperature and heated at different rates (c / min) according to ceramics manufacturers instructions (table 1). Five feldspathic ceramics shade a3 (n=20; table 2) were applied on the refractory dies of the four investment materials according to the manufacturers recommendations, resulting in 25 refractory dies for each investment, with 5 refractory dies for each ceramic evaluated . The discs were fired in a ceramic furnace (titan 99, edg equipments ltda, so paulo, brazil). Refractory dies were cooled and manually divested, and the internal surfaces were airborne - particle abraded (microjet iii; edg equipments ltda, so paulo, brazil) with 50-m aluminum oxide abrasive (aluminum oxide 50; almet gmbh, fellbach, germany). Ernst vetter gmbh, pforzheim, germany) and glazed according to manufacturers recommendations . For the control specimens, five ceramic specimens were fabricated with each ceramic using a circular stainless steel matrix, with a diameter of 11 mm and thickness of 1.5 mm without contact with any investment material . Specimens were removed from the matrix and placed in the porcelain - firing oven, fired, polished with silicone polishers, and glazed, as previously described . The evaluation of the color parameters was accomplished through the cie lab system of colors using a colorimeter (colour - guide, byk - gardner, columbia, md, usa). To simulate the color of an underlying dental structure, a background disc with 30-mm diameter was made with resin composite (filtek z-250, colour a3; 3m - espe, st paul, mn, usa). In the cie lab system, the place of a color in the space is defined through 3 coordinates: l , a , and b. The l coordinate is a measure of the lightness - darkness of the specimen; therefore, the greater the l , the lighter the specimen . The a coordinate is a measure of the chroma along the red - green axis . A positive a * relates to the amount of redness, and a negative a * relates to the amount of green of the specimen . The b * coordinate is a measure of the chroma along the yellow - blue axis, where a positive b * relates to the amount of yellowness, and a negative b * relates to the amount of blue of the specimen . The measure of the total difference of color among 2 objects is described by e . The formula used to calculate the e was: eab * = [(l ) 2 + (a ) 2 + (b ) 2]1/2 . The e values are graded as follows: e <1 = not appreciable, e <2 = clinically acceptable, e> 2 = clinically unacceptable, and e> 3.7 = clinically unacceptable with very poor match.2729 the lab color notation of each specimen was measured thrice consecutively, and an average was calculated to give the initial color of all substrates . The formula used to calculate the lightness is: l * = lf li, with f representing the final value of l * and i the initial value of l * (control group). Statistical analyses were done, with the significance level fixed at 5%, and analyzed by two - way anova, followed by tukey post hoc test . The influence of the different investments was analyzed on the final color of the ceramic specimens . The amount of alterations in a , b , and l * color parameters resulted in e chromatic alterations of the tested ceramics (table 3). Vision esthetic ceramic was the only ceramic to present statistically significant differences (p=.0001) in all color parameters (a , b , and l ) among the control and investments tested .. the exception for vision esthetic ceramic was for the investments fortune and vitadurvest for b and duravest for a , with no statistical difference (p>.05). Significant differences were detected for the ceramic vintage halo in a color parameter among the control group and duravest (p=.0009) and fortune (p=.0001) investments, with no statistical differences between the two investments . For vitadur alpha ceramic, a significant color difference also was found between the control group and fortune investment (p=.0029) for l * and b * color parameters, and control and duravest investment (p=.008) for b * color parameter . When we considered the control groups alone, without investments, significant differences were detected for both color parameters a * and b * between ceramics ips classic and vitadur alpha (p=.0155), and ips classic and vintage halo (p=.0015). The same trend also has occurred for vintage halo and vitadur alpha (p=.0001) and vintage halo and super porcelain ex-3 (p=.0001). When considering a * values only, comparison between ips classic and super porcelain ex-3 presented significant differences (p=.0001). Additionally, b * values also were different between ips classic and vision esthetic (p<.0001), and vintage halo and vision esthetic (p=.0001). For l * values, statistically significant differences were found between vitadur alpha and super porcelain ex-3 (p=.0526), and vision esthetic (p=.0179) and vintage halo (p=.001) (figures 1, 2, and 3). There is a concern on the final color of ceramics commonly used for the fabrication of dental veneers, that is, to allow a clinically stable and aesthetic final restoration . The results of this study support the rejection of the null hypothesis because statistical differences were observed in the color parameters of the ceramics promoted by the investment materials tested . In this study, the color of vision esthetic was overall influenced by all refractory investments used, resulting in color alterations considered unacceptable, as e values were higher than the visually perceptible limit (3.7 units).2729 in general, the ceramic specimens were less red and less yellow because of a decrease in the values of color parameters a * and b , respectively, when compared with the control group . Regarding the lightness variation, the l values for this ceramic were negative (figure 4), resulting in a darker color . Ceramic specimens of vintage halo using duravest and fortune were less red considering the small decrease in the a * values when compared with the control group . Vitadur alpha underwent significant alterations in a * and l * parameters when fortune was used . However, the alterations were restricted to b * values and only when duravest was used . The use of fortune resulted in more red and lighter ceramic discs (increase in parameters a * and l , figure 4) when compared with the control group . On the other hand, duravest resulted in less yellow ceramic discs (decreased b values) when compared with the control group . These findings support the idea that the color of ceramic is dependent on the choice of the investment material; therefore, a significant influence of the investment can be expected on the color of the ceramics, resulting in chromatic alterations that may lead to clinically unacceptable results . Although phosphate - bonded dental casting investment materials have been successfully used in dentistry for more than 40 years, they still present characteristics that remain unclear.24 dental refractory die products are used to construct models and dies for casting dental alloys and ceramic restorations.16 a variety of factors have an important role on the appropriate performance of the investment materials that include their setting and thermal expansion,20 mould temperature,30 air bubbles pores,25 casting procedures,31 investment technique,15 and deformation of investment materials at high temperatures.16 in relation to procedures for casting dental alloys, certain defects in the final metal dental alloy superstructures may result from the casting process, such as porosity due to poor filling, shrinkage, or dissolved gases; chemical segregation that results in nonuniform properties due to the physical chemistry of solidification; and contamination due to mould - casting interactions.32 despite the few studies in the literature that evaluated investment influence on ceramic properties, 18,26 the surface of investment materials are shown to react with the ceramics and affect the surface and shrinkage crystallization,26,33 surface roughness,17 biaxial flexural strength, and the fit of ceramic restorations.30 a rough or irregular texture surface will reflect an irregular and diffuse pattern of light, therefore modifying the color of aesthetic restorations.34 surface topography also can influence the color of the ceramic, especially the l * value.35 this pattern was verified in vision esthetic ceramic, which presented the higher e , resulted mainly from the variation of l. Hence, color alterations detected in the ceramics tested in our study can be attributed to possible alterations in ceramic surface topography from the interaction with the refractory investment materials in high temperatures . Despite all the laboratory and clinical factors that can interfere with the final color of ceramic restorations, color differences among ceramics with the same shade but from different manufacturers can occur.8 the results of this study corroborates these findings, as significant color differences were detected among the color parameters a , b , and l * when comparing the controls (no contact with the investment materials) of the five ceramics tested (table 3). In general, vintage halo ceramic used in the control group had the higher a * value (higher red value), whereas vision esthetic and ips classic ceramics showed the lower a * values, with all ceramics presenting positive values . Regarding parameter b , vintage halo ceramic showed the higher b values (more yellow), whereas vitadur alpha and ips classic showed the lower b * values (less yellow). When considering ceramics lightness, super porcelain ex, vision esthetic, and vintage halo were the lighter ceramics (higher l * values); however, vitadur alpha and ips classic were the darkest ceramics, presenting the smaller l * values . The choice of using a chromatic background was intended to simulate a typical clinical condition in which a chromatic underlying dental structure is present, and a ceramic veneer restoration is planned.36,37 the background color of an aesthetic restoration is more or less chromatic, and the thickness of the material can vary.36,38 the use of black and white backgrounds has been reported in several studies to standardize the collection of the color data, but this methodology can influence the final color of the ceramic.29 clinically, a composite restoration with a white cement base may appear lighter, more reddish, and more saturated than that with a dark background.38 this effect becomes smaller as the thickness of the sample increases.38 when increasing resin thickness, the resin may appear darker and less chromatic with a white background and lighter and more chromatic with a black background.39 therefore, a sample may be more or less chromatic depending on its thickness and whether the background is white or black.39 to enable the reproduction of indistinguishable restorations with a natural and esthetic aspect, mimicking the natural dentition, besides a perfect anatomy, texture, and marginal adaptation, the perfect final match in color must be achieved . Little consideration has been given to the refractory die materials and the laboratory techniques used during the construction of ceramic restorations . A wide range of chromatic color alterations on veneering ceramics occurring through refractory die methods with investments was found . During the laboratorial process of constructing metal - free ceramic restorations with refractory die techniques, intimate contacts of the investments occur with the ceramic surfaces7,15 in high temperatures, contact that varies from some hours to days . Therefore, it is interesting to develop studies that could explore the potential interactions that occur among these materials, especially the ones that can result in alterations of mechanical resistance, surface, and color of ceramics, which directly influence their quality and longevity . One of the limitations of this study is that there was no analysis of the composition and differences of thermal expansion coefficients among the investment materials and ceramics tested, which could have resulted in a better understanding of their influence on the performance of the unit investment - ceramic during the construction process of a ceramic restorations . In laboratorial practice, ceramics and refractory materials routinely used are usually purchased from independent manufacturers, with some combinations having different thermal expansion coefficients but generally not resulting in differences of performance from matched ceramic and refractory products.40 therefore, further investigations are necessary to evaluate the composition, structure, and performance relationships of these related systems because there are no recommendation protocols of combinations of these materials (ceramics versus investments).40 notwithstanding that such information would be important in understanding the processes involved in the construction of ceramic restorations, a guideline for daily practice for product selection, specifying comparative or standard compliance tests, are mandatory . There was a significant influence of the investment refractory materials on the color parameters a , b , and l * on the ceramics tested, resulting in chromatic alterations unacceptable for some of the ceramic groups tested.
Tiam1 is involved in a number of cellular processes including migration and the regulation of cell - cell adhesions and survival . Althoughtiam1 has been implicated in cellular proliferation, its role during the cell cycle is unknown . In mdck ii cells expressing either gfp - tagged tiam1 or ha - tagged tiam1, we were intrigued to observe a signal not only at cell - cell adhesions (as previously described) but also at the two microtubule (mt) asters surrounding the centrosomes . This localization was apparent both before nuclear envelope breakdown (nebd) in prophase cells (figure 1a and figure s1a, available online) and after nebd in prometaphase cells (figure 1b and figure s1b) and was dependent on mts because it was lost after nocodazole treatment (data not shown). However, in metaphase cells, tiam1 localization to centrosomal regions was no longer detected (right - hand cell, figure 1b). To investigate the potential function of tiam1 at centrosomes, we generated mdck ii cells expressing doxycycline - inducible short hairpin interfering rnas (shrnas) to tiam1 (two independent targets: rnai #1 and rnai #2) or control scrambled shrna (scr). Upon doxycycline addition, endogenous tiam1 protein was depleted from cells containing either tiam1 shrna target (figure 1c). In subcellular fractions enriched for centrosomes, endogenous tiam1 cosedimented with the centrosomal marker -tubulin and was depleted from these fractions after tiam1 shrna expression (figures s1c and s1d). Moreover, immunofluorescence analysis confirmed that endogenous tiam1 localized to centrosomal regions in early mitosis (figures 1d and s1e); this signal was depleted after tiam1 shrna expression (figure 1d). Because tiam1 is a rac - specific activator gfp - tagged rac was detected at cell - cell adhesions as shown previously; however, it also colocalized with the two mt asters in prophase (figure 1e) and prometaphase cells (figure s1f). Endogenous rac showed a similar localization (figure 1f and figure s1 g). Hence, tiam1 and rac localize to centrosomal regions in early mitosis . During prophase, duplicated centrosomes begin to separate around the nuclear envelope before nebd (prophase pathway), in preparation to form a bipolar spindle in prometaphase . Hence, we measured centrosome separation in control and tiam1-depleted prophase cells . Strikingly, the intercentrosomal distance increased by over 40% in tiam1-depleted cells (figure 2a). This defect was efficiently rescued by expressing rnai - resistant wild - type tiam1 (figure 2a) but not the gef mutant tiam1 (q1191a, k1195a), which is unable to activate rac (figure 2a). Treatment with a specific rac inhibitor (nsc-23766) also increased centrosome separation in prophase cells (figure s2a). After tiam1 depletion, a similar increase in centrosome separation was seen in mouse p1 cells (figures s2b and s2c). Thus, tiam1 functions via rac to oppose centrosome separation in prophase . Relatively little is known about centrosome separation during prophase, with conflicting evidence for the involvement of eg5 [13, 14]. Treatment with the eg5-specific inhibitor monastrol led to a significant decrease in the intercentrosomal distance of prophase mdck ii cells (figure 2b); similar results were obtained with a second eg5-specific inhibitor s - trityl - l - cysteine (stlc, data not shown). Tiam1 also localizes to the cortex in prophase cells (figure 1a and figure s1a), and cortical movements driven by myosin - ii activity (regulated by rho kinase [rock]) are required after nebd for centrosome separation via astral mt connections . To gain mechanistic insight into the regulation of centrosome separation in prophase, we treated cells with inhibitory concentrations of blebbistatin (myosin - ii inhibitor) or y-27632 (rock inhibitor) (figures s2d and s2e). However, neither treatment affected centrosome separation in control prophase cells (figure 2b) or the increased intercentrosomal distance in tiam1-depleted prophase cells (figure 2c). In contrast, low concentrations of taxol, a mt - stabilizing agent, although not effecting centrosome separation in control cells, prevented the increased centrosomal separation of tiam1-depleted prophase cells (figure 2c). These data suggest that in prophase, centrosome separation is driven by eg5 but is unlikely to involve cortical movements . Further, the effects of tiam1 depletion on centrosome separation, although probably independent of the cortex, may be mediated through microtubule stabilization, although how is currently unclear . A role for tiam1 in stabilizing mts during front - rear polarization in cell migration we detected no change in the localization of eg5 in tiam1-depleted cells (data not shown), suggesting that this is independent of tiam1 . Using time - lapse fluorescence microscopy of cells expressing -tubulin - gfp after tiam1 depletion, there was no significant difference in the period between the onset of centrosome separation and nebd compared with controls (figure 2d). However, the centrosome separation rate in prophase was significantly increased in tiam1-depleted cells compared to controls (figures 2d and 2e). Further, tiam1-depleted cells maintained their increased centrosome separation after nebd (figure s2f). Consistent with this, the intercentrosomal distance in prometaphase from fixed samples was also significantly increased after tiam1 depletion (figure s2 g) or rac inhibition (figure s2h). However, the rate of centrosome separation during prometaphase was markedly reduced compared to prophase and was unaffected by tiam1 depletion (figure 2e). To investigate further the role of tiam1 in centrosome separation in prometaphase, we arrested cells in a monopolar prometaphase state with monastrol, then released them to allow mitosis to proceed . We found that centrosomes in tiam1-depleted cells separate significantly further 10 min after washout compared to controls; however, at 20 min the increase was not significant (figure s2i). Thus, tiam1-rac affects centrosome separation apparently independently of the nuclear envelope and seemingly more potently when centrosomes are in close proximity . This property may account for the greater influence that inhibition of this pathway exerts during prophase compared to prometaphase . To examine whether tiam1 antagonizes eg5 during bipolar spindle formation, we treated control and tiam1-depleted cells for 18 hr with stlc or monastrol at concentrations that reduce but do not abolish eg5 activity . In controls, eg5 suppression resulted in mainly monopolar mitotic spindles (figure 3a): 60% in stlc - treated cells (figure 3b) and 70% in monastrol - treated cells (figure s3a). However, after tiam1 depletion, mitotic spindles were mainly bipolar (figure 3a), with significantly fewer monopolar spindles: 15% in stlc - treated (figure 3b) and 30% in monastrol - treated cells (figure s3a). We next confirmed that the bipolar spindles formed in tiam1-depleted cells, under conditions of eg5 suppression, were functional and able to segregate chromosomes later in mitosis . In control cells, the proportion of mitotic cells in anaphase or telophase significantly reduced from over 32% to 20% after stlc (figure 3c) and 12% after monastrol treatment (figure s3b). Conversely, in tiam1-depleted cells, eg5 suppression had little effect on the proportion of mitotic cells in anaphase or telophase: 32% after stlc (figure 3c) and over 25% after monastrol treatment (figure s3b). Consistent with this, in controls the mitotic index increased by over 4.5-fold after eg5 suppression (figure 3d and figure s3c) as a result of the monopolar spindles activating the spindle assembly checkpoint (sac), thereby delaying mitosis . However, tiam1 depletion prevented the accumulation of mitotic cells after eg5 suppression (figure 3d and figure s3c). To determine whether eg5 function is antagonized by rac signaling in vivo, we utilized conditional rac1 knockout mice, focusing on the intestinal epithelium . Epithelial cells within intestinal crypts are highly proliferative and arrest rapidly after administration of various drugs; this arrest is visualized via an accumulation of mitotic figures within the intestinal crypt . We first conditionally deleted rac1 within the intestinal epithelium (as described in the supplemental experimental procedures). Three days after cre induction, the intestinal epithelium from ahcre rac1 mice showed loss of rac1 (figures s3d and s3e). Deletion of rac1 at this stage did not affect proliferation (data not shown) or cellularity (figure s3f). We next treated mice with 25 mg / kg stlc and investigated mitotic arrest after 3 hr . In wild - type mice, stlc treatment induced a significant mitotic arrest (figures 3e and 3f; rac1 control versus rac1 stlc - treated). Remarkably, rac1 deletion completely rescued the induction of mitotic arrest (figures 3e and 3f; rac1 stlc - treated versus rac1 stlc - treated). Moreover, in wild - type mice the proportion of mitotic cells in anaphase was significantly reduced after stlc treatment, indicating an arrest prior to this stage (figure 3 g). Taken together, these data indicate that tiam1-rac signaling antagonizes eg5 function, both in vitro and in vivo; in their absence mitotic cells can assemble bipolar spindles and complete mitosis under conditions of reduced eg5 activity . We tested whether tiam1 depletion induced further mitotic defects by using time - lapse fluorescence microscopy of cells expressing histone-2b - gfp . In controls, chromosomes efficiently congressed to the metaphase plate after nebd and rapidly entered anaphase (figure 4a). In over 60% of tiam1-depleted cells, however, chromosomes spent over 15 min aligning on the metaphase plate (compared to less than 5% of controls), before eventually aligning and entering anaphase (figure 4a). The duration of early mitotic stages, calculated from numerous movies, indicated that tiam1 depletion specifically increased prometaphase duration (figure 4b). Analysis of fixed mitotic cells revealed that after tiam1 depletion, congression errors were over 2.5-fold higher than controls (figure 4c; see figure s4a for example images). Similar results were obtained after tiam1 depletion in p1 cells (data not shown). Importantly, this chromosome congression defect was efficiently rescued by expressing rnai - resistant wild - type tiam1, but not gef mutant tiam1 (figure 4c). Moreover, cells treated with rac inhibitor also displayed significantly increased congression errors compared to controls (figure 4d; see figure s4b for example images), consistent with a role for rac downstream of tiam1 . We confirmed that these congression errors observed in tiam1-depleted cells were due to an increased prometaphase duration (figure s4c). Hence, cells with compromised tiam1-rac signaling fail to efficiently align chromosomes to the metaphase plate, resulting in a prolonged prometaphase . This is probably due to maintained activation of the sac, which delays mitosis until all chromosome kinetochores are captured . Subsequently, we determined whether the increased centrosome separation of tiam1-depleted cells leads to the congression errors . Using time - lapse fluorescence microscopy of individual mitotic cells expressing -tubulin - gfp, we confirmed that in the presence of stlc, the majority of tiam1-depleted mitotic cells formed bipolar spindles (figure s4d); furthermore, the intercentrosomal distance in prometaphase was now similar to that found in untreated controls (figure 4e). Significantly, correcting the balance of forces during bipolar spindle formation completely rescued the chromosome congression errors resulting from tiam1 depletion (figures 4f and 4 g) and restored normal mitotic progression (figures s4e and s4f). Together, these data indicate that the defects in chromosome congression induced by tiam1 depletion are due to the increased centrosome separation . In conclusion, we have shown that tiam1, acting through rac, is a critical mediator of forces during bipolar spindle assembly . Tiam1-rac signaling counteracts eg5 during bipolar spindle formation in vitro and in vivo . To our knowledge, this is the first signaling module that retards centrosome separation during prophase in mammalian cells and the first demonstration of an increased rate of centrosome separation in prophase leading to chromosome congression defects . Importantly, we show that correcting the balance of forces, by attenuating eg5 activity in tiam1-depleted cells, completely rescues the defects in chromosome congression, allowing cells to satisfy the sac and progress through mitosis normally (for a model see figure 4h). There are numerous other pathways that promote [1, 3, 20] and oppose [13, 21] centrosome separation during bipolar spindle formation . This considerable overlap is not surprising given the importance of the bipolar mitotic spindle to the survival of eukaryotic cells, providing a number of fail - safes that decrease the risk of chromosome segregation errors during cell division.
Among adults with type 2 diabetes (t2 dm), suboptimal medication adherence is common (1,2) and is associated with higher healthcare costs and worse health outcomes (2,3). Few interventions have been designed to promote adherence to diabetes medications, resulting in limited knowledge about what content should be included in interventions to improve this outcome (4). To date, interventions that have effectively improved medication adherence in diabetes (510) have coupled content with reminder systems (6,7) and/or disseminated content using labor - intensive contacts with healthcare providers (5,6,810). Although much has been gleaned from these interventions, it is unclear what content has been more or less effective . The incorporation of theory - based behavior change content in interventions to improve medication adherence may offset the need for labor - intensive dissemination strategies, thereby increasing the likelihood of interventions being successfully implemented, adopted, and sustained . Furthermore, theories specifying modifiable and measurable determinants of behavior provide an opportunity to improve the mechanisms underlying behavioral performance, which allows for identifying why an intervention was more or less effective in changing behavior for future intervention efforts (11). However, to our knowledge, no theoretical framework detailing the content necessary for medication adherence promotion in diabetes has been validated, and no results from diabetes medication adherence promotion interventions with theory - based content have been published to date . In hiv, interventions based on the information motivation behavioral skills (imb) the imb model of adherence has been validated with cross - sectional data collected from diverse samples of hiv - infected persons (1720) and then used to design interventions that have successfully improved both medication adherence (1316) and clinical outcomes (14,16). Although medication adherence is just one of many recommended diabetes self - care behaviors, each recommended behavior (e.g., exercising and blood glucose testing) is complex, with specific determinants that should be identified and accounted for in interventions . The imb model is a behavior - specific theory that posits the performance of a behavior requires behavior - specific information, motivation, and behavioral skills . While the imb model has predicted and promoted adherence to exercise and dietary recommendations among adults with t2 dm (21,22), it has not yet been validated as a useful framework to promote diabetes medication adherence . According to the imb model of adherence, medication adherence is determined by the extent to which an individual is informed about his / her regimen, is motivated to adhere, and possesses the necessary behavioral skills to adhere in a variety of situations (12,1820) (fig . Adherence information includes accurate knowledge about a regimen (i.e., how and when to take medications and what dose to take), potential side effects and drug interactions (12,1820), and having accurate heuristics and theories that support consistent adherence (as opposed to inaccurate heuristics such as i only have to take medications when my blood sugar is high) (12). Adherence motivation is a function of personal and social motivation to adhere (12). Personal motivation to adhere reflects an individual s attitudes about adherence and is based on one s beliefs that medications are helpful, and not taking medications as prescribed would have undesirable consequences . Social motivation to adhere rests on one s perceptions of social norms endorsing adherence and/or social and instrumental support for adherence . Adherence behavioral skills includes objective and perceived abilities or self - efficacy to obtain, store, have accessible, and self - cue the use of medications as directed across situations and despite challenges (12). Solid lines indicate an effect between constructs, and the dashed line indicates a feedback loop in which health outcomes affect future levels of adherence information and motivation, which in turn affect adherence behavioral skills and subsequent diabetes medication adherence and health outcomes . The imb model of adherence (12) suggests that adherence information and motivation often covary (17,19) (i.e., more knowledge may lead to increased motivation and motivated individuals may possess more knowledge), but adherence motivation may be present in the context of inaccurate or insufficient adherence information and vice versa (18,20). Adherence information and motivation affect medication adherence primarily through the enactment of adherence behavioral skills used to initiate and maintain medication adherence (1820), but may also directly affect behavior (17) when complex behavioral skills are not required for the performance of the behavior (12). The model s primary outcome is adherence, but it also stipulates that consistent adherence should be associated with favorable health outcomes (e.g., glycemic control), which, in turn, contribute to high levels of adherence information and motivation overtime (12) (fig . Many factors consistently associated with adults diabetes medication adherence (23) are included in the imb constructs . Information includes comprehension of the treatment regimen; personal motivation includes perceptions of benefits and adverse effects of treatment; and behavioral skills include patients ability to overcome cost - related barriers to adherence . Additional evidence suggests deficits in medication adherence related information (24), motivation [i.e., personal attitudes (24,25) and social support (26)], and behavioral skills [i.e., objective skills and self - efficacy (24,25)] are associated with suboptimal adherence to diabetes medications and are more common among populations with lower socioeconomic status (ses) (24,27). Additional factors associated with diabetes medication adherence include insulin use (28), regimen complexity (23), and psychological distress (23). However, each of these factors alone cannot fully explain the presence, persistence, and complexity of nonadherence to diabetes medications . Thus, the imb model of adherence may provide a multivariate explanation of nonadherence and guide to promoting medication adherence among people with diabetes . Because populations with low ses are at increased risk of nonadherence to diabetes medications (24,27), we tested the imb model s predictions in a diverse, low ses sample from a federally qualified health center (fqhc). Consistent with the imb model, we hypothesized that adherence - related information and motivation would determine adherence - related behavioral skills, which, in turn, would determine adherence behavior, and behavior would determine glycemic control . We also tested whether insulin status and/or regimen complexity explained any additional variance in medication adherence or moderated associations between adherence - related information, motivation, or behavioral skills and adherence behavior . We recruited adults (18 years old) who were diagnosed with t2 dm and received outpatient care at an fqhc in nashville, tn, between june 2010 and november 2012 . Trained research assistants (ras) worked with clinic personnel to consecutively recruit eligible patients who arrived for a clinic appointment, approaching patients in the clinic waiting room to describe the study and advertising the study on flyers in the clinic to prompt self - referrals . Eligible patients were english- or spanish - speaking adults who were self - administering prescribed medications for t2 dm . All study materials were translated from english to spanish using the forward - backward technique by licensed translators (29). Ras consulted with clinic personnel to identify and exclude patients who did not have a social security number required for compensation or had an intellectual disability, unintelligible speech, a lack of orientation to person / place / time, or a severe hearing impairment . Participants provided informed consent and completed an interviewer - administered survey in a private room before and/or after their clinic appointment . To accommodate high rates of limited literacy among patients treated at fqhcs in the southeastern u.s . (30), ras read all items and response options aloud to ensure participants ability to read did not compromise their data . Ras also supplied a copy of each set of response options in large font, so participants could respond aloud and/or point to their response . Clinic nurses administered a point - of - care a1c test, and ras reviewed medical records . Demographic characteristics included self - reported age, sex, race, ethnicity, income, education, and insurance status . Clinical characteristics included self - reported duration of diagnosed diabetes in months and years and the number and type of prescribed diabetes medications obtained from the medical record . We dichotomized insulin status (prescribed insulin vs. oral agents only), and regimen complexity was indicated by the number of prescribed diabetes medications . Adherence information was assessed with the five - item diabetes medication knowledge questionnaire (dmkq), which assesses respondents knowledge of the: 1) name; 2) purpose; 3) dosing schedule; 4) side effects; and 5) appropriate management of a missed dose for a single diabetes medication in their regimen (31). To generate a more accurate estimate of adherence information, we administered the dmkq for each diabetes medication in a participant s regimen . In our sample, internal consistency reliability for the dmkq was unacceptable (= 0.36), so we examined pearson correlation coefficients between individual dmkq items and the measures of medication adherence (described below) to identify relevant adherence - related information . Significant dmkq items asked respondents about their dosing schedule (i.e., how and when to take a medication) and handling of missed doses . The internal consistency reliability of these items across diabetes medications was good (= 0.80), so we summed item scores for each medication and then averaged scores across medications to create a two - item dmkq composite . Adherence motivation was assessed with the medicines for diabetes questionnaire (mdq), which measures respondents beliefs about taking diabetes medications (32). The behavioral beliefs and normative beliefs subscales of the mdq were used to assess personal and social motivation to adhere, respectively . The seven - item behavioral beliefs subscale asks respondents to report the degree to which they believe taking diabetes medications would help me stay well or cause me to gain weight . The three - item normative beliefs subscale asks respondents the degree to which their doctor / nurse, family / relatives, or partner / spouse would approve of them taking diabetes medications regularly . For both subscales, response options range from 1, strongly disagree, to 5, strongly agree, and are averaged to create a composite score ranging from 15, with higher scores indicating greater adherence motivation . Adherence behavioral skills were assessed with the revised medication adherence self - efficacy scale (mases - r), which is a reliable and valid measure of medication adherence self - efficacy (33). The 13-item mases - r asks respondents to report their degree of confidence in taking their medications in various situations / circumstances such as when you are traveling or whatever they cost . We added the word diabetes to each item to specify diabetes medications (e.g., how confident are you that you can take your diabetes medications). Response options range from 1, not at all sure, to 4, extremely sure, and are averaged to create a composite score ranging from 14, with higher scores indicating greater adherence behavioral skills . Diabetes medication adherence was assessed with two reliable and valid self - report measures: the summary of diabetes self - care activities medications subscale (sdsca - ms) (34) and the adherence to refills and medications scale for diabetes (arms - d) (35). Although the sdsca - ms is the most widely used self - report measure of medication adherence in diabetes, the arms - d is more sensitive to nonadherence and more predictive of glycemic control (35). Including both measures the two - item sdsca - ms asks respondents, on how many of the last 7 days did you take this medication? And take the correct number of pills / injections for this medication? Requiring a 07 response for each item (34). We administered the sdsca - ms for each diabetes medication in a participant s regimen and then averaged these scores across medications to maintain the 07 scale, with higher scores indicating greater adherence . The sdsca - ms composite had good interitem reliability across medications (average r = 0.86). The 11-item arms - d asks respondents about their daily experiences, on average with taking their diabetes medications [e.g., how often do you forget to take your diabetes medicine(s) when you feel sick?] Or refilling [e.g., how often do put off refilling your diabetes medicine(s) because they cost too much money?] (35). Response options range from 1, none of the time, to 4, all of the time, and are summed to create a score ranging from 1144 . We reverse - scored the arms - d so higher scores indicate greater adherence (in the same direction as the sdsca - ms). Glycemic control was assessed with a nurse - administered valid and reliable point - of - care a1c (%) test (37). We used stata 12 to assess internal consistency reliability (i.e., cronbach and interitem correlation), calculate summary statistics, and explore correlations . We used amos 21, a structural equation modeling (sem) program, to test statistical assumptions and estimate the models . First, we assessed whether our data met the assumptions of maximum likelihood estimation sem . Multivariate nonnormality may result in an underestimate of data fit (i.e., inflated x values) and biased ses (36). Three data characteristics contribute to multivariate nonnormality (36): 1) the distribution of any single variable is not normal (i.e., variables are skewed and kurtotic); 2) the joint distribution of any pair of the variables is not bivariate normal [i.e., madia s coefficient> 1.96 (36)]; and 3) the presence of multivariate outliers [i.e., cases with an atypical pattern of scores; mahalanobis distance p <0.001 (36)]. Data were also multivariate nonnormal (mardia s coefficient, 29.3), and removal of five multivariate outliers improved, but did not correct, multivariate nonnormality (mardia s coefficient, 18.1). Therefore, we used bootstrapping to estimate robust ses and bias - corrected p values for parameter estimates and 95% cis for indirect effects (38). Stable parameter estimates are indicated by low bias (i.e., bootstrapped and maximum likelihood estimates are similar) and an unbiased good fitting model is indicated by a bollen - stine with p> 0.05 calculated using the bootstrapped distribution (38). We used 5,000 bootstrapped samples (38) to estimate an imb model of diabetes medication adherence with five multivariate outliers removed . Hypotheses regarding structural relations between the imb model constructs were evaluated with an inspection of the direction and magnitude of the path coefficients (direct effects) and indirect effects, which indicate mediation . Significant indirect effects occur when the relationship between a predictor and an outcome is due to the predictor being associated with a third variable (i.e., all or part of the direct effect of a on c is due to a relationship between a and b). Model fit was tested with the comparative fit index (0.95 indicates good fit), the root mean square error of approximation (0.06 with ci 0.000.08 indicates good fit), and the standardized root mean square residual (<0.08 indicates acceptable fit, and 0 indicates perfect fit) (36). Agreement between multiple indices provides the best support a model has good data fit (36). After evaluating the imb model of adherence, we conducted a series of additional sems to examine the effects of insulin status and regimen complexity on the imb model relationships . First, we added insulin status and, separately, regimen complexity to the model as predictors of adherence . Next, we evaluated each potential interaction with different models to assess whether insulin status or, separately, regimen complexity moderated associations between each of the imb constructs and adherence . Demographic characteristics included self - reported age, sex, race, ethnicity, income, education, and insurance status . Clinical characteristics included self - reported duration of diagnosed diabetes in months and years and the number and type of prescribed diabetes medications obtained from the medical record . We dichotomized insulin status (prescribed insulin vs. oral agents only), and regimen complexity was indicated by the number of prescribed diabetes medications . Adherence information was assessed with the five - item diabetes medication knowledge questionnaire (dmkq), which assesses respondents knowledge of the: 1) name; 2) purpose; 3) dosing schedule; 4) side effects; and 5) appropriate management of a missed dose for a single diabetes medication in their regimen (31). To generate a more accurate estimate of adherence information, we administered the dmkq for each diabetes medication in a participant s regimen . In our sample, internal consistency reliability for the dmkq was unacceptable (= 0.36), so we examined pearson correlation coefficients between individual dmkq items and the measures of medication adherence (described below) to identify relevant adherence - related information . Significant dmkq items asked respondents about their dosing schedule (i.e., how and when to take a medication) and handling of missed doses . The internal consistency reliability of these items across diabetes medications was good (= 0.80), so we summed item scores for each medication and then averaged scores across medications to create a two - item dmkq composite . Possible scores ranged from 03 with higher scores indicating greater adherence information . Adherence motivation was assessed with the medicines for diabetes questionnaire (mdq), which measures respondents beliefs about taking diabetes medications (32). The behavioral beliefs and normative beliefs subscales of the mdq were used to assess personal and social motivation to adhere, respectively . The seven - item behavioral beliefs subscale asks respondents to report the degree to which they believe taking diabetes medications would help me stay well or cause me to gain weight . The three - item normative beliefs subscale asks respondents the degree to which their doctor / nurse, family / relatives, or partner / spouse would approve of them taking diabetes medications regularly . For both subscales, response options range from 1, strongly disagree, to 5, strongly agree, and are averaged to create a composite score ranging from 15, with higher scores indicating greater adherence motivation . Adherence behavioral skills were assessed with the revised medication adherence self - efficacy scale (mases - r), which is a reliable and valid measure of medication adherence self - efficacy (33). The 13-item mases - r asks respondents to report their degree of confidence in taking their medications in various situations / circumstances such as when you are traveling or whatever they cost . We added the word diabetes to each item to specify diabetes medications (e.g., how confident are you that you can take your diabetes medications). Response options range from 1, not at all sure, to 4, extremely sure, and are averaged to create a composite score ranging from 14, with higher scores indicating greater adherence behavioral skills . Diabetes medication adherence was assessed with two reliable and valid self - report measures: the summary of diabetes self - care activities medications subscale (sdsca - ms) (34) and the adherence to refills and medications scale for diabetes (arms - d) (35). Although the sdsca - ms is the most widely used self - report measure of medication adherence in diabetes, the arms - d is more sensitive to nonadherence and more predictive of glycemic control (35). Including both measures reduces the influence of measurement error (36). The two - item sdsca - ms asks respondents, on how many of the last 7 days did you take this medication? And take the correct number of pills / injections for this medication? Requiring a 07 response for each item (34). We administered the sdsca - ms for each diabetes medication in a participant s regimen and then averaged these scores across medications to maintain the 07 scale, with higher scores indicating greater adherence . The sdsca - ms composite had good interitem reliability across medications (average r = 0.86). The 11-item arms - d asks respondents about their daily experiences, on average with taking their diabetes medications [e.g., how often do you forget to take your diabetes medicine(s) when you feel sick?] Or refilling [e.g., how often do put off refilling your diabetes medicine(s) because they cost too much money?] (35). Response options range from 1, none of the time, to 4, all of the time, and are summed to create a score ranging from 1144 . We reverse - scored the arms - d so higher scores indicate greater adherence (in the same direction as the sdsca - ms). Glycemic control was assessed with a nurse - administered valid and reliable point - of - care a1c (%) test (37). We used stata 12 to assess internal consistency reliability (i.e., cronbach and interitem correlation), calculate summary statistics, and explore correlations . We used amos 21, a structural equation modeling (sem) program, to test statistical assumptions and estimate the models . First, we assessed whether our data met the assumptions of maximum likelihood estimation sem . Multivariate nonnormality may result in an underestimate of data fit (i.e., inflated x values) and biased ses (36). Three data characteristics contribute to multivariate nonnormality (36): 1) the distribution of any single variable is not normal (i.e., variables are skewed and kurtotic); 2) the joint distribution of any pair of the variables is not bivariate normal [i.e., madia s coefficient> 1.96 (36)]; and 3) the presence of multivariate outliers [i.e., cases with an atypical pattern of scores; mahalanobis distance p <0.001 (36)]. Data were also multivariate nonnormal (mardia s coefficient, 29.3), and removal of five multivariate outliers improved, but did not correct, multivariate nonnormality (mardia s coefficient, 18.1). Therefore, we used bootstrapping to estimate robust ses and bias - corrected p values for parameter estimates and 95% cis for indirect effects (38). Stable parameter estimates are indicated by low bias (i.e., bootstrapped and maximum likelihood estimates are similar) and an unbiased good fitting model is indicated by a bollen - stine with p> 0.05 calculated using the bootstrapped distribution (38). We used 5,000 bootstrapped samples (38) to estimate an imb model of diabetes medication adherence with five multivariate outliers removed . Hypotheses regarding structural relations between the imb model constructs were evaluated with an inspection of the direction and magnitude of the path coefficients (direct effects) and indirect effects, which indicate mediation . Significant indirect effects occur when the relationship between a predictor and an outcome is due to the predictor being associated with a third variable (i.e., all or part of the direct effect of a on c is due to a relationship between a and b). Model fit was tested with the comparative fit index (0.95 indicates good fit), the root mean square error of approximation (0.06 with ci 0.000.08 indicates good fit), and the standardized root mean square residual (<0.08 indicates acceptable fit, and 0 indicates perfect fit) (36). Agreement between multiple indices provides the best support a model has good data fit (36). After evaluating the imb model of adherence, we conducted a series of additional sems to examine the effects of insulin status and regimen complexity on the imb model relationships . First, we added insulin status and, separately, regimen complexity to the model as predictors of adherence . Next, we evaluated each potential interaction with different models to assess whether insulin status or, separately, regimen complexity moderated associations between each of the imb constructs and adherence . Of the 588 patients with t2 dm who attended a clinic appointment during recruitment, ras approached 86.2% (507). Of these, as shown in table 1, the sample of participants included in analyses (with outliers removed; n = 309) was diverse (53% were african american / black, and 8% reported hispanic ethnicity), and 11 interviews were conducted in spanish . Nearly half (45%) reported incomes <$10,000, and 46% were uninsured . On average, participants were prescribed 1.6 0.7 diabetes medications (range 14), and 47% were prescribed insulin . Table 2 presents descriptive statistics for each variable of interest and bivariate correlations between these variables . Participant characteristics (n = 309) descriptive statistics and correlations between model variables (n = 309) first, we estimated an imb model with the measures of personal and social motivation to adhere as indicators of a latent variable: adherence motivation . However, estimated variances on these factor loadings were negative, which is theoretically impossible and results from having only two indicator variables with small factor loadings (i.e., a heywood case) (36). Therefore, we estimated a model in which personal and social motivation to adhere were permitted to covary, but were independent representations of diverse aspects of adherence motivation . 2) had excellent data fit: comparative fit index, 1.00; root mean square error of approximation, 0.00 (90% ci 0.000.04); and standardized root mean square residual, 0.02 . The parameter estimates were stable and unbiased by multivariate nonnormality (bias ranged from 0.001 to 0.003), and the bollen - stine (8, 309), p = 1.00 indicated an unbiased model . 2 shows the parameter coefficients and the proportion of variance explained in each endogenous variable (r). An empirical test of the imb model of diabetes medication adherence using sem with standardized path coefficients and bootstrapped bias - corrected p values . + p <0.08; * p 0.05; * * * p <0.001 . Paths from information to behavioral skills and from personal motivation to behavioral skills were significant and in the predicted direction . Behavioral skills were significantly related to medication adherence, and also mediated the effects of information (indirect effect 0.08 [ci: 0.010.15]) and personal motivation (indirect effect 0.12 [ci: 0.050.20]) on adherence . There was also a trend (p <0.08) toward a direct effect of information on adherence . The imb constructs explained 41% of the variance in adherence, and adherence explained 9% of the variance in glycemic control . Social motivation was significantly related to adherence (0.13; p <0.05), indicating participants with greater perceived social norms for adherence reported worse adherence . Given this unexpected finding, we examined post hoc models with each item separately (i.e., doctor / nurse, family / relatives, and partner / spouse) and found only the item about family / relatives was related to less adherence (0.15; p <0.01). Neither insulin status nor regimen complexity was associated with adherence (not shown in fig . 2), and no additional variance in adherence was explained by their inclusion in the model . Moreover, none of the relationships between information, personal or social motivation, or behavioral skills and adherence were moderated by insulin status or regimen complexity . The imb model of adherence (12), which has been largely used to conceptualize hiv medication adherence (1720), was applied to conceptualize the determinants of diabetes medication adherence among a low - income, diverse sample of adults with t2 dm . Consistent with the imb model s predictions, adherence information and motivation were associated with adherence behavioral skills, which were, in turn, associated with diabetes medication adherence, and adherence was associated with glycemic control . In short, the imb model was well positioned to explain the sample s medication adherence behavior, accounting for 41% of the variability in this outcome . Furthermore, none of the associations between the imb constructs and adherence were moderated by insulin status or regimen complexity . We used an objective assessment of adherence information and found that knowing how and when to take a medication and how to handle a missed dose were the types of information relevant to adherence . Consistent with other empirical tests of the imb model of adherence (1820), the effect of information on adherence was mediated by behavioral skills . We also found a trend toward a direct effect of information on adherence, which may be because we did not assess more complex knowledge (e.g., knowing how and when to get prescriptions refilled / reauthorized) that may affect adherence through the enactment of behavioral skills, nor did we assess heuristics or implicit theories that may also influence adherence (12,18). Due to internal consistency concerns with our measure of information, only limited knowledge about medications was accounted for . As others have found in the context of adherence to hiv medications (18,20), information was not associated with personal or social motivation to adhere, suggesting the assessed adherence information was insufficient to motivate participants to adhere, and being motivated to adhere did not ensure participants had accurate and sufficient adherence information . Personal motivation to adhere, which we operationalized with a measure of personal beliefs toward taking diabetes medications, was associated with more adherence behavioral skills as predicted . We operationalized social motivation to adhere with a measure of social norms for adherence and found that having greater social norms for adherence was not associated with behavioral skills and was negatively associated with adherence . The mdq normative beliefs subscale has not been validated and, while it assesses participants perceptions of others approval of adherence, it does not assess the degree to which participants desire such approval (i.e., valence). Post hoc analyses indicated the item inquiring about family / relatives approval for adherence was driving the significant negative association with adherence behavior . This may be because positive family support (e.g., my family members help me remember to take my medications) often co - occurs with negative family support (e.g., my family members nag / argue with me about my medications) (26,39), whereas only negative family support has been associated with less medication adherence (26). Thus, the validity of the item inquiring about family members approval for adherence may be affected by the complex relationships between positive and negative family support and adherence . Future studies should measure: 1) respondents perceptions of social norms to adhere from meaningful others; 2) respondents desire to please these referents; and 3) referents positive and negative support for medication adherence . Adherence behavioral skills had a strong relationship with medication adherence and mediated the effects of information and motivation on adherence . Like others (1720), we used a measure of self - efficacy to operationalize behavioral skills because of the impracticality of observing one s skills across situations and the high correlations between actual and perceived skills, but objective skills or factors that might serve as proxies (e.g., problem - solving) for behavioral skills may operate differently . The remaining 59% of unexplained variance in diabetes medication adherence may be attributed to unmeasured aspects of information, motivation, and behavioral skills and/or to unmeasured constructs previously associated with adherence . However, several other constructs previously associated with adherence [e.g., access to healthcare and depressive symptoms (23)] would be considered moderating factors in the imb model (12). Others (23,28) have found associations between diabetes medication adherence and insulin status and regimen complexity, but we did not find these associations in our sample, nor did we find these to be moderating factors . (28) reported that patients prescribed insulin upon diagnosis were more likely to quit taking diabetes medications, but all of our participants were taking prescribed diabetes medications as a condition of enrollment and therefore may represent a different patient population . Relationships between regimen complexity and nonadherence have been found using dosing schedules to assess regimen complexity and ecological momentary assessment or pharmacy records to assess adherence (23). Other factors that account for the remaining variance in glycemic control include (but are not limited to) adherence to other recommended self - care behaviors, the class and dose of antidiabetes medications, age, obesity, and diabetes - related complications (40). In general, our use of self - report measures may have introduced recall and social desirability bias . We selected these measures because they were the best available for assessing the imb constructs specific to diabetes medications (rather than general measures of diabetes knowledge, motivation, and self - efficacy) and captured previously identified barriers to diabetes medication adherence, but neither the dmkq nor the mdq have been formally validated . Furthermore, cross - sectional data prevent conclusions about true causal mediation and the ability to test the imb model s proposed feedback loop from health outcomes to information and motivation (12). Our estimated model was theoretically justified, but, statistically, there are competing models (including those with opposite directionality) that would have generated comparable path coefficients and a good data fit . Moreover, the relationships between the imb constructs may be different in other patient populations and when using other measures of adherence . Different measures of the imb constructs may be necessary for patients managing multiple medications on different refill / reauthorization schedules and for those who have to self - adjust their insulin dose . Finally, the imb model (12) suggests potential moderators (e.g., unstable living situation or depression) we did not explore . Our results suggest patients adherence - related information, motivation, and behavioral skills are important targets for interventions promoting adherence to diabetes medications, regardless of their medication regimen . Imb model - based intervention content might include strategies to ensure patients possess appropriate knowledge about their medication regimen (i.e., dosing schedules and handling missed doses) and skills to obtain adherence information when needed (e.g., to access trustworthy sources and ask providers about medications). Motivation may be enhanced by targeting negative or false beliefs about adherence, juxtaposing consequences of nonadherence with a patients goals and interests, and teaching patients how to elicit support for adherence (e.g., asking for dose reminders when their routine changes). Intervention content that helps patients identify strategies to adhere in a variety of situations (e.g., setting alarm reminders to take / refill medications while traveling) may increase adherence behavioral skills . Evaluations of imb model based interventions can and should identify the extent to which intervention content improved patients adherence - related information, motivation, and behavioral skills and, in turn, medication adherence to inform future intervention efforts . In general, our use of self - report measures may have introduced recall and social desirability bias . We selected these measures because they were the best available for assessing the imb constructs specific to diabetes medications (rather than general measures of diabetes knowledge, motivation, and self - efficacy) and captured previously identified barriers to diabetes medication adherence, but neither the dmkq nor the mdq have been formally validated . Furthermore, cross - sectional data prevent conclusions about true causal mediation and the ability to test the imb model s proposed feedback loop from health outcomes to information and motivation (12). Our estimated model was theoretically justified, but, statistically, there are competing models (including those with opposite directionality) that would have generated comparable path coefficients and a good data fit . Moreover, the relationships between the imb constructs may be different in other patient populations and when using other measures of adherence . Different measures of the imb constructs may be necessary for patients managing multiple medications on different refill / reauthorization schedules and for those who have to self - adjust their insulin dose . Finally, the imb model (12) suggests potential moderators (e.g., unstable living situation or depression) we did not explore . Our results suggest patients adherence - related information, motivation, and behavioral skills are important targets for interventions promoting adherence to diabetes medications, regardless of their medication regimen . Imb model - based intervention content might include strategies to ensure patients possess appropriate knowledge about their medication regimen (i.e., dosing schedules and handling missed doses) and skills to obtain adherence information when needed (e.g., to access trustworthy sources and ask providers about medications). Motivation may be enhanced by targeting negative or false beliefs about adherence, juxtaposing consequences of nonadherence with a patients goals and interests, and teaching patients how to elicit support for adherence (e.g., asking for dose reminders when their routine changes). Intervention content that helps patients identify strategies to adhere in a variety of situations (e.g., setting alarm reminders to take / refill medications while traveling) may increase adherence behavioral skills . Evaluations of imb model based interventions can and should identify the extent to which intervention content improved patients adherence - related information, motivation, and behavioral skills and, in turn, medication adherence to inform future intervention efforts.
Childhood nephrotic syndrome (ns) is the commonest glomerular lesion encountered in childhood [1, 2]. Although various histological features have been described, the most important determinant of outcome of this condition is steroid responsiveness which is, however, not uniformly distributed globally . High steroid responsiveness has traditionally been demonstrated in temperate regions of the world and, conversely, high steroid resistance in tropical regions of the world like nigeria [310]. Consequently, the black race is often considered an indication for kidney biopsy in children with ns . Recent reports however suggest changes in steroid responsiveness of nephrotic syndrome, with steroid resistance being increasingly reported in non - blacks while some regions have experienced increasing steroid sensitivity [1115]. Previous reports from nigeria demonstrated high steroid resistance ranging between 35% and 92% [69, 1518]. Cursory observations of the cohort of children receiving care in our centre suggested high steroid sensitivity, hence the need for this report . This study therefore evaluates the pattern of steroid sensitivity among a cohort of black children with childhood nephrotic syndrome . The study was conducted at the paediatric nephrology unit of the lagos university teaching hospital, a 760-bed tertiary hospital in southwest nigeria . The hospital is one of the two referral centres in the state providing renal care to children in lagos state and its environs . The records of all children managed for nephrotic syndrome between january 2008 and april 2013 were reviewed . Nephrotic syndrome was diagnosed based on the following: 24-hour urine protein> 40 mg / m / hr or spot urine protein: creatinine ratio> 200 mg / mmol, hypoalbuminemia (serum albumin <25 g / l), generalized oedema, and hypercholesterolemia (serum cholesterol> 5.2 the following results were retrieved: blood pressure, urine microscopy and culture, serum electrolytes, calcium, phosphate, urea and creatinine, blood film for malaria parasite, haemoglobin genotype, renal ultrasound scan and screening for hepatitis b, hepatitis c, and human immunodeficiency virus, antinuclear antibodies, anca, and complement levels . Microscopic haematuria was defined as> 5 red blood cells (rbcs) per high field of a centrifuged urine specimen and glomerular filtration rate (egfr) was estimated using the modified schwartz formula . Chronic disease was defined according to the kidney disease outcomes qualitative initiative (kdoqi) guidelines . Hypertension was defined as blood pressure> 95th centile for age, gender, and height on three consecutive occasions . Urinary tract infection was diagnosed in the presence of supportive urinalysis findings and significant growth of uropathogenic organism in an appropriately collected urine specimen . Systemic lupus erythematosus (sle) was diagnosed using the revised american college of rheumatology criteria . Socioeconomic classification was done using the classification by oyedeji which employs the educational status and occupation of parents . At first presentation, patients receive oral prednisolone at 60 mg / m daily for 46 weeks . Since 2012, following kdigo (kidney disease improving global outcomes) recommendations, we have extended treatment to 8 weeks to define steroid resistance . Following remission, the dose is reduced to 40 mg / m on alternate days for 4 weeks and gradually tapered over 35 months . Steroid dependence (sd) was treated with the addition of levamisole (2.5 mg / kg / day) on alternate days or cyclophosphamide (oral or intravenous). Steroid resistance was treated with oral (2 mg / kg / day for 8weeks) or intravenous (500 mg / m / month for 6 months) cyclophosphamide, angiotensin converting enzyme (ace) inhibitors, or cyclosporine . Since 2012, cyclosporine rather than cyclophosphamide has become the preferred drug for the management of steroid resistance . Renal biopsies were performed by the paediatric nephrologists for steroid resistant or secondary cases of ns . Terms are defined as follows: remission: nil or trace proteinuria <30 mg / dl for 3 consecutive days after commencing treatment; steroid resistant nephrotic syndrome (srns): failure to achieve remission after 68 weeks of daily prednisolone; relapse: recurrence of 100 mg / dl (2 +) proteinuria for 3 consecutive days after having been in remission; steroid dependent nephrotic syndrome (sdns): two consecutive relapses during alternate day steroid therapy or within 14 days after cessation of steroids; frequently relapsing nephrotic syndrome (frns): two or more relapses within 6 months of initial response or 4 relapses in any 12-month period . Continuous data were represented as mean or median while categorical data were presented as percentages . Significance between steroid sensitivity and some variables was determined using chi - square test while comparison of means was done with student's t - test we managed 108 children, 68 males and 40 females (m: f = 1.7: 1). Age range was 8 months to 15.4 years (median 5.9 years). Their age distribution is shown in figure 1, reflecting a bimodal distribution with a peak at 1 to 2 years and a slightly lesser one at about 9 - 10 years . Of 88 children with complete data on socioeconomic status, majority, 38 (45.2%), were from the lower socioeconomic class while 31 (36.9%) and 15 (17.9%) were from the middle and upper socioeconomic classes, respectively . G / l 0.82 with mean serum cholesterol of 10.4 4.4 mmol / l . Most of the children, 94 (87%), had normal creatinine levels at presentation . Haematuria was present in (48) 44%, hypertension in (46) 43%, while (27) 25% had urinary tract infection . A secondary cause was found in 10 children as follows: systemic lupus erythematosus (3), sickle cell disease (3), chronic glomerulonephritis (2), infantile ns (1) and down's syndrome with cyanotic glomerulopathy (1). Five died before steroid sensitivity could be determined while others were unavailable due to loss to follow - up . Steroid sensitivity among children with ins was 82.8% (72/87) but 75.9% (72/95) overall . Twelve (16.7%) of those with ssns were either steroid dependent or frequently relapsing, and 23 children (24.2%) were steroid resistant . Time to remission in steroid sensitive patients ranged from 3 to 38 days (median 7 days). Fifteen children were biopsied: 14 with srns of whom 8 had idiopathic srns and an 11-year - old male at presentation on account of age, macroscopic haematuria, and hypertension . Biopsy was not done in the others due to refusal to consent, transfer to another facility, or death . Histological findings were as follows: focal segmental glomerulosclerosis (fsgs) in 8 (53.3%), minimal change disease (mcns) in 3 (20%) and 1 (6.7%) each of membranous nephropathy, membranoproliferative glomerulonephritis (mpgn), diffuse proliferative glomerulonephritis, and class 3 lupus nephritis . Histologic patterns among the 8 with idiopathic srns were fsgs in 5 (62.5%), mcns in 2 (25%), and mpgn in 1 (12.5%). Lower serum albumin, higher serum cholesterol, older age, haematuria, and raised serum creatinine were all associated with steroid resistance . Seven children (6.5%), all with srns, developed chronic kidney disease (ckd) of whom 5 progressed to end - stage kidney disease (eskd) during the review period . One was with haemoglobin ss disease (hbss) presented in ckd while the others (sle: 2; fsgs: 2; idiopathic: 2) developed ckd within 2 years of diagnosis . There were 7 deaths as follows: complications of eskd in 3 (lupus nephritis: 1, fsgs: 1, and ins: 1); aki in 2 patients, one of whom had lupus nephritis, and 2 newly diagnosed patients with undetermined steroid sensitivity one of whom died from a cerebrovascular accident . Our study revealed high prevalence of steroid sensitivity among a cohort of black children with ns . In summary about 76% of the whole cohort and approximately 83% of the cohort with idiopathic childhood nephrotic syndrome achieved remission following treatment with steroids . The high proportion of steroid sensitivity in this study is remarkable because such high sensitivity has rarely been described in a large sample of black children and ranks similar to that described among children of other races [24]. Various studies describe ns in children of african descent as being predominantly steroid resistant and nonminimal change on histology . In nigeria, steroid resistance ranges between 35% and 92% among different geographical locations [69, 1517] table 2, higher than the 23% in the current study . Doe et al . In ghana reported steroid resistance of 50% amongst their cohort while bhimma's group in south africa reported about 86% steroid resistance among black children . In the latter study, steroid sensitivity among indian children in the same cohort was over 65% again highlighting racial differences in steroid sensitivity . This has led to the recommendation of a kidney biopsy as part of the initial evaluation of black children with ns . The reasons for this striking observation of high steroid sensitivity likely include the younger age structure of our cohort . The statistically significant lower median age of the steroid sensitive compared with steroid resistant patients supports this . A small study from southern nigeria that reported 80% steroid sensitivity had a young population with a mean age of 5.8 years . Asinobi's group also from nigeria reported higher steroid sensitivity but their cohort comprised only children aged 5 years . This age pattern is similar to that reported among caucasians and asian children where high steroid sensitivity has been reported [14]. For instance, in a report from the united kingdom, median age of children with ssns was 4.5 years compared with 6 years in those with srns . We therefore argue that age and race should be considered together as strong predictors of response to steroid rather than the sole reliance on race . Another plausible explanation for the increased sensitivity observed in our cohort is the absence of certain previously prominent secondary aetiologies of ns in our environment resulting in a relatively higher idiopathic pool, the majority of whom were steroid sensitive . This is a likely consequence of intensified efforts by the government to reduce the burden of some childhood conditions in the region . For instance, we did not diagnose any case of quartan malaria nephropathy (qmn), which was previously reported to be a leading cause of steroid resistant nephrotic syndrome in our environment . Previous nigerian authors [8, 27] have similarly alluded to the reduced incidence of qmn in the region . Malaria control programs have resulted in improved access to antimalarial drugs over the last two decades and this is likely to have contributed to the decline in associated nephropathy . Doe's group in ghana also found no evidence of a tropical form of nephrotic syndrome in their patients . In the same vein, in contrast with reports from south africa and ghana, we did not diagnose infections such as hepatitis and schistosomiasis, which we attribute to improved vaccination rates and access to portable water, respectively . These diseases have seemingly been replaced by others like sle and sickle cell anaemia, as also observed by olowu et al . . Recent availability of diagnostic facilities for the former and improved survival beyond early childhood for the latter could account for this observation . Contrariwise, on the global scene, the incidence of steroid resistant ns in various parts of the world such as the usa [11, 28], poland, india, and canada is reportedly on increase which has been linked to increasing fsgs among these populations . Although kim's group and bonilla - felix et al . Attributed their findings to the large proportion of the african - africans in their cohort, similar observations in homogenous or near homogenous caucasian populations [12, 28] suggest the role of other factors . Socioeconomic class is thought to play a role in ns although the exact associations are not very clear . The majority of our patients were from the lower socioeconomic class similar to a report from india where 84% of study participants were from the lower socioeconomic class . While this may suggest a role for infections which are more prevalent in this socioeconomic class of children, we did not find a high prevalence of infection associated ns in this study . We however also reported a significant proportion of children from the middle socioeconomic class suggesting the role of other factors which are not apparent from this study . The aetiology of ins remains of continued research interest globally with extensive research into genetic mechanisms howbeit largely in the developed world . Fsgs was also our predominant histologic finding, a finding consistent with other african studies [16, 24, 25], especially since the era when kidney biopsy was reserved commonly for those with clinical and treatment features not in keeping with minimal change disease . Since minimal change ns is predominantly steroid sensitive, it is safe to assume that if all the children in the present study were to undergo kidney biopsies, it would have been the most predominant histological pattern . Higher cholesterol levels, hypertension, and haematuria were associated with increased steroid resistance, consistent with published data . Our findings also support the reportedly high frequency of uti in ns, particularly srns, and emphasize the need for screening for this infection in affected children . As expected, progression to ckd and mortality were predominantly a function of with steroid resistance [1, 4, 7]; hence, our lower overall mortality of 6.5%, compared with 6.614.3% in other african reports [7, 16, 24], is not surprising . Mortality was particularly high among those with steroid resistant nephrotic syndrome who progressed to eskd . This is largely a consequence of a combination of factors which include high cost of care, inadequate facilities for long - term dialysis, and lack of transplant facilities . Late presentation, hence advanced disease in two patients one of whom presented with a cerebrovascular accident, also contributed to the high mortality reported . In conclusion, although its retrospective nature resulted in incomplete data in some cases, this was compensated for by its large sample size . This study and few others in the region [14, 15] suggest a changing pattern of steroid sensitivity of childhood ns which may reflect an adaptation to the changing epidemiology of some childhood diseases as alluded to by previous authors . Surveillance of the epidemiology of childhood ns and corresponding modifications in practice guidelines over time are therefore recommended.
In 1959 ernst mayr (figure 1) flung down the gauntlet at the feet of the three great population geneticists ra fisher, sewall wright and jbs haldane (figure 2): " but what, precisely, " he said, " has been the contribution of this mathematical school to the evolutionary theory, if i may be permitted to ask such a provocative question? " His skepticism arose in part from the fact that the mathematical theory at the time had little to say about speciation, mayr's major interest . A particular focus was the simplification that he called " beanbag genetics ", in which " evolutionary genetics was essentially presented as an input or output of genes, as the adding of certain beans to a beanbag and the withdrawing of others . " . Photograph reproduced with permission from the archives of the ernst mayr library of the museum of comparative zoology, harvard university . (a) r a fisher (18901962), (b) sewall wright (18891988) photograph reproduced with permission from the capitol times, and (c) j b s haldane (18921964). Mayr was, however, criticizing textbook simplifications, rather than the actual work of the three pioneers . Far from treating gene frequency changes as analogous to the consequence of beans jostling at random in a bag, both fisher and wright considered gene interactions in detail . Fisher (figure 2a) showed that, despite interactions between genes, natural selection acts on the additive component of the genetic variance . The beanbag criticism was particularly inappropriate for wright (figure 2b), who specifically devised his' shifting balance' theory as a way for a population to go from one harmonious gene combination (mayr would say " integrated genotype ") to another when intermediates were disadvantageous . Who was to answer mayr's criticism? Fisher was already dead, and in any case preferred attack to defense, and wright was too gentle though admittedly not always when mayr was involved: returning from italy where he had received the prestigious balzan prize in 1984, wright told me that the value of the prize was considerably diminished when he discovered that mayr had won it the year before . In the event, however, it was haldane (figure 2c) who took up the challenge . This was haldane at his best witty, spirited, informed, interesting and convincing . But the larger question remains: what indeed has been the contribution of mathematical theory to evolution? Mathematics is not central to evolution in the way it has been in theoretical physics . Solid advances have been made without using mathematics, much being due to mayr himself . And these continue . Yet, i shall argue that mathematical ideas have made important, and often essential, contributions, and still do . Many concepts that are now established were arrived at mathematically, although their origins have since been forgotten . For example, the idea that polymorphisms become stabilized in populations because heterozygotes are at an advantage is now found in elementary textbooks, but fisher was the first to formulate it . Loss of heterozygosity with inbreeding is also textbook knowledge, but it was not clear until wright developed the theory and invented a simple algorithm for quantifying it . Similarly, the idea that the impact of mutation on the population depends on the mutation rate rather than the magnitude of the mutant effect is now taken for granted, but that was not known until haldane showed it mathematically . One final example is the inheritance of the abo blood groups, which was in doubt from the time of their discovery at the turn of the twentieth century until bernstein's mathematical population analysis in 1924 . All of these applications used only elementary methods, and they must have been known to mayr . Often, concepts that were developed mathematically were later explained in intuitive, non - mathematical ways . It's a lot easier to find an intuitive explanation when you already know the answer . His theory of " genetic revolutions " assumed that from a well integrated population, genetic drift in a small founder offshoot will sometimes produce a population with a new set of genotypes integrated in a new way . Intuitively, a small founder population seemed a particularly unlikely place to find a new favorable gene combination, and this was indeed shown to be the case in a very detailed mathematical analysis by barton and charlesworth . If mayr had had more respect for mathematical population genetics, he never would have made what most theorists regard as the mistake of proposing that small founder populations are a likely source of major evolutionary changes by genetic drift . Partly this is due to skilled mathematicians entering the field and bringing new techniques with them; especially noteworthy are stochastic processes . An additional influence is the explosive growth of molecular data, which lend themselves to mathematical treatment . In the first half of the twentieth century, population genetics and evolution had a beautiful theory, but there were very limited opportunities to apply it . What are some of the newer developments in evolution that are owed to mathematical theory? Here are a few . One striking result in the post - mayr period was motoo kimura's neutral theory, independently developed in 1968 by him and by jack king and thomas jukes . These writers shocked the biological world by arguing that the bulk of molecular evolution is due to selectively neutral mutations driven by the mutation process rather than selection . I think it would please mayr that the general idea that the rate of evolution in the population is equal to the rate of mutation in a single individual can be derived by simple reasoning using school mathematics . Yet, in order to apply the idea, we need to know how long a time period must be observed . This depends on how long it takes for a lucky new mutant to increase in frequency and completely replace its predecessors . When selection and migration are taken into account, the theory is much more complicated . One contribution of the neutral theory has been to provide a rationale for a molecular clock . Essentially, all our estimates of evolution rates depend on the assumption that the molecular changes used in constructing the clock are mutation - driven . Fortunately, enough of the dna does not have an obvious function and can reasonably be supposed to be evolving by neutral kinetics, or near enough so that the neutral theory can be used in practice . And a second important attribute of the neutral theory is that it supplies a natural null hypothesis for the study of selection . And yet another outgrowth of the neutral theory is the view that much of the molecular polymorphism in natural populations is effectively neutral . This is especially useful now that variation in the frequencies of single - nucleotide polymorphisms (snps) is easily observed . The various measures that are used to quantify genetic variability are outgrowths of population genetics theory . One striking result of such theory is the realization that all of the worldwide human population is descended from africa, and moreover from a small area within africa . The evidence for this striking conclusion is that molecular variance is greater in african peoples than elsewhere . The molecular clock can be used as one measure of the time taken during various human migrations and, of course, homo sapiens is not the only species that can be studied in this way . A new favorable allele arises by mutation, spreads through the population and becomes fixed at a rate that is determined mainly by how favorable it is . A consequence of this fixation is that neutral or weakly selected alleles linked to the locus are swept along with it . Because of this, there is a region on either side of the selected locus that is deficient in genetic variability . Such regions of reduced variability are footprints of a selective sweep in the past and, remarkably, provide evidence for events that occurred long ago and which can no longer be observed . Although the basic idea is simple and requires no mathematics, an assessment of how much the variability is reduced and the linkage distance over which the reduced variability occurs depend on mathematical theory . An area of biology in which mathematics, and especially computers, have become absolutely essential is systematics, ernst mayr's own field . Formerly, assessing species relationships and building phylogenetic trees based mainly on morphological differences was a matter of intuition and judgment . A mammalian dna sequence supplies billions of bits of information, thus for the first time providing an opportunity for a procedure independent of personal judgments . In recent decades the preferred procedures, such as fisher's maximum likelihood, required a great deal of computation, and for a while this meant that large phylogenies were out of computer range . It is now de rigueur to do statistical tests of significance of the tree structure and parts thereof . Many of these involve permutation methods, which have the merit of requiring minimum assumptions . They are computation - intensive, but with modern computers this is no longer an impediment . One striking example from such studies, which came as a complete surprise to classical systematists, is the close relationship of the elephant to the shrew . Molecular analysis of dna sequences, using the newly developed theory, has shown that our closest relatives are chimpanzees . Furthermore, that we and the chimpanzees are 99% identical at the dna level came as a surprise to many . Equally surprisingly, we share some 90% of our dna with mice, rabbits, dogs, horses and elephants . These numbers are fully consistent with expectations based on mutation rates and the times involved . Finally, there is now help available in the form of computer programs that can work out phylogenies and display the information graphically (see). These not only eliminate a lot of tedious work, but place advanced methods in the hands of relative novices . Finally, there has been a major theoretical advance, coalescent theory . Instead of looking forward in time any two alleles or homologous nucleotides are ultimately derived from a single one; that is, looking backward, they coalesce . One problem for which coalescent theory provided at least an approximate answer is the question of whether there was any mating between our ancestors and contemporary neanderthals . Small amounts of admixture are not ruled out, but coalescent theory has shown that any substantial intermating is very unlikely, as discussed by john wakeley . My other examples have been relatively simple, but this one isn't, as is apparent from this discussion . Another example, also given by wakeley, is evidence for a selective sweep in drosophila simulans . Until recently, mayr emphasized allopatric speciation and the prevailing model, due to dobzhansky and muller, prevailed . Recent mathematical studies support it and favor the view that speciation genes correspond to normal genes, selected for their effects within the species . Thus, hybrid incompatibility is a by - product of ordinary selection in geographically isolated populations . There is no evidence that random drift plays an important part, so mayr's' genetic revolution' and similar ideas have little support . Yet it is important to point out that, aside from this, mayr has usually been right . The field of mathematical studies of speciation is barely started; it will surely increase . I have given only a few examples of the part that mathematical theory has played in evolution studies . There are many more, but these, i hope, constitute a convincing sample of the importance of mathematics in population genetics and evolution . I do not intend to imply that all evolutionary study need be mathematical and theory - driven . For example,' evo - devo' studies, looking at changes in development during evolution, have produced exciting results while largely ignoring population genetics . Another non - mathematical example is horizontal gene transfer brought about by transposable elements, which is especially important in the evolution of microorganisms . There is also abundant evidence for increases in genetic complexity by the accumulation of small duplications . And, as always, a lot of morphological and behavioral evolution is interesting in and of itself . Yet, my guess is that as these subjects become more quantitative, population genetic theory will play an increasing role . The rise of molecular methods has led to an increase in the importance of mathematics in population genetics and evolution . The abundance of data that require mathematical analysis has greatly increased . At the time of mayr's challenge now the situation is reversed: data appear faster than existing theory can deal with them . That mathematics will play i think these examples show not only that mathematical theory is helpful, but that it is often essential . He might have had a change of mind, but i doubt it . Knowing how much he enjoyed arguing, i suspect he would be quite critical of much that i have written . Unfortunately, although he lived to be 100, he was not immortal and died in 2005 . Were he still alive, i would surely hear from him and whatever his opinions, he would not keep them to himself . He would have enjoyed an argument, preferably over a glass of sherry . And so would i. i am indebted to bret payseur for reading the manuscript and offering some very useful suggestions.
Posterior reversible encephalopathy syndrome (pres) was first reported as reversible posterior leucoencephalopathy syndrome in 1996 . Initially, it was described in acutely ill patients who had a reversible syndrome of headache, altered mental function, seizure, and loss of vision associated with findings indicating predominantly posterior leucoencephalopathy on imaging studies . Literature consists of few case reports or few small series of patients in different clinical settings . The setting in which it was described included patients with hypertensive encephathalopathies, patients who were receiving immunosuppressive medicines after organ transplantation or other malignancies, patients with renal failure, and patients with eclampsia . Other comorbid conditions later described included children with nephrotic syndrome, acute nephritis, hemolytic uremic syndrome, systemic lupus erythematosus, and patients with volume and electrolyte disturbances . Pathogenetic mechanism is believed to be vasogenic edema and hyperperfusion of cerebral white matter affecting commonly occipito - parietal region due to autoregulatory failure or endothelial dysfunction . Predilection for posterior brain regions could be due to pial and intracerebral vessels in anterior circulation which have higher concentration of adrenergic nerves when compared with posterior hemisphere . Diagnosis was made in appropriate clinical setting with neurological symptoms supported by neurological imaging by computed tomography (ct) scan brain, magnetic resonance imaging (mri), and diffusion weighted images . We describe a series of children with kidney disease who developed pres and describe their clinical and laboratory features . Out of total 660 pediatric renal patients including renal transplant patients admitted at our institute during september 2010 to february 2012 (18 months), we identified 11 patients with characteristic neurological symptom complex in association with typical neurological images suggesting pres as described in introduction . All patients had undergone thorough evaluation clinically, neurologically, and biochemically to search for likely contributory factors with special attention to blood pressure, volume status, severity of anemia, presence or absence of hemolysis, and its severity by following peripheral smear, platelet count, ldh, level of renal function, electrolyte and acid base balance, severity of hypoproteinemia, character of underlying renal disease, and blood level of immunosuppressive medications . Hypertension was staged according to fourth task force report on high blood pressure in children and adolescents by comparing patients bp reading to bp level for boys and girls by age and height percentile in bp table prepared by national heart lung and blood institute (nhlbi). Grade 1 hypertension is defined as systolic or diastolic bp from 95 percentile to 99 percentile, + 5 mm hg for age and height for individual gender . Grade 2 hypertension is designated to bp levels that are more than 5 mmhg above 99 percentile for age and height for individual gender . Possibility of other neurological disorders like cerebral venous thrombosis, meningeal infection, intracranial hemorrhage or space occupying lesion were ruled out in each case with help of appropriate investigations . Initially, all patients were evaluated by in - house cranial ct scan without contrast . Mri with or without contrast was obtained in patients where ct brain alone was inconclusive . All patients received appropriate treatment for renal disease, adequate dialysis for renal failure, timely correction of fluid electrolyte and acid base balance, nitroglycerine drip and other anti - hypertensives for hypertension, iv albumin for hypoalbuminemia, plasma exchanges in cases of hus, and dose adjustment of the medication was made . Record of treatment received for renal disease and prevailing abnormal parameters and time frame of clinical and biochemical improvement was made and follow - up radio - images were compared (usually taken at 4 week interval and more frequently in selected cases) with original images . A total of 11 patients (eight boys and three girls, ranging in age from 3 years to 15 years) with pres, were evaluated and studied . Presenting symptoms were seizures in ten cases (91%), headache in eight cases (72%), altered mental status in five cases (45%), and dimness of vision or cortical blindness in three cases (27%) in different combinations . So far as underlying renal disease and setting of syndrome complex is considered among these patients, six patients (55%) had acute renal failure (four had hus, one had pauci - immune crescentic gn, one had solitary functioning kidney obstructed by a stone who developed pres in setting of post - obstructive diuresis and associated persistent hypocalcemia), four patients (36%) had normal renal function [one patient with diffuse proliferative lupus nephritis and uncontrolled hypertension, one patient of focal segmental glomerulosclerosis (fsgs) with high trough level of cyclosporine 300 ng / ml, one patient with mpgn who developed hypertensive encephalopathy due to non - compliance to medication, one with morbid nephrotic state with scrotal skin necrosis and severe hypoproteinemia (serum albumin 1.1 gm / dl)]. One patient of chronic renal failure (crf) who was on continuous ambulatory peritoneal dialysis (capd) developed pres after receiving inadvertent blood transfusion due to misjudged anemia and his hb at the time of pres was 12.5 gm / dl . Significant comorbid contributory factors found in these patients at the time of pres were uncontrolled hypertension in all 11 patients (100%), renal failure (55%), cni toxicity (9%), hypoproteinemia (9%), high hematocrit (9%), hypocalcemia (9%), steroid toxicity (9%), hus (36%), and lupus nephritis (9%) [figure 1]. 5 showing morbid nephrotic state with scrotal skin necrosis a five - year - old - male child weighing 14 kg was admitted with arf and serum creatinine of 6.4 mg / dl, and respiratory acidosis . He was prepared by acute peritoneal dialysis for pcn diversion of obstructed solitary functioning kidney . Clinically, his pulse and hydration was normal and bp was 130/90 mmhg (grade 2 ht) which was controlled with oral nifedipine . Biochemically, electrolytes were normal except for ionized serum calcium which was 2.26 mg / dl, phosphate 5.4 mg / dl, magnesium 1.77 mg / dl, ph of 7.32 and serum bicarbonate level was 17.5 patient was treated by sodium bicarbonate drip in one iv line and calcium gluconate drip in another iv line . In first 12 h the child received 15 ml of calcium gluconate and 10ml in next 12 h. he was given 0.5 ml of magnesium sulphate (i.e., 250 mg; 25% w / v) intramuscularly . The child's venous blood gas and electrolytes were studied every 6 hours and child persisted to be severely hypocalcemic though he received 15 ml of calcium gluconate on second day and 10 ml for further two days . So in total 50 ml of calcium gluconate was given in three days of pcn diversion in a 14 kg child . Childs hypocalcemia was within normal range on fourth day only when we reduced 24 h calcium dose in stepwise fashion . His bp was well controlled with oral nifedipine . Throughout the course, his urine output, serum urea, and serum creatinine were improving day by day after diversion . We measured 24 h urinary calcium also on 2 day after diversion which was 30 mg/24 h which was high in face of very low ionized serum calcium level . The amount of calcium gluconate the child required parenterally to correct hypocalcemia was surprisingly high which we have observed first time in such a setting . His ipth, five days after pcn diversion was 408 pg / ml which was 540 pg / ml when he presented with arf . Logically, we hypothesized that calcium gluconate given iv was increasingly being taken up by bone due to decreasing hyperparathyroidism in association with improving renal function and it was wasted to some extent in urine also because of defect in tubular reabsorption leading to persistent hypocalcemia . Calcium has unique intracellular messanger role and it is complexed with numerous ligands in cytosol, including adenosine and vitamin d dependent calcium binding protein, calmodulin . Persistent hypocalcemia may be expected to affect directly or indirectly mechanisms involved in autoregulation and endothelial function . An 8-year - old child, weighing 15 kg (crf on capd) was found to have pres with history of blood transfusion by private pediatrician . His hb was 12.5 g% with grade 2 ht (bp 120/80 mm hg) when he presented to us with pres . Initially, we also intensified pd with 2.5% dextrose capd fluid, used diuretics, and controlled ht with oral medication for two days . Child was on capd for 1.5 year without any neurological complaint . During his intensive pd with hypertonic fluid, child lost 300 g weight after 1 day and further 150 g on 2 day after admission . Child was euvolemic with normal x - ray chest and was not improving neurologically for two days . So, observing the temporal relationship of blood transfusion and development of pres, we tapped 100 ml of blood on 3 day by femoral route and pd was switched to 1.5% dextrose solution again . Temporal relationship of improvement with phlebotomy left no doubt about the fact that blood transfusion was causally related to pres in this child who was habituated to low hematocrit . Sudden rise in hematocrit might have resulted in increased viscosity of blood and rise in vascular resistance, superimposed on possibly sensitive cerebral vasculature, may have resulted in hypertensive encephalopathy like picture . Kdoqi clinical practice recommendation for anemia in ckd also have set target of hb in the range of 11 - 12 g / dl . Initial cranial imaging revealed fairly symmetrical areas of edema not only involving occipito - parietal white matter regions in majority of cases but also extended to cortical gray matter in nine cases (81%) and extended to fronto - temporal area in five cases (45%), involved basal ganglia also in two patients (case 4 and 11) whereas cerebellum and medulla were also involved in one patient (case 11) [figure 2]. Computed tomography / magnetic resonance imaging images of brain showing positive findings all patients had neurological recovery within 5 - 7 days except one patient of hus (case four) who persisted to have altered mentation even after a month of follow up, though his renal function and cranial imaging had recovered completely after a month . This was the patient who had neurological symptoms for six days before getting admitted in our hospital where specific management of peritoneal dialysis and plasma exchange was started . Follow - up cranial images were normal after four to five weeks in all cases . All patients had normal renal function after four weeks except in two cases, one with hus who was put on capd and one patient with obstructed solitary functioning kidney who had serum creatinine of 1.9 mg / dl at the end of one month when he was stone free . A five - year - old - male child weighing 14 kg was admitted with arf and serum creatinine of 6.4 mg / dl, and respiratory acidosis . He was prepared by acute peritoneal dialysis for pcn diversion of obstructed solitary functioning kidney . Clinically, his pulse and hydration was normal and bp was 130/90 mmhg (grade 2 ht) which was controlled with oral nifedipine . Biochemically, electrolytes were normal except for ionized serum calcium which was 2.26 mg / dl, phosphate 5.4 mg / dl, magnesium 1.77 mg / dl, ph of 7.32 and serum bicarbonate level was 17.5 patient was treated by sodium bicarbonate drip in one iv line and calcium gluconate drip in another iv line . In first 12 h the child received 15 ml of calcium gluconate and 10ml in next 12 h. he was given 0.5 ml of magnesium sulphate (i.e., 250 mg; 25% w / v) intramuscularly . The child's venous blood gas and electrolytes were studied every 6 hours and child persisted to be severely hypocalcemic though he received 15 ml of calcium gluconate on second day and 10 ml for further two days . So in total 50 ml of calcium gluconate was given in three days of pcn diversion in a 14 kg child . Childs hypocalcemia was within normal range on fourth day only when we reduced 24 h calcium dose in stepwise fashion . His bp was well controlled with oral nifedipine . Throughout the course, his urine output, serum urea, and serum creatinine were improving day by day after diversion . We measured 24 h urinary calcium also on 2 day after diversion which was 30 mg/24 h which was high in face of very low ionized serum calcium level . The amount of calcium gluconate the child required parenterally to correct hypocalcemia was surprisingly high which we have observed first time in such a setting . His ipth, five days after pcn diversion was 408 pg / ml which was 540 pg / ml when he presented with arf . Logically, we hypothesized that calcium gluconate given iv was increasingly being taken up by bone due to decreasing hyperparathyroidism in association with improving renal function and it was wasted to some extent in urine also because of defect in tubular reabsorption leading to persistent hypocalcemia . Calcium has unique intracellular messanger role and it is complexed with numerous ligands in cytosol, including adenosine and vitamin d dependent calcium binding protein, calmodulin . Persistent hypocalcemia may be expected to affect directly or indirectly mechanisms involved in autoregulation and endothelial function . An 8-year - old child, weighing 15 kg (crf on capd) was found to have pres with history of blood transfusion by private pediatrician . His hb was 12.5 g% with grade 2 ht (bp 120/80 mm hg) when he presented to us with pres . Initially, we also intensified pd with 2.5% dextrose capd fluid, used diuretics, and controlled ht with oral medication for two days . Child was on capd for 1.5 year without any neurological complaint . During his intensive pd with hypertonic fluid, child lost 300 g weight after 1 day and further 150 g on 2 day after admission . Child was euvolemic with normal x - ray chest and was not improving neurologically for two days . So, observing the temporal relationship of blood transfusion and development of pres, we tapped 100 ml of blood on 3 day by femoral route and pd was switched to 1.5% dextrose solution again . Temporal relationship of improvement with phlebotomy left no doubt about the fact that blood transfusion was causally related to pres in this child who was habituated to low hematocrit . Sudden rise in hematocrit might have resulted in increased viscosity of blood and rise in vascular resistance, superimposed on possibly sensitive cerebral vasculature, may have resulted in hypertensive encephalopathy like picture . Kdoqi clinical practice recommendation for anemia in ckd also have set target of hb in the range of 11 - 12 g / dl . Initial cranial imaging revealed fairly symmetrical areas of edema not only involving occipito - parietal white matter regions in majority of cases but also extended to cortical gray matter in nine cases (81%) and extended to fronto - temporal area in five cases (45%), involved basal ganglia also in two patients (case 4 and 11) whereas cerebellum and medulla were also involved in one patient (case 11) [figure 2]. Computed tomography / magnetic resonance imaging images of brain showing positive findings all patients had neurological recovery within 5 - 7 days except one patient of hus (case four) who persisted to have altered mentation even after a month of follow up, though his renal function and cranial imaging had recovered completely after a month . This was the patient who had neurological symptoms for six days before getting admitted in our hospital where specific management of peritoneal dialysis and plasma exchange was started . Follow - up cranial images were normal after four to five weeks in all cases . All patients had normal renal function after four weeks except in two cases, one with hus who was put on capd and one patient with obstructed solitary functioning kidney who had serum creatinine of 1.9 mg / dl at the end of one month when he was stone free . With easy availability of radio - imaging technique, mri in particular, there is increasing number of reports of pres in all age groups including pediatric patients over last ten years . With an objective of studying predisposing factors, clinical spectrum, cranial imaging, and course of pres in pediatric renal patients, renal patients are at particular risk of pres because renal disease and hypertension go hand in hand . Higher chances of volume perturbation, electrolyte disturbances and mechanism of renal disease itself, its treatment - associated drug toxicities, and other complications of management put these patients at special risk for pres . Table 2 shows comparison of our results with two other pediatric studies of pres in literature . It could be seen that in all three pediatric series there were more boys than girls with pres . Other two pediatric series, recently published, also showed more boys than girls . In adult series comparative data of pres in pediatric patients seizure as a presenting symptom was most common in ishikura's study (85%) as well as in our study (91%). All the series had different combination of four clinical characteristic symptoms as a presenting picture . So far as level of renal function was considered among these patients, we found six patients (55%) with acute renal failure . Other two studies have not specified about level of renal function . But more and more patients with hypertension and renal failure of different etiologies are reported with pres . Hypertension was almost always observed in pres but its level was not correlated to the severity of pres and cause effect relationship is yet to be established, as in many cases other comorbid conditions like drug toxicity, vacuities, auto - immune disease, or endothelial dysfunction from any cause may be involved . May have multiple factors involved because they are often hypertensive, receive steroid and cni, may have severe hypoproteinemia, and may have massive oedema with fluid retention and associated increased vascular permeability . Ishikura's series had seven cases (35%), kwon series had two cases (16%) whereas our study had two cases (18%) of nephrotic syndrome . Like our series (36% hus), series of kwon et al . Also had two cases of hus (16.5%) who developed pres . Here, pathological process itself in addition to renal failure and hypertension could be involved in pathogenesis like that of pre - eclampsia . Our patient with pauci - immune crescentic gn (case 1) had developed pres following three pulses of methylprednisolone ., in two cases (16%) of nephrotic syndrome, whereas no specific allegation to steroid high dose was made in ishikura series . Ishikura's series had ten patients (50%) with kidney transplantation on cni and kwon et al . 33% of cases on cni and our study had 9% (1 case) of pres which was associated with cni overdose . Immunosuppresive medicines and cni in particular are more frequently reported to be associated with pres in post - transplant scenario . After renal toxicity, vascular toxicity, and neurotoxicity are the most serious side effect of cni, affecting 25 - 59% of transplant patients . Reduction in drug dosage or prompt withdrawal of cytotoxic drugs is usually recommended in cases of pres . When same agent is re - introduced, patient must be followed closely as recurrence has been reported in this setting . Recently, de laat from netherlands described seven childhood cancer patients with clinical radiological findings of pres . He also reviewed 49 other documented cases of pres from literature during childhood cancer and concluded that hypertension and renal failure related to immunosuppressive treatment of leukemia and other solid tumor seems to be the most important triggers for occurrence of pres . Similarly, faruk inceik from turkey in 2009 described nine cases of pediatric pres of whom seven were receiving immunosuppressive treatment and two were hypertensive crisis patients . Involvement of occipital and parietal region was almost universal in all three studies and lesions were not restricted to white matter alone but involved gray matter also in majority of cases in all three studies . Other areas like frontal lobe, temporal lobe, basal ganglia, cerebellum, and medulla were involved in lesser frequency in all series with individual incidence . Follow - up cranial images were normal after four to five weeks in all cases in present study . One case of hus (9%) persisted to have altered mentation even after a month of follow - up, though his renal function and cranial imaging had recovered completely . In a study by ishikura, hypertension, renal disease, immunosuppression, and chemotherapy of malignancies are triggers for pres . It is important to consider this diagnosis in children presenting with seizure, visual disturbances, headache, and altered mentation in appropriate clinical setting.
Archival tissues of formalin - fixed, paraffin - embedded (ffpe) tumor blocks were retrieved from consecutively archived specimens . A set of 177 t / nk - cell lymphomas diagnosed in severance hospital from 1999 to 2013 were included . The subtypes of t / nk - cell lymphomas were as follows: extranodal nk / t - cell lymphoma, nasal type (nktl; n=60), peripheral t - cell lymphoma, not otherwise specified (ptcl - nos; n=44), angioimmunoblastic t - cell lymphoma (aitl; n=16), anaplastic large cell lymphoma (alcl; anaplastic lymphoma kinase [alk]-positive, n=16; alk - negative, n=17), t lymphoblastic leukemia / lymphoma (t - lbl; n=20) and other types (n=4; hepatosplenic t - cell lymphoma, n=2; subcutaneous panniculitis - like t - cell lymphoma, n=1; cutaneous gamma - delta t - cell lymphoma, n=1). A set of 151 diffuse large b cell lymphoma (dlbcl) and other types of 31 b - cell lymphomas, which were diagnosed in severance hospital from 2005 to 2011, were included . Other types of b - cell lymphomas were as follows: follicular lymphoma (n=14), mantle cell lymphoma (n=5), marginal zone b - cell lymphoma (n=7), and burkitt lymphoma (n=5). Hyperplastic palatine tonsil tissues of cancer - free individuals (n=20) were randomly retrieved . All tumor samples were reviewed by hematopathologists based on the current world health organization (who) criteria . Sections of ffpe tissues were prepared and stained with hematoxylin and eosin . Under the microscope, the representative area was confirmed and selected for a 3-mm - core sized tissue microarray (tma). One or two different representative areas per case were selected and 29 cores were embedded in a tma block . Immunohistochemistry of nova1 (1:500, abcam, cambridge, uk) was performed on 4-m tissue sections using the ventana benchmark xt autostainer (ventana medical systems, tucson, az, usa). Immunohistochemistry with other primary antibodies was performed for diagnostic purposes and the tested antibodies are listed in appendix 2 . Nova1 expression was semiquantitatively determined according to the h - score method with a total score range of 0300 . The dominant intensity score of nuclear staining (0, absent; 1, weak or barely detectable; 2, distinct brown; and 3, strong dark brown) was multiplied by the percentage of positive tumor cell nuclei . The nova1 protein expression score ranged from 0 to 300, as high (200<h - score300), intermediate (100<h - score 200), or low (0h - score100) expression . Jurkat, a t - lbl cell line, was maintained in rpmi1640 medium (22400 - 089, gibco, life technologies, carlsbad, ca, usa) supplemented with 10% heat - inactivated fetal bovine serum (fbs; 6000 - 044, gibco, life technologies). Hh, a cell line derived from cd30-positive cutaneous t - cell lymphoma, and mac1, a cell line derived from cd30-positive alk - negative alcl, were maintained in rpmi1640 medium supplemented with 20% heat - inactivated fbs . Snk6, derived from epstein - barr virus (ebv)-positive nktl, was cultured in rpmi1640 medium supplemented with 10% heat - inactivated human plasma and 700 u / ml of recombinant interleukin 2 . Yt, an ebv - positive human nk - like leukemic cell line, was cultured in iscove s modified dulbecco s medium (12440 - 046, gibco) supplemented with 20% heat - inactivated fbs . All the cell lines derived from t- or nk - cell lymphomas were kindly provided by prof . Toledo, a dlbcl cell line, was purchased from the american type culture collection (atcc, manassas, va, usa) and maintained in rpmi1640 supplemented with 10% heat - inactivated fbs . Rna isolation was performed using an rneasy plus mini kit (74134, qiagen, hilden, germany), and cdna was constructed using a cdna synthesis kit (11754 - 050, invitrogen, carlsbad, ca, usa). The taqman probes used were as follows: gapdh (hs99999905-m1, ab, life technologies) and nova1 (hs00359592-m1, ab, life technologies). Quantitative polymerase chain reaction was performed using abi steponeplus under the following conditions: 50c for 2 minutes, 95c for 5 minutes, 40 cycles of 95c for 15 seconds, and 60c for minutes . Relative mrna expression levels of nova1 were determined by the comparative method (2) against the reference gapdh . The kruskal - wallis (k - w), mann - whitney u (m - w), t and tests were used to analyze differences between the variables examined . Survival rates were analyzed with the kaplan - meier method, and differences were compared using the log - rank test . Archival tissues of formalin - fixed, paraffin - embedded (ffpe) tumor blocks were retrieved from consecutively archived specimens . A set of 177 t / nk - cell lymphomas diagnosed in severance hospital from 1999 to 2013 were included . The subtypes of t / nk - cell lymphomas were as follows: extranodal nk / t - cell lymphoma, nasal type (nktl; n=60), peripheral t - cell lymphoma, not otherwise specified (ptcl - nos; n=44), angioimmunoblastic t - cell lymphoma (aitl; n=16), anaplastic large cell lymphoma (alcl; anaplastic lymphoma kinase [alk]-positive, n=16; alk - negative, n=17), t lymphoblastic leukemia / lymphoma (t - lbl; n=20) and other types (n=4; hepatosplenic t - cell lymphoma, n=2; subcutaneous panniculitis - like t - cell lymphoma, n=1; cutaneous gamma - delta t - cell lymphoma, n=1). A set of 151 diffuse large b cell lymphoma (dlbcl) and other types of 31 b - cell lymphomas, which were diagnosed in severance hospital from 2005 to 2011, were included . Other types of b - cell lymphomas were as follows: follicular lymphoma (n=14), mantle cell lymphoma (n=5), marginal zone b - cell lymphoma (n=7), and burkitt lymphoma (n=5). Hyperplastic palatine tonsil tissues of cancer - free individuals (n=20) were randomly retrieved . All tumor samples were reviewed by hematopathologists based on the current world health organization (who) criteria . Sections of ffpe tissues were prepared and stained with hematoxylin and eosin . Under the microscope, the representative area was confirmed and selected for a 3-mm - core sized tissue microarray (tma). One or two different representative areas per case were selected and 29 cores were embedded in a tma block . Immunohistochemistry of nova1 (1:500, abcam, cambridge, uk) was performed on 4-m tissue sections using the ventana benchmark xt autostainer (ventana medical systems, tucson, az, usa). Immunohistochemistry with other primary antibodies was performed for diagnostic purposes and the tested antibodies are listed in appendix 2 . Nova1 expression was semiquantitatively determined according to the h - score method with a total score range of 0300 . The dominant intensity score of nuclear staining (0, absent; 1, weak or barely detectable; 2, distinct brown; and 3, strong dark brown) was multiplied by the percentage of positive tumor cell nuclei . The nova1 protein expression score ranged from 0 to 300, as high (200<h - score300), intermediate (100<h - score 200), or low (0h - score100) expression . Jurkat, a t - lbl cell line, was maintained in rpmi1640 medium (22400 - 089, gibco, life technologies, carlsbad, ca, usa) supplemented with 10% heat - inactivated fetal bovine serum (fbs; 6000 - 044, gibco, life technologies). Hh, a cell line derived from cd30-positive cutaneous t - cell lymphoma, and mac1, a cell line derived from cd30-positive alk - negative alcl, were maintained in rpmi1640 medium supplemented with 20% heat - inactivated fbs . Snk6, derived from epstein - barr virus (ebv)-positive nktl, was cultured in rpmi1640 medium supplemented with 10% heat - inactivated human plasma and 700 yt, an ebv - positive human nk - like leukemic cell line, was cultured in iscove s modified dulbecco s medium (12440 - 046, gibco) supplemented with 20% heat - inactivated fbs . All the cell lines derived from t- or nk - cell lymphomas were kindly provided by prof . Toledo, a dlbcl cell line, was purchased from the american type culture collection (atcc, manassas, va, usa) and maintained in rpmi1640 supplemented with 10% heat - inactivated fbs . Rna isolation was performed using an rneasy plus mini kit (74134, qiagen, hilden, germany), and cdna was constructed using a cdna synthesis kit (11754 - 050, invitrogen, carlsbad, ca, usa). The taqman probes used were as follows: gapdh (hs99999905-m1, ab, life technologies) and nova1 (hs00359592-m1, ab, life technologies). Quantitative polymerase chain reaction was performed using abi steponeplus under the following conditions: 50c for 2 minutes, 95c for 5 minutes, 40 cycles of 95c for 15 seconds, and 60c for minutes . Relative mrna expression levels of nova1 were determined by the comparative method (2) against the reference gapdh . The kruskal - wallis (k - w), mann - whitney u (m - w), t and tests were used to analyze differences between the variables examined . Survival rates were analyzed with the kaplan - meier method, and differences were compared using the log - rank test . The nova1 expression patterns in the cell nuclei were similar to those reported in a previous study . Almost all b cells of the germinal centers or outside the germinal centers were negative for nova1 . As for t cells, occasional expression of nova1 (intensity grade 23 with 5% or less percentage of t cells) was noted in the paracortical area (table 1). T / nk - cell lymphomas showed low to high nova1 expression and 99 (56.5%) showed high nova1 expression (table 1, fig . The expression level (score) of nova1 was higher in tumor cells of t / nk - cell lymphomas when compared to normal t cells of tonsil tissues (mean score, 209 vs. 7; p<.001; non - parametric m - w) (fig . . Within t / nk - cell lymphomas, precursor t - lbl showed a relatively higher level of nova1 expression than did nktl or mature (peripheral) t - cell lymphomas, although a statistically significant difference was not noted (p=.226; k - w) (fig . According to the specific subtypes of t / nk - cell lymphomas, the expression level of nova1 in tumor cells varied (p=.011; k - w). Aitl, alk - negative alcl, and t - lbl showed relatively higher nova1 expression levels when compared to other subtypes (table 1, fig . 2c). Specifically, alk - positive alcl revealed a lower level of nova1 expression when compared to alk - negative alcl (mean score, 156 vs. 259; p=.027; m - w) and alk - positive alcl showed the lowest expression level among subtypes of t / nk - cell lymphomas (table 1, fig . 2c). The rate of high, intermediate, or low expression of nova1 was also different according to subtype (p <.001); high expression was relatively more frequent in aitl, alk - negative alcl, and t - lbl cells, while low expression was relatively more frequent in nktl and alk - positive alcl (table 1, fig . The tested 151 dlbcl cases revealed an expression pattern with a score less than 10 . Furthermore, in the various types of other b - cell lymphomas, high nova1 expression was not noted (table 1, fig . Nova1 mrna was expressed only in the jurkat cell line, which corresponds to t - lbl . Nova1 mrna was not detected in other tested cell lines of hh (correspondent to peripheral, mature t cell lymphoma), mac1 (peripheral, mature t - cell lymphoma), snk6 (nktl), yt (nk cell leukemia), or toledo (dlbcl) (fig . The nova1 expression level in tumor cells was not different according to the organ / sites where lymphomas were growing (appendix 3). The known clinicopathological prognostic factors such as age, lactate dehydrogenase level, ann - arbor stage, or international prognostic index (ipi) risk scores showed no correlation with nova1 expression . In cases of mature (peripheral) t - cell lymphomas, high expression of nova1 tended to be related to shorter overall survival rate in comparison to low or intermediate nova1 expression, although statistical significance was not observed (fig . 5a, b). In other subtypes, such as nktl and t - lbl the nova1 expression patterns in the cell nuclei were similar to those reported in a previous study . Almost all b cells of the germinal centers or outside the germinal centers were negative for nova1 . As for t cells, occasional expression of nova1 (intensity grade 23 with 5% or less percentage of t cells) was noted in the paracortical area (table 1). T / nk - cell lymphomas showed low to high nova1 expression and 99 (56.5%) showed high nova1 expression (table 1, fig . The expression level (score) of nova1 was higher in tumor cells of t / nk - cell lymphomas when compared to normal t cells of tonsil tissues (mean score, 209 vs. 7; p<.001; non - parametric m - w) (fig . Within t / nk - cell lymphomas, precursor t - lbl showed a relatively higher level of nova1 expression than did nktl or mature (peripheral) t - cell lymphomas, although a statistically significant difference was not noted (p=.226; k - w) (fig . According to the specific subtypes of t / nk - cell lymphomas, the expression level of nova1 in tumor cells varied (p=.011; k - w). Aitl, alk - negative alcl, and t - lbl showed relatively higher nova1 expression levels when compared to other subtypes (table 1, fig . 2c). Specifically, alk - positive alcl revealed a lower level of nova1 expression when compared to alk - negative alcl (mean score, 156 vs. 259; p=.027; m - w) and alk - positive alcl showed the lowest expression level among subtypes of t / nk - cell lymphomas (table 1, fig . 2c) the rate of high, intermediate, or low expression of nova1 was also different according to subtype (p <.001); high expression was relatively more frequent in aitl, alk - negative alcl, and t - lbl cells, while low expression was relatively more frequent in nktl and alk - positive alcl (table 1, fig . The tested 151 dlbcl cases revealed an expression pattern with a score less than 10 . Furthermore, in the various types of other b - cell lymphomas, high nova1 expression was not noted (table 1, fig . Nova1 mrna was expressed only in the jurkat cell line, which corresponds to t - lbl . Nova1 mrna was not detected in other tested cell lines of hh (correspondent to peripheral, mature t cell lymphoma), mac1 (peripheral, mature t - cell lymphoma), snk6 (nktl), yt (nk cell leukemia), or toledo (dlbcl) (fig . The nova1 expression level in tumor cells was not different according to the organ / sites where lymphomas were growing (appendix 3). The known clinicopathological prognostic factors such as age, lactate dehydrogenase level, ann - arbor stage, or international prognostic index (ipi) risk scores showed no correlation with nova1 expression . In cases of mature (peripheral) t - cell lymphomas, high expression of nova1 tended to be related to shorter overall survival rate in comparison to low or intermediate nova1 expression, although statistical significance was not observed (fig . In other subtypes, such as nktl and t - lbl, no correlation was noted between nova1 expression and overall survival rate . In this study, we found that nova1 was frequently and highly expressed in various subtypes of t / nk - cell lymphomas, with about 60% of t / nk - cell lymphoma cases showing high nova1 expression, whereas only a small number of t cells expressed nova1 in the hyperplastic palatine tonsils . As for b - cell lymphomas, nova1 was negative in various types of b - cell lymphomas other than dlbcl, or normal b cells . In dlbcl, nova1 was expressed in a few cases; however, high expression was not noted in any cases . Our previous study revealed high expression of nova1 in activated t cells and the present study also confirmed that finding . Moreover, nova1 was upregulated in lymphomas derived from immature t cells such as precursor t - lbl and nktl . However, in contrast to t / nk cells, nova1 might not be involved in b cell lesions, either malignant lymphomas or non - neoplastic b cells, as shown in our studies . Among subtypes of t regarding the different biological characteristics and pathogenesis of each subtype of t / nk - cell lymphoma, nova1 expression may be affected by various intrinsic biological factors related to each subtype . The aitl, alk - negative alcl, and t - lbl subtypes, which showed distinctively high nova1 expression, are biologically characteristic entities . Interestingly, the nova1 expression pattern was different even within alcl specimens, according to alk expression status . Alk - negative alcl showed distinctively high nova1 expression while alk - positive alcl exhibited low nova1 expression . Both types of alcl share common morphologic features of cd30 positivity, large anaplastic hallmark cells, frequent loss of pan - t - cell antigens, and expression of cytotoxic molecules, and are classified as the same disease . However, alk - positive alcl and alk - negative alcl are thought to be different entities; the clinical features, prognosis, and gene expression signatures have been noted to be different therefore, the differences in nova1 expression may be related to the intrinsic biologic characteristics of these two types . There are no known studies that can explain the distinctively low nova1 expression in alk - positive alcl . We thought that nucleophosmin (npm), the fusion partner of alk - npm translocation / t(2;5) in alk - positive alcls, may be associated . Mutated npm molecules are abnormally localized in the cytoplasm because of impaired nuclear localization, and this abnormal localization of npm is thought to be associated with abnormal ribosome biogenesis . The aberrant function of mutated npm may be related to inhibiting nova1 function in the splicing process of pre - rna molecules via abnormal ribosome biogenesis . The proportion of cases showing an intermediate level of nova1 expression was relatively higher than that in other subtypes . This difference may be because ptcl - nos is a disease group of heterogeneous entities of mature (peripheral) t - cell lymphomas . These findings indicate that nova1 expression may be primarily dependent on the intrinsic characteristics of the tumor cell itself rather than extrinsic factors such as the organ - specific microenvironment, which is pre - formed before tumor growth . In association with patient prognosis, however, in mature (peripheral) t - cell lymphomas, high nova1 expression tended to be related to an inferior overall survival rate . In our previous study, high nova1 expression levels in reactive, non - neoplastic (mature) t cells, which accumulate within the tertiary lymphoid structures of gastric carcinoma microenvironment, were associated with favorable patient prognosis . Suppression of nova1 in such reactive t cells was related with poor patient prognosis, and this effect is thought to be related to the suppression of regulatory t cell functions . From these findings, the role of nova1 in tumor t cells of malignant lymphomas and non - tumor t cells of tissues seems to be different . In normal physiologic conditions, nova1 upregulation seems to be involved in normal immune regulation . In neoplastic t cells alternative splicing of pre - mrna is the main mechanism of transcriptome and proteome diversity, and the splicing program relies on the action of splicing factors like nova1 . The action of splicing factors is cell type - specific and independent of organ origin . Previous studies have suggested or shown that normal and tumorous hematopoietic cells are regulated by specific alternative splicing programs and alternative transcript variants such as c - myb, a transcription factor of hematopoietic cells; cd45, a transmembrane protein tyrosine phosphatase of t cells, b cells, and other hematopoietic cells; and cd20, a surface marker of mature b cells [15 - 17]. Likewise, nova1 may work as an alternative splicing factor in the lymphoreticular system and in lymphomagenesis of t / nk - cell lineage . Among the tested cell lines, only jurkat cells, which corresponded to immature t cells the functional role of nova1 in lymphoid cells and target genes regulated by nova1 in lymphomagenesis should be identified through further studies . In summary, nova1 was frequently upregulated in various subtypes of t / nk - cell lymphomas . Nova1 expression may be a new biomarker for t / nk - cell lymphomas and should be studied as a possible therapeutic target for this type of lymphoma.
