Simultaneous determination of emtricitabine, tenofovir alafenamide fumarate and dolutegravir sodium by validated stability-indicating RP-HPLC-DAD method.

OBJECTIVE: The purpose of this research was to develop and validate a stability-indicating RP-HPLC technique for simultaneous quantification of Emtricitabine (EMT), Tenofovir Alafenamide Fumarate (TEN), and Dolutegravir Sodium (DOL) in bulk and in their combined formulation. MATERIAL AND METHODS: The developed approach was done on Exterra C18 column (150×4.6mm, 5µm) and Methanol and Buffer (comprising 0.1 (v/v) of Triethylamine and o-phosphoric acid in water, pH 2.6) as mobile phase in the proportion of 75:25 (v/v), eluted at 1mL/min. The analytes were quantified using DAD detector at 265nm. RESULTS: The approach was validated in accordance with the ICH guidelines. Linearity, precision, accuracy, specificity, Limit of Detection (LOD), Limit of Quantitation (LOQ), and robustness were used to validate the proposed method. Linear response was found in the range of 500-1500µg/mL for EMT, 62.5-187.5µg/mL for TEN and 125-375µg/mL for DOL. The LOD values of EMT, TEN and DOL were found 91.78µg/mL, 10.47µg/mL and 19.28µg/mL correspondingly. The LOQ values of EMT, TEN and DOL were found and 278.11µg/mL, 31.74µg/mL and 58.42µg/mL correspondingly. The assay outcomes for all drugs were observed between 99.11-100.84%. To access the method's stability indicating capabilities, the drugs were exposed to various environmental (acid, alkaline, neutral, oxidative, photolytic and thermal) conditions. CONCLUSION: The established approach was considered to be accurate, linear, precise, specific, robust and it can be utilized to analyse the drugs mentioned in its tablet.

Chronic norovirus infection in primary immune deficiency disorders: an international case series.

OBJECTIVE: Predictive factors associated with clinical outcomes of chronic norovirus infection (CNI) in primary immunodeficiency diseases (PIDD) are lacking. METHOD: We sought to characterize CNI using a multi-institutional cohort of patients with PIDD and CNI using the Clinical Immunology Society's CIS-PIDD Listserv e-mail group. RESULTS: Thirty-four subjects (21 males and 13 females) were reported from centers across North America, Europe, and Asia. All subjects were receiving high doses (median IgG dose: 1200 mg/kg/month) of supplemental immunoglobulin therapy. Fifty-three percent had a complete absence of B cells (median B-cell count 0; range 0-139 cells/µL). Common Variable Immune Deficiency (CVID) subjects manifested a unique phenotype with B-cell lymphopenia, non O+ blood type, and villous atrophy (logistic regression model, P = 0.01). Five subjects died, all of whom had no evidence of villous atrophy. CONCLUSION: While Norovirus (NoV) is thought to replicate in B cells, in this PIDD cohort of CNI, B-cell lymphopenia was common, indicating that the presence of B lymphocytes is not essential for CNI.

Gender and age differences in cardiovascular complications in anorexia nervosa patients.

BACKGROUND: Anorexia nervosa (AN) is characterized by severe malnutrition and electrolyte imbalances. Differences in cardiovascular complications between males and females with AN have not been studied. Additionally, coronary artery disease (CAD) prevalence and cardiovascular complications in patients with AN >65years have not been reported. METHODS: AN patients>18years were identified in the Nationwide Inpatient Sample 2009-2010 database using the Ninth Revision of International Classification of Disease code 307.1. Demographics, risk factors, and cardiovascular event rates were compared to general population data. RESULTS: CAD incidence was lower in all AN patients compared to the general population (4.4% vs 18.4%, p<0.001). Despite AN males having higher prevalences of hypertension and diabetes than AN females, there was no difference in CAD. AN males had higher rates of cardiac arrests, arrhythmias, and heart failure. Interestingly, 4.3% of patients with AN were >65years old. When compared to the general population>65years, older AN patients had lower rates of CAD (35% vs 16%; p<0.001). Older AN patients still had higher CAD rates compared to the general population under 65 (8