The following terms were used to describe the different kinds of genetic maps used in this study, as suggested by hudson et al . (2012): 1) sex - averaged map: a consensus map for both parents of a pedigree; 2) consensus map: an integrated map based on segregation data from individual component maps; 3) composite map: an integrated map of different individual component maps built by a marker - merging method; and 4) component map: each of the maps used in the construction of a composite map . The graphics and the representations of genetic maps were produced with r 3.1.0 (r core team 2014). We used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . The first six maps were obtained from three controlled crosses (pedigrees #1, #2, and #3 in supplementary fig . S1, supplementary material online) between three different genotypes: corsica landes (cl), morocco landes (ml), and corsica morocco (cm). In total, 106, 117, and 94 full - sibs the regression mapping algorithm of joinmap 4.1 (van ooijen 2011) was used to produce two maps for each parental genotype (one per cross), according to a two - way pseudotestcross mapping strategy (grattapaglia and sederoff 1994), using testcross markers (i.e., segregating in a 1:1 mendelian ratio) only . The genetic maps were then combined into sex - averaged maps (corsica, landes and morocco, see supplementary fig . S1, supplementary material online) with the function combine groups for map integration of joinmap 4.1 . More details on the construction of the maps can be found in lagraulet (2015). The first population was a three - generation inbred pedigree consisting of an f2 population (#4 in supplementary fig . S1, supplementary material online) resulting from the selfing of an interprovenance tree (landes corsica). The second population was a three - generation outbred pedigree (g2, #5) resulting from a controlled cross of two intraprovenance hybrid trees (landes landes). For the f2 population, two different sets of individuals were used to generate two maps (f2_o and f2_n) with the record algorithm (van os et al . For the g2 population, one map for each parent (g2 m and g2f) was produced with the regression mapping algorithm of joinmap 4.1 (van ooijen, 2011). The f2_o, g2 m, and g2f maps included different marker types: aflp, single sequence repeat (ssr), expressed sequence tag (est), and snps from different arrays (chancerel et al . 2011, 2013), whereas the f2_n map contained only snps from the 9k snp - array (plomion et al . We made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map (referred to as f2c by plomion et al . The last four maps were generated from two different f1 crosses: c14c15 (#6 in supplementary fig . S1, supplementary material online) and gal1056oria6 (#7). From the initial parental maps of the c14c15 mapping population described by de miguel (2012), we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . (2011) and 273 snps from a 1,536 snp - array (sez - laguna et al . For both pedigrees, we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . The four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers (ssrs, ests, sampls, and snps). For all maps, genetic distances in centimorgans (cm) were calculated with the kosambi mapping function (kosambi 1943). We carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family, for which sequence information was publicly available . Eckert et al . (2010) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda (accession number: the white spruce pedigree was an f1 full - sib family, whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . Lind et al . (2014) provided the most saturated and gene - rich map to date for pic . This map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . A detailed list of mapping features for each component map included in this study is available in table 1 . A high - density linkage map for c. japonica was incorporated into this study, as a representative species from the cupressaceae family (moriguchi et al . This map was constructed from an f1 full - sib family (table 1), with the regression mapping algorithm implemented in joinmap v 3.0 . The 17 maps described above were mostly constructed with snp markers (100% of the markers for picea sp ., 98% for p. pinaster, and 90% for p. taeda). The flanking sequences of each snp marker were compared with the most recent unigene sets available for each species to obtain the sequence of unigenes containing the mapped snps: canales et al . This comparison was carried out with the blastn tool (the blast 1 step in fig . Unigenes with a percentage identity exceeding 95% with mapped snp flanking sequences were retained for the next step . (2014), pine v3, was then used as the reference for the identification of homologous unigenes within pinaceae species . A second sequence comparison (r - blast 2 in fig . 1a) was performed, between the mapped unigenes of each species and the unigene sequences of pine v3 . For this interspecific comparison, only sequences with a reciprocal best hit with a percentage identity exceeding 85%, an e value below e, and an alignment of more than 200 bp were retained as homologous unigenes . Homologous unigenes between different species were considered as orthologs if they were positioned in the same lg (i.e., syntenic unigenes). Identified orthologous unigenes were used as anchor markers to construct a composite linkage map for each genus (pinus and picea), as a preliminary step in the construction of a composite map for the pinaceae family including both genera . We used the r package lpmerge (endelman and plomion 2014) to integrate component linkage maps into a composite map without the use of segregation data . Lpmerge assessed the goodness of fit of the composite map by calculating a root mean squared error (rmse) per lg, by comparing the position (in cm) of all markers on the composite map with that on the component maps . We calculated this metric for different maximum interval sizes (parameter k in the algorithm), ranging from 1 to 10 . The value of k minimizing the mean rmse per lg was selected for construction of the composite map . This method was used for the construction of all the composite species maps reported here . Further details about the production of each composite map are described below . Before integrating the 14 base genetic linkage maps into a single composite map, we established consensus maps (supplementary fig . S1, supplementary material online) based on markers common to different accessions across pedigrees (corsica, landes, morocco genotypes for pedigrees #1, #2, and #3, respectively), or accessions within pedigrees (coca and gxo for pedigrees #6 and #7, respectively), or based on the merging of different data sets of the same pedigree (f2 for pedigree #4). This process, designed to increase the number of markers common to component maps, was facilitated by the use of the same 12k (chancerel et al . 2013) and 9k (plomion et al . 2015) snp - arrays for some pedigrees . The snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . This step, which was essential for the use of lpmerge (i.e., same marker name for orthologous markers), also made it possible to check the collinearity between maps . Thus, nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps (supplementary table s2, supplementary material online). Finally, lpmerge was used to create the composite map for p. pinaster . Given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps, each component map was assigned the same weight in lpmerge . The snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster (canales et al . We established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . This process was designed to increase the number of markers common to the component maps for each genus (pinus and picea). Lpmerge was used to build these two composite maps, following the same procedure as described for p. pinaster . We discarded nonsyntenic unigenes, except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map, from the construction of composite linkage maps . Noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . Abies (lind et al . 2014), when the map for this species was compared with that for pic . Abies lg7 (renamed lg2 after comparison with the p. pinaster reference map) was reconstructed from genotyping data provided as supplementary material by lind et al . (2014), using the same parameters described in lind s article and the same mapping software (joinmap v4.1). Two markers with a log10 (p)> 1 that produced a large number of double recombinants were excluded from this lg map . We were thus able to map 16 additional markers, and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . The available map for c. japonica consisted of 77% of snp markers (moriguchi et al . Sequences of unigenes containing the mapped snps were retrieved from the unigene set developed by ueno et al . 2014) mapped in the pinaceae composite map using tblastx (blast 3, fig . Different e - value cutoff for homologous unigenes identification between pinaceae and c. japonica was tested: lower than 1e and 1e . Selected homologs from the pinaceae and c. japonica linkage maps were used for comparative mapping . We established orthologous blocks within lgs where several homologous unigenes were shared between both families . Different thresholds were also tested to consider an orthologous block within an lg: blocks with at least four and six shared unigenes . The most parsimonious evolutionary model between pinaceae and cupressaceae considering the existence of the identified cars was proposed . Circular genetic maps used in interfamily comparative mapping were drawn with circos software (krzywinski et al . The following terms were used to describe the different kinds of genetic maps used in this study, as suggested by hudson et al . (2012): 1) sex - averaged map: a consensus map for both parents of a pedigree; 2) consensus map: an integrated map based on segregation data from individual component maps; 3) composite map: an integrated map of different individual component maps built by a marker - merging method; and 4) component map: each of the maps used in the construction of a composite map . The graphics and the representations of genetic maps were produced with r 3.1.0 (r core team 2014). We used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . The first six maps were obtained from three controlled crosses (pedigrees #1, #2, and #3 in supplementary fig . S1, supplementary material online) between three different genotypes: corsica landes (cl), morocco landes (ml), and corsica morocco (cm). In total, 106, 117, and 94 full - sibs the regression mapping algorithm of joinmap 4.1 (van ooijen 2011) was used to produce two maps for each parental genotype (one per cross), according to a two - way pseudotestcross mapping strategy (grattapaglia and sederoff 1994), using testcross markers (i.e., segregating in a 1:1 mendelian ratio) only . The genetic maps were then combined into sex - averaged maps (corsica, landes and morocco, see supplementary fig . S1, supplementary material online) with the function combine groups for map integration of joinmap 4.1 . More details on the construction of the maps can be found in lagraulet (2015). The first population was a three - generation inbred pedigree consisting of an f2 population (#4 in supplementary fig . S1, supplementary material online) resulting from the selfing of an interprovenance tree (landes corsica). The second population was a three - generation outbred pedigree (g2, #5) resulting from a controlled cross of two intraprovenance hybrid trees (landes landes). For the f2 population, two different sets of individuals were used to generate two maps (f2_o and f2_n) with the record algorithm (van os et al . For the g2 population, one map for each parent (g2 m and g2f) was produced with the regression mapping algorithm of joinmap 4.1 (van ooijen, 2011). The f2_o, g2 m, and g2f maps included different marker types: aflp, single sequence repeat (ssr), expressed sequence tag (est), and snps from different arrays (chancerel et al . 2011, 2013), whereas the f2_n map contained only snps from the 9k snp - array (plomion et al . We made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map (referred to as f2c by plomion et al . The last four maps were generated from two different f1 crosses: c14c15 (#6 in supplementary fig . S1, supplementary material online) and gal1056oria6 (#7). From the initial parental maps of the c14c15 mapping population described by de miguel (2012), we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . (2011) and 273 snps from a 1,536 snp - array (sez - laguna et al . For both pedigrees, we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . The four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers (ssrs, ests, sampls, and snps). For all maps, genetic distances in centimorgans (cm) were calculated with the kosambi mapping function (kosambi 1943). We carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family, for which sequence information was publicly available . Eckert et al . (2010) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda (accession number: the white spruce pedigree was an f1 full - sib family, whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . Lind et al . (2014) provided the most saturated and gene - rich map to date for pic . This map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . A detailed list of mapping features for each component map included in this study is available in table 1 . A high - density linkage map for c. japonica was incorporated into this study, as a representative species from the cupressaceae family (moriguchi et al . This map was constructed from an f1 full - sib family (table 1), with the regression mapping algorithm implemented in joinmap v 3.0 . We used 14 base maps generated from seven different crosses to generate a composite genetic linkage map for p. pinaster . The first six maps were obtained from three controlled crosses (pedigrees #1, #2, and #3 in supplementary fig . S1, supplementary material online) between three different genotypes: corsica landes (cl), morocco landes (ml), and corsica morocco (cm). In total, 106, 117, and 94 full - sibs the regression mapping algorithm of joinmap 4.1 (van ooijen 2011) was used to produce two maps for each parental genotype (one per cross), according to a two - way pseudotestcross mapping strategy (grattapaglia and sederoff 1994), using testcross markers (i.e., segregating in a 1:1 mendelian ratio) only . The genetic maps were then combined into sex - averaged maps (corsica, landes and morocco, see supplementary fig . S1, supplementary material online) with the function combine groups for map integration of joinmap 4.1 . More details on the construction of the maps can be found in lagraulet (2015). The first population was a three - generation inbred pedigree consisting of an f2 population (#4 in supplementary fig . S1, supplementary material online) resulting from the selfing of an interprovenance tree (landes corsica). The second population was a three - generation outbred pedigree (g2, #5) resulting from a controlled cross of two intraprovenance hybrid trees (landes landes). For the f2 population, two different sets of individuals were used to generate two maps (f2_o and f2_n) with the record algorithm (van os et al . For the g2 population, one map for each parent (g2 m and g2f) was produced with the regression mapping algorithm of joinmap 4.1 (van ooijen, 2011). The f2_o, g2 m, and g2f maps included different marker types: aflp, single sequence repeat (ssr), expressed sequence tag (est), and snps from different arrays (chancerel et al . 2011, 2013), whereas the f2_n map contained only snps from the 9k snp - array (plomion et al . We made use of the large number of common markers and the high level of collinearity between the two f2 maps to construct a composite map (referred to as f2c by plomion et al . The last four maps were generated from two different f1 crosses: c14c15 (#6 in supplementary fig . S1, supplementary material online) and gal1056oria6 (#7). From the initial parental maps of the c14c15 mapping population described by de miguel (2012), we mapped an additional set of 980 snps from the 12k snp - array described by chancerel et al . (2011) and 273 snps from a 1,536 snp - array (sez - laguna et al . For both pedigrees, we used the maximum - likelihood mapping algorithm of joinmap 4.1 to generate individual genetic maps and sex - averaged maps . The four maps from the c14c15 and gal1056oria6 crosses contained different types of molecular markers (ssrs, ests, sampls, and snps). For all maps, genetic distances in centimorgans (cm) were calculated with the kosambi mapping function (kosambi 1943). We carried out a literature review to identify previously published high - density gene - based linkage maps for members of the pinaceae family, for which sequence information was publicly available . Eckert et al . (2010) provided a sex - averaged linkage map for a two - generation outbred pedigree based on snps for p. taeda (accession number: tg091, http://dendrome.ucdavis.edu/cmap/, last accessed may 21, 2015). The map provided by pavy et al . The white spruce pedigree was an f1 full - sib family, whereas the black spruce pedigree was a backcross representing the hybridization species complex pic . Lind et al . (2014) provided the most saturated and gene - rich map to date for pic . This map was a sex - averaged map of the two parents of an f1 controlled cross and was also constructed with the regression algorithm of joinmap 3.0 . A detailed list of mapping features for each component map included in this study is available in table 1 . A high - density linkage map for c. japonica was incorporated into this study, as a representative species from the cupressaceae family (moriguchi et al . This map was constructed from an f1 full - sib family (table 1), with the regression mapping algorithm implemented in joinmap v 3.0 . The 17 maps described above were mostly constructed with snp markers (100% of the markers for picea the flanking sequences of each snp marker were compared with the most recent unigene sets available for each species to obtain the sequence of unigenes containing the mapped snps: canales et al . This comparison was carried out with the blastn tool (the blast 1 step in fig . Unigenes with a percentage identity exceeding 95% with mapped snp flanking sequences were retained for the next step . (2014), pine v3, was then used as the reference for the identification of homologous unigenes within pinaceae species . A second sequence comparison (r - blast 2 in fig . 1a) was performed, between the mapped unigenes of each species and the unigene sequences of pine v3 . For this interspecific comparison, only sequences with a reciprocal best hit with a percentage identity exceeding 85%, an e value below e, and an alignment of more than 200 bp were retained as homologous unigenes . Homologous unigenes between different species were considered as orthologs if they were positioned in the same lg (i.e., syntenic unigenes). Identified orthologous unigenes were used as anchor markers to construct a composite linkage map for each genus (pinus and picea), as a preliminary step in the construction of a composite map for the pinaceae family including both genera . We used the r package lpmerge (endelman and plomion 2014) to integrate component linkage maps into a composite map without the use of segregation data . Lpmerge assessed the goodness of fit of the composite map by calculating a root mean squared error (rmse) per lg, by comparing the position (in cm) of all markers on the composite map with that on the component maps . We calculated this metric for different maximum interval sizes (parameter k in the algorithm), ranging from 1 to 10 . The value of k minimizing the mean rmse per lg was selected for construction of the composite map . This method was used for the construction of all the composite species maps reported here . Further details about the production of each composite map are described below . Before integrating the 14 base genetic linkage maps into a single composite map, we established consensus maps (supplementary fig . S1, supplementary material online) based on markers common to different accessions across pedigrees (corsica, landes, morocco genotypes for pedigrees #1, #2, and #3, respectively), or accessions within pedigrees (coca and gxo for pedigrees #6 and #7, respectively), or based on the merging of different data sets of the same pedigree (f2 for pedigree #4). This process, designed to increase the number of markers common to component maps, was facilitated by the use of the same 12k (chancerel et al . 2013) and 9k (plomion et al . 2015) snp - arrays for some pedigrees . The snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . This step, which was essential for the use of lpmerge (i.e., same marker name for orthologous markers), also made it possible to check the collinearity between maps . Thus, nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps (supplementary table s2, supplementary material online). Given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps, each component map was assigned the same weight in lpmerge . The snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster (canales et al . We established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . This process was designed to increase the number of markers common to the component maps for each genus (pinus and picea). Lpmerge was used to build these two composite maps, following the same procedure as described for p. pinaster . We discarded nonsyntenic unigenes, except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map, from the construction of composite linkage maps . Noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . Abies (lind et al . 2014), when the map for this species was compared with that for pic . Abies lg7 (renamed lg2 after comparison with the p. pinaster reference map) was reconstructed from genotyping data provided as supplementary material by lind et al . (2014), using the same parameters described in lind s article and the same mapping software (joinmap v4.1). Two markers with a log10 (p)> 1 that produced a large number of double recombinants were excluded from this lg map . We were thus able to map 16 additional markers, and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . Before integrating the 14 base genetic linkage maps into a single composite map, we established consensus maps (supplementary fig . S1, supplementary material online) based on markers common to different accessions across pedigrees (corsica, landes, morocco genotypes for pedigrees #1, #2, and #3, respectively), or accessions within pedigrees (coca and gxo for pedigrees #6 and #7, respectively), or based on the merging of different data sets of the same pedigree (f2 for pedigree #4). This process, designed to increase the number of markers common to component maps, was facilitated by the use of the same 12k (chancerel et al . 2013) and 9k (plomion et al . 2015) snp - arrays for some pedigrees . The snp marker i d of each component map was replaced by the corresponding maritime pine unigene i d from canales et al . This step, which was essential for the use of lpmerge (i.e., same marker name for orthologous markers), also made it possible to check the collinearity between maps . Thus, nonsyntenic unigenes between different p. pinaster linkage maps were removed from the analysis with the exception of those validated for at least two other component maps (supplementary table s2, supplementary material online). Given that similar numbers of genotypes were used to obtain the base maps and the high degree of synteny between base maps, each component map was assigned the same weight in lpmerge . The snp marker i d of each species component map was replaced by the corresponding homologous unigene i d of p. pinaster (canales et al . We established composite maps at genus level before integrating the four genetic maps for each species into a single composite map for pinaceae . This process was designed to increase the number of markers common to the component maps for each genus (pinus and picea). Lpmerge was used to build these two composite maps, following the same procedure as described for p. pinaster . We discarded nonsyntenic unigenes, except for nonsyntenic unigenes validated by at least two component maps in the p. pinaster composite map, from the construction of composite linkage maps . Noncollinear unigenes with inversions of more than 15 cm were also excluded from the construction of the composite linkage map . Abies (lind et al . 2014), when the map for this species was compared with that for pic . Abies lg7 (renamed lg2 after comparison with the p. pinaster reference map) was reconstructed from genotyping data provided as supplementary material by lind et al . (2014), using the same parameters described in lind s article and the same mapping software (joinmap v4.1). Two markers with a log10 (p)> 1 that produced a large number of double recombinants were excluded from this lg map . We were thus able to map 16 additional markers, and a much higher degree of synteny and collinearity was found between the homologous lgs of pic . The available map for c. japonica consisted of 77% of snp markers (moriguchi et al . 2012). Sequences of unigenes containing the mapped snps were retrieved from the unigene set developed by ueno et al . 2014) mapped in the pinaceae composite map using tblastx (blast 3, fig . Different e - value cutoff for homologous unigenes identification between pinaceae and c. japonica was tested: lower than 1e and 1e . Selected homologs from the pinaceae and c. japonica linkage maps were used for comparative mapping . We established orthologous blocks within lgs where several homologous unigenes were shared between both families . Different thresholds were also tested to consider an orthologous block within an lg: blocks with at least four and six shared unigenes . The most parsimonious evolutionary model between pinaceae and cupressaceae considering the existence of the identified cars was proposed . Circular genetic maps used in interfamily comparative mapping were drawn with circos software (krzywinski et al . All the linkage maps described here are available from the pinus portal (a european genetic and genomic resource for pinus) through the pinusmap application (https://w3.pierroton.inra.fr/pinusportal/index.php). Accession numbers for marker sequences used in this study are available in supplementary table s3, supplementary material online . Supplementary figures s1s5 and tables s1s3 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).
Low - grade lymphomas are chronic diseases, and some patients may live for many years following initial diagnosis.1 therefore, for many patients it is currently regarded as appropriate not to treat immediately at diagnosis, but rather to watch and wait . Once patients do require treatment, there are many therapies available: chemotherapeutic agents known to be effective in low - grade lymphoma include alkylating agents, anthracycline - based regimens, purine analogs, and bendamustine . In addition, there is the established monoclonal antibody therapy rituximab, and newer antibodies such as ofatumumab . Patients may also undergo stem - cell transplantation either autologous or allogeneic with or without myeloablation preceding allogeneic transplantation . Finally, there is currently a small amount of experience available on the use of techniques such as dna vaccination or antisense therapy . Questions about the long - term outcome of following an initial watchful waiting approach have been raised by a recent study, which compared this approach with immediate treatment with rituximab in patients with stage ii, iii or iv asymptomatic follicular lymphoma (fl).2 the estimated median time to initiation of new therapy among patients in the watchful waiting group was 33 months, whereas among those receiving rituximab the median time was not reached at 4 years (p<0.001). There were also significant differences in progression - free survival between the observation and rituximab arms (p<0.001), although no difference in overall survival . These data indicate that initial treatment with rituximab significantly delays the need for new therapy . This may, in the future, change the management approach to patients with newly - diagnosed fl . It is unclear whether some patients with low - grade lymphoma can be treated with curative intent, particularly as patients at stages i and ii, and some at stage iii (for example, those with up to five lymph nodes involved), can live for a long time after diagnosis . Usually treatment is undertaken to manage disease - associated symptoms, such as b symptoms, hematopoietic insufficiency (e.g. Anemia, leukopenia, and/or thrombocytopenia), rapid tumor progression, or bulky disease . Some patients develop phenomena such as autoimmune hemolytic anemia or idiopathic thrombocytopenic purpura as a consequence of their disease, and others may have recurrent infections as a result of hypogamma - globulinemia, all of which require treatment . At this time, combined immuno - chemotherapy with rituximab followed by rituximab maintenance therapy for 2 years is the standard of care for patients with fl (note that the most appropriate maintenance therapy for all other low - grade lymphomas is currently under investigation). What is less clear is the best chemotherapy to combine with rituximab . Chemotherapy combinations comprising cyclophosphamide, doxorubicin, vincristine, and prednisone [chop; or cyclophosphamide, vincristine, and prednisone (cvp)] are well accepted by clinicians . In addition, chlorambucil - based chemotherapies such as mitoxantrone, chlorambucil, and prednisone (mcp), and some fludarabine - based therapies [e.g. Fludarabine, cyclophosphamide, and mitoxantrone (fcm)] are commonly used, and bendamustine therapy has been investigated with promising results in these patients . Maintenance therapy with rituximab is also the standard of care in patients with low - grade lymphoma . This has been known for some time to be the optimal approach for patients with relapsed disease, and the recent results of the prima study indicate that this is also the most effective treatment strategy for patients with fl after first - line treatment with rituximab plus chemotherapy.3 there are currently few data on the use of non - pegylated liposomal doxorubicin (npld) specifically in low - grade lymphoma . Some of the publications available are case series, studies involving only small numbers of patients, or studies of patient populations with aggressive as well as low - grade lymphomas . One retrospective study of 37 patients with lymphomas who were either elderly or had cardiac comorbidities included three with fl, two with chronic lymphocytic leukemia, and one with multiple myeloma.4 these patients received npld in combination chemotherapy, and experienced a high rate of remission for this poor - risk population [complete remission rates were 75% for diffuse large b - cell lymphoma and 55% for t / nk - cell neoplasm; overall response rates (orr) of 80% and 89%, respectively], with no major cardiac toxicities . Rates of hematologic toxicity were comparable to those for regimens containing conventional anthracyclines.4 similarly, in a prospective study of 35 elderly and frail patients, five patients with fl received r - comp (r - chop with npld substituted for conventional doxorubicin) as a first - line therapy: r - comp was effective and well tolerated, and no cardiac events have been observed.5 again, the hematotoxicity of this combination was the same as that seen with the r - chop regimen . A small study has also been conducted using a combination of the new proteasome inhibitor bortezomib with npld, fludarabine, and ritux - imab in 16 patients with refractory / relapsed mantle - cell lymphoma (mcl).6 there was a good orr of 74% and 9 of 15 patients experienced a complete response (cr) or unconfirmed cr . Rates of neutropenia (37.5%) and thrombocytopenia (31.2%) were high, as expected; the rate of car - diotoxicity was 12.5% (although no information is provided on how this was defined). The german study group for indolent lymphoma (stil) conducted a prospective, randomized phase iii trial (nhl 1 - 2003) to compare the efficacy of bendamustine plus rituximab (br) with the widely accepted r - chop regimen in patients with low - grade lymphomas . The results were presented at the 2009 american society of hematology meeting as an oral presentation.7 bendamustine (90 mg / m) was administered on days 1 and 2, with rituximab on day 1, every 4 weeks for a maximum of six cycles . R - chop was given every 3 weeks, for a maximum of six cycles . In total, 513 patients were evaluable for efficacy and toxicity, most of whom (54%) had fl, with the remainder having mcl (18%), marginal zone lymphoma (mzl; 13%), waldenstrm macroglobuline - mia (wm; 8%), and small lymphocytic leukemia (sll; 4%). . There was a clear difference between br and r - chop in the incidence of grade iii and iv hemato - toxicities . There were significantly fewer cycles of br, compared with r - chop, in which patients experienced grade iii or iv leucocytopenia (12.1% versus 38.2%, respectively), neutropenia (10.7% versus 46.5%), or required granulocyte colony - stimulating factor (4.0 versus 20.0; p<0.0001 in all cases). Br was associated only with grade 1 alopecia and there was a low incidence of paraesthesias and stomatitis compared with r - chop . On the other hand, almost all patients in the br treatment arm received the full dose of chemotherapy (96.1% of cycles), whereas a smaller proportion of cycles of r - chop were completed at the full dose of chemotherapy (88.8%). The orr was good in both groups: 93.8% for br and 93.5% for r - chop.7 the cr rate was significantly higher with br versus r - chop (40.1% versus 30.8%; p=0.0323). Progression - free survival (pfs) was significantly longer with br (median pfs 54.8 months) compared with r - chop (median 34.8 months) at a median observation period of 32 months [hazard ratio (hr) 0.5765; 95% confidence interval (ci) 0.4292 - 0.7783; p=0.0002]. Similarly, event - free survival was 54 months for br and 31 months for r - chop (hr 0.6014; 95% ci 0.4515 - 0.7845; p=0.0002). Another key measure was the time to next treatment, which had not been reached in the br arm versus a median of 40.7 months with r - chop (hr 0.5416; 95% ci 0.3897 - 0.7491; p=0.0002). In summary, this study demonstrated that br significantly improved pfs and cr rates, compared with r - chop, in patients with fl, mcl, and wm . The stil study is now underway to investigate the effect of duration of maintenance therapy with rituximab in fl, and the effect of ritux - imab maintenance therapy in other low - grade lymphoma entities . In the nhl 7 - 2008 (maintain) study (nct00877214), patients with fl will be randomized to either 2 or 4 years of rit - uximab maintenance therapy, every 2 months, following br induction therapy in addition, infectious complications can be studied prospectively in this patient population, which is important because it has been reported that rituximab treatment is associated with neutropenia and increases infectious complications.8 another part of the maintain study is designed to investigate the efficacy of maintenance therapy in other forms of low - grade lymphoma . Patients with wm, mzl, or mcl will be randomized to either 2 years of maintenance therapy with rit - uximab every 2 months, or 2 years of watch and wait, both following br induction . Studies with other agents are also ongoing . As discussed above, combination therapy with bortezomib, npld, fludarabine, and rituximab was shown in a small study to be effective in relapsed mcl.6 the stil nhl 8 - 2010 study plans to further investigate the efficacy of combination therapy with bortezomib in patients with relapsed low - grade lymphomas . Lenalido - mide is another promising therapy in low - grade lymphomas, which has demonstrated good results in patients with several lymphoma types, in hard - to - treat relapsed or refractory disease . Low - grade lymphomas will become chronic diseases, and many patients will require treatment over long periods of time, with many treatment cycles . R - chop is commonly used to treat low - grade lymphoma, though chlorambucil- and fludarabine - based regimens are also used . The efficacy and toxicity of r - chop may be improved by substitution of npld into this combination, although data on the use of liposomal doxorubicin are currently more focused on aggressive lymphomas . Br therapy has produced promising results in several types of low - grade lymphoma, both in terms of efficacy and safety outcomes, and is recommended in the national comprehensive cancer network (nccn) clinical practice guidelines in oncology (v 1.2011) as first - line therapy for indolent lymphoma.11 the use of rituximab maintenance treatment after br induction is currently under investigation in different lymphoma types . In addition, the efficacy of a combination treatment of bortezomib and br will be studied.
Morphologic variants of papillary thyroid carcinoma (ptc) have been described already, some of which have worse prognosis and more recurrence rate than conventional ptc . Interestingly, the first clinical presentation of the tumor may be a metastasis to a lymph node of the neck from a small occult primary tumor . Cystic change has been identified in many thyroid lesions including ptc but this change is far more common in nonneoplastic thyroid nodules than thyroid neoplasms . Fine - needle aspiration (fna) has become a useful, safe, and accurate tool for diagnosis of thyroid lesions . The main purpose of this study was to describe the cytological features of cptc and compare the findings with other thyroid lesions with cystic change . A 30-year (1980 - 2013) retrospective review of the cytology slides from our cytology division of pathology department revealed a total of 750 ptcs . Out of these, 73 cases of cptc, which were confirmed by histologic sections, were retrieved from the files: 55 females and 18 males, with an age range of 28 - 52 years . Aspiration was performed from the cysts tissue (42 cases under ultrasound guidance and the remaining without guidance). The cysts yielded 2 - 3 ml of the fluid that was hemorrhagic in 40 cases and clear yellow in color in the remaining cases . The air - dried smears were stained with the wright giemsa stain, and alcohol - fixed smears were stained with papanicolaou stain . Some selected cases were subjected to immunocytochemistry for thyroid transcription factor-1 (ttf-1, dako, clone 8g7g3/1, denmark), cytokeratin 19 (ck 19) (dako, clone rck108, denmark), and cluster of differentiation 68 (cd68) (dako, clone kp1, denmark). The fna smears of other cystic thyroid lesions were retrieved from the files for the comparative study . These cases included 300 colloid goiters, 290 adenomatoid nodules, 11 follicular neoplasms, and 9 hurtle cell neoplasms . The main cytologic findings of 73 cases of cptc were as follows: isolated plasmacytoid cells (71, 97% cases) (which showed deep basophilic dense cytoplasm and dusty chromatin), and some of them showed cytoplasmic vacuolization; signet - ring formation (65 cases, 89%); cytoplasmic blebs (pseudopods); intranuclear cytoplasmic inclusions (42 cases, 57%); and nuclear grooves (46 cases, 63%) [figure 1a and b]. Small groups of 5 - 10 cells with scalloped (well - defined) margins and distinct cell borders were frequently seen (72 cases, 98%). Small and large papillary clusters with infiltrative borders were seen after searching many fields in all cases . Some of them showed central cores of hyaline material, nuclear inclusion, and grooves . Small clusters with a radial distribution of cells (cartwheel pattern) were readily observed (61 cases, 83%). Small ball - like cellular clusters with a concentric arrangement of cells (cellular swirls) were also observed in 58 (70%) cases . (wright - giemsa, 1000) sticky (wispy) colloids (31 cases, 42%) and intracellular colloids (32 cases, 43%) were observed, but colloid balls were not noted . Anisonucleosis (69 cases, 94%), multinucleated giant cells (39 cases, 53%), hemosiderin - laden macrophages (73 cases, 100%), spindle cells (31 cases, 42%), and squamoid cells (30 cases, 41%) were also observed . Atypical follicular cells with mesothelial - like features, such as cytoplasmic blebs (pseudopods) and flat borders or windows (i.e. The cells separated by narrow slit - like spaces), were also seen in five cases [figure 2a c]. (a and b) isolated mesothelial - like cell with cytoplasmic blebs and nuclear groove and pseudo - inclusion . (wright - giemsa, 1000) (c) mesothelial - like cells separated by narrow slit - like space (window). (wright - giemsa, 1000) three cases of cptc were associated with hashimoto's thyroiditis and showed erythro- and lymphophagocytosis (follicular cells engulfing mature rbc and lymphocytes). Ttf-1, ck19 immunostaining of these cases, and negativity for cd 68 also showed the follicular epithelial origin of these cells [figure 3a c]. (b) neoplastic follicular cell with intranuclear inclusion shows engulfed lymphocyte and cytoplasmic vacuole . (ttf- ihc, 400) figure 4a and b shows corresponding histologic sections of a cptc . (a and b) histologic sections of a case of cptc with papillary configuration facing a cystic space with optically clear nuclei . (a: h and e, 250; b: h and e, 400) the comparative cytological study results of cptc with other cystic thyroid lesions are shown in table 1 . Comparative study of cytomorphologic findings in cystic thyroid lesions adenomatoid (nodular) colloid goiter is the most common cause of a nonneoplastic cystic lesion of the thyroid gland . It is estimated that about 23% of the thyroid cystic lesions are proved to be malignant . Among the neoplasms, the fna diagnosis of cystic thyroid lesions is very difficult due to low cellularity, presence of nuclear debris, and many hemosiderin - laden macrophages . In fna smears, the presence of histiocyte / macrophages is a surrogate marker for cystic degeneration but these cells may show nuclear atypia and prominent nucleoli mimicking thyroid malignancy, especially ptc . Moreover, the presence of squamous cells in benign cysts can lead to a misdiagnosis of a malignant tumor . On the other hand, follicular epithelial cells in cptc may show histiocytic morphology; therefore, differential diagnosis of histiocyte from histiocytoid ptc is of great importance . Careful screening of all smears and comprehensive knowledge of the cytologic findings of cystic thyroid lesions may improve the precision of the diagnoses and prevent false - negative and false - positive diagnoses . The cytological findings of these lesions have been described well in previously published papers and textbooks . With minimal effort, the cystic colloid goiter and other cystic neoplasms can be differentiated from cptc . By contrast, the differentiation of cptc from cystic adenomatoid nodules is very difficult because the two have many findings in common . Some findings of this study were common to all the cysts and were therefore not useful for differentiation, such as foamy macrophages, atypical histiocytes, spindle cells, squamous cells, anisonucleosis, multinucleated giant cells, and calcification . It has been stated that multinucleated giant cells in ptc are more irregular in shape and have more nuclei and dense cytoplasm, but these cells can be seen in inflammatory thyroiditis as well . Isolated plasmacytoid cells were frequently observed in all cases of cptc, but not in case of other cystic lesions, with the exception of cystic adenomatoid nodules . The isolated cells in cptc showed characteristic dense cytoplasm and dusty chromatin, whereas the plasmacytoid cells in cystic adenomatoid nodules showed fine granular cytoplasm and smooth chromatin . Various forms of colloid material (colloid balls, bubble gum, and intracytoplasmic colloid) were frequently seen in adenomatoid nodules, colloid goiter, cptc, and follicular neoplasms . However, the intracytoplasmic colloid was seen more frequently in cptc than in other lesions . Nuclear grooves and inclusions were readily observed in a large number of cases of cptc, but were only rarely seen in a few cases of adenomatoid nodules, follicular neoplasms, and hurtle cell neoplasm . Monolayer sheets and papillary clusters were noted in almost all cases, but the papillary clusters with central hyaline cores were seen only in cptc . The most important findings of this study specific to cptc were as follows: small clusters with scalloped margins, cellular swirls, and clusters with a cartwheel pattern . These findings were not noticed in other cystic lesions and therefore were useful for the differentiation of cptc from other cystic lesions . In this study, two interesting findings were noticed in cptc, but not in other cysts: hemophagocytic cells were found in three cases, which were associated with hashimoto's thyroiditis . The immunocytochemical study revealed positive reaction with ck19, and ttf-1 and negative reaction with cd68 . So, we believe that these cells are of follicular epithelial origin rather than macrophages.mesothelial-like cells with windows and cytoplasmic blebs were found in five cases . Hemophagocytic cells were found in three cases, which were associated with hashimoto's thyroiditis . The immunocytochemical study revealed positive reaction with ck19, and ttf-1 and negative reaction with cd68 . So, we believe that these cells are of follicular epithelial origin rather than macrophages . Stated that small cohesive sheets of epithelial cells with windows between cells may be seen in the cytology of benign thyroid cysts but the cells in cptc are crowded and overlapping . In conclusion, the cytologic differentiation of cptc from cystic adenomatoid nodules was very difficult . Small clusters with scalloped margins, cellular swirls, and clusters with a cartwheel pattern were seen only in cptc, which helped to differentiate cptc from adenomatoid nodule . Mesothelial - like cells and hemophagocytic cells were seen in a few cases of cptc.
In june 2006, a dutch farmer contacted the pig health unit of the veterinary faculty of utrecht university about an outbreak of exudative epidermitis among his swine . On his breeding farm (farm a), which had 200 sows (22.5 piglets / sow / year), a high preweaning mortality rate (20%) was caused by exudative epidermitis . Exudative epidermitis is a skin disease normally caused by s. hyicus and is usually an acute infection in suckling and weanling piglets . On farm a, a litter of 3-week - old piglets housed in a crate had clinical signs of exudative epidermitis . Other pigs on this farm had been unsuccessfully treated with ceftiofur, cefquinome, tylosin, and trimethoprim / sulfonamides . A skin lesion sample from 1 piglet was sent to the veterinary microbiological diagnostic center of utrecht university, where it was plated on sheep blood agar and macconkey agar and incubated at 37c for 24 h. no growth was seen on macconkey agar, but large numbers of hemolytic white colonies were found on the sheep blood agar . These colonies were identified as s. aureus by colony morphology examination, gram staining, catalase and coagulase testing, and api id32 staph (biomrieux, marcy - letoile, france). Antimicrobial drug susceptibility was determined by using an agar diffusion method with isosensitest agar (cm471, oxoid, basingstoke, uk) and neosensitab discs (rosco, taastrup, denmark). The breakpoints used were those recommended by the dutch committee on guidelines for susceptibility testing (4). The s. aureus was susceptible to enrofloxacin, trimethoprim / sulfamethoxazole, and fusidic acid and resistant to ampicillin, gentamicin, kanamycin, tetracycline, erythromycin, lincomycin, and tylosin . Because this s. aureus was resistant to multiple drugs, it was suspected of being mrsa, and presence of the meca gene was confirmed by pcr (5). Additional samples were taken from the skin lesions, nares, or both of 5 other 3-week - old piglets with exudative epidermitis from a different litter than the first piglet; from the nares of 1 healthy sow; and from the nares and throats of 2 veterinary students . The students had had contact with the pigs on the day the samples were taken and 1 week earlier . To investigate the source of the mrsa, samples were also taken from the nares of 12 healthy weanling pigs and 10 healthy gilts on a supplier farm (farm b) that had provided gilts for breeding to farm a. nares samples were also taken from 2 farmers on farm b (table). The samples were plated on sheep blood agar and incubated in tryptic soy broth, 4% saline, 1% mannitol, phenol red (16 g / ml), ceftizoxime (5 g / ml), and aztreonam (50 g / ml). After incubation at 37c for 48 h, broth cultures were plated on sheep blood agar and incubated at 37c for 24 h. suspected colonies were identified as mrsa, and antimicrobial drug susceptibility was determined as described above . Mrsa was cultured from the nares of 1 student (farm a), 1 farmer (farm b), 1 sow and 4 piglets (farm a), 20 pigs (farm b), and from skin lesions of 3 piglets (farm a). Susceptibility testing showed that all mrsa isolates were susceptible to fusidic acid, trimethoprim / sulfamethoxazole, and enrofloxacin and resistant to ampicillin, tetracycline, gentamicin, and kanamycin . Of the 32 isolates, 18 were susceptible to lincomycin, tylosin, and erythromycin, and 14 were resistant to these antimicrobial drugs . The phenotypic resistance of the isolates to oxacillin was confirmed by etest (abbiodisk, solna, sweden) according to manufacturer s guidelines . Mics were> 128 mg / l for all mrsa isolates . * for some,> 1 positive sample or 2 types of mrsa were obtained from the same pig or person . T, tetracycline; k, kanamycin; g, gentamicin; ty, tylosin; l, lincomycin; e, erythromycin; all isolates were also resistant to ampicillin . Mrsa isolates were genotyped by using pulsed - field gel electrophoresis (pfge) with smai according to the harmony protocol (6), spa typing (7), and multilocus sequence typing (mlst) (8). Typing of the staphylococcal cassette chromosome (sccmec) was performed by using pcr (911). Nontypeable mrsa associated with pig farming possesses dna methylase, which methylates the smai - recognition sequence and leads to uninterpretable results (12). Genotyping showed that all isolates had spa type t011 and mlst 398 and sccmec type iv (ccra / b gene type 2, mec complex non class a). Although 2 distinct resistance profiles were observed, all isolates belonged to the mlst / spa genotype associated with pigs in the netherlands . Because farm b regularly sells gilts to farm a, farm b is probably the source of the mrsa isolated from farm a. farm b is a closed farm that has not purchased pigs since 1996 . Further research is necessary to identify the source of mrsa on farm b. since 2002, human mrsa isolates sent to the national institute for public health and the environment by the regional laboratories are typed by pfge, and nontypeable mrsa in persons who are not in contact with pigs is rare . The mrsa - positive farmer and student had no other known risk factors for mrsa carriage . The differences in the resistance patterns may be caused either by erythromycin - lincomycin - tylosin resistance genes located on mobile elements such as plasmids or transposons (like tn554) or by differences in the expression of the resistance genes . Transmission of mrsa between pigs and pig farmers has been previously reported by voss et al . However, to our knowledge, ours is the first report of culturing mrsa from clinically diseased pigs . The infected piglets were only 3 weeks of age, which suggests that they might have been infected through contact with their mother . The isolation of mrsa from piglets with exudative epidermitis was unexpected . That large numbers of s. aureus but no s. hyicus were cultured from the skin lesions indicates clinical relevance . It was recently reported that 209 (39%) of 540 finishing pigs at dutch slaughterhouses were mrsa positive and that all mrsa had mlst 398 and were resistant to tetracycline (13). Farm a used many different antimicrobial drugs, including third - generation and fourth - generation cephalosporins, for treatment of exudative epidermitis; farm b regularly used amoxicillin . Currently, no precautions are taken before and during slaughter of mrsa - positive pigs . In conclusion colonization with mrsa seems to be widespread in dutch pigs; supplier farms that sell mrsa - colonized pigs to other farms play a role in spreading the organism . Because the netherlands exports pigs to other countries, further research on the prevalence of mrsa in pigs in foreign countries is warranted.
It is known that in patients with ischemic heart disease, anxiety and depression are predictive of adverse short- and long - term outcomes [1, 2]. In fact, patients who have anxiety or depression during hospital admission are at increased risk for higher rates of in - hospital complications such as recurrent ischemia, re - infarction and malignant arrhythmias [3, 4]. They also suffer higher mortality and re - infarction rates months to years after their initial cardiac event [4 - 7]. Anxiety disorders and depression are among the most prevalent psychiatric disorders . Given the prevalence of anxiety and depression in the general population and in patients with coronary heart disease [chd], the potential public health impact for preventing the development and progression of chd by appreciating the nature of the relationship between anxiety, depression and chd is enormous . Thus, it is clinically relevant in patient with cardiovascular disease to assess the psychological profile and treat emotional conditions that confer an increase risk of major adverse cardiovascular events . Anxiety is a negative affective state resulting from an individual s perception of threat and characterized by a perceived inability to predict, control or gain the preferred results in given situations . Anxiety seemed to be an independent risk factor for incident chd and cardiac mortality . The most recent meta - analysis of references [1980 to 2009] on prospective studies of nonpsychiatric cohorts of initially healthy persons, in which anxiety was assessed at baseline, has shown as anxious persons were at risk of chd [hazard ratio 1.26] and cardiac death [hazard ratio: 1.48], independently by demographic variables, biological risk factors, and health behaviors . In a much larger study, in subjects with no prior history of chd, a dose - response relationship was found between phobic anxiety and coronary heart disease mortality [relative risk 2.5, 95% confidence interval 1.00 to 5.96]. Patients who are too anxious frequently are unable to learn or act upon new information about necessary life - style changes . Moreover a recent swedish 37years longitudinal study which have investigated the long - term cardiac effects of depression and anxiety assessed at young age [18 - 20 years] according to international classification of diseases-8th revision [icd-8] criteria has been shown as anxiety is an independently predicted subsequent chd events . Observations have shown a hazard ratios associated with anxiety of 2.17 and 2.51 for chd and for acute myocardial infarction, respectively, against a corresponding hazard ratios associated with depression of 1.04 and 1.03 . Has examined, by a cross - sectional method, the effects of both depression and anxiety on platelet reactivity in a patient population with stable coronary artery disease . Findings show as subjects who were both depressed and anxious had significantly higher serotonin - mediated platelet aggregation compared with depressed - only subjects and subjects without affective symptoms . Anxiety and the mental stress associated with it contribute to excessive sns activation and catecholamine release . The bidirectional association between mood disorders and heart disease is multifaceted, involving an integration of several central and peripheral processes . On the base of the whole these data wittstein hypothesizes potential mechanisms of biological pathways between anxiety and serotonin to enhanced platelet activation . As indicated by dsm - tr depression is a clinical syndrome defined by the presence of five out of nine follow criteria during the same 2-week period: depressed mood, diminished interest or pleasure in daily activities, significant unintentional weight change, sleep disturbance, psychomotor retardation or agitation, fatigue or loss of energy, feelings of worthlessness or excessive / inappropriate guilt, decreased ability to concentrate, and thoughts of death or suicide . Depression is associated with a odd ratio of 2 to 7 fold elevated risk of subsequent cardiac events, which is comparable to traditional cardiovascular risk factors such as hypertension [19- 22] and this require a particular attention for the diagnosis . The diagnostic difficulty of the depression in cardiac patient is due to atypical clinical presentation of depression in this patients and this explains the elevated percentage under - diagnosis of depression among cardiac patients . In 2008 the american heart association recommended [and the american psychiatric association endorsed] that screening tests for depressive symptoms should be applied to identify patients who may require further assessment and treatment if appropriate referral for further depression assessment and treatment is available . Depressive symptom dimensions after myocardial infarction [mi] may be differently related to prognosis . Somatic / affective [e.g., fatigue, sleep problems, and poor appetite] dimensions appear to be associated with a worse cardiac outcome than cognitive / affective dimensions [e.g., shame, guilt and negative self - image]. Specifically somatic / affective symptoms of depression [odds ratio: 1.49] it has been observed to be related to a higher killip class and mortality during the follow - up [12 months] period [odds ratio: 1.92]. These findings, which are in concordance with earlier studies [24 - 26], put further light on possible behavioral pathways which could explain the relationship between depressive symptoms and prognosis in acs patients via unhealthy behavior, like smoking, less compliance, unhealthy diet and inactivity and lack of physical activity . Recently the american heart association [aha], the american college of cardiology, and the american college of physicians highlight the need to adopt validated and easily performed screening test for depressions and anxiety in patients with cardiovascular disease [29, 30]. Physicians have to be acknowledge that the psychological risk is not uniform across all patients and that psychological factors might cluster together within individuals . Currently, these individual differences are largely ignored in clinical research and practice, but they could be assessed with brief and standardized self - report measures and would do away with a single risk factor approach . The beck depression inventory [33, 34] is a self - report measure has 21 items grouped by diagnostic symptom [e.g., feelings of guilt, sadness, self - confidence and discouragement, loss of interest, crying, changes in appetite, sleep difficulties, suicidal ideation]. A cut - off score of 10 or greater identify the presence of depression . Scores at or above 10 are associated with poorer prognosis, whether for chd progression or hard medical endpoints such as death or myocardial infarction [36, 37]. Other symptom - based measure of depression used in cardiac patients are the centers for epidemiological studies - depression [ces - d] scale [38, 39], and the hospital anxiety and depression scale . To specifically assess exhaustion, it has been well studied in both screening for and follow - up of depression in primary care [42, 43]. The phq-2 consists of the two first questions of the phq-9, which deal with mood and lack of pleasure . A cut - off score of 3 or higher has a sensitivity of 83% and a specificity of 92%, fulfilling the need for a quick and reliable depression screening tool . The clinician can also ask for a yes - or - no answer to the two questions of the phq-2 . A yes to either of the two questions is up to 90% sensitive and 75% specific [43, 45]. Structured interviews are superior to questionnaires in differentiating dysthymia from minor depression . Based on the dsm- iv tr criteria, structured interviews have been developed to assess depression as structured clinical interview for dsm - iv tr axis i disorders [scid] and others [for review see]. Patients with acute coronary syndrome [acs] have many difficulties to emotional tolerate lengthy interviews specifically during the initial brief hospital stays . For this patients are indicated flexible clinical interviews conducted using an expressive - supportive method . The diagnostic interview and structured hamilton [dish] incorporates elements of other diagnostic and severity measures of depression, and is well suited for use with this population . The american heart association [aha] released a consensus document recommending that health care providers screen for and treat depression and anxiety in patients with coronary heart disease . Also pozuelo and colleagues recommend that clinicians systematically screen for it in their heart patients, in view of the benefits of antidepressant therapy . Pozuelo and colleagues think that clinicians should routinely screen for depression in cardiac patients and should not hesitate to treat it and eligible patients should routinely be referred to cardiac rehabilitation programs . As commented by dimsdale the recent observations on anxiety come at a time when psychiatry is once again redrawing diagnostic guidelines in the diagnostic and statistical manual . To evaluate anxiety, simple screening questionnaires that have high reliability and validity are available . The beck anxiety inventory, the zung self - rating anxiety scale, or hamilton anxiety scale can be used as part of the routine workup of every patient . The major disadvantages in using the above scales are the time required for administration [1525 minutes as part of a full battery], the need for scoring, and significant cost to purchase . A newly useful created instrument, the psychological general well being index - 6 [pgwbi-6] was designed specifically for the outpatient cardiology setting [54 - 56] (see table 1). This is a brief 6-item self - report measure that takes only minutes to perform and is highly correlated with other common scales and screens for anxiety, depressed mood, positive well - being, self - control, general health and vitality . The advantage of the pgwbi-6 is that only 1 scale is required to screen for all of these risk factors, and it can be filled out easily by the patient as part of the initial work up . The results can be quickly analyzed by a nurse or the cardiologist and is available for no charge . These scales provide data that can quickly lead to treatment decisions regarding mental health care . The screening can serve to delineate normal levels of anxiety and depression from more pathologic levels . Because anxiety can have neurobiologic, behavioral, and cognitive / psychologic correlates, it is imperative that cardiologists accurately elicit symptoms of anxiety in the history and via screening that draw upon these 3 domains . The treatment of depression is articulated in three phases: 1) acute phase; purpose: to reduce symptoms [6 - 12 weeks]; 2) continuation phase; purpose: prevent relapse [4 - 9 months]; and 3) maintenance phase; purpose: long - term treatment outcome . Pharmacological treatment of depression in patients with chd is complex and entails certain limitations and contraindications . Selective serotonin reuptake inhibitors [ssri] tend to be tolerated better than the traditional tricyclic antidepressants [tca]. However, most ssris should not be used in combination with class ic anti - arrhythmics and may also potentiate the effects of beta - adrenergic blocking agents . Furthermore, ssris appear to reduce depressive symptoms but depression tends not to completely remit in patients with cad [coronary artery disease]. The myocardial infarction depression intervention trial [mind - it] looked at whether the antidepressant mirtazapine [remeron] would improve long - term depression and cardiovascular outcomes in depressed post - mi patients . In 18 months of follow - up, neither objective was obtained . The cardiac randomized evaluation of antidepressant and psychotherapy efficacy [create] trial tested the efficacy of the ssri citalopram [celexa] and interpersonal therapy in a short - term intervention . Here, the antidepressant was superior to placebo in the primary outcome of treating depression, but interpersonal therapy had no advantage over clinical management, ie, a shorter, 20-minute supportive intervention . The main goal of this approach is to modify dysfunctional thoughts and emotions by structured and empathic questioning of patients perceptions and thought processes . The typical duration of psychological interventions in cad patients ranges from 8 to 12 weeks [65- 68]. Sebregts and colleagues intended to develop a relatively short intervention program of 8 weeks, accessible for a large group of ami [acute myocardial infarction] and coronary artery bypass grafting [cabg] patients . It was directed at the reduction of psychological stress / distress [type a behavior in particular], the reduction of excessive consumption of dietary fat, elevated serum cholesterol, lack of physical exercise, and although less explicitly smoking and insufficient social support through the involvement of the patients partners in the intervention . The results of this study show that type a behavior can be reduced in coronary patients through a relatively short intervention program aimed at behavior change and risk reduction . Although no favorable effects were found on depression or vital exhaustion . In view of the findings on the diagnosis of depression, researchers do not unequivocally advise the intervention to the general population of ami and cabg patients . Other behavioral medicine group programs have attempted to incorporate stress management, relaxation training / cognitive behavior therapy and lifestyle modification for heart disease including ornish's lifestyle heart trial [70, 71] and the recurrent coronary prevention project for reducing type - a behavior . In the case of the former, actual regression of coronary artery stenosis was achieved in 1-year, and 4- and 5- year follow - up studies in those participants who maintained the lifestyle changes . A recent study of an integrative medicine approach applied to cardiac risk reduction demonstrated that after 10 months of a personalized health plan consisting of mindfulness meditation, relaxation training, stress management, and health education and coaching, patients in the active treatment group had lowered risk, lost more weight, and exercised more frequently compared with the usual care group . Overall behavioral medicine group programs have been shown to reduce the cost of unnecessary medical procedures and work - ups [especially related to anxiety], improve complain with treatment, and reduce the risk factors of stress and an unhealthy lifestyle . As well because these are group interventions this helps patients overcome stigma, social isolation and learn how to better manage their anxiety and depression . Also for anxiety, cognitive behavior therapy, a well - documented evidence - based treatment, should be instituted at the beginning of treatment to ensure that patients understand their condition and that medication management is only one aspect of their treatment . In cognitive behavior therapy, patients are taught to restructure anxiety - provoking thoughts leading to panic attacks, are taught relaxation techniques to counteract stress and anxiety, and are given exposure therapy to desensitize themselves to stressful stimuli . Cognitive behavior therapy is also extremely useful and effective in teaching anger management skills when treating type a behavior pattern [tabp personality]. Cognitive behavior therapy conveys the message to the patient that it is possible to learn self - management techniques and methods that will most likely allow them to discontinue medications within 6 months to 1 year . Other forms of psychotherapy, such as psychoanalytic, interpersonal, and supportive therapies, can be effective as adjunctive therapies, but do not carry the wealth of evidence - based research demonstrating their effectiveness in the treatment of anxiety disorders . A new proposed anxiety and depression treatment algorithm is based on pgwb initial screening (see fig . (1)), when the screening identifies patients who have more than mild anxiety and depression and have failed 1 - 2 medication trials, have suicidal or homicidal ideation or a history thereof, alcohol / substance abuse, mania or psychosis, history of sexual, physical abuse, or severe psychosocial distress, a referral should be made to a mental health professional . Integrating ongoing assessments reduces the stigma of mental illness and leads to the greater likelihood of patients receiving adequate mental health treatment . This strengthens the treatment alliance and can improve compliance with medical treatment . Among the psychotherapies, cognitive behavior therapy [cbt] and interpersonal psychotherapy [ipt] are the most effective, in both acute and maintenance treatments . Interpersonal psychotherapy is a time - limited [12 - 16 weekly 1-hour sessions] manualized psychotherapy that was developed in the early 1970 s by gerald klerman and colleagues as a research intervention for outpatients with unipolar major depression . Interpersonal psychotherapy is based on the presumption that interpersonal issues affect mood and mood impairs interpersonal functioning . Interpersonal psychotherapy has been extensively researched and shown to be effective as both an acute and preventive treatment for major depressive disorder . Ipt conceptualizes clinical depression as having three component processes: symptom formation caused by biological and/or psychological mechanisms; social functioning [involving, obviously, social interactions with others]; and enduring personality traits . Ipt intervenes in symptom formation and social dysfunction but does not address enduring personality traits in view of the time limits of treatment, the relatively low level of psychotherapeutic intensity, the emphasis of treatment on the current depressive episode, and the difficulty in accurately evaluating personality in the midst of an axis i disorder . The brevity of the treatment imposes a structure that pressures the patient and therapist to work quickly to alleviate depressive symptoms and resolve the current interpersonal crisis linked with the depression, thus discouraging the patient s becoming dependent on the therapist . For each patient, therapy focuses on one or, at most, two interpersonal problem areas that are identified as precursors of the current depressive episode . These problem areas were derived from extensive research on the role of environmental influences on mood and are characterized as unresolved grief following the death of a loved one, role transitions [difficulty adjusting to changed life circumstances], interpersonal role disputes [conflicts with a significant other] and interpersonal deficits [impoverished social networks]. Maintaining the focus of treatment on a problematic interpersonal issue prevents the therapy from becoming too diffuse and forces the therapist and patient to discuss material that is relevant to the focal area and treatment objectives . Ipt also focuses on the here and now, that is, on current problematic interpersonal issues that are amenable to change, rather than on reconciling unresolved interpersonal problems having to do with the past . The focus on resolving current interpersonal problems and developing strategies for warding off future problems helps reduce the depressed patient s tendency to ruminate about past events and experiences that cannot be changed and which only serve to reinforce the patient s already low sense of self - esteem and dysphoria . The primary goal of ipt is to obtain the remission of depressive symptoms by facilitating resolution of a current interpersonal crisis . At one - year follow - up, patients who received interpersonal psychotherapy had better psychosocial functioning than those who did not receive this treatment . The national institute of mental health treatment of depression collaborative research program [nimh tdcrp] found that, in general, interpersonal psychotherapy is an effective monotherapy for patients with mild to moderate depression, but that antidepressant medication should be used as a first line treatment for severely ill patients . Pharmacological treatment of depression in patients with chd is complex and entails certain limitations and contraindications . Selective serotonin reuptake inhibitors [ssri] tend to be tolerated better than the traditional tricyclic antidepressants [tca]. However, most ssris should not be used in combination with class ic anti - arrhythmics and may also potentiate the effects of beta - adrenergic blocking agents . Furthermore, ssris appear to reduce depressive symptoms but depression tends not to completely remit in patients with cad [coronary artery disease]. The myocardial infarction depression intervention trial [mind - it] looked at whether the antidepressant mirtazapine [remeron] would improve long - term depression and cardiovascular outcomes in depressed post - mi patients . In 18 months of follow - up, neither objective was obtained . The cardiac randomized evaluation of antidepressant and psychotherapy efficacy [create] trial tested the efficacy of the ssri citalopram [celexa] and interpersonal therapy in a short - term intervention . Here, the antidepressant was superior to placebo in the primary outcome of treating depression, but interpersonal therapy had no advantage over clinical management, ie, a shorter, 20-minute supportive intervention . The main goal of this approach is to modify dysfunctional thoughts and emotions by structured and empathic questioning of patients perceptions and thought processes . The typical duration of psychological interventions in cad patients ranges from 8 to 12 weeks [65- 68]. Sebregts and colleagues intended to develop a relatively short intervention program of 8 weeks, accessible for a large group of ami [acute myocardial infarction] and coronary artery bypass grafting [cabg] patients . It was directed at the reduction of psychological stress / distress [type a behavior in particular], the reduction of excessive consumption of dietary fat, elevated serum cholesterol, lack of physical exercise, and although less explicitly smoking and insufficient social support through the involvement of the patients partners in the intervention . The results of this study show that type a behavior can be reduced in coronary patients through a relatively short intervention program aimed at behavior change and risk reduction . Although no favorable effects were found on depression or vital exhaustion . In view of the findings on the diagnosis of depression, researchers do not unequivocally advise the intervention to the general population of ami and cabg patients . Other behavioral medicine group programs have attempted to incorporate stress management, relaxation training / cognitive behavior therapy and lifestyle modification for heart disease including ornish's lifestyle heart trial [70, 71] and the recurrent coronary prevention project for reducing type - a behavior . In the case of the former, actual regression of coronary artery stenosis was achieved in 1-year, and 4- and 5- year follow - up studies in those participants who maintained the lifestyle changes . A recent study of an integrative medicine approach applied to cardiac risk reduction demonstrated that after 10 months of a personalized health plan consisting of mindfulness meditation, relaxation training, stress management, and health education and coaching, patients in the active treatment group had lowered risk, lost more weight, and exercised more frequently compared with the usual care group . Overall behavioral medicine group programs have been shown to reduce the cost of unnecessary medical procedures and work - ups [especially related to anxiety], improve complain with treatment, and reduce the risk factors of stress and an unhealthy lifestyle . As well because these are group interventions this helps patients overcome stigma, social isolation and learn how to better manage their anxiety and depression . Also for anxiety, cognitive behavior therapy, a well - documented evidence - based treatment, should be instituted at the beginning of treatment to ensure that patients understand their condition and that medication management is only one aspect of their treatment . In cognitive behavior therapy, patients are taught to restructure anxiety - provoking thoughts leading to panic attacks, are taught relaxation techniques to counteract stress and anxiety, and are given exposure therapy to desensitize themselves to stressful stimuli . Cognitive behavior therapy is also extremely useful and effective in teaching anger management skills when treating type a behavior pattern [tabp personality]. Cognitive behavior therapy conveys the message to the patient that it is possible to learn self - management techniques and methods that will most likely allow them to discontinue medications within 6 months to 1 year . Other forms of psychotherapy, such as psychoanalytic, interpersonal, and supportive therapies, can be effective as adjunctive therapies, but do not carry the wealth of evidence - based research demonstrating their effectiveness in the treatment of anxiety disorders . A new proposed anxiety and depression treatment algorithm is based on pgwb initial screening (see fig . (1)), when the screening identifies patients who have more than mild anxiety and depression and have failed 1 - 2 medication trials, have suicidal or homicidal ideation or a history thereof, alcohol / substance abuse, mania or psychosis, history of sexual, physical abuse, or severe psychosocial distress, a referral should be made to a mental health professional . Integrating ongoing assessments reduces the stigma of mental illness and leads to the greater likelihood of patients receiving adequate mental health treatment . This strengthens the treatment alliance and can improve compliance with medical treatment . Among the psychotherapies, cognitive behavior therapy [cbt] and interpersonal psychotherapy [ipt] are the most effective, in both acute and maintenance treatments . Interpersonal psychotherapy is a time - limited [12 - 16 weekly 1-hour sessions] manualized psychotherapy that was developed in the early 1970 s by gerald klerman and colleagues as a research intervention for outpatients with unipolar major depression . Interpersonal psychotherapy is based on the presumption that interpersonal issues affect mood and mood impairs interpersonal functioning . Interpersonal psychotherapy has been extensively researched and shown to be effective as both an acute and preventive treatment for major depressive disorder . Ipt conceptualizes clinical depression as having three component processes: symptom formation caused by biological and/or psychological mechanisms; social functioning [involving, obviously, social interactions with others]; and enduring personality traits . Ipt intervenes in symptom formation and social dysfunction but does not address enduring personality traits in view of the time limits of treatment, the relatively low level of psychotherapeutic intensity, the emphasis of treatment on the current depressive episode, and the difficulty in accurately evaluating personality in the midst of an axis i disorder . The brevity of the treatment imposes a structure that pressures the patient and therapist to work quickly to alleviate depressive symptoms and resolve the current interpersonal crisis linked with the depression, thus discouraging the patient s becoming dependent on the therapist . For each patient, therapy focuses on one or, at most, two interpersonal problem areas that are identified as precursors of the current depressive episode . These problem areas were derived from extensive research on the role of environmental influences on mood and are characterized as unresolved grief following the death of a loved one, role transitions [difficulty adjusting to changed life circumstances], interpersonal role disputes [conflicts with a significant other] and interpersonal deficits [impoverished social networks]. Maintaining the focus of treatment on a problematic interpersonal issue prevents the therapy from becoming too diffuse and forces the therapist and patient to discuss material that is relevant to the focal area and treatment objectives . Ipt also focuses on the here and now, that is, on current problematic interpersonal issues that are amenable to change, rather than on reconciling unresolved interpersonal problems having to do with the past . The focus on resolving current interpersonal problems and developing strategies for warding off future problems helps reduce the depressed patient s tendency to ruminate about past events and experiences that cannot be changed and which only serve to reinforce the patient s already low sense of self - esteem and dysphoria . The primary goal of ipt is to obtain the remission of depressive symptoms by facilitating resolution of a current interpersonal crisis . At one - year follow - up, patients who received interpersonal psychotherapy had better psychosocial functioning than those who did not receive this treatment . The national institute of mental health treatment of depression collaborative research program [nimh tdcrp] found that, in general, interpersonal psychotherapy is an effective monotherapy for patients with mild to moderate depression, but that antidepressant medication should be used as a first line treatment for severely ill patients . Despite the large amount of evidence supporting significant and independent associations between anxiety and depression and the pathogenesis of cardiovascular disease, the 2010 american college of cardiology foundation / american heart association guideline for assessment of cardiovascular risk in asymptomatic adults [84, 85] does not consider yet any of them . Further research is warranted to determine factors that may moderate anxiety in order to better understand the phenomenon among acute myocardial infarction patients and develop effective interventions . Future studies need to disentangle the cause effect relationships between depression, anxiety and adverse health behaviors in patients with coronary artery disease . As commented on by dimsdale the recent findings regarding anxiety come at a time when psychiatry is once again redrawing diagnostic guidelines in the diagnostic and statistical manual . For decades, the diagnostic and statistical manual has differentiated between anxiety disorders and depressive disorders but in clinical practice these disorders rarely occur in isolation and the distress associated with them increases synergistically when both sets of symptoms coexist . . It would be useful for clinical practice to develop treatments that combine strategies for anxiety and depression and evaluate whether such treatments would reduce cardiovascular risk . Also anxiety is common among cardiac patients and should be treated to enhance recovery and decrease patients risk of subsequent cardiac events . One of the most important areas for future research is elucidating the mechanisms whereby anxiety causes poorer outcomes in acute myocardial infarction patients . The mechanisms [either physiological or behavioral] whereby anxiety is related to poorer short and long term outcomes in acute myocardial infarction patients have yet to be elucidated . Research in this area is important to help clinicians determine the best ways to manage acute myocardial infarction patients to decrease the negative impact of anxiety . In future trials, it is important to pay particular attention to nonresponders to intervention, because these patients have been shown to be at a higher risk of late mortality compared with responders . If we are to continue lowering the rates of chd, we have to continue to emphasize prevention of the modifiable risk factors . This will require that cardiologists, cardiovascular surgeons and mental health professionals work together to provide comprehensive treatments that address not only chd but optimize the mental health of patients.
Mycobacterium tuberculosis isolates from all culture - positive tb patients in seattle and king county, washington, during 20032004 were genotyped by spacer oligonucleotide typing and mycobacterial interspersed repetitive unit methods . We included patients who had an isolate that matched the outbreak strain or who had a social link to an already included patient . Patient medical records were reviewed, and infectious periods were calculated . For sputum smear positive patients, the infectious period extended from 3 months before symptom onset or the first positive smear (whichever was earlier) until 2 weeks after the start of appropriate tb treatment or until the patient was placed into isolation or produced consecutively negative smears . For sputum smear negative patients, the infectious period extended from 1 month before symptom onset, the start of appropriate tb treatment, or the date that the patient was isolated (whichever was earlier), until 2 weeks after the start of appropriate tb treatment or until patient isolation (7). We interviewed patients to learn their contacts, activities, and locations frequented while they were contagious . Additional contacts were found by outreach workers and a disease intervention specialist from the east - african community who was hired to work in the neighborhoods frequented by the patients . While in these neighborhoods, outreach workers and the disease intervention specialist recruited persons seen with patients or their contacts to be evaluated for tb and latent tb infection . Contact activities, specifically those related to illicit drugs, were observed or self - reported . Friends were defined as contacts of patients who spent time within a close - knit network of young men who exhibited similar marijuana - using behavior . Other contacts were defined as the families and relatives of patients and those who were named but were not closely associated with this network . Contacts received a tb evaluation including a tuberculin skin test (tst) to detect infection . Patient 1 was first evaluated in december 2003, when a chest radiograph suggested pulmonary tb (i.e., upper lobe cavitary infiltrate). However, only clarithromycin was prescribed, and the patient was lost to follow - up . He was again seen in an emergency room in april 2004 after the infection evolved into bilateral extensive pulmonary tb . Eight patients were born in east africa; a median of 13 years (range 622) had passed since their arrival in the united states . Patient 5 was a white woman who received illicit drugs from patient 1 . * unknown for 1 patient . Patients were symptomatic and had findings indicating infectiousness: all had pulmonary tb, 7 had cavitary disease, and 8 had sputum that tested smear - positive for acid - fast bacilli . Consecutive chest radiographs indicated progression to cavitary disease in <75 days weeks in 3 patients and <121 days in another patient . Table 2 shows the dates of clear chest radiographs interpreted as normal and the first chest radiographs showing disease . While contagious, patients stayed in various locations, including cars, for most of the day . A single - bedroom apartment occupied by at least 1 patient while he was contagious was regularly visited by 2 other patients . Numerous members of the friend network slept there on any given night, and many others would regularly visit during a 10-week period beginning in april 2004 (figure). The occupants nailed boards over the apartment windows to conceal activities, primarily marijuana use, from outsiders . All reported frequent " hotboxing, " the practice of smoking marijuana with others in a vehicle with the windows closed so that exhaled smoke is repeatedly inhaled . Considerable overlap in infectious periods was noted, which highlights the potential for simultaneous contact with multiple contagious patients . At least 31 (57%) friend contacts spent time at the 1-bedroom apartment . After those with a past positive tst result were removed, 14 (64%) of 22 screened friends and 6 (23%) of 26 other contacts had a positive tst result . The risk for a positive tst result was 2.8 greater among friends than among other contacts (95% confidence interval = 1.36.0). Twenty - nine (54%) friend contacts self - reported or were observed hotboxing . Among the friends who reported or were observed hotboxing, 11 (79%) of 14 who received a tst had a positive result . Risk factors for tb include birth in a country with high tb prevalence (2) and incarceration (8). Although most patients in this outbreak were foreign - born and had histories of incarceration, genotyping results and epidemiologic findings suggest that tb was transmitted recently in the community rather than before immigration or during incarceration . Frequent marijuana use has been reported among tb outbreak patients (9) and was the behavior linking these patients together . Creative sharing of marijuana has been described recently as a factor for m. tuberculosis transmission . In australia, sharing a water pipe (i.e., " bong ") was linked to transmission (10). " Shotgunning " refers to inhaling smoke from illicit drugs then exhaling it directly into another's mouth (11) and was associated with m. tuberculosis transmission among a group of exotic dancers and their contacts (12). This investigation noted that a similar activity, hotboxing, might have contributed to transmission . One patient with smear - positive cavitary disease reported daily hotboxing with friends, often for most of the day . In addition, marijuana smoking might induce cough, creating an ideal environment for transmission . Many friends stayed and used marijuana at the single - bedroom apartment during the height of the outbreak . Furthermore, by nailing boards over the windows, ventilation was limited, creating an environment similar to that of hotboxing . Although progressive primary tb by nature is thought to be due to recent transmission, progressive primary tb with cavitation is uncommon (13). However, frequent marijuana use and the setting of intense exposure may have played a role . In addition, poor nutrition and unhealthy lifestyles might have predisposed these young men to more rapid progression of disease . While no laboratory investigation to assess genetic susceptibility or strain virulence was conducted, these factors might have also contributed to the development of cases . This outbreak resembles an outbreak reported among regular patrons of a neighborhood bar (14). Both were fueled by a highly infectious source patient who spent extended amounts of time indoors with 1 group of persons who regularly used substances (i.e., alcohol or marijuana). The result in both situations was a higher than expected incidence of tb disease and latent tb infection . In the outbreak reported in this article, however, the substance of choice was illicit and further complicated the control of this outbreak . Patients' illicit drug activities promoted a reluctance to name contacts at risk and locations frequented . Efforts had to revolve around meeting these young patients at times and locations convenient to the group . Then after gaining the groups' trust, outreach workers successfully found and screened contacts . Many successful screenings took place on street corners and in parking spaces throughout the community . Often outreach workers were successful only after spending hours driving throughout the community searching for patients and contacts . Alternative strategies to name - based contact investigations may become increasingly critical to tb control as tb recedes further from the general population, yet persists within smaller guarded groups (15).
The pcos is the most common cause of menstrual disorders, ovarian dysfunction, and infertility of women . Observational studies postulate that up to 15% of women suffer under this condition during their reproductive life . Pcos etiopathology is not clear, but most probably a strong genetic cause that is influenced by gestational environment and lifestyle seems to be the key factor . The most common features of pcos are hyperandrogenism, chronic anovulation, typical pcos ultrasound images, and skin issues such as acne, hirsutism, and seborrhea . Furthermore, recently it has been found that insulin resistance plays a key role in the clinical development of pcos in almost all the women . Severe disorders of the insulin sensitivity with a compensatory hyperinsulinemic state not only in obese pcos patients but also in lean women have been described, so that the hypothesis is strongly supported that the insulin resistance is independent of the weight . In particular, the related hyperinsulinemia could induce an excess of androgens production in pcos women through two different ways: first one is direct stimulation of ovaries to produce androgens, and the other one is the reduction of sex hormone binding globulin (shbg) serum levels . Due to the key role of insulin in the syndrome etiopathology, for many years, insulin sensitizers such as metformin, pioglitazone, or troglitazone have been considered as possible therapeutic options in the management of these problems . Metformin has been used in the last time on patients with a hyperinsulinemic status for the improvement of ovarian dysfunction with consecutive anovulation, irregular menstrual cycles, and infertility problems [3, 4]. Nevertheless metformin, when used in the therapeutic dose range, was shown to have several side effects such as flatulence, diarrhea, and nausea, so that many patients are unable to use this treatment option in gynecology for a longer period of time [5, 6]. Therefore, in parallel to the common use of metformin and other insulin sensitizer agents for the treatment of pcos, in the recent years, other therapeutic alternatives have been investigated . Myoinositol is one of the most interesting molecules that have been studied for the treatment of pcos . The substance inositol is a chemical compound with the formula c6h12o6 or (choh)6, a sixfold alcohol (polyol) of cyclohexane, with five equatorial and one axial hydroxyl group . Exist nine different stereoisomer forms, but myoinositol is the most common one found in nature . In fact, myoinositol is very often found in many plants and in tissues of animals . It is important to distinguish between the lecithin formulation that is bioavailable for the human and the phytate formulation of corns that are not bioavailable . Foods with the highest concentration of myoinositol are fruits, beans, corns, and nuts . Inositol was defined in the past as myometrial sugar, but it is indeed not a substance belonging to the carbohydrate group if we use modern definitions . Defining inositol as a vitamin b is also being discussed with controversy as inositol is not an essential substance and it can be produced in human cells from glucose . In fact, several studies have proved that the inositol molecule is directly involved in the insulin cellular signaling . Regarding pcos, several studies have shown that one of the mechanisms of insulin deficiency has its rise from the inositolphosphoglycan (ipg) mediator and that a deficiency of inositol in the inositolphosphoglycans is responsible for insulin resistance . It has demonstrated that the administration of d - chiro - inositol (intracellularly converted from myoinositol) could reduce the insulin resistance (see figure 1). Indeed, myoinositol, as a second messenger, plays an essential role for the signal pathways of cells . In particular, the action of myoinositol in a pcos pathway would be related to an improved insulin sensitivity and a sequent increased intracellular glucose uptake [2, 10]. All these pieces of evidence have opened a new clinical interest on myoinositol, as a potential insulin sensitizer agent to be used as safe and effective option in pcos patients, through the restoration of their metabolic profile and a consequent ovulation induction in infertile pcos patients . Studies report also a very good safety profile of the molecule, even when administered up to 12 grams / day, where only mild gastrointestinal side effects have been reported . The aim of this study was to determine the pregnancy rates under the use of a combination of myoinositol and folic acid in patients with a pcos in germany, to establish if this molecule can be used as a safer treatment option for the fertility improvement of this disease . A standardized questionnaire was created and a questionnaire (see appendix) was presented to 245 gynecologists present in germany, between june 2014 and march 2015 . During this time reports were generated of 3602 women with a pcos and infertility according to the rotterdam classification . The women started with the intake of myoinositol and folic acid at a dosage of 2 2000 mg myoinositol and 2 200 g folic acid per day and used it for at least 2 - 3 months . The primary outcome of the study was to determine the ovulatory function restoration and the pregnancy rate after treatment . The pregnancies were documented by the gynecologists and registered in a database, and these women were followed up during the whole pregnancy . Secondary outcome was the evaluation of side effects reported in those patients undergoing treatment . In a subgroup of patients, the values investigated were testosterone, free testosterone, and progesterone . In this group of patients the data of 3602 patients with a pco syndrome were evaluated . According to the obtained records 2520 women experienced an improvement of their menstrual cyclicity towards ovulatory cycles . Among them, a total number of 545 women became pregnant . The pregnancies occurred after the intake of two to three months of mayoinositol and folic acid . This means a ratio of 15.1% of the investigated women becomes pregnant during this observational study . No relevant side effects have been reported in the patients taking myoinositol and folic acid product . Figure 2 depicts the data . In the subgroup of 32 patients where hormonal values were evaluated a significant improvement of androgen levels and a rise in the progesterone values furthermore, out of these 32 women who became pregnant, 5 of them experienced an abortion, whereas the remaining 27 delivered healthy newborns . Despite the clear limitations of the observational study, there are reliable available data, since a wide range of patients can be analyzed . This study could show that a new treatment option for patients with a pcos and infertility is available . Furthermore, the achieved pregnancy rates are at least in a range equivalent to or even superior to those reported by the use of the insulin sensitizer metformin . Karimzadeh and javedani described a pregnancy rate of 14.4% in a cohort of 90 women and legro et al . The interesting results that the study has shown seem to be related to the mechanism of action of myoinositol . The administration of this molecule, acting as a direct messenger of insulin signaling and improving the glucose tissues uptake, could improve the insulin resistance status of pcos women, restoring indeed their hormonal status and restoring the ovulation process . Another important evidence is also related to the difference of myoinositol and metformin in terms of safety profile and compliance for patients . In patients under metformin, side effects have been commonly reported, in particular from mild up to severe gastrointestinal side effects, such as abdominal pain, nausea, and diarrhea . Only in rare cases,, myoinositol seems to be a safe and well - tolerated approach, anyhow able to give similar results of metformin in terms of clinical efficacy . In fact, many studies have demonstrated in the last months that an improvement in the rates of ovulation and regularization of menstrual cycles was obtained by the combined use of 4 g myoinositol with 400 g folic acid per day . Could show in a prospective study that the group of patients receiving myoinositol + folic acid experienced in 82% of the cases an ovulation, whereas this was only observed in 63% of the cases in the group of patients which received a placebo . By the same way 70% of the patients of the myoinositol group developed regular menstrual cycles after 16 weeks of treatment, whereas only 13% of the women did it in the placebo group . In a study of raffone et al ., where a comparison between the administration of myoinositol (2 2000 g + 200 g per day) and the administration of metformin (1500 mg per day) in women with a pco syndrome was performed, it could be shown that the number of pregnancies was clearly higher in the myoinositol group than in the metformin group of patients . Some other studies upon others have shown the efficacy of myoinositol in the improvement of the fertility of pcos patients due to its improvement of the insulin resistance of these women [1618]. Many studies have been performed that show that the treatment with myoinositol + folic acid in the classical dosage (2 2000 g myoinositol + 200 g folic acid per day) leads to significant positive changes of metabolic and hormonal parameters . . Could show in a double - blinded, placebo controlled study that myoinositol led to a statistically significant improvement of the blood pressure, triglycerides, cholesterol, glucose, and insulin values after a 75 mg oral glucose tolerance test . The evaluated hormonal values showed a significant decrease of the total and free testosterone serum levels and at the same time the progesterone levels, as a marker of ovulation, experienced a significant rise in the group that received myoinositol (see table 1). This could show that myoinositol did lead not only to positive changes in metabolic parameters but also to a reduction of elevated androgenic values and subsequently to an improvement of skin problems such as acne or hirsutism . These data can be supported by our own data as a rise of progesterone from a value of 2.1 ng / ml to a value of 12.3 ng / ml could be observed . By the same time a reduction in the levels of testosterone (from 96.6 ng / ml to 43.3 ng / dl) and free testosterone (from 1.2 ng / ml to 0.35 ng / ml) could also be observed . A meta - analysis of unfer et al . This study could also show that, under the investigated studies, where the dosage of 4000 g myoinositol + 400 mg folic acid was used, no side effects were observed, especially those which are seen when other insulin sensitizers like metformin are used in high levels of 1500 mg per day . Improvement in ovulation induction with myoinositol alone and in combination with clomiphene citrate in polycystic ovarian syndrome in patients with insulin resistance was also confirmed by kamenov et al . . Whether the addition of melatonin will represent a benefit must be confirmed by more studies but first data suggest this . This confirms that myoinositol is not only an effective alternative in the treatment of pcos patients but also a secure one as no side effects could be observed in the standard dosage . This is on the other side relevant as the compliance of the use rises resulting in better outcomes in the management of ovulation, hyperandrogenism, and metabolic parameters on patients with a pcos.
The relationship between pulp of the tooth and surrounding periodontium was first described in 1964, and since then the term endo - perio has become an integral part of the dental vocabulary . Unfortunately, this term has been used indiscriminately to categorize disease of either periodontal or endodontic etiology, with or without secondary involvement of the other . It conveniently provides a blanket diagnosis but could be misleading for any such lesion, regardless of its primary etiology . Although the role of pulpal pathology in the etiology of furcation involvement is still unclear, the high incidence of molar teeth with accessory canals supports such an association . Various treatment modalities have been proposed for the treatment of furcation involvement alone including open flap debridement, bio - modification of root surface, and various regenerative procedures including gtr and bone grafts . Bone grafts having a property of osteogenesis, osteoinduction, and osteoconduction have been used in the past . This case report attempts to utilize a bioactive glass with the property of osteostimulation as bone replacement graft in the treatment of a combined endo - perio lesion with furcation involvement . The periodontium and pulp have embryonic, anatomic, and functional interrelationship . The relationship between pulpal and periodontal disease can be traced to embryological development, since the pulp and the periodontium are derived from a common mesodermal source . At the stage of tooth development, the developing tooth bud pinches off a portion of mesoderm that becomes pulp, while the remaining mesoderm develops into the periodontium . Ectomesenchymal cells proliferate to form the dental papilla and follicle, which are the precursors of the periodontium and the pulp, respectively . This embryonic development may give rise to an anatomical connection between these two vital structures throughout the life of a tooth . Three main pathways have been implicated in the development of periodontal - endodontic lesions, namely: dentinal tubuleslateral and accessory canalsapical foramen lateral and accessory canals the most commonly used classification was given by simon et al . Primary endodontic lesionprimary periodontal lesionprimary endodontic lesion with secondary periodontal involvementprimary periodontal lesion with secondary endodontic involvementtrue combined lesion primary endodontic lesion primary periodontal lesion primary endodontic lesion with secondary periodontal involvement primary periodontal lesion with secondary endodontic involvement the periodontium and pulp have embryonic, anatomic, and functional interrelationship . The relationship between pulpal and periodontal disease can be traced to embryological development, since the pulp and the periodontium are derived from a common mesodermal source . At the stage of tooth development, the developing tooth bud pinches off a portion of mesoderm that becomes pulp, while the remaining mesoderm develops into the periodontium . Ectomesenchymal cells proliferate to form the dental papilla and follicle, which are the precursors of the periodontium and the pulp, respectively . This embryonic development may give rise to an anatomical connection between these two vital structures throughout the life of a tooth . Three main pathways have been implicated in the development of periodontal - endodontic lesions, namely: dentinal tubuleslateral and accessory canalsapical foramen lateral and accessory canals primary endodontic lesionprimary periodontal lesionprimary endodontic lesion with secondary periodontal involvementprimary periodontal lesion with secondary endodontic involvementtrue combined lesion primary endodontic lesion primary periodontal lesion primary endodontic lesion with secondary periodontal involvement primary periodontal lesion with secondary endodontic involvement a 22-year - old patient reported to the department of periodontics with a complaint of pain in the lower right back tooth region associated with pus discharge since 1 month . On intraoral examination, an intraoral sinus was found to be present in relation with 46 [figure 1]. A radiograph was taken with a gutta percha cone inserted in the sinus so as to gauge the path of the sinus tract as shown in figure 2 . Iopa also showed widening of periodontal ligament space in relation with the mesial root and radiolucency in the furcation area . The horizontal probing depth (hpd) with naber's probe and vertical probing depth (vpd) with the unc-15 probe were measured which were found to be 6 mm and 4 mm, respectively . Probing was standardized by fabricating an acrylic stent as shown in figures 1 and 3 . The patient was then referred to department of conservative dentistry to check for tooth vitality . Electric pulp testing was done to check for tooth vitality, which confirmed that the tooth was nonvital . Vpd of 4 mm with unc-15 probe radiograph showing gutta percha when inserted though the sinus tract extending till the mesial root of 46 hpd of 6 mm with naber's probe treatment planning was done taking into consideration that the tooth was nonvital with a prevailing sinus tract and grade ii furcation involvement as well . Endodontic treatment was taken up first and the patient was followed up for 3 months . At the end of third month, iopa was taken with 46 which showed that the furcation involvement still prevailed [figure 4]. On clinical examination, it was observed that there was no change in the soft tissue measurements . Therefore, periodontal regenerative surgery using dental putty was planned for treatment of furcation defect [figure 5 and 6]. Obturated 46 with radiolucency still evident after 3 month furcation defect with 46 graft material in place at the defect after taking care of asepsis and sterilization the surgery was planned . The area selected for surgery a full thickness flap was raised at the buccal aspect following intracrevicular incision and vertical releasing incision . A vertical releasing incision was placed extending into the alveolar mucosa not closer than one tooth to the involved area, i.e. 46 . Full thickness flap was raised till the base of furcation defect followed by split thickness flap beyond the mucogingival junction . This was done so as to facilitate the coronal positioning of flap, there by resulting in complete coverage of the defect and the material used . After reflection thorough degranulation and debridement was done at the defect area using gracey's curette #13 and 14 . Also thorough scaling and root planning was carried out on the exposed root surface area of the defect . Dental putty graft material with osteoconductive and osteostimulative properties was placed and stabilized in the furcation area . Primary soft tissue closure of the flap was done with nonresorbable black silk (30) suture using interrupted suturing technique [figure 7]. Immediate postoperative the patient was advised proper plaque control, and was prescribed 0.12% chlorhexidine mouthwash for rinsing twice daily . The sutures were removed 10 days after surgery and the patient was advised to brush at the surgical site using a postsurgical brush for 2 weeks . The patient was put on regular recall at 1, 3, 6, and 9 months . The post - op radiograph shows bone fill in the furcation defect as shown in figure 8 . After taking care of asepsis and sterilization the surgery was planned . The area selected for surgery a full thickness flap was raised at the buccal aspect following intracrevicular incision and vertical releasing incision . A vertical releasing incision was placed extending into the alveolar mucosa not closer than one tooth to the involved area, i.e. 46 . Full thickness flap was raised till the base of furcation defect followed by split thickness flap beyond the mucogingival junction . This was done so as to facilitate the coronal positioning of flap, there by resulting in complete coverage of the defect and the material used . After reflection thorough degranulation and debridement was done at the defect area using gracey's curette #13 and 14 . Also thorough scaling and root planning was carried out on the exposed root surface area of the defect . Dental putty graft material with osteoconductive and osteostimulative properties was placed and stabilized in the furcation area . Primary soft tissue closure of the flap was done with nonresorbable black silk (30) suture using interrupted suturing technique [figure 7]. The patient was advised proper plaque control, and was prescribed 0.12% chlorhexidine mouthwash for rinsing twice daily . The sutures were removed 10 days after surgery and the patient was advised to brush at the surgical site using a postsurgical brush for 2 weeks . The patient was put on regular recall at 1, 3, 6, and 9 months . The post - op radiograph shows bone fill in the furcation defect as shown in figure 8 . When a clinician cannot make a definitive diagnosis in the case of an endo - perio lesions, it may be prudent for him or her to initiate either of the treatment modalities and hope for repair . However, this could be overcome by proper history taking and sequential treatment planning . When the etiology is purely endodontic, calcium hydroxide can be used as an intracanal medicament . It is an excellent medicament in general, because it is bactericidal, anti - inflammatory and proteolytic; it inhibits resorption; and it favors repair . It is especially effective in endodontic lesions with extensive periapical pathology and pseudo pockets, because of its temporary obturating action which would inhibit periodontal contamination of the instrumented canals via patent channels of communication . However, lesions which are not true combined lesions, little or no improvement would be seen with the periodontal perspective after endodontic treatment, leaving a very poor and often hopeless prognosis . But with the advent of new regenerative materials, however, successful periodontal treatment of such lesions has been possible . In this report the pulp vitality test which showed the nonvital nature of the tooth was a pivoting finding suggesting the primary endodontic involvement . Generally, in a case of combined endo - perio lesion, an adequate endodontic therapy would result in healing of the endodontic component, and the prognosis would finally depend on the efficacy of periodontal repair / regeneration initiated by either of the treatment procedures . In this case, following endodontic treatment the periodontal lesion did reduce to an extent on radiographic evaluation after 3 months but did not subside completely with no change in the clinical parameters . It enhances bone formation by ionic dissolution of the ceramic particles such that the silica gel layer forms over the particles on contact with body fluids . Over this silica gel layer, a calcium phosphate layer forms, which is quickly converted into a hydroxycarbonate apatite layer this apatite layer has been shown to be identical to bone mineral and to provide the surface for osteoblast cell attachment and bone deposition . The results of this case report suggest that bioactive glass in the form of putty resulted in a significant amount of bone fill and reduction in hpd . This was in accordance with the studies by yukna et al . And anderegg et al . Using bioactive glass showed significant improvement in the clinical parameters when used in furcation defects . The healing of an endodontic lesion is highly predictable, but the repair or regeneration of periodontal tissues is questionable if associated with it . Endodontic therapy mostly should precede periodontal pocket elimination procedures in the case of a primary endo and secondary periodontal involvement; however, endodontic therapy would result only in resolution of the endodontic component of involvement and would have a little effect on the periodontal lesion . Therefore a thorough diagnostic examination usually will indicate the primary etiology and, thereby, direct the proper course of treatment plan as presented in this case . It could also be concluded that bioactive glass in the form of putty which was primarily due to 21% of glycerin added to 69% of bioactive glass and 10% of poly ethylene glycol, made it feasible for manipulation of the material and effective defect fill . Therefore within the limits of this study it can be concluded that bioactive glass is effective as a bone graft substitute in treatment of periodontal component of the endodontic lesion.
Inhalation of ambient particulate matter (pm) causes respiratory and cardiovascular health problems and mortality in humans . Pm may induce these effects by producing reactive oxygen species (ros), including hydrogen peroxide (hooh) and hydroxyl radical (oh), in the body . Though less reactive than oh, hooh is of interest because it is a signaling molecule in vivo, has a relatively long lifetime, can cross cell membranes, and is a precursor for oh . Once deposited in the lung lining fluid, redox - active species from inhaled pm can chemically produce hooh at levels that far exceed those originally present in the particles . Endogenous reductants such as ascorbate, and other reducing species such as dithiothreitol (dtt), increase the production of hooh and oh from pm or metal solutions; thus the interactive chemistry between endogenous reductants and deposited pm can increase the oxidant load in the body . However, adverse effects can occur when an overproduction of oxidants for example, upon pm exposure overwhelms the body s anti - oxidative defenses . It is unclear what concentration of hooh is necessary to elicit adverse effects in the lung, as hooh likely affects each cell type differently . Fibroblasts and human alveolar cells exposed to hooh in the range of 10400 m exhibit apoptosis, while higher concentrations induce necrosis . Human alveolar and bronchial epithelial cells released 40% of their lactate dehydrogenase (ldh) in the presence of 100 and 1000 m hooh, respectively, indicating alveolar cells may be more susceptible to hooh than bronchial cells . Both transition metals (cu, zn, fe) and quinones have been implicated in hooh formation from pm . Zn and fe were identified via correlation between hooh production and pm metal content; however, many trace metals and quinones are covariate, which confounds identifying the redox - active species responsible for ros generation . In addition, although total (acid - soluble) metals are typically measured in these studies, it is likely that the soluble metals drive the redox activity . For example, while ros production from particles using the dichlorofluorescein diacetate (dcf - da) assay showed a good correlation between ros production and soluble cu (r = 0.59), there was no correlation with total cu (r = 0.02). There is also quantitative, mechanistic evidence that specific particle components can generate hooh . Chung et al . Measured quinone concentrations and hooh production (in the presence of 100 m dtt) in ph 7.4 aqueous extracts of ambient fine particles (pm2.5). Response curves of the pure quinones, they concluded that the quinone content of ambient pm2.5 could account for all hooh produced by the particles . In contrast, shen et al . Found that soluble cu could explain essentially all hooh production from ambient fine and coarse pm in ph 7.3 aqueous extracts containing 50 m ascorbate . Additionally, desferoxamine, a strong metal chelator, halted hooh production, further indicating that hooh was produced by metals . Given the uncertainties in our understanding of hooh production from ambient pm, our purpose is to quantify hooh formation from transition metals and quinones (both individually and in mixtures) in a more representative (though still cell - free) surrogate lung fluid (slf). In this work we characterize an in vitro, cell - free assay to measure the rate of hooh production from pm . Given the important role of lung - lining fluid antioxidants in ros formation, we include typical lung concentrations of four antioxidants: ascorbate (asc), reduced glutathione (gsh), urate (ua), and citrate (cit). Asc, gsh, and ua are naturally occurring in the lung fluid, while cit is a good proxy for proteins that mobilize iron in the lung fluid . While we use ph and antioxidant conditions similar to lung lining fluid, it is impossible to reproduce the complexity of particle lung interactions using an in vitro assay . This technique is intended as a useful screening assay for the oxidative potential of ambient pm . It also allows us to identify the chemicals that can produce hooh in lungs and which of these redox - active species are likely most important for hooh production from inhaled ambient pm . Information about chemicals and their purities, metal and quinone stocks, detailed hooh measurement steps, and ambient pm samples is given in the supporting information, section s1 . Our slf consists of phosphate - buffered saline (pbs) with four antioxidants . The pbs contains 114 mm nacl, 7.8 mm sodium phosphate dibasic, and 2.2 mm potassium phosphate monobasic, ph 7.27.4, and is treated with chelex 100 resin (sodium form, bio - rad) to remove trace metals . Each batch of pbs is treated twice with chelex resin at a rate of one drop per four seconds . Antioxidant stock solutions, made fresh each day, are added to the pbs at the start of the reaction at final concentrations of 200 m l - ascorbic acid sodium salt (asc), 300 m citric acid (cit), 100 m reduced l - glutathione (gsh), and 100 m uric acid sodium salt (ua). Analytical details are given in the supporting information, section s1 . At time zero, we mix the redox - active species into 5.0 ml of slf in a 7.0 ml fep bottle, seal it, and agitate it on a shake table at setting 5 at room temperature . We measure the hooh concentrations in each reaction solution at 0, 0.5, 1, and 1.5 h. at each time point we remove a 0.50 ml aliquot, add 10.0 l of 5.0 mm desferoxamine (dsf) to chelate metals and help stabilize the hooh, and then immediately inject onto the hplc (50 l sample loop). Fe(ii) destroys hooh via the fenton reaction, and our method is especially sensitive to fe contamination because of the presence of asc, which cycles inactive fe(iii) into active fe(ii). To mitigate this effect, we adhere to rigorous cleaning methods to maintain background fe concentrations below 50 nm; however, fe likely destroys some hooh even at these low levels . We use only fep bottles (fisher scientific), and all are washed in a freshly made 1 m nitric acid bath before use . We calculate the rate of hooh production from the concentrations of hooh measured at 0, 0.5, 1, and 1.5 h. in many cases the rate of hooh production decreases with time, causing a slight curvature for most data (figure s1) and significant curvature for solutions with high concentrations of fe (figure s2). Similar behavior was observed by shen et al . For both ambient pm and cu stocks, and by wang et al . For hooh production from coarse mode pm . We calculate the initial rate of hooh production using the b term of a second - order polynomial: y = at + bt + c, where a, b, and c are fitted constants . The initial rate of production between 0 and 1.5 h would not be affected by the choice of reaction time for samples with slight curvature, but could be affected for highly curved samples (generally above 300 nm fe(ii), as shown in figure s2) if curvature occurs very early . However, significant curvature occurs only for laboratory samples where the rate of hooh production is essentially zero . Ambient samples did not show as much sensitivity to fe, as discussed below, and ambient rates should be less sensitive to the choice of time points . We measure hooh production in a positive control (250 nm cu(ii)) and blank (slf containing four antioxidants) on each experiment day . All sample rates are blank - corrected by subtracting that day s blank rate . If data have an error bar it indicates that two or more replicates were measured and the data are reported as the average standard deviation of the blank - corrected initial rates . A small subset of data do not have replicates and are reported without an error bar to identify that only one measurement was made . We could estimate the error from the standard error of the slope of the rate regression, but this under - predicts the actual variability of day - to - day replicates . We estimate that the typical relative standard deviation for our rates is 14% based on variability in the blank - corrected positive control, which has an average (1) initial rate of hooh formation of 1.99 0.28 m / h (n = 18), with a blank rate of 0.21 0.1 m / h . Statistical differences between means (where n 2) are calculated using the student s t - test, with p 0.05 as a first step in identifying the components in pm that can produce hooh, we start by screening hooh formation from individual chemicals at a concentration of 500 nm in the slf . As detailed in the supporting information, section s2, 500 nm is a reasonable concentration for fe and cu but is over an order of magnitude higher than expected for the quinones (table s2). We start with this relatively high concentration in order to identify any compound that can produce hooh under our reaction conditions . Of the 10 transition metals tested, only cu(ii) produces significant hooh under our slf conditions, while fe(ii) destroys background hooh, resulting in a slightly negative rate of hooh production (figure 1). Pb produces hooh at a rate statistically different than the blank, though extremely slowly . The other seven metals mn, co, v, ni, zn, cd, cr do not produce hooh, though there is some evidence that v can destroy hooh . Of the four quinones tested, three produce hooh phenanthrenequinone (pqn), 1,4-naphthoquinone (1,4-nqn), and 1,2-naphthoquinone (1,2-nqn)but benzoquinone (bqn) does not . Previous measurements of hooh production from 12 quinones in a ph 7.4 extract solution containing 100 m dtt as a reductant also found that the same three quinones produce hooh, while the nine other quinones did not . At 500 nm, 1,2-nqn produces 1633 times more hooh than an equal concentration of cu(ii), pqn, or 1,4-nqn (figure 1). Initial rates of hooh production from 500 nm concentrations of individual metals and quinones in a slf with four antioxidants . Asterisks mark rates that are statistically larger than zero (p <0.05). The rate for 1,2-nqn (44 4 m / h) is divided by 10 to fit on this scale . To quantify hooh production from cu and quinones at concentrations relevant to ambient pm, we next measured hooh rates as a function of concentration for the four active compounds . As shown in figure 2, the concentration responses of all three quinones are linear, with slopes (table 1) that indicate their relative ability to produce hooh . The relative reactivities of the quinones in our slf are 23:2:1, i.e., 1,2-nqn pqn> 1,4-nqn . A previous study by chung et al . Of hooh production from quinones in ph 7.4 phosphate buffer with 100 m dtt as a reductant showed a different relative reactivity, pqn> 1,4-nqn = 1,2-nqn . This difference is likely due to the difference in antioxidant composition and reductant . Our slf uses asc as the reductant and contains three other antioxidants, while chung et al . Used a pbs that contains only dtt . The reductive potential of dtt (0.33 v) is much stronger than that of asc (+ 0.105), and our antioxidant composition affects hooh production (see next section). We have previously shown that pqn is much more active in the dtt assay relative to cu than in our slf . When both species are at a concentration of 500 nm, pqn produces the same rate of hooh production as cu (figure 1), while at the same concentration pqn causes 9 times more dtt loss than cu . Response curves of the rates of hooh production as a function of concentration of redox - active species . Regression equations for the species are given in table 1 y is the initial rate of hooh production (m / h), and x is the concentration of chemical species (nm). Hooh production from cu goes to zero at 3.4 nm; therefore, hooh production should be assumed to be zero at cu concentrations below 3.4 nm . Unlike the quinones, cu shows a nonlinear concentration response with a fast initial increase in hooh production that begins to level off around 200 nm cu (figure 2). We believe this results from the loss of asc over time, which causes asc to become the limiting reactant . As we will describe below (figure 3) asc acts as a reductant and is necessary for formation of both hooh and oh in our slf . Once asc becomes limiting, addition of more cu causes only small increases in the rate of hooh production . A result of the nonlinear behavior is that the relative reactivity of cu compared to quinones changes depending on the concentrations of each compound in solution . We found a similarly nonlinear concentration response curve for cu in the dtt assay, which measures the oxidative potential of pm by monitoring the oxidation of dtt over time . Thus, this result is not limited to the slf experimental conditions in the hooh assay . Previous laboratory studies measured the concentration response curve for hooh production from cu(ii) in a slf containing only 50 m asc was linear through 400 nm cu but began to plateau at 600 nm cu, the maximum concentration tested . This indicates that the antioxidant mixture in the slf alters the concentration response behavior . One implication of the nonlinear cu curve in figure 2 is that after 200 nm, large increases in cu concentration cause only small increases in the rate of hooh production . At low concentrations (<50 nm), cu and 1,2-nqn have very similar reactivities, while at higher concentrations 1,2-nqn is much more reactive than cu . However, this is tempered by the differences in particle concentrations of these two species: for typical ambient conditions (table s2), the concentration of cu in a slf extract of pm will be approximately 1001000 times larger than that of 1,2-nqn . While filter - based quinone measurements may have both negative (volatilization) and positive (formation from ozone) artifacts, the magnitudes of these artifacts are unlikely to be large enough to make 1,2-nqn more significant than cu as a source of hooh for typical ambient pm . Effect of antioxidant composition on the rate of hooh production from 250 nm cu(ii) in ph 7.3 pbs . When present, the concentration of each antioxidant is constant for all experiments: asc is 200 m, cit is 300 m, and gsh and ua are each 100 m . The final solution composition, with all four antioxidants, is our slf condition used in all other figures . Each sample rate is corrected by a blank containing the same composition of antioxidants . Our slf includes four antioxidants: asc, cit, gsh, and ua . In previous work using the same slf we found that the antioxidant composition has a significant effect on oh production from transition metals . Because hooh is a precursor for oh we test different mixtures of antioxidants to better characterize our assay; however, the base case is a slf with all four antioxidants . As expected, hooh production by 250 nm cu is affected by the antioxidant composition (figure 3). Cu produces hooh in the presence of asc, and to a much smaller extent in the presence of gsh only, but not in the presence of cit only . Asc acts as the reductant in our system, cycling transition metals from their oxidized to reduced forms and thereby allowing oxidant production from cu(ii) via pathways such as1234although cu with gsh produces hooh (figure 3), this mixture does not form oh . Compared to asc only, the combination of asc and cit doubles the rate of hooh production, to 43 m / h for 250 nm cu(ii). Under the conditions with asc and cit as the only antioxidants, 100% of cu(ii) thus, the cu(ii)-citrate complex is apparently more reactive than free cu(ii), which is the dominant cu form in the asc - only condition . If we add gsh to the asc cit mixture, the production of hooh plummets by a factor of 20, to 2.2 m / h, which is similar to the hooh production rate from our slf case with all four antioxidants . Thus, ua does not affect hooh production from cu in the presence of the other antioxidants . Overall, hooh production in our mixture with all four antioxidants is substantially reduced because of gsh, likely because gsh binds to cu and reduces its reactivity . A similar suppression by gsh was observed for oh production from cu(ii) in the same slf, and also in other studies of oh from cu . Minteq speciation modeling of a similar slf with the same four antioxidants shows that gsh replaces cit as the primary ligand, and 100% of cu(ii) is bound to gsh under these conditions . Gsh is well known as an important antioxidant in vivo, and may be especially important in mitigating damage from hooh . Binding and deactivating cu may be one component of this protective effect of gsh . We also find that the antioxidant mixture affects the ability of quinones to generate hooh . As shown in figure s3, compared to the case of asc only, hooh production in the four antioxidant (slf) mixture is lower by factors of 2 and 6 for 500 nm pqn and 20 nm 1,2-nqn, respectively . While hooh formation from the quinones is less sensitive to antioxidant composition than is cu, the impact on quinones is surprising and more work is necessary to confirm this result and understand its mechanism . Ambient pm samples contain a complex mixture of chemical species that may produce hooh in a more complicated mechanism than in the pure laboratory solutions measured here . For example, quinones and cu can act synergistically to produce hooh under some conditions: semiquinone radicals can reduce cu(ii) to cu(i), producing superoxide that can react with cu(i) to make hooh . To examine this in our slf, we measured hooh production in mixtures of cu, quinones, and/or fe (figure 4). The gray bars in figure 4 represent the rate of hooh production measured from the species mixed in the same bottle, while the colored stacked bars are the sum of hooh production measured from the individual compounds . Initial rates of hooh production in laboratory mixtures of quinones and/or transition metals (gray bars) compared to the sum of the rates from the individual redox - active species (stacked colored bars). Error bars of the colored stacked bars are the propagated errors of the sum (all have replicate samples). The concentrations of metals and quinones are constant: cu, fe, and pqn are at 500 nm, and 1,2-nqn is at 20 nm . As shown by the first two sets of bars, the rate of hooh production in a mixture of a quinone and cu is the same as the sum of the rates of the individual species; i.e., hooh production from cu and either 1,2-nqn or pqn is additive (figure 4). Thus, reactions between cu and quinones appear to be negligible for hooh formation in this system . This may be due to the presence of asc, which rapidly cycles cu(ii) to cu(i) and is present at a concentration that is 40010,000 times higher than the quinones; we expect a similar asc dominance in lung lining fluid in vivo . This likely causes most cu(ii) to react with asc instead of a semiquinone radical . In contrast to the additive behavior of cu with quinones, the addition of 500 nm fe to slf containing cu and/or 1,2-nqn, greatly reduces hooh formation, to 2030% of the rate measured without fe (figure 4). This decrease in the rate of hooh formation might be because fe is suppressing the formation of hooh from cu or quinones, or because fe(ii) is destroying hooh and converting it to oh via the fenton reaction . We cannot deduce which mechanism is at work here, but plan to explore this in the future by looking at oh production from the same mixtures . Based on these results, fe may have an important role in suppressing hooh generation in ambient pm extracts since ambient concentrations of fe are high, resulting in slf concentrations up to a few micromolar under the sampling conditions described in the supporting information, section s2 . We further investigated the effect of fe by quantifying hooh production in 17 laboratory solutions containing mixtures of fe with cu, 1,2-nqn,1,4-nqn and/or pqn (summarized in table s3). Normalized hooh production rate, which is a value between 0 and 1 calculated according tor1the numerator is the measured hooh rate in the mixture (including fe), while the denominator is the hooh rate in the absence of fe, which was either measured directly or determined based on the cu and/or quinone concentrations in conjunction with the concentration response curves in table 1 . As an example, a normalized hooh production value of 0.7 indicates the hooh rate in the presence of fe is 70% of the rate in a similar solution without fe (i.e, . Impact of iron on hooh production in laboratory solutions and suppression of hooh production in extracts of ambient particles . Panel (a) shows the normalized hooh production, i.e., the measured rate of hooh formation in laboratory mixtures containing copper and/or quinones and iron divided by the expected (calculated) rate from just the copper and quinones . The rate of hooh production in the absence of fe is measured directly for all cases except the three diamonds marked with a bold outline, where hooh production was calculated based on the concentration of cu . Black lines in panel (a) are the model fits to the data, with equations noted on the lines . Panel (b) shows the normalized hooh production for ambient particle extracts from fresno, ca, where the expected rate was calculated using only the copper concentration . The last two panels compare the measured rates of hooh production in ambient pm extracts with (c) the calculated hooh rates based on the fit to the laboratory solutions in panel (a), and (d) the calculated rates based only on the concentrations of soluble cu in the ambient pm extracts . Figure 5a shows that fe has a similar effect on all of the laboratory mixtures: the normalized rate of hooh production decreases with increasing fe concentration between 0 and 270 nm and is relatively stable at approximately 20% of the rate in the absence of fe at higher fe concentrations . We applied the same analysis to 39 ambient samples collected in fresno, ca, during 2008 and 2009 . We measured both the concentration of soluble metals and the initial rate of hooh production from these samples, but did not measure the concentration of quinones (supporting information, section s1). We calculate the normalized rate of hooh production from the ambient extracts using the expected rate from soluble cu since quinone concentrations are unknown (figure 5b). Compared to the laboratory mixtures, fe appears to have nearly the opposite effect on hooh production in the ambient pm extracts: there is generally less suppression of hooh formation as the concentration of fe increases (figure 5b), but the relationship is very scattered and there is no strong trend . How could higher concentrations of fe lead to a smaller suppression of hooh production by fe? One possibility is that samples with higher fe concentrations also contain higher amounts of organic ligands, which are binding to fe and reducing its ability to reduce hooh formation . In our laboratory solutions cit is the dominant ligand for fe and the fe - cit complex clearly suppresses hooh formation (figure 5a). It is also possible that cu produces hooh less efficiently in ambient samples compared to in our laboratory solutions due to the presence of organic ligands (figure 2). If we apply the laboratory solutions model from figure 5a to ambient pm we can calculate the expected hooh production based on the concentration of cu and fe in each sample . When we plot the measured versus expected hooh production using this method (figure 5c), the modeled data are clustered around the 1:1 line, but the r value for this correlation is 0.01, indicating the model has no predictive ability . We obtain a much better relationship (r = 0.3) when we calculate the expected rate of hooh production based on only the concentration of soluble cu using the cu concentration response curve (figure 2), without considering suppression by fe (figure 5d). In this case the expected rate of hooh production is nearly always larger than the measured rate (i.e., points are almost all above the 1:1 line). This overestimate of hooh rates suggests hooh production is suppressed in ambient pm extracts, but that this suppression is not strongly tied to the soluble fe concentration . It is also possible that organic ligands from the particles are binding to cu and suppressing its ability to form hooh . If we assume that cu controls hooh formation in figure 5d (i.e., if the contribution from quinones is negligible), the average (1) suppression in hooh formation by cu in ambient pm extracts is 44 22% (median suppression = 40%; range = 3.793% . More work is needed to determine whether this suppression is due to fe or to a reduction in cu reactivity by organic ligands, but the chemistry of hooh production in ambient particles is clearly complicated . However, the similarity between the measured rate of hooh production and that predicted by cu (figure 5d) indicates that cu is likely a major contributor to hooh production . Because of the variable impact of fe on hooh production in ambient pm extracts, and the potential for particulate ligands to reduce cu reactivity, we cannot predict the absolute rate of hooh production based on the concentration of soluble metals in ambient pm . However, mixtures of cu and quinones (the only species able to make hooh based in figure 1) show that the rate of hooh production from these redox - active species is additive (figure 4) and that fe has a similar suppressive effect on hooh production from both species (figure 5a). Thus, in the case that fe is responsible for suppressing hooh in ambient pm extracts we can still estimate the unsuppressed rate of hooh production based on the reported range of ambient pm2.5 quinone and soluble cu concentrations (table s2). Under these conditions cu produces the largest rate of hooh production, 0.8 to 2.9 m hooh per hour, across the entire range of redox - active concentrations reported in the literature . Quinones, on the other hand, exhibit very low particle - phase concentrations and generally do not contribute significantly to hooh production . At the highest ambient concentrations, 1,2-nqn can produce on the order of 0.5 m hooh per hour, while the rates for 1,4-nqn and pqn are in the range 0.00.05 m / h, which is negligible compared to production from cu . If we consider the lowest, median, and highest concentrations of each redox - active species, cu accounts for nearly all hooh production (100%, 96%, and 84%, respectively), while 1,2-nqn accounts for 0%, 2%, and 14% of total hooh for these three scenarios, while the other quinones make up the remainder . Though the total hooh rate in ambient pm extracts will be somewhat lower than these calculated rates, we also consider the possibility that reduced cu reactivity (rather than reactions with fe) accounts for the hooh suppression we see in ambient pm extracts (figure 5). In this case we repeat the rate calculations above but with a 44% reduction in the rate of hooh formation by cu, which is the average reduction needed to explain figure 5d . In this case, cu is still the dominant source of hooh in pm extracts, accounting for 100%, 93%, and 75% of hooh formation for the lowest, median, and highest concentration scenarios . Thus, even if cu reactivity is suppressed in ambient pm, we expect soluble cu to dominate hooh production in most ambient pm samples . This result agrees with other studies that have identified cu as important for ros generation using a variety of techniques, including the dtt assay, a macrophage ros assay, and hooh and oh measurements . These results also agree with a recent epidemiological study that found the cu content of pm was associated with mortality in california . One important source of cu is likely traffic emissions, which have been consistently linked to adverse health effects . For example, rats instilled with particles from multiple sites showed a statistically significantly higher response for sites with higher traffic emissions and a higher concentration of cu, but no association with the pah content of pm . Taken together, these diverse studies provide consistent evidence that cu is an important component in the health effects from airborne particles.
Infestation with the liver flukes fasciola hepatica and fasciola gigantica, has traditionally been associated with substantial production and economic losses in livestock, particularly sheep and cattle (1). Several studies have shown that f. hepatica occurs in temperate areas, while f. gigantica occurs mainly in tropical zones, and both species overlap in subtropical areas (24). Fasciola hepatica and f. gigantica were previously reported from a range of domestic animals (buffalo, cattle, sheep, and goat) and humans based on morphological and some molecular features in iran (5, 6). The differential diagnosis between f. hepatica and f. gigantica infection is very important because of their different transmission and epidemiological characteristics . Fasciola hepatica and f. gigantica can generally be distinguished based on their morphology (3). However, it is usually difficult to discriminate accurately between f. hepatica and f. gigantica because of the high variations in their morphological characteristics . The two species can be discriminated by dna sequences of nuclear ribosomal internal transcribed spacer 1 (its1), its2, and 28s rdna regions and of mitochondrial nadh dehydrogenase i (nd1) and cyto - chrome c oxidase i (co1) genes (7, 8). As intraspecific genetic variations among liver flukes may reflect differences in virulence, host specificity and drug susceptibility or resistance (9), the population genetics of fasciola parasites in iran needs to be studied . The genetic variation within f. hepatica sequences of the mitochondrial dna and ribosomal regions has been reported previously from iran and several other countries (5, 6, 912). However, limited information is available about intraspecific genetic diversity among iranian f. gigantica isolates . Its1, its2, 18s rdna and co1 regions have been used as markers for genetic characterization of fasciola flukes from iran (9, 13, 14). In previous studies, there was little information available on the characterization of iranian f. gigantica existing genotypes based on variations in nadh dehydrogenase subunit 1 (nd1) dna sequences . The present study was designed to molecularly differentiate fasciola species and characterize genetic diversity of f. gigantica using nd1gene in the endemic areas of iran . Fasciola specimens (n=40) were collected at local abattoirs during post - slaughter inspection from livers of naturally infected sheep (n=15), cattle (n=15), buffalos (n=5) and camels (n=5) from four distinct areas in iran including fars (29 37n, 5231e), khorasan razavi (3617n, 5936e), gilan (3716n, 4934e) and khuzestan (32 22 n figure 1 shows the iran s map and the geographic locations where the parasitic flukes were collected . Fresh adult worms were washed thoroughly in physiological saline, fixed in 70% ethanol and stored at 20 until further use . The geographic locations where the parasitic flukes (fasciola spp .) Were collected from different hosts in iran individual worms were pressed slightly between two slides and were subjected to computerized morphometric examination using computer image analysis system (image analyzer version 1.34). Body length (bl), body width (bw), body area (ba) and body perimeter were measured and bl / bw ratio was calculated . The initial differentiation between f. hepatica and f. gigantica species was made based on the morphometric criteria according to standard taxonomic keys (15). Samples were transferred from fixative solution to sterile microtubes and left until complete evaporation of ethanol . Dna was extracted from a small part of the parasite apical zone, to avoid inclusion of female genitalia likely to include foreign sperm . Dna extraction was carried out using a commercial extraction kit (dneasy blood & tissue kit, qiagen, germany) in according to the manufacturer s instructions . The purity of the extracted dna samples was evaluated using spectrophotometric method (eppendorf, germany). The mitochondrial dna was amplified using specific primers for nadh dehydrogenase 1(nd1) (jb11: 5-aga ttc gta agg ggc cta ata-3 and jb12: 5-acc act aac taa cta attcac ttt c-3) (16, 17). The primers for amplification of the its2 region (5-tct tga acg cat att gcg gc-3 and 5-agt tca gcg ggt aat cac gt-3) were used as described by ghavami and others (11). The expected pcr amplicons are fragments of 534 bp from nd1 gene and fragments of 456 bp from its2 region . Reactions were carried out in a final volume of 50l containing 5l and 4l of genomic dna for ndi and its2 assays, respectively . Each its2 pcr reaction contained 50 mm kcl, 10 mm tris - hcl (ph=9.0), 1 mm mgcl2, 200 m dntps, 0.4 m of each primer, and 2.5 u taq dna polymerase (fermentas, usa . Each nd1 pcr reaction contained 50 mm kcl, 10 mm tris - hcl (ph=9.0), 1.5 mm mgcl2, 100 m dntps, 0.48 m of each primer, and 2.6 u taq dna polymerase (fermentas, usa). Pcr amplification was performed under the following conditions: initial denaturation at 94c for 5 min, followed by 35 cycles of denaturation at 94c for 60 s, annealing at 55c for 45 s (nd1) and at 60c for 45 s (its2), extension at 72c for 45 s, and a final synthesis at 72c for 7 min, in the mastercycler gradient eppendorf thermal cycler (germany). 5l of each reaction product was subjected to electrophoresis in tae buffer (40 mm tris base, 20 mm acetic acid,1 mm edta) using 1% agarose gels, stained with ethidium bromide, (0.5g / ml) and visualized on a uv transilluminator . Briefly, ten microliters of pcr products were incubated with 2 l of pagi restriction endonuclease (er1282, 00022771, fermentas, usa) and 2 l of supplied buffer at 37c for 16 hours . The final reaction volume was adjusted to 20 l by adding enough sterile distilled water . The digested dna was analyzed by electrophoresis in 2% agarose gels in tae buffer, visualized by ethidium bromide staining, and photographed . Lengths of resulting restriction fragments from f. hepatica and f. gigantica were predicted by means of the gene runner software v. 3.05 . Based on computerized cutting, this enzyme has no cutting site on its2 pcr amplicons for fasciola hepatica and one cutting site for f. gigantica, producing fragments of 165 and 291bp . The pcr products of ndi for f. gigantica parasites (characterized by morphological data and rflp analysis of its2) were directly sequenced using capillary dna analyzer (abi 3730, applied biosystems, foster city, ca, usa) after sequencing reactions with a big dye terminator v3.1 cycle sequencing kit (applied biosystems). Forward and reverse nucleic acid sequence data were used to construct a continuous sequence of inserted dna . Further comparison of the continuous ndi sequences was made with previously available sequences in national center for biotechnology information (ncbi) using basic local alignment search tool (blast) and compared with other homologues by means of neighbor joining and maximum parsimony trees reconstructed from kimura 2-parameter distances from 1,000 bootstrap replicates of multiple sequence alignments created using clustalw, as implemented by mega version 4.0 (18). This experiment was performed under the approval of the state committee on animal ethics, shiraz university, shiraz, iran . In addition, the recommendations of european council directive (86/609/ec) of november 24, 1986, regarding the protection of animals used for experimental purposes, were considered . Fasciola specimens (n=40) were collected at local abattoirs during post - slaughter inspection from livers of naturally infected sheep (n=15), cattle (n=15), buffalos (n=5) and camels (n=5) from four distinct areas in iran including fars (29 37n, 5231e), khorasan razavi (3617n, 5936e), gilan (3716n, 4934e) and khuzestan (32 22 n figure 1 shows the iran s map and the geographic locations where the parasitic flukes were collected . Fresh adult worms were washed thoroughly in physiological saline, fixed in 70% ethanol and stored at 20 until further use . The geographic locations where the parasitic flukes (fasciola spp .) Were collected from different hosts in iran individual worms were pressed slightly between two slides and were subjected to computerized morphometric examination using computer image analysis system (image analyzer version 1.34). Body length (bl), body width (bw), body area (ba) and body perimeter were measured and bl / bw ratio was calculated . The initial differentiation between f. hepatica and f. gigantica species was made based on the morphometric criteria according to standard taxonomic keys (15). Samples were transferred from fixative solution to sterile microtubes and left until complete evaporation of ethanol . Dna was extracted from a small part of the parasite apical zone, to avoid inclusion of female genitalia likely to include foreign sperm . Dna extraction was carried out using a commercial extraction kit (dneasy blood & tissue kit, qiagen, germany) in according to the manufacturer s instructions . The purity of the extracted dna samples was evaluated using spectrophotometric method (eppendorf, germany). The mitochondrial dna was amplified using specific primers for nadh dehydrogenase 1(nd1) (jb11: 5-aga ttc gta agg ggc cta ata-3 and jb12: 5-acc act aac taa cta attcac ttt c-3) (16, 17). The primers for amplification of the its2 region (5-tct tga acg cat att gcg gc-3 and 5-agt tca gcg ggt aat cac gt-3) were used as described by ghavami and others (11). The expected pcr amplicons are fragments of 534 bp from nd1 gene and fragments of 456 bp from its2 region . Reactions were carried out in a final volume of 50l containing 5l and 4l of genomic dna for ndi and its2 assays, respectively . Each its2 pcr reaction contained 50 mm kcl, 10 mm tris - hcl (ph=9.0), 1 mm mgcl2, 200 m dntps, 0.4 m of each primer, and 2.5 u taq dna polymerase (fermentas, usa . Each nd1 pcr reaction contained 50 mm kcl, 10 mm tris - hcl (ph=9.0), 1.5 mm mgcl2, 100 m dntps, 0.48 m of each primer, and 2.6 u taq dna polymerase (fermentas, usa). Pcr amplification was performed under the following conditions: initial denaturation at 94c for 5 min, followed by 35 cycles of denaturation at 94c for 60 s, annealing at 55c for 45 s (nd1) and at 60c for 45 s (its2), extension at 72c for 45 s, and a final synthesis at 72c for 7 min, in the mastercycler gradient eppendorf thermal cycler (germany). 5l of each reaction product was subjected to electrophoresis in tae buffer (40 mm tris base, 20 mm acetic acid,1 mm edta) using 1% agarose gels, stained with ethidium bromide, (0.5g / ml) and visualized on a uv transilluminator . Briefly, ten microliters of pcr products were incubated with 2 l of pagi restriction endonuclease (er1282, 00022771, fermentas, usa) and 2 l of supplied buffer at 37c for 16 hours . The final reaction volume was adjusted to 20 l by adding enough sterile distilled water . The digested dna was analyzed by electrophoresis in 2% agarose gels in tae buffer, visualized by ethidium bromide staining, and photographed . Lengths of resulting restriction fragments from f. hepatica and f. gigantica were predicted by means of the gene runner software v. 3.05 . Based on computerized cutting, this enzyme has no cutting site on its2 pcr amplicons for fasciola hepatica and one cutting site for f. gigantica, producing fragments of 165 and 291bp . The pcr products of ndi for f. gigantica parasites (characterized by morphological data and rflp analysis of its2) were directly sequenced using capillary dna analyzer (abi 3730, applied biosystems, foster city, ca, usa) after sequencing reactions with a big dye terminator v3.1 cycle sequencing kit (applied biosystems). Forward and reverse nucleic acid sequence data were used to construct a continuous sequence of inserted dna . Further comparison of the continuous ndi sequences was made with previously available sequences in national center for biotechnology information (ncbi) using basic local alignment search tool (blast) and compared with other homologues by means of neighbor joining and maximum parsimony trees reconstructed from kimura 2-parameter distances from 1,000 bootstrap replicates of multiple sequence alignments created using clustalw, as implemented by mega version 4.0 (18). This experiment was performed under the approval of the state committee on animal ethics, shiraz university, shiraz, iran . In addition, the recommendations of european council directive (86/609/ec) of november 24, 1986, regarding the protection of animals used for experimental purposes, were considered . Based on the morphometric criteria, 14 parasitic samples were initially identified to be f. gigantica while 26 samples were diagnosed to be f. hepatica . Morphometric data of the f. gigantica samples from different geographical locations and host origins captured in iran are present in table 1 . Morphometric data of adult liver flukes of fasciola gigantica form different geographical locations and host origins used for genotyping in iran the expected pcr products of 456bp were produced in all morphologically like f. gigantica samples using its2 gene primers independently from the different geographical origins and the different host species . Restriction fragment length polymorphism (rflp) patterns of parasitic flukes were obtained after digestion of the pcr products with pagi enzyme, in order to differentiate two fluke species (fig . 2b). Based on sequence differences of amplified portions of the its2 gene between f. gigantica and f. hepatica, the restriction enzyme pagi was expected to have one cutting site on the its2 pcr amplicons from f. gigantica . Accordingly, all of the its2 gene pcr amplicons of 456 bp from f. gigantica flukes were cut by the pagi endonuclease to fragments of 165 and 291bp . None of its2 pcr products from f. hepatica like samples was digested by the restriction enzyme treatment (fig . All samples confirmed to be f. gigantica (n=14) were subjected to pcr amplification of nd1 gene . The expected pcr products of 534 bp were produced in all reactions from f. gigantica samples (fig . Sequencing results of the nd1 pcr products were used to characterize the genotypic diversity of the f. gigantica flukes obtained from endemic areas of iran . Comparison of partial sequencing results of nd1 gene (410 bp) with previously available sequences in ncbi using blast showed that 13 out of 14 studied fasciola samples belonged to f. gigantica . Interestingly, one fluke sample (12 m: kf356181) that had been classified as f. gigantica according to morphometric criteria and its2 pcrrflp pattern, belonged to f. hepatica based on nd1 nucleotide sequence . Agarose gel electrophoresis; (a) ndi pcr products from fasciola samples compared with the molecular 100 bp weight marker . (b) pcr - rflp for its2 products using pagi (f.hep = f. hepatica, lane 4 and f. gig = f. gigantica, lanes 2, 5 and 6), lane 3=blank control the phylogenetic analysis of the nd1 sequence data revealed no host and geographic specificity among genotypes (fig . Twenty - six sites out of 410 base pairs of analyzed nd1 gene in 10 isolates of f. gigantica were diagnosed to be polymorphic denoting noticeable genetic diversity between different isolates of the parasite in iran . Nucleotide sequence variation among the iranian f. gigantica isolates for nd1 analyzed in this study ranged from 0% to 4.63% . From 26 polymorphism sites, only eight ones were leaded to the post - translational amino acid changes in nd1 gene product (fig . The nucleotide sequence data obtained in this study have been deposited in the genbank under accession numbers kf356168 to kf356181 . The ndi phylogenetic analysis, constructed according to maximum parsimony (mp) method to show the position of fasciola spp . Isolates in sheep, camel, cow and water buffalo in various locations in iran partial alignment of the nd1 amino acid sequences for fasciola gigantica (n=13 isolates) obtained from different geographical locations and host origins in iran . The species of genus fasciola (platyhelminthes: digenea) are the etiological agents of hepatic damage in a wide range of vertebrate hosts worldwide (4), resulting in profound economic losses (19) and public health challenge (9, 20). The present study characterized the genetic diversity of f. gigantica obtained from different hosts and different geographic locations in iran . Intraspecific genetic variations among liver flukes may reflect differences in virulence, host specificity and drug susceptibility or resistance (9). The differential diagnosis between f. hepatica and f. gigantica infection is very important because of their different transmission and epidemiological characteristics . Due to the limitations of morphological methods, various molecular approaches have been developed and used successfully for the identification and differentiation of fasciola species (21). Pcr analyses of ribosomal dna genes have been widely used for molecular identification of fasciolides . Previous studies have confirmed that the first and second internal transcribed spacers (its1 and its2) of rdna located between the nuclear small and large subunit rdna genes can provide genetic markers for species - level identification of fasciola . Several studies on species identification of fasciola flukes based on its2 sequence as molecular marker have been conducted (10, 12, 2225). In the current study, the application of pcr - rflp analysis of the its2 sequence was successful to differentiate fasciola species and results were in agreement with morphological classification of two fluke species . However, it seems that the accurate characterization of some types of intermediate form of fasciola needs both mitochondrial ndi and ribosomal its sequence data . The mitochondrial markers have been extensively used for phylogenetic studies and population differentiation because of its relativity rapid rate of evolution, importance in differentiation and discrimination of closely related organism, maternally inherited and does not undergo any recombination (26).for example, nadh dehydrogenase subunit 1 gene (nd1) have been shown to be well suited for investigating the population genetics of trematodes, i.e., f. hepatica (27), clonorchis sinensis (28) and opisthorchis viverrini (29). In the present study, mitochondrial nd1 gene was amplified using pcr from all f. gigantica samples and partial sequencing results were compared with previously available sequences in ncbi using blast and compared with other homologues by means of maximum parsimony trees . Nd1 gene sequence showed a considerable genetic variability among samples obtained from the different geographical origins and the different host species in iran . All of the isolates were classified as f. gigantica based on both its2 and nd1 gene sequences except for one sample, which categorized as f. hepatica based on nd1 gene sequence . The presence of intermediate forms of f. gigantica and f. hepatica has been reported from several countries . Previous studies has confirmed the existence of mixed infections of the both fluke species in the same animal, which imply that hybridization and/or introgression phenomena are possible cause of formation of intermediate forms (3, 6). Phylogenetic analysis revealed that it was impossible to identify accurately genotype using a host, suggesting that host associations are not likely to be useful characters for fasciola genotype classification . For example, geno - types of a and j were reported from different hosts . Similarly, no associations were found between geographic location and phylogenetic lineage, indicating that these characters were not useful for classification of f. gigantica . In addition, the results of this study showed heterogeneity might also exist in one location . The existence of the heterogeneity in one area may be due to traditional shepherding in iran, which includes regional movements between different pastures as well as transportation of sheep during slaughtering process . The amino acid substitutions identified in the nd1 peptide sequence of f. gigantica suggests that this polymorphism may exhibit considerable functional heterogeneity in nd1, which may be of importance to the parasite biological activities . Some authors believe that the capacity of fasciolids to adapt rapidly to new definitive hosts species and environments is most probably related to the high genetic variability of this parasite (30). The mitochondrial genetic variations among the iranian f. gigantica isolates (0% to 4.63%) were higher than those previously reported for f. hepatica in iran were . Formerly, moazeni et al . (2101) showed a limited genetic variation using mitochondrial co1 sequence (0% to 0.98%) among 19 different isolates of f. hepatica from cattle and sheep in different areas of iran (5). These authors only defined four different co1 haplotypes - based on five nucleotide substitutions - among this trematode isolates . The number of different nd1 genotypes obtained in this study demonstrates that, the mitochondrial gene is a suitable marker to show variability within f. gigantica isolates . Additional studies involving more isolates and other mitochondrial polymorphic genes, such as that for cytochrome c oxidase gene (co1), may help us to clarify genetic divergence of f. gigantica worldwide.
, this inoculation primarily infects the intervertebral disc and subsequently spreads to the adjacent vertebral structures4). Most patients with early postoperative spondylodiscitis can be treated with conservative methods, such as intravenous antibiotics, immobilization, and bracing . However, more aggressive approach or even surgical management is required in advanced or complicated cases11). Anterior lumbar interbody fusion (alif) may be an effective alternative method for the management of postoperative spondylodiscitis11). To date, however, there is no general consensus on the surgical approach in postoperative spondylodiscitis management . Since the clinical outcomes and complications after alif for postoperative spondylodiscitis are also poorly understood, we retrospectively analyzed the outcomes and complications of patients who received alif for the treatment of postoperative spondylodiscitis in this study . From january, 1994 to august, 2013, 13251 patients received spinal surgeries at our institute . Among them, 82 (0.62%) suffered postoperative spondylodiscitis, and alif was done in 13 patients . Therefore, the study population consisted of 13 consecutive patients with postoperative spondylodiscitis who were treated with alif . Postoperative spondylodiscitis was suspected when the patients had symptoms and signs suggestive of local infection, including fever, progression of local pain, and redness or edema at the operation site . Diagnostic measures are composed of laboratory findings, x - ray, and contrast - enhanced mri . To treat postoperative spondylodiscitis, we first tried non - operative methods such as administrating antibiotics for 6 - 8 weeks . However, whenever the infection was so progressive that it was insufficient to treat by antibiotics alone, such as epidural abscesses or neural compression, we operated as soon as possible . The oswestry disability index (odi) was used for assessment of quality of life, and the modified visual analogue scale (vas) for assessment of pain . Serial spine x - rays that included dynamic flexion - extension views were used to assess radiologic outcomes . The successful fusion was defined as: 1) absence of halo around the screws; 2) presence of bilateral continuous trabecular bone bridge between the fused segments on the anteroposterior plain film; and 3) lack of motion on the flexion - extension film . The successful fusion was confirmed by the attending surgeon using these parameters7). At our institute, we usually follow up patients at postoperative 1, 3, and 6 months and then annually thereafter . In patients with complications, additional outpatient visits or hospital admissions the follow - up data were collected by medical record review and direct telephone interview using a standardized form including information about demographics, medical history, clinical presentation, results of follow - up images, and adverse clinical events . In the cases with missing values in the variables before 2000, especially in the odi score, we approximated the values by extensive review of the previous medical records regarding patient's subjective complaint of symptoms . Patients were positioned supine on the operating table with their arms abducted on arm boards . After a paramedian " minilaparotomy " was performed, location of the aortic bifurcation, the vena cava, and iliac arteries and veins were identified after anterior retroperitoneal approach . The great vessels and their tributaries were mobilized to provide exposure of the disc space . For l5-s1 level, the midline sacral vessels were ligated, and the iliac arteries and veins were mobilized . For l3 - 4, l4 - 5 level, the iliolumbar and segmental vessels were ligated, and the aorta and vena cava were mobilized to expose the entire ventral surface of the disc space . After the infected and adhered anterior surface of the spine was exposed, a rectangular incision was made anteriorly in the annulus fibrosus, followed by a discectomy at that level . Once the ventral dura was exposed, infected tissue was debrided completely and irrigated sufficiently . After compete removal of the infected tissue, interbody fusion was performed with autologous tricortical iliac bone block or interbody fusion cages packed with autologous cancellous bone . All analyses were performed with statistical package for the social sciences (spss) version 18.0 (spss inc ., continuous variables are presented as meansd, and categorical variables are presented as frequencies . Paired t - tests and chi - square tests were used to compare pre- and post - operative values . All p - values are two - tailed, and p<0.05 was considered statistically significant . From january, 1994 to august, 2013, 13251 patients received spinal surgeries at our institute . Among them, 82 (0.62%) suffered postoperative spondylodiscitis, and alif was done in 13 patients . Therefore, the study population consisted of 13 consecutive patients with postoperative spondylodiscitis who were treated with alif . Postoperative spondylodiscitis was suspected when the patients had symptoms and signs suggestive of local infection, including fever, progression of local pain, and redness or edema at the operation site . Diagnostic measures are composed of laboratory findings, x - ray, and contrast - enhanced mri . To treat postoperative spondylodiscitis, we first tried non - operative methods such as administrating antibiotics for 6 - 8 weeks . However, whenever the infection was so progressive that it was insufficient to treat by antibiotics alone, such as epidural abscesses or neural compression, we operated as soon as possible . The oswestry disability index (odi) was used for assessment of quality of life, and the modified visual analogue scale (vas) for assessment of pain . Serial spine x - rays that included dynamic flexion - extension views were used to assess radiologic outcomes . The successful fusion was defined as: 1) absence of halo around the screws; 2) presence of bilateral continuous trabecular bone bridge between the fused segments on the anteroposterior plain film; and 3) lack of motion on the flexion - extension film . The successful fusion was confirmed by the attending surgeon using these parameters7). At our institute, we usually follow up patients at postoperative 1, 3, and 6 months and then annually thereafter . In patients with complications, additional outpatient visits or hospital admissions the follow - up data were collected by medical record review and direct telephone interview using a standardized form including information about demographics, medical history, clinical presentation, results of follow - up images, and adverse clinical events . In the cases with missing values in the variables before 2000, especially in the odi score, we approximated the values by extensive review of the previous medical records regarding patient's subjective complaint of symptoms . Patients were positioned supine on the operating table with their arms abducted on arm boards . After a paramedian " minilaparotomy " was performed, location of the aortic bifurcation, the vena cava, and iliac arteries and veins were identified after anterior retroperitoneal approach . The great vessels and their tributaries were mobilized to provide exposure of the disc space . For l5-s1 level, the midline sacral vessels were ligated, and the iliac arteries and veins were mobilized . For l3 - 4, l4 - 5 level, the iliolumbar and segmental vessels were ligated, and the aorta and vena cava were mobilized to expose the entire ventral surface of the disc space . After the infected and adhered anterior surface of the spine was exposed, a rectangular incision was made anteriorly in the annulus fibrosus, followed by a discectomy at that level . Once the ventral dura was exposed, infected tissue was debrided completely and irrigated sufficiently . After compete removal of the infected tissue, interbody fusion was performed with autologous tricortical iliac bone block or interbody fusion cages packed with autologous cancellous bone . All analyses were performed with statistical package for the social sciences (spss) version 18.0 (spss inc ., continuous variables are presented as meansd, and categorical variables are presented as frequencies . Paired t - tests and chi - square tests were used to compare pre- and post - operative values . All p - values are two - tailed, and p<0.05 was considered statistically significant . The baseline characteristics, surgical indications, and clinical outcomes are summarized in table 1, and a representative case is described in fig . 1 . Most patients were men (92.3%, 12/13), and the mean age was 54.5 years old (27 - 77 years). Seven out of 13 patients underwent spine surgery once, four underwent spine surgery twice, and two underwent spine surgery more than three times before alif at our institute . The previous surgical procedures were partial hemilaminectomy and discectomy in five patients, decompressive laminectomy in three patients, posterior lumbar interbody fusion in four patients, and artificial disc replacement in one patient . Among the 13 total patients, 10 had revision surgery within 1 year after the primary surgery; three had revision surgery 1 - 3 years after the primary surgery . The mean interval from the primary surgery to alif was 7.8 months (1 - 23 months), and the average duration of symptoms before alif was 2.1 months (1 - 5 months). The mean interval from the primary surgery to postoperative spondylodiscitis was 5.8 months (1 - 22 months). After alif, the patients were followed up for an average of 112 months (8 - 234 months). Eleven patients underwent single - level fusion, and two patients underwent two - level fusions . The levels fused included l5-s1 (4 cases), l4 - 5 (9 cases), and l3 - 4 (2 cases). Autologous tricortical bone block was used in cases with endplate destruction from the discitis, whereas interbody fusion cages were used in cases without endplate destruction after the antimicrobial therapy . The autologous iliac bone block was used as the interbody fusion material in 10 out of 13 cases . The most common pathogens were staphylococcus species, including one methicillin - resistant staphylococcus aureus and three methicillin - resistant coagulase - negative staphylococci . 12), vancomycin - sensitive enterococcus was initially identified, but candida albicans was also isolated thereafter . This patient was treated with combination of vancomycin and amphotericin b. in the laboratory findings, the initial average erythrocyte sedimentation rate (esr) level was 42.228.8 mm / h (0 - 15 mm / h), c - reactive protein (crp) level was 61.169.4 mg / l (0 - 5.3 initial intravenous (iv) antibiotic therapy was started immediately after the clinical diagnosis was made . The average duration of intravenous antibiotic therapy in the study population was 8 weeks (4 - 15 weeks) after the alif . After 6 months, inflammatory markers (wbc, esr, and crp) were within the normal range for all patients . Mean vas for back and leg pain before alif was 6.81.1, which improved to 3.22.2 after alif (p<0.001). The mean odi score before was 70.014.8, which improved to 34.227.0 at 6 months after alif . Bony fusion at 6 months after the surgery occurred in 11 out of 13 patients . The remaining two patients did not meet the definition of bony fusion which used for the assessment in this study; however, all patients had no related symptoms and could be managed conservatively . There were no specific post - alif complications during the follow - up in the study population . The baseline characteristics, surgical indications, and clinical outcomes are summarized in table 1, and a representative case is described in fig . 1 . Most patients were men (92.3%, 12/13), and the mean age was 54.5 years old (27 - 77 years). Seven out of 13 patients underwent spine surgery once, four underwent spine surgery twice, and two underwent spine surgery more than three times before alif at our institute . The previous surgical procedures were partial hemilaminectomy and discectomy in five patients, decompressive laminectomy in three patients, posterior lumbar interbody fusion in four patients, and artificial disc replacement in one patient . Among the 13 total patients, 10 had revision surgery within 1 year after the primary surgery; three had revision surgery 1 - 3 years after the primary surgery . The mean interval from the primary surgery to alif was 7.8 months (1 - 23 months), and the average duration of symptoms before alif was 2.1 months (1 - 5 months). The mean interval from the primary surgery to postoperative spondylodiscitis was 5.8 months (1 - 22 months). After alif, the patients were followed up for an average of 112 months (8 - 234 months). Eleven patients underwent single - level fusion, and two patients underwent two - level fusions . The levels fused included l5-s1 (4 cases), l4 - 5 (9 cases), and l3 - 4 (2 cases). Autologous tricortical bone block was used in cases with endplate destruction from the discitis, whereas interbody fusion cages were used in cases without endplate destruction after the antimicrobial therapy . The autologous iliac bone block was used as the interbody fusion material in 10 out of 13 cases . The most common pathogens were staphylococcus species, including one methicillin - resistant staphylococcus aureus and three methicillin - resistant coagulase - negative staphylococci . 12), vancomycin - sensitive enterococcus was initially identified, but candida albicans was also isolated thereafter . This patient was treated with combination of vancomycin and amphotericin b. in the laboratory findings, the initial average erythrocyte sedimentation rate (esr) level was 42.228.8 mm / h (0 - 15 mm / h), c - reactive protein (crp) level was 61.169.4 mg / l (0 - 5.3 initial intravenous (iv) antibiotic therapy was started immediately after the clinical diagnosis was made . The average duration of intravenous antibiotic therapy in the study population was 8 weeks (4 - 15 weeks) after the alif . After 6 months, inflammatory markers (wbc, esr, and crp) were within the normal range for all patients . Mean vas for back and leg pain before alif was 6.81.1, which improved to 3.22.2 after alif (p<0.001). The mean odi score before was 70.014.8, which improved to 34.227.0 at 6 months after alif . Bony fusion at 6 months after the surgery occurred in 11 out of 13 patients . The remaining two patients did not meet the definition of bony fusion which used for the assessment in this study; however, all patients had no related symptoms and could be managed conservatively . There were no specific post - alif complications during the follow - up in the study population . Early treatment of postoperative spondylodiscitis usually consists of bed rest, immobilization, and broad - spectrum antibiotics . For advanced disease, however, this procedure has some drawbacks, including failure to manage the infection components anterior to the vertebrae and postoperative instability12). This retrospective analysis revealed that alif led to successful management of postoperative spondylodiscitis in terms of infection control and symptom improvement . In addition, alif provided optimal symptomatic improvement and stabilization of the vertebrae in most cases5,11). Alif has many benefits in postoperative spondylodiscitis management of the previous infected vertebral structures compared to a posterior approach . Second, it provides surgeons with wide exposure of the entire disc space through unscarred passage . Third, it avoids dissection of perineural scar tissue, retraction of the scarred nerve root and dura, and removal of the facets usually associated with repeated discectomy3). Fourth, it allows more efficient restoration of intervertebral disc height and indirect decompression of the intervertebral foramen in case of foraminal stenosis3). Accessing the entire disc space allows for greater distraction, the placement of bigger interbody devices, and greater possibilities of arthrodesis14). In addition, alif preserves all remaining posterior paraspinal tissues (lamina, facets, ligaments, and muscle), thereby preserving spinal stability, which may help reduce the degeneration of adjacent motion segments3). From a biomechanical standpoint, the interbody fusion device can be placed at the weight - bearing center of the spine across the degenerated disc space, where 80% of axial load occurs13). Furthermore, disc height and sagittal balance can be more effectively restored in alif than in posterior lumbar surgery in terms of creating preferable fusion conditions by placing the interbody fusion device as extensively as possible under compression with a voluminous blood supply from the adjacent vertebral endplates1,3). In terms of onset of postoperative spondylodiscitis, it could be classified as early (<20 weeks) and late (> 20 weeks) infections2). In our series, although all patients were operated at the mean of 7.7 months after initial surgery, some patients received delayed operation due to late onset infection and they also successfully treated using alif . The goals of treatment for postoperative spondylodiscitis are as follow: 1) eradicate the infected tissue, 2) prevent neurological damage, 3) restore spinal balance, and 4) relieve pain6,8,9). Patients with mild infection can be managed with 6 - 8 weeks of intravenous antibiotics6). Moreover, if possible, surgery can be delayed for 2 weeks to decrease inflammation, which allows for better demarcation of tissue planes for surgical dissection and decreases the risks from anesthesia in acutely ill patients12). Regarding the timing of the surgery, there are several critical indications of emergent surgery, including progressive neurologic deterioration, epidural abscess, and symptoms of spinal cord compression10). As the patient's prognosis cannot be improved after 48 hours of established paralysis, prompt surgical intervention is warranted in these " emergent " cases10). For this reason, indications of surgery, antibiotic treatment, and general care of patients varied from surgeon to surgeon, and the independent impact of alif on the management of postoperative spondylodiscitis could not be verified . In addition, this study focused only on a descriptive analysis of alif without a control group; thus, we cannot firmly state that alif is better or even equivalent to traditional posterior approaches . Although the recruiting period spanned two decades, only 13 patients could be enrolled, which limits the generalization and the prospectiveness of this study . Alif has many benefits over the posterior approach, including lessening damage to the paraspinal structures, providing a wider fusion bed for better arthrodesis, and more effective restoration of the vertebral balance.
Many benign tumors can affect the orbit and, if symptomatic or cosmetically disfiguring, most can be removed via various cutaneous and bony approaches . Many of these approaches involve removal of the lateral orbital wall, with replacement after tumor excision [17]. This approach can produce excellent operative exposure of the lesion and allow for removal with minimal manipulation of the orbital contents . With interest in minimizing surgical risk, postoperative recovery and scarring, recent surgical trends favor minimally invasive techniques and local anesthesia where possible [1, 2]. Traditionally, orbital tumors located in the middle to posterior orbit are approached from a variety of soft - tissue incisions incorporating a bony lateral orbitotomy (marginotomy) of the zygoma [17]. The bony flap increases exposure of the deep orbit and provides the surgeon with added maneuverability for tumor removal [6, 7]. However, the bony marginotomy can be associated with longer operative time, increased postoperative pain, recovery time, temporalis muscle wasting, and external scar . Using modern imaging modalities in conjunction with minimal incision surgical techniques, we have found that bony marginotomy is rarely needed in order to access presumed benign tumors of the middle and deep orbit . In this study, we present our surgical experience in the removal of orbital tumors using a combination of soft - tissue approaches, without bony marginotomy, under monitored local anesthesia and general anesthesia . In this retrospective case series, electronic medical records of all patients with orbital tumors removed through the oculoplastics clinic of the jules stein eye institute between 1992 and 2013 were reviewed . The institutional review board at the university of california, los angeles approved the study protocol, and the tenets of the declaration of helsinki were followed . Data including snellen visual acuity, ocular motility, tumor size and type, follow - up duration, recurrence, and intra- and postoperative complications were recorded . Imaging was used to classify tumor location as posterior or anterior based on confinement to the anterior half of the orbit or extension into the posterior half, respectively . Surgeries were performed under general anesthesia . However, if the case was amenable to completion under local anesthesia, an array of orbital and regional nerve blocks was utilized . After adequate anesthesia is achieved, the appropriate incision is chosen based on location of the tumor within the orbit . Tumors inferior to the optic nerve are generally approached with a conjunctival incision 4 mm below the inferior tarsal margin through the lower eyelid retractors and into orbital fat (figures 1 and 2). The incision can be extended medially or laterally as necessary . When extending the incision laterally, the lateral canthal tendon can be loosened slightly by blunt spreading with scissors, avoiding complete release from the orbital rim . For tumors located superiorly, an incision is made through the upper eyelid crease 810 mm above the lid margin . The incisions were customized medially or laterally based on the location of tumor and the site needs to be exposed . An incision through the septum with medial or lateral displacement of the levator fibers allows blunt dissection through the intermuscular septum . The levator can be safely distracted medially or laterally and we have not found a vertical lid splitting procedure necessary to access the superior orbit . Working towards the anticipated 3-dimensional location of the tumor predicted by imaging studies, stevens scissors and peanut or cotton tipped applicators can be used to dissect fatty soft tissue off the face of the tumor . Once the face of the tumor is within view, the surgeon can assess the gross surgical pathology and confirm the diagnosis of a benign lesion . A half - circle needle can now be passed through the face of the tumor if appropriate, which if compressible will cause partial exsanguination and a decrease in tumor size . An additional pass of the needle is utilized to create a whip suture that will provide better control and forward traction . In the case of cavernous malformations, this stitch also aids in exsanguination of the lesion, decreasing its overall size for delivery anteriorly . Some tumors such as dermoid cysts or schwannomas can be additionally decompressed by suctioning the contents through a small anterior incision into the tumor . With the tumor partly exsanguinated or decompressed (if possible) and suture in place, blunt dissection (e.g., with a freer elevator) can now be carried back toward the posterior edge of the tumor with simultaneous forward traction . As the attachments are dissected, the tumor will move towards the surgical opening, revealing posterior attachments, which can be gently lysed with blunt and sharp dissection (figure 3). There are usually no major vascular stalks associated with cavernous hemangiomas, but any bleeding vessels are controlled with bipolar cautery (figure 4). Small conjunctival or eyelid crease incisions allow a relatively quick recovery with minimal ecchymosis and minimal visible scar (figure 5). Median age was 48.5 (range 3187) years old and median follow - up was 24.5 (range 4375) weeks (table 1). The average standard deviation tumor size, measured in its longest dimension based on preoperative imaging, was 22.6 6.5 mm (range 1051). Twenty - nine (out of 30) tumors were located in the posterior half of the orbit . Surgeries were performed through eyelid crease (17 eyes), conjunctival (9 eyes), lateral canthal (2 eyes), and transcaruncular (2eyes) approaches . Six cases were performed with monitored anesthesia care and local block, and 24 were performed under general anesthesia . Two patients (#16&29) had mild decrease in visual acuity at the last follow - up, which was related to ocular surface changes . Confirmed surgical pathology revealed several tumor types including 15 cavernous hemangiomas, 5 pleomorphic adenomas, 4 solitary fibrous tumors, 2 neurofibro- mas, 2 schwannomas, and 1 orbital varix . Middle to deep orbital tumors are most commonly removed through a bony lateral marginotomy [17]. This technique was first described by kronlein in 1889 for the removal of dermoid cysts, providing a relatively wide field of view to search for retrobulbar tumors . With the advent of modern imaging modalities and ability to distinguish tumor character and location preoperatively, other less invasive soft - tissue approaches have been described . All documented series have reported surgical excision under general anesthesia and typically involve a lateral canthotomy or rectus resection for exposure . Both kiratli et al . And cheng et al . Presented series of intraconal orbital cavernous hemangiomas removed via transconjunctival approach [10, 11]. Their series were limited to tumors touching or near the globe and necessitated removal of the medial rectus for larger tumors . Another 2004 paper by yan and wu presented results of removing 139 (out of 214) orbital cavernomas through an anterior orbitotomy . The remaining 75 tumors (out of 214) were removed successfully by standard lateral orbitotomy with bony marginotomy . The authors stated that traditional lateral orbitotomy must be used in cases where tumor dimensions exceed 3 cm, or the tumor extends to the orbital apex, or has imaging inconsistent with cavernous hemangioma . In another series limited to cavernous hemangiomas, pelton and patel excised 5 medial intraconal cavernomas through a superomedial eyelid crease . The authors concluded that the superomedial lid crease approach to the medial intraconal space has a number of advantages over the medial transconjunctival and lateral orbital approaches, including ease of dissection, incision - to - nerve distance, and angle of approach to the optic nerve . With appropriate screening and intraoperative flexibility, we believe the most benign intraconal tumors can be removed through one of many soft - tissue approaches, often under local anesthesia . If the lesion is compressible, for example, cavernous hemangioma or dermoid cyst, large size is not a contraindication to a small incision approach . . The anticipated pathology should be consistent with a benign tumor . Additionally ct or mri imaging modalities should demonstrate a well - defined mass, without tethering, infiltration into surrounding tissue or growth into bone or the sinuses . These features suggest that the tumor is likely benign in nature and distinct from the surrounding tissues making it possible to be completely resected . Tumors located in the inferior orbit are best approached through an inferior conjunctival incision . Most often a canthotomy is not required, but for more exposure, the canthus can be loosened with a small internal incision, leaving the skin and orbicularis intact . Occasionally a deep medial orbital tumor adjacent to the periosteum may be more easily accessed using a caruncular incision . Superiorly positioned orbital tumors are removed through an upper eyelid crease incision with appropriate medial or lateral orbital entry (working around the levator aponeurosis). Most benign tumors of the deep orbit can be safely reached through careful dissection with any of these incisional approaches, although tumors at the deepest apex, for example, those extending through the superior orbital fissure, generally require bone removal for optimal exposure . This is achieved through careful examination of fine cut orbital ct or mri images preoperatively in combination with adequate lighting and the use of intraoperative landmarks such as the bony rim, globe, and rectus muscles . Using this spatial knowledge along with palpation and ballottement of the tumor during dissection allows the surgeon to accurately focus the blunt and sharp dissection through the relatively small opening . Once the dissection has reached the face of the tumor, the gross pathologic appearance should be assessed . If visual inspection and palpation suggest an infiltrative process, the surgeon must be flexible enough to change the operative plan, for example, to biopsy the lesion or convert to a larger access procedure under general anesthesia . None of our cases necessitated conversion from monitored anesthesia care to general anesthesia or bone removal . The next critical point is the pass of the half - circle needle through the face of the tumor . In the case of a cavernous venous malformation (cavernous hemangioma), the resulting exsanguination of the tumor, which can be easily suctioned, shrinks the tumor in size and allows more space to maneuver . Additional passes of the needle create a whip suture, allowing forward traction for adequate dissection and expression of the tumor through the small soft - tissue incision . Local anesthesia is preferred by many patients as it shortens the operative time, avoids the discomfort and risks of general anesthesia, and allows the patient to return home sooner . We have found that benign orbital tumors can be removed under monitored local anesthesia in some patients . Patients who are overly anxious will do better under general anesthesia although moderate anxiety will respond to common anxiolytic agents used for monitored anesthesia [6, 7]. In our series, those who underwent monitored anesthesia did well with common anxiolytic agents . Intraoperatively, the orbit should become adequately anesthetized superficially and deep with blocks of the zygomaticotemporal, zygomaticofacial, infraorbital, and supraorbital nerves as well as along the orbital floor back to the level of the superior orbital fissure . It is important to maintain communication with the patient and provide more local anesthetic when necessary . More local anesthetic is often necessary in the central and medial orbit when dissecting down to the orbital rim, as there are many sensory nerves in this region . The retrobulbar block may dampen or eliminate the physiologic pupillary reaction of the ipsilateral eye . This should not affect operative decision - making: the surgeon always uses maximal care to minimize traction and pressure on nerves in the orbit . Pupillary dilation is more often related to the efferent nerves to the pupil and is not a reliable sign of optic nerve compression . Optic nerve function can be better assessed, if needed, by looking for a reverse afferent papillary defect in the opposite pupil . This includes a detailed history, physical exam, and careful study of appropriate imaging modalities . Combining this data with a thoughtful orbital differential diagnosis, appreciation for nuances of intraoperative gross surgical pathology, and readiness to convert to an open procedure if necessary allows the surgeon to safely approach most orbital tumors with the techniques described above . The desire to perform minimally invasive surgery should not be pursued to the extent that adequate exposure or patient safety is compromised in any way . However, within the constraints of good judgment and safety, it is appropriate to try to minimize the invasiveness of orbital surgery . In our experience, with appropriate preoperative evaluation and a creative flexible surgical approach, many benign orbital tumors can be safely approached through a minimally invasive soft - tissue approach, avoiding a bony marginotomy.
Patients with congestive heart failure (hf) are at high risk of dying from its progression as well from sudden cardiac death related to ventricular tachyarrhythmia . Over the last decade, cardiac resynchronization therapy (crt) and implantable cardioverter defibrillators (icds) have markedly improved the prognosis of hf patients, with prolongation of survival over and above that conferred by medical therapy alone . It has been well established in trials that in patients with severe left ventricular (lv) systolic dysfunction, new york heart association (nyha) class iii / iv symptoms, and wide qrs, crt improves symptoms / quality of life and also reduces mortality . Additionally, more recent trials have shown beneficial reverse lv remodelling even in patients with milder symptoms . This has resulted in a class i recommendation for crt in appropriately selected candidates in guidelines framed on both sides of the atlantic . Since most patients who are candidates for crt will have a lv ejection fraction (ef) of 35%, this automatically makes them candidates for an icd as well by the current guidelines, which makes the assumption that there is universally significant excess mortality due to sudden cardiac death (scd) among crt - p patients who can therefore definitively benefit from the added defibrillator . However, concrete evidence for such a premise in a contemporary crt - p population is lacking and in any population of this kind; competing risks for mortality need to be carefully considered . A few studies have attempted to directly compare outcomes between crt - p vs. crt - d subjects . Furthermore, such outcome comparisons based on observational studies have methodological limitations and may be biased . Current guidelines do not make firm recommendations in terms of the choice between crt - p vs. crt - d, leaving room for physician discretion . For instance, the proportion of crt implantations, which are crt - d, reaches> 90% in most practices in the usa, whereas it is relatively lesser across europe . The use of crt - d or crt - p in clinical practice is an important question with significant implications in terms of costs, as well as device - related complications . In this context, a better understanding of the relative contribution of scd as opposed to other competing causes of mortality in the crt population can be very informative . A cause - of - death analysis among crt - p vs. crt - d patients, may represent a novel approach to this problem . Using a large, multicentre study with prospective follow - up, we evaluated the characteristics of crt - p vs. crt - d patients in a real - world scenario and analysed to what extent crt - p subjects, as currently chosen in clinical practice, would have potentially additionally benefited from the presence of a back - up defibrillator . Certitude, a 2-year, prospective, multicentre registry launched in january 2008 and held under the direction of the working group on pacing and arrhythmias of the french society of cardiology, was funded and coordinated by the french society of cardiology . Its primary objective was to define the baseline characteristics and clinical outcomes of french patients who undergo implantation of crt systems . An analysis of the precise causes of death was planned at 2 years after device implantation . The 41 medical centres participating in the study (appendix) enrolled consecutive patients who, between 1 january 2008 and 31 december 2010, had undergone crt device implantations . The criteria for crt implantation were as per the 2007 guidelines of the european society of cardiology and european heart rhythm association, updated in 2010 . However, all crt recipients were enrolled, in order for the registry to reflect real - world medical practice . Each patient was then enrolled in a specific follow - up programme with clinical, ecg, echocardiographic, and device interrogation data collected every 6 months over the following 2 years (up to 1 january 2013). The study was conducted in accordance with good clinical practice, french law, and the french data protection law . The protocol was reviewed by the committee for the protection of human subjects in biomedical research (cctirs #08 - 522) and the data file was reported to, and authorized by, the commission nationale informatique et des liberts (french data protection committee, cnil #909048). Individual patient data were collected, using an electronic case report form created by the scientific committee to record, at each participating medical centre, the demographic and baseline clinical characteristics, and the implantation procedures and techniques . These data were regularly transferred (every 3 months) via an internet - based system to a central database created at the data management centre of the french society of cardiology in collaboration with the paris cardiovascular research center, european georges pompidou hospital, paris (inserm unit 970). All variables recorded before device implantation were defined and classified using standard clinical terminology, including gender, age (stratified as <60, 6074, and 75 years), and underlying heart disease (ischaemic vs. non - ischaemic). Renal clearance was estimated using the cockroft and gault's formula, and defined as severe renal insufficiency if <30 ml / min/1.73 m, and qrs duration was classified as 120, 121149, and 150 ms . Left ventricular ejection fraction was measured on transthoracic echocardiograms, using simpson's method, and recorded as a continuous variable and also stratified as 20, 2135, and> 35% . A history of atrial fibrillation (af) in addition to af and renal failure, other co - morbidities were systematically recorded, including cancer, chronic obstructive pulmonary disease, liver disease, diabetes mellitus, and cerebral vascular disease . The type of crt (crt - p or crt - d) implanted was recorded without the manufacturer's information . The complications recorded included infections, changes in capture threshold, lead dislodgement, haematomas, hf, fever, arrhythmias, pneumothorax, phrenic nerve stimulation, and death . Finally, drug regimens prescribed at the time of hospital discharge including beta - adrenergic blockers, anti - arrhythmics, digoxin, calcium antagonists, angiotensin - converting enzyme inhibitors or angiotensin ii receptor blockers, mineralocorticoid receptor antagonists, diuretics, and anticoagulants were recorded . Device programming was left to the discretion of the investigators at each centre, with the guiding principle being achievement of maximal biventricular pacing . All patients were followed at 6-month intervals for 2 years by the implanting centre till the close of study on 1 january 2013 . At each follow - up, the patients underwent clinical examination, ecg, transthoracic echocardiogram, and device interrogation . In addition, information on any intercurrent events (such as hospitalization) was also recorded in the file . The above data were systematically gathered at each follow - up visit from the date of device implantation to study closure or death or heart transplantation . The investigators at each enrolling centre recorded major clinical events, using a standardized form, and a clinical events committee verified their accuracy by contacting the attending physicians or the patients as required, on a yearly basis, focusing on the vital status and on the specific modes and causes of death and on major clinical events or interventions during follow - up, including changes in drug regimens, as well as interim hospitalizations . Sources to ascertain the vital status also included registries of the patients' birthplaces, the french national institute of health and medical research (inserm cpidc unit le kremlin - bictre, france), and the french national institute of statistics and economical studies . The cause - of - death were classified as sudden if the patient (i) died suddenly and unexpectedly within 1 h of symptoms in the absence of progressive cardiac deterioration, (ii) died unexpectedly in sleep, or (iii) died unexpectedly within 24 h after last being seen alive and in the usual state of health . Other cardiovascular deaths included myocardial infarction, hf, acute aortic syndrome, stroke, and pulmonary embolism . Fatal arrhythmias associated with end - stage hf were classified as non - sudden cardiovascular deaths . Deaths attributable to causes, such as cancer, infectious disease, or renal or respiratory failure, were classified as non - cardiovascular . When inadequate or no data were available, the cause of death was classified as unknown or unidentifiable . We used multiple sources to assess and finally adjudicate the cause of death, which included medical data obtained by the regional investigators, pathology report, emergency medical services report, as well as data from the french center on medical causes of death (inserm cpidc unit), which is able to provide the causes of death occurring in france . Continuous variables are presented as mean standard deviation and categorical variables are presented as numbers and percentages . Comparisons between groups (patients with crt - p vs. patients with crt - d) were made, using the or fisher's exact tests for discrete variables and with unpaired t - tests, wilcoxon signed - rank tests, or one - way analysis of variance for continuous variables . Factors associated with the implantation of crt - p were identified, using a multiple variable, stepwise, logistic regression analysis . Meier curves were constructed to estimate the 2-year survival, and crt - p and crt - d groups were compared using the log - rank test . For the cause - specific mortality, we used a competing risk analysis and estimated the cumulative incidence function . We then used gray tests to assess the difference between the crt - p and crt - d groups . A cox proportional hazards regression analysis was used to identify variables independently associated with overall mortality . The crude associations between mortality and different variables (listed in table 1) were first quantified by univariate cox regression . All covariates that reached a significance level of p <15% were then included in an initial multivariate regression model . A stepwise selection was applied to obtain a final model that included covariates with p <5% . Given the observational design of the study and minimization of indication bias for device implantation, propensity score analyses were conducted . We estimated the propensity score of receiving a crt - p therapy by fitting a logistic regression model using age, sex, af, lvef, aetiology of hf, nyha, and beta - adrenergic blockers as covariates . We then matched patients who received crt - d therapy with those who received crt - p in an 1: 1 ratio using a greedy matching algorithm with a maximum allowable difference of 0.05 (see supplementary material online, table s1 and figure s1). Then, the association between device type and mortality was repeated after propensity score matching (462 patients). All data were analysed at inserm, unit 970, cardiovascular epidemiology and sudden death, paris, using sas version 9.4 (sas institute, inc ., cary, nc, usa). Table 1characteristics of the entire registry sample and of the crt - p vs. crt - d recipientstotal (n = 1705)crt - d (n = 1170)crt - p (n = 535)p - valueage (years)68.8 11.165.6 10.475.9 9.0<0.0001men1317 (77.2)945 (80.8)372 (69.5)<0.0001heart disease ischaemic724 (47.0)556 (49.3)168 (40.7)0.0026 non - ischaemic816 (53.0)571 (50.7)245 (59.3)qrs duration mean (ms)157.7 27.1155.0 26.2160.8 29.00.0018left ventricular ejection fraction median,% 25.5 (10.0)25.5 (10.0)25.5 (10.0)0.084 20%484 (29.3)333 (29.2)151 (29.6)<0.0001 2135%1078 (65.3)764 (67.1)314 (61.5)> 35%88 (5.3)42 (3.7)46 (9.0)new york heart association functional class i16 (1.0)14 (1.3)2 (0.4)<0.0001 ii250 (16.0)194 (18.0)56 (11.7) iii1188 (76.2)824 (76.2)364 (76.0) iv106 (6.8)49 (4.5)57 (11.9)history of: atrial fibrillation445 (27.3)248 (22.1)197 (38.7)<0.0001 renal insufficiency211 (14.4)138 (13.0)73 (18.2)0.0128 copd264 (18.1)198 (18.7)66 (16.4)0.3120 cancer122 (8.4)88 (8.3)34 (8.5)0.9272 miscellaneous disorders266 (18.2)174 (16.4)92 (22.9)0.0043drug therapy at the time of implantation diuretics1045 (66.2)752 (69.2)293 (59.6)0.0002 ace inhibitors / arb1057 (66.9)792 (72.9)265 (53.9)<0.0001 mra404 (25.6)331 (30.5)73 (14.8)<0.0001 beta - adrenergic blockers945 (59.9)732 (67.3)213 (43.3)<0.0001 oral anticoagulant agent658 (41.7)438 (40.3)220 (44.7)0.0989 antiplatelet agents693 (43.9)512 (47.1)181 (36.8)0.0001values are means sd, median (iqr), or numbers (%) of observations.ace, angiotensin - converting enzyme; arb, angiotensin receptor blocker; copd, chronic obstructive pulmonary disease; mra, mineralocorticoid receptor antagonist; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Characteristics of the entire registry sample and of the crt - p vs. crt - d recipients values are means sd, median (iqr), or numbers (%) of observations . Ace, angiotensin - converting enzyme; arb, angiotensin receptor blocker; copd, chronic obstructive pulmonary disease; mra, mineralocorticoid receptor antagonist; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Certitude, a 2-year, prospective, multicentre registry launched in january 2008 and held under the direction of the working group on pacing and arrhythmias of the french society of cardiology, was funded and coordinated by the french society of cardiology . Its primary objective was to define the baseline characteristics and clinical outcomes of french patients who undergo implantation of crt systems . An analysis of the precise causes of death was planned at 2 years after device implantation . The 41 medical centres participating in the study (appendix) enrolled consecutive patients who, between 1 january 2008 and 31 december 2010, had undergone crt device implantations . The criteria for crt implantation were as per the 2007 guidelines of the european society of cardiology and european heart rhythm association, updated in 2010 . However, all crt recipients were enrolled, in order for the registry to reflect real - world medical practice . Each patient was then enrolled in a specific follow - up programme with clinical, ecg, echocardiographic, and device interrogation data collected every 6 months over the following 2 years (up to 1 january 2013). The study was conducted in accordance with good clinical practice, french law, and the french data protection law . The protocol was reviewed by the committee for the protection of human subjects in biomedical research (cctirs #08 - 522) and the data file was reported to, and authorized by, the commission nationale informatique et des liberts (french data protection committee, cnil #909048). Individual patient data were collected, using an electronic case report form created by the scientific committee to record, at each participating medical centre, the demographic and baseline clinical characteristics, and the implantation procedures and techniques . These data were regularly transferred (every 3 months) via an internet - based system to a central database created at the data management centre of the french society of cardiology in collaboration with the paris cardiovascular research center, european georges pompidou hospital, paris (inserm unit 970). All variables recorded before device implantation were defined and classified using standard clinical terminology, including gender, age (stratified as <60, 6074, and 75 years), and underlying heart disease (ischaemic vs. non - ischaemic). Renal clearance was estimated using the cockroft and gault's formula, and defined as severe renal insufficiency if <30 ml / min/1.73 m, and qrs duration was classified as 120, 121149, and 150 ms . Left ventricular ejection fraction was measured on transthoracic echocardiograms, using simpson's method, and recorded as a continuous variable and also stratified as 20, 2135, and> 35% . A history of atrial fibrillation (af) in addition to af and renal failure, other co - morbidities were systematically recorded, including cancer, chronic obstructive pulmonary disease, liver disease, diabetes mellitus, and cerebral vascular disease . The type of crt (crt - p or crt - d) implanted was recorded without the manufacturer's information . The complications recorded included infections, changes in capture threshold, lead dislodgement, haematomas, hf, fever, arrhythmias, pneumothorax, phrenic nerve stimulation, and death . Finally, drug regimens prescribed at the time of hospital discharge including beta - adrenergic blockers, anti - arrhythmics, digoxin, calcium antagonists, angiotensin - converting enzyme inhibitors or angiotensin ii receptor blockers, mineralocorticoid receptor antagonists, diuretics, and anticoagulants were recorded . Device programming was left to the discretion of the investigators at each centre, with the guiding principle being achievement of maximal biventricular pacing . All patients were followed at 6-month intervals for 2 years by the implanting centre till the close of study on 1 january 2013 . At each follow - up, the patients underwent clinical examination, ecg, transthoracic echocardiogram, and device interrogation . In addition, information on any intercurrent events (such as hospitalization) was also recorded in the file . The above data were systematically gathered at each follow - up visit from the date of device implantation to study closure or death or heart transplantation . The investigators at each enrolling centre recorded major clinical events, using a standardized form, and a clinical events committee verified their accuracy by contacting the attending physicians or the patients as required, on a yearly basis, focusing on the vital status and on the specific modes and causes of death and on major clinical events or interventions during follow - up, including changes in drug regimens, as well as interim hospitalizations . Sources to ascertain the vital status also included registries of the patients' birthplaces, the french national institute of health and medical research (inserm cpidc unit le kremlin - bictre, france), and the french national institute of statistics and economical studies . The cause - of - death were classified as sudden if the patient (i) died suddenly and unexpectedly within 1 h of symptoms in the absence of progressive cardiac deterioration, (ii) died unexpectedly in sleep, or (iii) died unexpectedly within 24 h after last being seen alive and in the usual state of health . Other cardiovascular deaths included myocardial infarction, hf, acute aortic syndrome, stroke, and pulmonary embolism . Fatal arrhythmias associated with end - stage hf were classified as non - sudden cardiovascular deaths . Deaths attributable to causes, such as cancer, infectious disease, or renal or respiratory failure, were classified as non - cardiovascular . When inadequate or no data were available, the cause of death was classified as unknown or unidentifiable . We used multiple sources to assess and finally adjudicate the cause of death, which included medical data obtained by the regional investigators, pathology report, emergency medical services report, as well as data from the french center on medical causes of death (inserm cpidc unit), which is able to provide the causes of death occurring in france . Continuous variables are presented as mean standard deviation and categorical variables are presented as numbers and percentages . Comparisons between groups (patients with crt - p vs. patients with crt - d) were made, using the or fisher's exact tests for discrete variables and with unpaired t - tests, wilcoxon signed - rank tests, or one - way analysis of variance for continuous variables . Factors associated with the implantation of crt - p were identified, using a multiple variable, stepwise, logistic regression analysis . Meier curves were constructed to estimate the 2-year survival, and crt - p and crt - d groups were compared using the log - rank test . For the cause - specific mortality, we used a competing risk analysis and estimated the cumulative incidence function . We then used gray tests to assess the difference between the crt - p and crt - d groups . A cox proportional hazards regression analysis was used to identify variables independently associated with overall mortality . The crude associations between mortality and different variables (listed in table 1) were first quantified by univariate cox regression . All covariates that reached a significance level of p <15% were then included in an initial multivariate regression model . A stepwise selection was applied to obtain a final model that included covariates with p <5% . Given the observational design of the study and minimization of indication bias for device implantation we estimated the propensity score of receiving a crt - p therapy by fitting a logistic regression model using age, sex, af, lvef, aetiology of hf, nyha, and beta - adrenergic blockers as covariates . We then matched patients who received crt - d therapy with those who received crt - p in an 1: 1 ratio using a greedy matching algorithm with a maximum allowable difference of 0.05 (see supplementary material online, table s1 and figure s1). Then, the association between device type and mortality was repeated after propensity score matching (462 patients). All data were analysed at inserm, unit 970, cardiovascular epidemiology and sudden death, paris, using sas version 9.4 (sas institute, inc ., cary, nc, usa). Table 1characteristics of the entire registry sample and of the crt - p vs. crt - d recipientstotal (n = 1705)crt - d (n = 1170)crt - p (n = 535)p - valueage (years)68.8 11.165.6 10.475.9 9.0<0.0001men1317 (77.2)945 (80.8)372 (69.5)<0.0001heart disease ischaemic724 (47.0)556 (49.3)168 (40.7)0.0026 non - ischaemic816 (53.0)571 (50.7)245 (59.3)qrs duration mean (ms)157.7 27.1155.0 26.2160.8 29.00.0018left ventricular ejection fraction median,% 25.5 (10.0)25.5 (10.0)25.5 (10.0)0.084 20%484 (29.3)333 (29.2)151 (29.6)<0.0001 2135%1078 (65.3)764 (67.1)314 (61.5)> 35%88 (5.3)42 (3.7)46 (9.0)new york heart association functional class i16 (1.0)14 (1.3)2 (0.4)<0.0001 ii250 (16.0)194 (18.0)56 (11.7) iii1188 (76.2)824 (76.2)364 (76.0) iv106 (6.8)49 (4.5)57 (11.9)history of: atrial fibrillation445 (27.3)248 (22.1)197 (38.7)<0.0001 renal insufficiency211 (14.4)138 (13.0)73 (18.2)0.0128 copd264 (18.1)198 (18.7)66 (16.4)0.3120 cancer122 (8.4)88 (8.3)34 (8.5)0.9272 miscellaneous disorders266 (18.2)174 (16.4)92 (22.9)0.0043drug therapy at the time of implantation diuretics1045 (66.2)752 (69.2)293 (59.6)0.0002 ace inhibitors / arb1057 (66.9)792 (72.9)265 (53.9)<0.0001 mra404 (25.6)331 (30.5)73 (14.8)<0.0001 beta - adrenergic blockers945 (59.9)732 (67.3)213 (43.3)<0.0001 oral anticoagulant agent658 (41.7)438 (40.3)220 (44.7)0.0989 antiplatelet agents693 (43.9)512 (47.1)181 (36.8)0.0001values are means sd, median (iqr), or numbers (%) of observations.ace, angiotensin - converting enzyme; arb, angiotensin receptor blocker; copd, chronic obstructive pulmonary disease; mra, mineralocorticoid receptor antagonist; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Characteristics of the entire registry sample and of the crt - p vs. crt - d recipients values are means sd, median (iqr), or numbers (%) of observations . Ace, angiotensin - converting enzyme; arb, angiotensin receptor blocker; copd, chronic obstructive pulmonary disease; mra, mineralocorticoid receptor antagonist; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Overall, a total of 1705 consecutive patients were enrolled in the study and received crt devices . The mean age of the overall population was 68.8 11.1 years, 33% were> 75 years of age, and 77% were men . The heart disease was ischaemic in 47% of patients and related to non - ischaemic dilated cardiomyopathy in 53% . Overall, 29% of patients presented with a lvef of 20% at the time of implantation . Overall, 13% of the crt - d group was implanted in the secondary prevention, following symptomatic ventricular tachycardia or sudden cardiac arrest . Patients with crt - p compared with crt - d were older (75.9 vs. 65.6 years, p <0.0001), less often male (69.5 vs. 80.8%, p <0.0001), more symptomatic (proportion of nyha class iii / iv, 87.9 vs. 80.8%, p = 0.0005), with less coronary artery disease (40.7 vs. 49.3%, p = 0.003), wider qrs (160.8 vs. 154.9 ms, p = 0.002), more af (38.7 vs. 22.1%, p <0.0001), and more co - morbidities (2 comorbidities, 16.9 vs. 12.9%, p = 0.04; table 1). Independent variables associated with crt - p (vs. crt - d) implantation are depicted in table 2 . Table 2independent variables associated with crt - p (vs. crt - d) implantationvariablesor95% cip - valueage1.171.141.19<0.0001female1.781.242.550.0018dilated non - ischaemic cardiomyopathy1.751.282.400.0005atrial fibrillation1.581.142.200.0062lvef <25%1.051.021.070.0001nyha iv2.821.614.920.0003no beta - adrenergic blockers2.401.763.26<0.0001lvef, left ventricular ejection fraction; nyha, new york heart association; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Independent variables associated with crt - p (vs. crt - d) implantation lvef, left ventricular ejection fraction; nyha, new york heart association; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Fatal periprocedural complications occurred in one patient, and death occurred before hospital discharge in five others (0.3%), due to severe cardiogenic shock . Overall, significant perioperative complications occurred in 133 subjects (7.8%) without significant difference between crt - d and crt - p (8.9 vs. 6.7%, p = 0.20). Pulse generator pocket haematoma (2.5%), lead dislodgment (1.6%), and phrenic nerve stimulation (1.6%) were the most frequent complications, and the need for new intervention during the same hospital stay was observed in 40 patients (2.3%). The 1705 consecutive patients enrolled in the study were followed for a mean of 665.6 173.8 days (1.0730.5 days). At 2-year follow - up (completed in 94.5% of subjects), 267 patients died, giving an overall annual mortality rate of 83.8% (95% ci 73.494.2) per 1000 person - years, with a higher rate among crt - p, compared with crt - d, patients [130.8 vs. 65.1 per 1000 year, respectively, relative risk (rr) 2.01, 95% ci 1.562.58, p <0.0001; figure 1a]. The incidence of scd was not statistically higher in the crt - p group compared with crt - d (rr 1.57, 95% ci 0.713.46, p = 0.42) (figure 1b). The rate of hospitalization for hf was not different between the crt - d vs. crt - p groups (19.6 vs. 22.0%, p = 0.28). Figure 1overall mortality incidence over time according to crt - p and crt - d groups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Overall mortality incidence over time according to crt - p and crt - d groups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . After considering potential confounding factors in a cox proportional hazards regression analysis, crt - p remained associated with increased mortality [hazard ratio (hr) 1.54, 95% ci 1.072.21, p = 0.0209], as well as the presence of co - morbidities (hr 1.98, 95% ci 1.342.92, p = 0.0006) and functional nyha class iv (hr 1.85, 95% ci 1.103.11, p = 0.0207). Using the propensity - matched cohort, crt - p was associated with increased mortality (rr 2.0, 95% ci 1.223.28, p = 0.01). Cardiac resynchronization therapy without defibrillator was not associated with a higher incidence of scd (rr 1.21, 95% ci 0.453.29, p = 0.70). Forest plots showing hazard ratios of crt - p vs. crt - d for mortality by different subgroups were represented in figure 2 . Figure 2forest plots showing unadjusted hazard ratios of crt - p vs. crt - d for mortality by different subgroups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Forest plots showing unadjusted hazard ratios of crt - p vs. crt - d for mortality by different subgroups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . However, when considering the specific cause - of - death analysis (table 3), the increase in mortality among crt - p patients was not related to that in scd, though scd incidence was higher in the crt - p group: 11.8 per 1000 among crt - p vs. 7.5 per 1000 among crt - d recipients (p = 0.26). The main reasons for the almost twice - higher risk of death in the crt - p group were an increase in non - scd cardiovascular mortality, mainly comprising progressive hf (rr 2.27, 95% ci 1.623.18) as well as other cardiovascular mortality (rr 4.40, 95% ci 1.2915.03). Overall, 95% of the excess mortality among crt - p recipients was not related to scd . Table 3incidence of specific causes of death among crt - p and crt - d recipientsincidences (per 1000 patient - years)crt - p (n = 535)crt - d (n = 1170)unadjusted risk ratio (95% ci)total mortality130.865.12.01 (1.562.58)cardiovascular heart failure75.433.32.27 (1.623.18) sudden death11.87.51.57 (0.713.46) others8.31.94.40 (1.2915.03)device - related1.22.80.42 (0.053.48)non - cardiovascular31.819.71.62 (1.002.62)crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Incidence of specific causes of death among crt - p and crt - d recipients crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Overall, a total of 1705 consecutive patients were enrolled in the study and received crt devices . The mean age of the overall population was 68.8 11.1 years, 33% were> 75 years of age, and 77% were men . The heart disease was ischaemic in 47% of patients and related to non - ischaemic dilated cardiomyopathy in 53% . Overall, 29% of patients presented with a lvef of 20% at the time of implantation . Overall, 13% of the crt - d group was implanted in the secondary prevention, following symptomatic ventricular tachycardia or sudden cardiac arrest . Patients with crt - p compared with crt - d were older (75.9 vs. 65.6 years, p <0.0001), less often male (69.5 vs. 80.8%, p <0.0001), more symptomatic (proportion of nyha class iii / iv, 87.9 vs. 80.8%, p = 0.0005), with less coronary artery disease (40.7 vs. 49.3%, p = 0.003), wider qrs (160.8 vs. 154.9 ms, p = 0.002), more af (38.7 vs. 22.1%, p <0.0001), and more co - morbidities (2 comorbidities, 16.9 vs. 12.9%, p = 0.04; table 1). Independent variables associated with crt - p (vs. crt - d) implantation are depicted in table 2 . Table 2independent variables associated with crt - p (vs. crt - d) implantationvariablesor95% cip - valueage1.171.141.19<0.0001female1.781.242.550.0018dilated non - ischaemic cardiomyopathy1.751.282.400.0005atrial fibrillation1.581.142.200.0062lvef <25%1.051.021.070.0001nyha iv2.821.614.920.0003no beta - adrenergic blockers2.401.763.26<0.0001lvef, left ventricular ejection fraction; nyha, new york heart association; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Independent variables associated with crt - p (vs. crt - d) implantation lvef, left ventricular ejection fraction; nyha, new york heart association; crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Fatal periprocedural complications occurred in one patient, and death occurred before hospital discharge in five others (0.3%), due to severe cardiogenic shock . Overall, significant perioperative complications occurred in 133 subjects (7.8%) without significant difference between crt - d and crt - p (8.9 vs. 6.7%, p = 0.20). Pulse generator pocket haematoma (2.5%), lead dislodgment (1.6%), and phrenic nerve stimulation (1.6%) were the most frequent complications, and the need for new intervention during the same hospital stay was observed in 40 patients (2.3%). The 1705 consecutive patients enrolled in the study were followed for a mean of 665.6 173.8 days (1.0730.5 days). At 2-year follow - up (completed in 94.5% of subjects), 267 patients died, giving an overall annual mortality rate of 83.8% (95% ci 73.494.2) per 1000 person - years, with a higher rate among crt - p, compared with crt - d, patients [130.8 vs. 65.1 per 1000 year, respectively, relative risk (rr) 2.01, 95% ci 1.562.58, p <0.0001; figure 1a]. The incidence of scd was not statistically higher in the crt - p group compared with crt - d (rr 1.57, 95% ci 0.713.46, p = 0.42) (figure 1b). The rate of hospitalization for hf was not different between the crt - d vs. crt - p groups (19.6 vs. 22.0%, p = 0.28). Figure 1overall mortality incidence over time according to crt - p and crt - d groups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Overall mortality incidence over time according to crt - p and crt - d groups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . After considering potential confounding factors in a cox proportional hazards regression analysis, crt - p remained associated with increased mortality [hazard ratio (hr) 1.54, 95% ci 1.072.21, p = 0.0209], as well as the presence of co - morbidities (hr 1.98, 95% ci 1.342.92, p = 0.0006) and functional nyha class iv (hr 1.85, 95% ci 1.103.11, p = 0.0207). Using the propensity - matched cohort, crt - p was associated with increased mortality (rr 2.0, 95% ci 1.223.28, p = 0.01). Cardiac resynchronization therapy without defibrillator was not associated with a higher incidence of scd (rr 1.21, 95% ci 0.453.29, p = 0.70). Forest plots showing hazard ratios of crt - p vs. crt - d for mortality by different subgroups were represented in figure 2 . Figure 2forest plots showing unadjusted hazard ratios of crt - p vs. crt - d for mortality by different subgroups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Forest plots showing unadjusted hazard ratios of crt - p vs. crt - d for mortality by different subgroups . Crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . However, when considering the specific cause - of - death analysis (table 3), the increase in mortality among crt - p patients was not related to that in scd, though scd incidence was higher in the crt - p group: 11.8 per 1000 among crt - p vs. 7.5 per 1000 among crt - d recipients (p = 0.26). The main reasons for the almost twice - higher risk of death in the crt - p group were an increase in non - scd cardiovascular mortality, mainly comprising progressive hf (rr 2.27, 95% ci 1.623.18) as well as other cardiovascular mortality (rr 4.40, 95% ci 1.2915.03). Overall, 95% of the excess mortality among crt - p recipients was not related to scd . Table 3incidence of specific causes of death among crt - p and crt - d recipientsincidences (per 1000 patient - years)crt - p (n = 535)crt - d (n = 1170)unadjusted risk ratio (95% ci)total mortality130.865.12.01 (1.562.58)cardiovascular heart failure75.433.32.27 (1.623.18) sudden death11.87.51.57 (0.713.46) others8.31.94.40 (1.2915.03)device - related1.22.80.42 (0.053.48)non - cardiovascular31.819.71.62 (1.002.62)crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . Incidence of specific causes of death among crt - p and crt - d recipients crt - d: cardiac resynchronization therapy with defibrillator; crt - p: cardiac resynchronization therapy without defibrillator . To the best of our knowledge, our study provides the first cause - of - death analysis comparing crt - p with crt - d patients in a real - world population . We demonstrate that crt - p patients, as chosen in routine clinical practice, were older, more likely to be female, with less ischaemic heart disease, more advanced hf, and greater co - morbidity burden compared with crt - d patients . These characteristics of the crt - p patients are in agreement with previous reports . At 2 years, the overall mortality in the crt - p group was greater than that in the crt - d group . However, importantly, this difference in mortality was mostly accounted for by an increase in non - scd . Since scd did not significantly contribute to the excess mortality in the crt - p group, it suggests that the presence of a back - up defibrillator would probably not have been beneficial in terms of improving survival for these patients . The rates of hf hospitalization were greater in the crt - p group, which is in line with the greater hf mortality in this group . This is likely related to a sicker population with more co - morbidity, older age, and potentially more severe hf and serves to highlight that progressive hf rather than scd may be the main driver of morbidity as well as mortality in the crt - p population . These results from a large, prospective cohort with robust cause - of - death adjudication need careful consideration in the context of the current controversy in the selection of crt - p vs. crt - d . Our study is not intended as a direct comparison of outcomes between crt - d and crt - p, and subgroup analyses should be interpreted with caution . While direct comparisons in observational studies may reveal differences in death rates, knowledge of what makes up this difference takes our understanding an important step further . Cause - of - death analysis, while being technically challenging to perform in a large population, represents an innovative, alternate approach to this problem . It also helps bring to the forefront the issue of competing risks for mortality in any population of this nature . With the exception of patients with af, where the evidence - base is admittedly weaker, crt represents an important therapeutic option for a growing segment of the hf population . Though the guidelines presently do not make definitive recommendations for crt - d vs. crt - p, in practice, many physicians may feel compelled to use crt - d, as a defibrillator is considered the effect of this choice may be even greater in light of the fact that many centres are exploring broader indications for crt in patients with milder symptoms and narrow qrs . This has important economic implications in that the incremental cost of crt - d over crt - p is significantly greater when compared with the cost over optimal medical therapy and this difference is even steeper in the older age group . The addition of a defibrillator lead can also contribute to additional adverse events and need for repeat procedures . Thus, there is a fairly urgent need for more data such as from the present study to tease out the putative benefits of an added defibrillator over crt - p, and to better define optimal criteria to select crt - p or crt - d . Since the crt - p group had a greater proportion of non - ischaemic cardiomyopathy where the benefit from primary preventive icd is lower, this could influence results as well . In the absence of proven superiority by trials and the small survival benefit, the 2013 european society of cardiology task force was of the opinion that no strict recommendations can be made, and has preferred to merely offer guidance regarding the selection of patients for crt - d or crt - p, based on overall clinical condition, device - related complications, and cost; factors favouring crt - p being advanced hf, co - morbidities, including frailty and cachexia . In contrast, factors favouring crt - d implantation are life expectancy> 1 year, stable hf, moderate functional status, ischaemic heart disease, and lack of comorbidities being in favour of crt - d implantation, and the french practice appears to be in agreement with this . The only randomized trial to have crt - p as well as crt - d arms the companion trial did not show a significant benefit of crt - d over crt - p for the primary endpoint . Non - randomized studies, which have compared outcomes for these two modalities, have yielded conflicting results . Using registry - based data, morani et al . Showed that among patients with an european society of cardiology class ia indication for crt, crt - d was associated with better survival than crt - p . Similar findings were reported from a us - based registry, which concluded that crt - d should be recommended to most congestive hf patients with indications for biventricular pacing. However, recent experience with reasonable numbers of patients emphasized the higher risk of mortality among crt - p patients compared with crt - d, indicating that long - term benefit of an additional defibrillator may be restricted to a selected subgroup . Furthermore, logistic regression models which are relied on in comparative studies to draw conclusions, may not adequately overcome the limitations in comparing heterogeneous groups . A bayesian network meta - analysis in 2007 concluded that evidence from randomized trials is insufficient to prove the superiority of crt - d over crt - p . Whether crt - p by itself reduces risk of arrhythmia is still a matter of some debate . Long - term data from care - hf show reduction in scd rates by crt . A mechanistic link is supported by the fact that scd is reduced in subjects with systolic hf and ventricular dyssynchrony . Recent analysis from the madit - crt trial showed that risk of ventricular arrhythmias was significantly reduced in crt patients with normalization of lvef . Importantly, risk of inappropriate icd therapy was unchanged, suggesting that these patients may be better served by a downgrade to crt - p at device change . Similarly, another study showed that based on lvef improvement, up to one - third of crt - d patients no longer had an ongoing indication for icd at the time of battery change and the rate of device therapy in this group was very low . By inducing favourable remodelling of the lv, crt may reduce the substrate for ventricular arrhythmias . Some patients experience rapid reverse remodelling (super responders), with major improvement in ef so that they are no longer icd candidates . Data suggest that such patients have excellent long - term prognosis . In anticipation of rapid improvement of lvef, it would seem logical to provide temporary protection against scd such as using a life vest rather than implanting a defibrillator . However, although predictors of super response have been proposed, it is still difficult to identify such super responders with a high degree of confidence; thus, more work may be needed in this regard . The rationale for an anti - arrhythmic effect of crt is also tempered to some extent by concerns over the pro - arrhythmic effects of lv pacing . Prospective follow - up of patients with crt - p has shown that the incidence was overall relatively low, and that sudden cardiac death events were likely to be preceded by recorded sustained ventricular arrhythmias, emphasizing the importance of regular crt - p device memory interrogation, as well as the potential benefit of remote monitoring in these patients, for possible urgent upgrading to crt - d . The possibility of accurate and continuous surveillance to detect life - threatening arrhythmias, with upgrading of crt - p patients to crt - d only after such objective documentation during follow - up, might represent a safe and cost - effective alternative to the practice of universal crt - d implantation in all crt candidates . Our results should not be interpreted as a general lack of benefit from crt - d vs. crt - p or vice versa . Rather, we demonstrate that given currently selected crt - p patients in the french population, addition of a defibrillator may not significantly add to survival . At least in a subset of the crt eligible hf population, competing risks of non - sudden death may diminish the incremental value of adding a defibrillator to crt; therefore, all patients eligible for crt cannot be automatically considered as requiring a crt - d . Thus, in a broader context, the requirement for crt - d in similar populations needs careful consideration of the putative risks and benefits . Relative strengths of the present study include the fact that it is prospective, multicentric with dedicated cause - of - death adjudication . First, the study was non - randomized and therefore, selection bias may have influenced results . The clinical decision concerning device type may affect subsequent management as well lead to variations in clinical care . On the other hand, randomized trials, while being a rigorous design, have rigid selection criteria, which often do not reflect real - world scenarios . Though the results need to be interpreted with caution in view of potential confounding, it reflects actual clinical practice . Secondly, follow - up was censured at 2 years, which can influence results as device utilization is a function of time; however, there were adequate events during follow - up to draw reasonable conclusions . Thirdly, information on qrs morphology was unavailable and the extent of lbbb in the two groups may have influenced crt outcomes . Finally, although our study suggests that mortality in this real - world crt - p population may not be improved by upgrade to crt - d, it does not address the question of whether, in the population implanted with a crt - d device, crt - p would perform just as well . Thus, this study was not intended to answer the question of whether crt - p is comparable with crt - d overall, but rather provides a real - world assessment of cause of death in a contemporary crt - p vs. crt - d population, which we believe can more meaningfully inform clinical practice . It should be borne in mind that these outcomes mainly pertain to a hf population with broad qrs . In this prospective, multicentre cohort study, crt - p patients were older, with more advanced hf, and co - morbidities when compared with crt - d recipients . At 2-year follow - up, crt - p patients had 2-fold higher mortality than crt - d . By cause - of - death analysis, the excess mortality among crt - p subjects was almost entirely related to non - scd . Our results indicate that crt - p patients, as currently selected in routine clinical practice, would potentially not benefit from addition of a defibrillator, emphasizing that there is still considerable room for crt - p in the present day hf treatment . : performed statistical analysis; j .- y.l.h . : handled funding and supervision; e.m ., g.m ., j .- c.d ., and j .- y.l.h . : conceived and designed the research; c.l ., p.c ., and p.m.: made critical revision of the manuscript for key intellectual content . Certitude was funded by grants from the french institute of health and medical research (inserm) and from the french society of cardiology . A specific research grant support was funded specifically for the certitude cohort study from biotronik, boston scientific, medtronic, sorin and st . Funding to pay the open access publication charges for this article was provided by the french society of cardiology . D.g . Is a consultant for medtronic, boston scientific, saint jude medical, and biotronik.
Studies of wld, an aberrant fusion protein with critical nicotinamide mononucleotide adenylyltransferase (nmnat) activity responsible for the slow wallerian degeneration phenotype, indicate this often shares molecular features with injury - induced axon degeneration (conforti et al ., 2014). New, endogenous regulators of wallerian degeneration have emerged, so establishing interactions among them will be important for developing appropriate therapies (conforti et al ., 2014; neukomm and freeman, 2014; pease and segal, 2014). While a conserved pathway is emerging, the relationship between nmnat2 and sterile alpha and tir motif containing 1 (sarm1), key regulators of axon degeneration with opposing influences, is still unknown (conforti et al ., 2014). Each robustly delays wallerian degeneration when re - localized or sufficiently overexpressed (mack et al ., 2001; milde et al ., 2013 2009), but only depletion of nmnat2, not nmnat1 or nmnat3, triggers wld - sensitive neurite degeneration in neuronal cultures (gilley and coleman, 2010). This suggests nmnat2 is the isoform that contributes most to axon survival in a physiological context . Drosophila nmnat ortholog, dnmnat, appears to have a similar role in flies (fang et al ., 2012). Early loss of short - lived nmnat2 in injured axons, prior to frank degeneration, has thus been proposed as a trigger for wld - sensitive axon degeneration and the point of wld influence (conforti et al ., 2014; gilley and coleman, 2010). This is supported by studies that show that a critical requirement for phr1/highwire ubiquitin ligase during wallerian degeneration may be linked to its regulation of nmnat2/dnmnat turnover (babetto et al ., 2013; xiong et al ., 2012) and the proposal that accumulation of the nmnat substrate nmn, a predicted consequence of nmnat2 loss, acts as a pro - degenerative signal (di stefano et al ., 2014). Severe truncation of peripheral nerve axons and cns axon tracts in nmnat2-deficient embryos, likely responsible for their perinatal lethality, is also consistent with a critical axon survival function for nmnat2 in vivo (gilley et al ., 2013; hicks et al ., 2012) importantly, though, this developmental phenotype appears to be primarily due to an initial outgrowth defect rather than dying - back degeneration of established axons (gilley et al ., 2013). It is required for the normal, rapid progression of degeneration after injury and other wld - sensitive insults (gerdts et al ., 2013; osterloh et al ., 2012). Multimerization, mediated by its sam (sterile motif) domain, appears to be required for a pro - degenerative function dependent on its tir (toll - interleukin-1 receptor) domain (gerdts et al ., 2013). Crucially, sarm1 depletion is currently the only known manipulation that delays wld - sensitive axon degeneration as robustly as expression of wld or other nmnats . In this study, we used axon injury in nerves and neurons from sarm1 mice to establish the relationship between nmnat2 loss and a sarm1-dependent function during wld - sensitive degeneration . Remarkably, we also find that mice lacking both nmnat2 and sarm1 are viable, enabling us to examine the mechanistic origins of the developmental axon outgrowth defect associated with constitutive nmnat2 deficiency . Nmnat2 levels have previously been found to decline rapidly in the distal stumps of transected wild - type scg and drg neurites prior to their degeneration at 8 hr (gilley and coleman, 2010; yang et al ., 2013). Here, we find equivalent loss of nmnat2 in wild - type and sarm1 scg neurites by 4 hr after cut (figure 1a), even though degeneration of transected sarm1 neurites is delayed for at least 3 days (osterloh et al ., 2012). Levels of the nmnat substrate, nmn, and product, nad, have also been shown to respectively rise and fall in the distal stumps of transected wild - type sciatic nerves (di stefano et al ., 2014) prior to axon degeneration, which occurs at around 36 hr (beirowski et al ., 2005). This is consistent with a predicted loss of nmnat2 in injured axons in vivo . Here, we find that these changes are not prevented in sarm1 nerves at 30 hr after lesion (figure 1b), despite sarm1 axons being preserved for at least 14 days (osterloh et al ., 2012). In fact, by 30 hr, nmn has risen significantly more than in wild - type nerves, and by 120 hr, nad levels have dropped below those in wild - type nerves at 30 hr (a short time before they degenerate). Together, these data suggest sarm1 promotes wallerian degeneration downstream or independently of nmnat2 loss and consequent changes in nmn and nad levels . Although nmnat2 loss has been strongly implicated as a trigger for wallerian degeneration, this is difficult to prove definitively, as injury induces many simultaneous changes . We therefore assessed whether sarm1 is required during related, wld - sensitive axon degeneration induced specifically by small interfering rna (sirna)-mediated depletion of nmnat2 (gilley and coleman, 2010). We found distal neurites of sarm1 neurons are completely protected from nmnat2 sirna - induced degeneration for at least 72 hr, and also from the later, slower loss of cell viability (figures 1c and 1d). Nmnat2 depletion can thus trigger sarm1-dependent axon degeneration in the absence of injury or other initiating stimuli supporting a situation in which either sarm1 itself, or a sarm1-dependent activity of a convergent pathway, promotes wld - sensitive axon degeneration downstream of nmnat2 loss . We next bred mice doubly homozygous for the nmnat2 gene trap allele (effectively a null allele; gilley et al ., 2013) and a sarm1 knockout allele (kim et al ., 2007) to investigate whether sarm1 might also contribute to axon defects associated with constitutive nmnat2 deficiency during development . Intriguingly, whole - mount labeling of distal phrenic nerve branches in the diaphragm and of intercostal nerves in nmnat2;sarm1 embryos / newborn pups revealed clear and reproducible rescue of the peripheral nerve axon truncation defect associated with nmnat2 deficiency in nmnat2 animals (figures 2a and 2b). The underlying cause of this defect in nmnat2 embryos is restricted axon extension (gilley et al ., 2013); therefore, to obtain a quantitative measure of rescue by sarm1 deficiency, we compared neurite outgrowth in scg explant cultures from nmnat2 and nmnat2;sarm1 embryos with that of relevant controls (figures 2c and 2d). Strikingly, nmnat2;sarm1 neurite outgrowth matched that in sarm1 and nmnat2;sarm1 littermate cultures, as well as in wild - type and nmnat2 cultures assessed in parallel . The axon extension defect associated with a lack of nmnat2 thus appears fully corrected in late - stage embryos and newborn mice deficient for sarm1 . Consistent with this, while nmnat2 (and nmnat2;sarm1) pups die at birth, nmnat2;sarm1 mice in contrast are viable and reach weaning at the expected mendelian ratios (figure 2e). Adult nmnat2;sarm1 mice are also fertile and, despite subtle gender- and sarm1-genotype - specific differences, their weights at 10 weeks are within a healthy range and similar to control groups (figure 2f). Crucially, rt - pcr and immunoblotting confirmed that the nmnat2 allele remains effectively silenced on a sarm1 background (figures 2 g and 2h). This rules out reduced efficacy of the gene trap as being responsible for rescue of the phenotype . As in injured nerves, sarm1 deficiency does not prevent expected changes to nmnat metabolites in the brains of embryos lacking nmnat2 (figure 3a). In fact, the relative increase in nmn in nmnat2;sarm1 brains is greater than in nmnat2 brains, presumably due to rescued outgrowth of cns axons, where changes, away from the influence of nuclear nmnat1, should be greatest . A small, but significant, reduction in nad is also evident in nmnat2;sarm1 brains . In contrast, heterozygosity for the nmnat2 allele does not significantly alter brain nmn or nad levels on either sarm1 background . Interestingly, there is a trend toward higher nmn and nad levels in sarm1 brains, independent of nmnat2-related changes, and the total adenylate pool (atp + adp + amp) is increased (1,764 113 nmol / g tissue in sarm1 brains compared to 1,314 97 in sarm1 brains; p = 0.009). Thus, sarm1 itself may have some influence over nad and/or energy metabolism . In case the complexity of whole brain concealed more marked changes specifically in axons, we also assessed nucleotide levels separately in drg neurites and ganglia (figures 3b and s1). Indeed, consistent with nmnat2 being the major axonal nmnat, we detected a greater relative increase in nmn and decrease in nad in nmnat2;sarm1 neurites than in brains . In contrast, changes in ganglia fractions were far more modest, indicating nmnat2 has less influence in the cell body (and associated proximal neurite stumps). Surprisingly, accumulation of nmn was not evident in the limited neurite outgrowth in nmnat2 cultures . However, the nmn / nad ratio is increased to the same extent as in nmnat2;sarm1 neurites, due to a much greater reduction in nad, consistent with comparable loss of nmnat activity . We speculate that, even though stunted nmnat2 neurites do not show signs of overt degeneration, their integrity could still be compromised, resulting in nmn and nad efflux and the lower than expected levels of both nucleotides . Low nad in nmnat2 ganglia fractions suggests this may also extend to proximal neurite stumps and possibly even cell bodies . Finally, as in brains, heterozygosity for the nmnat2 allele does not significantly alter nmn and nad levels in either drg fraction . Overall, these results indicate that sarm1 is critically involved in the manifestation of the phenotype of nmnat2 mice, with it, or a dependent activity, acting downstream of predominantly axonal alterations to nmn and nad resulting from the lack of nmnat2 . Crucially, this suggests significant mechanistic overlap between wld - sensitive axon degeneration and the axon outgrowth defect in these mice . We have previously implicated rising nmn, rather than declining nad, as a critical pro - degenerative event in injured axons, in part by showing that blocking nmn synthesis with nampt inhibitor fk866 and scavenging nmn via ectopic expression of bacterial nmn deamidase (converting it to namn) both delay wallerian degeneration (di stefano et al ., 2014). We therefore used these reagents to investigate whether nmn also limits outgrowth and survival of axons lacking nmnat2 . First, we tested the prediction that survival of nmnat2;sarm1 scg neurites should be impaired by ectopic re - expression of sarm1 due to re - activation of a sarm1-dependent degeneration pathway downstream of nmnat2 deficiency, and assessed whether these events are nmn - dependent using co - expression of nmn deamidase (figures 4a and 4b). While ectopic re - expression of sarm1 (c - terminal gfp tagged) in sarm1 neurons (with normal nmnat2) unexpectedly induced some neurite degeneration by 24 hr, it induced much more extensive degeneration of nmnat2;sarm1 neurites (lacking nmnat2) and, crucially, this was significantly reduced by co - expression of nmn deamidase (n - terminal gfp tagged). This suggests that, as well as promoting wallerian degeneration, nmn also drives sarm1-dependent degeneration in this context . Importantly, these experiments additionally confirm that rescue of nmnat2;sarm1 axons is due to sarm1 deficiency and not other, undefined changes resulting from mouse breeding . Next, we used fk866 to assess whether nmn accumulation also limits nmnat2 neurite outgrowth (figures 4c and 4d). Interestingly, fk866 stimulated additional outgrowth of nmnat2 drg neurites, but only for about 24 hr, after which neurites underwent complete degeneration . Because fk866 causes simultaneous reductions to both nmn and nad in neuronal cultures (di stefano et al ., 2014), we considered that this later degeneration could be due to an extreme reduction in nad in neurites now lacking both nampt and nmnat2 activities and/or secondary to neuronal death caused by reduced somal nad . To overcome this, we co - treated cultures with potential sources of nad . Nad itself was ineffective (figure s2), likely due to inefficient entry into neurons and/or failed conversion of more readily internalized nad precursors (generated by extracellular degradation) back into nad when nmnat2 is absent (di stefano et al ., 2014; nikiforov et al ., 2011), possibly even leading to further nmn accumulation in nmnat2 neurites . However, naad, an alternative source of nad via endogenous nad synthetase (di stefano et al ., 2014), did promote additional outgrowth and survival of nmnat2 neurites when added with fk866 (but, importantly, not when added alone). Together, these data suggest increased nmn does contribute to the restricted outgrowth of nmnat2 neurites, although rescue of nmnat2 neurite outgrowth by fk866 and naad still falls some way short of the outgrowth of wild - type neurites treated in the same way (despite this also being slightly inhibitory) (figure 4c). Therefore, either inhibition of nampt by fk866 or clearance of pre - accumulated nmn is relatively ineffective and/or outgrowth is not exclusively limited by nmn - dependent events . Despite their shared ability to profoundly influence axon survival after injury, no link between nad metabolism and sarm1 function had previously been firmly established . Our studies show not only that either sarm1 itself, or a sarm1-dependent convergent activity, acts downstream of nmnat2 loss and resulting changes to key nmnat metabolites to promote wld - sensitive axon degeneration but also that this also occurs downstream of nmnat2 deficiency and dependent changes during development to impair axon outgrowth and limit mouse survival . Intriguingly, an nmnat2 depletion - dependent accumulation of nmnat substrate, nmn, also appears to be a critical pro - degenerative signal in both situations (this study; di stefano et al ., 2014), strongly suggesting they are each mediated by activation of the same nmn- and sarm1-dependent degeneration pathway . Importantly, our data appear to rule out any lasting stabilization of nad or reduced accumulation of nmn as part of the mechanism underlying protection of sarm1-deficient axons, although intriguing increases in total adenylate pool and steady - state levels of nmn and nad in sarm1 brains may yet be found to be involved . Overall, our studies suggest either that changes resulting from nmnat2 loss directly activate a pro - degenerative sarm1 function in axons in a linear pathway or that parallel sarm1-dependent and nmnat2 loss - dependent signals merge at a common downstream step in a convergent pathway (figure s3). Importantly, the critical sarm1-dependent event in either model likely acts upstream of calpain activation (yang et al ., 2013). Given that nmnat2 depletion alone can activate sarm1-dependent neurite degeneration, the convergent pathway model would appear to require that sarm1 has constitutive pro - degenerative activity, with activation or disinhibition of a critical downstream step only occurring after nmnat2 loss . While a proposed regulatory role for the n - terminal domain of sarm1 (gerdts et al ., 2013) appears more consistent with activation of a pro - degenerative function in a linear pathway, our finding that exogenous expression of sarm1(-gfp) induces some degeneration even when nmnat2 is present (in sarm1 neurons) suggests it could have some constitutive pro - degenerative activity (although conditions in these assays could also influence this outcome). Understanding precisely how endogenous sarm1 functions in the context of nmnat2 loss and nmn accumulation during axon degeneration, and how this relates to other cellular roles of sarm1 (conforti et al ., we had previously proposed that the early, limited extension of axons lacking nmnat2 reflects restricted supply of an nmnat - activity - dependent molecule required for axon growth, with nad being a prime candidate (gilley et al ., 2013). However, initially improved outgrowth of nmnat2 neurites further deprived of nad by fk866 challenges this idea, and the involvement of sarm1 instead suggests these neurites stop extending at the point where sarm1-dependent degeneration becomes active . In light of our findings we predict that, due to a diminishing influence of nuclear nmnat1, nmn in nmnat2-deficient axons will rise as a function of distance from the soma, with some axon outgrowth possible up to the point at which a critical threshold is exceeded and sarm1-dependent degeneration is triggered . Nad plays a key role in many diverse cellular functions (di stefano and conforti, 2013), so the fact that significant reductions in nad do not prevent the delayed degeneration of cut sarm1-deficient axons or limit the normal growth and survival of intact axons lacking both nmnat2 and sarm1 (in culture and in mice) was somewhat surprising . This, together with the ability of fk866 to delay degeneration of cut axons (di stefano et al ., 2014) and temporarily rescue nmnat2 neurite outgrowth, despite further depletion of nad, clearly indicates that fairly substantial reductions in nad are not necessarily detrimental . However, it would be surprising if greater nad depletion did not limit axon survival in some situations . On its own, survival of nmnat2;sarm1 mice for several months with no overt problems suggests very strong correction of the defects associated with constitutive nmnat2 deficiency during early development . However, it will still be critical to establish the precise extent and duration of this rescue in an aging study . Nevertheless, these mice already represent a unique resource for the identification or confirmation of other early pro - degenerative events triggered by nmnat2 loss and nmn accumulation, given that our data suggest an activated degeneration pathway is paused at a sarm1-dependent step in nmnat2;sarm1 axons . For example, sarm1 appears to act downstream of reactive oxygen species accumulation, atp depletion, and ca influx when cell death and axon degeneration are induced by the mitochondrial toxin carbonyl cyanide m - chlorophenyl hydrazone (cccp) (summers et al ., 2014), but it will be important to establish whether these changes also occur downstream of nmnat2 loss during wld - sensitive degeneration . They should also be particularly useful for studying non - axonal phenotypes associated with nmnat2 deficiency that may be sarm1 independent, such as its role in cardiac hypertrophy (cai et al ., 2012). To conclude, these studies have now firmly established the order of two core events in an emerging wld - sensitive and sarm1-dependent axon degeneration pathway . As additional steps are added, it will become increasingly apparent how this relates to other cell - death programs and how it might be most effectively targeted in axonal disorders . Crucially, the lack of any overt defects in adult nmnat2;sarm1 mice suggest that if effective targeting of this pathway can be achieved, it could potentially provide long - term relief from symptoms without major side effects . Animal work was carried out in accordance with the animals (scientific procedures) act, 1986, under project license ppl 70/7620 . Explant cultures of superior cervical ganglia (scgs) from embryonic day 18.5 (e18.5) mouse embryos and postnatal day 0 (p0) to p2 pups and dorsal root ganglia (drgs) from e13.5/14.5 and e17.5/e18.5 embryos and p0 pups, and dissociated scg neuron cultures (p0p2), were set up as described previously (gilley et al . Neurite outgrowth assays were performed and quantified as described previously (gilley et al ., 2013). Nampt inhibitor fk866 (100 nm, generously supplied by the national institute of mental health), naad (250 m, sigma - aldrich), nad (1 mm, sigma - aldrich), or nmn (1 mm, sigma - aldrich) were added to e17.5/e18.5 nmnat2 drg explant cultures at 2 days in vitro as described . Whole brains from e18.5 mouse embryos and neurite fractions from scg explant cultures were processed as described previously for the generation of rna and/or protein extracts (gilley et al ., 2013; gilley and coleman, 2010). Primers for rt - pcr, and primary antibodies and concentrations for immunoblotting, are listed in the supplemental experimental procedures . Nmn and nad measurements in whole brains from e18.5 embryos or newborn (p0) pups, uncut or lesioned sciatic nerves from adult mice, and neurite and ganglia fractions from drg cultures (prepared as for scg cultures in gilley and coleman, 2010) were performed using uv - c18 high - performance liquid chromatography (hplc) and/or spectrofluorometric hplc analysis essentially as described previously (di stefano et al ., 2014). Injections and quantification of neurite / cell - body survival were performed essentially as described previously (gilley and coleman, 2010). Plasmids and sirnas injected are listed in the figure legends and in the supplemental experimental procedures . Anterograde labeling of intercostal nerve axons with the lipophilic dye dii (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) in fixed p0 mouse pups and whole - mount neurofilament light - chain immunostaining of fixed diaphragms from e17.5/e18.5 embryos were performed as described previously (gilley et al ., 2013). Phase - contrast and fluorescence images of primary cultures and stained / labeled tissues were acquired as described previously (gilley et al ., 2013). The student s t test, anova with tukey s or dunnett s post hoc correction (as applicable), and pearson s chi - square test were used in this study . A p value> 0.05 was considered not significant, and indicates p <0.05, indicates p <0.01, and indicates p <0.001 . Conceived the study . J.g . Designed and performed all experiments with the exception of nucleotide measurements, obtained by g.o ., and in vitro and in vivo neurite and nerve injury experiments, designed and performed by i.n .- f . J.g . And m.p.c.
The size and form of the dental arches vary among individuals according to tooth size, tooth position, pattern of craniofacial growth and by several genetic and environmental factors . Survey of arch size could help clinicians in the selection of stock trays, the size of artificial teeth, and the overall forms of artificial dental arch at the wax trial stage are amenable to modification by the dental surgeon and in orthodontic treatment . As orthodontics has advanced as a specialty and the number of adults seeking orthodontic care has increased, an understanding of the changes that normally take place in adult craniofacial structures becomes critical . A systematic and well - organized dental care program for any target population in a community requires some basic information, such as the prevalence of the condition . In the more developed parts of the world, where the specialty of orthodontics has been established, adequate baseline information is available . Despite efforts in the arab world during the last decades to make health systems more equitable access to dental healthcare is still far from adequate, especially in poor communities . In yemeni population hence, this study has been designed to provide a baseline data on the mandibular dimensions of yemeni adults with normal normal dento - skeletal relationship, aged (18 - 25) years . Ethical approval was obtained from both the ethics committee of sanaa university and the faculty of dentistry at the university of sanaa . A brief outline of the study was explained to all participants and consent was obtained prior to participation . The sample of this study consists of 214 adults aged between 18 and 25 years, among whom 113 were males and 101 females, selected from clinical examination of 765 yemeni adults, comprised of 387 males and 378 females . All subjects considered for the sample have the following dental and skeletal features full complement of normal shaped permanent teeth (excluding third molars) with no heavy fillings, congenital missing teeth, retained deciduous, and supernumerary teeth.class i molar and canine relationships and class i skeletal relationship, decided visually by using the two - finger technique.normal vertical and horizontal dental relationships (normal overjet and overbite).no previous orthodontic, orthopedic, facial surgical treatments and no history of bad oral habits such as thumb sucking or mouth breathing.well-aligned arches with less than 3 mm of spacing or crowding in either arch . Full complement of normal shaped permanent teeth (excluding third molars) with no heavy fillings, congenital missing teeth, retained deciduous, and supernumerary teeth . Class i molar and canine relationships and class i skeletal relationship, decided visually by using the two - finger technique . Normal vertical and horizontal dental relationships (normal overjet and overbite). No previous orthodontic, orthopedic, facial surgical treatments and no history of bad oral habits such as thumb sucking or mouth breathing . Well - aligned arches with less than 3 mm of spacing or crowding in either arch . All the individuals examined under natural light with interchangeable plane mouth mirrors . During this examination, each individual was seated on an ordinary chair with his head being positioned so that the frankfort horizontal plane is parallel to the floor . The selected individuals were subjected to a thorough clinical examination to reassure the fulfillment of the required sample specifications . Certain selected tooth - related points visible in an occlusal view were marked bilaterally with a sharp pencil in the mandibular study casts . Great care was taken to ensure that the landmarks were accurately located on the study casts . Measurements were taken from 214 mandibular dental casts, which were made of dental stone, with the base, made of plaster of paris . Dental arch dimension measurements were carried out using the modified sliding caliper gauge, which is accurate up to 0.02 mm . A - inter - canine distance: the linear distance from cusp tip of one canine to the cusp tip of the other . Dental model measurements, inter - canine width (a), inter - first molar width (b), inter - second molar width (c), anterior arch length (d), molar arch length (e), total arch length (f) b - inter - first molar distance: the distance from the mesiobuccal cusp tip of one first permanent molar, to the mesiobuccal cusp tip of the other . C - inter - second molar distance: the distance between the disto - buccal cusp tips of one second permanent molar, to the disto - buccal cusp tip of the other . D - anterior arch length: the vertical distance from the incisal point to the inter - canine distance at the cusp tip . E - molar arch length: the vertical distance from the incisal point perpendicular to a line joining the mesiolingual cusp tip of first permanent molars . F - total arch length: the vertical distance from the incisal point to the midpoint of a line joining the disto - buccal cusp tip of the second permanent molars . Descriptive statistics were performed for the calculation of the mean, standard deviation, minimum, maximum, range and coefficient of variation (cv). The t - test was applied to test the level of significance between the mean for males and females for all mandibular arch dimensions . The spearman's correlation coefficient (r) was also calculated between the measurement of arch widths and lengths in female and males . A - inter - canine distance: the linear distance from cusp tip of one canine to the cusp tip of the other . Dental model measurements, inter - canine width (a), inter - first molar width (b), inter - second molar width (c), anterior arch length (d), molar arch length (e), total arch length (f) b - inter - first molar distance: the distance from the mesiobuccal cusp tip of one first permanent molar, to the mesiobuccal cusp tip of the other . C - inter - second molar distance: the distance between the disto - buccal cusp tips of one second permanent molar, to the disto - buccal cusp tip of the other . D - anterior arch length: the vertical distance from the incisal point to the inter - canine distance at the cusp tip . E - molar arch length: the vertical distance from the incisal point perpendicular to a line joining the mesiolingual cusp tip of first permanent molars . F - total arch length: the vertical distance from the incisal point to the midpoint of a line joining the disto - buccal cusp tip of the second permanent molars . Descriptive statistics were performed for the calculation of the mean, standard deviation, minimum, maximum, range and coefficient of variation (cv). The t - test was applied to test the level of significance between the mean for males and females for all mandibular arch dimensions . The spearman's correlation coefficient (r) was also calculated between the measurement of arch widths and lengths in female and males . The means and standard deviations (mean sd) of the mandibular dimensions are shown in table 1 . The minimum and maximum values were recorded and expressed by the range . It can be noticed that the inter - second molar distances have the widest range while the lowest difference existing in the anterior arch length . Mean and standard deviation (sd) of mandibular arch widths (mm) and lengths for the sample table 2 depicts the mandibular arch dimensions according to gender . Overall, the male group displayed greater transverse and sagittal mandibular dimensions; however, this was only statistically significant for measurements of inter - first and second molar distances and anterior arch length (p <0.05). Mandibular arch dimensions (mm) according to gender the correlation coefficient was calculated between arch widths and lengths; some of them were highly significant, positive and direct relationship was noted, others showed moderate, weak or negative relationships [table 3]. Relationship between the mandibular widths and lengths shown with the corresponding spearman correlation coeficients (r) statistically significant correlations between the inter - canine distance and mandibular arch lengths were noted, but the r values were not greater than 0.5 . In the present study, the measurements taken for the dimensions of the mandibular arch, confirmed the accepted view that male dental arches are greater than that of females . In most studies, the arch dimensions depended on the gender of the subjects, with smaller values in females . This may be attributed to the smaller bony ridge and alveolar process of females, the average weakness of musculature in females that play an important role in facial breadth measurements, width and height of the dental arch and the later growth period in males than females . The findings of this study showed statistically significant difference between males and females for some measurements, this confirms those results previously published by many investigators . While it contradicted with other investigators . Kuntz reported that both sexes had nearly similar inter - canine distance and ismail et al . Reported that females had larger widths than males, but the difference was not statistically significant the largest difference between males and females existed in the inter - second molar distance, which might be attributed to the differences in arch form in the sample . In this study, it seems to indicate also that male group had greater mandibular dimensions in the sagittal direction than female group, this difference was statistically significant in the anterior arch length, which agree with the finding of raberin et al . And borgan . On the other hand, this difference was statistically insignificant in the molar arch length and total arch length, which does not agree with the finding of raberin et al . And borgan . The difference with the other studies may be attributed to different ethnic groups, sample sizes and environmental factors . The craniofacial and dental dimentions represent a highly complex interaction between numerous genetic and environmental factors . Overall, the correlation coefficient values were less than 0.5 indicating that the relationship is at best indicating a moderate linear relationship . Studying the descriptive analysis for the mandibular arch width revealed that the strongest relathinships were observed between the inter - canine distance and total arch length, molar arch length and anterior arch length . It could be also seen that the cv values for all measurements were nearly close to each other, with the anterior arch length distances showing the higher cv than the others . The anterior arch length values share the inter - canine distance in the contribution of determining all types of the dental arch forms . Thus, it is not unexpected for the anterior arch length to have the highest coefficient of variation among other dimensions . The craniofacial and dental dimentions represent a highly complex interaction between numerous genetic and environmental factors . Overall, the correlation coefficient values were less than 0.5 indicating that the relationship is at best indicating a moderate linear relationship . Studying the descriptive analysis for the mandibular arch width revealed that the strongest relathinships were observed between the inter - canine distance and total arch length, molar arch length and anterior arch length . It could be also seen that the cv values for all measurements were nearly close to each other, with the anterior arch length distances showing the higher cv than the others . The anterior arch length values share the inter - canine distance in the contribution of determining all types of the dental arch forms . Thus, it is not unexpected for the anterior arch length to have the highest coefficient of variation among other dimensions . Based on this study it could be said that among studied mandibular dimensions in subjects with normal dento - skeletal relationship, the inter - canine distance showed the strongest linear relationship with the mandibular arch sizes.
It is generally agreed that up to 30% of patients with igan eventually progress to end - stage kidney disease . The diagnosis of igan can be confirmed only by renal biopsy that shows mesangial cell proliferation and mesangial iga deposits . In the circulation, recently, the key feature has been considered to be the deficiency of galactose in the hinge region of the iga1 heavy chains and it is thought that these abnormal iga1 glycosylations reduce clearance of immune complexes which may then be sufficient to trigger glomerular mesangial deposition . With respect to complement deposition, c3 is highly detected in glomeruli and the detection rate is found in more than 90% of patients of igan [3, 4]. Since other complement molecules, such as properdin and c5, are colocated in the mesangial areas with iga, but c1q is absent and there is an increase of c3 breakdown products in the serum of patients with igan, we had recognized that alternative pathway (ap) activation is involved in the pathogenesis of igan . After discovering new molecules which belong to the lectin pathway (lp), we came to believe there is accumulating evidence to support a hypothesis of the occurrence of lp activation in the pathogenesis of igan . The issues surrounding these scenarios with lp activation are reviewed to summarize their potential significance in the pathogenesis of igan . In order to explain the updated knowledge in plain terms, the main body was separated into three parts, in circulation, in kidney, and infection and clinical course . We previously measured the serum concentrations of mbl in 20 healthy controls and in 80 patients with primary glomerular disease (figure 1). The serum concentrations of mbl from the patients presented a wider distribution than in healthy controls, but a comparison of the mean levels from each group showed no significant difference . Since mbl levels are altered by more than 3 - 4 times baseline levels during severe infection, production of mbl might be enhanced under some context, such as inflammation . Our colleague, onda, had reported that the patients with igan showed hypercomplementemia; the mean of ch50 titer and serum levels of c4, factor b and properdin in patients with igan were significantly higher than those in normal controls (table 1). Although hypercomplementemia is derived from chronic inflammation which accelerates the production of complement components in igan, it is interesting to note that serum concentrations of c3 and mbl were relatively lower than in normal controls . Also previously described no significant difference of mean levels of serum mbl between healthy controls and igan patients . This implies that consumption of c3 may increase by lp activation, thereby adding to ap activation . From the 1980s, researchers have explored the essential molecules in the pathogenesis of igan . . Deduced by an immunohistochemistry technique that the mesangial deposition of iga1 is critical for glomerular damage, and a subsequent study showed that the polymeric form of iga1 is predominantly eluted from renal tissues of igan patients [10, 11]. Conley et al . Also confirmed a more predominantly glomerular deposition of iga1 than that of iga2 . Recently, unique o - linked glycosylation sites at the hinge region of iga1 have received much attention . From the analysis of the circulating immune complex, the galactose deficiency (under - o - glycosylated iga) has been pointed out in the iga1 molecules of patients with igan [1315], although a high circulating overload alone does not induce the glomerular damage . As demonstrated in the current reviews, synthesis and binding of autoantibodies directed against galactose - deficient iga1 (galnac residues on the hinge region) are required for the formation of pathogenic iga1-containing immune complexes [2, 16]. Excesses of these relatively large complexes might lodge into the paramesangial area and might activate lp . Since under - o - glycosylated iga leads to polymerization of iga and purified polymeric iga from patients with igan can activate lp, an important question is whether aberrant iga can activate lp . Prominent ligands for mbl are mannose and n - acetyl - glucosamine (glcnac), and ligands for ficolins are glcnac . Because galactose and sialic acid are usually present in mammalian glycoproteins, they cannot be captured by mbl and ficolins . Although such an under - o - glycosylated iga1 from patients with igan could potentially interact with plant lectins, mbl and ficolins have no lectin activity for galnac residues . To the best of our knowledge, galactose - deficient iga1 itself is able to activate c3 directly but no direct clinical evidence from human studies is available which has demonstrated that mbl and ficolins can bind to aberrant iga1 in human studies . Secretory iga (siga) is the first line of defense in protecting the respiratory and intestinal tract, and the serum concentration of siga is very low (2 - 3 g / ml). Since the n - glycans on the heavy chains of both siga1 and siga2 present terminal glcnac and mannose residues clearly demonstrated glomerular staining of siga with mbl and c4d in the patients with igan . Further extended study is needed to clarify the pathological role of siga and complement activation . Functional mbl deficiency is probably the most common immunodeficiency in humans, involving 10 to 20% of the population . It is characterized by low levels of circulating functional multimers due to a number of genetic polymorphisms within the coding (codon 54 of exon 1 is most common in humans and it determines serum concentration and carbohydrate recognition ability) and promoter regions of the mbl2 gene . Our preliminary comparisons of the clinical backgrounds of mbl - sufficient patients of igan and mbl deficient patients of igan were determined by the analysis of polymorphisms of codon 54 (table 2). The means of urinary protein and glomerular filtration rate (gfr) in mbl deficient subjects were better than in mbl - sufficient subjects, but lacked statistical significance . Showed that polymorphism of codon 54 did not appear to have a primary involvement in the susceptibility and severity of igan in italian patients . They also reported a similar distribution of polymorphism frequency between healthy volunteers and igan patients . Nevertheless, the pirulli's data and ours could have been influenced by the low number of igan patients and this might be due to a racial difference . Of great interest is whether the difference can be found between mbl deficiency and mbl sufficiency at the onset and progression of igan . Endo et al . First demonstrated that glomerular deposition of mbl / masp-1 occurred in in the 25% of the cases of igan and that this was not observed in normal kidney and that the frequency was higher than that found in the other forms of glomerulonephritis . In a subsequent paper, lhotta et al . Also confirmed that 27% of patients with igan had mesangial deposits of mbl . Furthermore, the recent study by roos et al . Reported that the most mbl - positive patients presented mesangial deposits of l - ficolin and iga1 but not iga2 . These reports suggested that mbl binds to immune complexes which contain aberrant iga and/or igg . Showed that patients with mesangial deposits of iga1 and c3c showed no deposits of c4, mbl, and masp-1 . Moreover, patients with mesangial deposits of iga1, iga2, and c3c showed deposition of c4, mbl, and masp-1 . Furthermore they concluded that ap activation occurs as a result of mesangial deposition of iga1 and that lp activation is associated with mesangial deposition of iga2 . It is clear, however, that a determination of the initiator of lp activation in the mesangium has not yet been carried out to complete satisfaction . In igan, the protease - damaged surface of glomerular resident cells or apoptotic cells can be recognized by mbl . In regard to patients of lupus nephritis, we recently proposed that positive glomerular staining for annexin v would be seen in the majority of patients who had confirmed glomerular deposits of mbl, l - ficolin, and properdin . Further examinations will also be necessary to elucidate the initial activator of lp in situ . Endo et al . Reported that the patients with glomerular deposits of mbl / masp-1 deposition were young and that the duration of the disease prior to renal biopsy was short compared with that in patients without mbl / masp-1 deposition . In considering histological alterations, patients with glomerular deposits of mbl / masp-1 have a more distinct activity index than chronicity index . Roos et al . Compared igan patients who had negative glomerular staining for mbl with those who had positive staining for mbl . They concluded that the activation of the lp was associated with more severe renal damage, as demonstrated by proteinuria, decreased renal function, and more severe histological findings, such as mesangial proliferation, crescent formation, glomerular sclerosis, and interstitial fibrosis . Accordingly, it is possible to envisage a scenario in which glomerular activation of lp might play an enhancer of progression of glomerular damage . Thus, we should perform immunofluorescent analysis of c4 and/or c4 breakdown products (c4c, c4d), and this should be a routine procedure in histological work and the presence of mesangial deposits of c4 may help us to identify patients with worse prognosis . Showed that there were no differences in clinical and pathological severity parameters for the patients with mesangial deposits of mbl . In their paper, patients with mesangial deposits of mbl and iga2 constituted more than half of the patients examined, which was a higher proportion than those described in the reports of endo et al . And because igan is chronic disease of long duration and shows a broad spectrum of clinical presentations, it is difficult to plan to compare their clinical course . At any rate, previous reports had indicated that glomerular deposits of mbl were present in the patients of postinfectious diseases and that causative pathogens were variable, such as streptococcus [29, 30], propionibacterium, mucor, and hepatitis c virus . In igan, several pathogenic viral and bacterial antigens have been proposed as being responsible for the formation of mesangial deposits of iga and infection might trigger the onset and progression of the diseases [34, 35]. Up to now, there has benn only one report which has explored the association between mbl polymorphism and clinical course of igan . Gong et al . Described the clinical significance of mbl polymorphism of codon 54 in patients with igan . In this report, the patients carrying the variant allele (gac) had episodes of upper respiratory or gastrointestinal infections prior to the onset, or exacerbation of igan which are absent in wild homozygotes (ggc / ggc). The association between infection and progression of glomerular damage via lp activation is a promising area of research for obtaining a better understanding of the pathogenesis of igan . It appears that the activations of ap and lp are very much involved in the pathogenesis of igan . The traces of lp activation in the fluid phase and local glomerular tissues have been shown to accumulate in a subpopulation of igan . However, further studies will be essential to confirm the ligands for the mbl in the pathogenesis of igan.
Intraspinal juxtafacet cysts (commonly, synovial or ganglion cyst) can be an uncommon cause of lower and radicular pain, neurogenic claudication, and cauda equina syndrome2,9,10,13). Spinal synovial cysts generally arise from a degenerated facet joint and often presenting as a gradual onset of back pain and potentially, chronic progressive radiculopathy is a much less common occurrence, and patients may present with acute onset of symptoms3,13). We describe a case in which hemorrhage into a right l2 - 3 facet synovial cyst caused acute back pain and radiculopathy and required surgical excision . A 72-year - old - woman with a past history of hypertension presented to the clinic with acute back pain radiating to the anterior surface of the right thigh and leg . The right straight - leg - raise test elicited pain in the leg at 30 which was exacerbated further by ankle dorsiflexion . Magnetic resonance imaging (mri) revealed the presence of a cystic formation in the right l2-l3 facet joint with arthritis compressing the l3 right root and the dural sac (fig ., the mri displayed a hyperintense abnormality on t1-weighted images and hypointense on t2-weighted images consistent with hemorrhage . The posterior elements from l1 to l3 were exposed and right l2-l3 laminectomy and facetectomy were performed . A brownish mass was found in continuity with the right l2-l3 facet joint adherent to the dural sac . The extradural hematoma was visualized and evacuated, effectively decompressing the right 2 nerve root and sacral sac . Microscopically, there was a synovial cyst with synovial cell lining, neovascularization, hemosiderin microdeposits, and blood (fig . One month after the operation, the patient had no symptoms in her lower back and leg . Spinal synovial cyst can be defined as a soft tissue mass located extradurally along the medial border of a degenerated facet joint, especially l4 - 5 level1,4,8,11,12). These cysts are filled with clear or xanthochromic fluid and have a synovial - like epithelial lining with a demonstrable connection to a joint capsule3,11). If the synovial cell lining and the communication with a joint capsule are absent, the cyst is classified as ganglion11). Intraspinal synovial cysts are rare but have been documented with increasing frequency because of the improvement of neuroradiological imagings4). An mri is considered a good modality for diagnosis . On mri, cysts appear as well - circumscribed, epidural mass lesions that are hypo- and hyper - intense on t1 and t2 weighted images, respectfully . Bleeding into a synovial cyst may cause a sudden expansion of the cyst leading to compression of the epidural space result in root compression symptoms . Hemorrhage into a synovial cyst results in an acute increase in pain and radicular symptoms, including neurological deficits12). Hemorrhagic presentation can be caused by anticoagulation treatment, trauma, disc herniation, vascular anomaly, and neoangiogenesis in the cyst4). In addition, high vascularization of the cyst can cause hemorrhage in spite of minor trauma or just spinal instability . Synovial cyst hemorrhagic events in some cases can occur despite the absence of trauma or coagulopathy13). As a result, other yet to be determined risk factors for synovial cyst hemorrhage occurrence exist prompting the need for more studies . Non - hemorrhagic synovial cysts are occasionally treated by percutaneous aspiration with successful resolution of symptom6). The injection of corticosteroid agents into the facet joint may be an treatment option . Despite conservative treatment, recurrence of cyst with symptoms the natural history of spinal synovial cysts is unknown, but spontaneous remissions of a synovial cyst on follow up imaging with improved symptoms have been reported5) however, pain can be treated with steroid injections and bed rest in substantial number of patients . We report of a case in which hemorrhage into a right l2 - 3 facet synovial cyst causing an acute back pain and radiculopathy . Treatment by resection of the cyst and evacuation of the hematoma led to complete neurological recovery . Synovial cysts of the lumbar spine are an uncommon cause of back and radicular pain . Rarely, these cysts can go through hemorrhagic transformation by trauma, anti - coagulopathic state, or for unknown reasons . Hemorrhage into a cyst is an uncommon occurrence that can lead to acute symptom . In most cases, surgery is the treatment of choice for symptomatic hemorrhagic synovial cysts . Surgery involving the evacuation of the hematoma and/or cyst can result in complete resolution of acute symptoms and neurologic deficit.
Cell culture tissue culture reagents were purchased from commercial sources (invitrogen). The human pancreatic epithelial cancer cell lines, panc-1, aspc-1, capan-1, capan-2, bxpc-3, l3.6, miapaca-2, and cfpac-1, were obtained from american type culture collection and maintained according to the supplier's suggestions . All cells were grown at 37 c in a humidified incubator under 5% co2 . Plasmid construction standard molecular biology techniques were used to clone klf4, klf5, klf7, klf9, klf11, klf14, and klf15 into the pcdna3.1/his (invitrogen) and pcmvtag2 (stratagene) vectors for expression as his - tagged or flag - tagged proteins, respectively, as well as the truncated (263 bp) tgfrii promoter into the pgl3-lux vector (promega). The p3tp - lux reporter plasmid containing tgf-responsive elements anita roberts (national institutes of health) as a positive control for tgf1 stimulation experiments (data not shown). Semi - quantitative rt - pcr total rna was extracted from cells according to the manufacturer's instructions using an rneasy kit (qiagen), and 5 g was used for cdna synthesis using oligo(dt) primer using the superscript iii first - strand synthesis system for rt - pcr (invitrogen) per the manufacturer's protocol . Rt - pcr was performed using la taqdna polymerase with a gc buffers kit (takara) per the manufacturer protocols . Semiquantitative rt - pcr analysis was performed with the primer sets provided in supplemental table s1 . Amplification of four human housekeeping genes, gapdh (unigene hs.544577), 2-microglobulin (b2 m, hs.709313), 2-tubulin (tubb2, hs.300701), and hypoxanthine phosphoribosyltransferase 1 (hprt1, hs.412707) was used for all samples as an internal control . Densitometric values were obtained and normalized to the average of the housekeeping cdnas for each individual sample using scion image beta 4.02 software (scion corp . ). To represent the tgf inducibility of individual klf transcripts, each tgf-treated sample was compared with an untreated control that was originally plated and ultimately collected at the same time to minimize compounding factors, which can often influence gene expression (i.e. Cell cycle stage, cell density, potential unknown paracrine, and/or autocrine stimuli). At each time point, values were determined by first normalizing the tgf-treated sample (t, treated) to the average of the four aforementioned housekeeping genes in the same sample (tc, treated housekeeping gene control). This resultant value was divided by the corresponding untreated sample (ut, untreated) normalized in the same manner (utc, untreated housekeeping gene control), to express the fold of tgf induction ([t / tc]/[ut / utc] = fold tgf-induction). Another housekeeping gene, -actin (actb, hs.520640), was not used in our analyses, because it showed significant differences with tgf treatment over control values (data not shown). Total protein extracts were prepared by lysing cells in radioimmune precipitation assay buffer supplemented with complete protease inhibitor mixture (roche applied science). Cellular lysates were subjected to 10% sds - page and then separated proteins are transferred to polyvinylidene difluoride membranes (millipore). Membranes were incubated overnight at 4 c in blocking solution (tris - buffered saline solution containing 5% nonfat dried milk and 0.1% tween 20). Immune complexes were visualized by enhanced chemiluminescence (pierce) and exposed to x - ray film . Transcriptional reporter assays cells were transfected with specified reporter constructs along with expression constructs and/or empty vector using electroporation (2 10 cells/0.4-cm microcuvette, 360 v, and 10 ms) and subsequently serum - starved overnight . Transfection was performed with equimolar concentrations of dna, and expression was quantified with western blotting directed against epitope - tagged proteins as described . Cells were stimulated with tgf1 (r&d systems) as specified and assayed at various specified time points . At 24 or 48 h after transfection and treatment as noted, cells were lysed, and luciferase measurements were performed using a 20/20 luminometer (turner designs) according to manufacturer's suggestions (promega). Data were normalized as relative light units and normalized to the protein concentration as the mean s.d . Cells were transfected with flag - tagged constructs . At 24 h post - transfection, cells were washed and lysed in lysis buffer (150 mm nacl, 0.5% nonidet p-40, 50 mm tris - hcl, ph 7.5, 20 mm mgcl2) supplemented with complete protease inhibitor tablets (roche applied science) for 30 min at 4 c . Immunoprecipitations were performed using anti - flag m2 agarose - conjugated antibodies (sigma) for 2 h at 4 c . To detect interaction with endogenous co - repressors, immunocomplexes were collected by centrifugation, washed with lysis buffer, and analyzed by western blot as described above using anti - msin3a and hdac2 antibodies (santa cruz biotechnology). The following primer set for the 263-bp tgfrii promoter was used for pcr: 5-gca gat gtt ctg atc tac ta-3 (forward); 5-agc tgg gca gga cct ctc tc-3 (reverse) using takara la taq according to the manufacturer's protocol (mirus). Site - directed mutagenesis was performed with quikchange ii site - directed mutagenesis kits per the manufacturer's protocol (stratagene). All constructs were sequenced by the mayo clinic molecular biology core facility . Gst fusion the klf14 cdna fragment encoding amino acids 191323 corresponding to the dna - binding zinc finger region was cloned into the gst fusion vector pgex 5x-1 (amersham biosciences) using standard techniques . Gst fusion protein expression was induced in bl21 cells (stratagene) by the addition of 1 mm isopropyl - d - thiogalactopyranoside and incubation for 2 h. cells were lysed and subsequently purified by using glutathione - sepharose 4b affinity chromatography as previously described (15). Briefly, 1.75 pmol of double - stranded oligonucleotides were end - labeled with [-p]atp using 10 units of t4 polynucleotide kinase and appropriate buffer (700 mm tris - hcl, ph 7.6, 100 mm mgcl2, 50 mm dithiothreitol) according to the manufacturer's instructions (promega). The reaction was incubated at 37 c for 10 min and halted with addition of te plus edta (0.5 m edta, 1 m tris, ph 8.0, h2o). Protein lysates included 0.5 g of purified gst - klf14/zf fusion protein and in some experiments rhsp1 (promega) at the indicated dilutions . A 5 zncl2 buffer was used in this reaction (100 mm hepes, ph 7.5, 250 mm kcl, 25 mm mgcl2, 50 m zncl2, 30% glycerol, 1 mg / ml bovine serum albumin, 250 g / ml poly(di - dc), h2o) for 10 min at room temperature . The -p - labeled oligonucleotides were added for 20 min . In some cases, an excess of cold probe, at the indicated dilutions, was added concomitant with the addition of radiolabeled probe in addition to anti - sp1 polyclonal rabbit antibody purchased from commercial sources (millipore). The mixtures were electrophoresed in a 4% nondenaturing polyacrylamide gel in a hoeffer midi - gel using 0.5 tris borate - edta for 4 h at 160 v. gels were then transferred to blotting paper (whatman 3 mm), covered in plastic wrap, and vacuum dried for 1.5 h at 65 c . Dried gels were then analyzed using a storm scanner 860 phosphorimager (amersham biosciences). Sp1 consensus and sp1 mutant double - stranded oligonucleotides were obtained from commercial sources (santa cruz biotechnology) with the following sequences: sp1 consensus, 5-att cga tcg ggg cgg ggc gag c-3 (forward); 5-gct cgc ccc gcc ccg atc gaa t-3 (reverse) and sp1 mutant, 5-att cga tcg gtt cgg ggc gag c-3 (forward); 5-gct cgc ccc gaa ccg atc gaa t-3 (reverse). Figure 1.a, repression of the tgfrii promoter occurs after 24 h of tgf stimulation in panc1 cells . Panc1 cells were transfected with a full - length and a truncated 263-bp tgfrii promoter luciferase reporter and then stimulated with tgf1 after overnight serum starvation . Luciferase levels were obtained at specified time points after treatment and compared with untreated controls . Data are the mean s.d . From three independent experiments, with triplicates for each experiment . Binding sites . The diagram represents a schematic of the human tgfrii proximal promoter containing five gc - rich sp1-like elements . A, repression of the tgfrii promoter occurs after 24 h of tgf stimulation in panc1 cells . Panc1 cells were transfected with a full - length and a truncated 263-bp tgfrii promoter luciferase reporter and then stimulated with tgf1 after overnight serum starvation . Luciferase levels were obtained at specified time points after treatment and compared with untreated controls . Data are the mean s.d . From three independent experiments, with triplicates for each experiment . The diagram represents a schematic of the human tgfrii proximal promoter containing five gc - rich sp1-like elements . A yet undefined sp / klf repressor protein plays a role in silencing of the type ii tgf-receptor promoter previous studies have described the regulation of the tgfrii by tgf ligands, primarily showing a bimodal response consisting of an sp1-dependent up - regulation (14, 17) and a subsequent down - regulation of receptor transcript levels upon this stimulation (1820). Interestingly, although the activation of tgfrii promoter, in particular by sp1, has received precise attention, how this receptor is repressed remains poorly understood . Consequently, the major goal of the current study has been to characterize the role of a specific family of non - smad proteins (sp / klf transcription factors), which may functionally explain the down - regulation tgfrii through gc - rich sp1-like sequences . Our studies began with examination of the transcriptional activity of the tgfrii gene promoter in panc1 epithelial cells, a widely used model for studying tgf signaling . We have performed an initial series of reporter assays using full - length tgfrii and the previously described, tgf-sensitive, 263-bp core promoter, which is located 5 of the transcriptional start site (14). Treatment of panc1 cells with exogenous tgf1 leads to a marked reduction in activity of the full - length tgfrii reporter when compared with untreated control cells (fig . This silencing effect is recapitulated in the 263-bp tgfrii core promoter, suggesting that both the previously described activation pathway (14) and the negative transcriptional regulatory mechanism characterized further here (fig . 1a), are operational at this core promoter level . Using bioinformatics analyses (transfac public), we have identified five putative sp / klf binding sites within this tgf-sensitive, 263-bp core promoter region (fig . Four of these sites (#14) have been previously identified, although an additional site (#5) has not been previously reported (14, 21). Some of these previously identified sites have been shown to be activated by sp1 . However, whether novel sp / klf silencer proteins can also bind to this sequence to reverse the activation by sp1 remained unknown . Therefore, this analysis has led us to the hypothesis that a yet undefined sp / klf repressor protein plays a role in the silencing of this promoter . Novel tgf-inducible non - smad, sp / klf proteins are identified as candidate regulators of the type ii tgf-receptor to test our hypothesis, we have developed a four - tier screening approach . This approach includes first, testing which of the 24 known sp / klf proteins are expressed in tgf-sensitive epithelial cells and, thus, can be considered initial candidates to target the tgfrii promoter . Our experimental cell model, panc1, a human epithelial cell line, is an optimal model for our studies, because they have adequate expression of tgfrii mrna and display growth inhibition to exogenous tgf1 stimulation (22). In addition, as shown in fig . 2a, each of the 24 known sp / klf transcription factors are consistently expressed in these cells, which made it ideal for performing a comprehensive screen . Second, we determine whether any sp / klf genes are tgf-inducible with a kinetic that is consistent with playing a role in the down - regulation of the tgfrii . Third, by utilizing transfection studies combined with reporter assays, we test the potential of distinct members of this family to repress tgfrii promoter activity and then, by electromobility shift assays, determine which sites on the promoter are utilized by our candidate klf protein . Finally, we examine whether the repressor that is isolated according to these criteria binds to the endogenous tgfrii gene and can remodel chromatin on this target, suggesting the bona fide target status of the candidate klf protein . Thus, by applying this comprehensive screening, our study has been robust in evaluating the klf protein family in the tgf response and regulation of the tgfrii . The genomic axiom that functionally related genes follow a similar pattern of expression suggested that, hypothetically, the protein that represses the tgfrii may be expressed in a similar manner after tgf treatment, in particular, during the repression response to this cytokine . Consequently, we have evaluated which, if any, of these sp / klf members are inducible by tgf1 treatment . Thus, using rna from panc1 cells either untreated or treated with tgf1, we have performed rt - pcr at various time points (fig . 2b). As a positive control for tgf-mediated transcriptional induction, we monitor the expression of p21, a known tgf-inducible gene within the pathway . Of the 24 known sp / klf transcription factors, we have identified 7 that were markedly induced with exogenous tgf1 treatment, suggesting that these could be potential candidate transcriptional repressors of the tgfrii promoter (fig . These results are not only consistent with our previous work, which identified klf11 as a tgf-inducible gene (16, 23), but, more importantly, it characterizes previously unidentified klf targets for this cascade, namely klf4, -5, -7, -9, -14, and -15 . Thus, based upon their expression patterns, these seven genes are good candidates to further investigate their regulation of the tgfrii promoter . Figure 2.a, eight pancreatic cancer cell lines were screened by rt - pcr for expression of 24 members of the sp / klf family of transcription factors . The figure shows that all 24 sp / klf members are expressed in the tgf-sensitive panc1 cell line . Panc1 cells were serum - starved overnight, treated with 10 ng / ml tgf1, and screened for the expression of each klf gene by semi - quantitative rt - pcr . The expression levels for each klf gene from tgf1-treated samples were compared with untreated controls and normalized to housekeeping genes as described under material and methods . The expression of the p21 gene was used as a positive control for tgf1 treatment . Four different housekeeping genes, namely gapdh, b2 m, tubb2, and hprt1, were used as internal standards for normalization . C, quantification of tgf induction of transcript levels over time . At each time point, values were determined by first normalizing the densitometric measurement of the tgf-treated sample to the average of four housekeeping genes (gapdh, b2 m, tubb2, and hprt1) in the same sample . This resulting value was divided by the corresponding value of the untreated sample normalized in the same manner in order to express the -fold of tgf induction . A, eight pancreatic cancer cell lines were screened by rt - pcr for expression of 24 members of the sp / klf family of transcription factors . The figure shows that all 24 sp / klf members are expressed in the tgf-sensitive panc1 cell line . Panc1 cells were serum - starved overnight, treated with 10 ng / ml tgf1, and screened for the expression of each klf gene by semi - quantitative rt - pcr . The expression levels for each klf gene from tgf1-treated samples were compared with untreated controls and normalized to housekeeping genes as described under material and methods . The expression of the p21 gene was used as a positive control for tgf1 treatment . Four different housekeeping genes, namely gapdh, b2 m, tubb2, and hprt1, were used as internal standards for normalization . C, quantification of tgf induction of transcript levels over time . At each time point, values were determined by first normalizing the densitometric measurement of the tgf-treated sample to the average of four housekeeping genes (gapdh, b2 m, tubb2, and hprt1) in the same sample . This resulting value was divided by the corresponding value of the untreated sample normalized in the same manner in order to express the -fold of tgf induction . Figure 3.a, panc1 cells were transfected with klf(flag / his) epitope - tagged expression constructs or control empty vector to test tgfrii promoter activity . B, panc1 cells were transfected with indicated klf(flag / his) epitope - tagged expression constructs or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to test tgfrii promoter activity . C, panc1 cells were transfected with various concentrations of epitope - tagged klf14(flag) expression construct or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to observe the effect on tgfrii promoter activity . Western controls (flag / his) were shown for epitope - tagged klf expression in all experiments . Expression of flag - tagged klf4, klf7, klf14, and klf15 was confirmed by an anti - flag antibody (sigma), whereas expression of his - tagged klf5, klf9, and klf11 was verified by the omni d8 antibody (santa cruz biotechnology). Data are the mean s.d . From three independent experiments, with triplicates for each experiment . A, panc1 cells were transfected with klf(flag / his) epitope - tagged expression constructs or control empty vector to test tgfrii promoter activity . B, panc1 cells were transfected with indicated klf(flag / his) epitope - tagged expression constructs or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to test tgfrii promoter activity . C, panc1 cells were transfected with various concentrations of epitope - tagged klf14(flag) expression construct or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to observe the effect on tgfrii promoter activity . Western controls (flag / his) were shown for epitope - tagged klf expression in all experiments . Expression of flag - tagged klf4, klf7, klf14, and klf15 was confirmed by an anti - flag antibody (sigma), whereas expression of his - tagged klf5, klf9, and klf11 was verified by the omni d8 antibody (santa cruz biotechnology). Subsequently, we have tested the transcriptional activity of these candidates on the tgfrii promoter using reporter assays by co - transfecting the 263-bp core tgfrii promoter - luciferase construct with cdnas encoding each of the seven tgf-inducible klf candidates . Out of these, five candidates induced a marked decrease in tgfrii promoter activity, namely klf4, -7, -11, -14, and -15 (fig . Klf5 did not affect tgfrii promoter activity above control levels, whereas klf9 appeared to slightly activate this promoter, therefore these two proteins were not continued in subsequent experiments due to our objective of identifying tgfrii repressors . To confirm whether this observed repression of tgfrii promoter regulation is consistent with tgf pathway activation, these five proteins were further tested in panc1 cells treated with exogenous tgf1 treatment . Interestingly, upon treatment, these klf proteins were capable of further repressing tgfrii promoter activity, with the largest repression achieved by klf14 (fig . These data also indicate that a second tgf-dependent mechanism, directly (signal - induced klf expression or post - translational modifications) or indirectly (induction of another transcription factor), cooperates with klf proteins to additionally repress this gene . Upon increasing concentrations of klf14 cdna in transfection studies, we found a concentration - dependent repression of the core tgfrii promoter (fig . Together, these results strongly identify klf14 as a good candidate to be a regulator of tgfrii promoter activity and expression . Klf14, a novel non - smad protein, regulates the type ii tgf-receptor the gene encoding klf14 has been previously identified by our group and named bteb5 due to its sequence similarities to members of this subfamily of klf silencing proteins.5 although, recently, genetic studies have reported the genomic structure, intronless nature, and potential imprinted status of this gene (24), a functional characterization of this protein at the cellular and biochemical level has never been performed . Because our data indicate that klf14 appears to be a potent regulator of the tgfrii in promoter assays combined with this existing gap in knowledge on this klf family member and its targets, the choice to further investigate the role of this particular klf in tgfrii regulation would significantly expand the current knowledge on the functional properties of members of this family . Noteworthy, we have validated these in vitro reporter results in vivo by assessing whether this protein has a regulatory effect on endogenous tgfrii levels in panc1 cells . Initial correlative experiments demonstrate that the levels of tgfrii mrna levels decrease at a time in which the amount of klf14 increases (repression phase of the bimodal expression pattern of tgfrii in response to tgf), raising the possibility that klf14 is induced to subsequently down - regulate the tgfrii (fig . Mechanistically support this correlation, we have performed rt - pcr on cells overexpressing klf14 to determine tgfrii levels in comparison to mock transfected cells in the presence or absence of exogenous tgf1 stimulation (fig . 4b). Klf14 overexpression alone is sufficient to decrease tgfrii mrna, interestingly to the same extent as tgf1 stimulation alone . Furthermore, subsequent tgf1 treatment in cells transfected with klf14 leads to further down - regulation of tgfrii transcripts . Because klf14 is a tgf-inducible gene, the further decrease in tgfrii mrna levels observed upon tgf1 treatment likely results from the induction of endogenous sp / klf transcription factors by this cytokine . These results suggest that tgf down - regulates tgfrii transcripts through klf14 expression with subsequent negative regulation of promoter activity . Next, we have tested whether the effect of klf14 on tgfrii expression interfered with tgf-induced signals that target downstream genes, such as p21 (25). Indeed, as expected, tgf1 treatment increases p21 promoter activity, as observed via reporter assays, as well as its mrna levels (fig . 4, c and d), whereas klf14 reduces both p21 promoter activity and mrna levels upon tgf1 treatment (fig . These results suggest that klf14 interferes with the activation of downstream tgfsignaling effects, at least in part, by its ability to repress the tgfrii . Figure 4.a, tgfrii and klf14 transcript levels were analyzed by determination of density, and relative transcript levels were represented by the ratio tgfrii cdna / gapdh cdna and klf14 cdna / gapdh cdna . Panc1 cells were transfected with klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . Densitometry was performed after normalizing to the average of gapdh, b2 m, tubb2, and hprt1 levels and untreated control cdna . C, panc1 cells were transfected with klf14 flag epitope - tagged constructs or control empty vector along with a p21 promoter reporter construct and then stimulated with 10 ng / ml tgf1 after overnight serum starvation . Panc1 cells were transfected with flag - klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . Densitometry was obtained after normalizing to the average of gapdh, b2 m, tubb2, and hprt1 levels and untreated control cdna . A, tgfrii and klf14 transcript levels were analyzed by determination of density, and relative transcript levels were represented by the ratio tgfrii cdna / gapdh cdna and klf14 cdna / gapdh cdna . Panc1 cells were transfected with klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . Densitometry was performed after normalizing to the average of gapdh, b2 m, tubb2, and hprt1 levels and untreated control cdna . C, panc1 cells were transfected with klf14 flag epitope - tagged constructs or control empty vector along with a p21 promoter reporter construct and then stimulated with 10 ng / ml tgf1 after overnight serum starvation . Panc1 cells were transfected with flag - klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . Densitometry was obtained after normalizing to the average of gapdh, b2 m, tubb2, and hprt1 levels and untreated control cdna . Klf14 represses the tgfrii promoter via distinct sp1-like gc - rich sequences and competition with sp1repressor klf proteins, such as klf14, have been previously shown to compete with the canonical sp1 protein for overall transcriptional activity of a promoter, such as the cyp1a1 and ldlr promoters (26, 27). Therefore, herein we test the ability of klf14 to repress transcription of the tgfrii promoter in the presence of exogenous sp1 expression . Indeed, as shown in fig . 5a, the expression of exogenous sp1 relieves the repression mediated by klf14 in a dose - dependent manner . Due to the direct, sp1-dependent up - regulation of the tgfrii promoter as part of its bimodal response during activation, we dissect whether the repression of the tgfrii promoter via klf14 is also acting through a direct mechanism on the promoter rather than indirect due to klf14 mediating a secondary effector . Panc1 cells were transfected with klf14(flag) and sp1(his) epitope - tagged expression constructs or control empty vector as indicated to evaluate in vivo competition on tgfrii promoter activity . Western control (flag / his) is shown for epitope - tagged expression . B, klf14 binds to four of the putative sp1 sites and competes with sp1 on the tgfrii promoter . Electromobility shift assay was performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides for each of the five putative sp1 sites . Lane 1: control - sp1 consensus oligonucleotide; lane 2: control - sp1 mutant oligonucleotide; lane 3: sp1 consensus oligonucleotide and rhsp1 protein; lane 4: wt oligonucleotide; lane 5: mut#1 oligonucleotide; lane 6: mut#2 oligonucleotide; lane 7: wt oligonucleotide and 25 cold probe; lane 8: wt oligonucleotide and 100 cold probe; lane 9: wt oligonucleotide and anti - gst; lane 10: wt oligonucleotide and 1 rhsp1 protein; lane 11: wt oligonucleotide and 5 rhsp1 protein . C, klf14 utilizes four sp / klf sites to repress the tgfrii promoter in vivo . Panc1 cells were transfected with tgfrii - luciferase with mutated sp1 sites as indicated and stimulated with 10 ng / ml tgf1 after overnight serum starvation, and transcriptional activity was measured and normalized to control empty vector . Data are the mean s.d . From three independent experiments, with triplicates for each experiment . Panc1 cells were transfected with klf14(flag) and sp1(his) epitope - tagged expression constructs or control empty vector as indicated to evaluate in vivo competition on tgfrii promoter activity . Western control (flag / his) is shown for epitope - tagged expression . B, klf14 binds to four of the putative sp1 sites and competes with sp1 on the tgfrii promoter . Electromobility shift assay was performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides for each of the five putative sp1 sites . Lane 1: control - sp1 consensus oligonucleotide; lane 2: control - sp1 mutant oligonucleotide; lane 3: sp1 consensus oligonucleotide and rhsp1 protein; lane 4: wt oligonucleotide; lane 5: mut#1 oligonucleotide; lane 6: mut#2 oligonucleotide; lane 7: wt oligonucleotide and 25 cold probe; lane 8: wt oligonucleotide and 100 cold probe; lane 9: wt oligonucleotide and anti - gst; lane 10: wt oligonucleotide and 1 rhsp1 protein; lane 11: wt oligonucleotide and 5 rhsp1 protein . C, klf14 utilizes four sp / klf sites to repress the tgfrii promoter in vivo . Panc1 cells were transfected with tgfrii - luciferase with mutated sp1 sites as indicated and stimulated with 10 ng / ml tgf1 after overnight serum starvation, and transcriptional activity was measured and normalized to control empty vector . Data are the mean s.d . From three independent experiments, with triplicates for each experiment . Thus, to first delineate the dna binding properties of klf14 to the promoter of tgfrii in vitro, we characterize the five putative sp / klf sites (shown in fig . Radiolabeled oligonucleotides for each of the 5 sp1 sites have been created as well as mutants for each site containing either 2- or 4-nucleotide substitutions (mut#1 or mut#2, respectively) within the gc - rich binding sequence . Sp1-consensus oligonucleotides and respective mutants have been used as internal binding controls (fig . The interaction between a recombinant klf14 zinc finger dna - binding domain and four of the sp / klf site probes (#13 and 5) is specific as indicated by the fact that an excess of unlabeled probe competes with the radiolabeled probes (fig . Furthermore, we were able to abolish this binding by using antibodies against the recombinant klf protein (anti - gst antibody), thus confirming specificity of the complex (fig . 5b, lane 9). Further specificity is evidenced by the impaired ability of recombinant klf14 protein to bind to mutated sp / klf probes (fig . Visible but reduced binding is noted with the 2-bp mutant probes (mut#1, lane 5) for sites #13, however a significant loss of binding was observed with the 4-bp mutant probes (mut#2, lane 6) at all sites . Moreover, through addition of recombinant sp1 protein to the reaction, an increase in the concentration of sp1 diminished the binding of klf14, demonstrating that klf14 and sp1 compete for these gc - rich binding sites (fig . 5b, lanes 10 and 11), complementing the competition results we observed in vivo (fig . 5a). Only a weak complex was visualized with site #4, indicating that this site was less specific for klf14 binding than the other four sites . To functionally complement these studies, we have performed tgfrii reporter assays with both wild - type and mutant promoter - luciferase constructs . Site - directed mutagenesis has been utilized to create a 2-nucleotide substitution in each of the 5 gc - rich sp / klf sites (fig . 5c). These assays resulted in a marked loss of the repression observed with the wild - type tgfrii promoter upon klf14 overexpression with mutation in sp / klf sites #1, 2, 3, and 5, particularly with a significant gain of transcriptional activity upon mutating site #1 . These findings demonstrate that these four are operational to silence the transcriptional activity of the tgfrii promoter by klf14 . Furthermore, we did not observe a significant loss of repression with mutation of site #4 with klf14 overexpression, which supports our finding of negligible binding of klf14 to this specific sp / klf site (fig . 5b). All together, klf14 appears to bind and act on four of the gc - rich sites of the tgfrii promoter to repress its transcriptional activity, and this occurs through a mechanism that includes, at least in part, competition with sp1 . Repression of the tgfrii gene by klf14 occurs via mechanisms involving chromatin - modifying enzymes careful examination of klf14 sequence reveals a 26-amino acid domain that is highly similar to repression domains of klf9 (bteb1), klf13 (bteb3), and klf16 (bteb4) proteins (fig . 6a), which we have previously shown to repress transcription by recruiting sin3a via its paired amphipathic helix 2 domain, as well as hdac (15, 28). Interestingly, other studies have found that hdac inhibition leads to transcriptional activation of the tgfrii promoter in various cancer cell lines, raising the possibility that a repressor of this type may be operational to achieve this effect (29, 30). Thus, we have tested whether klf14 does indeed interact with sin3a and hdac by immunoprecipitating full - length flag - tagged klf14 from panc1 cells . The presence of sin3a and hdac complexed to klf14 is detected by western - blot analysis (fig . Thus, these results indicate that klf14 interacts with sin3a and hdac in mammalian cells, implying that these proteins are part of a repressor complex . Subsequently, we have investigated whether this repressor complex occupies the endogenous tgfrii promoter in vivo via chip assays . First, we find that klf14 indeed binds to the tgfrii promoter (fig . 6c), treatment with tgf1 increases the amount of klf14 bound to the tgfrii promoter . In addition, the treatment with tgf1 coincides with the appearance of sin3a on the same region of the promoter as klf14, further implicating this repressor complex in the mechanism of klf14 repression of the tgfrii promoter (fig . 6d). To gain insight into the type of chromatin remodeling that accompanies klf14 and sin3a occupation, we also have performed chip using antibodies to various histone marks . Interestingly, we find that, under native conditions, acetylated histones h3 and h4 are present on the tgfrii promoter; however, upon tgf1 treatment, these fall to negligible levels, indicating a change from a transcriptionally active, acetylated state to a relatively inactive, non - acetylated state (fig . Moreover, we observe that, although methylated k20 h4, a mark of repression (31), is not present under native conditions, it occupies the promoter upon tgf1 treatment along with sin3a (fig . These types of experiments are in agreement with the histone code hypothesis (32), revealing that the state of chromatin in the tgfrii promoter appears to switch from active to repressed upon klf14 occupation after treatment with tgf1, all consistent with the idea that klf14 binds to the tgfrii promoter to cause repression (fig . Therefore, both competition with sp1 and direct repression via the n - terminal domain, are likely to behave as a dual mechanism of repression that would make it more difficult to reverse than if one of them was operational . This redundancy would ensure that the promoter remains silent even under circumstances that inactivate either of the single mechanisms (fig . Figure 6.a, alignment displaying the 26-amino acid sin3-interacting domain of klf14 similar to other klf repressors, klf9, klf13, and klf16 . Panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and flag - immunoprecipitated . Panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and subject to chromatin immunoprecipitation . A 263-bp fragment of the tgfrii promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna samples . Note that the tgfrii promoter was amplified from anti - flag (-flag) but not mock immunoprecipitated samples (mock) transfected with klf14 . D, tgf1 leads to repressive chromatin modifications upon klf14 occupation of the tgfrii promoter . Panc1 cells were transfected with flag - tagged klf14 and treated with tgf1 and chromatin landscape chip assay was performed using specified antibodies . A, alignment displaying the 26-amino acid sin3-interacting domain of klf14 similar to other klf repressors, klf9, klf13, and klf16 . Panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and flag - immunoprecipitated . Panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and subject to chromatin immunoprecipitation . A 263-bp fragment of the tgfrii promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna samples . Note that the tgfrii promoter was amplified from anti - flag (-flag) but not mock immunoprecipitated samples (mock) transfected with klf14 . D, tgf1 leads to repressive chromatin modifications upon klf14 occupation of the tgfrii promoter . Panc1 cells were transfected with flag - tagged klf14 and treated with tgf1 and chromatin landscape chip assay was performed using specified antibodies . The current study provides us with several novel mechanistic insights on the regulation of the tgf pathway . For instance, the data reported here represent the first functional characterization of the klf14 protein, outline a novel pathway for the silencing of the tgfrii promoter, provide insight into the molecular mechanisms by which these phenomena are achieved, and report additional tgf-inducible, klf proteins that are good candidates to regulate this promoter . These findings are of significant relevance to the areas of klf proteins, tgf signaling, the maintenance of cell homeostasis, and their potential contribution to disease states . Tgf1 ligand itself may play a role in regulating signaling by down - regulating the cell surface receptor and thus abrogating downstream messages . Others have also shown that the tgf receptors are under autoregulation control by ligand stimulation (18). It has been previously shown that treatment with tgf1 leads to down - regulation of tgfrii levels (19). Derynck and colleagues (20) have previously described a process in osteoblastic differentiation in which at first there is a marked up - regulation of tgf1 and sensitivity followed by marked down - regulation of receptors and insensitivity to tgf1 . Furthermore, recent in vitro studies on the biosynthesis of the receptors indicate that the half - life of tgfrii is 60 min and is further reduced to 45 min in the presence of exogenous tgf1 (33). Therefore, understanding how this down - regulation of the tgfrii promoter occurs is of primary biological importance for better understanding tgf signaling . Activation of the tgfrii would occur via tgf1 ligand initiating an up - regulation of the tgfrii promoter by sp1 . In our model, activation of the tgf pathway also causes an induction of klf14 expression (a), which would, in turn, bind to the tgfrii promoter through its gc - rich sites (b), competing away sp1 (c), recruiting the sin3a / hdac complex (d), and remodeling chromatin from a transcriptionally active, acetylated state to an inactive state with silencing methyl marks (e). Thus, klf14 mediates a negative feedback loop to down - regulate the type ii tgf receptor upon tgf stimulation . Model of klf14-mediated tgfrii silencing . Activation of the tgfrii would occur via tgf1 ligand initiating an up - regulation of the tgfrii promoter by sp1 . In our model, activation of the tgf pathway also causes an induction of klf14 expression (a), which would, in turn, bind to the tgfrii promoter through its gc - rich sites (b), competing away sp1 (c), recruiting the sin3a / hdac complex (d), and remodeling chromatin from a transcriptionally active, acetylated state to an inactive state with silencing methyl marks (e). Thus, klf14 mediates a negative feedback loop to down - regulate the type ii tgf receptor upon tgf stimulation . In this regard, the current study reports several novel observations of significant biochemical relevance for extending our knowledge of tgf signaling regulation by non - smad proteins . For instance, this study represents the first functional characterization of klf14 as a novel tgf-inducible repressor protein that mediates silencing of the tgfrii, and the mechanisms by which this phenomenon occurs, in particular the role of corepressors and chromatin modifications on the tgfrii promoter . Previous promoter studies have primarily focused on the role of sp1 in the activation of this promoter . However, recent studies have uncovered a paradoxical behavior for this promoter, namely that histone deacetylase inhibitors are needed to activate this promoter (34, 35). These data point to the existence of a repressive state of this promoter, which is less likely to be regulated by sp1 but rather by sp / klf repressors of the type previously described by our laboratory (11). However, in the current study, rather than looking for a candidate gene within the family of klf repressors, we have performed an unbiased screening for the tgf inducibility of each of the 24 klf transcription factors and find that klf14 is up - regulated upon tgf treatment and also can efficiently repress the tgfrii . Interestingly, we demonstrate that there is a strong inverse correlation between expression patterns in a temporal manner of tgfrii mrna and klf14 mrna in human pancreatic cancer cells . Expression of exogenous klf14 decreases the level of transcription from the tgfrii promoter, implying a repressive role for klf14 in tgfrii expression . Further analysis reveals the mechanism by which this protein works, indicating that klf14 is a member of the sin3-dependent klf repressors . This is important in light of there being two types of repressors in this family as recently reviewed by us (11), the sin3a - dependent proteins, which include klf9, -10, -11, -16, and now -14, and the ctbp - dependent proteins, such as klf3 and -8 . This knowledge can now be very useful for performing rapid screening of the proteins that may be acting as repressors of particular genes, besides the traditional hdac inhibitor experiments . This screening, we propose, can utilize sirna to either target ctbp or sin3a . An abolition of the silencing activity would then rapidly focus the investigations on a reduced number of candidates belonging to each of these groups . In addition to the recruitment of a klf14-msin3a - hdac2 repressor complex to the tgfrii promoter, we observed a concurrent remodeling of chromatin, which involves loss of transcriptionally active acetylated histone marks and an increase in histone marks that associate with transcriptional silencing . Finally, we defined binding sites involved in klf14-mediated repression of the tgfrii promoter, namely sites -143/138 (#1), -101/-95 (#2), -59/52 (#3) and -11/-5 (#5), which are consistent with previously reported sp1 sites, and these same sites, when mutated, lose the repressive effect of klf14 . Overall, however, the major relevance of this study is the fact that it contributes to organize our thoughts on how tgf signaling is regulated . It has recently been appropriately proposed to classify these mechanisms by whether they are directly mediated by smads (the best studied to date) or those less understood events regulated by non - smad proteins (4). In fact, our laboratory has previously described a role for klf repressors, namely klf10 (tieg1) and klf11 (tieg2), as non - smad effectors of the tgf response in cell growth (9, 36). Now, due to the findings reported in the current study, we can propose a model wherein tgf cytokines activate and repress the tgfrii promoter using the very same gc - rich sites utilized by this family of proteins . Activation would occur in an initial phase via sp1, whereas inhibition would follow activation and require the action of klf14, competition of sp1, recruitment of sin3a, hdac, and distinct chromatin modifications on the promoter (see model in fig . Therefore, klf14 becomes another important non - smad protein, in which at least one of its functions is to silence the tgfrii . This novel transcriptional pathway thus becomes an important step for modulating the activity of tgf signaling.
Endometrial cancer (ec) is the most common gynecologic cancer in the united states . This year, ec was among the first malignancies to be linked with obesity, and there is consensus that excess adiposity is a major risk factor for the disease . Overweight and obese women are two to three times more likely to develop ec compared with women of normal weight . Given that 64% of american women are overweight or obese and therefore, at risk for ec, there is a critical need to better understand the molecular mechanisms and associated biomarkers linking excess adiposity with this prevalent disease . The higher concentrations of estrogen seen in overweight and obese postmenopausal women are recognized as a major risk factor for developing precancerous and cancerous lesions of the uterus . The results of several recent laboratory and epidemiological studies suggest that other endocrine - related mechanisms associated with adiposity may also be linked with ec . Insulin and leptin stimulate the proliferation of ec cells in vitro and positively correlate with endometrial cell proliferation rates in the normal tissue of women who are normal weight, overweight, and obese . In contrast, adiponectin, which is typically reduced with excess adiposity, may be protective against ec, by inducing cell cycle arrest and apoptosis . The hormonal disturbances associated with obesity can mediate cell proliferation via intracellular signaling pathways . At the molecular level, insulin and leptin both phosphorylate and activate akt, which in turn activates the mammalian target of rapamycin (mtor) complex 1 (mtorc1), a key pathway that links growth factors, hormones, and energy balance with cell proliferation . Conversely, adiponectin has been shown to inhibit mtorc1, partly through dephosphorylation of akt via the activation of adenosine monophosphate (amp)-activated protein kinase (ampk). One of the downstream targets of mtorc1 is the eukaryotic initiation factor 4e (elf4e)/eukaryotic initiation factor 4e binding protein-1 (4e - bp1) complex . When phosphorylated by mtorc1, 4e - bp1 releases eif4e, a key mediator of messenger ribonucleic acid (mrna) translation and protein synthesis, resulting in increased cell growth and proliferation . Thus, the upstream phosphorylation of akt by growth factors and the downstream phosphorylation of 4e - bp1 are biomarkers, not only of mtorc1 activity, but also of cell growth and proliferation . Previous studies have shown that activation of the akt / mtor pathway is involved in ec and that this pathway is a potential therapeutic target for the disease . Further, the activation of 4e - bp1 has been linked to poorer ec prognosis, rendering it a plausible candidate for biomarker development . However, the role that obesity, a modifiable risk factor for ec, plays in mtorc1 signaling is unclear . Thus, we performed an exploratory study to examine the associations between phosphorylated (p)akt, phosphorylated 4e - bp1 (p4e - bp1), and cellular proliferation rates (indicated by the cell marker antigen ki67), in women with and without disease, seeking to determine whether there were differences between obese and nonobese women . In addition, we used data available from the cancer genome atlas (tcga) to test whether there were differences in the gene expression of akt and 4e - bp1, between obese and nonobese women . This study was approved by the university of alabama at birmingham (uab) institutional review board and conforms to the code of ethics of the world medical association (declaration of helsinki). Women who underwent endometrial biopsy at uab hospital between 2009 and 2010 were identified via medical records . Patients with normal endometrium (benign), hyperplasia without cytologic atypia, complex hyperplasia with cytologic atypia (cah), and type 1 endometriod ec, international federation of gynecology and obstetrics (figo) stage i iii were identified . A uab gynecologic pathologist (ln) reviewed all the hematoxylin and eosin - stained (h&e) slides for concordance with the surgical pathology reports . The surgical pathology reports and h&e slides were obtained from hysterectomy tissues to confirm biopsy diagnoses . Height, weight, race, age, menstrual history, parity, medication use, and comorbid conditions were abstracted from the medical records . Height and weight were used to calculate body mass index (bmi) (kg / m). First, 45 m sections were cut from formalin - fixed paraffin - embedded tissue blocks . The slides were warmed at 60c for 2 hours, cooled to room temperature, and submerged in three baths of laboratory grade xylene and graded alcohols, for 5 minutes each . The antigen retrieval was performed in a pressure cooker, in citric acid ph 6 for the phosphoproteins and in ethylenediaminetetraacetic acid (edta) buffer ph 9 for the ki67 . The endogenous peroxidases were quenched with 3% h2o2, and nonspecific antigen binding was blocked with 3% goat serum . Cell proliferation was determined with anti - ki67 (catalog number: pima515690; thermo fisher scientific inc, waltham, ma, usa). The antibody was diluted 1:2000 in phosphate - buffered saline with 1% bovine serum albumin, 1 mm edta, and 1.5 mm sodium azide, ph 7.6 (pbe), applied to the slides and incubated for 1 hour at room temperature . The activation of akt and 4e - bp1 was determined using antibodies specific for pakt (phospho - akt [ser473] [d9e] xp rabbit mab #4060; cell signaling technology inc, danvers, ma, usa) and p4e - bp1 (phospho-4e - bp1 [thr37/46] [236b4] rabbit mab #2855; cell signaling technology inc). The antibodies for the pakt and p4e - bp1 were diluted 1:50 and 1:1000 in pbe, respectively, and incubated overnight at 4c . A rabbit horseradish peroxidase conjugated secondary antibody diluted 1:200 was applied for 40 minutes at room temperature, and diaminobenzidine tetrahydrochloride was used to develop a brown stain to indicate the antigen antibody complex . The sections were counterstained with hematoxylin, and the slides were subject to graded alcohol and xylene baths and then cover - slipped . For each case, a quality control or delete slide was processed without primary antibody but following the same procedure as above to confirm the nonspecific binding of the secondary antibody and background staining . The slides were evaluated and graded by a gynecologic pathologist (ln). For ki67, the percentage of cells that stained positive was determined . For the cytoplasmic and nuclear staining of p4e - bp1 and pakt, semiquantitative immunostaining scores were calculated . In this scoring method, the intensity of the staining (on a scale of 03, where 0= no staining and 3= highest intensity) and the proportion of cells stained were determined . The percentage of cells at each intensity was multiplied by the corresponding intensity value, and the scores were added together to obtain overall mean immunostaining scores . Like others who have found that the presence of atypia is a defining characteristic that cosegregates with more aggressive disease, we too observed that the tissue biomarker staining was more similar in the slides of women diagnosed with cah and ec than with normal endometrium or hyperplasia without cytologic atypia . Therefore, we proceeded with an analysis of two groups: benign / hyperplasia (without cytologic atypia) and hyperplasia with cytological atypia / carcinoma . The between - group differences in the characteristics of the study sample were tested with independent t - tests, fisher exact tests, or chi - square tests . Wilcoxon rank - sum tests were used to compare the biomarkers according to lesion type . Additionally, we used unpaired t - tests on data from tcga to determine whether the gene expression of 4e - bp1 and akt differed between overweight and obese women . Using the illumina genome analyzer iix (illumina inc, san diego, ca, usa) for rna sequencing, level 3 gene expression data were available from the primary tumors of 228 subjects diagnosed with stage i iii endometrioid adenocarcinoma . Data analyses were performed using sas version 9.3 (cary institute inc, cary, nc, usa). This study was approved by the university of alabama at birmingham (uab) institutional review board and conforms to the code of ethics of the world medical association (declaration of helsinki). Women who underwent endometrial biopsy at uab hospital between 2009 and 2010 were identified via medical records . Patients with normal endometrium (benign), hyperplasia without cytologic atypia, complex hyperplasia with cytologic atypia (cah), and type 1 endometriod ec, international federation of gynecology and obstetrics (figo) stage i iii were identified . A uab gynecologic pathologist (ln) reviewed all the hematoxylin and eosin - stained (h&e) slides for concordance with the surgical pathology reports . The surgical pathology reports and h&e slides were obtained from hysterectomy tissues to confirm biopsy diagnoses . Height, weight, race, age, menstrual history, parity, medication use, and comorbid conditions were abstracted from the medical records . Height and weight were used to calculate body mass index (bmi) (kg / m). First, 45 m sections were cut from formalin - fixed paraffin - embedded tissue blocks . The slides were warmed at 60c for 2 hours, cooled to room temperature, and submerged in three baths of laboratory grade xylene and graded alcohols, for 5 minutes each . The antigen retrieval was performed in a pressure cooker, in citric acid ph 6 for the phosphoproteins and in ethylenediaminetetraacetic acid (edta) buffer ph 9 for the ki67 . The endogenous peroxidases were quenched with 3% h2o2, and nonspecific antigen binding was blocked with 3% goat serum . Cell proliferation was determined with anti - ki67 (catalog number: pima515690; thermo fisher scientific inc, waltham, ma, usa). The antibody was diluted 1:2000 in phosphate - buffered saline with 1% bovine serum albumin, 1 mm edta, and 1.5 mm sodium azide, ph 7.6 (pbe), applied to the slides and incubated for 1 hour at room temperature . The activation of akt and 4e - bp1 was determined using antibodies specific for pakt (phospho - akt [ser473] [d9e] xp rabbit mab #4060; cell signaling technology inc, danvers, ma, usa) and p4e - bp1 (phospho-4e - bp1 [thr37/46] [236b4] rabbit mab #2855; cell signaling technology inc). The antibodies for the pakt and p4e - bp1 were diluted 1:50 and 1:1000 in pbe, respectively, and incubated overnight at 4c . A rabbit horseradish peroxidase conjugated secondary antibody diluted 1:200 was applied for 40 minutes at room temperature, and diaminobenzidine tetrahydrochloride was used to develop a brown stain to indicate the antigen antibody complex . The sections were counterstained with hematoxylin, and the slides were subject to graded alcohol and xylene baths and then cover - slipped . For each case, a quality control or delete slide was processed without primary antibody but following the same procedure as above to confirm the nonspecific binding of the secondary antibody and background staining . The slides were evaluated and graded by a gynecologic pathologist (ln). For ki67, the percentage of cells that stained positive was determined . For the cytoplasmic and nuclear staining of p4e - bp1 and pakt, semiquantitative immunostaining scores were calculated . In this scoring method, the intensity of the staining (on a scale of 03, where 0= no staining and 3= highest intensity) and the proportion of cells stained were determined . The percentage of cells at each intensity was multiplied by the corresponding intensity value, and the scores were added together to obtain overall mean immunostaining scores . Like others who have found that the presence of atypia is a defining characteristic that cosegregates with more aggressive disease, we too observed that the tissue biomarker staining was more similar in the slides of women diagnosed with cah and ec than with normal endometrium or hyperplasia without cytologic atypia . Therefore, we proceeded with an analysis of two groups: benign / hyperplasia (without cytologic atypia) and hyperplasia with cytological atypia / carcinoma . The between - group differences in the characteristics of the study sample were tested with independent t - tests, fisher exact tests, or chi - square tests . Wilcoxon rank - sum tests were used to compare the biomarkers according to lesion type . Additionally, we used unpaired t - tests on data from tcga to determine whether the gene expression of 4e - bp1 and akt differed between overweight and obese women . Using the illumina genome analyzer iix (illumina inc, san diego, ca, usa) for rna sequencing, level 3 gene expression data were available from the primary tumors of 228 subjects diagnosed with stage i iii endometrioid adenocarcinoma . Data analyses were performed using sas version 9.3 (cary institute inc, cary, nc, usa). This study included 43 women with a mean age of 56.712.21 years who underwent endometrial biopsy as part of standard clinical care for abnormal uterine bleeding . The tumor characteristics and the characteristics of the study population are shown in tables 1 and 2, respectively . Overall, there were no significant differences in the study characteristics between the benign / hyperplasia (n=18) and atypia / carcinoma (n=25) groups . Overall, cytoplasmic pakt and nuclear pakt were highly correlated, and both were significantly and positively correlated with cytoplasmic and nuclear p4e - bp1 . Cytoplasmic p4e - bp1 and nuclear p4e - bp1 were highly correlated, and both were significantly and positively correlated with cellular proliferation as indicated by ki67 staining . As shown in table 4, ki67 and the nuclear and cytoplasmic p4e - bp1 were significantly higher in the atypia / carcinoma cases compared with the benign / hyperplasia cases . We observed no differences in pakt between the groups . When stratified by bmi status, this same trend continued, with the obese women with atypia / carcinoma having significantly higher ki67, and cytoplasmic and nuclear p4e - bp1 . In the nonobese women, ki67 also was significantly higher in the atypia / carcinoma group compared with the benign / hyperplasia group; however, unlike the obese women, no statistically significant differences in p4e - bp1 were found between the benign / hyperplasia versus atypia / carcinoma groups . The roles of menopause and diabetes status on the marker expression levels are presented in table 5 . Ki67, and nuclear and cytoplasmic p4e - bp1 were significantly higher in postmenopausal and in nondiabetic women with atypia / carcinoma than in the women with benign / hyperplasia . No differences in gene expression of 4e - bp1 and akt were observed between overweight versus obese women in the dataset available through tcga (data not shown). Similarly, no differences in the expression of these genes were observed in the analyses after excluding the pre- and perimenopausal women or when the women were stratified by race . This exploratory study provides evidence that the ec - related expression level of p4e - bp1, a downstream target of mtorc1, may differ between obese and nonobese women . To our knowledge, these findings are among the first to implicate obesity as a factor associated with higher activation of the mtorc1 signaling pathway in situ in ec . These results suggest that adiposity should be considered in future studies evaluating the utility of p4e - bp1 as an ec biomarker . Given that the mtorc1 pathway is currently considered an important therapeutic target for ec treatment and that there are several clinical trials currently underway to test the effectiveness of mtorc1 inhibitors in ec treatment (nct01420081, nct00703807, and nct01205672), these results also suggest that adiposity is a potential modulating factor in the inhibition of 4e - bp1 . Notably, the effect of obesity on the mtorc1 pathway appears to occur at the posttranslational level, through phosphorylation, and not at the gene expression level, as indicated by our results, using data from tcga . Overall, we found strong positive correlations between pakt, p4e - bp1, and ki67 . Our data support previous findings that the phosphoinositide 3-kinase (pi3k)/akt / mtorc1 pathway is a critical intracellular signaling pathway in ec cells . Over 80% of type 1 endometrioid ecs, which account for the preponderance of ec cases (70%80%), pi3k / akt / mtorc1 signaling is less dominant in the other ec tumor subgroup, type 2 nonendometrial ec, in which p53 mutation, human epidermal growth factor receptor 2 (her2)/neu overexpression, and p16 inactivation are most frequent . Nonetheless, it is noteworthy that mtorc1 activity has been linked to aggressive disease and poorer prognosis, regardless of the carcinoma type, suggesting that genetic alteration is not the only factor behind ec - associated mtorc1 signaling . Interestingly, we observed that akt was not directly associated with ki67, nor was it higher in the atypia / carcinoma versus the benign / hyperplasia group . These findings differ from those of abe et al who reported that nuclear pakt was higher in ec versus normal endometrium and hyperplasia, and that nuclear pakt was higher in well - differentiated carcinoma versus moderately and poorly differentiated cancer . In contrast, another study, among patients with advanced ec, found that pakt was positively correlated with ki67 and reduced survival . The discrepancy between our findings and these other studies may be due to differences in sample characteristics . For instance, our study sample was comprised of both pre- and postmenopausal women with early - stage disease, whereas other studies were conducted primarily on premenopausal women and those with advanced ec . Thus, tissue biomarkers may vary across populations due to the hormonal fluctuations associated with menstruation, adiposity, and/or stage of disease . The activation of both cytoplasmic and nuclear akt was highly correlated with p4e - bp1 . P4e - bp1 was significantly higher in atypia / carcinoma versus benign / hyperplasia and was significantly correlated with ki67 . It is thus plausible that the activation of akt stimulates mtorc1 signaling and the activation of 4e - bp1, resulting in increased cellular proliferation . Similar to the findings by castellvi et al, we observed high cytoplasmic p4e - bp1 staining in atypia / carcinoma and light staining in benign / hyperplasia . We also observed positive nuclear staining for p4e - bp1 in both benign / hyperplasia and atypia / carcinoma, although it was significantly higher in atypia / carcinoma . When stratified by weight status, as expected, we observed that obese women diagnosed with atypia / carcinoma had significantly higher cytoplasmic and nuclear p4e - bp1 than did obese women with benign / hyperplasia . However, in nonobese women, we did not observe significant differences in either cytoplasmic or nuclear p4e - bp1staining between women with atypia / carcinoma compared with benign / hyperplasia . This observation is particularly notable given that both obesity and the activation of 4e - bp1 are factors linked with poor ec prognosis . Based on these findings, we speculate that the biological factors associated with obesity, such as hormonal disturbances, may be a driving force in the activation of 4e - bp1 in obese women with ec and that such forces are absent in the nonobese . However, given our results that 4e - bp1 expression levels also differed by menopause and diabetes status in the women with atypia / carcinoma versus benign / hyperplasia, we cannot rule out the possibility that diabetes, a consequence of obesity, or menopause - related hormonal changes, which may alter mtorc1 signaling, underlie this relationship . We acknowledge that a main limitation of this study was its relatively small sample size . Given that we had difficulty discovering patients who were not overweight or obese, this was particularly so in the normal body weight group . Relatedly, we categorized the diagnosis at endometrial biopsy into two groups, benign / hyperplasia and atypia / carcinoma, whereas a larger study population would have allowed for analysis of each diagnosis separately . However, atypia and carcinoma was a logical pairing grounded in previous work and our own observations of similarities in tissue biomarker staining . In addition, while we chose to analyze the p4e - bp1 expression level based on the literature indicating that this form of the protein is, not only highly correlated to mtor pathway activity, but also associated with ec aggressiveness and survival, the characterization of total 4e - bp1 protein or mrna would be of interest in future study . Due to the retrospective design of our study, a further limitation was that we were unable to obtain additional, potentially useful data, such as the stage of menstrual cycle at biopsy, for the premenopausal women in our study population . We also acknowledge that the archival tissue specimens could be subject to variations in processing and storage, thus potentially affecting marker expression . However, we believe that using clinical biopsy tissue specimens that were directly submerged into formalin fixative and then embedded in paraffin adequately preserved the tissue and biomarkers of interest, and there was no suggestion that data were affected by the length of storage . Further, we used tissue samples from within a 2-year period to reduce the potential that time and storage could cause alterations in biomarkers . To preserve the immunoreactivity of the tissue overall, a key strength of this study is its novelty, as it is among the first to consider obesity status as a factor associated with mtorc1 signaling in situ . Our findings confirm that activation of 4e - bp1 is higher in preneoplastic and neoplastic lesions, and suggest that an elevated activation of 4e - bp1 is more characteristic of obese versus nonobese women with these lesions . Larger samples sizes and further investigation is necessary as additional insight into these associations may provide new opportunities for prevention, treatment, and control of ec.
Diseases that affect the buccal cavity are considered a serious public health problem . Among the more prevalent periodontal diseases and dental caries are usually caused by the presence of biofilm as a result of ineffective mechanical oral hygiene practices . For this reason, chemical agents are important to reduce gingivitis, periodontal disease, and dental caries.1 candida albicans has already been isolated from the dental biofilm, caries, and the periodontal pockets, which together with streptococcus mutans has more severe consequences.2 there are also several clinical issues associated with c. albicans which represent serious problems for treatment and prophylaxis.3 therefore, the development of more effective therapies to treat these diseases is one of the major public dental health challenges . Chlorhexidine gluconate (chx) is an antiseptic agent widely used to inhibit the formation of gingivitis and periodontitis.4 this cationic biguanidine interacts with the bacterial anionic surface and alters the integrity of the bacterial cell membrane leading to cytoplasm precipitation.5 however, besides the usual stability problems, this drug presents frequent side effects including taste perception alteration and an increase in tooth discoloration.6,7 for these reasons, several cyclodextrin (cd) inclusion complexes have been developed to increase stability and improve antimicrobial activity.4,8 the synergic association of chx and nystatin (nys) is a good option for the simultaneous control of c. albicans and s. mutans . Like chx, nys has an unpleasant flavor that leads to nausea during oral application, limiting its use and reducing patient compliance despite its undoubted effectiveness in the control of candidiasis and other buccal cavity disorders.9 the formulation of nanostructured nys and chx could overcome the low solubility and stability problems of these drugs, producing a controlled release system with new improved antimicrobial activity . Previous reports have shown that a nys and chx -cyclodextrin (cd) inclusion can improve stability and solubility.4,10 however, better results can be obtained using nanocarriers with sustained release properties, such as sodium montmorillonite (mmtna), the main constituent of bentonite (60%). This nanocarrier may present some synergistic effect due to the capability of these materials to adsorb and fix bacteria and fungi, immobilizing their toxins,11 and also presents parallel lamellae with internal anionic surfaces and interlamellar cations for equilibrium and charge stabilization . Therefore, inclusion processes can occur with several different cations or small molecules, leading to substance encapsulation in the interlamellar space, increasing system tortuosity and water uptake.12 the use of mmtna for acetate chlorhexidine inclusion has been previously reported,11 as has the use of cd for chx and nys encapsulation.4,10 however, the use of water - soluble methyl--cyclodextrin (mcd) and hydroxypropyl--cyclodextrin (hpcd) for chx, and mmtna for both drugs, are other possibilities to be studied in order to obtain stable formulations with tolerable taste.13 although there are several techniques to obtain cd complexes, such as co - precipitation, paste complexation, extrusion, spray drying, and kneading,6,1315 considering these drugs, most of the authors show only the application of solution technique . Taking into account the industrial applicability, it has become necessary to evaluate other options, as kneading, due the feasibility and low cost . The aim of this study is the development of new nano - structured drug - delivery systems with industrial applicability containing classical drugs for control of buccal pathologies, nys and chx, in order to increase the drug stability and improve the taste and antimicrobial action . Prepared and tested were inclusion complexes of nys and chx with mmtna, and chx with cd and two derivatives, mcd and hpcd . Ethanol 95% (merck, darmstadt, germany), mmtna (acros chemical co, pittsburgh, pa), cd, mcd, and hpcd (wacker gmbh, munich, germany), nys and chlorhexidine (sigma, st louis, mo) were pharmaceutical grade . Solutions were prepared with purified water obtained using a milli - q system (millipore, bedford, ma). The inclusion complexes chx: cd were prepared by solubilization - freeze - drying and kneading at molar ratios of 1:1, 1:2, and 1:3, using cd, mcd, and hpcd . Physical mixtures were prepared by mixing together cd and freeze - dried chx in a mortar at the same molar ratios . Using the kneading method, cd and chx an ethanol: water (70:30; v / v) solution was added, and the system mixed for 30 minutes to obtain a homogeneous paste . The paste was dried under reduced pressure and the granulometry adjusted using a 40 mesh sieve . Using the solution method, the appropriate proportions of chx and cd were mixed in 20 ml of distilled water using a magnetic stirrer for 72 hours . Nys and chx: mmtna nanosystems were prepared with the solution method using different cation exchange capacity (cec) values: 100%, 80%, and 60% of the total mmtna . The cec value considered was 100 meqv of cation to 100 g of mmtna.12 the inclusion reactions were performed in triplicate for different periods (1, 18, 24, and 48 hours), stirring at room temperature . The reaction mixtures were centrifuged at 4000 rpm for 40 minutes, and the precipitates dried in a vacuum desiccator . These nanosystems were characterized by x - ray powder diffraction (xrpd), fourier transform infrared spectroscopy (ftir), and differential scanning calorimetry (dsc). Xrpd patterns of nanosystems, physical mixtures, and pure substances were recorded with a rigaku miniflex diffractometer bd11197 (rigaku, tokyo, japan) using cuk radiation with a current of 30 ma, voltage of 40 kv, and a 2 angle between 2 and 20. ftir spectra were collected by an ir prestige-21 shimadzu a210045 (shimadzu, kyoto, japan) spectrometer using 2% kbr pellets and wavenumber between 4000 and 400 cm . Dsc analyses were carried out with dsc 882e mettler - toledo equipment (mettler - toledo, greifensee, switzerland) using hermetically sealed aluminum pans under a nitrogen flow of 28 mlmin and heating rate of 10c min . The s. mutans (atcc25175) and c. albicans (atcc36901) strains were grown at 37c for 24 hours in the presence of the two drugs being tested and the isolated carriers (cd and mmtna), with simultaneous comparison with the chx and mmtna nanosystems and chx 0.12% solution formulations . The inoculum containing 10 cells / ml was uniformly seeded on plates containing solid brain heart infusion broth . After drying the surface, 10 l of each sample was placed on the culture medium surface and incubated for 24 and 48 hours at 37c . The inhibition effect was verified by the presence of inhibition zones around the area on the plate where the solution was deposited and sized for analysis and comparison . Mouthwash formulations were developed with chx (0.120% w / v) or with cd inclusion complexes . Water - soluble mint essence (0.1% v / v), ethanol (0.050% v / v), polysorbate 20 (0.500% v / v), sorbitol (70% w / w aqueous solution, 15.0% v / v), menthol (0.010% w / v), and water (20%) were mixed until complete dissolution, and the volume was adjusted to 100% with water . The formulations were assayed by high efficiency liquid chromatography (hplc) as described below . The hplc system used was a lc-10 a / vp shimadzu (shimadzu, kyoto, japan) with a chromatography c18 column (250 mm 4.6 mm, 5 m particle size), the mobile phase was methanol: water with triethylamine 0.4% (63:37; v / v) with a 0.8 ml min flow rate . The analysis was performed at room temperature, with a 20 l injection volume and monitored at a wavelength of 240 nm . The running time was established at 15 minutes.6,16 the stability tests were performed based on international conference on harmonization guidelines17 with chx alone and inclusion complex the chx:c, chx: mcd, and chx: hpcd . All the inclusion complexes were at a molar ratio of 1:1 and obtained by kneading . The formulations were conditioned in a nova tica (so paulo, brazil) climatic chamber set to a relative humidity of 45c 2c and 75% 5% . The chx: hpcd inclusion complex preparation, formulation, and stability evaluation have not yet been described in the literature . The chx assay was carried out according to the usp 34.6,16 one - way analysis of variance and wilcoxon matched pairs tests were used to analyze all data obtained in this study using statsoft statistica (statsoft inc, tulsa, ok) software . Ethanol 95% (merck, darmstadt, germany), mmtna (acros chemical co, pittsburgh, pa), cd, mcd, and hpcd (wacker gmbh, munich, germany), nys and chlorhexidine (sigma, st louis, mo) were pharmaceutical grade . Solutions were prepared with purified water obtained using a milli - q system (millipore, bedford, ma). The inclusion complexes chx: cd were prepared by solubilization - freeze - drying and kneading at molar ratios of 1:1, 1:2, and 1:3, using cd, mcd, and hpcd . Physical mixtures were prepared by mixing together cd and freeze - dried chx in a mortar at the same molar ratios . Using the kneading method, cd and chx an ethanol: water (70:30; v / v) solution was added, and the system mixed for 30 minutes to obtain a homogeneous paste . The paste was dried under reduced pressure and the granulometry adjusted using a 40 mesh sieve . Using the solution method, the appropriate proportions of chx and cd were mixed in 20 ml of distilled water using a magnetic stirrer for 72 hours . The inclusion complexes chx: cd were prepared by solubilization - freeze - drying and kneading at molar ratios of 1:1, 1:2, and 1:3, using cd, mcd, and hpcd . Physical mixtures were prepared by mixing together cd and freeze - dried chx in a mortar at the same molar ratios . Using the kneading method, cd and chx were mixed in a mortar for 5 minutes . An ethanol: water (70:30; v / v) solution was added, and the system mixed for 30 minutes to obtain a homogeneous paste . The paste was dried under reduced pressure and the granulometry adjusted using a 40 mesh sieve . Using the solution method, the appropriate proportions of chx and cd were mixed in 20 ml of distilled water using a magnetic stirrer for 72 hours . Nys and chx: mmtna nanosystems were prepared with the solution method using different cation exchange capacity (cec) values: 100%, 80%, and 60% of the total mmtna . The cec value considered was 100 meqv of cation to 100 g of mmtna.12 the inclusion reactions were performed in triplicate for different periods (1, 18, 24, and 48 hours), stirring at room temperature . The reaction mixtures were centrifuged at 4000 rpm for 40 minutes, and the precipitates dried in a vacuum desiccator . These nanosystems were characterized by x - ray powder diffraction (xrpd), fourier transform infrared spectroscopy (ftir), and differential scanning calorimetry (dsc). Xrpd patterns of nanosystems, physical mixtures, and pure substances were recorded with a rigaku miniflex diffractometer bd11197 (rigaku, tokyo, japan) using cuk radiation with a current of 30 ma, voltage of 40 kv, and a 2 angle between 2 and 20. ftir spectra were collected by an ir prestige-21 shimadzu a210045 (shimadzu, kyoto, japan) spectrometer using 2% kbr pellets and wavenumber between 4000 and 400 cm . Dsc analyses were carried out with dsc 882e mettler - toledo equipment (mettler - toledo, greifensee, switzerland) using hermetically sealed aluminum pans under a nitrogen flow of 28 mlmin and heating rate of 10c min . The s. mutans (atcc25175) and c. albicans (atcc36901) strains were grown at 37c for 24 hours in the presence of the two drugs being tested and the isolated carriers (cd and mmtna), with simultaneous comparison with the chx and mmtna nanosystems and chx 0.12% solution formulations . The inoculum containing 10 cells / ml was uniformly seeded on plates containing solid brain heart infusion broth . After drying the surface, 10 l of each sample was placed on the culture medium surface and incubated for 24 and 48 hours at 37c . The inhibition effect was verified by the presence of inhibition zones around the area on the plate where the solution was deposited and sized for analysis and comparison . Mouthwash formulations were developed with chx (0.120% w / v) or with cd inclusion complexes . Water - soluble mint essence (0.1% v / v), ethanol (0.050% v / v), polysorbate 20 (0.500% v / v), sorbitol (70% w / w aqueous solution, 15.0% v / v), menthol (0.010% w / v), and water (20%) were mixed until complete dissolution, and the volume was adjusted to 100% with water . The formulations were assayed by high efficiency liquid chromatography (hplc) as described below . The hplc system used was a lc-10 a / vp shimadzu (shimadzu, kyoto, japan) with a chromatography c18 column (250 mm 4.6 mm, 5 m particle size), the mobile phase was methanol: water with triethylamine 0.4% (63:37; v / v) with a 0.8 ml min flow rate . The analysis was performed at room temperature, with a 20 l injection volume and monitored at a wavelength of 240 nm . The running time was established at 15 minutes.6,16 the stability tests were performed based on international conference on harmonization guidelines17 with chx alone and inclusion complex the chx:c, chx: mcd, and chx: hpcd . All the inclusion complexes were at a molar ratio of 1:1 and obtained by kneading . The formulations were conditioned in a nova tica (so paulo, brazil) climatic chamber set to a relative humidity of 45c 2c and 75% 5% . The chx: hpcd inclusion complex preparation, formulation, and stability evaluation have not yet been described in the literature . The chx assay was carried out according to the usp 34.6,16 one - way analysis of variance and wilcoxon matched pairs tests were used to analyze all data obtained in this study using statsoft statistica (statsoft inc, tulsa, ok) software . One - way analysis of variance and wilcoxon matched pairs tests were used to analyze all data obtained in this study using statsoft statistica (statsoft inc, tulsa, ok) software . Xrpd patterns of lyophilized chx, cd, and chx:cd complex are presented in figure 1 . As expected, lyophilized chx shows an amorphous pattern after water uptake, while cd shows a highly crystalline pattern . The xrpd patterns of the inclusion complexes show greater amorphous characteristics than pure cd and physical mixtures . This is evidence of inclusion, as this disorder phenomenon has already been described by corts and colleagues.18 these amorphous patterns are characterized by the disappearance and decrease of peak intensity a decrease of 70%80% with the kneading method and 80%90% with the solution method . These amorphous patterns were observed at molar ratios of 1:1, 1:2, and 1:3 . The ftir spectra of the cd inclusion complex (data not shown) showed the characteristic peaks of chx at 17001500 cm, corresponding to aromatic ring c = c stretching which is different from the cd peaks . The spectra of both the physical mixture and the complex illustrated the typical peaks of each material of which the inclusion complex is composed, with the presence of significant frequency shifts . These results demonstrate the efficacy of the inclusion process with a chx presence.18 as observed for cd, mcd presents a crystalline pattern (xrpd) and inclusion is evidenced by sample amorphization . The peak decrease was observed in samples prepared by both methods and at all molar ratios: 1:1, 1:2, and 1:3 . In this case this lack of crystallinity indicates inclusion complex formation as shown by figueiras and colleagues.19 the characteristic peaks of both chx and mcd can be seen in the ftir spectra of the inclusion complexes with significant shifts, confirming the presence of chx in the inclusion processes . The characteristic peaks of hpcd are at 2 angles of 11.4 and 19.3. a 60% peak reduction can be observed in the xrpd pattern of the complex produced by kneading at a 1:1 molar ratio . The samples produced by the solution method did not show significant peak reduction, meaning that the method employed is determinative to this complex formation . Wang and colleagues observed the same characteristics producing trans - ferulic acid complexes.20 the characteristic peaks of both chx and hpcd were demonstrated by the ftir spectra of the inclusion complexes with significant shifts, confirming the effect of the presence of chx in the production processes . Thermal analysis, which is based on the comparison of the thermal behavior of single components, their physical mixture, and inclusion complexes, is the usual technique for qualitative investigation of cd inclusion complex formation . 21 dsc curves for the pure substances and inclusion compounds are shown in figure 2 . The dsc curve of chx shows stability from 25c to 174c, after which, a sequence of thermal decomposition events were observed . Dsc curves of inclusion compounds show that the chx degradation peak temperature was higher for all inclusion complexes obtained with cd and its synthetic derivatives (mcd and hpcd). This evidences a new supramolecular compound formation characteristic of inclusion complexes with improved stability . The disappearance or flattening of the drug melting point peak are considered conclusive evidence of inclusion compound formation.21 yallapu and colleagues have observed these same patterns for curcumin complexation, where the melting endothermic peak of the drug completely disappeared in the dsc curves.22 in all cd derivatives studied, the kneading method was chosen to prepare inclusion complexes of chx, cd, mcd, and hpcd at a 1:1 molar ratio, due its low cost and industrial applicability . Mmtna was used as a nanocarrier due to its sustained release properties and synergistic antimicrobial activity . The nanosystems produced were characterized by xrpd, dsc, and ftir . A decrease in the 2 value observed in the xrpd analysis is an indicative factor of the inclusion process since a reduction in this angle is associated with an increase in basal spacing, which is related to drug inclusion.23 the chlorhexidine nanosystem obtained showed a 2 value decrease, described in table 1, when compared with pure mmtna, indicating the success of the inclusion process . However, there is no significant difference between all the proposed conditions presented in table 1 (p> 0.05). Chx molecule rearrangement should also be considered since, like an alkyl ammonium chain, chx may have a particular conformation that expands basal spacing in low concentration between lamellae . Absorption of chx can occur when a high concentration is used for inclusion . In order to avoid this, a 60% cec was used (24 hours). Under these conditions, the inclusion complex presented an average lamellar spacing of 16.8 1.3 and an inclusion yield of 70.0% 0.02% . These results corroborate those observed by meng and colleagues11 and yang and colleagues,24 who reported an interlamellar space increase from 15.1 to 19.4 and 14.5 to 16.6, respectively, for chx acetate . These authors have observed that the chx molecular volume is around 0.5 nm, slightly less than the 0.69 nm calculated by subtracting the mmtna monolayer size (0.96 nm) from the basal spacing measurement (1.65 nm). This larger molecular volume may be due to a different conformation assumed by chx in mmtna lamellae.11 dsc curves for the pure substances and nanosystems are shown in figure 3 . Nanosystem c3 shows an endothermic peak between 200c and 220c, which may indicate the release of chx from lamellar spacing . This result may be evidence that substances intercalated between clay lamellae can be observed through the calorimetric patterns of nanosystems at different temperatures.24 ftir spectra of pure chx, mmtna, physical mixtures, and nanosystems present characteristic stretching bands due to water absorption at 3462 cm, 3400 cm, and 3420 cm . The hydroxyl group from the al - oh bond presents a stretching band at around 3620 cm . Vibration bands of silicate between montmorillonite lamellae are shown at 1114 cm and 1047 cm for the si - oh bond.11,25 regarding the spectra of chx, this shows a shift of the stretching bands for the aromatic n - h and c - h bonds at 3396 cm and 3226 cm respectively . It is important to note that the spectra of the physical mixture, which simulates c3, showed differences when compared with the spectra of the inclusion process, indicating their contrasting profiles and confirming the inclusion process . The nys: mmtna inclusion studies showed a 2 value decrease in relation to pure mmtna, which is evidence of nanosystem formation under all tested conditions (table 2). However, the 24-hour inclusion period produced a larger basal spacing with all tested cec . The nanosystem n3 (60% cec) was chosen for characterization due to the higher basal spacing compared with 100% cec and the economic aspect . This nanosystem presented an average lamellae spacing of 16.63 0.33 (figure 4) and an indirect inclusion yield of 54% 0.01% . Xrpd profiles for the pure substances and nanosystem n3 at different cec values are shown in figure 4 . This result is evidence of inclusion complex formation in the clay lamellae . The calorimetric pattern of nys derivatives are shown in figure 5 . The characteristic thermal event related to nys, its melting point, occurs between 160c and 180c, but this is not registered on the dsc curve of the nys: mmtna 60% cec this pattern is similar to the pure mmtna with an endothermic peak at 100c due to water loss . The absence of nys endothermic melting point is evidence of inclusion, since the presence of the drug in the nanosystem was confirmed by ftir analysis (data not shown). The inhibition halo technique has the greatest applicability in the evaluation of antimicrobial activity of biologically active montmorillonite chlorhexidine nanocomposite.24,26 therefore, the inhibition halo technique was used to evaluate and compare the activity of the nanosystems and pure drugs and the possible synergy of the drugs with clays and cd . These nanosystems were prepared by kneading, using chx and cds at a 1:1 molar ratio . The inhibition halo diameters observed for c. albicans and s. mutans are shown in table 3 . All samples showed very similar activity and were quite similar to the chx aqueous solution against gram - positive bacteria and fungi . However, corts and colleagues18 have shown that a chx:cd inclusion complex demonstrated modified antimicrobial activity when its minimum inhibitory concentration was measured, increasing its efficacy against pathogenic bacteria . Herein, contrary results were obtained with no statistical difference in the samples tested (p = 0.07653). The halo diameters of the mmtna nanosystems are represented in table 4 and figure 6 . The mmtna suspension did show any inhibition activity, which corroborates results shown by other authors.11,24,26,27 these authors not only tested sodium but also calcium montmorillonite . Chx aqueous solution 0.12% and the c3 derivative showed a large growth inhibition zone for both microorganisms . Halos for the chx solution against s. mutans were between 26 and 27 mm, while the c3 derivative presented halos of around 2122 mm . Furthermore, halos against c. albicans of 18, 19, and 26 mm were observed using the chx solution, and 11, 12, and 20 mm with the c3 derivative . The smaller halo diameters presented by the c3 derivative were expected since it is an inclusion product with known prolonged release characteristics.24,27 yang and colleagues24 studied the antimicrobial activity of chlorhexidine acetate: mmtna nanosystems and found that chx was released slowly and the inhibition halo could be seen after 1 year . However, the results of this present study show that the chx c3 derivative presented acceptable immediate release properties without total drug release, which is ideal for the treatment of oral diseases . Pure nys, an antifungal agent, did not show any inhibition activity against s. mutans, as expected, but presented halos ranging from 16, 19, and 27 mm against c. albicans strains (table 4, figure 6). These results are similar to those found by carrillo - muoz and colleagues of 20 mm.28 the nys nanosystem was expected to show the same prolonged release profile as c3, but did not present any initial inhibition, indicating that the release is possibly slower than chlorhexidine due to lower nys solubility in water and saliva . However, the large fluid volume in vivo could lead to a more effective release, justifying the use of this nanosystem in prolonged release tablets, which could be a therapeutic alternative to more complex pharmaceutical forms . Initially, the chx stability in a mouthwash formulation was investigated because chx has several incompatibilities with common excipients, such as some ions and organic compounds . These incompatibility reactions lead to the formation of the degradation product p - chloroaniline and its subsequent precipitation.29,30 no precipitation or incompatibility was observed between chx and formulation excipients 48 hours after formulation preparation with pure chx . Mouthwash formulations were produced with pure chx and the inclusion complexes using 0.12% w / v chx content . These samples were stored in a climatic chamber at 45c 2c and 75% 5% relative humidity (rh) in order to evaluate the thermal stability . The results of the stability study are presented in figure 7, and a great increase in the stability of all nanosystems studied can be seen, indicating the potential for the commercial application of these new cd derivatives . Xrpd patterns of lyophilized chx, cd, and chx:cd complex are presented in figure 1 . As expected, lyophilized chx shows an amorphous pattern after water uptake, while cd shows a highly crystalline pattern . The xrpd patterns of the inclusion complexes show greater amorphous characteristics than pure cd and physical mixtures . This is evidence of inclusion, as this disorder phenomenon has already been described by corts and colleagues.18 these amorphous patterns are characterized by the disappearance and decrease of peak intensity a decrease of 70%80% with the kneading method and 80%90% with the solution method . These amorphous patterns were observed at molar ratios of 1:1, 1:2, and 1:3 . The ftir spectra of the cd inclusion complex (data not shown) showed the characteristic peaks of chx at 17001500 cm, corresponding to aromatic ring c = c stretching which is different from the cd peaks . The spectra of both the physical mixture and the complex illustrated the typical peaks of each material of which the inclusion complex is composed, with the presence of significant frequency shifts . These results demonstrate the efficacy of the inclusion process with a chx presence.18 as observed for cd, mcd presents a crystalline pattern (xrpd) and inclusion is evidenced by sample amorphization . The peak decrease was observed in samples prepared by both methods and at all molar ratios: 1:1, 1:2, and 1:3 . In this case this lack of crystallinity indicates inclusion complex formation as shown by figueiras and colleagues.19 the characteristic peaks of both chx and mcd can be seen in the ftir spectra of the inclusion complexes with significant shifts, confirming the presence of chx in the inclusion processes . The characteristic peaks of hpcd are at 2 angles of 11.4 and 19.3. a 60% peak reduction can be observed in the xrpd pattern of the complex produced by kneading at a 1:1 molar ratio . The samples produced by the solution method did not show significant peak reduction, meaning that the method employed is determinative to this complex formation . Wang and colleagues observed the same characteristics producing trans - ferulic acid complexes.20 the characteristic peaks of both chx and hpcd were demonstrated by the ftir spectra of the inclusion complexes with significant shifts, confirming the effect of the presence of chx in the production processes . Thermal analysis, which is based on the comparison of the thermal behavior of single components, their physical mixture, and inclusion complexes, is the usual technique for qualitative investigation of cd inclusion complex formation . 21 dsc curves for the pure substances and inclusion compounds are shown in figure 2 . The dsc curve of chx shows stability from 25c to 174c, after which, a sequence of thermal decomposition events were observed . Dsc curves of inclusion compounds show that the chx degradation peak temperature was higher for all inclusion complexes obtained with cd and its synthetic derivatives (mcd and hpcd). This evidences a new supramolecular compound formation characteristic of inclusion complexes with improved stability . The disappearance or flattening of the drug melting point peak are considered conclusive evidence of inclusion compound formation.21 yallapu and colleagues have observed these same patterns for curcumin complexation, where the melting endothermic peak of the drug completely disappeared in the dsc curves.22 in all cd derivatives studied, the kneading method was chosen to prepare inclusion complexes of chx, cd, mcd, and hpcd at a 1:1 molar ratio, due its low cost and industrial applicability . Mmtna was used as a nanocarrier due to its sustained release properties and synergistic antimicrobial activity . The nanosystems produced were characterized by xrpd, dsc, and ftir . A decrease in the 2 value observed in the xrpd analysis is an indicative factor of the inclusion process since a reduction in this angle is associated with an increase in basal spacing, which is related to drug inclusion.23 the chlorhexidine nanosystem obtained showed a 2 value decrease, described in table 1, when compared with pure mmtna, indicating the success of the inclusion process . However, there is no significant difference between all the proposed conditions presented in table 1 (p> 0.05). Chx molecule rearrangement should also be considered since, like an alkyl ammonium chain, chx may have a particular conformation that expands basal spacing in low concentration between lamellae . Absorption of chx can occur when a high concentration is used for inclusion . In order to avoid this, a 60% cec was used (24 hours). Under these conditions, the inclusion complex presented an average lamellar spacing of 16.8 1.3 and an inclusion yield of 70.0% 0.02% . These results corroborate those observed by meng and colleagues11 and yang and colleagues,24 who reported an interlamellar space increase from 15.1 to 19.4 and 14.5 to 16.6, respectively, for chx acetate . These authors have observed that the chx molecular volume is around 0.5 nm, slightly less than the 0.69 nm calculated by subtracting the mmtna monolayer size (0.96 nm) from the basal spacing measurement (1.65 nm). This larger molecular volume may be due to a different conformation assumed by chx in mmtna lamellae.11 dsc curves for the pure substances and nanosystems are shown in figure 3 . Nanosystem c3 shows an endothermic peak between 200c and 220c, which may indicate the release of chx from lamellar spacing . This result may be evidence that substances intercalated between clay lamellae can be observed through the calorimetric patterns of nanosystems at different temperatures.24 ftir spectra of pure chx, mmtna, physical mixtures, and nanosystems present characteristic stretching bands due to water absorption at 3462 cm, 3400 cm, and 3420 cm . The hydroxyl group from the al - oh bond presents a stretching band at around 3620 cm . Vibration bands of silicate between montmorillonite lamellae are shown at 1114 cm and 1047 cm for the si - oh bond.11,25 regarding the spectra of chx, this shows a shift of the stretching bands for the aromatic n - h and c - h bonds at 3396 cm and 3226 cm respectively . It is important to note that the spectra of the physical mixture, which simulates c3, showed differences when compared with the spectra of the inclusion process, indicating their contrasting profiles and confirming the inclusion process . The nys: mmtna inclusion studies showed a 2 value decrease in relation to pure mmtna, which is evidence of nanosystem formation under all tested conditions (table 2). However, the 24-hour inclusion period produced a larger basal spacing with all tested cec . The nanosystem n3 (60% cec) was chosen for characterization due to the higher basal spacing compared with 100% cec and the economic aspect . This nanosystem presented an average lamellae spacing of 16.63 0.33 (figure 4) and an indirect inclusion yield of 54% 0.01% . Xrpd profiles for the pure substances and nanosystem n3 at different cec values are shown in figure 4 . This result is evidence of inclusion complex formation in the clay lamellae . The calorimetric pattern of nys derivatives are shown in figure 5 . The characteristic thermal event related to nys, its melting point, occurs between 160c and 180c, but this is not registered on the dsc curve of the nys: mmtna 60% cec this pattern is similar to the pure mmtna with an endothermic peak at 100c due to water loss . The absence of nys endothermic melting point is evidence of inclusion, since the presence of the drug in the nanosystem was confirmed by ftir analysis (data not shown). The inhibition halo technique has the greatest applicability in the evaluation of antimicrobial activity of biologically active montmorillonite chlorhexidine nanocomposite.24,26 therefore, the inhibition halo technique was used to evaluate and compare the activity of the nanosystems and pure drugs and the possible synergy of the drugs with clays and cd . These nanosystems were prepared by kneading, using chx and cds at a 1:1 molar ratio . The inhibition halo diameters observed for c. albicans and s. mutans are shown in table 3 . All samples showed very similar activity and were quite similar to the chx aqueous solution against gram - positive bacteria and fungi . However, corts and colleagues18 have shown that a chx:cd inclusion complex demonstrated modified antimicrobial activity when its minimum inhibitory concentration was measured, increasing its efficacy against pathogenic bacteria . Herein, contrary results were obtained with no statistical difference in the samples tested (p = 0.07653). The halo diameters of the mmtna nanosystems are represented in table 4 and figure 6 . The mmtna suspension did show any inhibition activity, which corroborates results shown by other authors.11,24,26,27 these authors not only tested sodium but also calcium montmorillonite . Chx aqueous solution 0.12% and the c3 derivative showed a large growth inhibition zone for both microorganisms . Halos for the chx solution against s. mutans were between 26 and 27 mm, while the c3 derivative presented halos of around 2122 mm . Furthermore, halos against c. albicans of 18, 19, and 26 mm were observed using the chx solution, and 11, 12, and 20 mm with the c3 derivative . The smaller halo diameters presented by the c3 derivative were expected since it is an inclusion product with known prolonged release characteristics.24,27 yang and colleagues24 studied the antimicrobial activity of chlorhexidine acetate: mmtna nanosystems and found that chx was released slowly and the inhibition halo could be seen after 1 year . However, the results of this present study show that the chx c3 derivative presented acceptable immediate release properties without total drug release, which is ideal for the treatment of oral diseases . Pure nys, an antifungal agent, did not show any inhibition activity against s. mutans, as expected, but presented halos ranging from 16, 19, and 27 mm against c. albicans strains (table 4, figure 6). These results are similar to those found by carrillo - muoz and colleagues of 20 mm.28 the nys nanosystem was expected to show the same prolonged release profile as c3, but did not present any initial inhibition, indicating that the release is possibly slower than chlorhexidine due to lower nys solubility in water and saliva . However, the large fluid volume in vivo could lead to a more effective release, justifying the use of this nanosystem in prolonged release tablets, which could be a therapeutic alternative to more complex pharmaceutical forms . Initially, the chx stability in a mouthwash formulation was investigated because chx has several incompatibilities with common excipients, such as some ions and organic compounds . These incompatibility reactions lead to the formation of the degradation product p - chloroaniline and its subsequent precipitation.29,30 no precipitation or incompatibility was observed between chx and formulation excipients 48 hours after formulation preparation with pure chx . Mouthwash formulations were produced with pure chx and the inclusion complexes using 0.12% w / v chx content . These samples were stored in a climatic chamber at 45c 2c and 75% 5% relative humidity (rh) in order to evaluate the thermal stability . The results of the stability study are presented in figure 7, and a great increase in the stability of all nanosystems studied can be seen, indicating the potential for the commercial application of these new cd derivatives . Xrpd, ftir, and dsc analysis were used to confirm the proposed nanosystems formation and the suitability of the production methods . However, the nanosystems that showed best antimicrobial effect were chx and cd inclusion complexes and chx: mmtna 60% cec 24 hours . Although nys nanosystems presented no antimicrobial activity with the technique applied here, this does not preclude their use since this method does not represent in - vivo conditions . Moreover, these nanosystem formulations present the following improvements: higher stability for all chlorhexidine inclusion complexes compared with pure chlorhexidine under 40c 2c and 75% 5% rh and being a sustained release system, the potential for nys nanosystems to mask the bitter taste, justifying subsequent in - vivo studies . For these reasons,
The inevitable exposure of salivary glands to radiation occurs frequently during radiotherapy of the head and neck region, which results in decreased saliva secretion, called xerostomia, shortly after a few radiation fractions . This may persist for the rest of the patient's life, contributing to oral infections, caries and reduction in taste, and has been shown to be very prejudicial to the quality of life4 . Of the cells that comprise the salivary glands, the secretory cells are the most radiosensitive, especially the serous secretors4,7 . The submandibular gland has two types of secretory cells, serous and mucous cells, and it is frequently exposed during radiotherapy of the head and neck . In rodents, the submandibular serous cells are confined to the convoluted granular tubules, and the mucous cells are found in the acini1 . Different methods have been used to estimate the impact of various ionizing radiation doses on secretory cells, such as qualitative descriptions of acute light and electron microscopic alterations7,9,15,22,23 . In order to overcome the influence of fibrosis and different degrees of atrophy in different cells, morphometric determinations of different cell types have been applied at the level of light microscopy . However, such measurements do not reflect subtle alterations in the morphology of individual cell types7 . Unfortunately, there is no adequate treatment for the deleterious effects of radiation on the salivary glands . Therefore, research has been undertaken on the administration of substances called radioprotectors that may inhibit or attenuate these effects . In a series of experiments demonstrating the radioprotecting effects of wr-2721, isoproterenol18 and camp17, the weight of the salivary glands was the only factor used to determine the relative radiation injuries under different experimental conditions . Among the substances that promote radioprotection, selenium is a mineral essential to the organism and has been shown to have a radioprotective action in rat intestines10, to increase survival in animals24 and cell cultures14,20,25 . Therefore, the aim of this study was to perform an ultrastructural evaluation of the radioprotective effect of sodium selenite on the damage caused by gamma radiation on the submandibular gland secretory cells in rats . Fifty - seven 3-month - old male albino wistar rats, weighing 250 - 300 g were used . The rats were housed in polycarbonate cages (5 - 6 rats per cage) under a light / dark (14/10 h) cycle . The entire experiment was performed in agreement with the ethical principles for animal research established by the brazilian college for animal experimentation (cobea). The project was approved by the institutional committee for ethics in animal research (state university of campinas, unicamp) on march 11, 2004 . The rats were randomly assigned to 4 groups: control group, irradiated group, sodium selenite group and sodium selenite / irradiated group . The other groups were divided into 6 sub - groups in accordance with the time of removal of the submandibular gland after irradiation: 4, 8, 12, 24, 48 and 72 h. the animals belonging to the sodium selenite and sodium selenite / irradiated groups received 0.5 mg / kg body weight of sodium selenite (merck kgaa, darmstadt, germany) intraperitoneally and saline was administered to the others . Twenty - four hours after administration of sodium selenite, all rats were anesthetized by intraperitoneal injection of sodium pentobarbital (nembutal, 30 mg / kg body weight). The animals in the irradiated and sodium selenite / irradiated groups had only the head and neck region irradiated with a single, fixed nominal dose of 15 gy of gamma radiation co. limitation of the exposed area was obtained by collimating the apparatus . The treatment distance to the focal point on the skin was 80 cm, and the apparatus used was an alcion cgr ii model with a yield of 1.07 gy / min, with an average of 1.25 mv . At the previously established times, the animals were anesthetized by intraperitoneal injection of sodium pentobarbital (nembutal, 40 mg / kg body weight), for surgical removal of the right submandibular gland . Subsequently, the rats were sacrificed under general anesthetic with sodium pentobarbital (nembutal). The glands were sectioned into fragments of approximately by 1 mm and fixed by immersion in 2.5% glutaraldehyde at ph 7.3, 0.1 m sodium cacodylate buffer and 0.1 m sucrose for 24 h at 4c . The specimens were postfixed by immersion for 1 h in 1% osmium tetroxide, 0.1 m buffered in a 0.1 m phosphate buffer (ph 7.3) at 25c . They were then dehydrated in a graded acetone series (50%, 70%, 80%, 90%, 100%) and embedded in araldite resin8 . For light microscopy, 1-m - thick sections were cut on an mt2b sorvall porter blum ultramicrotome and stained with toluidine blue . After light microscopy field selection (area with terminal secretory portions and convoluted ducts), ultrathin sections (60 nm) were cut with an mt2c ultramicrotome for transmission electron microscopy . These sections were stained with uranyl acetate and lead citrate and examined with a zeiss em-10 transmission electron microscope (zeiss, . Only alterations that were observed in 3 selected rats of each sub - group were considered . The figures presented in the results section are representative of these 3 rats per sub - group . Four hours after irradiation, vascularization, cytoplasmic and nuclear alterations were observed in both types of secretory cells in the irradiated group . Diminished concentration, union, undefined limits and alterations in the electrodensity of serous granule components (figure 1b) and the presence of electrodense content inside some mucous granules (figure 1d) were found in the secretion granules . With regard to the nuclei, pleomorphism, chromatin condensation, thickening and rupture of the membrane were observed . Around these, altered and destroyed organelles, such as endoplasmic reticulum and mitochondria, were found (figure 1 g). Due to the rupture of the rough endoplasmic reticulum in all the studied groups, the mucous cells showed greater integrity than the serous cells in the first time period studied (figure 2a, b). However, the behavior among the groups and times was similar for the two types of cells . In the mucous secretory cells, the sodium selenite and sodium selenite / irradiated groups presented the same alterations, but with less intensity than that found in the irradiated group, mainly in the sodium selenite / irradiated group . In the serous secretory cells, the intensity of the alterations was similar to that found in the irradiated group, except for the greater integrity of the secretion granules in the sodium selenite / irradiated group (figure 2c, h after irradiation, the intensity of the alterations in the two types of cells in the irradiated group, was greater, with more tissue destruction and disorganization (figure 3a). Both types of cells in the sodium selenite and sodium selenite / irradiated groups presented with diminished alterations at 8 h after irradiation (figure 3b, c). Regression of the alterations only occurred in the irradiated group from 24 h (figure 3d), while the sodium selenite and sodium selenite / irradiated groups presented greater integrity (figure 3e, f). After 48 and 72 h, the alterations diminished in all groups; the sodium selenite group, and especially the sodium selenite / irradiated group presented greater similarity to the control group than the irradiated group (figure 4). These latter times were characterized by an increase in the size of the nuclei and the number of organelles, mainly the rough endoplasmic reticulum . Gamma radiation (15 gy) caused degenerative processes in both types of secretory cells, but with greater destruction of the serous cells, in agreement with the findings of stern, et al.19 (1976) and vissink, et al.22 (1991). The greater radiosensitivity of serous cells is explained by the hypothesis put forward by abok, et al.1(1984) according to which, the serous secretion granules have proteolytic and metallic transmission enzymes, while the mucous secretion granules mainly contain glycoproteins . The transmission materials are known to promote the induction of oxidative stress, potentiating the damage to the serous granule membranes . As a result, the proteolytic enzymes may infiltrate and damage the cytoplasm, causing autolysis and cellular death . Later research has confirmed the involvement of the serous secretion granules in the increased damage caused by ionizing radiation4,2,11 . The alterations found in the present study are consistent with those observed in similarly investigations15,21 . The rupture of the rough endoplasmic reticulum increased the amount of free polyribosomes in the cytoplasm, as previously reported elsewhere15,19,22 . The radiosensitivity of the mitochondria has also been observed in other studies involving submandibular glands9,15,19,22,23 . The destruction and decrease in the number of secretion granules observed right from the first assessment after irradiation have been previously observed2,15,19,22 . In the mucous granules, in addition to these alterations, electrodense fibrils were also found, corroborating the findings of vissink, et al.22 (1991), who suggested they were a sign of repair and regeneration . Nuclear alterations were noticed in the present study and in other ultrastructural studies15,19,21 on the secretory cells of submandibular glands . Among these alterations, nuclear rupture and less electrodensity of the euchromatin were found as well, characterizing cellular necrosis according to rafferty, et al.13 (2003). In both types of secretory cells, the alterations found in the present research after 4 h were similar to those observed in other investigations15,22,23 but were in disagreement with the evaluation by stern, et al.19 (1976), who observed the first alterations as from the fourth day after exposure to 2 gy of neutron radiation . In relation to the time of greatest destruction, there was greater intensity after 8 and 12 h for both cells, but stern, et al.19 (1976), found that the first great alterations occurred on the fourth day . Reade and steidler15 (1985), using an 8 gy dose of x radiation, reported greater alterations after 48 h, while vissink, et al.22 (1991) observed the greatest changes by light and electron microscopy at 72 h. with respect to the period at which destruction diminished, indications of recovery were observed from 24 h, with decrease in damage at the last time assessed . This result differed from those of other studies, in which the indications of recovery appeared from the sixth day15,22 . With the aim of avoiding or attenuating the effects of ionizing radiation, radioprotective substances are used, which act by means of antioxidant action18,11 . According to aruoma3 (1996) animals have lines of defense like antioxidant enzymes that are able to inhibit the production of free radicals . Manganese superoxide dismutase, and copper and zinc superoxide dismutase enzymes are considered to be agents in the first line of defense against free radicals, removing the hydrogen superoxide and peroxide3 . But the most important hydrogen peroxide removal is done by the peroxidase glutathione enzyme, which acts in the presence of selenium3,21 . Its probable mechanism of action occurs by means of its covalent binding to proteins, forming selenoproteins, with emphasis on peroxidase glutathione3,5,13,14,20,21 and thioredoxin reductase, which have similar antioxidant properties5,19,21 . Among the selenium based components, inorganic selenium components, such as sodium selenite, sodium selenite diminished the effects of radiation at all the times studied in both secretory cells, but did not prevent the sodium selenite / irradiated group from undergoing alterations similar to those of the irradiated group . Four hours after irradiation, the sodium selenite / irradiated group differed from the irradiated group only by the larger number of whole secretory granules . At the following times, the sodium selenite / irradiated group presented significant and progressive tissue integrity and organization in relation to the irradiated group . In view of these observations, sodium selenite probably helped to maintain the integrity of the secretion granules initially, preventing leakage of their contents into the cellular cytoplasm, with the consequent rupture of the organelles and cell destruction found with greater frequency in the irradiated group . A similar effect of sodium selenite was found by tuji, et al.21 (2005) in wound healing in irradiated rats . In spite of having antioxidant action, selenium in excess has toxic activity, as demonstrated by studies on cell cultures14,20,25 and in vivo studies5,6 . Its toxicity is characterized by promotion of oxidative lesions in dna14, cell death14, inhibition of cellular proliferation6 and morphologic alterations5 . The cytotoxicity of selenium is associated with the oxidation of glutathione and other thiols, giving rise to selenodiglutathione and selenopersulfide glutathione which promote the formation of hydrogen superoxide and peroxide radicals14,24,25 . The sodium selenite dose used in the present study (0.5 mg / kg body weight) was chosen because it promoted a radioprotector effect on wound healing in rats in a previous study21, without causing histomorphologic damage in the rats treated only with sodium selenite . Although this dose promoted radioprotection in the present study, this same dose in the sodium selenite group caused degenerative processes similar to those caused by the radiation in the irradiated group . This difference in the results can be explained by the fact that the work of tuji, et al.21 (2005) was a histomorphologic analysis and the present study is an ultrastructural analysis . However, it is worth pointing out that during the course of the present study, no animal treated with sodium selenite died . Ionizing radiation results in a decrease in the accumulation of selenium by breaking its bond to proteins, involving the union of sulfur groups24 . In addition, this may also explain the decreased concentration of selenium in the cell, used to diminish the concentration of hydrogen peroxide as a result of the ionizing radiation, as opposed to its greater accumulation in the sodium selenite group and the resultant oxidation of thiols . Thus, it is suggested that the difference in the results between the sodium selenite and the sodium selenite / irradiated groups is due to the changes in protein metabolism after irradiation, leading to the reduced accumulation of selenium in the cells . Nordman, et al.12 (1976) stated that selenite uptake is around 5 - 7 times greater in malignant cells than in normal tissues . Therefore, in view of the dichotomy of sodium selenite acting as a radioprotective substance, and as a toxic substance at greater concentrations, in vivo studies about the toxicity of sodium selenite in various organs are suggested, in order to find doses that may simultaneously cause toxicity in neoplastic cells, accentuate the deleterious effects of ionizing radiation, and lastly, provide radioprotection to normal cells . Under this experimental conditions it was concluded that despite the alterations observed in the sodium selenite group, sodium selenite has a radioprotective action on the secretory cells of submandibular glands.
Root canal dressings are used usually during the endodontic treatment of teeth with apical periodontitis . Calcium hydroxide is considered the standard intracanal medication because of its antibacterial effect . However, clinical studies have shown that it is not possible to sterilize the root canals in necrotic teeth even using calcium hydroxide dressings . For these reason, new compounds as chlorhexidine, antibiotics, and natural compounds as propolis has been suggested to be used as alternative intracanal medications . Propolis in its crude form, as an extract, is not ideal for the endodontic use, therefore shows characteristic resinous and sticky . (2009) reached a propolis paste formulation for endodontic use using the lowest concentration of crude extract of propolis which retains its biological activity . Other authors have shown that propolis can be useful as a root canal dressing due to its low toxicity and broad antibacterial spectrum . In addition to the antimicrobial properties, intracanal medications must be easily removed from the root canal walls . The obstruction of dentinal tubules, by either smear layer or intracanal medication can impair the performance of the endodontic sealers . The penetration of the sealers into dentinal tubules improves the seal by increasing the contact surface to the root canal walls . The purpose of this study was to evaluate the cleaning of the root canal walls after the placement of calcium hydroxide- or propolis - based intracanal medications, using scanning electron microscopy (sem). Twenty freshly extracted single - rooted human teeth from the department of dental materials, restorative dentistry and endodontics, bauru school of dentistry, brazil, were used . Conventional pulp chamber access procedures using spherical diamond and endo z drills (dentsply ind . E com . Petrpolis, rj, brazil) were used and the working length determination was established at 1 mm from the apical foramen using a 10 k - file . Cleaning and shaping of the root canals was performed until the diameter of a 60 k - file using 1 ml of 1% sodium hypochlorite . A final rinse with 2 ml of 17% edta for 3 min and finally 1 ml of 1% sodium hypochlorite were used . The root canals were dried using conventional paper points, and the teeth were divided into four experimental groups (n=5). Group i - no drug (control), group ii - calcium hydroxide / distilled water paste, group iii - propolis paste a70d, group iv - propolis paste d70d . The pastes were introduced into the root canals using an engine driven lentulo instrument (dentsply ind . E com ., the medications were removed using a k- file and 5 ml of 1% sodium hypochlorite . Finally, the canals were flushed with 2 ml of 17% edta for 3 min, and final irrigation was performed with 1 ml of saline . For sem analysis, the crowns of teeth were removed and the roots were cleaved using a hammer and chisel . The root segments were sputter - coated with gold in a sputter - coating unit (denton vacuum inc ., morristown, nj, usa) and the middle third of the root canal walls examined at 750x with a scanning electron microscope (jsm, 220a, jeol tokyo, japan). Cleaning of the root canal walls was analyzed with regard to the number of patent dentinal tubules using the image tool 3.1 software (uthscsa, san antonio, texas, usa) (figure 1). Statistical analysis was performed to show differences in the number of open dentinal tubules among the four analyzed groups using anova and tukey's test (p<0.05). Red points: open dentinal tubules (image tool software). Scanning electron microscopy cleaning of the root canal walls was indicated by the mean of number of patent dentinal tubules . The non - medicated control group was significantly cleaner than the others, with a larger number of patent dentinal tubules (229.842.04) (p<0.05). There was no statistically significant difference (p>0.05) among the other groups - group ii (calcium hydroxide: 142.367.02), group iii (propolis paste a70d: 12711.69) and group iv (propolis paste d70d: 147.336.51) -showing that cleaning of the root canal walls decrease after the use of the intracanal medications . A panel of representative sem micrographs of the four experimental groups is shown in figure 2 . Scanning electron microscopy micrographs representative of the four groups: a control (no drug); b interference of the smear layer or intracanal medications decrease the sealing ability of endodontic sealers, and it is basically due to the difficulty to remove them from the root canal walls, as demonstrated by porkaew, et al . (1990) and calt & serper (1999). As propolis paste is an experimental drug, it was necessary to know the performance of this material, after its introduction and removal from root canals . The choice for the use of sodium hypochlorite and edta, as a final rinse, was based on previous studies . The analysis of the middle root canal third was defined by the findings of other authors, who described the facility to view the dentinal tubules at the middle and cervical third due to the larger these regions in comparison to the apical third, allowing greater movement of irrigating solutions facilitating the removal of smear layer and consequently the intracanal medication . The results of this study confirmed the findings of the literature about the difficulty of cleaning the root canal walls after use of calcium hydroxide dressing . In contrast, when the root canals did not receive the intracanal medication, only final cleaning with sodium hypochlorite and edta, the dentinal tubules were cleaner without the presence of smear layer in comparison to the groups that received medication . The difficulty to eliminate the experimental propolis pastes was similar to that observed for calcium hydroxide and, in some cases, it was observed the presence of precipitations, as reported by giamalia, et al . It should be noted the effect of edta on the intracanal medicaments because the findings of a pilot - study showed that without its use the canal walls were completely smear - covered with no patent dentinal tubules . Since there was no difference between the two propolis pastes in this experimental study, the election of one of the pastes over the other will depend on their performance in biological studies, which are currently being developed . Removal of experimental propolis pastes from the root canals was difficult and similar to that observed for calcium hydroxide paste, which is a standard intracanal dressing . Therefore, the experimental propolis pastes a70d and d70d presented acceptable physical characteristics to be used as intracanal medicaments.
Caesarean section may sometimes be the only means to save the life of the mother and/or foetus . Current estimates in cameroon put the national caesarean section rate at about 2%, with the lowest rate of 0.4% being reported in the far north region . This is lower than the national rate of 515% of the estimated live births, currently recommended by the united nations . A stillbirth rate of 7% to 12% was reported at the university hospital, cameroon [3, 4]. A recent study reported poor foetal outcome of ftus delivered through caesarean section in far north cameroon region and revealed that one of three caesarean deliveries ended up in foetal death . A study in nigeria revealed a higher perinatal mortality of 34% in women who refused elective caesarean delivery compared to 5% for those who accepted the procedure . Refusal of caesarean delivery might be due to the lack of detailed information about the procedure . In order to perform caesarean section at right time for safety of the mother and her child, counseling on cesarean delivery the ultimate decision is based on the woman's obstetric history and the anticipated mode of delivery . The aim of this study therefore was to identify the possible risk factors of caesarean delivery that can be used as content of prenatal counselling in cameroon . We conducted this retrospective case - control study to identify possible risk factors of delivery through caesarean section that can be used as content of prenatal counselling in cameroon . We conducted a retrospective case - control study of caesarean delivery outcomes in the provincial hospital, maroua, cameroon . Data was collected retrospectively from delivery and operating room registers between 01/01/2003 and 31/12/2004 inclusive . There were 144 cases of caesarean deliveries during the study period . After excluding cases of multiple pregnancies (n = 10) and cases where complete information was lacking (n = 9), we were left with 125 cases for analysis . After identification of each case of cs, data from the next successive 2 vaginal deliveries that followed were collected . Since it was possible to have consecutive cs before two vaginal deliveries, we finally compared 125 cases of cs with 244 cases of vaginal deliveries . The maternal variables studied included marital status, number of prenatal visits, route of delivery, and maternal death . Exact gestational age was not studied because it was not recorded in such cases . In maroua, women are admitted for delivery irrespective of whether they have had prior antenatal care . The chi - square test was used to compare the distribution of the various variables in the two study populations . The odds ratio and the corresponding 95% confidence intervals were used to measure the relationship between mothers' characteristic and risk of delivery by caesarean section . The sociodemographic characteristics of women delivered by cs (n = 125) and those delivered vaginally (n = 244) are presented in table 1 . The marital status is similar among the two study populations . Compared to women delivered vaginally, women delivered by cs were more likely to be adolescents less than 17 years (6% versus 12%) or to be 40 years old and above (3.7% versus 7.2%). Women delivered by caesarean section are more likely to be nulliparous (26% versus 40%), to be grand multiparous (13% versus 25%), and to have less than 4 prenatal visits (23.2% versus 31.6%) or no prenatal visit at all (2% versus 8%). The foetal weight at index delivery is presented in table 3 . Women delivered by caesarean section are more likely to have a foetus of 4000 grams and above (2% versus 8.8%). Risk factors associated with cesarean section were maternal age less than 17 years (or: 3.55, 95%ci: 1.468.64), maternal age over 39 years (or: 3.55, 95%ci: 1.1710.75), nulliparity (or: 2.72, 95%ci: 1.594.66), grand multiparty (or: 3.43, 95%ci: 1.796.57), and macrosomia (or: 4.82, 95%ci: 1.4916.44). The marital status in this study is consistent with the previous findings that almost all women who deliver in far north cameroon are married . This study shows that when the parturient's age is less than 17 years, the rate of delivery by caesarean section is doubled and the risk of caesarean delivery is tripled compared to women aged 2029 . Documented a higher rate of caesarean section in teenagers compared to women in their twenties . Lao and ho found that incidence of caesarean section was lower in the teenage mothers . They attributed the good results observed to the free and readily available prenatal care and the quality of support from the family or welfare agencies that were involved with the care of teenage mothers . However, these studies had some limitations; they included all teenagers until 19 years and are therefore a heterogeneous population [9, 10]. In the present study the proportion of teenagers aged 1719 is similar in both study populations (19% versus 20%). We found that women delivered by cs were more likely to be aged 40 and above (3.7% versus 7.2%) and the risk of caesarean delivery is more than threefold when compared to women aged 2029 years . In a previous study at the university centre hospital, we found that the risk of delivery by caesarean section for women in their forties (16.1%) was significantly higher compared to women in their twenties (10.0% versus 16.1%) hr: 1.7; 95%ci: 1.12.8; p = 0.027). Other studies found an increased risk of caesarean delivery in nulliparous and multiparous women aged 40 years and above [12, 13]. The grand multiparity observed in caesarean delivery group of our study (13% versus 25%) could have contributed to the risk of caesarean delivery . Women delivered by caesarean section are more likely to be nulliparous (26% versus 40%) and the risk of caesarean delivery is about 3 times for nulliparous women compared to those with parity of 15 . Grand multiparous women were mostly in the caesarean delivery group (13% versus 25%), with the caesarean delivery risk increased by 3 times among grand multiparous women . Nulliparous women in maroua are more likely to be teenagers aged less than 17 years and multiparous women are more likely to be 40 and above . Women delivered by caesarean section were more likely to have had less than 4 prenatal visits (23.2% versus 31.6%) or no prenatal visit at all (2% versus 8%). However, only a weak association was found between the antenatal care and the risk of caesarean delivery . This observation underscores that the anc is not effective as such in preventing cesarean delivery in the far north region of cameroon . Our findings corroborate with results from the moma group who failed to identify a strong anc parameter to predict the occurrence of dystocia in west africa . However, in spite of the weakness of the positive predictive value, there is a close relationship between severe maternal morbidity and many sociodemographic and obstetric characteristics of the patients . In the present study the frequency of anc care was about one visit per month this could lead to overload of work which in turn limits the possibility of health workers to correctly identify potential risk factors and ensure the continuity of the care till the delivery . However, studies from west africa revealed a high incidence rate of dystocia (18%) and severe maternal morbidity (6.7%) and underscore the need to be alert during anc and delivery . Ten risk factors for severe maternal morbidity were identified, and health workers need to be aware of them during delivering anc . These risk factors are hemorrhage during pregnancy, antecedent of cs, high blood pressure, antecedent of multiple pregnancy, height less than 150 cm, lack of fetal movements, history of stillborn, age over 35 years, nulliparity, and presence of disease during pregnancy . The antenatal visit is the right moment to provide the counselling related to this mode of delivery . The basic component of the new world health organization (who) antenatal care model prescribes reduced number of clinic visits at 4 and limited investigations for low - risk pregnant women [16, 17]. The inadequate antenatal care in our population could have influenced the caesarean delivery because the anc is also the opportunity to anticipate macrosomia and induce labour before the expected delivery date and avoid excessive foetal growth . First, this is a retrospective study and is therefore subjected to some information bias . In addition, due to the limited number of cases in different categories, we did not perform adjusted analysis . However, this study provides baseline information on the topic and gives the opportunity for further prospective studies . Caesarean delivery in cameroon is associated with extreme ages of reproductive life, macrosomia, nulliparous and grand multiparous status . These findings suggest that some of the caesarean section cases could be avoided while others could be anticipated . The study provides relevant information for counselling of pregnant women on timely caesarean section when indicated . We strongly recommend that these issues be taken into consideration in order to strengthen the mother and child health programs in cameroon.
Pig eyes were obtained from a local abattoir . Only eyes that could be identified as right eyes were used . Within 2 hours of death, the bulbar conjunctiva was preserved, while the palpebral conjunctiva was removed together with adnexal tissues . Eyes irrigated with pbs were placed with the optic nerve into a cryogenic vial cup (cryoelite cryogenic vial #w985100; wheaton science products, millville, nj, usa) for compression free mount as described before . 1a), eyes were positioned under a surgical microscope looking up with the temporal side of the eye directed toward the surgeon . A clear corneal incision was fashioned with a 1.8-mm keratome approximately 2 mm anterior to the temporal limbus while the inside was slightly flared for a striae - free visualization during the procedure . Tustin, ca, usa) was placed on the cornea to visualize the chamber angle (fig . The tip of a microsurgical handpiece (#600018; neomedix, inc .) That was connected to a standard microsurgical system (trabectome system #600026; neomedix, inc .) Was inserted and advanced to the opposite nasal angle of the anterior chamber . Since only right eyes were used in this study, this would represent the anatomic 3 o'clock position . After gentle goniosynechiolysis with the side of the instrument's tip to disengage pectinate ligaments along the nasal angle, tm ablation (fig . 2 with insert) was continued toward the left by 45. the tip was then turned around inside of the eye, the tm was engaged again, and ablation continued by 45 in the opposite direction . (a) (1) trabectome inserted through a clear corneal incision ablates tm that is engaged in between footplate and bipolar electrodes . (2) handpiece and magnified view of tip . (3) stand, operating console with peristaltic pump and high frequency generator and footswitch . (b) direct, gonioscopic view of ablation in porcine eye immediately before engaging the tm (left) and with tip obscured by tm during ablation (right). Microspheres were tested for a size that is known to not pass through human tm within the perfusion time used here (fig . 3a) while allowing visualization of the collector channels after the tm was removed (fig . We chose 0.5 m diameter carboxylate - modified microspheres (fluospheres, 0.5 m, yellow - green fluorescent [505/515], 2% solids; thermo fisher scientific, eugene, or, usa). The fluorescent microspheres were diluted 100-fold with phenol red free dulbecco's modified eagle's media (dmem) to make the perfusate . Following the intervention described below, ait, a 30-gauge needle was inserted through the nasal cornea 2 mm anterior to the limbus with the tip of the needle positioned in the center of the anterior chamber . The head (fluid meniscus) was positioned at 204 mm height above the needle, the equivalent of 15 mm hg, and the stopcock was opened to start the gravity - based infusion . Fluorescence was visualized with a stereo dissecting microscope equipped for fluorescent imaging (olympus szx16 with gfp filter cube and dp80 monochrome / color camera; olympus corp . Images during time lapse analysis (cellsens; olympus life science, tokyo, japan) were acquired every 20 seconds for 15 minutes using 2 2 pixel binning and a resulting resolution of 580 610 pixels . To better obtain a contiguous image of the microbead distribution, subsequently, the entire uvea, including ciliary body (cb) and iris, was gently detached and the anterior segment was inspected with the dissecting microscope detailed above . (a) perfusion of eye with fluorescent microspheres that cannot pass through the tm in a control eye (top left, inside view after removal of uvea and iris), but light up the nasal outflow tract . There is additional flow beyond the ablation ends along the circumferential drainage elements and away from the eye along collector channels (dashed arrow). (b) preferentially nasal outflow system filling is observed (anterior chamber subtracted using baseline). (c) short filling times after ait nasal quadrants with occasional flow into adjacent quadrants . Filling times of st and it quadrants without nonfilling eyes was 67 22 seconds and 46 29 seconds, respectively . A fluorescent tracer reperfusion technique was used in six eyes to compare outflow changes before and after ait detailed below using a quantitative canalography method we recently developed . Reperfusion canalograms to analyze results of trabecular ablation required two different water soluble dyes because of partial diffusion into the extravascular space over time that makes displacement or washout attempts incomplete . Use of colloids (more viscous solutions) would have changed the canalogram dye flow behavior and made it less similar to that of aqueous humor . We selected two fluorescent dyes that can be readily detected with common fluorescent filter cubes used in epifluorescent microscopes and have a reasonably low toxicity to be useful in future in vivo experimentation, fluorescein and texas red . A 30-gauge needle was inserted through the nasal cornea as previously described and used to remove 0.2 ml of anterior aqueous humor from the whole eye's anterior segment . A 30-gauge needle was then placed in the anterior segment using the exact same entry site and media with fluorescein (ak - fluor 10%, fluorescein injection, usp, 100 mg / ml, ndc 17478 - 253 - 10; akorn, lake forest, il, usa), at a concentration of 0.017 mg / ml, was infused via gravity . The outflow pattern was imaged every 20 seconds for 15 minutes using a stereo dissecting microscope equipped for fluorescent imaging (fig . Following ait, a new 30-gauge needle was placed in the anterior segment using the same entrance wound, and media with 0.28 mg / ml texas red (sulforhodamine 101 acid chloride, 10 mg; alfa aesar, ward hill, ma, usa) was subsequently infused via gravity . Pre- and post - ait canalogram frames are matched (also see supplementary movie s1). To determine differences in chromophore detection sensitivity, a hemocytometer chamber was filled with 10 l each of fluorescein and texas red at the previously stated concentrations . Proper exposure times of 15 ms for fluorescein and 10 ms for texas red were determined with the fluorescence equipped stereo dissecting microscope above . Initially, six eyes were perfused with fluorescein first immediately followed by texas red without ait . Another six eyes were perfused with texas red first followed by fluorescein to determine whether the order of perfusion would have any effect . We chose the order to be fluorescein followed by texas red to avoid a type i error (false positive flow enhancement) because texas red had a slightly slower filling average that was not statistically significant in pilot eyes (p = 0.06, n = 12). As described before, we used a program written in r to automatically compute the focal outflow changes (fig . 5, left) and convergent perilimbal aqueous flow (fig . 5, right) using the eye - canalogram package to process the image datasets, the source code of which we made available for download (r package eye - canalogram, provided in the public domain at https://github.com/enbrown/eye-canalogram/tree/06461498c8). Quantitative canalograms with individual intensity fits (left) and circumferential flow rates (right). Both are increased in most eyes, even in quadrants away from the nasal ait site . Briefly, for each image set, the cornea was first manually delineated and masked to exclude the bright fluorescence in the anterior chamber from the image analysis . This step was neutral to the mathematical analysis of fluorescence changes along the outflow tract but considerably improved how observers were able to see and interpret subtle filling of small caliber vessels (fig . Larger pixels corresponded to more intense fluorescein signals while red dots corresponded to faster flow . In addition, all regions (clock hour and three radial rings) were fit to a single gam using smoothing terms for the radial ring, clock hour, and frame number . Pre- and posttreatment image sets for each eye were registered using the clock hour and radial distance to compute the change in fit metrics (fig . Similarly, all eyes were warped to a common reference frame and averaged to produce figure 6b . (a) quantitative change analysis after ait . Each eye produces a pair of images: change in fluorescence intensity (left image) and change in rate of fluorescence uptake (right image). Red (positive values) indicates greater intensity or rate of uptake following ait; blue (negative values) indicates less intensity or reduced uptake . Each quadrant was also analyzed for first detection of fluorescence in an outflow structure downstream of schlemm's canal segments . Timestamps from time - lapse recordings were summarized as graphs (average sd) for the inferonasal (in), superonasal (sn), superotemporal (st), and inferotemporal (it) quadrants . Student's paired two sample t - test was used to compare outflow changes in the same eyes before and after each intervention and to compare same eye quadrants . Tests were two - tailed and results were reported as mean sd . Segments were removed from the perfusion dish, rinsed in pbs, cut into quarters, and fixed with 4% paraformaldehyde and pbs for 48 hours before being placed in 70% ethanol . A corneoscleral wedge was taken from the lateral side near the incision and one from the nasal side where the ait had been performed (fig . 2). The section was paraffin - embedded for histological processing, cut at 6-m thickness, and stained with hematoxylin and eosin . Pig eyes were obtained from a local abattoir . Only eyes that could be identified as right eyes were used . Within 2 hours of death, the bulbar conjunctiva was preserved, while the palpebral conjunctiva was removed together with adnexal tissues . Eyes irrigated with pbs were placed with the optic nerve into a cryogenic vial cup (cryoelite cryogenic vial #w985100; wheaton science products, millville, nj, usa) for compression free mount as described before . 1a), eyes were positioned under a surgical microscope looking up with the temporal side of the eye directed toward the surgeon . A clear corneal incision was fashioned with a 1.8-mm keratome approximately 2 mm anterior to the temporal limbus while the inside was slightly flared for a striae - free visualization during the procedure . Tustin, ca, usa) was placed on the cornea to visualize the chamber angle (fig . The tip of a microsurgical handpiece (#600018; neomedix, inc .) That was connected to a standard microsurgical system (trabectome system #600026; neomedix, inc .) Was inserted and advanced to the opposite nasal angle of the anterior chamber . Since only right eyes were used in this study, this would represent the anatomic 3 o'clock position . After gentle goniosynechiolysis with the side of the instrument's tip to disengage pectinate ligaments along the nasal angle, tm ablation (fig . 2 with insert) was continued toward the left by 45. the tip was then turned around inside of the eye, the tm was engaged again, and ablation continued by 45 in the opposite direction . (a) (1) trabectome inserted through a clear corneal incision ablates tm that is engaged in between footplate and bipolar electrodes . (2) handpiece and magnified view of tip . (3) stand, operating console with peristaltic pump and high frequency generator and footswitch . (b) direct, gonioscopic view of ablation in porcine eye immediately before engaging the tm (left) and with tip obscured by tm during ablation (right). Microspheres were tested for a size that is known to not pass through human tm within the perfusion time used here (fig . 3a) while allowing visualization of the collector channels after the tm was removed (fig . We chose 0.5 m diameter carboxylate - modified microspheres (fluospheres, 0.5 m, yellow - green fluorescent [505/515], 2% solids; thermo fisher scientific, eugene, or, usa). The fluorescent microspheres were diluted 100-fold with phenol red free dulbecco's modified eagle's media (dmem) to make the perfusate . Following the intervention described below, ait, a 30-gauge needle was inserted through the nasal cornea 2 mm anterior to the limbus with the tip of the needle positioned in the center of the anterior chamber . The head (fluid meniscus) was positioned at 204 mm height above the needle, the equivalent of 15 mm hg, and the stopcock was opened to start the gravity - based infusion . Fluorescence was visualized with a stereo dissecting microscope equipped for fluorescent imaging (olympus szx16 with gfp filter cube and dp80 monochrome / color camera; olympus corp . Images during time lapse analysis (cellsens; olympus life science, tokyo, japan) were acquired every 20 seconds for 15 minutes using 2 2 pixel binning and a resulting resolution of 580 610 pixels . To better obtain a contiguous image of the microbead distribution, whole eyes were processed as previously described . Briefly, after removing all adnexal structures, globes were hemisected along the equator . Subsequently, the entire uvea, including ciliary body (cb) and iris, was gently detached and the anterior segment was inspected with the dissecting microscope detailed above . (a) perfusion of eye with fluorescent microspheres that cannot pass through the tm in a control eye (top left, inside view after removal of uvea and iris), but light up the nasal outflow tract . There is additional flow beyond the ablation ends along the circumferential drainage elements and away from the eye along collector channels (dashed arrow). (b) preferentially nasal outflow system filling is observed (anterior chamber subtracted using baseline). (c) short filling times after ait nasal quadrants with occasional flow into adjacent quadrants . Filling times of st and it quadrants without nonfilling eyes was 67 22 seconds and 46 29 seconds, respectively . A fluorescent tracer reperfusion technique was used in six eyes to compare outflow changes before and after ait detailed below using a quantitative canalography method we recently developed . Reperfusion canalograms to analyze results of trabecular ablation required two different water soluble dyes because of partial diffusion into the extravascular space over time that makes displacement or washout attempts incomplete . Use of colloids (more viscous solutions) would have changed the canalogram dye flow behavior and made it less similar to that of aqueous humor . We selected two fluorescent dyes that can be readily detected with common fluorescent filter cubes used in epifluorescent microscopes and have a reasonably low toxicity to be useful in future in vivo experimentation, fluorescein and texas red . A 30-gauge needle was inserted through the nasal cornea as previously described and used to remove 0.2 ml of anterior aqueous humor from the whole eye's anterior segment . A 30-gauge needle was then placed in the anterior segment using the exact same entry site and media with fluorescein (ak - fluor 10%, fluorescein injection, usp, 100 mg / ml, ndc 17478 - 253 - 10; akorn, lake forest, il, usa), at a concentration of 0.017 mg / ml, was infused via gravity . The outflow pattern was imaged every 20 seconds for 15 minutes using a stereo dissecting microscope equipped for fluorescent imaging (fig . A new 30-gauge needle was placed in the anterior segment using the same entrance wound, and media with 0.28 mg / ml texas red (sulforhodamine 101 acid chloride, 10 mg; alfa aesar, ward hill, ma, usa) was subsequently infused via gravity . Again pre- and post - ait canalogram frames are matched (also see supplementary movie s1). To determine differences in chromophore detection sensitivity, a hemocytometer chamber was filled with 10 l each of fluorescein and texas red at the previously stated concentrations . Proper exposure times of 15 ms for fluorescein and 10 ms for texas red were determined with the fluorescence equipped stereo dissecting microscope above . Initially, six eyes were perfused with fluorescein first immediately followed by texas red without ait . Another six eyes were perfused with texas red first followed by fluorescein to determine whether the order of perfusion would have any effect . We chose the order to be fluorescein followed by texas red to avoid a type i error (false positive flow enhancement) because texas red had a slightly slower filling average that was not statistically significant in pilot eyes (p = 0.06, n = 12). As described before, we used a program written in r to automatically compute the focal outflow changes (fig . 5, left) and convergent perilimbal aqueous flow (fig . 5, right) using the eye - canalogram package to process the image datasets, the source code of which we made available for download (r package eye - canalogram, provided in the public domain at https://github.com/enbrown/eye-canalogram/tree/06461498c8). Quantitative canalograms with individual intensity fits (left) and circumferential flow rates (right). Both are increased in most eyes, even in quadrants away from the nasal ait site . Briefly, for each image set, the cornea was first manually delineated and masked to exclude the bright fluorescence in the anterior chamber from the image analysis . This step was neutral to the mathematical analysis of fluorescence changes along the outflow tract but considerably improved how observers were able to see and interpret subtle filling of small caliber vessels (fig . Larger pixels corresponded to more intense fluorescein signals while red dots corresponded to faster flow . In addition, all regions (clock hour and three radial rings) were fit to a single gam using smoothing terms for the radial ring, clock hour, and frame number . Pre- and posttreatment image sets for each eye were registered using the clock hour and radial distance to compute the change in fit metrics (fig . Similarly, all eyes were warped to a common reference frame and averaged to produce figure 6b . Each eye produces a pair of images: change in fluorescence intensity (left image) and change in rate of fluorescence uptake (right image). Red (positive values) indicates greater intensity or rate of uptake following ait; blue (negative values) indicates less intensity or reduced uptake . Each quadrant was also analyzed for first detection of fluorescence in an outflow structure downstream of schlemm's canal segments . Timestamps from time - lapse recordings were summarized as graphs (average sd) for the inferonasal (in), superonasal (sn), superotemporal (st), and inferotemporal (it) quadrants . Student's paired two sample t - test was used to compare outflow changes in the same eyes before and after each intervention and to compare same eye quadrants . Segments were removed from the perfusion dish, rinsed in pbs, cut into quarters, and fixed with 4% paraformaldehyde and pbs for 48 hours before being placed in 70% ethanol . A corneoscleral wedge was taken from the lateral side near the incision and one from the nasal side where the ait had been performed (fig . 2). The section was paraffin - embedded for histological processing, cut at 6-m thickness, and stained with hematoxylin and eosin . Visualization and tm ablation could be achieved with a standard microsurgical system (neomedix, inc .) And the kit included, modified swan jacob goniolens (fig . 1a). The footplate encountered more stops than typical for human eyes when sc segment ends were reached . The histologic analysis showed that the relatively prominent tm and common sc segments had been ablated with a few exceptions of sc segments too small to enter with the trabectome tip (fig . 2, eye 1, shown to scale). No coagulative damage was observed . Eyes without ait that were infused with fluorescent spheres showed fluorescence only in the tm . When those were hemisected and the vitreous and iris removed, sphere distribution appeared relatively even throughout the tm circumference (fig . In contrast, canalograms with fluorescent spheres obtained after ait experienced fast filling of proximal and distal parts of the outflow system along the ablation site followed by circumferential and then centrifugal filling of adjacent quadrants . Filing of nasal quadrants was nearly 10 times faster than that of temporal quadrants (fig . 4 and supplementary movie s1) had a 17 5-fold increase in filling in, 14 3-fold increase sn, and also an increase in the adjacent quadrants, with a 2 1-fold increase st and 3 3 it . The superonasal quadrant was the fastest to fill (p <0.05) followed by the in, st, and it quadrants (figs . 4, 7). Although fluorescent dyes used in these differential canalograms were not blocked by the tm like the fluorescent spheres, st and it quadrants often filled circumferentially from the site of ait . Outflow change summary after ait . Short bars indicate fast filling not only in the nasal quadrants (in and sn quadrant), but also in the temporal quadrants (st and it quadrants) following ait . Individual fit curve analysis indicated a shift in filling speed toward the nasal side and an overall faster filling throughout the remainder of the outflow system in most eyes (fig . This can be seen by a shift toward red in the color coded analysis indicating a shorter time toward peak fluorescence, as well as the increased bubble size that describes the peak fluorescence obtained . With the exception of eye 1, all eyes post - ait showed an initial uptake centered on the in and sn quadrants as expressed by the red color coding that spreads circumferentially from here (fig . Changes from pre- to post - ait fluorescence intensity changes are summarized in figure 6 . In average of all six eyes, peak intensity increased in the in quadrant and in the sn quadrant, as well as in the remainder of the circumference (fig . In contrast, flow rate changes from pre- to post - ait were more substantial in the nasal quadrants (fig . 6b). These changes from pre- to post - ait were statistically significant in all quadrants but more pronounced in the in and sn quadrants . There was no difference between the st and it quadrants pre- or post - ait . Focal changes of conventional aqueous humor outflow are difficult to examine, yet are crucial to better understand how elements downstream of the tm may influence outflow . There is ample new clinical evidence that very significant outflow resistance exists downstream of the trabecular meshwork as trabecular ablation rarely achieves the theoretically lowest intraocular pressure, which would be equal to that of episcleral venous pressure according to the goldmann equation . The lack of an inexpensive, readily available, and high quality outflow model has impeded glaucoma research and training of surgery on this submillimeter structure alike . Here we address both with a differential canalography technique that allows examination of the effect of trabectome - mediated ait on focal outflow in porcine eyes . Visualization of the chamber angle and ablation of tm by ait was quite similar to surgery in human patients . Angle surgery can be practiced in human eyes, but costs of those are high and the corneal clarity is often too compromised to see the angle . As a result, artificial eyes were preferred by trainees . The histology following ait shows that despite the anatomical differences between the pig and human chamber angle, an extensive ablation of tm can be achieved without an obvious coagulative thermal effect on adjacent tissues . Different from coagulation devices, the instrument used here generates plasma to ionize tissue and has a highly confined heat dissipation cone that is similar to photodisruptive lasers . The initial canalograms obtained with fluorescent microspheres demonstrated that tm must be removed before they can enter the conventional outflow system . As has been demonstrated before in human and ungulate trabecular meshwork, the size of 0.5 m used here prevents spheres from moving into schlemm's canal within the perfusion time used while preventing fluid blockage that can be observed with 10 m spheres . Our choice of relatively small beads had the advantage that smaller collector channels could be visualized . Although the angular aqueous plexus of pigs does not have a continuous schlemm's canal as primate eyes do, but rather multiple sc - like segments, we observed circumferential flow that extended far beyond the ablation site . This indicates that canal segments are connected and that supraphysiological flow from the site of ablated tm is displacing the normal flow that is still occurring through the nonablated tm . The perilimbal flow analysis of differential canalograms showed high - flow areas near the in and sn angle . In pig eyes, this matches the areas in between the recti muscles, where larger collector channels reside (fig . 4). The circumferential flow patterns observed here contradicts the assumption of noncontinuous sc segments in the pig . A three - dimensional reconstruction at a higher resolution than conventional histological sections by sd - oct may be necessary . Such an approach has enabled discovery of previously overlooked valve - like elements in human eyes . We provide a heatmap summary image that combines fluorescence and flow rate of all six eyes . This allows us to visualize how flow can be enhanced in nonglaucomatous eyes to levels above the physiological flow rate and beyond the tm ablation area . In this constant pressure perfusion system, ait led to increased flow and peak fluorescence in all quadrants, beyond the nasal site where ait was performed . This has practical implications for patient care and suggests that it may not be necessary to obtain a very extensive ablation or circumferential access to the outflow tract . Carefully observing glaucoma surgeons these appeared to be limited to the site of ablation when observed through an operating microscope . Such visualization may relatively underestimate the amount of flow that could be detected with more sensitive, fluorescent dyes as used here . There are well established angiography methods for organs larger than the anterior segment of the eye, such as the heart . Characteristic for such angiography is that vessels branch off large primary vessels to subsequently smaller ones, and typically without collaterals . The outflow tract of the eyes is different and has a much more diffuse and connected nexus of vessels with variable caliber . The drainage system just distal to the outer wall of the sc is more similar to the honeycomb pattern of a capillary network, but its vessels are larger and collapsible depending on the perfusion pressure . This makes it challenging to use doppler strategies to compute flow speeds in single vessels . The data presented here validate the use of fluorescence change as a surrogate to tracking reflective particles . In conclusion, we developed a method to quantify outflow enhancement from plasma - mediated ait and show that nasal ablation of trabecular meshwork increased outflow not only locally but circumferentially.
Urinary tract infections (uti) are the most common infectious complications in patients receiving renal transplantation for end stage kidney disease . Uti could be associated with an increased morbidity and mortality risk, can worsen the graft and patient survival in renal transplant recipient . A significant proportion of kidney transplant recipients with utis may develop acute pyelonephritis, which is an independent risk factor for deterioration of graft function . Some recent studies suggest routine prophylactic stenting of ureterovesical anastomosis at the time of graft implantation to reduce the incidence of early postoperative major urologic complications, as urinary leaks and stenosis [6 - 8]. Others advocate the use of stent only in selective, difficult cases while routine stenting could be associated with specific complications as haematuria, migration, malposition or complications of removal . It has been shown, that urinary stenting could lead to a significant increase of infections of lower and upper urinary tract in immunocompromised patients . Although, whether universal routine stenting of the ureteroneocystostomy is a real risk factor for the development of severe urinary tract infection (uti) after renal transplantation is still controversial . Therefore, to help further clarify this issue, we compared the frequency of uti in our cohort of deceased donor kidney transplant recipient transplanted with or without ureteric stent implantation . This is a retrospective, observational study of deceased donor renal allograft recipients who were consecutively transplanted at university hospital essen, germany between january 2002 and december 2006 (n = 310). Data for this study were obtained from our transplant database and review of electronic and paper - based medical records . All adult (> 18 years) deceased donor kidney transplantations, except combined multiorgan transplants were considered . Living donor transplantations double - j ureteral catheters were placed intraoperatively per the surgeon's discretion based on ureter, bladder and anastomotic characteristics until june 2004 and on routine basis thereafter . 157 patients were enrolled to a stent- (st) and 153 patients to a no - stent (nst) group . Recipient and donor demographic and laboratory data, at the time of transplantation and afterwards were reviewed . Urinary tract infection was defined as the patient having one of the following symptoms dysuria, fever, urgency, frequency, suprapubic tenderness, and positive urine culture with 10microorganism / cmor two of the above signs and pyuria (> wbc / mm) or <105 microorganism / cmif patient was on antibiotics . Intravenous cefazoline (2 g) was given once perioperatively within 30 minutes prior to skin incision and repeated once when operation exceeded 3 hours . A dose of single strength trimetoprim / sulfametoxazole (tmp / smx) was started on day 2 or 3 and continued every second day for four to six months . All stents were removed by cystoscopy under aseptic conditions 4 - 6 weeks after transplantation . Immunosuppression consisted of methylprednisolone (500 mg given intraoperatively, followed by sequential tapering to daily oral prednisone 30 mg by one week, 10 mg at one month and 5 mg at 6 - 12 months), mycophenolate - mofetil (2 g / d postoperatively with dose adjustment for side effects), calcineurininhibitors (tacrolimus or cyclosporin - a started within 24 hours after surgery) and/or il-2 receptor blockers (basiliximab, daclizumab). Estimated glomerular filtration rate (egfr) was calculated using the mdrd formula: egfr (ml / min/1.73 m2) = 186 crs-1.154 age-0.203 0.742 (if female). " Extra risk " recipients were defined as recipients older than 60 years or older than 50 years with at least one of the following risk factors: coronary heart disease, peripheral arterial disease (pad, grade iia or higher), diabetes mellitus . Patients with findings suggestive of uti were hospitalized for further work - up and treatment, if necessary . The technique of the ureteroneocystostomy consisted of an end - to - side extravesical implantation of the ureter onto the anterior bladder wall, modified from the lich - gregoire procedure . In summary, a cc . 1.5 cm incision of detrusor was made on the posterolateral aspect of bladder, followed by sharp dissection of mucosa . The anastomosis was constructed between the spatulated distal donor ureter and a small bladder mucosal nick made after myotomy incision . A watertight mucosa - to - mucosa anastomosis, using 5/0 or 6/0 absorbable running sutures was performed of the spatulated ureter to a distal mucosal opening, followed by the creation of submucosal tunnel by approximation of seromuscular layers using absorbable sutures . Data are given as counts (with percentages) or mean values (with sds). Medians (with interquartile ranges [iqr]) are reported for non - normally distributed data . For univariate comparisons between the groups, we used chi - square or fisher's exact test for categorical variables, and by student's t or mann whitney rank sum test for continuous variables . A logistic regression model was developed to predict urinary tract infection, using various demographic and medical variables as covariates . By stepwise backward selection, non - predictive variables were eliminated using a threshold of p <0.1 . The explanatory power of the final model was examined by calculating nagelkerke's rcoefficient, which ranges between 0 (no predictive power at all) and 1 (uti incidence predictable with 100% certainty). This is a retrospective, observational study of deceased donor renal allograft recipients who were consecutively transplanted at university hospital essen, germany between january 2002 and december 2006 (n = 310). Data for this study were obtained from our transplant database and review of electronic and paper - based medical records . All adult (> 18 years) deceased donor kidney transplantations, except combined multiorgan transplants were considered . Living donor transplantations double - j ureteral catheters were placed intraoperatively per the surgeon's discretion based on ureter, bladder and anastomotic characteristics until june 2004 and on routine basis thereafter . 157 patients were enrolled to a stent- (st) and 153 patients to a no - stent (nst) group . Recipient and donor demographic and laboratory data, at the time of transplantation and afterwards were reviewed . Urinary tract infection was defined as the patient having one of the following symptoms dysuria, fever, urgency, frequency, suprapubic tenderness, and positive urine culture with 10microorganism / cmor two of the above signs and pyuria (> wbc / mm) or <105 microorganism / cmif patient was on antibiotics . Intravenous cefazoline (2 g) was given once perioperatively within 30 minutes prior to skin incision and repeated once when operation exceeded 3 hours . A dose of single strength trimetoprim / sulfametoxazole (tmp / smx) was started on day 2 or 3 and continued every second day for four to six months . All stents were removed by cystoscopy under aseptic conditions 4 - 6 weeks after transplantation . Immunosuppression consisted of methylprednisolone (500 mg given intraoperatively, followed by sequential tapering to daily oral prednisone 30 mg by one week, 10 mg at one month and 5 mg at 6 - 12 months), mycophenolate - mofetil (2 g / d postoperatively with dose adjustment for side effects), calcineurininhibitors (tacrolimus or cyclosporin - a started within 24 hours after surgery) and/or il-2 receptor blockers (basiliximab, daclizumab). Estimated glomerular filtration rate (egfr) was calculated using the mdrd formula: egfr (ml / min/1.73 m2) = 186 crs-1.154 age-0.203 0.742 (if female). " Extra risk " recipients were defined as recipients older than 60 years or older than 50 years with at least one of the following risk factors: coronary heart disease, peripheral arterial disease (pad, grade iia or higher), diabetes mellitus . Patients with findings suggestive of uti were hospitalized for further work - up and treatment, if necessary . The technique of the ureteroneocystostomy consisted of an end - to - side extravesical implantation of the ureter onto the anterior bladder wall, modified from the lich - gregoire procedure . In summary, a cc . 1.5 cm incision of detrusor was made on the posterolateral aspect of bladder, followed by sharp dissection of mucosa . The anastomosis was constructed between the spatulated distal donor ureter and a small bladder mucosal nick made after myotomy incision . A watertight mucosa - to - mucosa anastomosis, using 5/0 or 6/0 absorbable running sutures was performed of the spatulated ureter to a distal mucosal opening, followed by the creation of submucosal tunnel by approximation of seromuscular layers using absorbable sutures . Data are given as counts (with percentages) or mean values (with sds). Medians (with interquartile ranges [iqr]) are reported for non - normally distributed data . For univariate comparisons between the groups, we used chi - square or fisher's exact test for categorical variables, and by student's t or mann whitney rank sum test for continuous variables . A logistic regression model was developed to predict urinary tract infection, using various demographic and medical variables as covariates . By stepwise backward selection, non - predictive variables were eliminated using a threshold of p <0.1 . The explanatory power of the final model was examined by calculating nagelkerke's rcoefficient, which ranges between 0 (no predictive power at all) and 1 (uti incidence predictable with 100% certainty). There was no significant difference between the stented (st) and non - stented (nst) groups with respect to underlying kidney disease, body mass index (bmi), gender or hla mismatches . The patients from the st group were older, the cold ischemia time was longer and these patients were dialysed longer before transplantation . There was also no significant difference with regard to gender, relevant co - morbidities, cause of death, median length of icu stay or kidney function . One hundred twenty - nine infection episodes of urinary tract infections were diagnosed in 310 (41.6%) recipients . The rate of uti at three months was similar in the st group compared to nst group (43.3% vs. 40.1%), but without reaching significance (p = 0.65). The overall rate of urinary tract complications in the two groups were also summarized in table 3 . Anastomotic stenosis rate (1.3%) was similar in the nst versus the st group, urinary leakage occurred in 6.8% of all recipients, 8.3% in st- vs. 5.9% in nst group, respectively . We observed fewer episodes of acute rejection in st group (22.9% vs. 34.6%, p = 0.024). Wound in multivariate analysis (table 4) the following categorical variables had no impact on the rate of uti: age at transplantation, diabetes mellitus, bmi, duration of pretransplant dialysis, duration of surgery and cold ischemia time . However, female gender significantly (p <0.001) affected the incidence of urinary tract infection (54.0% vs. 33.5%, relative risk 1.4, 95%-ci 1.2 to 1.8). Graft function did not differ significantly between the two groups of recipients (figure 2). Clinical results in the two groups . Results of multivariate regression analyses of predictors for uti . Explanatory power of the final model was limited, as nagelkerke's r2 was 0.08 . Recipient egfr (in ml/1.73 m) at transplantation and after 1, 4 and 52 weeks in the two groups . Renal transplantation is a widely accepted treatment for patients with end - stage kidney disease . Despite of well established surgical technique and a continuous improvements in patient and graft survival over the past two decades, the rate of urinary tract complications remains still considerable common and may vary between 2 - 15% . These include urinary leak, obstruction, stenosis, necrosis and vesicoureteral re - flux . These complications increase morbidity, delay graft function, and occasionally may lead to graft and/or patient loss . Recently, many studies, including a prospective randomized trial, advocate stenting of the ureterovesical anastomosis to reduce the rate of these complication [15 - 18]. Opponents counter that the stent can migrate, could have bad permeability, could contribute to postoperative pain and stone formation . Others advocate that stent can increase postoperative infection risk, can erode the lumen, can exacerbate long - term stricturing of the anastomosis and the use of stenting should be restricted to difficult ureterovesical anastomosis . Currently, no controlled randomized study evaluated the rate of urinary tract infections with and without ureteral stent implantation, and there is no clearly defined consensus among transplant surgeons with regard to routine stenting of the vesicoureteral anastomosis during renal transplantation . Therefore, to further evaluate this issue we conducted a comparative - retrospective study to analyse the frequency of uti in a cohort of deceased donor kidney transplant recipients who were operated either with or without double - j placement in our center . We also investigated the influence of stenting on renal function and major ureteric complications (leakage, obstruction). Differences in the definition, follow - up period, immunosuppression and the use of antimicrobial prophylaxis could explain this wide range . A recent report showed that stenting of the vesicoureteral anastomosis is a predictor factor for uti after kidney transplantation . Our study has also failed to demonstrate an increased risk of uti in stented recipients . Other factors that have been also associated with a higher risk of uti in renal transplant recipients include female gender, age, diabetes mellitus the function of the transplanted kidney and prolonged dialysis pre - transplantation . However, except female gender, none of them was independently associated with a higher risk of uti in our study . We did also not find that advanced age would increase the risk of uti in our renal transplant recipients . Although earlier reports have noted a higher incidence of uti - related complications such as pyelonephritis and urosepsis, we did also not observe such an increase . We observed a significant decrease in acute rejection rate in the stented patient group (22.9% vs 34.6%). One speculative explanation could be that stenting allows early decompression of the anastomosis to ensure free drainage despite mucosal oedema, although auxiliary studies could further evaluate this issue . Previous published studies report conflicting results whether a history of diabetes increases or does not influence the renal transplant patient's risk of developing a uti . Diabetic patients have been shown to have a higher incidence of significant bacteriuria therefore we expected this trend to continue after renal transplantation . However, we did not find pre - transplant diabetes to be associated with an increased incidence of uti in our renal transplant population . The lack of impact may be due to the overall low incidence of diabetic recipients (12.9%) in our study population . The pathogens isolated from renal transplant recipients with uti have been previously reported to be similar to those causing uti in the general population . A renal transplant series reported recently, that escherichia coli would be the most common uropathogen (32%) and enterococcus isolated in 18% . These observations are in correlation with the data of alangaden et al . Identifying first that enterococcus spp first, due to retrospective comparative data analyses and relatively small number of patients we hesitate to draw firm conclusion from the observed associations . Secondary, in the first study period ureterovesical stent were rather implanted on a subjective basis when the transplant surgeon experienced an unfavourable anatomy and expected complications . Despite these biases, we would conclude that routine use of double - j stents for prophylactic stenting of the ureteroneocystostomy does not affect significantly the rate of urinary tract infection in deceased donor renal transplant recipient population . Whether longer duration of stenting, immunosuppression with induction therapy by multiple transplants or living donor transplantation has a further influence on incidence of uti is still unclear . It is therefore prudent to continue monitoring possible relationship between double - j implantation and uti in renal transplant recipients.
Postoperative vomiting places undue pressure on the repaired diaphragm and gastric wrap, risking early failure of the surgery.1 as such, patients undergoing laparoscopic fundoplication need effective, parenteral, nonopioid analgesia . Lidocaine is an inexpensive and widely accessible local anesthetic that possesses analgesic, anti - inflammatory, and antihyperalgesic properties.24 while most commonly used in infiltration and for central neuraxial and peripheral nerve blocks, lidocaine can also be given intravenously (iv) to treat acute perioperative pain4 and chronic neuropathic pain.5 the current evidence for using iv lidocaine to treat perioperative pain is based on 45 clinical trials contributing to five systematic reviews.4,69 these studies found that iv lidocaine infusion in the perioperative period decreases pain intensity, opioid requirements, the duration of postoperative ileus, and opioid - related side - effects, such as postoperative nausea and vomiting . These studies concluded that further research is needed to determine the optimum dose, timing, and duration of infusion of lidocaine in this setting . The effect of iv lidocaine infusion has not been studied in patients undergoing laparoscopic fundoplication surgery . This randomized controlled trial tested the primary hypothesis that iv lidocaine decreases postoperative pain intensity (at rest and during movement) after laparoscopic fundoplication . In order to verify the safety of iv lidocaine infusion for analgesia, the following secondary outcomes were measured: nausea and vomiting, opioid requirements, adverse events, serum lidocaine concentration, and length of hospital stay . This was an equally randomized, double - blind, placebo - controlled, parallel - group, single - center trial conducted at the sydney adventist hospital, wahroonga, sydney, australia . Approval was received from the adventist healthcare limited human research ethics committee (ec00141) and written informed consent was obtained from all patients . The study was registered with the australian and new zealand clinical trials registry (actrn12613000440729). All adults (age> 18 years) undergoing laparoscopic fundoplication surgery by a single surgeon (glf) were eligible to participate in the study . Exclusion criteria were allergies to local anesthetics, chronic use of analgesics or corticosteroids, impaired hepatic function (any single liver function test 20% normal reference range), epilepsy or other seizure disorder, severe cardiac failure (left ventricular ejection fraction 0.35) or cardiac arrhythmias and pregnancy . General anesthesia was standardized using midazolam 2.5 mg iv, effect - site target controlled infusions of propofol and remifentanil . Tracheal intubation was facilitated by rocuronium 1.2 mg / kg, and the lungs were ventilated with 33% oxygen in air using a circle system . Intraoperatively, all patients received iv granisetron 3 mg and dexamethasone 8 mg as prophylaxis against nausea and vomiting and parecoxib 40 mg for analgesia . Postoperative analgesia was commenced with fentanyl 1 g kg iv at the cessation of the remifentanil infusion . The diaphragmatic crura and port sites were infiltrated with 20 ml ropivacaine 0.2% by the surgeon . A patient - controlled analgesia (pca) device administering iv fentanyl was provided (10 g / ml, 10 g bolus, 5 minute lockout, no background) and pca usage was recorded . Acetaminophen (1 g iv every 6 hours) and indomethacin (100 mg per rectum every 12 hours) were administered to provide multi - modal analgesia . Rescue antiemetics (ondansetron 4 mg sublingual and droperidol 0.5 mg / kg iv) were offered to any patient who experienced nausea or vomiting . Patients were randomly assigned to study groups in fixed blocks of 12 using a computer - generated table of random numbers through the use of the randomization.com program . The patients in the intervention group received 1 mg / kg iv lidocaine bolus at induction, followed immediately by an infusion at 2 mg / kg / h for 24 hours . The patients in the control group were treated likewise using 0.9% sodium chloride in a double - blind fashion . Lidocaine was acquired as xylocard 500 ampoules (astrazeneca pty ltd, north ryde, nsw, australia), containing 500 mg of lidocaine hydrochloride in 5 ml of water . Lidocaine study drug was made to a concentration of 0.5% (5 mg / ml) and supplied in 1000 ml flasks compatible with imed gemini (imed corporation, san diego, ca, usa) infusion pumps and giving sets . No patient, research nurse, investigator, or any other medical or nursing staff was aware of the treatment assignments for the duration of the study . The randomization schedule was stored in a locked cupboard that was only accessible by the randomization authority (thus concealed from all care providers and other research personnel). When a patient was recruited into the study the study drug was given to the anesthetist accompanied by a sealed, opaque, tamper - proof envelope containing the treatment allocation . This envelope was kept in the patient file at all times in case serious adverse event required the knowledge of treatment allocation . Envelopes were examined at the completion of the trial to ensure that they were unopened . The patients were asked to score their current pain on two occasions: at rest and on mobilization from supine to sitting upright . Pain scores were obtained every 4 hours, for 30 hours following commencement of the trial drug infusion . The 11-point numeric rating scale is a validated, sensitive tool for assessing postoperative pain intensity.10,11 all trial data were entered directly into the electronic medical record, which had been adapted for the study and mandated the completion of all parameters . Secondary outcomes were nausea and vomiting, opioid requirements, adverse events, serum lidocaine concentration, and length of hospital stay . Patients were asked if they experienced any nausea or vomiting in the preceding 4 hours when pain scores were obtained . Responses were recorded as yes or no and this outcome was treated as binary data . Opioid requirements were recorded as the dose of fentanyl delivered (rather than total demands / attempts for analgesia). Only adverse events that required the patient to be discontinued from the trial were interpreted . Venous blood samples were collected from all patients every 4 hours for the duration of the trial . Serum was immediately separated and frozen, then later analyzed to determine the serum lidocaine concentration length of hospital stay was recorded as the number of nights the patient was in hospital . A priori sample size was estimated on the basis of an absolute reduction in pain score . A reduction of two in the nrs-11 was reported to be the amount needed to cause a clinically significant reduction in a person s experience of postoperative pain.12,13 a sample size of 18 patients in each group was calculated to be sufficient to detect a difference of two in the mean pain score, assuming a standard deviation of 212 with a significance level =0.05 and a power of 90% . The study would be stopped early when there was sufficient evidence to claim superiority (net benefit) or inferiority (net harm), or futility (little chance of achieving statistical significance) if the futility index was found to be> 0.8.14 statistical analyses were performed using ibm spss statistics v21.0 (ibm corporation). Sample size calculations and conditional power analyses were performed using pass v13 (ncss, llc). The comparison between the intervention and control groups was conducted using an independent samples t - test, mann whitney wilcoxon u - test, chi - squared test, or fisher s exact test as appropriate . Normally distributed data are presented as mean standard deviation, nonnormally distributed data are presented as medians (interquartile range), and categorical data are presented as raw data and as frequencies . This was an equally randomized, double - blind, placebo - controlled, parallel - group, single - center trial conducted at the sydney adventist hospital, wahroonga, sydney, australia . Approval was received from the adventist healthcare limited human research ethics committee (ec00141) and written informed consent was obtained from all patients . The study was registered with the australian and new zealand clinical trials registry (actrn12613000440729). All adults (age> 18 years) undergoing laparoscopic fundoplication surgery by a single surgeon (glf) were eligible to participate in the study . Exclusion criteria were allergies to local anesthetics, chronic use of analgesics or corticosteroids, impaired hepatic function (any single liver function test 20% normal reference range), epilepsy or other seizure disorder, severe cardiac failure (left ventricular ejection fraction 0.35) or cardiac arrhythmias and pregnancy . General anesthesia was standardized using midazolam 2.5 mg iv, effect - site target controlled infusions of propofol and remifentanil . Tracheal intubation was facilitated by rocuronium 1.2 mg / kg, and the lungs were ventilated with 33% oxygen in air using a circle system . Intraoperatively, all patients received iv granisetron 3 mg and dexamethasone 8 mg as prophylaxis against nausea and vomiting and parecoxib 40 mg for analgesia . Postoperative analgesia was commenced with fentanyl 1 g kg iv at the cessation of the remifentanil infusion . The diaphragmatic crura and port sites were infiltrated with 20 ml ropivacaine 0.2% by the surgeon . A patient - controlled analgesia (pca) device administering iv fentanyl was provided (10 g / ml, 10 g bolus, 5 minute lockout, no background) and pca usage was recorded . Acetaminophen (1 g iv every 6 hours) and indomethacin (100 mg per rectum every 12 hours) were administered to provide multi - modal analgesia . Rescue antiemetics (ondansetron 4 mg sublingual and droperidol 0.5 mg / kg iv) were offered to any patient who experienced nausea or vomiting . Patients were randomly assigned to study groups in fixed blocks of 12 using a computer - generated table of random numbers through the use of the randomization.com program . The patients in the intervention group received 1 mg / kg iv lidocaine bolus at induction, followed immediately by an infusion at 2 mg / kg / h for 24 hours . The patients in the control group were treated likewise using 0.9% sodium chloride in a double - blind fashion . Lidocaine was acquired as xylocard 500 ampoules (astrazeneca pty ltd, north ryde, nsw, australia), containing 500 mg of lidocaine hydrochloride in 5 ml of water . Lidocaine study drug was made to a concentration of 0.5% (5 mg / ml) and supplied in 1000 ml flasks compatible with imed gemini (imed corporation, san diego, ca, usa) infusion pumps and giving sets . No patient, research nurse, investigator, or any other medical or nursing staff was aware of the treatment assignments for the duration of the study . The randomization schedule was stored in a locked cupboard that was only accessible by the randomization authority (thus concealed from all care providers and other research personnel). When a patient was recruited into the study the study drug was given to the anesthetist accompanied by a sealed, opaque, tamper - proof envelope containing the treatment allocation . This envelope was kept in the patient file at all times in case serious adverse event required the knowledge of treatment allocation . Envelopes were examined at the completion of the trial to ensure that they were unopened . The patients were asked to score their current pain on two occasions: at rest and on mobilization from supine to sitting upright . Pain scores were obtained every 4 hours, for 30 hours following commencement of the trial drug infusion . The 11-point numeric rating scale is a validated, sensitive tool for assessing postoperative pain intensity.10,11 all trial data were entered directly into the electronic medical record, which had been adapted for the study and mandated the completion of all parameters . Secondary outcomes were nausea and vomiting, opioid requirements, adverse events, serum lidocaine concentration, and length of hospital stay . Patients were asked if they experienced any nausea or vomiting in the preceding 4 hours when pain scores were obtained . Opioid requirements were recorded as the dose of fentanyl delivered (rather than total demands / attempts for analgesia). Only adverse events that required the patient to be discontinued from the trial were interpreted . Venous blood samples were collected from all patients every 4 hours for the duration of the trial . Serum was immediately separated and frozen, then later analyzed to determine the serum lidocaine concentration length of hospital stay was recorded as the number of nights the patient was in hospital . A priori sample size was estimated on the basis of an absolute reduction in pain score . A reduction of two in the nrs-11 was reported to be the amount needed to cause a clinically significant reduction in a person s experience of postoperative pain.12,13 a sample size of 18 patients in each group was calculated to be sufficient to detect a difference of two in the mean pain score, assuming a standard deviation of 212 with a significance level =0.05 and a power of 90% . The study would be stopped early when there was sufficient evidence to claim superiority (net benefit) or inferiority (net harm), or futility (little chance of achieving statistical significance) if the futility index was found to be> 0.8.14 statistical analyses were performed using ibm spss statistics v21.0 (ibm corporation). Sample size calculations and conditional power analyses were performed using pass v13 (ncss, llc). The comparison between the intervention and control groups was conducted using an independent samples t - test, mann whitney wilcoxon u - test, chi - squared test, or fisher s exact test as appropriate . Normally distributed data are presented as mean standard deviation, nonnormally distributed data are presented as medians (interquartile range), and categorical data are presented as raw data and as frequencies . The three patients who did not complete the study were withdrawn due to adverse events suspicious of lidocaine toxicity (treatment allocation was not known until after withdrawal from the trial). The participant flow diagram is presented (figure 1). A planned interim analysis was performed when recruitment was 66% complete . At this point, the trial was stopped early on the basis of futility, prior to reaching the target sample size of 36 patients (18 per group). The futility index was 99%, that is, there was a 1% chance of rejecting a false null hypothesis at the end of the study given the data that had emerged.15 the demographic and clinical characteristics of both groups were similar (table 1). There was no statistically significant difference in pain scores between treatment groups either at rest or during mobilization when summarized over the 30-hour postoperative period . At rest, the mean pain score was 2.0 2.7 in the lidocaine group and 2.1 2.4 in the control group (p=0.286). With movement, the mean pain score was 2.0 2.6 in the lidocaine group and 2.6 2.7 in the control group (p=0.487). Box and whisker plots are presented for pain scores at rest (figure 2) and during mobilization (figure 3) for lidocaine and control groups stratified at 6-hour intervals over the 30-hour postoperative period . The incidence of nausea in the lidocaine group (50%) was similar to that in the control group (33%) (p=0.408). Three patients (25%) in the lidocaine group vomited, whereas one patient (8%) in the control group vomited (p=0.273). The average total dose of fentanyl administered was 124 165 g for the lidocaine group and 344 426 g for the control group over the 30-hour postoperative period (p=0.117). There were three clinically significant adverse events in the lidocaine group (25% of patients) compared with none in the control group, however, this difference was not statistically significant (p=0.064). One adverse event, severe bradycardia causing loss of consciousness, responded rapidly to external cardiac massage . The other adverse events were neurological symptoms (perioral paresthesia and restless legs) that are associated with early local anesthetic toxicity . The maximum serum lidocaine concentrations in these patients were 4.3, 2.7, and 3.5 mg / l . The serum concentration time profile of 12 patients who received the iv lidocaine infusion is presented in figure 4 . There was no statistically significant difference in the mean length of hospital stay between groups; lidocaine group 2.5 days (95% confidence interval [ci] 2.072.93) and control group 2.25 days (95% ci 1.962.54). This study used a high dose and long duration of intravenous lidocaine infusion in an attempt to identify a concentration - or time - dependent analgesic effect . Despite this, a clinically significant benefit of lidocaine was not demonstrated . This study cannot reject the possibility that a small amount of analgesia is achieved with intravenous lidocaine . A mean reduction of 0.54 (95% ci: 1.56, 0.48) in pain score during mobilization was found in this study . This effect size is similar to that reported in the meta - analysis,4 which showed intravenous lidocaine to reduce pain scores by 1.05 (95% ci: 1.68, 0.42) during activity at 6 hours after surgery and by 0.4 (95% ci: 0.8, 0.009) at 24 hours after surgery . This study was underpowered to detect such a small difference in pain scores between groups . Intravenous lidocaine infusion has only shown benefit (in reducing pain, nausea, opioid consumption, bowel function, and reducing hospital stay) in patients following surgery of the bowel or gall bladder,8 with the exception of radical prostatectomy.16 the studies of intravenous lidocaine for patients undergoing abdominal hysterectomy,17 orthopedic surgery,18 tonsillectomy,19 or coronary artery bypass20 could not identify any benefits of lidocaine . Based on the available evidence, it appears that the analgesic efficacy of lidocaine is dependent on the surgical procedure performed . We propose that the analgesic efficacy of lidocaine relates to the afferent (sensory) innervation of the manipulated tissues and the type of nociceptive pain associated with this innervation . It would seem that intravenous lidocaine has the potential to improve postoperative analgesia following abdominal surgical procedures associated with visceral pain or postoperative ileus . Fundoplication predominantly involves surgical manipulation of the diaphragm, which receives somatic sensory innervation via the phrenic nerve . The pneumoperitoneum and tissue combustion plume that are associated with laparoscopic surgery may also irritate the parietal peritoneum . Since there seems to be a greater proportion of somatic pain than visceral pain associated with laparoscopic fundoplication, this may explain why an analgesic benefit of lidocaine was not observed in this study . No correlation was found between the occurrence of adverse events and serum lidocaine concentration, however, this is not unusual, as local anesthetic toxicity is described as being insidious and often unexpected.21 clinical signs and symptoms of lidocaine toxicity can occur below the much quoted toxic threshold of 5 mg / l, as there are inter - individual differences between lidocaine serum concentration and its therapeutic or toxic effects.22 the greater number of adverse events seen in this study may be partially explained by the additional monitoring that the patients received . This unpredictability in safety may limit the clinical utility of intravenous lidocaine for postoperative analgesia . The patients in this study were found to have greater serum lidocaine concentration at steady - state than was anticipated . The clearance of lidocaine in this study population was 7.9 1.32 ml / kg / min, ~20% less than the estimate used when formulating the dosage regimen.23 this study found that an infusion rate of 33 g / kg / min (2 mg / kg / h) resulted in mean steady state serum concentration of 4.1 mg / l . An infusion rate of 26 g / kg / min (1.6 mg / kg / h) would have been required to result in the intended steady state serum concentration of 3.3 mg / l in this population . The weakness of this study is the small sample size because the trial was stopped early on the grounds of futility . We acknowledge that this trial has a higher risk of type ii error and it cannot exclude the possibility of a true effect size less than the minimum effect size of interest stipulated in the power calculation, however, we believe that it would have been an improper use of resources and unacceptable risk to patient safety in continuing the trial given the improbability of achieving statistical significance even if the entire a priori sample size was recruited . The strengths of this study are the validity of the randomized controlled trial methodology, the integrity of data collection, and close monitoring of patients for adverse events . As such, this well - designed, albeit small, study could contribute to future systematic reviews and meta - analyses in this field . Further investigation is warranted to define the optimal dosage regimen for intravenous lidocaine to balance analgesic efficacy and patient safety . Only when an evidence - based dosage protocol has been derived should further studies be performed to identify which surgical procedures or patient populations may benefit from intravenous lidocaine . This study does not support the use of intravenous lidocaine infusion for analgesia in patients undergoing laparoscopic fundoplication surgery . Intravenous lidocaine cannot be recommended for analgesia until a safe dosage regimen has been determined and the surgical procedures for which patients can benefit from intravenous lidocaine infusion have been identified.
The distal intestine is populated by a stunning quantity of bacteria, comprised of relatively few phyla that are highly diverse at the species level . This heterogeneous composition presents a vibrant metabolically active life - form, sharing the same habitat as our enteric organs . Realistically, enteric microbiota are a continuum, with members existing somewhere along the spectrum between symbiosis and pathogenicity . As the microbiota co - evolved with humans, they have provided us with genetic and metabolic characteristics that we have not had to develop on our own, such as enabling us to be able to harvest otherwise inaccessible nutrients and synthesizing vitamins (o'keefe et al ., 2009). Resistant starch and non - starch polysaccharides that evade human digestion provide energy for commensal bacteria . In turn, microbes metabolize these undigested nutrients to produce short - chain fatty acids (scfa), including butyrate, the primary nourishment for colonocytes which possesses potent anti - proliferative and anti - inflammatory properties (pryde et al ., 2002). Alternatively, enteric microbes may enhance disease pathogenesis at the cellular and molecular level via pro - inflammatory mechanisms (table 1). Microbial colonization of the gastrointestinal tract begins at birth, changes rapidly during the first year of life, and thereafter remains fairly constant (abraham and cho, 2009). During infancy, symbiotic bacteria have the capacity to promote growth and healing, induce angiogenesis, optimize nutrient absorption, attenuate intestinal inflammation, and prime the innate and the adaptive arms of the immune system (vaarala, 2003). Aberrations in the biodiversity of enteric microbiota can contribute to individual differences in immunologic behavior during and subsequent to childhood . Western nations, for instance, have experienced consistent increases in the incidence of allergic diseases in the past few decades, which may be due to a lack of microbial exposure during infancy the hygiene hypothesis or adoption of a western diet (strachan, 1989; wang et al ., 2008). A synchronous decrease in the incidence of infectious diseases in developed countries has occurred subsequent to the implementation of antibiotics, vaccination, and improvements in hygiene (bach, 2002). Mounting evidence suggests that diet and the microbiota, independently or in conjunction, influence the risk of developing atopic disease . The constitution of enteric microbiota may be a consequence of one's country of origin . Infants living in developing countries have been shown to be colonized at younger ages with fecal bacteria and have more rapid transfer of enteric microbial strains than infants living in developed countries (adlerberth et al ., 1998). Diversity is considerably greater in rural african than european children (figure 1), with a predominance of resistant polysaccharide hydrolyzers (prevotella and xylanibacter) and relative absence of inflammatory enterobacteriaceae (de filippo et al ., 2010). Compared to children without atopic illnesses, microbial species of children who manifest atopic sensitization demonstrate a reduced ratio of bifidobacteria to clostridia when they are infants (kalliomaki et al ., 2001a). Important determinants of enteric microbiotic composition in infants appear to be the mode of delivery, maternal diet, type of infant feeding (breast milk or formula), gestational age, infant hospitalization, antibiotic use by the infant, and the presence of siblings (penders et al ., 2006). One study comparing the enteric microbial species constitutions of 7-year - old children noted that those delivered by cesarean section had significantly lower quantities of clostridia and bifidobacteria (salminen et al ., 2004). Additionally, infants who are treated frequently with antibiotics have a heightened likelihood of developing asthma (marra et al ., 2009). (a, b) pie charts of median values of bacterial genera present in fecal samples of burkina faso (bf) and european union (eu) children (> 3%) found by rdp classifier v. 2.1 . Rings represent corresponding phylum (bacteroidetes in green and firmicutes in red) for each of the most frequently represented genera . (c) dendrogram obtained with complete linkage hierarchical clustering of the samples from bf and eu populations based on their genera . The subcluster located in the middle of the tree contains samples taken from the three youngest (12 years old) children of the bf group (16bf, 3bf, and 4bf) and two 1-year - old children of the eu group (2eu and 3eu). (d) relative abundances (percentage of sequences) of the four most abundant bacterial phyla in each individual among the bf and eu children . Blue area in middle shows abundance of actinobacteria, mainly represented by bifidobacterium genus, in the five youngest eu and bf children . (e) relative abundance (percentage of sequences) of gram - negative and gram - positive bacteria in each individual . Different distributions of gram - negative and gram - positive in the bf and eu populations reflect differences in the two most represented phyla, bacteroidetes and firmicutes . Adapted from de filippo et al . Breast feeding is associated with a diminished risk of atopic diseases, and alteration of the mother's diet to avoid n6 saturated fat and include supplementation with lactobacillus rhamnosus gg (atcc 53103), has been shown to reduce risk by 50% (kalliomaki et al ., 2001b). For instance, infants fed a strictly organic diet were found to have a significantly lower likelihood of developing eczema (kummeling et al ., 2008). Dietary lipids, particularly n6 long - chain polyunsaturated fatty acids, have a pro - inflammatory effect through the increased synthesis of prostaglandin e2; their consumption can contribute to the development of allergies and asthma (black and sharpe, 1997). N3 polyunsaturated fatty acids, on the other hand, possess a demonstrated ability to decrease the inflammatory response via their reduction in prostaglandin e2 levels (kobayashi et al ., diet may, thus, affect the development of allergic disease both directly by its effect on inflammation and indirectly through its influence on the composition and activity of the microbiota . Robust experimental and clinical evidence demonstrates that chronic inflammation increases the risk of neoplastic transformation (ekbom et al ., 1990; baron and sandler, 2000; coussens and werb, 2002; garcea et al ., 2005). Not surprisingly, studies have linked colonic carcinogenesis to chronic inflammation generated by pathogenic bacteria . Although specific bacterial species have yet to be proven to be the cause of colon cancer, the minor gut colonizer streptococcus bovis has been implicated in some clinical studies . Experimental investigations into its carcinogenic potential showed that bacterial proteins upregulated cox-2 production and promoted cellular proliferation by triggering mitogen - activated protein kinases which can increase the incidence of cell transformation as well as the rate of acquiring genetic mutations (baron and sandler, 2000; biarc et al ., 2004). Pro - inflammatory cytokines act as tumor promoters by regulating cytokines, chemokines, adhesion molecules, and angiogenesis (coussens and werb, 2002). A recent study of multiple intestinal neoplasia mice showed that infection with enterotoxin - producing bacteroides fragilis organisms that produce inflammatory diarrhea in humans triggered colitis and strongly induced colonic tumors (wu et al ., 2009). Clinical support for the importance of chronic inflammation is provided by the observation that colon cancer risk is increased five - fold in patients with chronic ulcerative colitis (ekbom et al ., furthermore, the risk of cancer in inflammatory bowel disease (ibd) is considerably higher if the disease affects the colon, rather than the small intestine, indicating that the carcinogenic effects of bacteria and inflammation may be additive . Finally, it has been clearly established that the use of anti - inflammatory medications suppresses colon cancer risk (baron and sandler, 2000). Substantial epidemiological evidence also supports a primary role for diet in the genesis of colorectal carcinoma (doll and peto, 1981). Comparative studies from high and low risk populations indicate that elevated intake of red meat and animal fat increase colon cancer risk, while greater consumption of fiber reduces risk . The effect of diet on colonic carcinogenesis may be mediated by the microbiota, which can either maintain colonic health or promote chronic inflammation (o'keefe et al ., 2009). Fermentation of indigestible carbohydrate, such as fiber and resistant starch, maintains colonic health by producing scfa such as acetate, propionate, and butyrate . Unlike most other cells in the body, furthermore, butyrate appears to be fundamental in the maintenance of cellular homeostasis and a normal colonocyte phenotype (topping and clifton, 2001). Scfas have anti - inflammatory effects, either directly by regulating the release of prostaglandin e2, cytokines, and chemokines from human immune cells (cox et al ., 2009), or indirectly by their ability to support the growth of probiotic species, such as bifidobacteria and lactobacilli (wang et al ., 2002; delcenserie et al ., scfas also possess immune - modulating and anti - inflammatory actions by binding certain g - coupled receptors which may help to stimulate the normal resolution of inflammatory responses in colonocytes (maslowski et al ., 2009). Colonic blood flow as well as fluid and electrolyte uptake are also enhanced by scfa production (topping and clifton, 2001). Evidence shows that risk reduction may be related to the effects of butyrate on activating the apoptosis cascade and inducing growth arrest of tumors by histone hyperacetylation (avivi - green et al ., 2000). In vitro, butyrate demonstrates numerous additional anti - inflammatory properties, including decreasing the effects of pro - inflammatory cytokines and their expression by inhibiting nf kappa b activation and abolishing lipopolysaccharide (lps)-induced expression of cytokines by peripheral blood mononuclear cells (segain et al ., 2000). Butyrate also inhibits the in vitro preneoplastic hyperproliferation - induced tumor promoters and arrests the growth of neoplastic colonocytes (pryde et al ., 2002). A fascinating recent study provides experimental evidence that butyrate maintains colonic motility, and therefore housekeeping, by modifying histone acetylation in the colonic myenteric plexus, leading to an increased proportion of cholinergic neurons and accelerated colonic transit (soret et al ., 2010). Excess hydrogen can damage living cells by impairing nad regeneration and inhibiting respiration (gibson et al ., this would not only injure the colonic mucosa (in the extreme case causing pneumatosis intestinalis), but would suppress the bacterial population size, thereby inhibiting its potential to feed itself and the colonic mucosa . The microbiota have evolved to circumvent this problem by expanding their diversity to contain organisms archaeal hydrogenotrophic methanogens (e.g., methanobrevibacter smithii) specialized to consume hydrogen to produce the non - cytotoxic end - product methane (wolin, 1976). The net effect is an enormous increase in the generation capacity of scfas from complex carbohydrates which ensures the health of both the microbiota and the colonic epithelium . As evidence, we observed that methanogenesis was considerably higher in low cancer risk rural africans consuming high complex carbohydrate diets compared to high risk americans who eat little fiber (o'keefe, 2001). Industrialized man's diet has changed from containing mainly unprocessed vegetables and grains to favoring meat, animal products, and refined cereals . This evolution reduces the quantity of complex carbohydrates or fiber that reaches the colon and alters the composition of the residue to contain more sulfur . The alteration in colonic milieu affects the diversity of the microbiota as sulfate stimulates the alternative growth of sulfur - reducing bacteria (srbs, e.g., desulfovibrio vulgaris), another form of hydrogenotrophic bacteria, which outcompete methanogens for hydrogen under these conditions (gibson et al ., 1988). Unfortunately, the metabolic end - product of srbs, hydrogen sulfide (h2s), differs from methane, being inflammatory and genotoxic . H2s impairs cytochrome oxidase, suppresses butyrate utilization, inhibits the synthesis of mucus and the methylation of dna (gibson et al ., 1993), and is genotoxic via the generation of free radicals (attene - ramos et al ., 2007). (1992) demonstrated that 70% of british fecal samples contained srbs and the remainder produced methane, whereas the reverse was the case with rural black south africans (gibson et al ., the link to diet was illustrated by further studies showing that a high meat diet increased fecal sulfide (magee et al ., 2000), and increasing colonic sulfide suppressed methanogenesis (christl et al ., 1992). The clinical significance of this finding is exemplified by observing that colonic overgrowth with srbs exists, together with suppression in butyrate production in patients with ulcerative colitis (roediger, 1993), another condition that increases colon cancer risk . Interestingly, ulcerative colitis is also rare in native africans (o'keefe et al ., 1983). While srbs play an essential role in the external environment by suppressing the release of methane gas one of the greenhouse gases from sewers and marshes into the atmosphere (guisasola et al ., 2008), it seems that humans, unlike the carnivores, have had insufficient exposure time to a westernized diet to evolve mechanisms for preventing h2s injury . A high meat diet is usually associated with elevated fat intake, and fat itself has been shown to influence cancer risk (ferguson, 2010). Fat stimulates the synthesis and enterohepatic circulation of primary bile acids, such as cholic acid and chenodeoxycholic acid, which are mostly reabsorbed, although a fraction reaches the colon and this quantity increases on a high fat diet (reddy, 1981). If the colonic microbiota contains 7-dehydroxylating bacteria (chiefly clostridial species), the primary bile acids are converted to the secondary bile acids, namely deoxycholic acid (dca) and lithocholic acid, which are well recognized co - carcinogens with lithogenic properties (nagengast et al ., 1995; higher fecal and plasma levels of dca have been observed in patients with colon cancer (hori et al ., 1998), and our studies have shown that levels were considerably higher in healthy, high risk african americans compared to low risk healthy rural africans (ou et al ., 2011). Our recent studies have confirmed that 7-dehydroxylating bacteria are less common in developing countries, where gallstones and colon cancer are rare (wells et al ., 2000; o'keefe et al . Figure a1 displays the relationship between food, the enteric microbiota, and the intestinal mucosa in regards to macronutrient metabolism and inflammation . Finally, an abundant supply of colonic carbohydrate residues can stimulate the synthesis of vitamins by the resident microbiota . In our studies in rural africans, we measured constituents of colonic evacuants following polyethylene glycol (peg) consumption (o'keefe et al ., 2007). As expected, total colonic scfa and butyrate were significantly higher in rural africans than in caucasians and african americans . More surprising was that colonic folate and biotin content was similar in all three groups despite considerably lower dietary intake in africans . Considering that content was higher than normal dietary intake, this indicated avid net synthesis by the microbiota . Folate is synthesized by some strains of specific microbes, e.g., bifidobacteria, from glycolysis and tricarboxylic acid cycle intermediates (watson, 2006). Studies have shown that increasing colonic carbohydrate, in the form of oligosaccharides or resistant starch, enhances the production rate of folate by microbiota (pompei et al ., 2007). The importance of luminal folate and biotin is evident in recent studies demonstrating that active colonic mucosal transport mechanisms exist for these vitamins and that both vitamins play key roles in dna synthesis and repair (o'keefe et al ., inflammatory bowel disease comprises two distinct, but overlapping, entities crohn's disease and ulcerative colitis . There are approximately 1.4 million people living in the united states with ibd and most cases are diagnosed in individuals in their teens or twenties (loftus jr . The etiology of ibd has been described as on - going intestinal mucosal inflammation that hinges on microbial or environmental factors and immune deficiencies in genetically predisposed individuals (abraham and cho, 2009). Etiologically, immune system dysfunction, an abnormally permeable mucosal barrier, or imbalances in beneficial and harmful species of enteric bacteria are hypothesized as contributing to ibd risk . Clinical and epidemiologic data demonstrate that a network of inflammatory components interacts to incite ibd (abt and artis, 2009). Examples of the influence of microbially perpetuated inflammation include the association of il-10 mutations with ulcerative colitis (franke et al ., 2008) and elevated levels of mucosal igg directed against the normal microbiota in ibd patients (cummings et al ., 2003) additionally, mutations in genes involved in bacterial peptidoglycan recognition (nod2/card15) are known risk factors for crohn's disease (abraham and cho, 2009) and recent studies have shown that the microbiota can mediate the effects of these genetic defects (rogler, 2010). The incidence of crohn's disease nearly tripled in northern europe (farrokhyar et al ., 2001). This hypothesis posits that the growing reliance on antibiotics and the sometimes obsessive focus on maintaining an anti - bacterial environment for children may be triggers for ibd on a population level . Studies of migrants, for instance, have shown that relocating from a developing to a developed nation increases the risk of ibd and risk is greatest if relocation occurs during infancy or early childhood (bernstein and shanahan, 2008). Regional differences in rates of ibd indicate that its etiology is exceptionally complex, with rates in the southern united states being lower than other us regions, but those of olmstead county (minnesota) being more similar to rates in wales (uk) rather than neighboring canadian provinces (bernstein et al . The incidence of ibd in african and asian countries, such as korea, is only a fraction of what it is in the united states and the united kingdom (yang et al ., 2008). Clinical observations of ibd patients and animal models highlight the role of bacteria, viruses, and mycobacteria in intestinal inflammation, although no specific microbe or profile of microbes have to date been proven to be causative and it must be remembered that the disease itself and its treatment may also disturb the microbiota . Murine models of colitis require bacteria for inflammation to occur, while other animal models have shown that antibiotics that target anaerobes and gram - positive organisms such as enterococci are protective against ulcerative colitis particularly when they are administered prior to the induction of inflammation (cummings et al ., 2003). Antibiotics can also be effective treatments for some ibd patients (elson et al ., 2005). Il-2 deficient, germ - free rats develop colitis due to a non - pathogenic e. coli mpk strain but not with exposure to b. vulgatus (waidmann et al ., 2003). Dysbiosis may also play a role in the etiology and severity of ibd, as some patients demonstrate lower enteric bacterial diversity with reduced proportions of butyrate - producers (frank et al ., 2007) and protective bacteria such as lactobacilli and bifidobacteria (eckburg and relman, 2007). This suggests that the mucosa is at risk, exemplified by the heightened incidence and severity of clostridium difficile colitis among ibd patients both in hospitalized and outpatient settings (ananthakrishnan et al . C. difficile thrives in a dysbiotic, permissive, environment and is suppressed by metabolically active butyrate - producers . However, butyrate deficiency may not provide the whole explanation as there is evidence for decreased butyrate utilization in ulcerative colitis and restoration of butyrate - producers may not reverse the inflammation (thibault et al ., 2010). Other microbial products may better explain the ability of butyrate - producers contained within clostridium cluster iv and xiva to decrease intestinal inflammation and necrosis in the mouse model (sokol et al ., 2008). Sokol et al . (2008) showed that the supernatant of a culture of a member of cluster iv, faecalibacterium prausnitzii, reduced colitis and mortality in the mouse model, even if administered orally or intraperitoneally . A japanese study found a strong correlation between increased intake of animal protein and n6 polyunsaturated fatty acids, and decreased intake of n3 polyunsaturated fatty acids over the course of 20 years with crohn's disease (shoda et al . Researchers in spain studied 43,013 patients aged 2074 years who had experienced an episode of acute infectious gastroenteritis and found that the estimated incidence rate of ibd was 68.4 per 100,000 person - years after an episode of gastroenteritis but 29.7 per 100,000 person - years in an age-, sex-, and calendar - matched controls (garcia rodriguez et al ., 2006). There was an especially greater risk of developing crohn's disease during the first year after the infective episode (hazard ratio, 6.6; 95% ci, 1.922.4) although confounding of crohn's symptoms with a bout of gastroenteritis might explain this observation . Ibd studies also note that patients have greater risk of developing gastrointestinal infections; an ibd twin study noted greater rates of self - reported enteric infections among patients than their non - affected twin (halfvarson et al ., 2006). Nonetheless, environmental triggers could be as important as genetic ones in the etiology of ibd . Studies of monozygotic twins in which only one twin is affected with ibd note that the unaffected twin may have altered mucosal glycosylation, indicative of preinflammatory nfkappab activation which would be acquired rather than inherited (bodger et al ., 2006). Enteric organisms share a common environment with the mucosa and have evolved in tandem to ensure survival of what has been described as the superorganism . In health, the microbiota prevents overgrowth with pathogens and simultaneously educates the gut immune system in immunotolerance and defense . Disturbance of the microbiota by diet, hygiene, antibiotics, or disease leads to dysbiosis, increasing the potential for mucosal injury and inflammation which, with time, may trigger a variety of mucosal and systemic diseases depending upon the host's genetic susceptibility . The final result of the soluble protein products of inflammatory cells is tissue damage, which can manifest in a variety of disease states, including eczema, colonic neoplasia, or ibd . Each of these entities requires a number of interactive factors to evolve and a prerequisite genetic susceptibility . Reproductive and lifestyle factors in developed nations, including advancements in public healthy hygiene measures, could help foster an environment that diminishes the protective effects of the enteric microbiota . Nonetheless, we can make a concerted effort to write fewer antibiotic prescriptions for infants, support breastfeeding for new mothers and discourage elective c - sections unless conditions necessitate them . For instance, encouraging patients to include ample fiber or complex carbohydrates in their diet, supplementing with probiotics, choosing healthy n3 fats over pro - inflammatory n6 fats, and limiting meat consumption, may optimize health and longevity . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . Dca, deoxycholic acid; h2s, hydrogen sulfide; ibd, inflammatory bowel disease; lps, lipopolysaccharide; nsaids, non - steroidal anti - inflammatory drugs; peg, polyethylene glycol; scfa, short - chain fatty acids.
Mood disorders are one of the most common psychiatric disorders that are expressed as diminished mood during periods of depression and elevated mood during periods of mania . Bipolar disorder is a complex chronic condition characterized by interactive periods of depression and mania or hypomania . The incidence of recurrent periods of depression and mania may have a deep impact on different aspects of life quality, including social, occupational, functional, and emotional well - being . The main features of these disorders are inability to cope with desire and power, interest and willingness to perform singular acts that are harmful to oneself or others, and feeling pleasure or relief during extreme behavioral tendencies such as excess in gambling, shopping, internet usage, romantic relationships, work, physical activities, and even eating . Recent studies have suggested that a large percentage of bipolar patients show a grave prognosis, high recurrence rate, residual symptoms, functional - cognitive deficits, psychosocial disabilities, and chronic addictive behaviors (overeating, extreme libido, watching television, gambling, impulsiveness, impulsive shopping, and work alcoholism, etc . ). The relationships between smoking and alcohol consumption, being exposed to violence and alcohol consumption, or early sexual contact and substance abuse have been repeatedly studied . Among these high - risk behaviors, sexual behaviors because of their irreversible consequences such as pregnancy, infectious diseases, and human immunodeficiency virus infection has been overemphasized in recent years . Other risky behaviors such as smoking, alcohol consumption, and drug abuse are proposed predictors of sexual risk - taking . Have shown that addictive behaviors in patients with mental disorders, such as schizophrenia and bipolar patients, are observed in a high level . Various researches have declared that the rate of drug - abuse, smoking, and addictive behavior in bipolar patients is higher than the other patients . Cerebrobehavioral systems are among the variables that probably play a role in the appearance of addictive behaviors . With the introduction of gray's theory, many psychiatric disorders are described in terms of two activation systems . In response to environmental stimuli, the theory viewing personality through a psychophysiological approach basically considers two activation systems namely behavioral activation system (bas) and behavioral inhibition system (bis), which has a neurological basis . Bas is responsible for the activation behaviors in response to reward (positive affection) and bis is responsible for the inhibitory behaviors in response to the threat and punishment (negative affection). In other words, bas makes individual potentially sensitive to the rewards, and creates incentives to explore them, whereas bis impacts individual's sensitivity toward punishment and makes them potentially sensitive to the punishments and avoiding them . Various findings have shown that an increase, decrease, or loss of balance in the operation of these systems is associated with a reduced life quality and several psychiatric and psychosomatic disorders . For example, an increase in bas activity influences substance abuse and antisocial behaviors, reduction of bas activity impacts depression, and its imbalance effects bipolar disorders . Studies have also shown that high bas sensitivity is associated with admission to addictive behaviors . In a comparative study between students with internet addiction and alcoholism, it was shown that students with internet addiction had higher scores in bis, whereas students with alcoholism had higher scores in response to stimulation and happiness and their scores were lower in bis . Meerkerk showed that drug abuse is more associated with bis rather than bas, however, yen et al . Reported contradictory results, that is, high bas and looking for entertainment characteristic are concluded to be among the risk factors leading to addiction . Kim and lee showed that morbid gambling behaviors are related to low scores in bis and high scores in bas . It has also been suggested that sometimes bas alongside low bis plays an important role in other addictive behaviors such as morbid gambling, which has similar features with internet addiction . In addictive behaviors, the person becomes obsessed (constantly thinks of) the object, activity, or substance . They will seek it out or engage in a behavior even though it causes harm (physical problems, poor work or study performance, problems with friends, family, and fellow workers). In addition, the person does not appear to have control regarding when, how long, or how much he or she will continue the behavior (loss of control). Another problems that are possible as a result of addictive behaviors include denying the problems, low self - esteem, and depression . In general, the present research is necessary considering the high prevalence of this disorder in our society, the role of bas and bis as key factors in generating success, promoting health, reducing psychological problems, and addictive behaviors, along with the lack of research in this field and the application of the results of the present study in the field of bipolar disorder patient's pathology . Therefore, the present study aimed to examine the role of bas and bis in predicting bipolar patients addictive behaviors . The population of this study consisted of all male patients with bipolar disorder who consulted psychiatrists or referred to the roozbeh psychiatric hospital, iran in 2014 . The sample comprises 80 male patients with bipolar disorder selected by a convenience sampling method . Considering that the sample should consist of at least 3050 participants in descriptive (correlative) studies, to increase the external validity of the study, 80 participants were selected . In addition, using the inclusion and exclusion criteria, the benchmark of participant's homogeneity was taken into consideration . Inclusion criteria for study participants were having at least junior high school education; having no cerebral palsy, mental retardation, or active hallucinations and delusions; being in partial remission period; being 2245 years old; and taking medicine during diagnosis and study project periods . Exclusion criteria included having severe comorbid disorders and confusion, severe depression, drug intoxication, or being in withdrawal period . This study recruited the persian translated version of comprehensive international diagnostic interview (cidi2.1) which provides the possibility of diagnosis of lifetime psychiatric disorders according to the diagnostic and statistical manual of mental disorders (dsm - iv - tr) and international classification of diseases (icd-10). Cidi consists of 14 sections, each marked with a letter (from a to x) and covers 17 major diagnostic fields: psychosis / mania module consists of f, g, and p, which is distinctively applied for the assessment of psychotic and bipolar disorders . In cases where psychosis / mania module was used alone, some schizophrenia diagnostic criteria for ruling out other mental disorders are not assessable . According to the cidi guideline, cidi's complete version or a special module can be applied . This self - report questionnaire developed by carver and white (1994), includes 24 items . The subscale of bis consists of seven items that measure the sensitivity of the bis . The subscale of bas includes 13 items measuring the sensitivity of bas which includes the following three subscales: response to drive containing four items, response to reward containing five items, and looking for entertainment containing four items . Four additional items are included in the scale having no effect on the assessment of bas / bis . Each item is rated on a four - point likert - type rating scale ranging from strongly agree to strongly disagree . As reported by carver and white, the internal stability of bis is 0.74 and the internal consistency of bas is 0.71 . Cronbach's alpha coefficients of bas subscales have been reported to be 0.73, 0.76, and 0.66, respectively . Retest reliability for bas scale to be 0.78 and for bis scale to be 0.81 . To determine the frequency of addictive behaviors (e.g., eating addiction, drug and alcohol addiction, and addiction to television and cell phone), lesieur and blume questionnaire can be used . This questionnaire had 28 items, each of which can be responded based on a likert scale (it does not describe me very well to it describes me very well). Thus, for each hypothesis, the questions were distributed among experts to consider their comments . After gathering their views and comments, the final questionnaire was prepared . To measure the internal consistency of the questionnaire, have reported the cronbach's alpha coefficient of the questionnaire to be 0.68 . In the present study, cronbach's alpha for eating addiction was 0.72, alcoholism, 0.84, television addiction 0.72, smoking addiction 0.76, and phone addiction was 0.82 . After coordinating with the roozbeh psychiatric hospital and gaining the satisfaction of the participants, as well as identifying men with bipolar disorder, the first aim of this study was explained to them . The participants were asked to read the questions carefully and choose the desired responses according to their own characteristics and answer as many questions as possible . Data were collected on an individual basis at related centers . Finally, the collected data were analyzed using pearson correlation and multiple regression statistical techniques . Moreover, the ethical issues including ensuring the participants about the confidentiality of their information and freedom of choice for participation in the study project observed in this research were explained to the participants . Ethical issues including ensuring the participants about the confidentiality of their information and freedom of choice for participation in the study project observed in this research were explained to the participants . This study recruited the persian translated version of comprehensive international diagnostic interview (cidi2.1) which provides the possibility of diagnosis of lifetime psychiatric disorders according to the diagnostic and statistical manual of mental disorders (dsm - iv - tr) and international classification of diseases (icd-10). Cidi consists of 14 sections, each marked with a letter (from a to x) and covers 17 major diagnostic fields: psychosis / mania module consists of f, g, and p, which is distinctively applied for the assessment of psychotic and bipolar disorders . In cases where psychosis / mania module was used alone, some schizophrenia diagnostic criteria for ruling out other mental disorders are not assessable . According to the cidi guideline, cidi's complete version or a special module can be applied . This self - report questionnaire developed by carver and white (1994), includes 24 items . The subscale of bis consists of seven items that measure the sensitivity of the bis . The subscale of bas includes 13 items measuring the sensitivity of bas which includes the following three subscales: response to drive containing four items, response to reward containing five items, and looking for entertainment containing four items . Four additional items are included in the scale having no effect on the assessment of bas / bis . Each item is rated on a four - point likert - type rating scale ranging from strongly agree to strongly disagree . As reported by carver and white, the internal stability of bis is 0.74 and the internal consistency of bas is 0.71 . Cronbach's alpha coefficients of bas subscales have been reported to be 0.73, 0.76, and 0.66, respectively . Retest reliability for bas scale to be 0.78 and for bis scale to be 0.81 . To determine the frequency of addictive behaviors (e.g., eating addiction, drug and alcohol addiction, and addiction to television and cell phone), lesieur and blume questionnaire can be used . This questionnaire had 28 items, each of which can be responded based on a likert scale (it does not describe me very well to it describes me very well). Maximum and minimum score of this scale is 112 and 28, respectively . Thus, for each hypothesis, the questions were distributed among experts to consider their comments . After gathering their views and comments, abolghasemi et al . Have reported the cronbach's alpha coefficient of the questionnaire to be 0.68 . In the present study, cronbach's alpha for eating addiction was 0.72, alcoholism, 0.84, television addiction 0.72, smoking addiction 0.76, and phone addiction was 0.82 . After coordinating with the roozbeh psychiatric hospital and gaining the satisfaction of the participants, as well as identifying men with bipolar disorder, the first aim of this study was explained to them . The participants were asked to read the questions carefully and choose the desired responses according to their own characteristics and answer as many questions as possible . Finally, the collected data were analyzed using pearson correlation and multiple regression statistical techniques . Moreover, the ethical issues including ensuring the participants about the confidentiality of their information and freedom of choice for participation in the study project observed in this research were explained to the participants . Ethical issues including ensuring the participants about the confidentiality of their information and freedom of choice for participation in the study project observed in this research were explained to the participants . The results showed that, among patients with bipolar disorder, 43% had high school education, 19% had diploma education, and 38% had upper diploma education . In addition, 50% of patients were hospitalized for 1 to 15 days, 25% for 15 to 30 days, 17% for 1 month, and 8% were hospitalized for more than 1 month . As it can be seen in table 1, the mean [standard deviation (sd)] of the total score of behavioral activation was 41.83 (4.14), and the mean (sd) of the total score of addictive behavior was 14.55 (3.15). Descriptive statistics of variables the results of table 2 show that there is a significant relationship between behavioral activation and addictive eating (r = 0.21), addiction to alcohol (r = 0.32), television addiction (r = 0.31), cigarette addiction (r = 0.34), mobile addiction (r = 0.34), and the score of all additive behaviors (r = 0.32). In fact, the greater level of behavioral activation in patients with bipolar disorder is associated with more addictive behaviors . In addition, there is a significant relationship between behavioral inhibition with addictive eating (r = 0.28), addiction to alcohol (r = 0.29), television addiction (r = 0.90), cigarette addiction (r = 0.38), mobile addiction (r = 0.25), and the score of all additive behaviors (r = 0.31). In fact, the greater level of behavioral inhibition in patients with bipolar disorder are associated with less addictive behaviors . The correlation coefficient between behavioral activation and inhibition systems with addictive behaviors as it can be seen in table 3, the observed f was significant and the results show that 47% of the variance of addictive behaviors in bipolar patients can be explained by reply to reward, derive, fun, bas, and bis . Given the amount of, reply to reward (= 0.38), derive (= 0.24), fun (= 0.24), bas (= 0.27), bis (= 0.21) are the most powerful variables to predict addictive behaviors in bipolar patients . In addition, the summary results of the multivariate regression model showed that behavioral activation and inhibition systems could predict 47% of the variance of addictive behaviors in patients with bipolar disorder . According to the results of the durbin watson test, that is equal to 1.88, the assumptions of normality of data, has also been considered . Summary results of standard and non - standard coefficients for the prediction of addictive behaviors based on behavioral activation and inhibition system this study aimed to assess the role of gray's activation / inhibition system in predicting addictive behaviors of bipolar patients . According to the results, there is a negative relationship between the bis and bas in bipolar patients, i.e. The higher level of behavioral inhibition is, the lower are the addictive behaviors . These findings are consistent with the studies of meerkerk et al ., kim and lee, and park et al ., whereas it is inconsistent with the studies of taylor et al . And staiger et al ., which showed that bis activity in addicted smokers is more than that in the non - addicted ones; that is, in the absence of reward, avoidance of negative outcomes, and unpleasant and severe conditions, individuals grow a tendency toward drug and alcohol consumption . Furthermore, bis is a neurological system which with its weakness, pleasure - feeling symptoms, becomes activated and creates a sense of euphoria and liberty . Low sensitivity of bis may create an increasing euphoria feeling and a tendency toward lack of inhibition, and therefore readiness to experience drugs and other deviations . One of them is related to the sensitivity of the bas and the uncontrollable desire for alcohol's rewarding . And the other is the uncontrollable desire for alcohol after leaving drug abuse, which is related to the sensitivity of the bis . Bipolar disorder and its features such as hopelessness, impulsivity, emotional instability, and interpersonal disturbances are significantly correlated with addictive and self - injuring behaviors . Considering that bipolar disorder is associated with negative emotions, the individual with the disorder when coping with adverse and stressful events is always looking for a way to escape from painful feelings . Such individuals may use different methods to minimize their pain including addictive behaviors and self - injury . A weak bis and a tendency toward lack of inhibition, that prevents the inhibition of inappropriate behaviors in different situations, causes the individual to have intense attachment to an object, behavior, or addictive behavior as a defense against depression and low mood, when experiencing negative emotions and stressful events . There is a positive relationship between bas and addictive behaviors in bipolar patients . In other words, the higher the level of behavioral activation is, the more addictive behaviors will be experienced . Mitchell and nelson - gray, and hundt et al . In explaining the results, it can be said that the lack of behavioral inhibition, experience seeking, risk - taking, and sensibility to monotony are effective factors on addictive behaviors . The desire to experience increased levels of risk for joy, especially in manic phases, is of higher risk - taking levels so that individuals in order to seek more pleasure or to escape monotony take the risks of consuming drugs and alcohol or perform other risky behaviors . On the other hand, they estimate the activation of risks and negative consequences of such behaviors as low and insignificant and much lower than the actual rate for themselves; for example, despite their occasional use of alcohol or drugs or engaging in risky sexual behaviors, they consider the consequences and risks as negligible for themselves, and with irrational beliefs, such as the recreational drug use is not harmful or addictive, or i use it only a few times and consider themselves strong in the face of these risks . Moreover, with the examination of the bas components, the results showed that there is a relationship between bas drive, response to reward, entertainment seeking, and addictive behaviors of bipolar patients . These results are consistent with the studies of park et al . And kim and lee . Bipolar patients, because of a lack of emotional awareness and cognitive capability of processing their emotions, have a limited ability to cope with stressful situations . As a result, the psychological component of emotional expression and psychological distress systems, namely depression, mania, and anxiety increases, which can be expressed as bas excitation . Based on the outcome of bas activity, the pleasure - seeking personality, which plays an important role in the development of addictive behavior, it can be concluded that those bipolar patients have high levels of sensitivity toward bas reward who have high enjoyable activities, looking for new experiences, are unable to control themselves, and make impulsive decisions as a result of the tendency toward high - risk and addictive behaviors . Finally, the small sample size and lack of a comparative study of women with bipolar disorder it is recommended that the authorities with their emotional support provide proper conditions for these patients to express their feelings and emotions in a suitable manner, and facilitate the emotional, cognitive, and psychiatric compatibility of these patients.
The unfortunate complication of a colotomy resulting from a colonoscopic polypectomy can be disastrous . Using the versatility of laparoscopic surgery we report a case of cecal perforation, in a 70 year old female, following colonoscopic polypectomy that was treated successfully with laparoscopic application of an endo - gia linear stapler . Four months following staple resection of her cecal perforation, the patient is asymptomatic and has undergone a repeat colonoscopy without any sequelae . Laparoscopic techniques can be safely applied in the treatment of colonic perforations following therapeutic polypectomies . A suggested management algorithm is provided highlighting the role laparoscopy may provide in selected patients . Colon perforation during therapeutic colonoscopy has been estimated to occur at a rate of approximately 0.1% to 0.3% . Perforation may occur through a variety of mechanisms, including energy application at the polyp site, direct mechanical trauma from the tip of the endoscope, lateral pressure from the side of a bowed endoscope, and pneumatic injury from overinsufflation . Several case reports advocating laparoscopic repair of colonic perforation following colonoscopy have been described in the literature . In our present case report, we discuss the single application of an endoscopic linear stapler to close the perforation while preserving colonic lumen integrity in the presence of endoscopically placed mucosal clips . A 70-year - old caucasian female with a past medical history significant for sigmoid diverticulosis and previous benign polyps of the colon was found to have a lipomatous lesion in the ascending colon during routine surveillance colonoscopy . A complete resection of the polyp was performed using a 15-ml saline injection with india ink lift and a hot snare . After resection, the mucosal biopsy site was larger than anticipated, and it became difficult to maintain insufflation . The procedure was halted secondary to progressive abdominal pain and the clinical suspicion of a colonic perforation . The perforation was confirmed with the presence of substantial free air on radiographs . In this rare situation of early discovery and hemodynamic stability, the patient was taken immediately to the operating room for surgical repair . In the operating theater, pneumoperitoneum was established with a veress needle, and a 5-mm camera with an optical access trocar was placed in the left lower quadrant . Of note, the abdominal pressure at initial access was measured at 10 mm hg, consistent with pneumoperitoneum from insufflation through the colotomy . A 5-mm anterior perforation of the cecum was clearly visible with an area of circumferential coagulation extending approximately 2 mm (figure 1a). The cecum was noted to be adhered to the side wall, and hence one additional 5-mm port and one 10-mm port were placed for mobilization in the midline . The ports were placed in the umbilicus and the suprapubic position . After a sufficient segment of the cecum was freed, a single 30 silk stitch was placed to provide temporary closure and to provide a handle for elevation of the cecum . An endo - gia 60-mm blue load was applied to preserve the lumen of the cecum while incorporating the entirety of the defect including the area of coagulation necrosis (figure 1b). The suture line incorporated healthy pink mucosa with preservation of the colonic lumen (figure 1c). The patient had an uncomplicated recovery with discharge on hospital day 4 . At the 4-month follow - up, the patient is without any complaints, tolerating a regular diet, and at baseline health . She has had an interval surveillance colonoscopy without any abnormalities and a well - healed polypectomy / surgical site . A: anterior cecal perforation with surrounding circumferential area of necrosis and endoscopically placed clips (white arrows). B: endo - gia 60 mm stapling with careful preservation of colonic lumen with incorporation of the entirety of the defect and area of coagulation necrosis . Colotomy that occurs during colonoscopy is a specialized subset of colon perforations for a variety of reasons . To begin with, in many cases the patient is under the care of a physician in a monitored setting with established intravenous access . Although there may be some question as to the precise benefits of mechanical preparation in elective colorectal surgery, in the emergent setting it may be advantageous in minimizing the amount of gross spillage . In addition, the endoscopist may provide localization of the suspected injury, allowing for more directed treatment . It has been advocated by some that colonoscopic polypectomy should be performed in conjunction with laparoscopy when a particularly difficult or suspicious polyp is anticipated . In keeping with the success of previous case reports and advances in minimally invasive techniques, we propose an active role of laparoscopy in complex colonoscopies as outlined in figure 2 . Perforations, depending upon their size, may be treated with direct suturing, staple resection or segmental resection . Our approach to this cecal perforation was fundamentally the same as that for an appendectomy . The technical skills involved in the procedure are well within the capabilities of practicing general surgeons, but several technical tips merit further comment . In this case, primary repair with intracorporeal suturing was technically possible . However, it seemed ill advised, given the visible zone of coagulation necrosis surrounding the defect . In regards to the surgical stapler, it would have been technically easier to place a single firing across the cecum rather than 2 angled applications; however, 2 factors suggested multiple stapling: the endoscopically placed clips may have interfered with proper staple line creation, and a single firing raised the concern for luminal compromise . It may be argued that in this case the procedure was effective, but that minimally invasive techniques are not broadly applicable . However, in the analogous situation of perforated appendicitis, there have been multiple studies in which laparoscopy does not have an increased rate of complications over open laparotomy . Conceptually, it seems there is little harm in starting with a diagnostic laparoscopy and proceeding based on the nature of the injury and the experience of the surgeon . Many general surgeons will be confronted with the problem of colon perforation during colonoscopy . In the case presented here, we modified the established technique of laparoscopic appendectomy to the specific problem of cecal perforation and have demonstrated the feasibility of such an approach.
Bednar tumor or pigmented dermatofibrosarcoma protuberans (dfsp) is a rare, aggressive, cutaneous tumor that constitutes 1 - 5% of all dfsps . A 40-year - old female presented with right shoulder swelling present since few months and gradually increasing in size . A complete excision was performed without any prior fine needle aspiration cytology or needle biopsy . The tumor was just beneath the skin, fairly circumscribed, non - capsulated extending into the subcutaneous fat and measuring 5 4 4 cm . Grossly, focal areas of pigmentation were noticed towards the periphery of the tumor (fig . The tumor was a spindle cell lesion abutting the skin and invading the underlying subcutaneous fat . 2). On higher magnification, the spindle cells were uniform appearing with a moderate amount of eosinophillic cytoplasm and plump nuclei with tapering edges . The pigment - laden cells showed coarse brown black pigment obscuring the nuclei and positivity for masson - fontana stain . It is considered to be a tumor of intermediate grade and very infrequently reported in the literature . It is seen in young to middle - aged adults in the fourth decade with very occasional cases in the pediatric age group . The preferred sites are the shoulder region as in our case as well as the trunk, extremities and the head and neck . Grossly, the tumors have been described to be multilobular with pigmentation, irregular surface and firm nodules growing deep within the subcutaneous tissue . In this case, the cut surface of the fairly circumscribed tumor showed trabeculations and peculiar pigmentation in the subepidermal portion, which has not been described in the literature . It has to be distinguished from other pigmented cutaneous spindle cell lesions like pigmented neurofibroma, psammomatous melanotic schwannoma, neurocristic cutaneous hamartoma and desmoplastic malignant melanoma . However, our case had classic features of dfsp, and the above lesions were ruled out on histopathology itself . Neuro - ectodermal differentiation or melanocytic colonization are the two proposed theories for histogenesis for the bednar tumor . It has also been reported in association of dermal melanocytosis (nevus of ito), and based on the immunohistochemistry, the cell of origin is thought to be a neuromesenchymal cell . Bednar tumor can rarely undergo malignant transformation in form of fibrosarcoma with repeated recurrences and distant metastasis . Hence, a close follow - up of these cases is always necessary, which is being done in our case . To conclude, though rare, it is important for the histopathologists to be aware and recognize this unusual entity and distinguish it from other pigmented spindle cell lesions . Ethics committee approval and patient consent has been obtained . Published research complies with the guidelines for human studies and animal welfare regulations . The patient has given informed consent, and the study protocol has been approved by the institute's committee on human research.
Colon cancer (cc) is the third most common cancer in both genders worldwide and the second cause of cancer - related death in western countries, only overcome by lung cancer [1, 2]. In cuba, cc is one of the most frequent tumors and the third cause of death by cancer in both men and women . Despite the significant advances in the treatment and knowledge of the colon tumor biology over the last decade, the 5-year survival after surgery varies from 90% to 10%, depending on stage and progression of disease [2, 4]. Therefore, it is important to increase our understanding of the molecular changes leading to development, spread, and metastasis and to identify potential prognostic and predictive markers for the disease . To date, tnm (tumor - node metastasis) staging, based on the american joint committee on cancer (ajcc) and the international union against cancer (uicc) classifications, has been the most important prognostic marker in cc . However, this classification provides limited information, since cancer outcomes vary significantly among patients within the same stage . Regard, ganglioside is one of the molecules under evaluation as biomarker and target for therapy . Gangliosides are sialic acid - containing glycosphingolipids expressed in the plasmatic membrane of vertebrate's cell . N - acetylneuraminic acid (neuac) is the most common in humans, while n - glycolylneuraminic acid (neugc) is not usually detected in normal tissues due to a specific inactivating mutation in the cytidine monophospho - n - acetylneuraminic acid hydroxylase (cmp - neuac hydroxylase) gene [8, 9]. However, the expression of n - glycolyl - containing gangliosides has been found in a variety of human malignancies [10, 11], suggesting its possible role in oncogenic process and becoming in attractive targets for cancer immunotherapy . The expressions of neugcgm3 ganglioside have been previously reported in different tumors using 14f7 mab [1215], a murine igg1 highly specific against the n - glycolyl - gm3 ganglioside produced by the center of molecular immunology, havana, cuba . Blanco et al . Evaluated the 14f7 mab immunostaining in digestive system tumors, including some specimens of colon adenocarcinoma . The objective of this study was to assess the prognostic role of 14f7 mab immunoreactivity in patients with cc and evaluate the relationship between its expression and clinicopathological features . We studied tumor specimens from 50 patients diagnosed with colon cancer who underwent tumor surgical resection at the national institute of oncology, havana, cuba, between 2004 and 2008 . All routinely stained sections were reviewed by two pathologists, who did all histopathologic classifications including stage, grade of differentiation, tumor type (mucinous or nonmucinous), peritumoral inflammation, degree of cell pleomorphism, and mitotic index; these last two were previously described . Briefly, 4 m thick sections were cut from formalin - fixed and paraffin - embedded blocks of surgical specimens . Subsequently the sections were dewaxed in xylene and rehydrated through a graded series of ethanol . Endogenous peroxidase activity was inhibited by incubating the sections in 0.3% hydrogen peroxide in methanol for 10 minutes followed by tris - buffered saline wash . Nonspecific labeling was blocked with bovine serum albumin for 30 min . As primary antibody, we used the 14f7mab, a murine igg1 highly specific against n - glycolyl - gm3 ganglioside . Sections were incubated with 14f7 mab (12 g / ml) for 30 minutes at room temperature . Then a kit universal dako lsab+system - hrp (code k0679, dako, denmark) was applied to all sections according to the manufacturer's recommendations . Finally, the sections were counterstained with mayer's hematoxylin, dehydrated through ascending ethanols to xylene, and mounted with eukitt (kinder gmbh & co., freiburg, germany). As a negative control, immunohistochemical results were evaluated according to proportion of stained cells and intensity of 14f7 mab reactivity . The proportion of stained cells was graded on a scale of 03 (0, no staining; 1, 150%; 2, 5175%; and 3, 76100%). The staining intensity was graded on a scale of 03; 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining . Subsequently, an immunoreactive scoring (irs) was obtained by multiplying the two previously mentioned parameters . The cutoff level of 14f7 immunostaining was defined as a dichotomous variable of low level (irs <6) or high level (irs 6). All slides were assessed by two trained observers who did not have knowledge of clinical characteristics or outcomes . The relationships between 14f7 mab immunoreactivity and clinicopathologic variables were analyzed using the chi - square test . Survival rates were estimated by the kaplan - meier method and compared with the log - rank test . For multivariate analyses, the cox regression model was used to identify independent prognostic factors for overall survival (os) and disease - free survival (dfs). The model included all variables to have significant prognostic value in univariate analysis (log - rank test). Os was measured from the date of surgery to death for any cause or last follow - up and was calculated for all patients . Dfs was measured from the date of surgery to the date of second cancer, locoregional recurrence, distant metastases, and death for any cause or last follow - up and was calculated in patients with disease stages i iii . All data on survival and disease - free survival were updated in july 2013 . The study population had a median age of 63 years (range: 3685 years). According to histopathological type, all tumors were adenocarcinomas and the majority was of a moderate histological grade (62%). The immunostaining was observed in all cases, with variable intensity and proportion of stained cells . Most specimens had strong intensity (68%) and more than 75% of positive cells (62%) as shown in table 2 . A moderate correlation was found between percentage of positive tumor cells and staining intensity (spearman's correlation coefficient 0,606; p = 0,000). According to irs, the reactivity was observed on both membrane and cytoplasm of tumor cells with a staining pattern finely granular . In a few cases (six specimens) no significant differences were observed with age, sex, tumor location, grade of differentiation, mucinous type, mitotic index, pleomorphism grade, peritumoral inflammation, or lymphovascular invasion . However, the level of immunoreactivity showed statistical correlation with tnm stage (p = 0,025 and spearman r = 0,317). When cases were analyzed independently, according to intensity or extent of staining (data not shown), no significant associations with clinicopathologic features were noted, except for the positive relation between proportion of stained cells and tnm stage (p = 0,038). In survival analysis, there was a significant difference in the 5-year os rates between high and low 14f7 mab immunostaining (40% versus 86,7%; p = 0,002). Furthermore, patients with high level of 14f7 mab immunoreactivity had significantly impaired 5-year dfs (p = 0,046) than those with low level (60,9% versus 92,3%). Immunostaining was associated significantly with os (p = 0,0078) while no significant relation was demonstrated with dfs, although a trend existed (p = 0,0745). Univariate analysis showed that level of 14f7 mab immunostaining (p = 0,0078), tnm stage (p = 0,0007), and lymphovascular invasion (0,027) were significant prognostic factors for os . Among these variables, level of 14f7 mab immunostaining (hr = 0,268; 95% ci 0,0780,920; p = 0,036) and tnm stage (hr = 0,249; 95% ci 0,0660,932; p = 0,039) were independent prognostic factors on multivariate analysis . For dfs, tumor location was significant prognostic factor (p = 0,036) since patients with sigmoides tumor had poor survival . However, a trend existed for the level of 14f7 mab immunostaining (p = 0,074). As only one variable was significant in univariate analysis, multivariate analysis was not performed . Given the limited impact of conventional factors in cc, it is necessary to identify new prognostic biomarkers that provide information concerning the natural history of this disease . The present study is the first to evaluate the prognostic significance of 14f7 mab immunostainingin patients with colon adenocarcinoma . The 14f7 mab immunoreactivity, against neugcgm3, has been previously reported in some tumors including breast carcinoma, skin neoplasms, lung cancer, and neuroectodermal tumors . In our research, we used formalin - fixed and paraffin - embedded tissues, which is common in retrospective and long - term survival studies . However, as the routine tissues processing could damage the structure of gangliosides, further studies in frozen samples are recommended to confirm these results . Although the presence of neugcgm3 in tumors has been demonstrated, the mechanisms that support its expression have been controversial . Some studies suggest that its presence in human cancer is due to metabolic incorporation of dietary neugc, related with changes in the metabolism of tumor cells . It is well described that cells can process exogenous sialic acids from the extracellular environment and use them for their own glycoconjugates [18, 19]. Furthermore, our data showed a moderate 14f7 mab reaction in some normal glands surrounding the tumor . This is in line with previous studies that reported a limited recognition of 14f7 mab in normal tissues [1214, 16]. A possible mechanism for this finding is that normal eukaryotic cells were able to take in a portion of ingested neugc and process it for their own glycoconjugates [18, 20], although other researches are needed in this regard . In this study, high 14f7 mab immunostaining was significantly associated with advanced tnm stage (p = 0,025), which is characterized by the presence of cancer cells in regional lymph nodes and evidence for metastases . Although our findings suggest the role of neugcgm3 ganglioside in the oncogenic process, tumor growth and progression, further studies in larger sample with better distribution by each stage are required . Our data is consistent with a previous report of scursoni et al ., who found a correlation between the expression of neugc - gm3, using 14f7 mabs and more aggressive disease, in neuroectodermal tumors . Previous studies have shown the relevance of this neugcgm3 in cancer progression and its capability of modulating cd4 expression on t cells [2123]. Some properties of this ganglioside have been described including significant inhibition of human dendritic cells differentiation by inducing apoptosis in precursor cells, modification of cd4 expression, and promotion of th2 differentiation pattern in t lymphocytes . In fact, this molecule has been described as one of the most immunosuppressive gangliosides . Showed that uptake of neu5gc into human tumor cells in vitro and delivery into the cytosolic compartment via a lysosomal transporter are enhanced by high cell growth rates . The tumor progression by stimulating inflammation via binding to neu5gc - positive tumor cells is another mechanism described . Hedlund et al . Found that the combination of tumor - associated neu5gc and circulating anti - neu5gc antibodies promotes tumor growth, by inducing weak inflammation, causing infiltration of inflammatory cells and enhanced angiogenesis . In survival analysis, we reported that higher 14f7 mab immunostaining was significantly associated with impaired os (in univariate and multivariate analysis) and dfs (in univariate analysis), suggesting its potential use in the prognostic estimate of colon adenocarcinoma . No similar studies have been previously described in this regard . Only few reports in lung cancer found that neugc - gm3 expression was associated with better survival in non - small - cell lung cancer (nsclc), while hayashi et al . One possibility is the differences in predominant histological type, as in the first two cases was it squamous subtype, while in the third it was adenocarcinoma, resembling our study . Other factors like the number of patients evaluated and the distribution according to clinical stage as well as the specificity of the antibodies might lead to differences in results . In current research, univariate survival analysis confirmed the poor os associated with tnm stage and lymphovascular invasion, two putative pathologic markers of invasion and adverse outcome in cc . Gangliosides have been considered attractive targets for cancer immunotherapy based on their higher abundance in tumors when compared with the corresponding normal tissue . In cuba, several clinical trials have been performed with the anti - neugc - containing gangliosides anti - idiotype monoclonal antibody racotumomab (also known as 1e10) and the conjugated neugcgm3/vssp (very small size proteoliposomes) nanoparticle vaccine for immunotherapy of melanoma, breast, and lung cancer . Both vaccines targeted to neugc - gm3 ganglioside have acceptable safety outcomes and are able to induce specific humoral and cellular immune responses . Preliminary evidence suggested that these vaccines may have a positive influence on survival in patients with immune response to neugcgm3 antigen [7, 3436]. This study is the first approach on the prognostic significance of 14f7 mab immunoreactivity, against n - glycolyl gm3 ganglioside, in patients with colon adenocarcinoma . The immunostaining correlated significantly with tnm stage and it was an independent prognostic indicator of overall survival in multivariate survival analysis . The current research suggests that assessment of 14f7 reactivity might be used in the prognostic estimate of colon adenocarcinoma, although our results need to be validated in a larger sample and prospective studies . Furthermore, the role of neugcgm3 in tumor biology and its differential expression in tumor cells suggest its potential use as target for immunotherapy.
The study was designed as a multicenter, retrospective collection of cases with using a case reporting form . Only patients who underwent an ia recanalization procedure due to acute anterior circulation ischemia in the internal carotid artery (ica) and the middle cerebral artery (mca), and this was performed within the past five years, were included in this review . This form included the basic clinical information, the initial imaging results, the angiography and procedure - related information, the follow - up imaging results and the clinical outcome of each patient together with the actual imaging data . We requested that the clinical and procedure - related parts of the form be filled out by the radiologist at each center . The basic clinical information included age, gender, national institute of health stroke scale (nihss) score at the time of presentation and the presence of underlying diabetes, hypertension and/or heart disease . Selection of the initial imaging modality, the time from symptom onset to the time of imaging, the time from symptom onset to the initiation of angiography (the first angiographic run), and the time of day of the procedure (i.e., during or after working hours) were analyzed . The pre- and post - procedure ct / mr images and angiographic findings were evaluated by the board - certified radiologist who had more than five years of experience with ia thrombolysis . The ct results were analyzed for the presence of early ct signs, involvement of basal ganglia and the presence of hyperdense mca signs . The mr findings were analyzed to determine the extent of the lesion on the diffusion - weighted images (dwi), involvement of the basal ganglia, the presence of perfusion / diffusion mismatch and presence of microbleeds . The angiographic results were analyzed for the anatomic location of the culprit steno - occlusive lesions and the initial and post - procedure thrombolysis in myocardial infarction (timi) grades . We regarded a post - procedure timi grade of ii or iii as indicative of successful recanalization . The immediate post - procedure ct was reviewed for the presence of hemorrhages and parenchymal hyperdensity that was observed due to the contrast media used during the thrombolytic procedure . The follow - up ct or mr findings were analyzed for occurrence of hemorrhagic transformation, which was evaluated by the european cooperative acute stroke study (ecass) classification method . We calculated the rates of occurrence of any hemorrhage and any radiologically significant hemorrhage, with the latter regarded as hemorrhage with a mass effect, such as parenchymal hematoma type 2 or some cases of hemorrhagic infarction type 2 . The clinical outcome analysis was performed using the modified rankin scale (mrs), which was recorded at various time points after the procedure (at 1 - 12 months, except for the patients who died in the interim). If the clinical outcome data were not available, the case was dropped from the clinical outcome analysis . We calculated the percentage of patients with each mrs score, as well as the mortality rate, which was defined as the rate of procedure - related deaths within one month . Univariate logistic regression methods were used to analyze the factors that had an influence on successful recanalization, significant hemorrhagic transformation and poor functional outcome results . The proportion of patients with a mrs of more than 3 was used for the latter . Variables with p values less than 0.05 on the univariate analyses were chosen as the variables for the multivariate logistic regression analysis . In both analyses, the basic demographic characteristic of the 163 patients from seven domestic institutes are summarized in table 1 . The mean time interval from the symptom onset to the initial ct scanning was 139145 minutes and the mean time interval from the symptom onset to the first angiography was 280178 minutes (table 2). Out of the 149 patients for whom we were able to obtain the time of day of their angiography, 92 (62%) had the procedures performed during normal working hours (09:00 - 18:00). The initial imaging modalities were ct in 46 patients (28%), mr in 63 (39%), and both ct and mr in 54 (33%). We were able to review the ct images of 69 of the 100 (69%) patients who initially underwent ct . The basic initial ct and mr findings are summarized in tables 3 and 4, respectively . The site of arterial stenosis (timi grade 1, n = 18) or occlusion (timi grade 0, n = 145) was the ica, including the carotid' t' occlusion, in 62 patients (38%), the mca, including m2 occlusion, in 99 (61%) and the anterior cerebral artery in two patients (1.2%) (table 5). Before the initiation of ia thrombolysis, 73 patients (45%) were administered intravenous tissue plasminogen activator (t - pa, 0.6 or 0.9 mg / kg). Urokinase at a median dosage of 400,000 iu (range 40,000 - 1,000,000 iu) was the ia fibrinolytic agent in 139 patients (85%), t - pa at a median dosage of 10 mg (range 5 - 20 mg) was used in 11 patients (7%), and the procedure was performed without ia fibrinolytics in 13 patients (8%). In the latter case, mechanical methods were primarily applied, except for one patient, who received an ia infusion of abciximab . Various mechanical methods were applied, along with local infusion of fibrinolytics, in 82 patients (50%), including those patients who were treated with primary mechanical thrombolysis . The methods employed included mechanical disruption of the clot using a micro - guidewire (n = 49, 60%), stenting of the steno - occlusive lesion using various kinds of metallic stents (n = 17, 21%), balloon catheter angioplasty or clot disruption (n = 12, 14%) and aspiration of the clot using a large - bore guiding catheter (n = 4, 5%). A combination of these methods was used in patients for whom the primary mechanical method was ineffective in recanalization . The final timi grades were zero in 21 patients (14%), one in 23 (15%), two in 31 (20%) and three in 77 (51%). Various patterns of parenchymal high densities were observed in 54 patients (47%), and 24 (21%) of them showed obvious hematoma formation and/or high density with a mass effect . Hemorrhagic transformation of the lesion was evaluated in 155 patients whose follow - up ct and/or mr images were available for the review, and 55 (35%) of these patients showed variable amounts of hemorrhage . The hemorrhage pattern was hemorrhagic infarction (hi) type 1 in 15 patients (27%), hi type 2 in six (11%), parenchymal hematoma (ph) type 1 in 17 (31%) and ph type 2 in 17 (31%). The follow - up period varied from one to 12 months, except for those patients who died in the interim . We noted mrs scores of zero in 17 patients (11%), one in 27 (17%), two in 19 (12%), three in 20 (13%), four in 33 (21%) and five in 24 (15%); these findings were comparable with those of other major thrombolysis trials (fig . 1). Out of the 158 patients, 18 died, yielding a mortality rate of 11% . Among the various factors considered, an initial nihss score of less than 15 was the only factor that positively influenced successful recanalization (p = 0.001). Table 6 shows the univariate analysis of the factors that influenced significant hemorrhage after the procedure, and table 7 shows the factors that influenced the poor functional outcome . Subsequent multivariate analyses failed to show any statistically significant variables both for significant hemorrhage and for a poor functional outcome . The basic demographic characteristic of the 163 patients from seven domestic institutes are summarized in table 1 . The mean time interval from the symptom onset to the initial ct scanning was 139145 minutes and the mean time interval from the symptom onset to the first angiography was 280178 minutes (table 2). Out of the 149 patients for whom we were able to obtain the time of day of their angiography, 92 (62%) had the procedures performed during normal working hours (09:00 - 18:00). The initial imaging modalities were ct in 46 patients (28%), mr in 63 (39%), and both ct and mr in 54 (33%). We were able to review the ct images of 69 of the 100 (69%) patients who initially underwent ct . The basic initial ct and mr findings are summarized in tables 3 and 4, respectively . The site of arterial stenosis (timi grade 1, n = 18) or occlusion (timi grade 0, n = 145) was the ica, including the carotid' t' occlusion, in 62 patients (38%), the mca, including m2 occlusion, in 99 (61%) and the anterior cerebral artery in two patients (1.2%) (table 5). Before the initiation of ia thrombolysis, 73 patients (45%) were administered intravenous tissue plasminogen activator (t - pa, 0.6 or 0.9 mg / kg). Urokinase at a median dosage of 400,000 iu (range 40,000 - 1,000,000 iu) was the ia fibrinolytic agent in 139 patients (85%), t - pa at a median dosage of 10 mg (range 5 - 20 mg) was used in 11 patients (7%), and the procedure was performed without ia fibrinolytics in 13 patients (8%). In the latter case, mechanical methods were primarily applied, except for one patient, who received an ia infusion of abciximab . Various mechanical methods were applied, along with local infusion of fibrinolytics, in 82 patients (50%), including those patients who were treated with primary mechanical thrombolysis . The methods employed included mechanical disruption of the clot using a micro - guidewire (n = 49, 60%), stenting of the steno - occlusive lesion using various kinds of metallic stents (n = 17, 21%), balloon catheter angioplasty or clot disruption (n = 12, 14%) and aspiration of the clot using a large - bore guiding catheter (n = 4, 5%). A combination of these methods was used in patients for whom the primary mechanical method was ineffective in recanalization . The final timi grades were zero in 21 patients (14%), one in 23 (15%), two in 31 (20%) and three in 77 (51%). Various patterns of parenchymal high densities were observed in 54 patients (47%), and 24 (21%) of them showed obvious hematoma formation and/or high density with a mass effect . Hemorrhagic transformation of the lesion was evaluated in 155 patients whose follow - up ct and/or mr images were available for the review, and 55 (35%) of these patients showed variable amounts of hemorrhage . The hemorrhage pattern was hemorrhagic infarction (hi) type 1 in 15 patients (27%), hi type 2 in six (11%), parenchymal hematoma (ph) type 1 in 17 (31%) and ph type 2 in 17 (31%). The clinical follow - up data were available for 158 patients . The follow - up period varied from one to 12 months, except for those patients who died in the interim . We noted mrs scores of zero in 17 patients (11%), one in 27 (17%), two in 19 (12%), three in 20 (13%), four in 33 (21%) and five in 24 (15%); these findings were comparable with those of other major thrombolysis trials (fig . 1). Out of the 158 patients, 18 died, yielding a mortality rate of 11% . Among the various factors considered, an initial nihss score of less than 15 was the only factor that positively influenced successful recanalization (p = 0.001). Table 6 shows the univariate analysis of the factors that influenced significant hemorrhage after the procedure, and table 7 shows the factors that influenced the poor functional outcome . Subsequent multivariate analyses failed to show any statistically significant variables both for significant hemorrhage and for a poor functional outcome . The results of our analysis provide an overview on the current practice status of ia thrombolysis in korea . Although more than 30 centers in korea actively perform neurointerventional procedures (see the 2005 member list of the korean society of interventional neuroradiology), only seven of these centers participated in this study . It is likely that many of the other centers have been reluctant to perform ia thrombolysis, primarily due to a shortage of trained personnel . In western countries also, ia thrombolysis is performed less frequently than the iv method due to a shortage of interventional specialists (15). We found that from the symptom onset, the mean time before the imaging was 2.3 hours, and the mean time before the treatment was 4.7 hours; these results are comparable with those reported in the proact ii trial (7). The mean delay between the ct and mr imaging was about 60 min, which was one of the in - hospital delay factors for ia thrombolysis (18). About 40% of these procedures were performed before or after normal working hours, and the functional outcome results of these patients were generally poorer than those of the patients who were treated during normal working hours . One of the primary reasons for the poorer outcome in the former group of patients may be the in - hospital delay . A well - organized stroke team and an on - call system may improve the results for these patients . One notable finding of our survey was the preference for mr as the initial imaging modality . Mr was the sole initial imaging modality in 39% of patients, and it was combined with the ct imaging in 33% . Although mr has the potential disadvantage of time delay, it has shown an advantage for the initial evaluation of acute ischemic stroke (19 - 22). However, a delay in the initiation of the optimal treatment due to logistic limitations of mr imaging cannot be justified . Many centers use multi - detector ct imaging to overcome the inevitable logistic limitation of mr imaging (21, 23). It was notable that about 10% of the cases in the study were in violation of the' one - third rule', which is one of the most important exclusion criteria of the european thrombolysis trial (24) and the proact ii trial (7). Out of all the patients evaluated by the mr imaging, 26% showed dwi lesions covering more than half of the involved vascular territory . This was probably due to a lack of certain inclusion or exclusion criteria for mr imaging in respect to the patient selection for ia thrombolysis . Although we limited our study to patients with anterior circulation infarction and so increasing the homogeneity of the study group, we found a wide variation in the occlusion site, from the proximal ica to the proximal portion of the mca . Our observed recanalization rate, 71%, was slightly higher than that reported in the proact ii trial (7), which may have been due, at least in part, to the liberal use of mechanical methods (14). We were disappointed to find that only one factor, the lower initial nihss score, had a significant effect on the successful recanalization rate . In contrast to reports on the combined or sequential thrombolytic treatments (25 - 27), we found that the use of iv thrombolytics prior to the ia procedure did not affect the recanalization rate . However, in many of these patients, however, the initial iv administration of thrombolytics was ineffective . About half of the patients received a combination of ia and mechanical thrombolysis, although the use of mechanical methods failed to improve the recanalization rate (p = 0.083). One of the major concerns after the ia thrombolytic treatment is hemorrhagic transformation of the affected lesion (28, 29). We found that the rate of any amount hemorrhagic transformation was 35%, and the rate of the significant hemorrhagic transformation was 13% . The univariate analysis showed that several factors that had influence on significant hemorrhagic transformation, including the presence of hypertension, severe initial neurological symptoms, involvement of the basal ganglia on ct / mr and the performance of procedures after normal working hours . In addition, the risk of significant hemorrhage was increased by the presence of any contrast density on the ct obtained immediately after the thrombolysis procedure . Violation of the one - third rule on the initial ct tended to increase the occurrence of significant hemorrhage (p = 0.08), whereas extensive involvement of the affected vascular territory on the dwi (p = 0.51) and the combined use of iv thrombolytics and mechanical methods had no effect . Although direct comparison is not possible because of the differences in the patient groups, the functional outcome in our series of patients was similar to that of other major thrombolysis trials (7, 30). Among the factors that were found to influence the poor functional outcome were the old age, severe initial neurological symptoms, more extensive initial dwi lesions and involvement of basal ganglia on mr, failed recanalization, any contrast staining on ct immediately after the procedure and the presence of significant hemorrhagic transformation . Other studies have found that these same factors were predictive of poor prognosis (8, 31, 32). The primary limitations of this analysis were its retrospective design and the collection of data from several centers, all of which had different clinical and imaging indications for the procedure and different clinical settings . In addition, the data we collected were not representative of practice throughout korea because only a few active centers contributed to the study . Due to our inability to collect some data from the participating centers, we were unable to perform appropriate multivariate analyses . This could be one of the possible reasons for our failure to determine any significant variables that affected the results of significant hemorrhagic transformation and poor functional outcomes . Furthermore, our analysis of the imaging data was limited due to the between - center differences in the imaging protocols, including the use of contrast enhancement, mr pulse sequences and the angiographic filming techniques . Nevertheless, the results presented here may be representative of the nation - wide data on current ia thrombolytic treatment, and they could serve as a basis for clinical practice guidelines, as well as for designing a prospective study on the basis of this protocol . In conclusion, our multicenter, retrospective analysis of ia thrombolysis showed an increased dependency on mr imaging during the initial patient evaluation and increased application of combined pharmacologic / mechanical thrombolysis in korea . Both our imaging and clinical outcome results were comparable with those of the other major thrombolytic trials . Our results could be used as the basis for clinical practice and for future prospective studies.
A 46-year - old man was transferred to our hospital for further evaluation and management of a gastric lesion that had been detected by gastroscopy during a medical checkup at a local clinic . The gastroscopy showed an approximately 2.5-cm sized submucosal mass with two active ulcers on the mucosal surface (fig . The diagnostic impression of the referring physician in the local clinic was a gist or lymphoma . After a gastroscopic biopsy of the mucosal ulcer, the gastric lesion was diagnosed pathologically as an adenocarcinoma at this clinic . A contrast - enhanced ct of the abdomen using a 64-mdct scanner (lightspeed vct, ge healthcare, milwaukee, wi) was performed at our hospital . The ct images revealed a 1.5 2.5-cm ovoid mass with contrast enhancement at the lesser curvature wall of the gastric high body near the cardia (fig ., the mass was initially thought to be a submucosal tumor such as a gist . There was no evidence of perigastric infiltration, enlarged lymph nodes, or distant metastasis on ct . After reviewing the clinical information of the biopsy - proven adenocarcinoma, a gastric carcinoma was included in the differential diagnosis on the radiological report . However, because the enhanced mass in the submucosal layer was not the usual ct finding for a gastric adenocarcinoma, comparison with the clinical findings of gastroscopy and pathology was recommended . The resected specimen appeared as a 2.0 2.5-cm submucosal mass with small elevated lesions and central ulcerations on the mucosa . On light microscopy, poorly differentiated adenocarcinoma with extensive lymphocytic reaction in the mucosa that invaded the submucosa and subsequently formed a submucosal mass was confirmed (fig . The tumor invaded the subserosal adipose tissue, and was of the diffuse histological type (lauren's classification). The presence of ebv in gastric tumor cells was confirmed by performing ebv - encoded rna-1 (eber-1) in situ hybridization . An etiologic association between ebv and gastric carcinoma has been demonstrated by the uniform expression of ebv in all carcinoma cells and its absence in normal epithelium or dysplastic lesions (1, 2). Ebv - associated gastric carcinomas have an approximately 9% overall prevalence and, tend to be located in the cardia and body of the stomach when compared with conventional gastric adenocarcinoma (3, 4). These tumors are closely related to postsurgical gastric stump / remnant cancers (3, 4). In addition, approximately 15 - 25% of ebv - associated gastric carcinomas exhibit the lymphoepithelioma - like pattern, whereas 86 - 91% of lymphoepithelioma - like gastric carcinomas were ebv positive (3, 4). Carcinomas with morphologic features similar to undifferentiated nasopharyngeal carcinomas are designated as lymphoepithelioma - like carcinomas and occur in the stomach, salivary gland, lung, and thymus . The pathologic findings indicate that the tumor cells, which are arranged in sheets or nests or as isolated cells, are surrounded by a dense population of non - neoplastic small lymphocytes (6). Ebv - associated gastric carcinomas can present as two histomorphological forms of a lymphoepithelioma - like carcinoma and a glandular adenocarcinoma, and these tumors are characteristically accompanied by prominent lymphocyte infiltration; ebv has been thought of as being the main cause for the lymphocytic response (1, 2, 8, 9). The intramucosal lace pattern with lymphocyte infiltration has been reported to be the most characteristic morphologic feature of ebv - associated gastric carcinomas . In addition, carcinomas with submucosal invasion had a lymphoepithelioma - like pattern of the submucosa in 42% of cases (1). The pathogenesis of the abundant lymphoid stroma in the submucosal layer remains unclear, but it is presumed that carcinomas with a lymphocytic reaction in the mucosa may invade the submucosa, and this lesion in the submucosa then grows due to the factors associated with lymphocytic induction, resulting in the formation of a submucosal mass (1). Lymphoepithelioma - like gastric carcinomas have a better prognosis than other forms of ebv - associated gastric carcinomas and conventional gastric carcinomas (1, 2, 9, 10). This is because the spread of tumors through the gastric wall and to the lymph nodes or remote organs may be prevented by the abundant lymphocytic reaction; that is, the patient's inflammatory response may contribute to a better prognosis in patients with ebv - associated gastric carcinoma (1, 9). Despite its distinct clinicopathologic features, lymphoepithelioma - like gastric carcinomas are not familiar to most radiologists . In a ct study including 13 ebv - associated gastric carcinomas, authors concluded that the carcinomas were generally located in the upper gastric region, with a large thickness - to - width ratio, or with a bulky mass projecting from the wall (11). However, the ebv - associated gastric carcinomas had various appearances on ct including focal mucosal thickening, marked wall thickening with contrast enhancement, and bulky mass formation . These appearances on imaging may overlap with cases of early gastric carcinoma, advanced gastric carcinoma, and lymphoma . In another study describing the endoscopic ultrasonographic findings of four ebv - associated early gastric carcinomas, hypoechoic submucosal nodules that corresponded to lymphoid stroma composed of carcinoma cells and infiltrating lymphocytes were observed in three cases that invaded the submucosa (7). It is difficult to differentiate lymphoepithelioma - like gastric carcinoma presenting as a submucosal mass from other gastric submucosal tumors such as a gist, neurogenic tumor, and glomus tumor by imaging alone . However, as noted in the case reported here, a discrepancy between imaging findings suggestive of a submucosal tumor and the biopsy results of the adenocarcinoma may suggest the diagnosis present in this case . Although mucosal ulceration can be present in both ebv - associated gastric carcinomas and submucosal tumors, the ulcerated shape of the advanced ebv - associated lesions has been thought to be related to the superficial depressed shape of the early lesions (12). In conclusion, the mdct findings of an ebv - associated lymphoepithelioma - like carcinoma that developed in the gastric body presented as an enhancing submucosal mass with abundant lymphoid stroma admixed with adenocarcinoma . Because the tumors have unique clinicopathologic features, understanding the imaging findings and clinical features of ebv - associated lymphoepithelioma - like carcinomas is important in the preoperative diagnosis and in differentiating this entity from other submucosal tumors.
Interhospital transport of critically ill patient may be indicated if additional care, whether technical, cognitive, or procedural, is not available at the existing location . Regionalization of intensive care medicine in centers with high patient volumes appears to improve outcome of patients and therefore may further increase the need for these transports [24]. The risks associated with interhospital transport should be weighted against its potential benefit for each individual critically ill patient [57]. The use of specialized teams and appropriate equipment might reduce these risks [8, 9]. Although guidelines have been developed to increase the safety of interhospital transport of critically ill patients, clinical evidence is lacking on factors determining the transportability of these patients [1, 4]. Decision - making in interhospital transport involves appraisal of several determinants including patient characteristics, indication for transport, level of escort, and transport facilities . The process of appraisal of these variables, however, has never been studied . The aim of the present study was to assess the relative importance of clinical and transport - related determinants influencing physicians' decision - making in interhospital transport of critically ill patients . We sent a national questionnaire survey with paper case descriptions, so - called clinical vignettes, to the medical heads (intensivist or supervising consultant) of all 95 intensive care units (icus) in the netherlands . Questionnaires were anonymous but coded, and therefore so nonresponders could be followed up with a postal reminder 2 months later . A prepaid envelope was included for its return, and a web - based version was available for digital responses . Of the 95 questionnaires 78 (82%) were returned and all were suitable for analysis . Respondents' mean age was 45 6.6 years (table 1). Most (n = 66, 86%) were intensivists with either anesthesiology or internal medicine as medical specialty . The median number of interhospital transport leaving their icu was one per month, with a considerable range (0.0112). Table 1characteristics of the 78 responding intensive care physicians and their hospitalsmean age (years)45(6.6)medical speciality (%) intensive care medicine66 (86%) anesthesiology37 (48%) internal medicine34 (44%) surgery1 (1%) other5 (7%)type of hospital academic medical center13 (17%) teaching hospital, nonacademic34 (44%) regional public hospital30 (38%)number of beds in icu, median (range)8 (242)number of interhospital transport per month median (range)1 (0.0112)multiple specialities per physician possible characteristics of the 78 responding intensive care physicians and their hospitals multiple specialities per physician possible the interhospital critical care transport system in the netherlands is diverse . The majority of the transports are by ground (standard) ambulances escorted by an advanced life - support paramedic and occasionally complemented by the sending physician . Only a few regions use a dedicated, fully equipped mobile icu with an escorting team of intensive care (ic) physician and ic nurse . The questionnaire consisted of two parts: (a) characteristics of the respondent and its icu including frequency of interhospital icu transport from their hospital; (b) 16 clinical vignettes . We identified eight potential determinants in decision making of ic transport which are known from clinical studies and critical care transport experience from the authors [1, 69, 11, 12]. The determinants were incorporated in the clinical vignettes: (a) age (30 vs. 60 vs. 80 years); (b) arterial oxygenation pressure (7.5 vs. 16.5 kpa); (c) level of positive expiratory pressure (peep) (8 vs. 18 cmh2o); (d) dose of noradrenaline infusion (0.12 vs. 0.60 g / kg per minute); (e) arrhythmia (self - terminating ventricular tachycardia <24 h vs. no arrhythmia within 6 h); (f) transport facility (fully equipped mobile icu, i.e., ic ventilator, ic monitor including invasive blood pressure monitoring and capnography, sufficient number of syringe pumps) vs. standard ambulance (i.e., transport ventilator without ic performance, no invasive and capnography monitoring); (g) escorting personnel paramedic (advanced life support paramedic characterized by, e.g., protocolized advanced life support with medication, cardiopulmonary resuscitation intubation) vs. ic physician and paramedic vs. ic nurse and paramedic vs. team of ic physician and ic nurse and paramedic; (h) indication for transport (shortage of icu beds in referring hospital vs. essential intervention not available in referring hospital). Basic structure of each case vignette: patient admitted to icu after initial presentation in the emergency department with severe sepsis (probably pneumococcal), acute physiology and chronic health evaluation ii of 18, mean arterial pressure of 70 mmhg after adequate fluid - resuscitation, endotracheally intubated and mechanically ventilated with 50% fio2 and after 6 h in the icu need for interhospital transport [vt, ventricular tachycardia (self terminating); micu, mobile icu]patients characteristicstransport conditionsage (years)pao2 (kpa)peep (cmh2o)noradrenaline (g / kg per minute)arrhythmiaequipmentescorting personnelindication for transport13016.5180.12vt <24 hmicu trolleyic nurselack of icu beds23016.580.12nonebasic ambulanceic physician and ic nurseintervention elsewhere33016.5180.6nonebasic ambulanceparamediclack of icu beds4307.580.6nonebasic ambulanceic physicianlack of icu beds5307.580.12vt <24 hmicu trolleyic physician and ic nurselack of icu beds6807.580.6vt <24 hbasic ambulanceic nurseintervention elsewhere78016.580.12vt <24 hbasic ambulanceparamediclack of icu beds8607.580.12nonemicu trolleyparamedicintervention elsewhere9307.5180.12nonebasic ambulanceic nurseintervention elsewhere10307.5180.60vt <24 hmicu trolleyparamedicintervention elsewhere113016.580.60vt <24 hmicu trolleyic physicianintervention elsewhere126016.5180.60vt <24 hbasic ambulanceic physician and ic nurseintervention elsewhere138016.5180.12nonemicu trolleyic physicianintervention elsewhere14607.5180.12vt <24 hbasic ambulanceic physicianlack of icu beds156016.580.60nonemicu trolleyic nurselack of icu beds16807.5180.60nonemicu trolleyic physician and ic nurselack of icu beds the 16 case vignettes . Basic structure of each case vignette: patient admitted to icu after initial presentation in the emergency department with severe sepsis (probably pneumococcal), acute physiology and chronic health evaluation ii of 18, mean arterial pressure of 70 mmhg after adequate fluid - resuscitation, endotracheally intubated and mechanically ventilated with 50% fio2 and after 6 h in the icu need for interhospital transport [vt, ventricular tachycardia (self terminating); micu, mobile icu] as 768 case descriptions were needed to present all possible combinations of the eight determinants and their levels, the number of representative clinical vignettes were reduced to 16 using an orthogonal main - effects design . This approach permits the statistical testing by conjoint analysis of a suitable fraction of all possible combinations of the factors (determinants) and their levels . Respondents were asked to rate the degree of transportability, defined as their personal clinical decision, whether they would let this patient be transported, for each of the 16 critically ill patients described in clinical vignettes . A seven point likert scale was used ranging from 1 (entirely not transportable) to 7 (definitely transportable). It was emphasized that no true or false answers were sought but their clinical judgment . The means and standard deviations for continuous variables and distributions for frequency of categorical variables were summarized using descriptive statistics . Conjoint analysis was performed with transportability as dependent variable to calculate the relative weights for each level of the determinants . This results in a utility score for each determinant level expressed in with 95% confidence interval . These utility scores, estimated by least squares regression analogous to regression coefficients, provide a quantitative measure of the preference for each determinant level, with larger values corresponding to greater preference . Considering the individual respondents as random effects took into account that the preference score originating from 16 repeated measurements . Determinants with a negative indicated preference against transportability . The reference value, by definition = 0, was defined as the optimal conditions for critical care transport (youngest age, highest pao2, lowest dose of noradrenaline, no arrhythmia, fully equipped mobile icu ambulance, escorting team of ic physician and ic nurse, intervention required not available in own facility). The conjoint analysis was repeated in relation to (a) type of hospital the respondents were working in regional hospital or teaching / university hospital, (b) speciality of the respondent, and (c) the method of data collection, either paper or online questionnaire . The questionnaire consisted of two parts: (a) characteristics of the respondent and its icu including frequency of interhospital icu transport from their hospital; (b) 16 clinical vignettes . We identified eight potential determinants in decision making of ic transport which are known from clinical studies and critical care transport experience from the authors [1, 69, 11, 12]. The determinants were incorporated in the clinical vignettes: (a) age (30 vs. 60 vs. 80 years); (b) arterial oxygenation pressure (7.5 vs. 16.5 kpa); (c) level of positive expiratory pressure (peep) (8 vs. 18 cmh2o); (d) dose of noradrenaline infusion (0.12 vs. 0.60 g / kg per minute); (e) arrhythmia (self - terminating ventricular tachycardia <24 h vs. no arrhythmia within 6 h); (f) transport facility (fully equipped mobile icu, i.e., ic ventilator, ic monitor including invasive blood pressure monitoring and capnography, sufficient number of syringe pumps) vs. standard ambulance (i.e., transport ventilator without ic performance, no invasive and capnography monitoring); (g) escorting personnel paramedic (advanced life support paramedic characterized by, e.g., protocolized advanced life support with medication, cardiopulmonary resuscitation intubation) vs. ic physician and paramedic vs. ic nurse and paramedic vs. team of ic physician and ic nurse and paramedic; (h) indication for transport (shortage of icu beds in referring hospital vs. essential intervention not available in referring hospital). Basic structure of each case vignette: patient admitted to icu after initial presentation in the emergency department with severe sepsis (probably pneumococcal), acute physiology and chronic health evaluation ii of 18, mean arterial pressure of 70 mmhg after adequate fluid - resuscitation, endotracheally intubated and mechanically ventilated with 50% fio2 and after 6 h in the icu need for interhospital transport [vt, ventricular tachycardia (self terminating); micu, mobile icu]patients characteristicstransport conditionsage (years)pao2 (kpa)peep (cmh2o)noradrenaline (g / kg per minute)arrhythmiaequipmentescorting personnelindication for transport13016.5180.12vt <24 hmicu trolleyic nurselack of icu beds23016.580.12nonebasic ambulanceic physician and ic nurseintervention elsewhere33016.5180.6nonebasic ambulanceparamediclack of icu beds4307.580.6nonebasic ambulanceic physicianlack of icu beds5307.580.12vt <24 hbasic ambulanceic nurseintervention elsewhere78016.580.12vt <24 hbasic ambulanceparamediclack of icu beds8607.580.12nonemicu trolleyparamedicintervention elsewhere9307.5180.12nonebasic ambulanceic nurseintervention elsewhere10307.5180.60vt <24 hmicu trolleyparamedicintervention elsewhere113016.580.60vt <24 hmicu trolleyic physicianintervention elsewhere126016.5180.60vt <24 hbasic ambulanceic physician and ic nurseintervention elsewhere138016.5180.12nonemicu trolleyic physicianintervention elsewhere14607.5180.12vt <24 hbasic ambulanceic physicianlack of icu beds156016.580.60nonemicu trolleyic nurselack of icu beds16807.5180.60nonemicu trolleyic physician and ic nurselack of icu beds the 16 case vignettes . Basic structure of each case vignette: patient admitted to icu after initial presentation in the emergency department with severe sepsis (probably pneumococcal), acute physiology and chronic health evaluation ii of 18, mean arterial pressure of 70 mmhg after adequate fluid - resuscitation, endotracheally intubated and mechanically ventilated with 50% fio2 and after 6 h in the icu need for interhospital transport [vt, ventricular tachycardia (self terminating); micu, mobile icu] as 768 case descriptions were needed to present all possible combinations of the eight determinants and their levels, the number of representative clinical vignettes were reduced to 16 using an orthogonal main - effects design . This approach permits the statistical testing by conjoint analysis of a suitable fraction of all possible combinations of the factors (determinants) and their levels . Respondents were asked to rate the degree of transportability, defined as their personal clinical decision, whether they would let this patient be transported, for each of the 16 critically ill patients described in clinical vignettes . A seven point likert scale was used ranging from 1 (entirely not transportable) to 7 (definitely transportable). It was emphasized that no true or false answers were sought but their clinical judgment . The means and standard deviations for continuous variables and distributions for frequency of categorical variables were summarized using descriptive statistics . Conjoint analysis was performed with transportability as dependent variable to calculate the relative weights for each level of the determinants . This results in a utility score for each determinant level expressed in with 95% confidence interval . These utility scores, estimated by least squares regression analogous to regression coefficients, provide a quantitative measure of the preference for each determinant level, with larger values corresponding to greater preference . Considering the individual respondents as random effects took into account that the preference score originating from 16 repeated measurements . The reference value, by definition = 0, was defined as the optimal conditions for critical care transport (youngest age, highest pao2, lowest dose of noradrenaline, no arrhythmia, fully equipped mobile icu ambulance, escorting team of ic physician and ic nurse, intervention required not available in own facility). The conjoint analysis was repeated in relation to (a) type of hospital the respondents were working in regional hospital or teaching / university hospital, (b) speciality of the respondent, and (c) the method of data collection, either paper or online questionnaire . The impact of the determinants in the decision making on transportability is displayed in fig . 1 . Those with the largest negative effects on preference for transportability were the type of escorting personnel [paramedic only: = 3.1 (3.7 to 2.5); ic nurse and paramedic: = 2.1 (2.5 to 1.7); team of ic physician, nurse, and paramedic: = 1.0 (1.2 to 0.8); standard ambulance: = 1.21 (1.7 to 0.8)]. Determinants reflecting the critically ill patient's condition and intensity of treatment were scored to be of relative minor importance [dose of noradrenaline: = 0.6 (1.0 to 0.1); arterial oxygenation = 0.8 (1.3 to 0.4); level of peep age [60 years: = 0.1 (0.2 to 0.3); 80 years: = 0.1 [0.4 to 0.7)], cardiac arrhythmia [= 0.1 (0.4 to 0.5)], and the indication for transport (= 0.3 (0.8 to 0.1)] had no significant effect on the preference for transportability (fig . 1). 1relative weight (expressed in, 95% confidence interval) of determinants influencing the decision on interhospital ic transport . Ref, reference value; peep, positive end - expiratory pressure; ventric, ventricular; ic, intensive care relative weight (expressed in, 95% confidence interval) of determinants influencing the decision on interhospital ic transport . Ref, reference value; peep, positive end - expiratory pressure; ventric, ventricular; ic, intensive care repeated analyses did not demonstrate significant differences in relative weights of the determinants in relation to respondents' working location (regional hospital vs. large teaching hospital or academic medical center), type of medical speciality, or method of data collection (paper vs. online). Decision - making in interhospital transport involves appraisal of several determinants such as patient characteristics, indication for transport, level of escort, and transport facilities . The present study shows that the level of escorting personnel is an important determinant in decision - making in interhospital transport of a critically ill patient . Additionally, transport facilities are perceived as most important by the majority of medical heads of dutch icus . Neither characteristics of the patient's condition nor the level of supportive care seems to be of significance in this process . The large number of publications on interhospital transport reflects the interest in this complex part of ic medicine but are descriptive and mainly focuses on the technical and organizational aspects of transport [1, 9, 12, 14]. The use of specialized transport teams and appropriate equipment may result in a decrease in transport associated morbidity and mortality by creating an intensive care environment in a vehicle - ground ambulance or aircraft [8, 9, 1517]. Despite the growth in interhospital transport due to regionalization of intensive care medicine the process by which ic physicians identify patients transportability is not well known [3, 10, 18]. Transportability as a result of a professional analysis of the balance between risks and potential benefits of an individual transport is hard to define . The accumulating literature on improved outcome associated with icus treating larger volumes of patients (e.g., with severe sepsis or mechanical ventilation) is not adequately accompanied by research on clinical parameters determining transportability in such conditions [19, 20]. A study by lee et al . Used a questionnaire with clinical scenarios before and after a program, including a 15-min training in the use of interhospital transfer rules . After the start of the program clinical staff were able to make appropriate decisions using these guidelines focusing on diagnosis and physiology . To our knowledge, however, no study has mimicked decision making in interhospital transport with appraisal of several realistic and detailed determinants as in daily clinical practice (i.e., as those in tested in this conjoint analysis) by experienced intensivists who endorse such transports . Age is an important prognostic factor, for mortality rates are higher in elderly than in younger icu patients . This has not been studied in transported ic patients, but it is conceivable that intensivists would weigh this determinant in their transportability decision . The finding of the present study that age does not influence decision making for transportability is remarkable . The same holds true for the level of peep, which seems representative for severity of oxygenation and is known to be a critical factor in transport . Ic physicians, however, seem to consider factors associated with severity of illness (age, peep, noradrenaline dose, oxygenation) to be less important than to transport conditions . International guidelines underline the importance of these conditions, but clinical transport studies and recommendations are lacking to address the issue of transport - related morbidity and mortality of extreme critical ill patients despite optimal expertise and equipment [79, 17, 22]. One of the limitations of this study is the intrinsic shortcoming of the vignette method . Paper case descriptions, so - called clinical vignettes, have been recognized as a valid policy capturing tool to assess preferences in clinical practice [18, 23]. However, it is impossible to overcome the sentinel effect in which the physicians know they are being evaluated . Due to this there may be a discrepancy between physicians' decisions in practice and their answers to vignettes with hypothetical patients . Another limitation is the choice of content of the vignettes with eight determinants of transportability . The content of vignettes survey is limited to a number of determinants with their corresponding levels as an intrinsic element of conjoint analysis to generate an optimal number of vignettes a respondent would still adequately evaluate . The set of determinants used in this study is based only on literature and critical care transport experience and could therefore be biased [1, 69, 11, 12]. Those factors would only be revealed in clinical transport studies documenting all clinical parameters and relate them with clinical outcome after transport . Finally, this national questionnaire survey is limited by the dutch situation, where due to geography interhospital transport is carried out by ground ambulance without air medical transport . It is conceivable that the choice of vehicle is a crucial determinant in the decision making in combination with the interhospital distance . This policy observing study indicates the importance of optimal escorting and transport facilities in interhospital transport as appreciated by ic physicians . These conditions are considered to be essential and enable even severe critically ill patients to be transported . Further clinical (transport) research should reveal which levels of expertise and transport facilities are indicated for which category of critically ill patients to tailor the use of expensive resources required for those inevitable road trips [9, 17].
Mhc class ii molecules are heterodimeric cell surface glycoproteins that bind exogenously derived antigenic peptides and present them to cd4 t cells 12 . Class ii and chains are translocated into the endoplasmic reticulum (er), where they form nonamers with invariant (ii) chain 3 . Ii chain prevents the binding of immunogenic peptides due to the presence of a 14amino acid domain (clip) that occupies the peptide - binding groove of / dimers 3 . After ii degradation in the endocytic pathway, the mhc - encoded molecules hla - dm (or h2-m in the mouse) and hla - do (h2-o) facilitate the removal of clip from / dimers, allowing peptide binding 456 . Ii chain has been implicated in functions such as er export, endosome targeting, and even b cell maturation 37 . Two alternatively spliced ii isoforms exist (p31 and p41), distinguished by a 64-residue domain in the lumenal portion of p41 8 . The isoforms are expressed differently in various apcs and regulate the presentation of certain antigen epitopes in b cells 9 . This difference may reflect protease inhibition by the amino acid insertion in p41, as it has been shown to inhibit the lysosomal cysteine protease cathepsin l both in vitro and in vivo 910 . Therefore, ii chain may contribute to the modulation of the proteolysis in the endocytic pathway and thus modulate antigen processing indirectly 1112 . We demonstrate here that ii chain deletion leads to the lysosomal degradation of h2-mb in apcs, suggesting that ii chain is required to prevent the proteolysis of h2-m and perhaps of other proteins . This feature may help explain how ii chain expression affects t cell selection and b cell maturation independently from its effect on mhc class ii traffic 131415 . C57bl/6 (control) and ii, ii p31 1416, class ii, and class ii / ii mice (the gift of p. marrack, university of colorado health sciences center, denver, co) were kept in a pathogen - free environment for 78 wk before killing . Splenocytes were obtained as described 7 . Bone marrow derived dendritic cells (dcs) were cultured as described 17 . After purification, immature dcs were characterized by immunofluorescence and processed in parallel with the lps - treated dcs . The primers used here to detect i - a, h2-m, and h2-m are identical to the primers described previously 19 . 3 10 late dcs were pulse labeled with 7.5 mci / ml of [s]methionine translabel (icn) and chased as described 17 . /2 h2-m was immunoprecipitated with the rat mab 2c3a (a gift of l. karlsson, r.w . Murine i - a was detected using rivoli, a rabbit polyclonal antibody directed against the conserved class ii i - a chain cytoplasmic tail 17; h2-mb was detected by immunofluorescence and immunoblots with the rabbit polyclonal antibodies anti ulm 17, k553 (19; a gift of l. karlsson) and anti h2-mb cytoplasmic tail (a gift of s. amigorena, institut curie, paris, france). Rabbit polyclonal anti murine lgp - b / lamp-2 and ii chain were detected using the rat mabs gl2a7 17 and in1 20 . For immunofluorescence, cells were fixed in 3.5% paraformaldehyde (in pbs) and permeabilized as described 17 . The cathepsin s specific inhibitor lhvs (a gift of hidde ploegh, harvard medical school, boston, ma) was added to the culture medium of late dcs (preincubated with lps for 18 h) at the concentration of 2 nm and 1 m . E64 was obtained from sigma chemical co. and used at 20 m . C57bl/6 (control) and ii, ii p31 1416, class ii, and class ii / ii mice (the gift of p. marrack, university of colorado health sciences center, denver, co) were kept in a pathogen - free environment for 78 wk before killing . Splenocytes were obtained as described 7 . Bone marrow derived dendritic cells (dcs) were cultured as described 17 . After purification, immature dcs were characterized by immunofluorescence and processed in parallel with the lps - treated dcs . The primers used here to detect i - a, h2-m, and h2-m are identical to the primers described previously 19 . 3 10 late dcs were pulse labeled with 7.5 mci / ml of [s]methionine translabel (icn) and chased as described 17 . /2 h2-m was immunoprecipitated with the rat mab 2c3a (a gift of l. karlsson, r.w . Murine i - a was detected using rivoli, a rabbit polyclonal antibody directed against the conserved class ii i - a chain cytoplasmic tail 17; h2-mb was detected by immunofluorescence and immunoblots with the rabbit polyclonal antibodies anti ulm 17, k553 (19; a gift of l. karlsson) and anti h2-mb cytoplasmic tail (a gift of s. amigorena, institut curie, paris, france). Rabbit polyclonal anti murine lgp - b / lamp-2 and ii chain were detected using the rat mabs gl2a7 17 and in1 20 . For immunofluorescence, cells were fixed in 3.5% paraformaldehyde (in pbs) and permeabilized as described 17 . The cathepsin s specific inhibitor lhvs (a gift of hidde ploegh, harvard medical school, boston, ma) was added to the culture medium of late dcs (preincubated with lps for 18 h) at the concentration of 2 nm and 1 m . E64 was obtained from sigma chemical co. and used at 20 m . The cathepsin b specific inhibitor ca074me used at 5 m was obtained from bachem . Ii chain is responsible for the correct folding and transport of mhc class ii in apcs . However, discrepancies between class ii transport and t cell selection in ii mice suggested that ii chain deletion may epistatically affect another critical component of antigen processing, such as h2-m . To investigate a possible relationship between ii chain deletion and h2-m expression, we isolated splenocytes from wild - type and ii mice and probed for h2-m by western blot . 1 a, greatly decreased steady - state levels of h2-mb (<5% of control) were detected in ii splenocytes, whereas similar levels of cathepsin b were detected . We next asked if the decrease in h2-m might somehow reflect the er accumulation of misfolded class ii molecules that occurs in the absence of ii chain 16 . Misfolded class ii might interact with h2-m resulting in er retention and degradation . However, h2-m levels were reduced in splenocytes derived from ii and class ii double deficient mice (fig . 1 b). Thus, ii chain affected h2-m expression independently of its interaction with mhc class ii . To determine if h2-m disappearance correlated with the amount of ii chain expressed, we quantified h2-mb protein in splenocytes from ii mouse reconstituted with the p31 isoform of ii chain at very low level of expression (p31). Surprisingly, even small amounts of p31 (1% of wild - type) disproportionately protected against h2-m disappearance, allowing its accumulation at much higher levels (20% of control; fig . 1 b), despite the fact that ii expression in p31 splenocytes was insufficient to restore proper class ii transport 14 . Mrna levels of mhc class ii and h2-m molecules were determined by reverse transcription (rt)-pcr (fig . No differences in the levels of i - a, h2-m, or h2-m mrna could be detected in mice lacking ii chain (ii), mice expressing low levels of the p31 (p31) chain 14, or control c57bl/6 mice (fig . Therefore, the absence of ii chain had no effect on the transcription of mhc class ii and h2-m, suggesting that h2-mb downregulation occurred posttranslationally . Dcs activate ii chain degradation during maturation 20, suggesting that the absence of ii chain might somehow render h2-m more susceptible to degradation . H2-m distribution was examined in immature and mature (lps - treated) bone marrow derived dcs from control and ii mice . Dcs were stained for h2-m and the lysosomal marker protein lgp - b / lamp-2 and observed by confocal microscopy . In immature dcs, still in clusters (fig . 2 a), the staining intensity of h2-mb and its colocalization with lgp - b / lamp-2 were similar in cells obtained from control or ii mice . This was confirmed by staining immature langerhans cells in situ, in which similar amounts of h2-m were detected in ii and control epidermis (fig thus, in immature dcs, the absence of ii chain did not affect delivery of h2-m to the lysosomes 21 or lead to its downregulation . However, in mature dcs h2-m was barely detectable in cells from ii mice (fig . The lgp - b / lamp-2 staining in these cells was indistinguishable from controls and exhibited a distinct perinuclear pattern characteristic of mature dcs 17 . As expected from the western blot experiments, h2-m was not noticeably downregulated in class ii dcs (fig . Control and ii mature dcs were radioactively labeled for 30 min and chased for various lengths of time . Since the h2-mb band was most readily quantifiable, we determined its half - life at 42 h in controls but only 10 h in the ii cells (fig . 4 b). The rapid degradation of h2-mb in the ii cells could be at least partially slowed by adding 1 m lhvs, a cysteine protease inhibitor, during the chase period (fig . 4 b). However, the effect was only evident at longer time points, possibly reflecting the time required for the lhvs to reach inhibitory concentrations within endocytic organelles . Thus, the disappearance of h2-m upon maturation of ii dcs appears to reflect h2-m degradation by lysosomal cysteine proteases . No differences were observed in acquisition of endo h resistance, confirming that ii chain was not required for transport through the golgi complex 1921 . As previously observed in b cells 22, in dcs h2-m could be precipitated with ii chain after a 30-min pulse, reflecting the presumptive interaction of these molecules in the er (fig . Although both ii chain splice forms were coimmunoprecipitated, approximately fivefold more p41 than p31 was detected, suggesting a higher affinity of p41 for h2-m . This preference is even far greater, as dcs express about three times more p31 than p41 (fig . 4 d). To further demonstrate the involvement of cysteine proteases in h2-mb downregulation, early and late dcs from control and ii mice were incubated with cysteine protease inhibitors and analyzed by immunoblot (fig . 5). Although h2-m was slightly (20%) reduced in early ii dcs, a striking decrease was observed in the lps - treated mature cells where h2-m was found at only 5% of the amount in controls . When the mature ii dcs were also treated with the inhibitor e64 (20 m) or lhvs (1 m) 11, h2-m levels were partially rescued to 50% of control (fig . 5a and fig . This protective effect was not observed at 2 nm lhvs where only cathepsin s was inhibited 20, suggesting that other cysteine proteases were responsible for h2-mb degradation . When 5 m of the cathepsin b inhibitor ca074me 9 was used, a slight rescue of h2-mb was observed, suggesting that cathepsin b might play a role (fig . 5 c). To rule out the possible degradation of h2-m in the cytosol, as occurs for misfolded proteins in the er, mature ii dcs were incubated with lactacystin . Lactacystin is a proteasome inhibitor known to prevent degradation of free er mhc class i 23 and mhc class ii 24 . 50 m of lactacystin somewhat enhanced the accumulation of aggregated mhc class ii but had no effect on h2-m disappearance in mature ii dcs (fig . The regulation of the degradation of h2-m, through the inhibition of lysosomal cathepsins, suggests that ii chain may function as a protease inhibitor that may help to protect h2-m and possibly other proteins against proteolysis in dc lysosomes . This role for ii chain has been previously suggested on the basis of two observations . L is mediated by the 64amino acid segment encoded by the alternatively spliced exon of p41 910 . Second, ii chain shares significant amino acid sequence homology (4045%) with various cysteine protease inhibitors, the cystatins 25 . Cathepsin l is present at minimal levels in dcs, and its activation by the absence of ii or by dc maturation was not detected (not shown). However, p41 seems to be associated preferentially with h-2 m, and its protease inhibitory activity could be relevant for the protection of the dimer . However, in mice expressing a low level of p31 (p31) h2-m degradation was at least partially reduced . Thus, the inhibitory effect of ii chain on degradation is not solely dependent on p41 and is consistent with the fact that p31 bears significant sequence homology to the cystatin superfamily 25 . Interestingly, transfection of ii p31 in fibroblasts resulted in the formation of enlarged endosomes and a delay in transport from endosomes to lysosomes 26 . A similar phenomenon has also been observed in cells treated with the protease inhibitor e64 or leupeptin, further supporting a protease inhibitory function for ii chain . We demonstrated recently that members of family 2 cystatins control the proteolytic endocytic environment of immature dcs 20 . In immature ii dcs, the lack of ii chain might be compensated by antiprotease effects of cystatins, which normally control lysosomal cathepsins in these cells . Upon activation by lps, downregulation of cystatins combined with the absence of ii chain thus, b cells may not exhibit the type of developmental regulation of protease activity seen in dcs, or b cells may express a different complement of proteases to which h2-m is more susceptible . Ii chain has been implicated in modulating b cell maturation 715 as well as t cell selection 131415 . Surprisingly, significant amounts of peptide - loaded mhc class ii have been detected at the surface of ii apcs, especially dcs expressing the i - a and i - a haplotypes 1315 . These observations suggest that in some strains of ii mice, impaired t cell selection is not the direct consequence of an abnormal transport of mhc class ii molecules . Our results suggest that h2-m function and therefore the peptide repertoire presented may be altered in such mice . As a consequence, h2-m degradation could contribute to the observed decrease in cd4 t cells in the thymus of all ii chain deficient strains and the milder phenotype for maturation of cd4 t cells in the periphery of the ii balb / c (i - a) mice 15 . The precise contribution of h2-m disappearance to the phenotypes observed in ii apcs is difficult to establish because the dependency of mhc class ii on both ii and h2-m varies greatly according to the haplotype 27 . The fact that mice lacking both ii and h2-m exhibit a nearly complete inhibition of cd4 t cell selection supports the idea that ii and h2-m deletions are synergistic 28 . The partial rescue of h2-m by low level ii chain would still explain why reconstituted mice (p31) have normal cd4 t cell positive selection despite the fact that mhc class ii traffic is still strongly inhibited 1415 . In any event, the fact that ii chain deletion can indirectly cause h2-m downregulation will indicate that results obtained with ii mice should be carefully controlled for the possible contribution of h2-m dysfunction.
Subjects with chronic insomnia have a higher risk for hypertension [13] and u - shaped associations have also been demonstrated between sleep duration and hypertension . Studies have reported that insomnia is associated with an overall hypersecretion of acth and cortisol, suggesting an activation of the hypothalamic - pituitary - adrenal (hpa) axis in these patients [58]. Given the well - established association of hypercortisolemia with significant medical morbidity (eg, hypertension, metabolic syndrome, osteoporosis), insomnia can induce hypertension through an activation of the hypothalamic - pituitary - adrenal (hpa) axis . On the other hand, it has also be confirmed that subjects with hypertension are more likely to report emotional disorders such as anxiety, depression, and sleep disorders, which are usually viewed as one of their clinical features, indicating that hypertension is a risk factor for sleep disorders . It is well known that both sleep quality and hypertension are strongly influenced by lifestyle . It has been confirmed that there is a u - shaped association between alcohol consumption and hypertension or chronic insomnia . Insomnia is a risk factor for hypertension [13], and individuals with hypertension are more likely to have sleep disorders . Some behaviors (eg, exercise, smoking, alcohol consumption) are risk factors for both sleep disorders and hypertension [1315], sleep disorders and hypertension are both risk factors for mortality in the elderly [4,1619], and hypertension and sleep disorders are both related to age . From all of these, we can conclude that in very elderly subjects (aged 90 years or more), there is a close association between sleep quality and arterial blood pressure, which may be different from that in he general population of older adults (aged 60 years or more). However, to our knowledge, no population - based study has evaluated the association between sleep quality and arterial blood pressure in the very elderly . In this study we examined the association between sleep quality and arterial blood pressure in the very elderly subjects using data from a sample of chinese nonagenarians and centenarians, the methods were reported previously . In brief, on the basis of the dujiangyan (located in sichuan province, southwest china) 2005 census, a cross - sectional study for age - related diseases was conducted in 870 very elderly subjects (> 90 years), which was a part of the project of longevity and aging in dujiangyan (plad). The plad aimed to investigate the relationship between environments, lifestyle, genetics, longevity and age - related diseases . Volunteers were examined by trained professional physicians according to basic health criteria and the results were entered into standard forms, especially the questionnaire on lifestyles (including the pittsburgh sleep quality index (psqi)). In this analysis, subjects were excluded who were using medicine for treating sleep problems of hypertension, were bedridden, had cancer, a history or clinical evidence of stroke, secondary hypertension, or were receiving anti - hypertensive treatment, had terminal stage of physical disease such as respiratory system disease, cardiovascular disease, kidney disease, dementia or cognitive decline . Overall, 21 men and 26 women were not eligible for the study because they had already died or moved away from the area . Of the 262 men and 561 women who were interviewed, 46 men and 117 women were excluded for the reasons above . Informed consents were obtained from all participants (as well as their legal proxies). The research ethics committee of the sichuan university approved the study . To assure reliability of this information, during the course of interviewing, at least 1 family member, who usually lived with the participant, took part in the interviewing and checked the completed questionnaire for accuracy . The methods were reported previously . Sitting or recumbent position, right arm blood pressure (bp) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer (phases i and v of korotkoff) by trained nurses or physicians . The mean value of 2 measurements was used to calculate systolic bp (sbp) and diastolic bp (dbp), and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . The mean of 2 readings was used for classification of bp according to joint national committee (jnc)vii criteria into normal (sbp <120mmhg and dbp <80mmhg), pre - hypertension (sbp 120139 mmhg and/or dbp 8089 mmhg), stage 1 hypertension (sbp 140159 mmhg and/or dbp 9099 mmhg), stage 2 hypertension (sbp> 160 mmhg and/or dbp> 100 mmhg), systolic hypertension was defined as a sbp> 140 mmhg, the isolated systolic hypertension (ish) was defined as a sbp> 140 mmhg and a dbp<90mmhg, diastolic hypertension was defined as a dbp> 90 mmhg, and the isolated diastolic hypertension (idh) was defined as a sbp <140 mmhg and a dbp> 90 mmhg . Hypertension was defined as a sbp> 140 mmhg and/or a dbp> 90 mmhg and/or receiving anti - hypertensive treatment . Subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . The pittsburgh sleep quality index (psqi) the psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale (03) to obtain 7 subcategories: sleep duration, sleep disturbances, sleep latency, daytime dysfunction, sleep efficiency, sleep satisfaction, and medication use . The sum of all subscores represents the total sleep quality score, ranging between 021, with higher scores representing lower sleep quality . The individuals were categorized as follows: good sleep quality (scores between 0 and 5); fairly good sleep quality (scores between 6 and 10); and poor sleep quality (scores between 11 and 21). Psychometric properties have demonstrated good reliability (internal consistency: 0.89; test retest reliability: 0.85) and good construct validity for the english language version . The psqi is a widely used tool in research studies and clinical trials, and has been translated into several languages including chinese, spanish, german, and hebrew, with comparable reliability and validity values . Internal consistency in the present study was =0.69 . When excluding the medication use subscale due to a low rate of medication users in this sample, internal consistency increased to =0.76 . Outcome measures included the total psqi score as well as self - report of sleep latency (sl) (question 2: how long (in minutes) has it usually taken you to fall asleep each night? ), and sleep efficiency percentage (se), computed as the ratio between the hours of actual sleep (question 4: how many hours of actual sleep did you get at night?) And total time in bed (hours computed based on reported bedtime in question 1: what time have you usually gone to bed at night? And reported wake - time in question 3: what time have you usually gotten up in the morning? ), multiplied by 100 . The baseline examination included information on age (years), sex (male / female), smoking habits (yes or no), alcohol consumption (yes or no), tea consumption (yes or no), exercise (yes or no), serum lipid / lipoprotein levels (including serum triglyceride (tg), total cholesterol (tc), high - density lipoprotein (hdl) cholesterol, and low - density lipoprotein (ldl) cholesterol), fasting blood glucose (fbg), serum uric acid (sua), and body mass index (bmi). The bmi was calculated as body weight in kilograms divided by height in meters squared . Serum lipid / lipoprotein levels, fbg and sua were determined by standard laboratory techniques (performed by a technician in the biochemistry laboratory of sichuan university). Behaviors of smoking, alcohol consumption, tea consumption, and exercise, which included former and current status, were collected by using a general questionnaire . In the questionnaire, subjects were asked whether they had ever had behaviors of smoking, alcohol consumption, tea consumption, and exercise, and 1 of 3 answers were recorded: never, did in the past, or currently . Among those who did, currently or in the past, information was obtained on the average frequency of smoking, using alcohol, drinking tea, and doing exercise, and on the number of years they had engaged in the behavior . The subjects, who did almost everyday during the past year, were classified as those with these habits current, otherwise as without . The subjects, who had done behaviors almost everyday for more than 2 years as of a year before, were classified as those with these habits previously, otherwise as without . Alcohol consumption included spirits, liqueurs, wine, sherry, martini, beer, lager, hard cider, stout, and so on . All of the statistical analyses for this study were performed with the spss for windows software package, version11.5 (spss inc, chicago, illinois, usa). Baseline characteristics were compared between subjects with and without hypertension, between subjects with and without systolic hypertension, between subjects with and without diastolic hypertension using pearson chi - square or fisher s exact test (where an expected cell count was <5) for categorical variables and unpaired student s t test for continuous variables . Baseline characteristics were also compared among different sleep quality groups using one - way for analysis of variance (anova) for continuous variables and pearson chi - square or fisher s exact test (where an expected cell count was <5) for categorical variables . Multiple logistic regression was used to estimate the odds ratio (or) and 95% confidence interval (ci) of sleep quality as a function of increased risk for hypertension, systolic hypertension or diastolic hypertension . The following items were adjusted: age, sex, serum lipid / lipoprotein, body mass index, blood glucose level, smoking, alcohol consumption, tea consumption, and exercise . A p value <0.05 was considered to be statistically significant, and all of the p values were 2-sided . The methods were reported previously . In brief, on the basis of the dujiangyan (located in sichuan province, southwest china) 2005 census, a cross - sectional study for age - related diseases was conducted in 870 very elderly subjects (> 90 years), which was a part of the project of longevity and aging in dujiangyan (plad). The plad aimed to investigate the relationship between environments, lifestyle, genetics, longevity and age - related diseases . Volunteers were examined by trained professional physicians according to basic health criteria and the results were entered into standard forms, especially the questionnaire on lifestyles (including the pittsburgh sleep quality index (psqi)). In this analysis, subjects were excluded who were using medicine for treating sleep problems of hypertension, were bedridden, had cancer, a history or clinical evidence of stroke, secondary hypertension, or were receiving anti - hypertensive treatment, had terminal stage of physical disease such as respiratory system disease, cardiovascular disease, kidney disease, dementia or cognitive decline . Overall, 21 men and 26 women were not eligible for the study because they had already died or moved away from the area . Of the 262 men and 561 women who were interviewed, 46 men and 117 women were excluded for the reasons above . Informed consents were obtained from all participants (as well as their legal proxies). The research ethics committee of the sichuan university approved the study . To assure reliability of this information, during the course of interviewing, at least 1 family member, who usually lived with the participant, took part in the interviewing and checked the completed questionnaire for accuracy . The methods were reported previously . Sitting or recumbent position, right arm blood pressure (bp) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer (phases i and v of korotkoff) by trained nurses or physicians . The mean value of 2 measurements was used to calculate systolic bp (sbp) and diastolic bp (dbp), and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . The mean of 2 readings was used for classification of bp according to joint national committee (jnc)vii criteria into normal (sbp <120mmhg and dbp <80mmhg), pre - hypertension (sbp 120139 mmhg and/or dbp 8089 mmhg), stage 1 hypertension (sbp 140159 mmhg and/or dbp 9099 mmhg), stage 2 hypertension (sbp> 160 mmhg and/or dbp> 100 mmhg), systolic hypertension was defined as a sbp> 140 mmhg, the isolated systolic hypertension (ish) was defined as a sbp> 140 mmhg and a dbp<90mmhg, diastolic hypertension was defined as a dbp> 90 mmhg, and the isolated diastolic hypertension (idh) was defined as a sbp <140 mmhg and a dbp> 90 mmhg . Hypertension was defined as a sbp> 140 mmhg and/or a dbp> 90 mmhg and/or receiving anti - hypertensive treatment . Subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . The pittsburgh sleep quality index (psqi) the psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale (03) to obtain 7 subcategories: sleep duration, sleep disturbances, sleep latency, daytime dysfunction, sleep efficiency, sleep satisfaction, and medication use . The sum of all subscores represents the total sleep quality score, ranging between 021, with higher scores representing lower sleep quality . The individuals were categorized as follows: good sleep quality (scores between 0 and 5); fairly good sleep quality (scores between 6 and 10); and poor sleep quality (scores between 11 and 21). Psychometric properties have demonstrated good reliability (internal consistency: 0.89; test retest reliability: 0.85) and good construct validity for the english language version . The psqi is a widely used tool in research studies and clinical trials, and has been translated into several languages including chinese, spanish, german, and hebrew, with comparable reliability and validity values . When excluding the medication use subscale due to a low rate of medication users in this sample, internal consistency increased to =0.76 . Outcome measures included the total psqi score as well as self - report of sleep latency (sl) (question 2: how long (in minutes) has it usually taken you to fall asleep each night? ), and sleep efficiency percentage (se), computed as the ratio between the hours of actual sleep (question 4: how many hours of actual sleep did you get at night?) And total time in bed (hours computed based on reported bedtime in question 1: what time have you usually gone to bed at night? And reported wake - time in question 3: what time have you usually gotten up in the morning? ), multiplied by 100 . The methods were reported previously . Sitting or recumbent position, right arm blood pressure (bp) was measured twice to the nearest 2 mmhg using a standard mercury sphygmomanometer (phases i and v of korotkoff) by trained nurses or physicians . The mean value of 2 measurements was used to calculate systolic bp (sbp) and diastolic bp (dbp), and the sbp and dbp were calculated as the mean of right and left arm values in exceptional subjects . The mean of 2 readings was used for classification of bp according to joint national committee (jnc)vii criteria into normal (sbp <120mmhg and dbp <80mmhg), pre - hypertension (sbp 120139 mmhg and/or dbp 8089 mmhg), stage 1 hypertension (sbp 140159 mmhg and/or dbp 9099 mmhg), stage 2 hypertension (sbp> 160 mmhg and/or dbp> 100 mmhg), systolic hypertension was defined as a sbp> 140 mmhg, the isolated systolic hypertension (ish) was defined as a sbp> 140 mmhg and a dbp<90mmhg, diastolic hypertension was defined as a dbp> 90 mmhg, and the isolated diastolic hypertension (idh) was defined as a sbp <140 mmhg and a dbp> 90 mmhg . Hypertension was defined as a sbp> 140 mmhg and/or a dbp> 90 mmhg and/or receiving anti - hypertensive treatment . Subjects with confirmed hypertension and no identified cause of secondary hypertension were diagnosed with essential hypertension . The pittsburgh sleep quality index (psqi) was used for the subjective assessment of sleep quality . The psqi is a questionnaire consisting of 19 items which are coded on a 4-point scale (03) to obtain 7 subcategories: sleep duration, sleep disturbances, sleep latency, daytime dysfunction, sleep efficiency, sleep satisfaction, and medication use . The sum of all subscores represents the total sleep quality score, ranging between 021, with higher scores representing lower sleep quality . The individuals were categorized as follows: good sleep quality (scores between 0 and 5); fairly good sleep quality (scores between 6 and 10); and poor sleep quality (scores between 11 and 21). Psychometric properties have demonstrated good reliability (internal consistency: 0.89; test retest reliability: 0.85) and good construct validity for the english language version . The psqi is a widely used tool in research studies and clinical trials, and has been translated into several languages including chinese, spanish, german, and hebrew, with comparable reliability and validity values . Internal consistency in the present study was =0.69 . When excluding the medication use subscale due to a low rate of medication users in this sample, internal consistency increased to =0.76 . Outcome measures included the total psqi score as well as self - report of sleep latency (sl) (question 2: how long (in minutes) has it usually taken you to fall asleep each night? ), and sleep efficiency percentage (se), computed as the ratio between the hours of actual sleep (question 4: how many hours of actual sleep did you get at night?) And total time in bed (hours computed based on reported bedtime in question 1: what time have you usually gone to bed at night? And reported wake - time in question 3: what time have you usually gotten up in the morning? ), multiplied by 100 . The baseline examination included information on age (years), sex (male / female), smoking habits (yes or no), alcohol consumption (yes or no), tea consumption (yes or no), exercise (yes or no), serum lipid / lipoprotein levels (including serum triglyceride (tg), total cholesterol (tc), high - density lipoprotein (hdl) cholesterol, and low - density lipoprotein (ldl) cholesterol), fasting blood glucose (fbg), serum uric acid (sua), and body mass index (bmi). The bmi was calculated as body weight in kilograms divided by height in meters squared . Serum lipid / lipoprotein levels, fbg and sua were determined by standard laboratory techniques (performed by a technician in the biochemistry laboratory of sichuan university). Behaviors of smoking, alcohol consumption, tea consumption, and exercise, which included former and current status, were collected by using a general questionnaire . In the questionnaire, subjects were asked whether they had ever had behaviors of smoking, alcohol consumption, tea consumption, and exercise, and 1 of 3 answers were recorded: never, did in the past, or currently . Among those who did, currently or in the past, information was obtained on the average frequency of smoking, using alcohol, drinking tea, and doing exercise, and on the number of years they had engaged in the behavior . The subjects, who did almost everyday during the past year, were classified as those with these habits current, otherwise as without . The subjects, who had done behaviors almost everyday for more than 2 years as of a year before, were classified as those with these habits previously, otherwise as without . Alcohol consumption included spirits, liqueurs, wine, sherry, martini, beer, lager, hard cider, stout, and so on . All of the statistical analyses for this study were performed with the spss for windows software package, version11.5 (spss inc, chicago, illinois, usa). Baseline characteristics were compared between subjects with and without hypertension, between subjects with and without systolic hypertension, between subjects with and without diastolic hypertension using pearson chi - square or fisher s exact test (where an expected cell count was <5) for categorical variables and unpaired student s t test for continuous variables . Baseline characteristics were also compared among different sleep quality groups using one - way for analysis of variance (anova) for continuous variables and pearson chi - square or fisher s exact test (where an expected cell count was <5) for categorical variables . Multiple logistic regression was used to estimate the odds ratio (or) and 95% confidence interval (ci) of sleep quality as a function of increased risk for hypertension, systolic hypertension or diastolic hypertension . The following items were adjusted: age, sex, serum lipid / lipoprotein, body mass index, blood glucose level, smoking, alcohol consumption, tea consumption, and exercise . A p value <0.05 was considered to be statistically significant, and all of the p values were 2-sided . Among the 660 participants, mean age was 93.52 years, 69 were centenarians and 444 were women . There were 150 (22.7%) subjects with normal arterial blood pressure, 133 (20.1%) with pre - hypertension, 216 (32.7%) with stage 1 hypertension, 161(24.5%) with stage 2 hypertension, 364 (55.2%) with systolic hypertension, and 86 (13.0%) with diastolic hypertension (tables 1 and 2). There were 385 (58.4%), 128 (19.4%) and 147 (22.2%) with good, fairly good, and poor sleep quality, respectively . The subjects with good sleep quality were younger in age (93.323.32, 93.953.59, 93.633.14, among subjects with good, fairly good, poor sleep quality respectively, p=0.016). The mean sleep quality score was 6.842.15, the mean sl was 45.8916.72 min, and the mean se was 76.538.78% (tables 1 and 2). Among the different sleep quality groups, none of the differences in levels of sbp, dbp, the prevalence of hypertension, systolic hypertension and diastolic hypertension was significant (sbp: 140.2123.20, 138.3522.24, 140.3722.78, p=0.703; dbp: 72.7712.30, 72.5010.92, 72.2312.65, p=0.889; hypertension: 57.66%, 56.25%, 57.14%, p=0.847; systolic hypertension: 55.32%, 55.47%, 54.42%, p=0.836; diastolic hypertension: 13.50%, 8.59%, 15.64%, p=0.334 among subjects with good, fairly good, and poor quality sleep, respectively) (table 1). Between subjects with and without hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.672.59 vs. 6.972.66, p=0.139; sleep latency: 44.1013.17 vs. 47.2318.11, p=0.235; sleep efficiency percentage: 79.1712.64 vs. 74.5614.36, p=0.473; prevalence of poor sleep quality: 22.34% vs. 22.22%, p=0.778 in subjects without and with hypertension, respectively). Between subjects with and without systolic hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.712.71 vs. 6.952.48, p=0.172; sleep latency: 44.3513.42 vs. 47.1417.99, p=0.347; sleep efficiency percentage: 79.2111.91 vs. 74.3513.44, p=0.469; prevalence of poor sleep quality: 22.64% vs. 21.98%, p=0.813 in subjects without and with systolic hypertension, respectively). Between subjects with and without diastolic hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.773.01 vs. 7.312.67, p=0.212; sleep latency: 45.9913.57 vs. 45.2211.65, p=0.881; sleep efficiency percentage: 76.342.25 vs. 77.792.81, p=0.786; prevalence of poor sleep quality: 26.74% vs. 21.60%, p=0.207 in subjects without and with diastolic hypertension, respectively). Neither the unadjusted nor the adjusted multiple logistic regression showed that good quality sleep had a function of decreasing the risk for hypertension (unadjusted odds ratio (or): 0.963, 95% confidence interval (ci): 0.6541.417; adjusted or: 0.981, 95% ci: 0.7361.328), systolic hypertension (unadjusted or: 0.968, 95% ci: 0.6741.497; adjusted or: 0.976, 95% ci: 0.6151.541) or diastolic hypertension (unadjusted or: 0.821, 95% ci: 0.5431.205; adjusted or: 0 . All of these showed that among very elderly subjects there was no association between sleep quality and arterial blood pressure . Among the 660 participants, mean age was 93.52 years, 69 were centenarians and 444 were women . There were 150 (22.7%) subjects with normal arterial blood pressure, 133 (20.1%) with pre - hypertension, 216 (32.7%) with stage 1 hypertension, 161(24.5%) with stage 2 hypertension, 364 (55.2%) with systolic hypertension, and 86 (13.0%) with diastolic hypertension (tables 1 and 2). There were 385 (58.4%), 128 (19.4%) and 147 (22.2%) with good, fairly good, and poor sleep quality, respectively . The subjects with good sleep quality were younger in age (93.323.32, 93.953.59, 93.633.14, among subjects with good, fairly good, poor sleep quality respectively, p=0.016). The mean sleep quality score was 6.842.15, the mean sl was 45.8916.72 min, and the mean se was 76.538.78% (tables 1 and 2). Among the different sleep quality groups, none of the differences in levels of sbp, dbp, the prevalence of hypertension, systolic hypertension and diastolic hypertension was significant (sbp: 140.2123.20, 138.3522.24, 140.3722.78, p=0.703; dbp: 72.7712.30, 72.5010.92, 72.2312.65, p=0.889; hypertension: 57.66%, 56.25%, 57.14%, p=0.847; systolic hypertension: 55.32%, 55.47%, 54.42%, p=0.836; diastolic hypertension: 13.50%, 8.59%, 15.64%, p=0.334 among subjects with good, fairly good, and poor quality sleep, respectively) (table 1). Between subjects with and without hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.672.59 vs. 6.972.66, p=0.139; sleep latency: 44.1013.17 vs. 47.2318.11, p=0.235; sleep efficiency percentage: 79.1712.64 vs. 74.5614.36, p=0.473; prevalence of poor sleep quality: 22.34% vs. 22.22%, p=0.778 in subjects without and with hypertension, respectively). Between subjects with and without systolic hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.712.71 vs. 6.952.48, p=0.172; sleep latency: 44.3513.42 vs. 47.1417.99, p=0.347; sleep efficiency percentage: 79.2111.91 vs. 74.3513.44, p=0.469; prevalence of poor sleep quality: 22.64% vs. 21.98%, p=0.813 in subjects without and with systolic hypertension, respectively). Between subjects with and without diastolic hypertension, none of the differences in sleep quality scores, sleep latency, sleep efficiency percentage and prevalence of poor sleep quality was significant (sleep quality scores: 6.773.01 vs. 7.312.67, p=0.212; sleep latency: 45.9913.57 vs. 45.2211.65, p=0.881; sleep efficiency percentage: 76.342.25 vs. 77.792.81, p=0.786; prevalence of poor sleep quality: 26.74% vs. 21.60%, p=0.207 in subjects without and with diastolic hypertension, respectively). Neither the unadjusted nor the adjusted multiple logistic regression showed that good quality sleep had a function of decreasing the risk for hypertension (unadjusted odds ratio (or): 0.963, 95% confidence interval (ci): 0.6541.417; adjusted or: 0.981, 95% ci: 0.7361.328), systolic hypertension (unadjusted or: 0.968, 95% ci: 0.6741.497; adjusted or: 0.976, 95% ci: 0.6151.541) or diastolic hypertension (unadjusted or: 0.821, 95% ci: 0.5431.205; adjusted or: 0 . All of these showed that among very elderly subjects there was no association between sleep quality and arterial blood pressure . This study evaluated the association between sleep quality and arterial blood pressure in very elderly subjects . In the present study, arterial blood pressure included systolic blood pressure, diastolic blood pressure, hypertension, systolic hypertension and diastolic hypertension . In the cross - sectional observations, there was no association between sleep quality and arterial blood pressure . Several studies have shown that there was a strongly significant association of sleep quality with arterial blood pressure, or with the risk of hypertension among children, adolescents, and adults (aged 60 years or younger). However, many previous studies showed that there was no association of sleep quality with arterial blood pressure in the elderly aged 60 years or older [2830]. In the previous studies, participants were all less than 90 years of age . In our study, participants were all aged 90108 years . Our finding provided the evidence from very elderly subjects and extended this association of sleep quality with arterial blood pressure to very elderly people . Combining the results of previous studies and our finding in the present study, we should conclude here that the association between sleep quality and arterial blood pressure changed with age; that in a population aged less than 60 years it was significant, but that in a population aged more than 60 years (including general elderly aged 60 years or older and very elderly aged 90 years or older) it was non - significant . Progressive arterial stiffness, as a result of structural and functional changes within the vessel wall, is a feature of aging and may precede the onset of hypertension [2631]. Isolated systolic hypertension, defined as raised sbp but normal dbp, is the predominant form of hypertension in elderly people (65 years); systolic - diastolic hypertension is not as common in this population, as dbp stabilizes or even declines with advancing age [26,3234]. In our study, in very elderly chinese, there were high prevalences of hypertension (57.2%), systolic hypertension (55.2%), and isolated systolic hypertension (44.2%), and low prevalences of diastolic hypertension (13.0%), systolic - diastolic hypertension (11.0%) and isolated diastolic hypertension (2.0%). The prevalence of hypertension, systolic hypertension and isolated systolic hypertension in the present study was higher than that in the elderly aged 60 years or older from the previous reports, but the prevalence of diastolic hypertension, systolic - diastolic hypertension and isolated diastolic hypertension were lower than that in the elderly aged 60 years or older from the previous reports . This was consistent with previous findings that with advancing age, progressive arterial stiffness results in sbp increase and dbp decline . These indicate that systolic and diastolic blood pressure has different clinical significance in the elderly, especially in very elderly subjects . Considering this, we further independently assessed the association of systolic and diastolic blood pressure with sleep quality when the data were analyzed . All the results showed that there was no association between sleep quality and arterial blood pressure (including sbp and dbp) in very elderly subjects; this strongly confirmed the non - significant association between sleep quality and arterial blood pressure in very elderly subjects . Among children, adolescents, and general adults, habitually sleep problems could lead to the development and maintenance of hypertension through elevated sympathetic nervous system activity, waking physical and psychosocial stressors, increased salt retention and changed exposure to raised 24-hour blood pressure and heart rate . Extended exposure to these forces could lead to the entrainment of the cardiovascular system to operate at elevated pressure equilibrium through structural adaptations such as arterial and left ventricle hypertrophic remodeling . Habitual sleep problems could also contribute to hypertension by disrupting circadian rhythmicity and autonomic balance . Dramatic alterations in these parameters in modern industrialized society are theorized to cause the environment sensed by the brain to become metabolically flattened and arrhythmic, disturbing the circadian rhythmicity of blood pressure in susceptible individuals . Hypertension is characterized by a disturbance in the circadian rhythmicity of many physiological variables, such as a shifting of the daily blood pressure profile to higher values, an increased prevalence of the non - dipping pattern, increased blood pressure variability, and disturbances in the diurnal rhythm of cardiac output . Hypertensive subjects have been shown to have reductions of> 50% in the 3 main neuronal populations of the scn in comparisons between normal subjects and hypertensive subjects who died of myocardial infarction or brain hemorrhage . There was the difference between the elderly and younger adults, children or adolescents, and there was no association of sleep with hypertension in the elderly . The mechanism of the difference is unclear and should be further explored, and might be related with the following . Firstly, there was a high mortality rate among the elderly, specifically among the every elderly, and both sleep problem and hypertension were related with high mortality rate . The high mortality in the elderly more likely removed those both with sleep problem and hypertension, and left the one or the other, and removed the association between sleep problem and hypertension . Secondly, degradation of arterial wall structure with age resulted in elevated arterial blood pressure, a high risk for hypertension, and insensitivity for sympathetic hormone . In the present study, there was a high prevalence of hypertension (57.19%) in the sample, and provide an evidence for a high prevalence of hypertension in the elderly [26,3134]. Activation of the sympathetic nervous system is an important mechanism that links sleep disorders with combined systolic and diastolic hypertension, which is common in middle - aged hypertensive patients . However, no mechanistic link has been identified between sleep disorders and isolated systolic hypertension that results from age - dependent loss of arterial compliance and accounts for nearly 60% of hypertension in elderly populations [3035]. Thirdly, advanced age is associated with changes in sleep architecture, with increased difficulties in sleep initiation and maintenance . Elderly subjects, who are often retired, also have more opportunities to take naps during the day [3638]. First, 870 subjects aged 90 years or older volunteered for the plad study, and among these 870 volunteers only 660 were included in the statistical analysis . There might be selection biases . Because subjects with cancer, a history or clinical evidence of stroke, terminal stage of physical disease such as of respiratory system disease, cardiovascular disease, and kidney disease, usually have symptoms which could lead to insomnia and being bedridden . However, as our sample was from the community, not from hospital or nursing home, the information from them (excluded from the sample) might be unable to influence the practical implications of the present study . Second, because of the cross - sectional nature of this study, the subjects might change their diets and the conditions related to elevated hypertension and habits of sleep . However, the lifestyles, habits of sleep and food habits of the nonagenarians / centenarians were relatively stable and similar . Third, since this was a part of the plad, there might be a survival bias . Finally, we did not adjust for other potential confounding factors, such as socio - economic status and family history of sleep disorders . To our knowledge, this is the first study to demonstrate the association between sleep quality and arterial blood pressure in long - lived subjects, but the association was non - significant . This is different from that in children, adolescents, and general adult (60 years or younger), and is similar with that in the general elderly (60 years or more). In very elderly chinese, there was high prevalence of hypertension, especially systolic hypertension and isolated systolic hypertension.
Bariatric surgery is most often performed with the laparoscopic roux - en - y gastric bypass (lrgb). Lrgb has favourable effects such as weight loss and correction of metabolic disturbances, but is also associated with serious complications . It is a severe complication, which can lead to massive bowel necrosis that may result in short bowel syndrome (sbs) and sometimes death . Pregnant women, who have previously undergone lrbg, represent a diagnostic challenge when presenting with acute abdomen and internal hernia in this setting might be particularly risky . In this case, we present a 22-year - old woman, who had undergone lrgb and who suffered a massive internal hernia during pregnancy leading to short bowel syndrome . In january 2012, at 26 weeks of gestation, a 22-year - old woman was admitted to our surgical department due to sudden onset of upper abdominal pain . The patient was otherwise healthy and had at the time she became pregnant lost 50 kg following a lrgb done two years and 11 months earlier . Following admission, an acute ultrasound showed gall bladder stones, and a mri scan showed no definite signs of internal hernia but could not demonstrate gallstones . Due to severe pain a trans - cystic balloon dilation of the papilla of vater was done as the cholangiogram demonstrated poor drainage caused by small gallstones in the common hepatic bile duct . On the 5th postoperative day she was re - operated due to cholascos with renewed cystic duct ligation and drainage . On the following day, endoscopy was performed because of hematamesis, with the finding of blood in the jejunum, but no specific bleeding site was identified . The clinical condition worsened and she was transferred to the intensive care unit (icu) with severe septicaemia and acute respiratory distress syndrome (ards). On day 13, a laparotomy was performed . An internal hernia through petersen's space with necrosis of 1 m jejunum starting 10 cm below the pouch - enteric anastomosis a vital premature girl of 1020 g was delivered by caesarean and the necrotic intestine was removed . Second and third look operations were performed the next days with further small bowel resections leaving her with a saliva fistula 10 cm below the pouch - enteric anastomosis, a jejunostomy 15 cm from the ligament of treitz and a blind closed ileum 35 cm from the ileo - coecal valve (fig . 1). After 3 months in the icu and surgical ward with several other complications, such as infectious complications, pneumothorax, thrombosis of the superior mesenteric vein as well as the iliac veins and a exposed bowel parquet covered with split skin, the patient had a good appetite and enjoyed the meals, but obviously she had no absorption of food due to the very short fistula below the pouch - enteric anastomosis . Through a central venous line (hickmann catheter) she initially received 2200 kcal and later when she increased her daily activities this was increased according to 2700 kcal given as smof kabiven (fresenius) added with vitamins and trace elements, and in addition 3 l of isotonic saline to maintain the fluid balance . Also in this phase complications occurred, and the hickmann catheter eventually had to be removed due to a yeast infection with candida albicans . The nutrition was thereafter delivered through a peripherally inserted central venous catheter (piccline). After four months of hospitalization the patient was discharged with home parenteral nutrition . In november 2012, ten month after the internal herniation, intestinal continuity was reconstructed, and a defect having a width of up to 12 cm in the anterior abdominal wall was closed with bilateral partial component separation technique ad modum ramirez . The patient insisted on trying without parenteral nutrition, and 10 weeks later, she was doing well . Her weight had increased 2 kg since the operation, she had 12 bowel movements a day and blood values for haemoglobin and albumin had increased as well . This patient suffered from massive small bowel internal hernia with extensive bowel resection leading to sbs dependent on parenteral nutrition during her pregnancy . The patient was left with an intact duodenum, 15 cm of jejunum, 35 cm of ileum and her colon . Initially she was treated with 100% parenteral nutrition, but after reconstructive surgery with re - established intestinal continuity the amount of parenteral nutrition was rapidly reduced, and after six weeks she was weaned off parenteral nutrition . Several cases of internal hernias during pregnancies have been reported and three maternal deaths have been described . Describe severe internal hernias in pregnant women, involving the entire small intestine, parts of the colon and in renault et al . She underwent extensive bowel resection leading to an acute intestinal failure associated with severe complications (type 2 intestinal failure). After treatment in a specialized intestinal failure unit, she underwent a long and complicated rehabilitation period and was discharged with a short bowel syndrome treated with home parenteral nutrition and fluids (type 3 intestinal failure), and she survived . To our knowledge, no pregnant woman with lrgb has previously survived an internal herniation with such massive bowel necrosis as in this case, see table 1 . The university of nebraska medical center describes in a series of papers that 5 out of 210 patient with postoperative sbs had previous gastric bypass operation, and thereafter they scrutinized 9 patients with sbs that had bariatric surgery performed . In a more recent paper, they reviewed 9 cases with massive bowel resection during pregnancy leading to small bowel syndrome . It is believed to be caused by the growing uterus, which increases the intra - abdominal pressure, or postpartum as the uterus rapidly decreases in size . The surveys of series of patients with previous bariatric surgery demonstrate that up to 16% of the patients develop internal herniation . Most bariatric surgeries have been performed in women of reproductive age, and as lrgb increases fertility, complications during pregnancy are to be expected . It is suggested that the changed anatomy during pregnancy disposes for herniation to occur . During pregnancy, the cause of abdominal pain can be difficult to diagnose, with potentially fatal consequences for both mother and foetus . It is most important that relevant tools such as mri or ct scans are not postponed . Interpretation of mri and ct scan can be difficult especially in pregnant women, thus laparoscopy should be performed on a wide indication, when there is clinical suspicion of internal hernia and laparotomy performed if the anatomy is not clear . In the present paper the literature provides other successful reconstructions after lrgb complicated with internal obstruction . A case report of a 44-year - old woman, who suffered from volvulus after lrgb, leaving her with an intact duodenum the younger age of our patient leaves her with an even better chance to stay off parenteral nutrition . Since internal hernia is potentially life threatening for mother and foetus, both obstetricians and abdominal surgeons must be aware of this condition and surgery should be performed on a wide indication . When bowel necrosis is found it should be resected and in case of extensive bowel resection the patient should start parenteral nutrition, and an evaluation should be made in centres specialized in intestinal failure . All authors have contributed to acquisition of data, analysis and interpretation of data, writing, drafting the article and revising it critically for important intellectual content and approved of the final version . Written informed consent was obtained from the patient for publication of this case report and case series and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request.
Type 2 diabetes mellitus (t2 dm) is a common chronic health condition in the elderly . The number of elderly t2 dm patients has been growing worldwide, especially in upper - middle income countries such as thailand . Based on the findings of the fourth thai national health examination survey in 2009, diabetes was most prevalent in women, the elderly, and urban areas . The prevalence of impaired fasting glucose and undiagnosed diabetes increased with age, peaking at age 75 years and 5564 years, respectively.1 diabetes in the elderly is associated with a greater risk of t2dm - related micro- and macrovascular complications, cognitive disorders, physical disability, morbidity, and mortality;25 the selection of antidiabetic treatment for elderly t2 dm patients poses many challenges for a number of reasons . First, elderly t2 dm patients have a greater incidence of hypoglycemia6 which can precipitate serious events such as falls and accompanying fractures . The study by zhao et al7 showed that hypoglycemia patients had higher rates of fall - related fractures than those without hypoglycemia, within 30 days and 1 year (0.64% vs 0.02% and 2.11% vs 0.50%, respectively). Second, elderly t2 dm patients are more likely to have comorbidities with their diabetes, leading to the use of polypharmacy.4,8,9 third, chronic kidney disease often occurs in elderly t2 dm patients;10 the prevalence of chronic kidney disease among t2 dm patients in australia,11 india,12 finland,13 singapore,14 and the us15 ranged from 40% to 70% . With these associated challenges for elderly t2 dipeptidyl peptidase-4 (dpp-4) inhibitors show particular promise for treating elderly t2 dm patients because they have excellent tolerability profiles, low risk of hypoglycemia, and little effect on body weight.4,16,17 therefore, this study evaluated the cost - effectiveness of dpp-4 inhibitor monotherapy compared with sulfonylurea (sfu) monotherapy or metformin monotherapy for treating elderly t2 dm patients in the thai context . From a thai health care system perspective, we conducted a cost - utility analysis and used a validated ims core diabetes model (cdm), version 8.5, to estimate long - term costs and outcomes associated with each treatment over a lifetime horizon . Details of this model are described elsewhere.18,19 a 3% discount rate per annum was applied to both costs and outcomes in line with the thai health technology assessment (hta) guideline.20 the cohort population was thai people with t2 dm aged at least 65 years . Table 1 presents the baseline demographics, risk factors, and clinical complications of the cohort, which were obtained from published data and hospital databases in thailand.2128 the all - cause mortality rate was also adjusted with the age - specific mortality rate of thai people.29 utility values used in the cdm were based mostly on published studies conducted in other countries.3034 this study was approved by the buddhachinaraj regional hospital ethics committee on august 8, 2014 . As the patient data is de - identified patient consent was not required . These medications were administered as a monotherapy and then compared with either metformin monotherapy or sfu monotherapy . Metformin and sfu are considered as usual care for elderly t2 dm patients in thailand . Second, we convened a panel of stakeholders to discuss the scope and appropriate comparators of the study, including endocrinologists, and policy makers, and then followed the consensus of the meeting . This study used the normal daily dose of each treatment option: saxagliptin (5 mg), sitagliptin (100 mg), vildagliptin (100 mg), glipizide (10 mg), and metformin (2,000 mg). Only the direct medical costs, such as cost of intervention, concurrent medications, diabetic screening, management, and treatment complications, were included in the cost - effectiveness analysis . Cost data were derived from the published literature and retrospective hospital database analyses (table 2).3641 all costs were inflated using thailand s consumer price index42 and presented in the year 2014 thb value . Costs were converted to us$at a rate of thb32.96 per us$as of december 30, 2014.43 the cost of dpp-4 inhibitors was proposed to the subcommittee for the development of the national list of essential medicine by the pharmaceutical companies . Total cost per year of saxagliptin, sitagliptin, and vildagliptin was thb13,492 (us$409.34), thb16,570 (us$502.73), and thb15,900 (us$482.40), respectively . Glipizide and metformin we used a median of the median prices of those generic products44 as recommended by the thai hta guideline.45 the annual total costs of metformin and sfu were thb496 (us$15.05) and thb149 (us$4.52), respectively . Due to limited evidence of treatment efficacy in thailand, we performed a systematic review and meta - analysis to estimate the pooled efficacy of dpp-4 inhibitor monotherapy compared to sfu monotherapy and metformin monotherapy in elderly t2 dm patients . The medline, embase, and clinicaltrial.gov databases were systematically searched from their inception to august 2014 . We found only one study46 that indicated noninferiority of alogliptin compared to glipizide in hba1c reduction (the weighted mean difference 0.09; 95% ci, to 0.06), substantially lower risk of hypoglycemia (risk ratio [rr] 0.21; 95% ci, 0.110.41), lower risk of severe hypoglycemia (rr 0.23; 95% ci, 0.031.99), and no weight gain with dpp-4 inhibitor monotherapy compared to glipizide monotherapy in elderly t2 dm patients (table 3). Three studies5,47,48 compared metformin with dpp-4 inhibitor monotherapy and concluded that dpp-4 inhibitor was an effective and well - tolerated treatment option for elderly t2 dm patients . In addition, reduction in hba1c after treatment with dpp-4 inhibitors in elderly t2 dm patients was not significantly different from those in younger patients.49 therefore, we decided to systematically search a meta - analysis study that compared dpp-4 inhibitor monotherapy with metformin monotherapy in t2 dm patients, and found two eligible studies.50,51 both were high quality studies (with scores of at least 9 of 11) based on the assessment of multiple systematic reviews, we decided to use wu et als51 meta - analysis as it was the most up - to - date . The efficacy of hba1c reduction from the baseline of metformin monotherapy was estimated from the pooled analysis of seven studies48,5257 included in the meta - analysis of wu et al.51 of those studies,48,5257 severe hypoglycemia was presented in two studies,52,55 for which the rr was estimated . The calculation details are shown in table 3 . To determine the robustness of the findings, we undertook a probabilistic sensitivity analysis and presented the relationship between the probability of favoring dpp-4 inhibitors and the value of the willingness to pay for an additional unit of quality - adjusted life year (qaly) as a cost - effectiveness acceptability curve . The current acceptable thai ceiling threshold of thb160,000/qaly (us$4,854.37/qaly) was recommended by the subcommittee for the development of the universal health coverage benefit package and service delivery in thailand.58 a series of one - way sensitivity analyses were also performed to determine the effect of hba1c change, risk of hypoglycemia, drug cost, and discount rate . From a thai health care system perspective, we conducted a cost - utility analysis and used a validated ims core diabetes model (cdm), version 8.5, to estimate long - term costs and outcomes associated with each treatment over a lifetime horizon . Details of this model are described elsewhere.18,19 a 3% discount rate per annum was applied to both costs and outcomes in line with the thai health technology assessment (hta) guideline.20 the cohort population was thai people with t2 dm aged at least 65 years . Table 1 presents the baseline demographics, risk factors, and clinical complications of the cohort, which were obtained from published data and hospital databases in thailand.2128 the all - cause mortality rate was also adjusted with the age - specific mortality rate of thai people.29 utility values used in the cdm were based mostly on published studies conducted in other countries.3034 this study was approved by the buddhachinaraj regional hospital ethics committee on august 8, 2014 . As the patient data is de - identified patient consent was not required . These medications were administered as a monotherapy and then compared with either metformin monotherapy or sfu monotherapy . Metformin and sfu are considered as usual care for elderly t2 dm patients in thailand . Second, we convened a panel of stakeholders to discuss the scope and appropriate comparators of the study, including endocrinologists, and policy makers, and then followed the consensus of the meeting . This study used the normal daily dose of each treatment option: saxagliptin (5 mg), sitagliptin (100 mg), vildagliptin (100 mg), glipizide (10 mg), and metformin (2,000 mg). Only the direct medical costs, such as cost of intervention, concurrent medications, diabetic screening, management, and treatment complications, were included in the cost - effectiveness analysis . Cost data were derived from the published literature and retrospective hospital database analyses (table 2).3641 all costs were inflated using thailand s consumer price index42 and presented in the year 2014 thb value . Costs were converted to us$at a rate of thb32.96 per us$as of december 30, 2014.43 the cost of dpp-4 inhibitors was proposed to the subcommittee for the development of the national list of essential medicine by the pharmaceutical companies . Total cost per year of saxagliptin, sitagliptin, and vildagliptin was thb13,492 (us$409.34), thb16,570 (us$502.73), and thb15,900 (us$482.40), respectively . Glipizide and metformin we used a median of the median prices of those generic products44 as recommended by the thai hta guideline.45 the annual total costs of metformin and sfu were thb496 (us$15.05) and thb149 (us$4.52), respectively . Due to limited evidence of treatment efficacy in thailand, we performed a systematic review and meta - analysis to estimate the pooled efficacy of dpp-4 inhibitor monotherapy compared to sfu monotherapy and metformin monotherapy in elderly t2 dm patients . The medline, embase, and clinicaltrial.gov databases were systematically searched from their inception to august 2014 . We found only one study46 that indicated noninferiority of alogliptin compared to glipizide in hba1c reduction (the weighted mean difference 0.09; 95% ci, to 0.06), substantially lower risk of hypoglycemia (risk ratio [rr] 0.21; 95% ci, 0.110.41), lower risk of severe hypoglycemia (rr 0.23; 95% ci, 0.031.99), and no weight gain with dpp-4 inhibitor monotherapy compared to glipizide monotherapy in elderly t2 dm patients (table 3). Three studies5,47,48 compared metformin with dpp-4 inhibitor monotherapy and concluded that dpp-4 inhibitor was an effective and well - tolerated treatment option for elderly t2 dm patients . In addition, reduction in hba1c after treatment with dpp-4 inhibitors in elderly t2 dm patients was not significantly different from those in younger patients.49 therefore, we decided to systematically search a meta - analysis study that compared dpp-4 inhibitor monotherapy with metformin monotherapy in t2 dm patients, and found two eligible studies.50,51 both were high quality studies (with scores of at least 9 of 11) based on the assessment of multiple systematic reviews, we decided to use wu et als51 meta - analysis as it was the most up - to - date . The efficacy of hba1c reduction from the baseline of metformin monotherapy was estimated from the pooled analysis of seven studies48,5257 included in the meta - analysis of wu et al.51 of those studies,48,5257 severe hypoglycemia was presented in two studies,52,55 for which the rr was estimated . To determine the robustness of the findings, we undertook a probabilistic sensitivity analysis and presented the relationship between the probability of favoring dpp-4 inhibitors and the value of the willingness to pay for an additional unit of quality - adjusted life year (qaly) as a cost - effectiveness acceptability curve . The current acceptable thai ceiling threshold of thb160,000/qaly (us$4,854.37/qaly) was recommended by the subcommittee for the development of the universal health coverage benefit package and service delivery in thailand.58 a series of one - way sensitivity analyses were also performed to determine the effect of hba1c change, risk of hypoglycemia, drug cost, and discount rate . In the base - case scenario, all three dpp-4 inhibitors incurred higher costs and yielded fewer qalys (5.965 qalys vs 5.986 qalys). In other words, all dpp-4 inhibitors were dominated, making metformin monotherapy a cost - saving treatment in elderly t2 dm patients in thai context (table 4). All three dpp-4 inhibitors were more effective (equal 0.031 higher qalys) but more costly than sfu . Saxagliptin yielded the lowest incremental cost per qaly, followed by vildagliptin and sitagliptin (thb3,632,604/qaly or us$110,212.50/qaly, thb4,335,273/qaly or us$131,531.34/qaly, and thb4,530,556/qaly or us$137,456.19/qaly, respectively). With the current thai threshold of thb160,000/qaly (us$4,854.37/qaly), dpp-4 inhibitors were not cost - effective compared to sfu for treating elderly t2 dm patients in the thai context (table 4). As vildagliptin and sitagliptin were dominated by saxagliptin, the results of one - way sensitivity analysis, therefore, were displayed on saxagliptin compared to sfu . The change in hba1c from the baseline of dpp-4 inhibitors, discount rate, risk of severe hypoglycemia, and cost of saxagliptin had some effect on the incremental cost - effectiveness ratio (icer) (figure 1). The greater the effect of dpp-4 inhibitors on the reduction of hba1c from baseline, the lower icer (figure 1). Based on the cost - effectiveness acceptability curve (figure 2), all dpp-4 inhibitors were not a cost - effective treatment compared to sfu at the ceiling threshold of 160,000 thb / qaly . Compared to metformin, the probability of dpp-4 inhibitors being cost - effective was even smaller than being compared to sfu . In the base - case scenario, all three dpp-4 inhibitors incurred higher costs and yielded fewer qalys (5.965 qalys vs 5.986 qalys). In other words, all dpp-4 inhibitors were dominated, making metformin monotherapy a cost - saving treatment in elderly t2 dm patients in thai context (table 4). All three dpp-4 inhibitors were more effective (equal 0.031 higher qalys) but more costly than sfu . Saxagliptin yielded the lowest incremental cost per qaly, followed by vildagliptin and sitagliptin (thb3,632,604/qaly or us$110,212.50/qaly, thb4,335,273/qaly or us$131,531.34/qaly, and thb4,530,556/qaly or us$137,456.19/qaly, respectively). With the current thai threshold of thb160,000/qaly (us$4,854.37/qaly), dpp-4 inhibitors were not cost - effective compared to sfu for treating elderly t2 dm patients in the thai context (table 4). As vildagliptin and sitagliptin were dominated by saxagliptin, the results of one - way sensitivity analysis, therefore, were displayed on saxagliptin compared to sfu . The change in hba1c from the baseline of dpp-4 inhibitors, discount rate, risk of severe hypoglycemia, and cost of saxagliptin had some effect on the incremental cost - effectiveness ratio (icer) (figure 1). The greater the effect of dpp-4 inhibitors on the reduction of hba1c from baseline, the lower icer (figure 1). Based on the cost - effectiveness acceptability curve (figure 2), all dpp-4 inhibitors were not a cost - effective treatment compared to sfu at the ceiling threshold of 160,000 thb / qaly . Compared to metformin, the probability of dpp-4 inhibitors being cost - effective was even smaller than being compared to sfu . Elderly patients with diabetes have an increased risk of t2dm - related morbidity and mortality . The treatment goal for elderly t2 dm patients is to optimize glycemic control while minimizing the risk of drug - associated adverse events . Thus, this study was conducted to generate economic evidence of dpp-4 inhibitors for t2 dm treatment in response to a request by the subcommittee for the development of the national list of essential medicine in 2014 . The findings of the study were submitted and presented to the subcommittee in 2015 to justify policy decision in terms of the value for money . This cost - effectiveness study followed the thai national hta guideline.59 our findings indicated that dpp-4 inhibitor monotherapy was not a cost - effective treatment for elderly t2 dm patients in thailand compared to either sfu monotherapy or metformin monotherapy . Efficacy in hba1c reduction, risk of severe hypoglycemia, and cost of dpp-4 inhibitors play an important role in the findings of the study . We are not aware of other studies evaluating the cost - effectiveness of dpp-4 inhibitor monotherapy in elderly t2 dm patients . Geng et al60 conducted a systematic review of cost - effectiveness of dpp-4 inhibitors for treating t2 dm; the eleven included studies assessed dpp-4 inhibitors as an add - on therapy . Of those, seven studies compared dpp-4 inhibitors and metformin with sfu and metformin . Six studies concluded that dpp-4 inhibitors were cost - effective compared to sfu for treating t2 dm patients for whom metformin monotherapy failed to achieve glycemic control . Our study was strengthened by incorporating input parameters, such as costs, baseline cohort characteristics, and adverse events, from data sources that were reliable and relevant to the thai context . . First, based on our systematic review, we found only one study46 that evaluated the efficacy and safety of a dpp-4 inhibitor monotherapy compared to sfu monotherapy in elderly t2 dm patients . This study indicated noninferiority in hba1c reduction but a lower risk of hypoglycemia and no weight gain with the dpp-4 inhibitor monotherapy compared to sfu monotherapy . Only three studies5,47,48 compared dpp-4 inhibitor monotherapy with metformin monotherapy in elderly t2 dm patients . However, hba1c reduction after treatment with dpp-4 inhibitors was not significantly different in elderly t2 dm patients vs younger t2 dm patients.49 we addressed this limitation by the pooled analysis of seven studies48,5257 included in the meta - analysis study by wu et al51 that compared metformin monotherapy with dpp-4 inhibitor monotherapy in t2 dm patients . Second, we tried our best to use resource utilization and cost data from thailand . Some cost data were not available, such as the cost of an infected ulcer; we assumed them equal to zero . We reanalyzed our base - case analysis, assuming the cost of an infected ulcer similar to an uninfected ulcer . The results showed a slightly lower icer from thb3,632,604/qaly (us$110,212.50/qaly) to thb3,630,697/qaly (us$110,154.64/qaly) comparing saxagliptin to sfu . Metformin monotherapy was still a dominant treatment compared to dpp-4 inhibitor monotherapy, when assuming equal cost of an infected and uninfected ulcer . Even though costs, baseline cohort characteristics, and adverse events were obtained from published studies or hospital databases in thailand, this study relies on utility values and transition probabilities within the cdm from studies conducted in other countries . Given these limitations, the confirmation of this study s however, the findings indicate that treating elderly t2 dm patients using dpp-4 inhibitor monotherapy in a thai context may not be cost - effective . For treating elderly t2 dm patients in thailand, dpp-4 inhibitor monotherapy is not a cost - effective treatment compared to metformin monotherapy . In addition, dpp-4 inhibitor monotherapy is not a cost - effective treatment compared to sfu monotherapy at the current thai threshold of thb160,000/qaly . The high acquisition cost of dpp-4 inhibitors is one of the key factors in the findings of this study.
Organic molecules containing bulky alkyl groups have shown great potential in drug discovery and medicinal chemistry . Sterically demanding alkyl substituents such as adamantyl or tert - butyl are often introduced into pharmaceuticals to enhance lipophilicity and/or improve the drug s metabolic stability by shielding adjacent functional groups or reactive sites from enzymatic degradation . Aminoadamantanes themselves have been examined and used as antiviral drugs; however, aryl aminoadamantane derivatives and other anilines based on hindered amines such as 35 (figure 1) remain largely unexplored, presumably due to difficulty in their preparation . Mechanism of pd - catalyzed arylation of primary amines and examples of bulky,,-trisubstituted primary amines . Successful strategies that have previously been used to synthesize these bulky anilines employ an electrophilic amination approach . Amines 1 and 3 have been arylated through a titanium - mediated coupling of the corresponding n - chloroamines with grignard reagents . Additionally, there are examples of transition - metal - free amination of arylboroxines and copper - catalyzed amination of organozinc reagents using 3 . Recently, lalic reported an elegant synthesis of hindered tertiary anilines through the copper - catalyzed coupling of aryl boronic esters with o - benzoyl hydroxylamines . While these methods are efficient, the electrophilic amine must be separately prepared, and many of the nucleophiles that are employed are moisture - sensitive . A useful alternative is the palladium - catalyzed c n cross - coupling an operationally simple and widely used reaction in both industrial and academic settings . Although advances in ligand design have overcome many challenges, only a few examples of the n - arylation of hindered primary amines have been reported . Amines 1 and 2 have been previously cross - coupled with catalysts with either phosphines or n - heterocyclic carbenes as supporting ligands . However, most of these reactions require moderate catalyst loadings (15 mol%) and elevated temperatures (90135 c) and, most importantly, are limited with regard to the substrate scope . In addition, there are no examples using more hindered and challenging amine substrates such as 35 . The availability of a general method to obtain a broad range of hindered anilines by a pd - catalyzed c herein, we describe the development of two related catalyst systems that demonstrate high activity for the coupling of,,-trisubstituted primary amines 15 with a variety of (hetero)aryl halides . These complementary systems were developed through an initial ligand screen followed by a rational ligand design based on the results of the kinetic analysis of the reaction (figure 1). In 2008 we reported brettphos (l1; table 1), a biaryl(dialkyl)phosphine ligand for the pd - catalyzed arylation of primary amines . Brettphos - based catalyst systems often promote amination reactions at low catalyst loadings and exhibit an exceptionally broad scope for both the aryl halide and the primary amine . However, when we evaluated the (2-aminobiphenyl)palladium methanesulfonate precatalyst of l1 (p1), for the coupling of 3,5-dimethoxychlorobenzene (6) with tert - octylamine (3), the product 7 was formed in low yield (table 1, entry 1). Given the size of both l1 and 3, we felt that either amine binding (iii, figure 1) or amine deprotonation (iii iv, figure 1) could be problematic . Therefore, we hypothesized that the use of a smaller ligand would allow for more effective coordination to the pd(ii) center and/or facilitate the approach of the base . Indeed, the yield was significantly improved with the less sterically demanding and less electron - rich phbrettphos, l2 (table 1, entry 2). Furthermore, the use of l3, which lacks methoxy groups on the phosphine - containing arene ring, was even more effective (table 1, entry 3). The use of a ligand in which the distal arene of l3 had been modified by replacing the triisopropyl substituents with two dimethylamino groups (l4) also gave the desired product in good yield (table 1, entry 4). The catalyst derived from the corresponding dicyclohexylphosphino analogue, cphos (l5), gave no product, suggesting that the relatively smaller and less electron - donating phenyl groups on the phosphine atom are required to promote the desired transformation . Reaction conditions: aryl halide (0.5 mmol), amine (0.6 mmol), nao - t - bu (0.6 mmol), 1,4-dioxane (0.5 ml). The most effective precatalysts, p3 and p4, were further evaluated in reactions between 3 and more complex aryl halides, and the use of p4 initially gave the best results (table 2). Reaction conditions: aryl halide (0.5 mmol), amine (0.6 mmol), nao - t - bu (0.6 mmol), 1,4-dioxane (0.5 ml). Ad = adamantyl reaction conditions: 1% p, 80 c, 90 min . Reaction conditions: 2% p4, 120 c, 24 h. reaction conditions: 1% p4, 100 c, 12 h. while the results with p4 were promising, relatively high temperatures (100120 c) and catalyst loadings (13 mol%) were required for the reaction to reach completion . To design an improved catalyst system, we set out to qualitatively explore the reaction rate dependence on each substrate using reaction progress kinetic analysis (rpka). As described by blackmond, rpka is a simple, systematic method to obtain a complete picture of a reaction s kinetic profile from a limited number of experiments performed under synthetically relevant conditions . This method has been successfully used in a number of laboratories to elucidate the reaction mechanism of various catalytic processes . The key parameter utilized in rpka is excess, which refers to the difference between the initial concentrations of the two reactants (eq 1), and the kinetic information is obtained from reactions run under different excess conditions.1 we chose the model reaction between aryl chloride 6 and amine 3 for our kinetic analysis using precatalyst p4 . To broaden the study, we also explored the reaction with the corresponding aryl bromide 12 . The reactions were monitored in situ by reaction calorimetry along with gc analysis to support the calorimetry results (see the supporting information). The reaction rate progress over time profiles for both aryl halides are shown together in figure 2 . It is immediately apparent that the shape of the curves is different for each aryl halide and that the reaction for arbr is notably faster than that for arcl . These observations suggest that the nature of the aryl halide plays a key role in the kinetics of the reaction . Following the rpka method, the data may be replotted as rate vs [substrate] to determine the rate orders of each substrate (only the most informative graphs are shown for each case; all graphs are included in the supporting information). Temporal reaction rate profiles for the reaction of amine 3 (1.0 m) and arcl 6 or arbr 12 (concentrations defined by [excess] given in legend; see eq 1) catalyzed by p4 (0.02 m). Figure 3 shows the rate vs [arcl] plot for the two different excess experiments shown in figure 2 over the range of arcl concentrations common to both reactions . At any given value of [arcl], the concentration of amine is different for the two kinetic profiles, as illustrated by the dashed line (when [arcl] = 0.3 m, [amine] = 0.8 and 0.3 m for the blue and red curves, respectively). An overlay of the curves at different amine concentrations indicates that the rate is independent of the concentration of amine for this range of concentrations . This behavior was unexpected given the steric encumbrance of 3, which we initially predicted to bind to the pd(ii) center with difficulty and therefore be involved in the rate - determining step . However, the linear decay of the curves indicates that the reaction has a positive order in [arcl] and that oxidative addition is (at least partially) rate - determining . The fact that the reactions reached different maximum rates when starting at different arcl concentrations (figure 2) provides additional evidence for a positive order in aryl halide (since the maximum rates do not differ by a factor of 2, the order in arcl is fractional). The use of a ligand (l4) with phenyl groups as the phosphine substituents could explain the relatively slow rate of oxidative addition (computational evidence suggests that l2 has a higher energy barrier than its alkyl analogue l1 for this step). Reaction progress kinetic profiles for the reaction of arcl 6 with amine 3 plotted as rate vs [arcl] over the range of concentrations common to the two reactions . The concentration of amine 3 at the point in the reaction progress marked by the dashed line is shown for each reaction . Further probing of the reaction mechanism is possible by exploring the behavior of aryl bromide 12 . Since the rate of oxidative addition is determined by the strength of the c x bond, it was not surprising that the reaction of 12 was ca . 4 times faster than that of the aryl chloride 6 (t1/2 = 10 min vs 46 min for [arx]0 = 0.5 m). In this case, plotting the data of the different excess experiments as rate vs [amine] for the concentrations common to the two reactions provides insights into the aryl bromide reaction kinetics (figure 4). First, the overlay between the curves reveals identical rates at different arbr concentrations (as illustrated by the dashed line: when [amine] = 0.75 m, [arbr] = 0.25 and 0.75 m for the blue and red curves, respectively), indicating zeroth - order kinetics in aryl halide, in contrast to the arcl case . This observation is confirmed by the fact that the reactions achieve identical maximum rates (0.03 m / min) for different initial concentrations of aryl bromide (figure 2). Second, the rate appears to be relatively insensitive to the concentration of amine . The nearly horizontal curves show the rate slightly decreasing over the range of amine concentrations from 0.9 to 0.5 m, suggesting zeroth - order kinetics in [amine]. Since oxidative addition of aryl bromides is faster, the rate - determining step of the reaction has shifted to a subsequent step in the catalytic cycle, which is independent of both [arbr] and [amine]. Therefore, for the reaction of aryl bromide 12 and amine 3 with precatalyst p4, reductive elimination is most likely the rate - determining step . Reaction progress kinetic profiles for the reaction of arbr 12 with amine 3 plotted as rate vs [amine] over the range of concentrations common to the two reactions . The concentration of arbr 12 at the point in the reaction progress marked by the dashed line is shown for each reaction . At approximately 40% conversion, the initial zeroth - order regime shown in figure 4 gives way to a slow linear decay in reaction rate for the [excess] = 0 m reaction (equal amounts of aryl bromide 12 and amine 3; see the supporting information for the full graph). This behavior corresponds to initial saturation kinetics in [amine]. As the concentration of amine 3 decreases, amine binding to the pd(ii) center becomes slower than reductive elimination and, therefore, rate - determining . The kinetic analysis of the reactions with two different aryl halides suggests that, to improve the catalyst system, oxidative addition and reductive elimination need to be accelerated . Modifying the ligand to make it both more electron - rich (faster oxidative addition) and larger (faster reductive elimination) might result in faster rates for each step . We reasoned that switching from a biaryl(diaryl)phosphine - type ligand to the (dialkyl)phosphino analogue (figure 5) would accomplish both requirements . However, our initial ligand screen (table 1) showed that cphos (l5) gave no product, suggesting this ligand was too large and/or too electron - rich . We thus prepared ligands with one phenyl group and one alkyl group (figure 5). By incorporating one large alkyl group, we could increase the electron density and size of the ligand, while keeping the smaller aryl group should still allow amine binding and deprotonation to proceed at a rapid rate . We synthesized two biaryl(alkyl)phenyl ligands in which one of the phenyl groups on the phosphine of l4 is replaced with either a cyclohexyl (l6) or a tert - butyl (l7) group (table 3; see the supporting information for details on the preparation of the ligands). The corresponding precatalysts p6 and p7 were prepared and examined under identical conditions used in our previous ligand assessment (table 1). As hypothesized, the hybrid (alkyl)phenyl phosphines l6 and l7 provided superior catalysts . A system based on ligand l7 proved optimal, giving full conversion of 6 in less than 30 min at 70 c (table 3, entry 3). Reaction conditions: aryl halide (0.5 mmol), amine (0.6 mmol), nao - t - bu (0.6 mmol), 1,4-dioxane (0.5 ml). To understand the difference between l4 and l7, we investigated the kinetics of the reactions between aryl halides 6 and 12 and amine 3 using a catalyst based on l7 under the same reaction conditions used for p4 (2% pd, 70 c). The use of ligand l7 dramatically accelerated the rate of the reaction: t1/2 = 2.6 and 1.9 min for 6 and 12, respectively, whereas the half - lives with p4 were 46 and 10 min for 6 and 12 ([arx]0 = 0.5 m; see the supporting information for details). To perform accurate calorimetric kinetic studies of the improved system, we adjusted the reaction conditions by reducing the catalyst loading (from 2 to 0.5 mol%) and the temperature for the aryl bromide (from 70 to 60 c). More consistent results were observed at lower catalyst loadings through in situ generation of an oxidative addition complex from (cod)pd(ch2tms)2, ligand l7, and aryl halide than through the use of precatalyst p7 as the pd source . In addition, it was found that better results were obtained in the presence of an extra equivalent of l7 (with respect to the palladium catalyst), which was extended to the general reaction conditions (table 4). Reaction conditions: aryl halide (1 mmol), amine (1.2 mmol), nao - t - bu (1.2 mmol), 1,4-dioxane (1 ml). Even with the sharply reduced catalyst concentration (and reduced temperature for the arbr substrate), the rate of these reactions was ca . 1 order of magnitude greater than that of the reactions with precatalyst p4 (figures 2 and 6) temporal reaction rate profiles for the reaction of amine 3 (1.0 m) and arcl 6 or arbr 12 (concentrations defined by [excess] given in legend; see eq 1). Note a: pd(l7) is an oxidative addition complex generated in situ from (cod)pd(ch2tms)2, l7, and 12 (see the supporting information for details). Figure 7 shows the data from the different excess experiments for aryl chloride 6 as rate vs [arcl] over the range of arcl concentrations common to both reactions . Although the reaction has a more complex profile than with p4, the nonhorizontal shape of the curves and the different maximum rates (0.124 and 0.02 m / min for the blue and the red curves, respectively) imply that there is a positive order in aryl chloride and thus that oxidative addition remains, at least partially, the rate - determining step (the reaction has a fractional order in [arcl]). In contrast to the arcl reactions with p4, the two curves are not overlaid, which could indicate a positive reaction order in [amine] (the reactions have different rates at different amine concentrations). However, the convex shape of the rate curves (particularly explicit for the red curve) is typically a hallmark of catalyst deactivation . The likelihood of rate dependence on [amine] vs catalyst deactivation can be deconvoluted with a quantitative assessment . We can consider relative rates of the two reactions at any given concentration of arcl and amine, with x and y as the reaction orders in [arcl] and [amine], respectively, and kobs as the observed rate constant, which contains the catalyst concentration (eq 2). Choosing [arcl] = 0.5 m, we see from figure 7 that [amine] = 1.0 m for the blue curve (the beginning of the reaction) and 0.5 m for the reaction in red (50% conversion) and that their relative rates differ by a factor of 5 (eq 3). If kobs is identical for the two runs, y is calculated to be greater than 2 (eq 4a), which is not mechanistically reasonable . If the reaction is in fact zeroth - order in [amine] (y = 0), eq 4b indicates that kobs for the red curve has been deactivated by a factor of 5 at 50% conversion . Although this mathematical argument cannot entirely exclude rate dependence on [amine], catalyst deactivation is a more likely explanation on the basis of the shape of the curves and the fact that different maximum rates are obtained for different arcl concentrations . Reaction progress kinetic profiles for the reactions of arcl 6 with amine 3 plotted as rate vs [arcl] over the range of concentrations common to the two reactions . The concentration of amine 3 at the point in the reaction progress marked by the dashed line is shown for each reaction . Note a: pd(l7) is an oxidative addition complex generated in situ from (cod)pd(ch2tms)2, l7, and 12 (see the supporting information for details). Lastly, the data for the cross - coupling between aryl bromide 12 and amine 3 with a catalyst based on l7, shown in figure 6, reveal the fastest reactions run in this work (even at 60 c). The curves show an uncommon shape in which the rates reach their maximum at ca . 2535% conversion, which suggests a delay in the catalyst entering the catalytic cycle . In addition, as in the reaction with aryl chloride 6, unusual behavior is observed at the end of the reactions: the curves decay, displaying a convex shape (see the supporting information for the full graph), presumably due to catalyst decomposition . Thus, it seems that the catalyst based on l7 has a transient nature in such a fast reaction and does not establish a persistent steady - state kinetics behavior to allow further rpka analysis . However, since both different excess reactions with aryl bromide 12 reach the same maximum rate independent of their initial concentration, it can be inferred that the reaction is likely zeroth - order in arbr as with precatalyst p4 . To gain a deeper insight into the differences between l4 and l7, we prepared oxidative addition complexes [l4pd(4-cnph)br] (13) and [l7pd(4-cnph)br] (14) as air - stable yellow solids and analyzed them by single - crystal x - ray crystallography (figure 8). Similar to what has been previously observed with biaryl(dialkyl)phosphine ligands, the complexes possess a slightly distorted square planar geometry at pd . As has been observed with l5, the dimethylamino substituents were not coplanar with the aryl ring to which they are attached, indicating that no conjugation of the nitrogen lone pairs with the aromatic -system is occurring . Thus, the dimethylamino groups should be considered as overall electron - withdrawing by induction rather than electron - donating . This could assist the binding of bulky and electron - rich amines by rendering the pd center more electrophilic . X - ray structures of oxidative addition complexes 13 and 14 and relevant bond lengths . Br angle (), which decreased from 86.5 to 83.2. we infer that this geometric difference also applies to the amido complexes iv (figure 1), bringing the two groups to be connected closer in 14 than in 13 and therefore facilitating reductive elimination . We next set out to examine the substrate scope of the reaction with p7 . We were able to reduce catalyst loadings from 12% p4 to 0.5 mol% p7 and the temperature from 100120 to 80 c for many substrates (table 4). More challenging substrates required 1 mol% pd and 100 c (e.g., with 30 or 33). A variety of electron - deficient, electron - rich, and heteroaryl chlorides and bromides esters and nitriles (32, 34) were tolerated under the reaction conditions, although free n h heterocycles and enolizable ketones were not compatible . Protic substrates such as phenols, carboxylic acids, and amides were also successfully coupled by using 2 equiv of base (23, 25, and 29). As previously reported, nitrogen - containing five - membered heterocycles are challenging, albeit important, substrates . However, replacing the methyl group with a trityl protecting group led to the clean formation of 31 . Reactions using ortho - substituted substrates and/or tritylamine 5 were sluggish with p7, and the use of p4 provided better results in these cases (table 5). This system allowed for the preparation of extremely hindered secondary anilines as 2,6-disubstituted aryl halides (35) and substrates bearing large ortho substituents (36) were successfully coupled in high yields . Additionally, certain heteroaryl halides such as 5-chloro-2,1,3-benzothiadiazole and 4-methyl-3-bromothiophene gave better results using p4 (40, 42; 1015% yields were obtained using p7). For activated 2-halopyridines and pyrazine (41, 44, 45) the corresponding aro - t - bu byproduct was also observed but was easily separated from the desired compound . Weaker inorganic bases (cs2co3, k3po4, or k2co3) that could circumvent this issue were found to be ineffective in promoting the desired transformation . Nonetheless, a wide variety of substrates, including aryl halides containing base - sensitive functional groups, were coupled with deliberate choice of the supporting ligand (l7 or l4) reaction conditions: aryl halide (1 mmol), amine (1.2 mmol), nao - t - bu (1.2 mmol), 1,4-dioxane (1 ml). In summary, we have developed a general method for the cross - coupling of hindered primary amines with a range of (hetero)aryl chlorides and bromides . This transformation occurs under mild conditions and provides the desired products in high yields utilizing two catalyst systems . Ligand l7 was designed after analysis of the reaction mechanism using the rpka method . Rather than amine binding, it was found that oxidative addition for aryl chlorides and reductive elimination for aryl bromides with the initial catalyst based on ligand l4 were rate - determining . These observations lead to the development of l7, a biaryl(alkyl)arylphosphine ligand that greatly accelerated the reaction with both aryl halides . L7 is the first example of a hybrid (alkyl)aryl ligand that provides superior results as compared to its analogues with identical alkyl- or arylphosphorus substituents, offering another tunable site for better control of catalyst activity . We anticipate that this methodology will find applications in both academic and pharmaceutical settings, and we expect the new class of biarylphosphine ligands reported here to influence our future design of ligands.
This investigation includes a series of blood investigations and imaging; when these investigations found no evidence for a demyelinating disorder (through mri features principally), systemic autoimmune condition (e.g., antinuclear antibodies, dna, aquaporin-4, and myelin oligodendrocyte glycoprotein antibodies), or infectious or metabolic causes, a granulomatous cause was considered, and further investigations were undertaken . Those in whom systemic features of sarcoidosis were identified during investigation and those already known to have active systemic sarcoidosis, in whom the diagnosis of a granulomatous optic neuropathy was considered by the authors to be highly probable, were recorded prospectively and included in the study . The records were kept according to data protection regulations in the neuroophthalmology departments and collated by 2 of us (b.j.b . And d.p.k . ). Criteria for the diagnosis of sarcoidosis involved histologic proof (in 40 cases) of a granulomatous disorder currently active or requiring continuing treatment or (in 12 cases) adequate proof on clinical grounds that no alternative explanation existed for the systemic disorder in the minds of the treating physicians: for example, clear radiologic evidence for lung and mediastinal involvement together with clear evidence for another characteristic feature of the disease, such as hypercalcemia, raised serum angiotensin - converting enzyme (ace) or other tissue involvement, associated with a typical pattern of isotope uptake using gallium scintigraphy or pet . Ethical approval for the study was obtained, and patients gave informed consent to be included . Ethical approval for the study was obtained, and patients gave informed consent to be included . Twenty - four were caucasian, 24 african or caribbean, and 4 were from south asia . The diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . The remaining 18 had already been diagnosed, between 6 months and 20 years before the onset of the optic neuropathy . A histologic diagnosis was made from respiratory tissue (transbronchial or mediastinal lymph gland biopsy) in 28 cases, lymph gland biopsy in 3, liver biopsy in 1, orbital biopsy in 3, and neurologic tissue in 5 . All fulfilled the criteria for the highly probable diagnosis of the wasog (world association of sarcoidosis and other granulomatous diseases) sarcoidosis organ assessment instrument . In 12 cases, the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians, with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . The predominate systemic clinical syndrome was respiratory in 38 cases, the orbit and lacrimal glands in 6, cervical lymph glands in 4, the skin in 3, the nose and sinuses in 3, and the liver and joints in 1 each . The serum ace was elevated in 24 (57%) of available data at the time of onset of the optic neuropathy; the median serum ace was 111 (18205) u / l (normal range 850 gallium scans were undertaken in 10 instances, in which 1 was normal, 6 showed a panda pattern of uptake in the lacrimal and parotid glands, 1 a lambda pattern of mediastinal uptake, and 2 a combination of both patterns . All patients presented with symptoms and signs of an optic neuropathy, defined as a reduction in central visual acuity and color vision, reduction in the visual field, and when seen, an abnormality of the pupillary response . Patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course, in which the symptoms evolved over weeks . The former were considered to show an optic neuritis syndrome, the latter a progressive syndrome . Forty - three cases showed an optic neuritis syndrome, of which 27 were unilateral . Twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . The median (range) acuity at presentation was 0.05 (01.0) in unilateral cases and 0.13 (01.0) in bilateral involvement . Mri showing (a) swelling and high signal within the nerve, and (b) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . With oral corticosteroid treatment (c) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . (d, e) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . A wide range of visual field defects was noted; most had central or centrocecal scotomas, but hemianopic and altitudinal defects were also seen . Clinical characteristics and ocular findings at presentation in the 52 patients studied intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases (36%): a granulomatous anterior uveitis was seen in 10 cases, an isolated vitritis in 1, a panuveitis in 1, isolated choroidal involvement in 1, features of a retinal vasculitis in 2, cotton wool spots in 2, macular exudates in 1, and episcleritis in 1 . In no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . One patient underwent ct; the remainder had mri studies of the orbits and brain, of which 13 were adjudged to be normal . Twenty patients had abnormalities restricted to the optic nerve or sheath, of whom 14 showed enhancement of the optic nerve and sheath (figure 1) with intrinsic lesions of one or both optic nerves, while the remainder showed high signal within the nerve without enhancement . In addition, 3 cases (and the one who had ct) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . The chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . Both patients with a perineuritis showed features of this, i.e., the predominate feature was of enhancement and thickening of the sheath, not the whole complex (figure 2). (b, c) optic perineuritis: t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . This was the presenting feature of the disease, which was diagnosed subsequently on histologic analysis of a cervical lymph node . A minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease: 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves, enhancement of the complex, which also involved the orbital apex (5 cases) (figure 3a), the floor of the middle cranial fossa (4 cases) (figure 3b), and the frontal lobes (1 case). (a, b) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs, mediastinum, and heart . (c, d) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . Despite intensive treatment with corticosteroids and immunosuppression, in general, the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen (figure 4). Histogram showing the relationships between mri features at onset / diagnosis (abscissa) and initial (nadir) visual acuity then best acuity after treatment (ordinate). Those whose clinical syndrome was of a progressive optic neuropathy (6 cases) all showed meningeal enhancing lesions that had formed masses, in each case at the intracranial portion of the orbital apex (figure 3). Csf results were available in 33 cases, including 2 with mass lesions and one with perineuritis . Seven (21%) showed csf protein levels greater than 0.6 g / dl (median 0.95 g / dl, range 0.791.92). Five (15%) showed a leukocyte pleocytosis (> 5 cells / cm) (median 19, range 670). Twenty - eight (85%) had no oligoclonal bands, and 5 showed matched bands in both csf and serum . Patients were treated in an individual way in 3 different hospitals so there was a wide range of medications, dosage regimens, and treatment durations . Forty - nine were treated with corticosteroids, of whom 14 commenced with iv methylprednisolone followed by oral prednisolone, while the remainder received treatment with oral steroids from the outset . The daily dose of prednisolone varied between 30 and 80 mg per day . The duration of treatment varied greatly (4320 weeks). The median (range) length of follow - up was 8 (020) years . The median (range) change in visual acuity (defined as the best acuity following treatment, even after relapse) was + 0.46 (0.01 to + 1.22) in bilateral cases and + 0.175 (0.05 to + 1.25) in unilateral cases . The median final acuity in these 2 groups was 0.7 (01.25) and 0.5 (01.25). Those with a subacute syndrome showed a median improvement of + 0.225 (range 0.01 to + 1.22). Those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . While there was a suggestion that mri abnormalities at onset (figure 4) correlated with nadir acuity and an improvement following treatment, this was not significant (p> 0.3). Considering treatment in those without a progressive disease course or perineuritis, 12 were treated with iv then oral steroids; median (range) acuity was 0.02 (01) and increased to 0.75 (0.021), median change + 0.3, and those treated with oral steroids at onset 0.1 (01) increasing to 0.75 (01.25), and median change + 0.5 (p = 0.77). For the most part, this occurred early on; for example, patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg, and the signs settled with an escalation of treatment . Immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . The patient treated with methotrexate had had repeated relapses, which stopped on the immunosuppressive drug . One patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened; she was treated with cyclophosphamide then infliximab . In those who relapsed, the improvement in acuity following treatment was + 0.25 (0.15 to + 1.2), and relapse did not appear to influence the outcome in any way . We have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace (median 100 [range 80178] u / l). The clinical syndrome was not different from the others described above; half were caribbean, half caucasian, and half were bilateral . The median (range) acuity in each eye was 0.1 (01.0). In each case, the affected discs were seen to be swollen . Imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . Csf in 4 cases showed normal protein and negative oligoclonal bands in 3; in the other case, matched serum and csf bands were found . In this case one was considered to have a disc granuloma and was watched without treatment; he improved over 2 years . These cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . They appear to have neither sarcoidosis nor multiple sclerosis . Twenty - four were caucasian, 24 african or caribbean, and 4 were from south asia . The diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . The remaining 18 had already been diagnosed, between 6 months and 20 years before the onset of the optic neuropathy . A histologic diagnosis was made from respiratory tissue (transbronchial or mediastinal lymph gland biopsy) in 28 cases, lymph gland biopsy in 3, liver biopsy in 1, orbital biopsy in 3, and neurologic tissue in 5 . All fulfilled the criteria for the highly probable diagnosis of the wasog (world association of sarcoidosis and other granulomatous diseases) sarcoidosis organ assessment instrument . In 12 cases, the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians, with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . The predominate systemic clinical syndrome was respiratory in 38 cases, the orbit and lacrimal glands in 6, cervical lymph glands in 4, the skin in 3, the nose and sinuses in 3, and the liver and joints in 1 each . The serum ace was elevated in 24 (57%) of available data at the time of onset of the optic neuropathy; the median serum ace was 111 (18205) u / l (normal range 850 gallium scans were undertaken in 10 instances, in which 1 was normal, 6 showed a panda pattern of uptake in the lacrimal and parotid glands, 1 a lambda pattern of mediastinal uptake, and 2 a combination of both patterns . All patients presented with symptoms and signs of an optic neuropathy, defined as a reduction in central visual acuity and color vision, reduction in the visual field, and when seen, an abnormality of the pupillary response . Patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course, in which the symptoms evolved over weeks . The former were considered to show an optic neuritis syndrome, the latter a progressive syndrome . Forty - three cases showed an optic neuritis syndrome, of which 27 were unilateral . Twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . The median (range) acuity at presentation was 0.05 (01.0) in unilateral cases and 0.13 (01.0) in bilateral involvement . Mri showing (a) swelling and high signal within the nerve, and (b) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . With oral corticosteroid treatment (c) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . (d, e) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . A wide range of visual field defects was noted; most had central or centrocecal scotomas, but hemianopic and altitudinal defects were also seen . Intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases (36%): a granulomatous anterior uveitis was seen in 10 cases, an isolated vitritis in 1, a panuveitis in 1, isolated choroidal involvement in 1, features of a retinal vasculitis in 2, cotton wool spots in 2, macular exudates in 1, and episcleritis in 1 . In no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . One patient underwent ct; the remainder had mri studies of the orbits and brain, of which 13 were adjudged to be normal . Twenty patients had abnormalities restricted to the optic nerve or sheath, of whom 14 showed enhancement of the optic nerve and sheath (figure 1) with intrinsic lesions of one or both optic nerves, while the remainder showed high signal within the nerve without enhancement . In addition, 3 cases (and the one who had ct) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . The chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . Both patients with a perineuritis showed features of this, i.e., the predominate feature was of enhancement and thickening of the sheath, not the whole complex (figure 2). The lesion was biopsied and granulomatous inflammation identified . The optic neuropathy improved with steroids . (b, c) optic perineuritis: t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . This was the presenting feature of the disease, which was diagnosed subsequently on histologic analysis of a cervical lymph node . A minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease: 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves, enhancement of the complex, which also involved the orbital apex (5 cases) (figure 3a), the floor of the middle cranial fossa (4 cases) (figure 3b), and the frontal lobes (1 case). (a, b) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs, mediastinum, and heart . (c, d) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . Despite intensive treatment with corticosteroids and immunosuppression, neither case showed a meaningful improvement in visual acuity . In general, the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen (figure 4). Histogram showing the relationships between mri features at onset / diagnosis (abscissa) and initial (nadir) visual acuity then best acuity after treatment (ordinate). Those whose clinical syndrome was of a progressive optic neuropathy (6 cases) all showed meningeal enhancing lesions that had formed masses, in each case at the intracranial portion of the orbital apex (figure 3). Csf results were available in 33 cases, including 2 with mass lesions and one with perineuritis . Seven (21%) showed csf protein levels greater than 0.6 g / dl (median 0.95 g / dl, range 0.791.92). Five (15%) showed a leukocyte pleocytosis (> 5 cells / cm) (median 19, range 670). Twenty - eight (85%) had no oligoclonal bands, and 5 showed matched bands in both csf and serum . Patients were treated in an individual way in 3 different hospitals so there was a wide range of medications, dosage regimens, and treatment durations . Forty - nine were treated with corticosteroids, of whom 14 commenced with iv methylprednisolone followed by oral prednisolone, while the remainder received treatment with oral steroids from the outset . The daily dose of prednisolone varied between 30 and 80 mg per day . The duration of treatment varied greatly (4320 weeks). The median (range) length of follow - up was 8 (020) years . The median (range) change in visual acuity (defined as the best acuity following treatment, even after relapse) was + 0.46 (0.01 to + 1.22) in bilateral cases and + 0.175 (0.05 to + 1.25) in unilateral cases . The median final acuity in these 2 groups was 0.7 (01.25) and 0.5 (01.25). Those with a subacute syndrome showed a median improvement of + 0.225 (range 0.01 to + 1.22). Those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . While there was a suggestion that mri abnormalities at onset (figure 4) correlated with nadir acuity and an improvement following treatment, this was not significant (p> 0.3). Considering treatment in those without a progressive disease course or perineuritis, 12 were treated with iv then oral steroids; median (range) acuity was 0.02 (01) and increased to 0.75 (0.021), median change + 0.3, and those treated with oral steroids at onset 0.1 (01) increasing to 0.75 (01.25), and median change + 0.5 (p = 0.77). For the most part, this occurred early on; for example, patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg, and the signs settled with an escalation of treatment . Immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . The patient treated with methotrexate had had repeated relapses, which stopped on the immunosuppressive drug . One patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened; she was treated with cyclophosphamide then infliximab . In those who relapsed, the improvement in acuity following treatment was + 0.25 (0.15 to + 1.2), and relapse did not appear to influence the outcome in any way . We have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace (median 100 [range 80178] the clinical syndrome was not different from the others described above; half were caribbean, half caucasian, and half were bilateral . The median (range) acuity in each eye was 0.1 (01.0). In each case, the affected discs were seen to be swollen . Imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . Csf in 4 cases showed normal protein and negative oligoclonal bands in 3; in the other case, matched serum and csf bands were found . In this case one was considered to have a disc granuloma and was watched without treatment; he improved over 2 years . These cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . This work forms the largest series of patients with optic neuropathy in sarcoidosis identified and each has been followed prospectively . The age range was wide but the condition was more prevalent in the first half of life . The majority of patients were women and there were equal numbers in caucasian and african or caribbean ethnic groups . According to the epidemiologic findings in access, the ethnic subgroups in london are 60% caucasian, 14% african and caribbean, and 18% south asian, and our results therefore suggest a greater prevalence of the disease in african and caribbean people who live in london . In general, the clinical syndrome resembled an optic neuritis, although in some, the disease course was more slowly progressive . Pain was a less prevalent feature than is seen in demyelinating optic neuritis, and the median visual acuity at nadir was equivalent, although more were no perception of light (table 2). Sixty - four percent had a unilateral involvement, 28% presented at a later time with involvement of the second eye, while synchronous bilateral involvement was less common (9%). It is noteworthy that there was evidence for concurrent intraocular inflammation in only a minority of cases . Predominately this was mild (although more severe cases had been excluded because of the uncertainty of the pathogenesis of the optic neuropathy). Indeed, in 64% of patients, there was no history of ocular involvement at any stage of the disease to date . Imaging showed clear abnormalities, which accounted for the clinical features in 75% of cases . Comparison of clinical features with results of the onsg spontaneous improvement occurred (as has been noted previously), although the majority of patients were treated with steroids . Following this, an often substantial improvement in vision took place, even after several weeks without treatment . Relapse occurred in 25% of cases, necessitating recommencement or an escalation of treatment, and in the majority, this occurred early, as steroids were withdrawn . Six cases (11%) had a more progressive disease course and were shown to have a more widespread neurologic involvement, each showing evidence for adjacent meningeal inflammation . Visual outcome in this subgroup was poor, even though the disease process required, in general, a higher and more prolonged dose of steroids, concurrent immunosuppression, and in 2 cases, a requirement for biological agents with refractory disease . Evidence for neurosarcoidosis defined as the presence of intracranial meningeal enhancement arose in 16 cases overall all of those with a progressive course and 10 with a subacute course . Involvement of the optic nerve in sarcoidosis may arise through a variety of mechanisms: inflammation of the nerve itself, compression or infiltration by an inflammatory mass adjacent to the anterior visual pathway (e.g., the orbital apex or pituitary gland), secondary involvement through ischemic complications of retinal and choroidal inflammation and glaucoma, granulomas within the disc itself, meningeal inflammation within the optic nerve sheath leading to an optic perineuritis, and finally as a consequence of hydrocephalus . It was seen in 5% of one series of 202 patients with ocular disease in sarcoidosis, but only one in a series of 649 patients with systemic sarcoidosis and none in another of 285 . In the literature before 2000, 57 cases had been published, of which 35 involved the optic nerve itself, 9 compression due to an intracerebral lesion, 6 due to hydrocephalus, and 6 due to the complications of uveitis . Many noted that bilateral involvement occurred and that imaging showed enlargement of the optic nerve others noted that the acuity was lower than that seen in a demyelinating optic neuritis . One study described 24 cases with optic neuropathy: 71% had not been known previously to have systemic sarcoidosis and 42% showed previous or current intraocular inflammation . Another showed that 14 of 67 patients known to have neurologic sarcoidosis had some form of optic neuropathy . We have identified a small subgroup of patients in whom the serum ace is elevated at presentation and then subsequently normalizes without relapse . On further investigation, these patients do not have sarcoidosis and on follow - up show no sign of transformation into a more widespread disease . The serum ace appears to increase in a nonspecific way and appears not to indicate a systemic granulomatous disease . It is important to identify this subgroup, which requires no additional treatment, merely close follow - up and observation . This large series of patients with optic neuropathy in conjunction with sarcoidosis has shown that more than half are not previously diagnosed with sarcoidosis, only a third have concurrent intraocular inflammation, and the clinical characteristics and in most cases the imaging features are not substantially different from that seen in a regular demyelinating optic neuritis . However, patients should be identified early because the response to treatment may lessen with time, and the identification of more widespread disease, particularly within the nervous system, will require prolonged, often high - dose treatment with steroids, immunosuppressive drugs, and modern biological therapies . Modern imaging investigations including pet may make identification of these cases easier, but a high index of clinical suspicion is still important, particularly in non - caucasian patients presenting with a painless optic neuritis . Once identified, we recommend that patients be treated with high - dose (oral or iv) corticosteroids, followed by a slow taper watching carefully for relapse as the dose is reduced . Repeated relapses, particularly preventing steroid dose reduction, should be treated with an additional immunosuppressive agent . The presence of neurosarcoidosis requires more prolonged higher - dose treatment with steroids and immunosuppression from the outset, with early recourse to biological agents should the clinical and radiologic features of the disorder fail to improve quickly . Further study is required to identify precisely which treatments are best and over what period of time . Dr . Kidd was responsible for the study design, data acquisition, analysis and interpretation, and writing the manuscript . Burton served on the scientific advisory board for bayer, novartis, received travel funding from novartis, bayer, is subeditor for eastern european journal of neurology, received research support from novartis alcon.
There is a subgroup of patients with temporomandibular disorder who nomadically pass from one dentist to another seeking bite correction . Their occlusal discomfort is characterized by an uncomfortable sensation mainly affecting corrected dentition, crowns, or dentures in which no abnormality is clinically detectable . This phenomenon is known as the phantom bite syndrome (marbach 1976, 1978; marbach et al 1983; jagger and korszun 2004). Despite repeated failures of dental surgery, these individuals persist in seeking bite correction from a succession of dentists . The condition is refractory to most treatments including psychotherapy (marbach 1976, 1996). We have reported previously that tricyclic antidepressants can be effective in treating phantom bite syndrome (toyofuku 2000). However, the patients non - acceptance of the anticholinergic side - effects of these drugs usually prevents high enough doses being achieved . Milnacipran is an antidepressant which, similarly to the tricyclics, inhibits the reuptake of serotonin and norepinephrine but without the side - effects of the older antidepressants . Recently it has been reported that milnacipran has been used to treat various oral psychosomatic disorders such as glossodynia and temporomandibular disorder (toyofuku 2003; toyofuku et al 2003; toyofuku and miyako 2004). We present here a preliminary study of the efficacy and safety of milnacipran in a series of patients complaining of occlusal discomfort and diagnosed as suffering from phantom bite syndrome . We report here the results with 7 patients who came to the department of dentistry and oral surgery at the fukuoka university hospital in japan in 20032004 complaining of occlusal discomfort and diagnosed as suffering from phantom bite syndrome . Six patients were outpatients and one was hospitalized for severe fatigue and because of difficulty of attending the hospital since she lives on a remote island . All patients met the following criteria; preoccupation with their dental occlusion and a false belief that their dental occlusion was abnormal, a long history of repeated dental surgery treatment failures with persistent requests for the occlusal treatment that they are convinced they need, no history of significant psychiatric illness, absence of obvious psychosocial problems . They all had a relatively high intelligence and socioeconomic status which enabled then to undergo endless costly and time - consuming dental treatments . Milnacipran was initially administered at 30 mg / day on a twice daily schedule and adjusted weekly according to clinical symptoms and tolerance . The effect on occlusal discomfort was measured using a 100 mm visual analogue scale (vas) where baseline severity was arbitrarily set to 100 . Severity of depression was assessed using the zung self - rating scale for depression (sds). (marked improvement with only occasional mild symptoms), mild (some improvement but with considerable residual symptoms), or poor (little or no improvement). One patient was lost to follow - up after 2 weeks because he refused to continue, stating that he was feeling well . Five of the remaining 6 patients were improved at the end of the 4-week treatment period, 3 markedly, and 2 mildly improved . Patient 6 was unco - operative and refused to complete the vas and sds self - rating evaluations at the end of the study . She insisted, however, that she had had no improvement at all over the 4 weeks of the study . During the study she refused any increase of the dose of milnacipran, insisting on dental surgery bite correction instead of medication . Scores of 0 improvement were therefore used for this patient for the sds and vas analyses (table 2). Overall, the occlusal discomfort felt by the patients was considerably decreased between baseline and week 4 (table 2). The level of depressive symptoms was also decreased as indicated by a 47.8% decrease in sds . There was, however, no correlation between individual reductions in sds and vas values . Three patients reported adverse effects (headache, dizziness, and nausea, table 2). All effects, however, were slight and transient, disappearing within a few days . Patients exhibiting the phantom bite syndrome are considered to be refractory to most dental treatment . They become increasingly difficult to manage after repeated failures of dental surgery resulting in frustration for both the dentist and the patient who is usually convinced of the incompetence of their dentist and moves on to another dentist . The present study suggests that milnacipran may be a beneficial treatment for such oral psychosomatic disorder patients with occlusal discomfort known as phantom bite syndrome . This is a small number, it is the largest study on phantom bite to have been reported to date . The department of dentistry and oral surgery at fukuoka university has an excellent reputation in the treatment of dental psychosomatic disorders and for this reason patients are referred to this department by psychosomatic doctors from all over southern japan . In addition 40% of patients in the present study came spontaneously to the department because of its reputation among patients . In addition to the small number of patients, the obvious weakness of the study resides in its open - label design . However, the refractory nature of the disorder (the average duration at the beginning of the study was over 3 years) gives some credence to the observed results . This technique probably lacked precision, however, since some of the patients said they had difficulty in expressing quantitatively their symptom on a visual analogue scale . The development of other patient - rated measures such as a modified faces scale as used to quantify pain in children (bieri et al 1990) would clearly be helpful for studying this and similar oral psychosomatic conditions . None of the patients had any history of significant psychiatric illness, indeed they were all strongly resistant to the idea of psychiatric referral or treatment . Six of the 7 patients were, however, mildly depressed as rated by the zung sds scale and one more moderately depressed . In 5 out of 6 patients completing the study, the depression scores improved considerably to a level considered to be asymptomatic . In general, however, there was no correlation in this study between the decrease in sds score and decrease in occlusal discomfort as indicated by the vas . This result and clinical observations suggests that the efficacy of milnacipran in reducing occlusal discomfort is more likely to be a direct effect on the somatosensory system rather than on any associated depression . Milnacipran and other serotonin - norepinephrine reuptake inhibitors have been shown to be effective on a variety of pain disorders both associated with, and independent of, depression (briley 2003, 2004). Milnacipran is not, however, an analgesic as such and these effects have been suggested to involve an action on serotonin and norepinephrine neurotransmission (stahl and briley 2004). The action of milnacipran appears to be to correct the erroneous integration of peripheral signals by the central nervous system . A further example of milnacipran correcting erroneous integration of peripheral signals is in fibromyalgia where normally non - painful stimuli are perceived as pain signals (allodynia) (vitton et al 2004). It would seem likely that the effect of milnacipran on phantom bite seen in the present study is another manifestation of this activity on the two monoamines systems where the occlusal discomfort experienced by the patient is the result of an erroneous integration of peripheral signals . In conclusion, after 4 weeks treatment, 5 of the 6 patients completing the study responded to milnacipran with clinically significant improvements in their occlusal discomfort . This suggests that milnacipran may be an effective and well tolerated treatment for phantom bite in oral psychosomatic disorder patients . Phantom bite is, however, a chronic syndrome and the promising effects seen here after 4 weeks need to be followed up over a much longer time period.
This clinical case report described a three - month - old mongrel dog that had the urethral orifice opening 3 cm from the tip of the penis and lacked a completely formed preputial sheath . Urinary incontinence stopped soon after surgery, suggesting that it had been probably due to an ascending urethritis . Hypospadias is a congenital developmental anomaly of the external genitalia in male animals and man, in which the external urethral orifice is on the ventral surface of the penis rather than at the tip of the glans . To a variable extent in individual cases, there is failure of fusion of the urogenital folds and incomplete development of the penile urethra . The urethra may open at any level on the ventral surface of the penis somewhere between the normal location and the ischiatic arch or on the surface of the perineum . In severe cases, lesions such as failure of the two halves of the scrotum to fuse, underdevelopment or absence of the penis, and failure of the urethra to close in the perineal area the urethral meatus may be located along the scrotal raphe . In one report hypospadias was seen in association with unilateral renal agenesis, whilst in another report there was underdevelopment of the penis, fusion failure of the urethra, prepuce and scrotum . Other abnormalities associated with hypospadias are retained testicles, bone or anorectal defects, umbilical hernia, hydrocephalus, and urinary incontinence . There are relatively few reports of this condition in the veterinary literature but this may not reflect the true prevalence of the condition . Its aetiology is unclear; it may be due to inadequate production of androgens by the foetal testes or to inadequate numbers of androgen receptors on the urethral folds . This case report describes a three - month - old mongrel dog that had hypospadias, unilateral retained testicle, urinary incontinence, and absence of a fully formed preputial sheath which has not been described before . A three - month - old mongrel dog, weighing 12 kg, was admitted to the veterinary hospital with urinary incontinence and an exposed and dry penis that appeared unsightly (figure 1). The referring veterinarian had informed the owner that the dog had severe paraphimosis and that the penis could not be readily replaced into the preputial sheath . On physical examination, there was urine scald of the skin on the ventral abdomen and urinary incontinence . The preputial sheath was absent from the bulbus glandis to the tip of the penis (figure 2) but the dorsal mucosa of the incompletely formed sheath was present on the ventral abdominal wall . The dog was anaesthetised using intravenous thiopentone (10 mg / kg) and intubated; anaesthesia was maintained with halothane and oxygen . A lateral incision was made in the mucocutaneous junction on either side of the midline up to the bulbus glandis . After reconstruction of the preputial sheath, a paramedian celiotomy was performed to locate and to remove the intraabdominal testicle . A small, nodular, vestigial testicle, 0.5 cm in diameter, was found and removed . The animal was kept for observation and it was noticed that urinary incontinence had resolved after 14 days . The surgical wounds had healed and the cosmetic appearance was satisfactory (figure 3). The prepuce has been reconstructed, leaving a wide preputial orifice . This report describes a case of hypospadias in a three - month - old dog with an incompletely formed preputial sheath . The cause of hypospadias is not known; it is thought that the affected foetus may secrete inadequate quantities of testosterone or that there may be inadequate conversion of testosterone to dihydrotestosterone in the target tissues of the urogenital sinus and external genitalia . A spectrum occurs from mild hypospadias, in which the urinary orifice is located in the glans penis to severe hypospadias where the orifice is at the penoscrotal junction, scrotum, or the perineum . In the dog, exposure of the male foetus to progesterone or anti - androgens during gestation, especially between day 30 and day 44 of pregnancy, may be responsible . Severe genetic defects including cryptorchidism, absence of the scrotum, bifid scrotum, and persistent mllerian structures have been observed in dogs . In many cases in animals and man, surgical correction is usually not attempted because the urethra cranial to the abnormal orifice is deficient . In the present case although the uretheral opening was 3 cm from the tip of the penis, it was patent up to the urinary bladder . One author unsuccessfully attempted to reconstruct the urethra and, subsequently carried out amputation of the penis whilst others recommended the removal of the open prepuce, partial penile amputation and prescrotal or perineal urethrostomy and castration as the treatment for hypospadias . When there are severe urethral defects, excision of the external genitalia and urethrostomy is the treatment of choice . In the present case the preputial sheath was easily reconstructed to cover the exposed penis . It was not necessary to modify the preputial orifice since, after surgery, the preputial opening created was sufficiently large and long enough to allow outflow of urine without scalding the ventral abdominal skin . It is thought that initially the penis appeared dry because of exposure to the air and it was soiled whenever the dog lay down . This must have caused irritation and probable ascending urethritis from bacteria entering through the abnormally located urethral opening . Urinary incontinence without neurological deficits is a frequent observation in dogs with hypospadias . In the present case it is presumed that the constant penile irritation and ascending urethritis could have been responsible for the urinary incontinence . In some animals penile and preputial disorders may be associated with pain, incontinence or reluctance to breed . In the present case, the owner was not interested in breeding the dog, and was satisfied with the outcome of surgery . It is concluded that in the less severe cases of hypospadias an attempt should be made to surgically reconstruct the affected parts . Although the cause of hypospadias is not known, affected dogs should not be used for breeding purposes.
Obesity is one of the major cardiovascular disease (cvd) risk factors and is a common health problem both in adults and in children . Not only do obese children have a higher risk of becoming obese adults, childhood bmi is independently associated with later coronary heart disease as well . Visceral fat in particular is related to an adverse metabolic profile and increased cvd risk through the production and secretion of metabolic active compounds like adipokines and cytokines, with effects on insulin sensitivity, lipid metabolism, and inflammation among others . From a cvd prevention point of view, early prevention of intra - abdominal fat accumulation growth patterns in early life have been suggested to be important for later fat distribution . Several studies determined an association between weight gain in the first 2 - 3 years of life and central adiposity, mostly assessed by larger waist circumference, in both adulthood and early childhood [813]. Imaging techniques such as magnetic resonance imaging, ultrasonography [10, 11], or computed tomography have seldom or never been used to assess the association between postnatal growth and later central adiposity . While in these studies weight gain in the first years of life was studied, the importance of particularly the first 3 months of life in the development of central adiposity was addressed in other studies in children and adults [1517]. Weight gain in the first 3 postnatal months is thought to comprise predominantly fat mass accumulation; fat mass as percentage of body weight increases until 36 months of age, followed by a gradual decline . Most of the previous studies did not account for length gain in infancy, while weight gain relative to length gain might reflect adiposity better than weight gain alone [19, 20]. Whether particularly rapid weight gain for length gain in the first 3 postnatal months, so excess weight gain relative to length gain, is related to differences in intra - abdominal fat, measured ultrasonographically, in childhood we set out to measure intra - abdominal and subcutaneous fat ultrasonographically in healthy 5-year - old children, to study whether differences in growth in the first 3 postnatal months, in particular increased weight gain accounting for length gain, are associated with increased central adiposity in early childhood . The present study is part of the wheezing illnesses study leidsche rijn (whistler) study, an ongoing population - based birth cohort on determinants of wheezing illnesses, initiated in 2001 . Healthy newborns in leidsche rijn, a new residential area near utrecht city, were enrolled . Exclusion criteria were gestational age <36 weeks, major congenital abnormalities, and neonatal respiratory disease . All five - year - olds (n = 1124 on april 26th, 2011) were invited according to the last - known telephone number and address, for follow - up measurements . 215/1124 (19%) subjects were lost to follow - up, due to incorrect telephone numbers and addresses, and 54/1124 (5%) were not yet contacted . Of the remaining 855 subjects, 285 (33%) declined to take part and 570 (67%) were willing to participate of whom 517 were measured before april 26th 2011 . Abdominal ultrasonography was performed and intra - abdominal fat and subcutaneous fat were measured successfully in 434/517 (84%) and 463/517 (90%) subjects, respectively . In the remaining participants the focus was solely on respiratory measures . Complete data on postnatal weight gain relative to length gain and abdominal fat distribution were available for 360/517 children (70%), and in addition on birth size for 316/517 (61%). Whistler - cardio was approved by the paediatric medical ethical committee of the university medical center utrecht . Parents visited the clinic when their offspring was approximately four weeks of age for lung function measurements, not further described here . Birth weight in 359/360 (99.7%) children and birth length in 317/360 (88%) children were measured standardized in the hospital or by midwifes using a standard electronic scale and an infant stadiometer . This questionnaire inquired for maternal age at childbirth and gestational age among others as well . In the netherlands infants we asked parents to report these measurements in monthly questionnaires for a period of 12 months . Exclusively breastfed if the only milk their infant had received during the month of interest was breast milk . Data on parental characteristics, like parental bmi, ethnicity and level of education among others, were obtained from the linked database of the utrecht - health - project (uhp), a large health monitoring study in leidsche rijn . Weight, height, and waist circumference were measured with the participants wearing indoor clothes without shoes . Standing with the feet slightly spread waist circumference was measured in duplicate at the level midway the lowest rib border and the iliac crest and hip circumference at the widest level over the major trochanter to the nearest mm . In addition, intra - abdominal and subcutaneous fat were measured using ultrasound according to a previously described procedure [26, 27] with a picus pro system (esaote, italy), using a ca 421 convex transducer . For intra - abdominal fat, the distances between the posterior edge of the abdominal muscles and distances were measured from 3 different angles: medial, left, and right lateral, with the transducer placed longitudinally on a straight line drawn between the left and right midpoint of the lower rib and iliac crest . Placing the probe transversely at the level of the umbilicus subcutaneous fat was measured with electronic callipers, from the external face of the rectus abdominis mucle (linea alba) to just below the skin . All measurements were performed by two of the investigators and a trained research nurse all blinded to infancy weight and length gain . Intraclass correlation coefficients (icc) based on measurements by one observer in 10 and, respectively, 11 subjects on 2 different occasions for intra - abdominal and subcutaneous fat were 0.67 and 0.96, respectively . Iccs for subcutaneous fat on the three measurements per child on the same occasion were 0.94, 0.94, and 0.97 for the three observers . In addition, information on child and parental characteristics, like smoking habits among others, with respect to the previous years was collected by questionnaires . We used weight gain rate, length gain rate, and particularly weight gain rate adjusted for length gain rate as measures of growth in the first 3 postnatal months . These measures were studied separately, as length gain might reflect lean mass accumulation, while weight gain, and particularly weight gain accounting for length gain, might reflect fat mass accumulation . For each child with at least two anthropometric measurements available in the first 3 postnatal months, weight gain and length gain rates were estimated using the monthly anthropometrics, to reduce an effect of measurement error and individual variation over time (regression to the mean). Since the number of weight and length measurements (median count: 3) and the age at which these were measured differed per child, the association between age, and both weight and length was assessed using linear mixed modelling, allowing for individual variation in birth weight or height and growth rate . Subsequently, to obtain weight gain rates for each child individually, linear regression modelling on the predicted weight and length values of the linear mixed model was performed stratified by child . Because of the small time window, weight and length gain rate were assumed constant in the first 3 postnatal months . Wlg was assessed by deriving z - scores internal to our study population for weight gain, conditioned on length gain, by using the standardized residuals from the linear regression model with weight gain as the dependent variable and length gain as the independent variable . A z - score of + 1 sd wlg indicates that the weight gain of a certain child is one standard deviation larger than the mean weight gain in the population based on the length gain of that child . Furthermore, size at birth was assessed by calculating internal z - scores for birth weight, adjusted for birth length, gestational, age and gender . Means and dispersion measures of parent and child characteristics were calculated by tertiles of z - score wlg . Differences by tertiles of z - score wlg were tested using analysis of variance, or kruskal wallis test in case of skewed data, for continuous variables and chi - squared tests or fisher's exact tests for frequencies (table 1). For analyzing the associations between postnatal growth and fat distribution we used generalized linear modelling . Bmi, waist circumference, intra - abdominal fat, and subcutaneous fat were used as dependent variables in separate models . Weight gain rate, length gain rate, and z - score wlg were used as independent variables in separate models . After univariable analysis, adjustments for age at follow - up and gender were made . In the analyses of intra - abdominal and subcutaneous moreover, adjustments for current height were made, as achieved height can be considered as confounder when studying the association between early weight gain and wlg and fat distribution . Parental characteristics, like smoking and bmi, and infant nutrition, were not considered as confounders, as to our view these factors might explain the associations between growth and later fat distribution and might therefore be in the causal pathway . Since an association between postnatal growth and fat distribution might be modified by fetal growth as well, we analyzed whether interaction on an additive scale was present between birth size, as proxy for fetal growth, and early postnatal growth on fat distribution . We therefore imputed birth length using multiple imputation technique in spss version 17.0 for windows and repeated the analyses on the 10 imputed datasets as sensitivity analysis . All results are expressed as linear regression coefficients with 95% confidence intervals (95%-ci) and corresponding p values . Median bmi, waist circumference, subcutaneous fat, and mean intra - abdominal fat were 15.1 kg / m (interquartile range (iqr): 14.316.1), 52.5 mm (iqr: 50.554.8), 6.3 mm (iqr: 5.07.9), and 36.4 6.5 mm, respectively . No differences in infant feeding type and parental characteristics were determined across growth tertiles, except for parental smoking habits and maternal gestational diabetes: parents of children with lower wlg smoked more often in the 5 years after birth and all three infants of the mothers who have had gestational diabetes belonged to the highest z - score wlg tertile . Postnatal wlg was positively associated with weight and height of the children at the follow - up visit at 5 years of age (table 1). In table 2 the associations between early postnatal growth and body fat distribution at the age of 5 years are shown . After correcting for confounders, size at birth was positively associated with bmi and waist circumference, but not with intra - abdominal and subcutaneous fat . With respect to postnatal growth, both weight gain and z - score wlg were significantly positively associated with bmi, waist circumference, and subcutaneous fat at the age of 5 years . No significant associations between both weight gain and wlg with intra - abdominal fat were observed . Size at birth did not modify the associations between postnatal growth and body fat distribution . After multiple imputation of birth length, to have complete data on size at birth for all 360 children in which an ultrasound was successfully performed and growth data was available, the associations between postnatal wlg and bmi, waist circumference, and subcutaneous fat strengthened . Regarding intra - abdominal fat, a trend towards significance was observed: per 1 unit increase in z - score wlg, intra - abdominal fat was 0.51 mm higher (95%-ci: 0.14, 1.2, p value: 0.12). Again, no interaction between size at birth and postnatal growth on body fat distribution was present . The present study contributes to current knowledge in showing that excess weight gain relative to length gain in the first 3 months after birth is associated with central adiposity . In addition, these results suggest an increase in intra - abdominal fat as well . Some remarks have to be made . Whistler is a population - based cohort and families with a healthier lifestyle might be more willing to participate . Moreover, children with follow - up measurements tended to have a higher birth weight and were larger at birth (data not shown) compared to those lost to follow - up . Since data on birth size was not available for all children, birth length was imputed using multiple imputation . Missing birth length was associated with lower gestational age and the imputed birth lengths were smaller than those nonimputed . Multiple imputation strengthened the observed associations between postnatal growth and adiposity measures in young childhood, indicating an effect of selection on the results from the complete case analyses . We consider our study population to be representative of a healthy pediatric population, as the mean weights and lengths in infancy are in line with the who growth charts . We derived internal z - scores for weight gain rate conditioned on length gain rate, as references values for comparable z - scores are not available . Since data on fetal growth have not been collected as part of the present study, birth size was used as proxy . Therefore, birth weight adjusted for birth length and gestational age might be a better indicator of intrauterine exposures . Besides age, gender, current height, and sonographer, we did not consider other factors, like parental bmi, infant feeding, tobacco smoke exposure among others as confounders, as these factors might precede the studied association and might therefore be in the causal pathway . Whether or not to adjust for achieved height is topic of discussion [29, 30]. However, when comparing absolute fatness of individuals of different heights, adjustment for height is indicated [19, 20]. Since the ultrasonography was performed blinded to infancy characteristics, the probability of information bias to have occurred is negligible . Additionally, we adjusted for sonographer, to further rule out the possibility of observer bias . Although ct is currently the most accurate method to measure intra - abdominal fat, ultrasonography is a good, child friendly, and practical alternative in population studies, which has been shown to correlate well with results from ct and mri . When the data collection of the present study started, we chose to use ultrasonographically measured intra - abdominal depth to assess visceral fat [26, 27]. More recently, validation studies of this method in children provided conflicting results [32, 33] and another ultrasonographic method for measuring intra - abdominal fat has been described [34, 35] and validated in infants . Although we previously found an association between intra - abdominal depth and vascular characteristics in the 5-year - olds of our study population as well, it should be kept in mind that consensus regarding the best, feasible method for measuring intra - abdominal fat in children, has not yet been reached . Previously, associations between weight gain in the first 2 years of life and childhood bmi [8, 9], waist circumference [8, 12, 13], intra - abdominal fat, and both subcutaneous and preperitoneal fat have been described . In the present study, we focused on excess weight gain relative to length gain the first 3 postnatal months, as the importance of particularly this period in the development of increased waist circumference in adulthood [1517] and childhood was addressed in previous studies . Associations between excess early postnatal weight gain and bmi, waist circumference, and abdominal subcutaneous fat were indeed observed in this study, and some evidence for an association with intra - abdominal fat was found as well . The difference in precision of the ultrasonographic fat measurements might explain why the association with intra - abdominal fat was less pronounced than the association with subcutaneous fat . The observed differences in adiposity associated with excess weight gain in early infancy might be explained by increased accumulation of fat mass in the early postnatal period . Weight gain for length gain in the first months after birth primarily reflects accumulation of fat mass, since fat mass increases from approximately 13% of total body weight to 31% between 0.5 and 36 months of age . According to the developmental origins of human health and disease hypothesis [37, 38] increased postnatal weight gain for length gain might be a reflection of metabolic programming following a period of relative growth impairment . However, in the present study no interaction between postnatal weight gain for length gain and size at birth was observed in the associations with adiposity at 5 years of age . This indicates that the association between increased weight gain for length gain and fat distribution is not only present for those infants who were growth restricted in pregnancy, but also for infants with normal fetal growth . We found some evidence for an association between excess early postnatal weight gain and intra - abdominal fat . Besides these differences in quantity of intra - abdominal fat, the previously observed adverse metabolic consequences later in life associated with increased postnatal growth, adipose tissue dysfunction might be even more important than quantity alone in the development of cvd . Further research is needed to study whether excess weight gain in early infancy leads to changes in adipose tissue functioning in early childhood . While the observed differences in fat distribution are relatively small from an individual perspective, they are relevant from a population perspective . However, we should be careful in extrapolating these finding to growth interventions, as rapid postnatal growth might have benefits as well [3941]. Moreover, to date it remains unknown how to modify infant growth in a way that avoids adverse outcomes . Based on current knowledge, prevention of known causes of impaired fetal growth and rapid postnatal growth, such as maternal smoking during pregnancy, is indicated . In conclusion, variations in postnatal growth are associated with abdominal fat in early childhood . Over the whole range of birth size, excess early postnatal weight gain relative to length gain is associated with increased general and central adiposity, characterized by higher bmi, larger waist circumference, more abdominal subcutaneous fat, and likely more intra - abdominal fat at 5 years of age.
Amoebiasis is the second leading cause of death from parasitic disease worldwide.1 amoebic liver abscess (ala) is the most common extra - intestinal manifestation of entamoeba histolytica infestation, and it is associated with significant morbidity and mortality.2 the common complications of ala include rupture into pleural cavity and peritoneal cavity; rarely, vascular complications are seen in ala in the form of thrombosis or compression resulting in either hepatic venous outflow obstruction or inferior vena cava (ivc) obstruction . There have been only a few case reports of ala with ivc obstruction.3 - 7 we present three cases of ala where the patients presented with signs of ivc obstruction and radiological confirmation of the ivc obstruction by thrombosis and/or external compression . A 24-year old man presented with pain in right upper abdomen with fever for five days . Hematological investigations revealed a hemoglobin (hb) level of 11.2 g / dl and white blood cell (wbc) count of 18,400/mm (normal range, 4,000 - 11,000/mm). The biochemical investigations showed a blood urea nitrogen (bun) of 34 mg / dl (normal range, 15 - 40 mg / dl), serum creatinine of 1.1 mg / dl (normal range, <1 mg / dl). Liver functions tests showed a total bilirubin of 1.5 mg / dl (normal range, <1 mg / dl), serum alkaline phosphatase (alp) of 178 iu / l (normal range, 40 - 280 iu / l (normal range, <40 iu / l), alanine aminotransferase (alt) 48 ultrasound at admission showed a large (8.67.36.2 cm), volume 202 ml, heterogeneously hypoechoic lesion with internal echoes in the caudate lobe suggestive of liver abscess . On ultrasonography, the abscess was compressing the intra - hepatic ivc with abnormal color fill (slowed down) and flow pattern . Magnetic resonance imaging (mri) with magnetic resonance cholangiopancreatography (mrcp) was performed which confirmed 6.96 cm abscess in the caudate lobe causing eccentric compression over the intrahepatic part of ivc (fig . 1, fig . 2). There was a mass effect with compression of the main portal vein and the common hepatic duct just after the ductal confluence, however, the right and left hepatic ducts and intra - hepatic biliary radicals were not dilated . The patient tested positive for amoebic antibody by enzyme - linked immunosorbent assay (elisa) test . After initiating the treatment, there was a gradual amelioration in the fever and abdominal pain . Repeat ultrasound by the end of first week revealed a gradual reduction in the size of the abscess to 50% of its original size and the compression on ivc was also relieved as confirmed by doppler scan . Follow - up investigations showed wbc of 8,800/mm, and liver functions tests showed a total bilirubin of 0.5 mg / dl, serum alp of 145 iu / l, ast 40 iu / l, alt 45 iu / l . Patient was followed up for three months and repeat ultrasound done at the end of three months showed no residual abscess and ivc was normal in caliber, color fill and flow pattern . A 21-year old male presented with upper abdominal pain for 15 days accompanied by a gradually increasing abdominal girth and swelling of the feet with low grade fever for one week . On examination, he was normo - tensive and showed pitting pedal edema . There was a tender hepatomegaly extending three fingers below the costal margin in the mid - clavicular line . The biochemical investigations showed a raised bun of 65 mg / dl and serum creatinine of 1.5 mg / dl . Liver functions tests showed a total bilirubin of 0.6 mg / dl, serum alp of 247 iu / l, ast 40 iu / l and alt 80 iu / l . Ultrasonography of the abdomen showed an abscess in the right lobe of liver on its postero - superior surface measuring 1.411 cm with internal echoes and moderate ascites . Hepatic ivc was seen displaced and compressed by the large abscess cavity but the flow was normal . The doppler findings were suggestive of ivc obstruction due to the liver abscess with proximal dilatation . A contrast enhanced ct scan confirmed the findings of thrombus in the ivc (fig . The liver abscess was aspirated twice under ultrasonic guidance within a period of two days yielding 500 ml and 360 ml of pus . The pus was thick, creamy and brownish in color and did not grow any organisms on culture . Doppler scan repeated four days later showed a decrease in the infra - hepatic ivc dilatation . Hepatic portion of the ivc was normal in caliber and fully patent in subsequent color doppler examination performed twelve days after the aspiration of pus . The biochemical renal parameters improved and two weeks after aspiration of the liver abscess, the bun was 32 mg / dl and serum creatinine was 1.1 mg / dl . Patient was followed up for three months, and repeat ultrasonography done was essentially normal . A 61-year old man presented with high grade fever for eight days, vomiting for five days associated with right upper abdomen pain, yellowish discoloration of eyes for three days, and swelling of the feet for three days . He was icteric with a pulse rate of 96/min and blood pressure of 126/80 mmhg . Ultrasonography revealed multiple abscesses in the liver, the largest one 87.26.8 cm in the right lobe with mild pleural effusion . Hematological investigations revealed a hb of 9.4 gm / dl and wbc 29,900/mm with predominantly polymorphic leukocytosis . The biochemical investigations showed a raised bun of 59 mg / dl, serum creatinine of 1.2 mg / dl . Liver function tests showed total bilirubin of 4.9 mg / dl, direct bilirubin of 3.8 mg / dl, alp of 1,055 iu/ l, ast of 99 iu / l and alt of 108 iu / l . Mri with mrcp revealed four abscesses, two in the right lobe, one in the left lobe and one in the caudate lobe (fig . 4). The largest one was 7.57.27 cm in size, in the right lobe . The abscess in the caudate lobe was compressing the porta hepatis and ivc (fig . Catheter drainage was done from the largest abscess and the patient was started on intravenous metronidazole and broad spectrum antibiotics . After five days of admission the pedal edema reduced markedly and after ten days it subsided completely . Liver function tests returned to normal range with total bilirubin 1.1 mg / dl, direct bilirubin 0.3 mg / dl, alp 22 iu / l, ast 42 iu / l and alt 20 iu / l . The patient was discharged in good condition and followed up for three months and repeat ultrasound done at the end of three months showed no residual abscess and ivc was normal in caliber, color fill and flow pattern . A 24-year old man presented with pain in right upper abdomen with fever for five days . Hematological investigations revealed a hemoglobin (hb) level of 11.2 g / dl and white blood cell (wbc) count of 18,400/mm (normal range, 4,000 - 11,000/mm). The biochemical investigations showed a blood urea nitrogen (bun) of 34 mg / dl (normal range, 15 - 40 mg / dl), serum creatinine of 1.1 mg / dl (normal range, <1 mg / dl). Liver functions tests showed a total bilirubin of 1.5 mg / dl (normal range, <1 mg / dl), serum alkaline phosphatase (alp) of 178 iu / l (normal range, 40 - 280 iu / l (normal range, <40 iu / l), alanine aminotransferase (alt) 48 ultrasound at admission showed a large (8.67.36.2 cm), volume 202 ml, heterogeneously hypoechoic lesion with internal echoes in the caudate lobe suggestive of liver abscess . On ultrasonography, the abscess was compressing the intra - hepatic ivc with abnormal color fill (slowed down) and flow pattern . Magnetic resonance imaging (mri) with magnetic resonance cholangiopancreatography (mrcp) was performed which confirmed 6.96 cm abscess in the caudate lobe causing eccentric compression over the intrahepatic part of ivc (fig . 1, fig . 2). There was a mass effect with compression of the main portal vein and the common hepatic duct just after the ductal confluence, however, the right and left hepatic ducts and intra - hepatic biliary radicals were not dilated . The patient tested positive for amoebic antibody by enzyme - linked immunosorbent assay (elisa) test . After initiating the treatment, there was a gradual amelioration in the fever and abdominal pain . Repeat ultrasound by the end of first week revealed a gradual reduction in the size of the abscess to 50% of its original size and the compression on ivc was also relieved as confirmed by doppler scan . Follow - up investigations showed wbc of 8,800/mm, and liver functions tests showed a total bilirubin of 0.5 mg / dl, serum alp of 145 iu / l, ast 40 iu / l, alt 45 iu / l . Patient was followed up for three months and repeat ultrasound done at the end of three months showed no residual abscess and ivc was normal in caliber, color fill and flow pattern . A 21-year old male presented with upper abdominal pain for 15 days accompanied by a gradually increasing abdominal girth and swelling of the feet with low grade fever for one week . On examination, he was normo - tensive and showed pitting pedal edema . There was a tender hepatomegaly extending three fingers below the costal margin in the mid - clavicular line . The biochemical investigations showed a raised bun of 65 mg / dl and serum creatinine of 1.5 mg / dl . Liver functions tests showed a total bilirubin of 0.6 mg / dl, serum alp of 247 iu / l, ast 40 iu / l and alt 80 iu / l . Ultrasonography of the abdomen showed an abscess in the right lobe of liver on its postero - superior surface measuring 1.411 cm with internal echoes and moderate ascites . Hepatic ivc was seen displaced and compressed by the large abscess cavity but the flow was normal . The doppler findings were suggestive of ivc obstruction due to the liver abscess with proximal dilatation . A contrast enhanced ct scan confirmed the findings of thrombus in the ivc (fig . The liver abscess was aspirated twice under ultrasonic guidance within a period of two days yielding 500 ml and 360 ml of pus . The pus was thick, creamy and brownish in color and did not grow any organisms on culture . Doppler scan repeated four days later showed a decrease in the infra - hepatic ivc dilatation . Hepatic portion of the ivc was normal in caliber and fully patent in subsequent color doppler examination performed twelve days after the aspiration of pus . The biochemical renal parameters improved and two weeks after aspiration of the liver abscess, the bun was 32 mg / dl and serum creatinine was 1.1 mg / dl . Patient was followed up for three months, and repeat ultrasonography done was essentially normal . A 61-year old man presented with high grade fever for eight days, vomiting for five days associated with right upper abdomen pain, yellowish discoloration of eyes for three days, and swelling of the feet for three days . He was icteric with a pulse rate of 96/min and blood pressure of 126/80 mmhg . Ultrasonography revealed multiple abscesses in the liver, the largest one 87.26.8 cm in the right lobe with mild pleural effusion . Hematological investigations revealed a hb of 9.4 gm / dl and wbc 29,900/mm with predominantly polymorphic leukocytosis . The biochemical investigations showed a raised bun of 59 mg / dl, serum creatinine of 1.2 mg / dl . Liver function tests showed total bilirubin of 4.9 mg / dl, direct bilirubin of 3.8 mg / dl, alp of 1,055 iu/ l, ast of 99 iu / l and alt of 108 iu / l . Mri with mrcp revealed four abscesses, two in the right lobe, one in the left lobe and one in the caudate lobe (fig . 4). The largest one was 7.57.27 cm in size, in the right lobe . The abscess in the caudate lobe was compressing the porta hepatis and ivc (fig . Catheter drainage was done from the largest abscess and the patient was started on intravenous metronidazole and broad spectrum antibiotics . After five days of admission the pedal edema reduced markedly and after ten days it subsided completely . Liver function tests returned to normal range with total bilirubin 1.1 mg / dl, direct bilirubin 0.3 mg / dl, alp 22 iu / l, ast 42 iu / l and alt 20 iu / l . The patient was discharged in good condition and followed up for three months and repeat ultrasound done at the end of three months showed no residual abscess and ivc was normal in caliber, color fill and flow pattern . Amoebiasis occurs in 10% of the world's population and is most common in tropical and subtropical regions.1 humans are the principal hosts and reservoir and get infected by ingestion of contaminated food and water containing amoebic cysts.2 ala develops in less than 1% of patients infested with e. histolytica, but this still represents a large number of patients.2 the disease should be suspected in anyone with an appropriate exposure history (residency or travel in an endemic area) and fever, right upper quadrant pain, and substantial hepatic tenderness . The diagnosis of ala relies on identification of space occupying lesion of the liver and positive amoebic serology.1 amoebic serology is highly sensitive (> 94%) and highly specific (> 95%) for the diagnosis of amoebic liver abscess.8 antibodies develop in 90 to 100% of patients with ala and become detectable at seven to ten days after the onset of symptoms . Indirect hemagglutination and enzyme - linked immunosorbent assay are widely used because of their high sensitivity.2 computed tomography is an ideal tool to detect liver abscesses, particularly smaller lesions and its associated complications . Its sensitivity for detecting liver abscess is as high as 97%.9 the rate of various complications described in an ala was reported to be 10.3%.10 complications associated with liver abscess include rupture into pleural, pericardial & peritoneal cavity, vascular thrombosis, and rupture into the bile ducts.11 early diagnosis and prompt treatment are essential to reduce the morbidity and mortality associated with a liver abscess . The mortality rate being low when the abscess is limited to liver but it increases as the abscess gets complicated . Vascular complications of hepatic abscess, such as thrombosis of the portal vein, occlusion of hepatic vein, or inferior vena cava obstruction, are rare and they have been previously described mostly in autopsy studies.12 this was a large study by krishnan et al12 where retrospective analysis of 95 autopsies on ala was done and ivc thrombosis was seen in 8% of the cases in combination with hepatic vein in all but one . Apart from this study, there have been very few case reports in which ivc obstruction has been described in patients with ala.3 - 7 huddle3 reported a case of 26 year old man with ala where it was complicated with extrinsic compression of the ivc by an abscess in the caudate lobe of the liver at the level where it passes through the liver . Hodkinson et al4 reported another case of 50 year old man with ala where the patient developed ivc obstruction due to a thrombus which extended up to the right atrium . Another case was reported by sharma and sarin5 where a 30 year old man presented with ala with signs of hepatic venous outflow obstruction, later found to be due to compression of the ivc by a large and tense abscess in the caudate lobe of the liver . Sodhi et al6 reported a case where amoebic liver abscess in a 57 year old man was complicated by hepatic vein and ivc thrombosis . Schmid et al7 reported another case of a 19 year old man with ala where the patient had signs if ivc obstruction due to external compression of the ivc and not due to thrombosis . In another study by yang et al,11 a retrospective analysis of 81 patients with pyogenic liver abscess, three patients had vascular complications . Of these three patients, two had portal vein thrombosis only and one had extrinsic compression of the ivc associated with portal vein thrombosis . There were only three cases of our report described where the ala has led to extrinsic compression of the ivc in absence of any thrombosis so far . This complication can be managed effectively by catheter drainage or percutaneous needle aspiration combined with antibiotics . In all the three cases of ala with ivc compression mentioned above, percutaneous needle aspiration of the abscess was required to relieve the compression and all the patients were discharged in good condition . In our cases, two patients required percutaneous drainage and in one patient, ivc compression was relieved with antibiotics alone . The management of ala with ivc thrombosis again mainly includes antibiotics and drainage of the abscess, but in few cases, anticoagulation therapy has also been added to achieve complete resolution . We report three cases of ala causing ivc obstruction which completely resolved by the percutaneous drainage and appropriate antibiotic therapy.
In this paper, we specify parasites as eukaryotic pathogens that largely include protozoa and helminths and survive off their host partly or completely for their life cycle . Innate immunity rarely eliminates parasites but can successfully inhibit growth while they recruit antigen - specific t and b cells to differentiate into effector cells that thwart the infection . For an effective parasite survival, evasion of adaptive immunity remains the key . In this scenario, this balance is accomplished by complex alteration of the innate and acquired immune response of the host where regulatory t (tregs) cells play an important role . Understanding the complex cellular and molecular mechanism that regulates the host immune response to parasitic infections still remains a key issue in immunology . The crippling effect of host immunity on onset of an infection is due to the fact that parasites induce tregs that in turn suppress antiparasite effector cells . The primary role of tregs is active suppression of several pathological and physiological immune responses in the host, thereby contributing to the maintenance of immune homeostasis [57]. Although tregs are defined as t cells with suppressive activity on immune responses, it had been documented that regulatory t cell populations remain diverse; a few of them are induced in response to infectious challenge and the others are considered as natural regulators . Parasites can ably manipulate natural tregs by amending the t cell immune response at the infection site to an extent that could lessen the infection burden, thereby prevailing in the host for a longer time frame . The well - characterized tregs are cd4cd25 population and represent about 10% of peripheral cd4 t cells both in mice and humans . Tregs are considered as negative regulators of t cell immune response and these natural tregs originate during thymic development and appear first in the fetal circulation . The suppressor activity is enriched in naturally occurring tregs such as cd4cd25 that plays a vital role in the initiation and orchestration of immune responses [13, 14]. The cd4cd25 population reveals a high expression of foxp3 transcription factor which is vital for differentiation and function of naturally occurring treg cells and for programming the suppressor t cell function [16, 17]. Foxp3 tregs play an essential role in controlling the voracity of the response as they generally strike a balance that limits potentially harmful immune - mediated pathology to the host while still allowing sufficient immune pressure against the pathogen . T - cell receptors remain the key to trigger suppressive function in both naturally occurring and induced tregs . The regulation of t cells is either by contact - dependent regulation or by soluble factors such as immunosuppressive cytokines . To date, no precise mechanism has been clearly postulated to explain the suppressor function exhibited by tregs . Many different hypotheses have demonstrated how tregs are regulated based on the contact - dependent suppressive mechanism . One mechanism is the interaction of t effector ligand cd80 and cd86 with cytotoxic - t - lymphocyte - associated protein (ctla-4). This interaction triggers the transmission of immunosuppressive signals on t effector cells thus inhibiting effector t - cell function (figure 1(a)). Ctla-4 is expressed at high levels on cd4cd25 tregs, and there is substantial evidence that ctla-4 expressed by natural tregs has a key role in treg - mediated suppression both in vivo and in vitro [6, 21, 22]. In another model, the costimulatory molecules cd80 and cd86 expressed in antigen - presenting cells (apcs) interact with ctla-4 leading to consequential signalling and activation of ido (indoleamine 2,3 dioxygenase) in dendritic cells (dcs), an enzyme responsible for immune tolerance on effector t cells (figure 1(b)). Ido catalyzes the conversion of tryptophan to kynurenine that provides immunosuppressive effects in the local environment of dcs by cytotoxicity or by de novo generation of tregs . Studies have reported decreased activation of t cells and t cell deletion in association with reduced tryptophan concentration in cultures [23, 24]. Studies have also demonstrated that human adaptive tregs preferentially expressed granzyme b and are capable of killing allogenic tumour cells in a perforin - dependent manner . In line with these studies, it is demonstrated that both subtypes cd4cd25 tregs exhibit perforin - dependent cytotoxicity against a variety of autologous target cells including cd4, cd8, cd14 monocytes and dendritic cells . In contrast to contact dependent suppressive mechanism, reports indicate that cytokines such as il-10 and transforming growth factor (tgf -) are needed for in vitro mediating suppression [27, 28]. Several in vivo studies have indicated the role of immune suppressive cytokines in suppression . In this model, treg - dependent inhibition of tumor - specific cd8 t cell - mediated cytotoxicity requires expression of the tgf - receptor by cd8 cells thereby demonstrating a specific role of tgf - signaling in the inhibition of cytotoxicity independent of cellular proliferation (figure 1(c)). The il-10 cytokine hampers the antigen presenting ability by downregulating the mhc class ii and costimulatory molecules on dcs thereby preventing the maturation and activation of dendritic cells both in humans and in mice . Tgf - also downregulates the mhc class ii expression and costimulatory molecules on dcs . In addition, in mouse models, a recent study had demonstrated that helminth parasites have evolved a novel mechanism to directly expand foxp3 tregs which may be a key part of the parasite's strategy to survive in the host for a longer time . On infection with intestinal helminth h. polygyrus, the h. polygyrus excretory - secretory antigen (he s) induced tregs and was demonstrated to suppress in vitro effector cell proliferation . The hypothesis proposed that he s ligated the transforming growth factor tgf - receptor and promoted smad2/3 phosphorylation . The foxp3 induction by he s was lost in dominant - negative tgf - rii cells and was eliminated by the tgf - signaling inhibitor . Tregs can reduce injurious host inflammatory and immune responses through mechanisms of cell - to - cell contact, inhibitory cytokines, and cytokine deprivation . This prevents an overexuberant immune response with bystander tissue damage during the host response to infections . However, tregs may also blunt th2 responses such as il-5-dependent eosinophil activation required to kill parasites . The interplay and balance among host th1, th2 and tregs responses is crucial in the defense against a parasitic infection . Some of the earliest studies emphasized that natural tregs help control the extent of immune - mediated pathology . During malarial infection increased numbers of cd4cd25foxp3 t cells have been found in both human and murine malaria infection [35, 36]. Evidences of the role of tregs as suppressors of t - cell responses in malaria was initially demonstrated in murine models, where tregs are known to be associated with increased or delayed parasite growth [37, 38]. Higher tregs numbers are associated with increased parasite load and development of human infection caused by p. falciparum [39, 40]. Given these associations between severe disease and exacerbated immune pathology, a number of studies have explored the role of cd4cd25 foxp3cd127 tregs in determining the outcome of malaria infection . In a study conducted among gambian children with severe, uncomplicated clinical malaria and with healthy (controls), tregs were unable to control the inflammation during acute and severe p. falciparum infections, suggesting that this component may be rapidly overwhelmed by virulent infections . Tregs may be beneficial to the host in the later part of the infection when parasitemia is being cleared by downregulating the inflammatory response and thereby preventing immune - mediated pathology . On the other hand, if tregs mediate their suppressive effects too early, this could hamper the responses required for initial control of parasitemia, permitting unbridled parasite growth, which may also lead to severe disease . It was also demonstrated that patients with acute p. vivax infection presented a significant augmentation of circulating tregs producing anti - inflammatory (il-10 and tgf -) as well as pro - inflammatory (ifn-, il-17) cytokines, which were further positively correlated with circulating parasites . Malaria - specific induction of tregs has been observed in a variety of experimental malaria infections in mice [36, 43], but their role in preventing severe malarial pathology is still unclear . Tregs are widely believed to be involved in silencing the immune response during chronic stages of any filarial infection . Although patients with chronic onchocerciasis (river blindness) posses higher worms burden, they reveal little / no signs of dermatitis . Studies have argued that tr1 (a subset of cd4 t cells) induce a substantial increase in il-10, il-5, and ifn- levels conferring an immunosuppressive effect in chronic onchocerciasis individuals, whereas in animal models studies had demonstrated that a subtle immune response is mediated by female worms [45, 46] and is dependent on tgf- and il-10, two cytokines closely implicated in the activity and induction of tregs [4749]. In litomosoides sigmodontis model, the infective l3 stage induces the proliferation of cd4foxp3 tregs, which translates to an increased percentage of cd4 t cell population at the site of infection expressing foxp3 cells within 7 days after infection . The cd4 regulatory t cell population has also been found significantly higher in several other filarial infections, including brugia malayi, the gastrointestinal nematode heligmosomoides polygyrus [51, 52], and the gut / muscle - dwelling nematode trichinella spiralis . Chagas, a tropical disease caused by trypanosoma cruzi is known to cause cardiomyopathy, an inflammatory response in the heart . The occurrence of larger proportion of foxp3 population has been demonstrated to control the inflammatory responses in the heart during chagas disease . Therefore, foxp3 cells may be involved in a possible mechanism to prevent exacerbation of the inflammatory responses [54, 55]. A similar pattern of tregs role was established with african trypanosomiasis in a mouse models where naturally occurring foxp3 tregs induce il-10 production with subtle ifn - gamma response by cd4 and cd8 effector t cells . In addition, tregs also downregulate classical activation of macrophages resulting in decreased activity of tnf - alpha . The treg activity is believed to decrease the tissue damage in the host cells suggesting a cardinal role for naturally occurring tregs in the development of a tolerant phenotype during african trypanosomiasis . The immune response against t. gondii, an intracellular parasite and the etiological agent of toxoplasmosis, has been largely characterized and demonstrated that cellular immunity plays a vital role in controlling infections . Of which, tregs were demonstrated to modulate the protective immune response against t. gondii, thereby driving a powerful th1 immune response [59, 60]. Also, it is believed that the absence of tregs in toxoplasmosis may induce an uncontrolled inflammatory response . Foxp3-expressing tregs have been implicated in parasite - driven inhibition of host immunity during chronic infection . One of the major effects of chronic helminth infections is induction of t - cell hypo responsiveness . The mechanisms involved have been thought to be multiple and the involvement of natural and inducible tregs in down regulating effector t cell responses upon chronic infection has been proposed . The cytokines il-10 and tgf - have been associated with down regulation, indicating that regulatory populations are activated during infection . The importance of il-10 as a crucial mediator of regulation in parasite infections has been well recognized both in humans and murine [6365]. In human filariasis, heavily infected individuals have high il-10 levels and il-10 messenger rna production which was inversely correlated with t cell proliferation [34, 66]. Similarly infection in experimental schistosoma mansoni was shown to be associated with immunoregulatory mechanisms, including treg that may help control morbidity and dampen resistance to reinfection . Treg responses control both th1 and th2 responses in an il-10 independent manner [67, 68] and are associated with regulation of granuloma formation in chronic infections . In a study conducted among patients infected with s. mansoni in kenya, it was revealed that few patients had higher proportions of cd3/cd4/cd25 tregs that subsequently decreased after treatment with praziquantel . The study concluded that not all schistosoma mansoni - infected individuals develop high percentages of circulating tregs . The effective treatment decreases the proportion of tregs and their phenotypes, possibly because of the removal of constant exposure to antigens from intravascular, egg - producing adult worms . In a nod mice model, treatment with s. mansoni egg antigen (sea) was shown to upregulate tgf - on t cells and th2 cells resulting in the expansion of foxp3 that remain crucial in determining the sea - mediated diabetes outcome . Also, hsp60 peptide (sjmhe1) of s. japonicum induces cd4cd25foxp3 tregs both in vivo and in vitro resulting in subsequent release of il-10 and tgf - . Nematode infections have been shown to induce regulatory cell expansion in both mice and humans . In a study conducted in lima, peru, among human t lymphocyte virus (htlv-1) patients with or without strongyloides infection, increased proportions of cd4cd25foxp3 tregs were observed in patients with s. stercoralis and htlv-1 coinfection in comparison to normal controls . Furthermore, those with increased proportions of cd25foxp3 cells had decreased antigen driven production of il-5 and lower eosinophil counts . This reduced response is inversely correlated with the proportion of cd4 cells, which are cd4cd25foxp3, suggesting a role for these cells in blunting antigen - driven protective responses . Visceral leishmaniasis (vl) represents a parasitic disease that has been shown not to induce expansion of natural tregs . In a study conducted among patients presenting with symptoms of kala - azar, frequencies of foxp3 cells in patient with vl before and after treatment did not increase, neither were they elevated when compared to endemic controls . It was therefore concluded that active vl is not associated with increased frequencies of peripheral foxp3 treg or accumulation at the site of infection . While active vl does not induce expansion of treg, it has been shown in animal models that treg is directly responsible for its re - activation . During the primary infection, l. major can disseminate to other tissues within the body which may persist after parasite reduction and healing [76, 77]. These persistent parasites are associated with the establishment of strong immunity against reinfection, a state that is referred to as concomitant immunity [75, 78, 79]. Tregs have been found abundantly at these reinfection sites while it reduces at the site of initial infection confirming its importance in reactivation of vl infection . Significant increase in il-10 production by dermal and ln cd4 t cells has been shown during the reactivation process confirming a role of il-10 mediated leishmaniasis both in susceptible and resistant individuals [80, 81]. In macaque (macaca mulatta) model, l. braziliensis strain that produces self - healing dermal lesions was used to characterize the systemic and local cell - mediated immune responses that led to controlled growth of granulomas in the infected host . Resolution of infection was observed to be dependent on concomitant recruitment of interleukin- (il-)-10-producing cd4cd25 regulatory t (treg) cells that suppress the effector t - cell - mediated inflammatory response . Parasites exert a selection pressure on their hosts and are accountable for driving diversity within gene families and immune gene polymorphisms in a host population . The pathogen driven selection on immune genes can potentially alter the primed sequence and can direct to substantial changes in gene expression . Genes such as il-2, il-4, il-10, il-13, stat-4, stat-6, gitr, tlrs, and foxp3 are established as key players in regulating tregs . The investigation of human polymorphisms in loci associated with treg activity may underlie both susceptibility to infection and level of treg expression . Many of these polymorphisms evolved and are maintained in a human population exposed to infectious diseases . Genotype associations may predict likely susceptibility and allow identification of subjects at the risk of developing the disease and may be subjected to therapeutic treatment . Investigation of human polymorphisms in immune relevant genes has been used to determine the level of treg expression and thereby the extent of susceptibility to parasitic infection . The single - nucleotide polymorphisms (snps) in the promoter region of the genes such as stat6, foxp3, and tnfrsf18 were well characterized for their functional role [4, 10, 83]. One gene of interest that plays a key role in the function of tregs is the il-10 gene locus . In populations exposed to leishmania braziliensis in bahia, brazil, genetic analysis of the il-10 - 819c / t snp polymorphism, located in the il-10 promoter, showed that the c allele increased the risk of lesions . The il-10 - 819 c / c genotype was associated with higher levels of il-10 than c / t and t / t genotypes demonstrating a vital role for il-10 in skin lesions in humans infected with l. braziliensis . Also, il-10 promoter polymorphism was recently shown to influence nonspecific total ige levels, but not schistosomiasis - specific immunity . In chagas disease low il-10 expression was associated with cardiac function and it was demonstrated that the polymorphic allele, which correlates with lower expression of il-10, was associated with the development of chagas disease cardiomyopathy . Also, in malaria infection it was shown that common il-10 promoter haplotypes condition susceptibility to severe malaria anemia and functional changes in circulating il-10, tnf - alpha, and il-12 levels in children with falciparum malaria . In a study among subjects infected with urinary schistosomiasis in mali, an association was found between stat6 (rs324013) gene polymorphism and infection level in subjects under 20 years while the same study did not observe any association with il-4 and il4r polymorphism . In summary, the field of infectious disease immunology is at an exciting intersection with new concepts in regulation of immune responsiveness . Despite extensive studies, there is still much that remains unclear about the mechanism and activities of treg . A more comprehensive understanding of the mechanisms and gene - expression pathways that underlie the treg activities will be essential if new therapeutic strategies are to be developed . The ability of the treg cells to control many facets of the immune response makes them an interesting model to study possible immune - modulatory intervention.
Study design and enrolled cats: nine domestic short - haired cats living at a research facility were enrolled; mean age was 6.11 1.71 years, and body weight was 3.51 0.42 kg . All cats were selected from a research colony maintained under standard laboratory conditions at the tottori university . None of the cats had received antimicrobials for at least six months prior to the study, and all cats were deemed healthy based on a physical examination and a hematological examination . The cats were randomized into three groups (groups a, b and c) of three cats each . The cats in groups a and b received a daily oral dose of 5 and 2.5 mg orb / kg (ds pharma animal health, osaka, japan), respectively, for 7 consecutive days, as approved dosages in japan . Further, the cats were fed commercial dry cat food and received no medication other than orb during the study . All cats were housed in separate cages located in one room, and direct contact among the cats was prevented during the study . Only when administered with orb, the cats were separately led out of each cage . This study was approved by tottori university animal use committee (approval number, 13-t-29). Isolation and identification of fecal coliforms: stool samples were collected from each of the 9 cats on days 1, 3, 5, 7, 9, 11, 13, 15 and 17 . On day 1, serial 10-fold dilutions were then prepared from 1 g of each stool sample in 0.1% peptone water . Once the appropriate dilution was prepared, 0.1 ml was plated onto eosin methylene blue (emb) agar (nissui pharmaceutical co., ltd ., tokyo, japan) containing either no drugs or orb at the concentration of 2 g / ml, which was defined as the concentration of intermediate susceptibility in to the clinical and laboratory standards institute guidelines . On emb agar, coliform colonies develop a metallic luster, other gram - negative bacteria appear colorless, and gram - positive bacteria cannot grow . The inoculated plates were incubated at 35c for 24 hr, and the number of coliform bacteria was enumerated as colony - forming units (cfu)/g of feces . A maximum of 10 coliform colonies per cat per sampling were picked up and subjected to identification of bacterial species . Isolates were confirmed to be e. coli by gram staining, the typical colony shape on deoxycholate hydrogen sulphide lactose agar (nissui pharmaceutical co., ltd .) And detection of the uid gene by pcr . When isolates were not identified as e. coli, they were identified using the api 20e kit (sysmex biomrieux co., ltd . The isolates were stored in 10% skim milk at 80c for antimicrobial susceptibility testing and genetic analysis . The remaining stool samples were frozen at 80c until high performance liquid chromatography (hplc) analysis . Antimicrobial susceptibility testing: susceptibility testing against orb was conducted using the agar dilution method, according to the clinical and laboratory standards institute guidelines . In addition, susceptibilities to ampicillin (amp), cefazolin (cez), tetracycline (tet), chloramphenicol (chl), kanamycin (kan) and trimethoprim / sulfamethoxazole (sxt) were determined using the disk diffusion method . Cez - resistant isolates, ampc -lactamase and extended - spectrum -lactamase (esbl) were phenotypically screened using cefoxitin disks (30 g / disk) and cefotaxime (3 g), respectively; the results were considered positive, if the inhibition zone diameters were 14 and 27 mm, respectively [10, 28]. Further, ampc - positive isolates were defined as derepressed ampc mutants or inducible ampc producers, as previously described . Analysis of the mechanism of fq resistance: isolates with an orb mic of 1 g / ml were assessed for the presence of mutations of the quinolone resistance - determining region (qrdr) and pmqr determinants . The qrdr of the gyra gene was amplified by pcr with previously described primers . The qrdr of the parc gene was also amplified and sequenced using previously described primers, when qrdr mutations were detected in gyra . Pcr products were then randomly selected and bidirectionally sequenced with the same primers for confirmation . Enterobacterial repetitive intergenic consensus sequence - based pcr: enterobacterial repetitive intergenic consensus sequence - based pcr (eric - pcr) was carried out to investigate epidemiologic relationship among the pmqr - positive isolates . The procedure was slightly modified from the previous studies [27, 31]. Briefly, the pcr reaction was set up in a 20 l reaction volume containing 2 l of a 10 extaq buffer, 1 u of extaq dna polymerase (takara bio inc ., otsu, japan), 0.25 mm each of the dntps, 1030 ng of bacterial dna and 20 pmol of each primer (i.e. Eric1r and eric2). Dna amplifications were performed with an initial denaturation (7 min at 94c) followed by 30 cycles of denaturation (1 min at 94c), annealing (1min at 52c) and extension (8 min at 65c) with a final extension (15 min at 65c). Measurement of orb concentration in feces: stool samples were obtained from cats in groups a and b on days 1, 3, 5, 7 and 9 and from cats in group c on days 1 and 9 . The concentration of orb in feces was determined at the research institute for animal science in biochemistry and toxicology (sagamihara, japan). In brief, orb in stool samples was extracted with acetonitrile containing 1% formic acid and purified via liquid liquid partition, salting - out and a mini - column (oasis max, nihon waters k.k ., orb was analyzed using an hplc system (shimadzu corporation, kyoto, japan) equipped with a binary pump, an autosampler, a column heater and a fluorescence detector . For separation, supelcosil abzplus (sigma aldrich co., llc, tokyo, japan) was concurrently used with lichrocart 4 - 4 lichrosher 100 rp-18 guard column (merck, tokyo, japan). The quality control was carried out by analyzing stool samples together with samples of known concentrations (i.e. 0.2 and 2 g / g) every time . Kramer multiple comparison test was used to compare the mean age and body weight of the enrolled cats, and mean cfu / g of stool samples among groups a, b and c. the mann whitney u test was used to compare mean concentrations of orb in feces between groups a and b. fisher s exact test was used to compare rates of antimicrobial resistance between groups and periods (i.e. Before and after treatment). A p value of <0.05 was considered as statistically significant in all analyses . Enrolled cats: there were no differences in age or body weight among the three groups (p>0.05). Total number of coliforms in cats: the total number of coliforms during the test schedule is shown in fig . 1fig . 1.the means of the total number of coliforms and orb concentration in the feces of cats in groups a, b and c during the test schedule . In groups a and b, orb was administered orally daily at a dose of 5.0 and 2.5 mg / kg, respectively, on days 17 (). See supplemental figs . 1 and 2 for the total number of coliforms and orb concentration, respectively significantly lower number of coliforms in groups a and b, respectively, compared with group c (p<0.05). * there were significant differences between groups a and b (p<0.05). And supplemental fig . 1 . According to the one - way analysis of variance and the tukey kramer multiple comparison test, pre - treatment coliform counts in groups a, b and c were not significantly different (7.08 0.53, 5.39 0.72 and 6.55 1.11, respectively). The means of the total number of coliforms and orb concentration in the feces of cats in groups a, b and c during the test schedule . In groups a and b, orb was administered orally daily at a dose of 5.0 and 2.5 mg / kg, respectively, on days 17 (). See supplemental figs . 1 and 2 for the total number of coliforms and orb concentration, respectively, in each cat . Significantly lower number of coliforms in groups a and b, respectively, compared with group c (p<0.05). * there were significant differences between groups a and b (p<0.05). In groups a and b, within 3 days of orb administration, the number of fecal coliforms decreased rapidly and continued to be significantly lower than that of group c (p<0.05), until day 9 or 7, respectively . After cessation of orb treatment, coliform counts in the cats of groups a and b mostly reached detectable levels by days 9 (cat 3) and 11 (cats 1 and 2) and by days 7 (cat 5) and 9 (cat 6), respectively . There were no significant differences in coliform counts between groups a and c from day 11 (p>0.05). On the other hand, significant differences were found in coliform counts between groups b and c (p<0.05) on days 13, 15 and 17, because of extremely low counts of coliform in cats 4 and 6 . Significant differences between groups a and b were not seen during the study period (p>0.05). No remarkable fluctuations were observed in coliform counts in the cats of group c during the study period . Rates of antimicrobial resistance of coliforms in cats: by antimicrobial susceptibility testing, orb resistance was not detected in groups a and b before the treatment . After the treatment, orb resistance was found in group b (6.0%); however, there was no significant difference in the resistance rates before and after the treatment (p>0.05). On the other hand, compared to before treatment, rates of amp resistance were significantly high in groups a and b after the treatment (0% vs. 26.2% and 0% vs. 76.2%, respectively, p<0.01); rates of cez resistance were also significantly high in these groups after the treatment (0% vs. 26.2% and 0% vs. 76.2%, respectively, p<0.01). There were significant differences in rates of resistance to the two drugs between groups a and b after the treatment (p<0.01). In group c, resistance to any antimicrobials tested was not detected during test period . Changes in the composition of the bacterial population of fecal coliforms: coliform species are shown in table 1table 1.bacterial species of coliforms from stool samples obtained from cats during the test schedulegroupcatdate of sampling (no . Of isolates) 1357911131517a1eccfececec2ececec / cfcfec3ececececec / cfec / cfb4eccf5eccfcfec / cfececcf6eccfcfcfecc7ececececececececec8ececececececececec9ecececececececececa) groups a and b were treated with 5 and 2.5 mg orb / kg, respectively . C) the isolates were also detected using emb agar containing orb (2 g / ml) and exhibited low (mic: 1 g / ml) or intermediate susceptibility to orb (mic: 24 g / ml).. on day 1, all isolates from cats in groups a, b and c were identified as e. coli . A) groups a and b were treated with 5 and 2.5 mg orb / kg, respectively . C) the isolates were also detected using emb agar containing orb (2 g / ml) and exhibited low (mic: 1 g / ml) or intermediate susceptibility to orb (mic: 24 g / ml). After orb treatment, the isolates of citrobacter freundii were detected in all cats in groups a and b. in group a, c. freundii isolates were detected on days 11, 13, 15 and/or 17, whereas in group b, the bacteria were detected on days 7, 9, 11, 13 and/or 17 . Furthermore, e. coli isolates were detected alone or along with c. freundii isolates in all cats except cat 4 (from group b), in whom coliforms were not detected until day 15 after treatment initiation, and only c. freundii isolates were detected on day 17 . Using emb agar containing orb, coliforms were detected with 10 cfu / g in cat 3 (from group a) on day 15 and with 10 cfu / g in cat 4 (from group b) on day 17 after treatment initiation; these isolates were identified as e. coli and c. freundii (cat 3) and c. freundii alone (cat 4). No growth of coliforms was detected in the other cats by using orb - containing emb agar . Coliform bacteria other than e. coli or c. freundii were not identified during the test schedule . Resistance mechanisms in e. coli and c. freundii: as for orb resistance, e. coli isolates were highly susceptible (mic, 0.0310.25 g / ml) in cats 1 and 2 (from group a), and 4, 5 and 6 (from group b) not only before treatment but also after treatment (table 2table 2.antimicrobial susceptibility and fq resistance mechanisms of coliforms from cats before and after orb administrationgroupcatperiodisolatesmic range of orbifloxacin(g / ml)qrdr mutationpmqrsusceptibility to other antimicrobialsspeciesngyraparca1pre - treatmentec100.063susceptiblepost - treatmentec300.0630.125susceptiblecf104noneqnrbamp cez2pre - treatmentec100.0630.125susceptiblepost - treatmentec220.0310.125susceptiblecf184noneqnrbamp cez3pre - treatmentec100.063susceptiblepost - treatmentec50.0310.063susceptible391s83lnonenonesusceptiblecf64noneqnrbamp cezec412s83lnonenonesusceptiblecf64noneqnrbamp cezb4pre - treatmentec100.0630.125susceptiblepost - treatmentcf104noneqnrbamp cezcf104noneqnrbamp cez5pre - treatmentec100.063susceptiblepost - treatmentec90.0630.25susceptiblecf254noneqnrbamp cez6pre - treatmentec100.063susceptiblepost - treatmentec100.0630.125susceptiblecf254noneqnrbamp cez58noneqnrbamp cezc7allec890.0630.125susceptible8allec900.0630.125susceptible9allec870.0630.125susceptiblea) groups a and b were treated with 5 and 2.5 mg orb / kg, respectively . C) the isolates were detected using emb agar containing orb (2 g / ml). D) s83l: codon position 83, serine - to - leucine mutation . ). On the other hand, isolates with low or intermediate susceptibility (mic, 12 g / ml) were predominantly detected in cat 3 (from group 1) after treatment, and these isolates had a single qrdr mutation in gyra at codon position 83 (serine to leucine: s83l). In group c, all e. coli isolates were highly susceptible to orb (mic, 0.0630.125 g / ml) during the test schedule . All c. freundii isolates from cats 15 demonstrated intermediate susceptibility (mic, 4 g / ml), whereas the resistant isolates (mic, 8 g / ml) were barely detectable only in cat 6 on days 9, 11 and 13 . According to pcr and sequencing results, none of the c. freundii isolates had qrdr mutations in gyra, but carried qnrb, one of the pmqr genes . A) groups a and b were treated with 5 and 2.5 mg orb / kg, respectively . C) the isolates were detected using emb agar containing orb (2 g / ml). D) s83l: codon position 83, serine - to - leucine mutation . All e. coli isolates were susceptible to the 5 tested antimicrobials, whereas all c. freundii isolates were resistant to amp and cez . All c. freundii isolates were phenotypically confirmed to be inducible ampc producers, but not esbl producers . Genetic relationship between c. freundii isolates from cats: four c. freundii isolates each from cats 16 (from groups a and b) were selected and subjected to eric - pcr . As the result, all of these isolates had the identical banding pattern (data not shown). Orb concentration in feces of orb - treated cats: the changes in orb concentration in feces are shown in fig . 1 and supplemental fig . Orb was detected in cats of groups a and b between days 3 and 9 after treatment initiation . Highest concentration of orb was determined in cats 2 and 6 of groups a and b, respectively, between days 3 and 7 . The peaks of mean concentration in groups a and b were observed on day 3 (141.0 and 61.3 g / g, respectively). On days 5 and 7, significantly higher concentrations of orb were detected in group a than in group b (p<0.05). On the other hand, orb was not detected in all of cats in group c on days 1 and 9 . To the best of our knowledge, this is the first study to evaluate the effects of oral fq on the fecal flora of cats . We have demonstrated that the counts of coliform bacteria significantly decreased during and after the period for administration of orb . A similar reduction in the number of coliforms was reported in dogs treated with enrofloxacin . Thus, these data indicate that fq administration reduces the number of coliforms in the fecal flora of cats and dogs . As for the composition of the bacterial population of the fecal coliform bacteria, only e. coli was detected in all cats on day 1, whereas isolates of c. freundii and e. coli were detected as a dominant species of all cats in groups a and b after cessation of orb treatment . Few reports have addressed the effect of antimicrobial treatment on the composition of the bacterial population of fecal or gut flora in animals . Johnson et al . Detected the emergence of streptococcus spp . And corynebacterium spp . In cats after the administration of metronidazole . Confirmed the increase in the number of enterococcal cells in dogs after the administration of cefovecin . Although there were several differences in test condition between studies, our and their results suggest that administration of antimicrobials can cause increase, in addition to decrease, of specific bacterial species of fecal or gut flora, which may be explained by microbial substitution . Pharmacokinetics of orb has not yet been evaluated in animals treated with different doses of this antibiotic . In this study, we found clear differences in orb concentrations in feces between the two doses (5.0 and 2.5 mg / kg). This dose - dependent kinetics of orb should be considered when administering this drug . Moreover, our results reveal remarkably high concentrations of orb in feces of cats receiving either the high - dose or low - dose treatment . Thus, orb is likely to be mostly excreted into the feces after oral administration: a notion supported by a previous study, revealing that a higher concentration of orb is present in bile acid than in serum of cats after orb treatment . These pharmacokinetic properties of the drug may be responsible for the significant reduction of fecal coliforms in cats after treatment . Our study showed some variations in the numbers of coliforms and orb concentrations in feces between cats administered with the drug, suggesting that effect of orb treatment on fecal coliforms and pharmacokinetics of the drug may vary by individual . Such interindividual variations should be taken into account when administering the drug for cats . Susceptibility testing revealed that no orb - resistant e. coli appeared because all isolates failed to develop more than one qrdr mutation: the necessary condition for the acquisition of fq resistance ., wherein all fecal e. coli isolates exhibit high - level resistance to enrofloxacin after treatment of dogs with the drug . In general, the concept of a mutant selection window has considerable implications for the acquisition of fq resistance, and antimicrobials at concentrations beyond the mutant prevention concentration (mpc) can prevent the development of fq - resistant mutants . In our study, the fecal orb concentration in cats treated with the doses of 2.5 and 5.0 mg / kg far exceeds the mpc value of e. coli (0.532 g / ml); this result has been confirmed by another study . These findings suggest that oral orb poses a low risk of selection of highly fq - resistant mutants among fecal e. coli isolates in cats because of high gut levels . Compared with e. coli isolates, c. freundii isolates show higher orb mic values . Such low susceptibilities to fqs may give a competitive advantage to c. freundii when administered with orb . Most strains of c. freundii maintained intermediate sensitivity to orb . Among the tested pmqr genes, this finding can be explained by a study by jacoby et al ., revealing that chromosomal qnrb is prevalent in c. freundii isolates . On the other hand, no qrdr mutations of gyra, the basis of the fq resistance mechanism, were identified in c. freundii isolates . Cesaro et al . Reported that qrdr mutations can be more effectively suppressed in qnr - positive e. coil strains than in qnr - negative e. coli strains; this property of qnr may elucidate our present results, namely that qnrb - positive c. freundii isolates fail to acquire strong orb resistance . It should be considered that gut flora of cats can act as a reservoir of qnr - positive bacteria, which are possibly selected as a result of fq use . In coliform - positive samples after orb treatment, qnr - positive c. freundii isolates were detected more frequently in group b (8 of 11 samples) than in group a (5 of 13 samples). This finding implies that low - dose treatment of orb might facilitate the selection of qnr - positive bacteria, compared with high - dose treatment . However, to clarify this point, further large - scale studies would be needed . As for susceptibilities to the antimicrobials other than fqs, all e. coli isolates exhibited susceptibility to all the tested antimicrobials both before and after orb treatment . As a result, multidrug - resistant e. coli isolates, which were reported in dogs after enrofloxacin treatment, were not detected in the present study . On the other hand, all c. freundii isolates exhibited resistance to amp and cez by the production of ampc, but not esbls . This finding resulted in the significant increase of resistance rates to amp and cez in isolates of e. coli and c. freundii after orb treatment . Similarly, the high prevalence of ampc in this bacterial species was previously found in human isolates in a study by kanamori et al . . Our study suggests that fq use poses a risk of coselection of ampc - producing c. freundii isolates in feces of cats . Eric - pcr revealed that c. freundii isolates, which were qnrb - positive and ampc - producing, from cats 16 (from groups a and b) were clonal or genetically identical . This result indicates that c. freundii isolates spread clonally in the cats; however, the cause of the clonal spread could not be identified . One hypothesis is cross - contamination between the cats during the study, despite deliberate efforts to prevent this . Another is that orb treatment might select c. freundii isolates that had spread horizontally among enrolled cats before the study . In either case, more aggressive and effective measures to prevent any transfer of bacteria would be needed for future studies . Firstly, this study was carried out as a pilot study by using a small number of cats, and thus, the present results might be somewhat biased . Secondly, the effect of orb treatment remains to be clarified in cats with clinical signs or household cats, because we used healthy experimental cats in research settings . Fecal or gut flora in cats is composed of a variety of aerobic and anaerobic bacteria, in addition to coliform bacteria . Therefore, this study could not entirely clarify an effect of orb treatment on fecal flora of cats . Nevertheless, we have described the effects of orb administration on fecal coliforms in healthy cats . We revealed alterations in bacterial composition, e.g. Selection of qnrb- and ampc - positive c. freundii isolates, in addition to a decrease in the total number of coliforms . Moreover, we could not detect strongly fq - resistant mutants among isolates of e. coli and c. freundii . Further studies using household cats with and without clinical signs are required to assess clinical and public health implications of the effects of fq use on the fecal flora of cats.
In korea, the incidence rate of prostate cancer was 32.5 per 100,000 during 2012 and has increased annually with the widespread use of prostate - specific antigen (psa) screening and transrectal ultrasound (trus)-guided prostate biopsy . The current trend in trus - guided prostate biopsy involves the use of an extended number of biopsy cores (10- to 12-core biopsy), because this scheme results in a higher cancer detection rate . However, as the number of biopsy cores has increased, many patients have described this procedure as uncomfortable and painful . A randomized trial demonstrated that intrarectal 2% lidocaine gel before prostate biopsy was simple, safe, and effective for providing satisfactory anesthesia during prostate biopsy . In addition, the combination of lidocaine suppository and periprostatic nerve block (ppnb) was effective in controlling pain during trus - guided prostate biopsy . Intravenous sedation with an opioid drug was also shown to reduce the discomfort and pain caused by prostate biopsy . Despite the use of these techniques to reduce pain, many patients still experience a considerable degree of pain during trus - guided prostate biopsy . Some studies have reported that a patient's pain perception during prostate biopsy is related to the patient's position, such as the left lateral decubitus (lld) or lithotomy position, although this is controversial . Homogeneous data with regard to pain perception during trus - guided prostate biopsy are currently sparse, especially in asian populations . In this background, we retrospectively investigated the effect of the patient's position (lld vs. lithotomy position) on pain perception during trus - guided prostate biopsy performed as an extended 12-core biopsy with the intrarectal administration of 2% lidocaine gel . We also analyzed the clinical factors that affected pain during trus - guided prostate biopsy in a korean population . This study was approved by the institutional review board of the yeungnam university medical center (approval no . 2015 - 03 - 028). A total of 128 men who underwent trus - guided prostate biopsy from july 2012 to june 2013 at a single center were included . The indications for biopsy were an abnormal digital rectal examination or an elevated psa level . Exclusion criteria were anal or rectal pathologies, chronic pelvic pain syndrome, the presence of urinary tract infection, a history of prostate biopsy, and contraindication for the lithotomy position . After informed consent was obtained, patients underwent prostate biopsy in the lithotomy position (n=70, wednesday and friday) performed by a qualified urologist in an operation room or prostate biopsy in the lld position (n=58, tuesday and thursday) performed by a qualified radiologist in the radiology department . All patients underwent 12-core biopsy with the same protocol (2 from the base, 2 from the mid lobe, 1 from the apex, and 1 from the transitional zone). With the use of an 18-gauge 20-cm disposable needle, all biopsies were performed with the hitachi high vision 5500 ultrasound machine with the ust-675p prostate probe (hitachi aloka medical ltd ., antibiotic prophylaxis with 250-mg intravenous ciprofloxacin was started on the night before and morning of the procedure . All biopsies were performed by using intrarectal application of 10 ml of 2% lidocaine gel 10 minutes before the procedure . Immediately after the procedure, all patients were asked to complete a questionnaire addressing pain perception that evaluated the level of pain associated with the procedure . Pain was evaluated by using a 10-point visual analogue scale (vas) from 0 to 10 (0=painless, 10=intolerable pain). The clinical variables included age, international prostate symptom score (ipss), psa levels, prostate volume, body mass index (bmi), diabetes mellitus (dm), cancer detection rate, microscopic hematuria, and pyuria . Results were expressed as mean and standard deviation for age, ipss, psa levels, prostate volume, bmi, cancer detection rate, and vas . Statistical analysis was performed by using the spss ver . 17.0 (spss inc ., chi - square test and paired t - test were used for comparison of clinical variables between the lithotomy and lld position . Pearson correlation coefficients and a multiple linear regression model were used for analysis of relationships between clinical variables and vas . This study was approved by the institutional review board of the yeungnam university medical center (approval no . 2015 - 03 - 028). A total of 128 men who underwent trus - guided prostate biopsy from july 2012 to june 2013 at a single center were included . The indications for biopsy were an abnormal digital rectal examination or an elevated psa level . Exclusion criteria were anal or rectal pathologies, chronic pelvic pain syndrome, the presence of urinary tract infection, a history of prostate biopsy, and contraindication for the lithotomy position . After informed consent was obtained, patients underwent prostate biopsy in the lithotomy position (n=70, wednesday and friday) performed by a qualified urologist in an operation room or prostate biopsy in the lld position (n=58, tuesday and thursday) performed by a qualified radiologist in the radiology department . All patients underwent 12-core biopsy with the same protocol (2 from the base, 2 from the mid lobe, 1 from the apex, and 1 from the transitional zone). With the use of an 18-gauge 20-cm disposable needle, all biopsies were performed with the hitachi high vision 5500 ultrasound machine with the ust-675p prostate probe (hitachi aloka medical ltd ., antibiotic prophylaxis with 250-mg intravenous ciprofloxacin was started on the night before and morning of the procedure . All biopsies were performed by using intrarectal application of 10 ml of 2% lidocaine gel 10 minutes before the procedure . Immediately after the procedure, all patients were asked to complete a questionnaire addressing pain perception that evaluated the level of pain associated with the procedure . Pain was evaluated by using a 10-point visual analogue scale (vas) from 0 to 10 (0=painless, 10=intolerable pain). The clinical variables included age, international prostate symptom score (ipss), psa levels, prostate volume, body mass index (bmi), diabetes mellitus (dm), cancer detection rate, microscopic hematuria, and pyuria . Results were expressed as mean and standard deviation for age, ipss, psa levels, prostate volume, bmi, cancer detection rate, and vas . Statistical analysis was performed by using the spss ver . 17.0 (spss inc ., chi - square test and paired t - test were used for comparison of clinical variables between the lithotomy and lld position . Pearson correlation coefficients and a multiple linear regression model were used for analysis of relationships between clinical variables and vas . The patients' mean age and mean psa levels were 66.99.4 years and 15.5716.89 ng / ml, respectively . Similar oncologic outcomes of the biopsies were observed between the lithotomy and the lld positions . The cancer detection rate was 32.8% and 34.4% in the lithotomy and lld positions, respectively (p=0.941). No statistically significant differences related to other clinical variables, including age, bmi, prostate volume, psa level, microscopic hematuria, pyuria, ipss, and dm, were observed between the lithotomy and lld positions . Vas score showed a significant correlation with dm (r=-0.141, p=0.038), psa level (r=0.183, p=0.019), and lithotomy position (r=-0.225, p=0.011) (fig . However, there were no significant correlations with age, microscopic hematuria, pyuria, ipss, prostate cancer, or bmi . Result of the multiple linear regression analysis for the correlation between vas score and clinical variables are shown in table 3 . Vas score showed a significant correlation with lithotomy position (=-0.772, p=0.003) and dm (=-0.803, p=0.033). The final regression equation was as follows: vas=3.351 - 0.772"lithotomy position"-0.803"dm " (r=0.085). Rectal bleeding, hematuria, and hematospermia were observed in the lithotomy and lld positions, all of which resolved within 2 weeks after biopsy without proper treatment . As the overall number of trus - guided prostate biopsies performed for the diagnosis of prostate cancer increases, it is essential for urologists to understand the patient tolerance associated with this procedure . Approximately 65% to 90% of patients report pain of moderate to severe intensity, and 20% do not want to repeat the examination . Pain during trus - guided prostate biopsy is generated by transducer insertion and needle penetration in the rectal wall, prostate capsule, and periprostatic tissue . By focusing on this mechanism, several techniques and agents for the alleviation of pain associated with prostate biopsy have been reported . Among the techniques to relieve pain during trus - guided prostate biopsy, ppnb is the most effective method . In a meta - analysis, compared with no anesthesia, placebo, and intrarectal lidocaine gel injection, ppnb significantly reduced the pain arising from trus - guided prostate biopsy . However, ppnb can have several complications resulting from repeated injections during the biopsy, systemic lidocaine toxicity, urinary incontinence, and degradation of the image resolution as the result of anesthetic injection . In addition, several studies have reported that patients who underwent periprostatic local anesthetic infiltration felt that the pain of probe introduction was as bad as or worse than the needle biopsies themselves . Therefore, other methods to relieve pain during trus - guided prostate biopsy should be considered . In the current study, patients in the lithotomy position experienced less pain during trus - guided prostate biopsy than did those in the lld position . Regarding the anatomy of the pelvic floor, we hypothesized that the lithotomy position may result in release of the external anal sphincter as the pelvic floor muscles are relaxed . Some studies have evaluated the activity of the pelvic floor muscle resting tone in different positions . Resende et al . Conducted a study comparing three different positions (lithotomy, lld, and supine position) to evaluate the myoelectrical activity of the pelvic floor muscle resting tone . The lld position presented a significantly greater myoelectrical signal of pelvic floor resting tone compared with the lithotomy and supine positions . Therefore, we anticipate that pain experienced while undergoing trus - guided prostate biopsy from the insertion of the ultrasound probe may be relieved in the lithotomy position . In addition, we hypothesized that the lithotomy position may be more acceptable than the lld position regarding communication between patients and physicians . Some studies have reported that pain perception during prostate biopsy was related to the level of anxiety . Bruyere et al . Published a study comparing the pain experienced during prostate biopsy in two different positions: lithotomy and lld . The results of that study suggested that the lld position may be less acceptable and more humiliating than the lithotomy position because of less eye contact and less gentle handling than in the lithotomy position . Although the relationship between the level of anxiety and the patient's position during trus - guided prostate biopsy was not analyzed from a psychobehavioral aspect besides the vas score in our study, we anticipate that the lithotomy position may be more acceptable than the lld position because patients in the lithotomy position can see the process of the procedure and the physician's movements . In contrast to our results, kilciler et al . And lodeta and lodeta reported that pain perception during trus - guided prostate biopsy is less profound in patients in the lld position than in the lithotomy position . Those studies suggested that the lithotomy position may be more unpleasant or embarrassing than the lld position in terms of psychological phenomenon, because the lld position is a more relaxed and physiological position typically used for sleep and rest . By using an adjustable stirrup in the operation room, however, we minimized the physical discomfort induced by the lithotomy position during trus - guided prostate biopsy . Also, our study was the first to report that the lithotomy position was a significant independent factor related to pain during trus - guided prostate biopsy, compared with other factors related to pain, such as psa, age, and ipss . Our study also demonstrated that pain scores during trus - guided prostate biopsy are lower in dm patients than in patients without diabetes . Chronic elevation of blood glucose levels leads to damage to blood vessels and results in diabetic neuropathy . We hypothesize that pain scores during trus - guided prostate biopsy are lower in patients with dm because sensation is significantly decreased in these patients . First, because this study was conducted retrospectively in a single center and included a small number of subjects, we did not investigate other questionnaires regarding anxiety and depression related to prostate biopsy . Therefore, additional prospective studies with larger numbers of patients should be conducted to further evaluate this finding . Second, we did not evaluate pain scores at each step, such as insertion of ultrasound probe and penetration of the needle . Nevertheless, to the best of our knowledge, this series was the first report to investigate the effect of the patient's position on pain during trus - guided prostate biopsy in asian men . In addition, it demonstrated that the patient's positioning during trus - guided prostate biopsy is a significant independent factor related to pain and that patients in the lithotomy position experienced less pain than did patients in the lld position . Patients who underwent trus - guided prostate biopsy in the lithotomy position experienced less pain than did those in the lld position, without a significant difference in the prostate cancer detection rate between the two positions . We suggest that the lithotomy position is the proper way to perform trus - guided prostate biopsy to reduce the patient's pain perception, especially in asian men.
It is surprising that a medline search reveals less than 10 articles on topics concerning adult thalassemia . There is a severe resource data crunch at a time when information needs to be readily available to the physicians, the emphasis being on evidence - based medicine . This study aims to evaluate the clinical profile of thalassemia major patients surviving into adulthood so as to orient the physicians and the general practitioners to adult thalassemia patients . The study included all adult patients of thalassemia (> 18 years old) who are attending the adult thalassemia care unit (department of medicine) for their management . The department of pediatrics at our institute, which happens to be a tertiary institute, runs a thalassemia day care unit, wherein about 250 patients regularly receive transfusion and all patients who attain 18 years of age are referred to adult thalassemia care unit (department of medicine) for management, from where these data are being reported . All patients were evaluated clinically and investigated for complete blood counts, serum urea, creatinine, bilirubin (total and direct), alanine and aspartate transaminases, alkaline phosphatase, total protein and albumin, fasting and postprandial plasma glucose, serum ferritin, and thyroid hormone levels . All the patients were receiving packed cell transfusions for at least 15 years with the aim to keep the hemoglobin levels at about 10 g / dl . Unpaired student's t - test was used to ascertain difference in clinical features between male and female thalassemia patients . A total of 19 thalassemia patients were above the age of 18 years and were availing regular transfusion services at fortnightly intervals . As is evident from the table, the height in both the male and female subjects was quite lower than the usual height in indian adults . Only three males and one female had a height exceeding 150 cm, the maximum height achieved by a male subject being 168 cm and for the female 151 cm . The average height in male thalassemics was significantly higher than that in females (unpaired t - test, p=0.023). Despite the fact, the height was below average, the bmi was <18.5 in eight thalassemia patients (42.1%) and only one (5.3%) had a bmi>25 i.e. 25.44 . Clinical features of thalassemia patients nearly half of the patients (47.4%) had undergone splenectomy by the time they reached their adulthood . Interestingly, 72% males had undergone splenectomy as compared to 12.5% female thalassemics (unpaired t - test, p=0.005). Average hemoglobin was similar in both male and female subjects but six (31.6%) had their hemoglobin levels <8 g / dl . Average leukocyte counts were higher in males (unpaired student's t - test, p=0.0058). Moreover, two (18.2%) males and four (50%) females had total leukocyte counts <4000/mm . Surprisingly, no patient had achieved the recommended serum ferritin levels of <1000 ng / ml . In fact, serum urea, creatinine, total proteins, and albumin levels were within the normal range for all patients . Four patients (21.1%) had serum bilirubin levels> 1.5 mg/ dl with a predominant indirect hyperbilirubinemia . One patient was positive for both hepatitis b and c virus infections and three others were only positive for hcv and one other harbored hiv infection . In total five (26.3%) patients were harboring at least one of the three infections . Four (21.1%) of the patients were receiving a combination of two iron - chelating drugs - deferiprone and desferrioxamine, while the remaining were receiving only deferiprone . Average dose of deferiprone being administered to each patient was 958 mg / kg body weight daily in divided doses . Thalassemia is an autosomal recessive disorder and thalassemia major manifests as severe anemia in infancy or childhood and used to be incompatible with life in the absence of regular red cell transfusions . However, hematopoietic stem cell transplantation (hsct) has offered a prospect of cure with a normal life expectancy and quality of life, relief from life - long blood transfusions and its attendant complications . Short of hsct, thalassemia major patients need a two - pronged therapy in the form of transfusion it is only thalassemia minor / trait which may be diagnosed in adulthood and does not carry a great clinical significance being asymptomatic in majority of individuals . However, with increasing number of thalassemia major patients surviving through their adolescence into adulthood, adult physicians should brace themselves to face the challenge of dealing with thalassemia in their adult patients . What adult thalassemics will be like has been presented in this article . In spite of improvements in transfusion practices and availability of effective iron chelators, the life of thalassemia patients the complications relate to inadequate transfusions, inadequate chelation, transfusion - transmitted viral infections, allosensitization, iron overload and its consequences of endocrine, cardiac, hepatic disturbance; iron - chelator - related drug toxicities; osteoporosis and psychosocial problems. [24] lack of proper and trained medical expertise, periodic requirement of safe and adequate packed red cell concentrates high cost of in - line leukocyte - depleting filters and iron - chelator drugs; and poor compliance with blood transfusions, periodic monitoring, and regular chelation pose other major hurdles for the thalassemics . Inadequate transfusions are a rule rather than the exception in india resulting in hypersplenism and greater rates of splenectomy . A total of 31.6% patients had hemoglobin levels <8 g / dl and 47.4% adult thalassemics had undergone splenectomy in the present study . However, this finding could not be corroborated because of lack of similar published studies . The high total leukocyte counts observed in males can be explained as a consequence to splenectomy . The low average height, weight, and the bmi in both males and females are signs of growth failure commonly seen in inadequately chelated beta - thalassemics . Normal serum protein and albumin levels rule out protein energy malnutrition as a cause of growth failure . Serum ferritin levels were high in all patients, a testimony to inadequate chelation in this group . This is despite the fact that all thalassemics were receiving iron chelation, either deferiprone alone or in combination with desferrioxamine . Even the average dose of deferiprone while being used as monotherapy was 95 mg / kg / day (range being 85114 mg / kg / day) against the recommended dose of 75 mg / kg / day in divided doses . In fact, one study citing that deferiprone was not found sufficiently effective at the recommended dose of 75 mg / kg / day, tried a high dose of 100 mg / kg / day for 2 years and found it to be safe and effective . However, in the present study in adult thalassemics, deferiprone is not effectively chelating even at this high dose, so a higher dose may need to be tried or patients shifted to alternative iron chelators like deferasirox . Repeated blood transfusions are associated with the serious risk of transmission of chronic viral infections, which were observed in 26.3% of the cases in the present series . Antibodies to hepatitis c virus were observed in 21% cases and the same figure has been reported from indian thalassemics . Average serum transaminase values are higher than normal in the present study and can be attributable to chronic viral hepatitis . The absence of diabetes, hypothyroidism, cardiac arrhythmias in the present cohort can possibly be explained by the young age (early adulthood), since these are a consequence of iron overload and the prevalence therefore increases with the number of blood transfusions and so with the age . Indeed, one indian study did not report diabetes or impaired glucose tolerance in chronically transfused patients of thalassemia major between 8 and 15 years of age . However, cardiac involvement cannot be entirely ruled out since it requires echocardiography and mri to see for the functional and structural effects of iron overload, respectively . Osteoporosis and osteopenia are extremely frequent in thalassemia affecting from 52% to 96% of patients and the prevalence rises with age . Echocardiography, mri, and dexa scan were not performed for the thalassemia patients in the preceding 1 year and could not be done as part of the study in view of cost constraints . This study presents a profile of the adult thalassemia subjects and provides insight into the problems and complications faced by them despite several medical advances . The onus of dealing with these lies squarely with the adult physician and it is imperative for one and all to be aware of these.
In bioinformatics, the sequence alignment has become one of the most important issues . When the biologists get an unknown sequence, in general they would compare this unknown sequence (denoted as query sequence) with the known database of sequences (denoted as database sequences) to find the similarity scores and then identify the evolutionary relationships among them . Needleman and wunsch proposed a dynamic programming method (abbreviated to nw algorithm) to solve the global alignment problem between two sequences in 1970 . The smith - waterman (abbreviated to sw) algorithm, which was proposed by smith and waterman in 1981, is designed to find the optimal local alignment, and it is enhanced by gotoh in 1982 . Although hirschberg's algorithm can be used for these algorithms above to reduce the memory space requirement, the computing time increases by a factor of two . When the lengths of two sequences are m and n, respectively, the time complexities of both nw and sw algorithms are o(mn), respectively, and their space complexities by adapting hirschberg's algorithm can be both reduced from o(mn) to o(m), where m is assumed to be larger than n. though the nw and sw algorithms guarantee the maximal sensitivity for the alignment, the cost is still expensive, especially for the computation time . Several fast heuristic methods such as fasta and blast [6, 7] have been devised to reduce the computation time at the expense of sensitivity . However, the exponential increase in the number of known sequences has increased the search time for querying against a database . Recently many core architectures, such as fpga [810], cell / bes [1113], and graphics processing units (abbreviated to gpus) [14, 15], have gradually become more popular in bioinformatics . These new architectures make it possible to enhance the performance of sequence alignments by using the parallel computing technologies . The potential advantage of gpu is its thousands of cores, with their total computing power exceeding the architecture with few cpus . Also nvidia released the compute unified device architecture (abbreviated to cuda) model to allow the programmers to use the commonly used programming languages, such as c / c++, to develop the applications . Thus many efforts have been made to accelerate the sw computations by cuda on gpu . In 2008, manavski and valle presented the first sw algorithm by cuda for the protein database search on gpu . The proposed algorithm was enhanced by the gsw algorithm proposed by striemer and akoglu in 2009 . Ligowski and rudnicki also presented another sw algorithm for the protein database search on gpu in 2009 . Liu et al . Proposed cudasw++1.0 and cudasw++2.0 for protein database search in 2009 and 2010, respectively . In cudasw++1.0, they defined the intertask parallelization (abbreviated to ite) and intratask parallelization (abbreviated to itr) techniques for the relationships of task - thread and task - thread block on gpu, respectively . In general, the performance of the ite technique is better than that of the itr technique; however the ite technique requires more memory space and then it is suitable for short sequences . Khajeh - saeed et al . Proposed a cuda - based sw algorithm (abbreviated to cuda - ssca#1) by using the itr technique and multiple gpus in 2010 . In 2011, hasan et al . Proposed a gpu - based sw algorithm (abbreviated to hka algorithm) for the protein database search by using new sequence database organization and several optimizations to reduce the number of memory accesses . Developed new ite and itr kernels of sw algorithm for the protein database search in 2011 . Sandes and de melo proposed cudaling1.0 and cudaling2.0 for comparing two huge genomic sequences on gpu in 2010 and 2011, respectively . Liu et al . Presented cudasw++3.0 in 2013 for the protein database search by coupling the cpu and gpu simd instructions and doing the cpu and gpu computations concurrently . With the development of next - generation sequencing (ngs) techniques, the ngs machines can generate more than 1000 million nucleotide short reads (dna, mrna, and small - rna) of lengths around 30~50 bps or more in a single run . For several ngs applications, such as metagenomics, these short reads will be assembled into possible contigs with lengths of tens to hundreds . These contigs are then used to match several well - known databases, such as ncbi - nt database, in order to classify or filter out these contigs . For coding dna or mrna short reads, they also can be translated to proteins for the proteomic research or be used for the analysis procedure of transcriptome . Now, several databases have more than hundreds of thousands protein (or nucleotide) sequences with lengths of tens to hundreds (or hundreds to tens of thousands). However, the previous works mentioned above are not suitable for the protein database search with a lot of database sequences . One is that most of these works are suitable for short query and database sequences by using the ite technique . The other is that these works all do the sw computations one by one (seen as the brute force search), and the computation time will be large for comparing a query sequence with a lot of database sequences under the limited hardware resources . Therefore, it is important to provide new concepts and procedures for the protein database search problem . There is a possible method to compare a query sequence with a lot of database sequences, called cuda - swf proposed by lee et al . In 2013 . Cuda - swf used the frequency distance filtration scheme (abbreviated to fdfs) in the run - time to filter out the unnecessary alignments, and then the computation time by cuda - swf is improved up to 41% . The fdfs in cuda - swf is to calculate the frequency vectors of query and database sequences on gpu at first; then it calculates the frequency distance for each pair of query and database sequences, and after that the database sequences that need to be compared (denoted as selected database sequences) should be transferred from gpu to cpu . Finally, these selected database sequences are sorted according to their lengths and then retransferred from cpu to gpu in order to do the sw computations . Hence, the computation time by cuda - swf may be large for a lot of database sequences . In this paper, we will propose an efficient sw alignment method, called cuda - swfr, for the protein database search by using the itr technique based on a cpu - gpu collaborative system . In order to avoid the unnecessary alignments, fdfs is also applied to cuda - swfr to enhance the computation performance . For most of bioapplications, the used database can be predownloaded and preprocessed according to the application requirements . Therefore, the frequency vectors of database sequences can be precalculated and then stored in the database . Before doing the sw computations on gpu, fdfs is executed on cpu by calculating the frequency distances for each pair of frequency vectors from a query and database sequences . After that, the query and selected database sequences are transferred from cpu to gpu . The experimental results indicate that cuda - swfr runs 9.6 times and 96 times faster than the cpu - based sw method without and with fdfs, respectively . These results indicated that cuda - swfr is suitable for the protein database search with a lot of database sequences . Section 2 briefly describes the background knowledge of cuda - swfr, including the sw algorithm, the cuda programming model, the frequency vector and frequency distance, and the related works of sw algorithm by cuda on gpu . Section 3 then introduces cuda - swfr consisting of the implementations of fdfs on cpu and the sw computations on gpu . Section 4 gives the experimental results to evaluate cuda - swfr without and with fdfs . The sw algorithm is designed to identify the optimal local alignment between two sequences (query and database sequences). The sw computation needs a substitution matrix, such as a series of blosum or pam matrices, and a gap - penalty function, such as the constant gap penalty or the affine gap penalty . The sw algorithm adopted in cuda - swfr is the same as that used in cuda - ssca#1 with the affine gap penalty . Given two sequences s1 and s2 with lengths l1 and l2, respectively, the sw algorithm computes the similarity score in an alignment matrix h(i, j) of these two sequences ending at positions i and j of sequences s1 and s2, respectively . The alignment matrix h(i, j) is computed according to (1)hi, j = maxmaxhi1,j1+sij,0max0<k <ihik, jgs+kgemax0<k <jhi, jkgs+kge, where 1 i l1, 1 j l2, and sij is the score in a substitution matrix, which is extracted according to a residue at position i in sequence s1 and another residue at position j in sequence s2 . Gs is the gap opening penalty, ge is the gap extension penalty, and k is the number of the extended gaps . The maximum value of alignment matrix h(i, j) indicates the similarity score between two sequences . The dependency of calculating an alignment matrix h(i, j) is shown in figure 1 . As mentioned in the literature, formula (1) is the native concept of the sw algorithm . In order to improve the sw computation, formula (2) is more suitable for the parallel computing and the details of sw algorithm can be found in the literature: (2)ei, j = maxei, j1,hi, j1gsge, fi, j = maxfi1,j, hi1,jgsge, hi, j = maxhi1,j1+sij, ei, j, fi, j,0 . Cuda is an extension of commonly used programming languages, such as c / c++, in which users can write scalable multithreading programs for various applications . In general, the cuda program is implemented in two parts: host and device . The host part is executed by cpu, and the device part is executed by gpu . The function executed on the device part is called a kernel . The kernel can be invoked as a set of concurrently executing threads (abbreviated to tds). These tds are grouped into a hierarchical organization which can be combined into thread blocks (abbreviated to tbs) and grids (abbreviated to gds). A gd is a set of independent tbs, and a tb contains many tds . The size of gd is the number of tbs per gd, and the size of tb is the number of tds per tb . Tds within a tb can communicate through a per - tb shared memory (abbreviated to sm), whereas tds in the different tbs fail to communicate or synchronize directly . Besides sm, five memory types are per - td private local memory (abbreviated to lm), global memory (abbreviated to gm) for data shared by all tbs, texture memory (abbreviated to tm), constant memory (abbreviated to cm), and registers (abbreviated to rg). Of these memory types, cm and tm can be regarded as fast read - only caches; the fastest memories are the register and sm . The gm, lm, tm, and cm are located on the gpu's memory . Besides sm accessed by a single tb and rg only accessed by a single td, the other memory types can be used by all tds . The caches of tm and cm are limited to 8 kb per streaming multiprocessor (abbreviated to sm). In the kepler architecture, sm the cache of tm is designed for tds in order to improve the efficiency of memory access . The fermi and kepler architectures have real configurable l1 per sm and unified l2 caches among sms . Hence, l2 caches can be accessed by gm and each sm can use the l1 caches and sm . The basic processing unit in nvidia's gpu architecture is called the streaming processor (abbreviated to sp). In the fermi and kepler architectures, several sps can be integrated into a sm according to various architectures, such as 32 and 192 sps per sm for the fermi and kepler architectures, respectively . While the program runs the kernel, the device schedules tbs for the execution on the sm . The single instruction multiple thread (abbreviated to simt) scheme refers to tds running on the sm in a small group of 32, called a warp (abbreviated to wp). The frequency vector and frequency distance are proposed by kahveci et al . In 2004, and they are used to the whole genome alignment problem . In the literature, the frequency distance is also used to remove the sequences which are dissimilar between the query and subject sequences before performing the sequence alignment . In 2013, lee et al . Used the frequency vector and frequency distance to remove the unnecessary sw alignments between the query and database sequences . Assuming that a query or database sequence s is composed of n kinds of nucleotides / amino acids (denoted as the alphabet set), the frequency vector (abbreviated to fv) of s is defined as follows:(3)fvs = fs=1,2,3,,n, where n represents the number of nth alphabets appearing in the sequence s. assume that there are two dna sequences u and v; the frequency distance (abbreviated to fd) of these two sequences is defined as follows:(4)fdfu, fv = fufv = auav+tutv+gugv+cucv . The fd is calculated from fvs of sequences u and v, which is the sum of differences for fvs of the alphabet set . For the biological sequences, the edit distance (abbreviated to ed) is a commonly used measurement to represent the difference between two sequences . In other words, a low edit distance means a high similarity score . The relation between fd and ed for sequences u and v is listed as follows:(5)fdfu, fvedu, v . In several bioapplications, a threshold as a lower bound of similarity score for example, in the homology modeling application, the similarity score should be larger than 35% between the template and target sequences . Moreover, in general, the sequence coverage ratio should be larger than 60% between the template and target sequences . Therefore, this threshold could be used as a factor to filter out unnecessary alignments between a query and database sequences . When we want to find the very similar sequences in a database for a query sequence, the value of ed is set to small . If a fd of a pair of query and database sequences is larger than ed, it means that this database sequence may be not similar to the query sequence . It is worth to note that a fd will be influenced by the sequence lengths between query and database sequences . When the length of a database sequence is longer than that of query sequence, a fd of these two sequences may be large due to their length difference . However, the possible subsequences in this database sequence may be similar to the query sequence . Hence, fdfs cannot be applied to the database sequence with a length longer than that of the query sequence in order to avoid the possible false negatives . Recently, many works have been proposed in the past to implement the sw algorithm on gpu . In the following, the brief descriptions of selected works have been made by considering the implementations and performance . In 2006, liu et al . Proposed the hardware implementation of the double affine smith - waterman (dasw) algorithm on a graphics card by using graphic api (opengl + glsl). By only computing alignment scores, dasw achieved 24 m (millions or mega) dp cells per second on single nvidia geforce 7800 gtx gpu card . Liu et al . Also presented an approach for the protein database scanning by using a graphics card (opengl + glsl) to gain a high performance at the low cost . The proposed approach achieved more than 650 m cell updates per second (abbreviated to cups) on single nvidia geforce 7800 gtx gpu card . Moreover, it ran 9 times and 15 times faster than ssearch and osearch, respectively . The above works are both proposed based on gpu by opengl (a gpgpu programming), not cuda . The sw - cuda precomputed a query profile stored in the tm to replace the query sequence and the substitution matrix . In sw - cuda, each td in a gd is used to do a sw computation by using the query profile and a database sequence . Hence, sw - cuda preordered the database sequences according to their lengths in order to balance the computing workload of each td in a tb . The alignment matrix is computed column by column and the similarity score is stored in the lm . The sw - cuda only calculated the similarity score for a sw computation and achieved 1830 mcups (= 1.8 giga - cups) and 3480 mcups (= 3.48 gcups) on single and dual - nvidia geforce 8800 gtx gpu cards, respectively, and it ran 2 to 30 times faster than any previous implementation of sw on gpu . Gsw algorithm pointed out that the design of query profile is not suitable for gpu due to the limited size of tm on gpu . They still used the query sequence and the substitution matrix both stored in the cm to do the sw computations by using the ite technique . They also proposed an efficient function with an ascii code table to access the score in the substitution matrix . The database sequences are stored in the gm and the similarity scores are stored in the sm . The gsw algorithm ran 10 times faster than ssearch on single nvidia tesla c870 gpu card; however, it is slower than farrar's implementation . Another sw algorithm by using the ite technique achieved 7.5 and 14.5 gcups on single and dual - nvidia geforce 9800 gtx gpu cards, respectively . In cudasw++1.0, the ite technique means that each sw computation consisting of a pair of query and database sequences (denoted as a task) is assigned to one td; the itr technique means a task is assigned to one tb . Due to the limited memory on gpu, each td can only process a pair of short sequences in a tb with many tds by using the ite technique . Conversely, a pair of long sequences can be processed by a tb with many tds by using the itr technique . However, the ite technique can achieve better performance than the itr technique due to more tasks concurrently executed on gpu . The alignment matrix is computed according to the diagonal direction in cudasw++1.0 . For the ite technique therefore, a preordered database is also used in cudasw++1.0 as the previous works above . However, these preordered database sequences should be rearranged and then stored in the gm . In cudasw++1.0, a threshold is set to 3072 for the length of database sequence . If the length of database sequence is less than the threshold, the sw computation is done by the ite technique, otherwise by the itr technique . Cudasw++1.0 achieved an average performance of 9.5 gcups and 14.5 gcups on single nvidia geforce gtx 280 gpu card and dual - nvidia geforce gtx 295 gpu card, respectively . However, the lengths of few sequences in the test swiss - prot protein database (release 56.6) are larger than the threshold . Cudasw++2.0 further optimized the performance of cudasw++1.0 based on the simt abstraction of cuda - enabled gpus and achieved 17 gcups and 30 gcups on single nvidia geforce gtx 280 gpu card and dual - nvidia geforce gtx 295 gpu card, respectively . Cudasw++3.0 achieved the maximum performance of 119.0 and 185.6 gcups on single nvidia geforce gtx 680 gpu card and dual - gpu geforce gtx 690 gpu card, respectively . The ssca#1 benchmark was used to evaluate 5 kernels that are various permutations of the sw algorithm, including saving the alignment results or not . In order to save the alignment result of a sw computation, cuda - ssca#1 needs to spend more memory space by using a traceback procedure . The hka algorithm processed the sw computations by using the ite technique and achieved 1.13 times better than cudasw++2.0 in terms of gcups . Hains et al . Found that cudasw++1.0 only achieved 1.5 gcups when comparing the same query and database sequences on single nvidia tesla c1060 gpu card by using the itr technique . The implementation of cuda - swfr can be divided into two parts: fdfs executed on cpu and the sw computations executed on gpu . The details of these two parts are described in the following sections, respectively . In cuda - swfr there are four steps in this procedure: (1) construct the preorder database sequences with fvs, (2) calculate the fv of query sequence, (3) calculate the fds for each pair of query and database sequences, and (4) collect the query and selected database sequences and then they are transferred from cpu to gpu . In the following, step 1 (construct the preorder database sequences with fvs). In the previous works in section 2.4, the database sequences should be preordered according to their lengths due to the ite technique . However, the following calculation of fd will be influenced by the sequence lengths between the query and database sequences as mentioned in section 2.3 . The database sequences are sorted and then stored in a database with their fvs in order to accelerate the computations of step 3 . The computation time of this step is omitted in the experimental test as the previous works in section 2.4 . In the previous works in section 2.4, the database sequences should be preordered according to their lengths due to the ite technique . However, the following calculation of fd will be influenced by the sequence lengths between the query and database sequences as mentioned in section 2.3 . The database sequences are sorted and then stored in a database with their fvs in order to accelerate the computations of step 3 . The computation time of this step is omitted in the experimental test as the previous works in section 2.4 . When the biologists want to use cuda - swfr for a query sequence, the fv of query sequence is calculated in the run - time by using formula (3). When the biologists want to use cuda - swfr for a query sequence, the fv of query sequence is calculated in the run - time by using formula (3). Moreover, step 3 (calculate the fds for each pair of query and database sequences). As mentioned in section 2.3, fdfs cannot be applied to the database sequence with a length longer than that of the query sequence in order to avoid the possible false negative . Hence, when a database sequence has the length longer than that of the query sequence, it needs to be compared in the second part of cuda - swfr and it is not necessary to calculate the fd with the query sequence . When the length of query sequence is short, this way will reduce a lot of time for step 3 . However, when a database sequence has the length shorter than or equal to the query sequences, the fd should be calculated by using formula (4) with the query sequence . When the calculated fd is larger than a threshold of ed, this database sequence is not selected according to formula (5). One is the length of query sequence and the other is the threshold of ed . When the length of query sequence is short, the lengths of most of database sequences may be longer than it, and the number of selected database sequences is large; in other words, the effect of fdfs is small . Similarly, when the threshold of ed is set to large, the fds of most of database sequences may be smaller than this threshold, and then the effect of fdfs is small; see the experimental results in section 4 . As mentioned in section 2.3, fdfs cannot be applied to the database sequence with a length longer than that of the query sequence in order to avoid the possible false negative . Hence, when a database sequence has the length longer than that of the query sequence, it needs to be compared in the second part of cuda - swfr and it is not necessary to calculate the fd with the query sequence . When the length of query sequence is short, this way will reduce a lot of time for step 3 . However, when a database sequence has the length shorter than or equal to the query sequences, the fd should be calculated by using formula (4) with the query sequence . When the calculated fd is larger than a threshold of ed, this database sequence is not selected according to formula (5). One is the length of query sequence and the other is the threshold of ed . When the length of query sequence is short, the lengths of most of database sequences may be longer than it, and the number of selected database sequences is large; in other words, the effect of fdfs is small . Similarly, when the threshold of ed is set to large, the fds of most of database sequences may be smaller than this threshold, and then the effect of fdfs is small; see the experimental results in section 4 . Step 4 (collect the query and selected database sequences and then they are transferred from cpu to gpu). In this step, the fvs of query and selected database sequences and fds of a pair of query and selected database sequences are not needed to be transferred from cpu to gpu . By fdfs, the transmission data (especially for the selected database sequences) can be reduced, and then the transmission time is decreased according to two factors mentioned above . One is to filter out the unnecessary alignments and the other is to reduce the transmission time . Moreover, the time by steps 3 and 4 can be overlapped with the time of sw computations executed on gpu . After this step, the second part of cuda - swfr is used to do the sw computations on gpu . In this step, the fvs of query and selected database sequences and fds of a pair of query and selected database sequences are not needed to be transferred from cpu to gpu . By fdfs, the transmission data (especially for the selected database sequences) can be reduced, and then the transmission time is decreased according to two factors mentioned above one is to filter out the unnecessary alignments and the other is to reduce the transmission time . Moreover, the time by steps 3 and 4 can be overlapped with the time of sw computations executed on gpu . After this step, the second part of cuda - swfr is used to do the sw computations on gpu . After the first part of cuda - swfr, the selected database sequences are stored in the gm and the query sequence is stored in the cm . When the length of query sequence is larger than the size of the cm, it is stored in the gm . In the fermi architecture, it may be fast to access the query sequence in the gm due to the l2 caches . In cuda - swfr, the sw computations are made by using the itr technique, and it means that the database sequences do not need to be rearranged before being stored in the gm . For the sw algorithm, a substitution matrix is needed . Hence, a substitution matrix is also stored in the cm . In order to access the score in a substitution matrix efficiently, the function with an ascii code table presented by striemer and akoglu is also used in cuda - swfr . By the itr technique, all tds in a tb are used to do a sw computation; it is a dynamic programming algorithm . According to the previous works and the simt scheme on gpus, there are eight implementation types of dynamic programming by using the itr technique on gpu . These eight implementation types are synchronous row single thread (srst), synchronous row multiple threads (srmt), asynchronous row single thread (arst), asynchronous row multiple threads (armt), synchronous diagonal single thread (sdst), synchronous diagonal multiple threads (sdmt), asynchronous diagonal single thread (adst), and asynchronous diagonal multiple threads (admt). Cuda - swfr adopts the srmt type of dynamic programming to implement a sw computation by an assignment method, in which one row is assigned to a tb until all rows are assigned . For a sw computation in a tb, according to formulas (1) and (2) and the dependency shown in figure 1, this process can be divided into two stages . In the first stage, the value of each cell (h(i, j)) in a row is calculated according to the upper cell, upper - left cell and 0 . Assume that the length of a row is n and the number of tds in a tb is t; the values of (n / t) cells are calculated by a td, where n is larger than t in general . However, these values are only intermediate values without considering the left cell according to formula (1) and (2)., only a td in a tb is used to correct the value of each cell in a row by considering its left cell . These two stages will be done repeatedly until all rows are assigned to this tb . The highest value among all cells in all rows is the similarity score for a query and a selected database sequence . For a query sequence, all of similarity scores or the best score will be copied from the sm to the gm, and then they could be transferred from gpu to cpu according to the user's requirement . In cuda - swfr, the number of tds in a tb is set to 256 and the number of tbs in a gd is equal to the number of selected database sequences (or partial of them in order to overlap the computation time between fdfs and sw computations). The goal of this paper is to accelerate sw computations for the protein database search by using fdfs based on a cpu - gpu collaborative system . As the previous works [1619] in the past, the alignment quality is not the consideration in this work . Only similarity scores are calculated for each pair of query and database sequences by using the sw algorithm . The query and selected database sequences can be aligned again by using other alignment algorithms or tools with specific substitution matrices in order to obtain the good alignment results or other simulation results, such as the homology modeling . In this work, cuda - swfr is implemented by c+cuda on single nvidia tesla c2050 gpu card, with 448 sps cores and 3 gb gddr3 ram . The host device is intel xeon e5506 cpu of 2.13 ghz with 12 gb ram running ubuntu 10.04 operation system . In order to evaluate the effect of fdfs in cuda - swfr, a test query sequence with a length of 1028, np_001116291, is a human protein sequence downloaded from the ncbi website (http://www.ncbi.nlm.nih.gov/). Three subsequences are extracted randomly from it with the lengths of 256, 512, and 768, respectively . These three subsequences and the original query sequence are used as the query sequences in the following tests . Based on these query sequences, 12 synthetic databases are generated and used in the experimental tests . These 12 synthetic databases can be classified into 4 groups according to the lengths: 256, 512, 768, and 1028 . For each group, there are three synthetic databases with 10000, 20000, and 40000 database sequences . For each database sequence in a group, it is generated according to the corresponding query sequence with the same length . For each synthetic database, it has database sequences with 0%~100% mutations of its corresponding query sequence . For example, assume that a synthetic database has 40000 database sequences and the length is 1028 . There are 4000 database sequences with 0%~10% mutations of query sequence, 4000 database sequences with 10%~20% mutations of query sequence, and so on . When we want to generate one database sequence with 0%~10% mutations of query sequence, a number t is randomly selected between 0 (1028 0%) and 102 (1028 10%). If t is 57, it means that 57 positions in the query sequence are randomly selected at first, and then the residues in these 57 positions are changed to others by adding a constant (also randomly selected) with its original ascii code . This work is done repeatedly until 40000 database sequences are generated for this synthetic database . In the following tests, if a threshold is set to 90%, it means that the selected database sequence may be very similar to the query sequence . Figure 3 shows the overall computation time (unit: second) by cuda - swfr with various thresholds for 12 synthetic databases . The threshold none means the sw computations by cuda - swfr without fdfs . From figure 3, the computation time decreases when the threshold increases for 12 synthetic databases . When the threshold is set to 90%, the computation time by cuda - swfr is fastest for all of 12 synthetic databases . From figure 3, it also shows that the computation time increases when the sequence length or the number of database sequences increases . In order to observe the relationship between the threshold and the number of selected database sequences, figure 4 shows the ratio (%) by dividing the number of selected database sequences by the number of original database sequences for 12 synthetic databases with various thresholds . From figure 4, the ratio decreases when the threshold increases for 12 synthetic databases . When the threshold is 90%, in figure 4, only 10% database sequences are selected for the sw computations on gpu for 12 synthetic databases . These results explain why the computation time by cuda - swfr is fastest when the threshold is set to 90% . From figure 4, it also shows that the ratio is not influenced by the number of database sequences for 12 synthetic databases . Besides, when the length of query sequence is equal to that of database sequences, the ratio is also not influenced by the sequence length for 12 synthetic databases . However, the ratio will be influenced by the sequence length when the length of query sequence is not equal to that of database sequences . As mentioned in section 2.3, when the length of database sequence is larger than that of query sequence, this database sequence should be compared with the query sequence . Hence, when database sequences have various lengths, for a query sequence, the ratio will increase when the number of database sequences, each having a length larger than that of query sequence, increases . Figure 5 shows the overall speedups by comparing cuda - swfr with the cpu - based sw method under various thresholds for 12 synthetic databases . From figure 5, the best speedup by cuda - swfr with fdfs achieved 96 times . From figure 5, the speedup increases when the threshold increases for 12 synthetic databases . From figure 5, it also shows that the speedup is not influenced by the number of database sequences for 12 synthetic databases . Besides, when the length of query sequence is equal to that of database sequences, the speedup is also not influenced by the sequence length for 12 synthetic databases . The observations in figure 5 match those of figures 3 and 4 . Unlike the previous works that use the gcups to evaluate the performance, cuda - swfr only considered the computation time and the speedups . First, the overall computation workload (dp cells) by cuda - swfr is difficult to be estimated due to filtering out the unnecessary alignments by fdfs . Second, the overall computation time by cuda - swfr consisted of fv and fd calculations (steps 2 and 3 in section 3.1), sw computations (section 3.2), and transmission time (step 4 in section 3.1 and section 3.2). Cuda - swfr achieved the peak performance of 2.133 gcups by using the itr technique . When the length of query sequence is less than the length of database sequences, figure 6 shows the overall speedup by comparing cuda - swfr with the cpu - based sw method for various combinations . The query sequence of length 256 was used to compare with the synthetic database of lengths 512, 768, and 1028, respectively; the query sequence of length 512 was used to compare with the synthetic database of lengths 768 and 1028, respectively; the query sequence of length 768 was used to compare with the synthetic database of length 1028 . Since the length of database sequence is larger than that of query sequence, all of database sequences should be compared with the query sequence . These results are similar to those (8.5~9.6 times) by cuda - swfr without fdfs as shown in figure 3 . From figure 6, the reason is that the overall computation workload increases when the length of database sequences increases . In this work, we did not use the real biological sequences to evaluate the effect of fdfs . The first one is the lengths of real biological sequences are various . In order to avoid the possible false negative, fdfs cannot be applied to the database sequence with a length longer than that of the query sequence . Hence, it is hard to choose the suitable set of real biological sequences and then to prove the relationships of ratio - threshold and ratio - speedup on gpu . The second one is the range of similarity scores among real biological sequences is large . In practice however, in the experimental tests, it is hard to choose the suitable set of real biological sequences to prove the relationships of threshold - speedup on gpu . In this paper, we presented a gpu - based sw alignment method, cuda - swfr, with fdfs by using the itr technique . The fdfs is executed by the cpu capability to filter out the unnecessary alignments and the sw computations are made by the gpu capability . From the experimental tests, cuda - swfr ran 9.6 times faster than the cpu - based sw method without fdfs; it ran 96 times faster than the cpu - based sw method with fdfs . The effect by fdfs is influenced greatly by the threshold . When the threshold as the sequence identity is large, the number of selected database sequences is small which means that the effect by fdfs is visible . In other words,, the effect by fdfs also could be influenced by the number of database sequences; each has a length larger than that of query sequence . Cuda - swfr is suitable for the protein database search with a lot of database sequences, and the proposed fdfs can be integrated into other research works, such as huang et al . And feng et al ., in recent years.

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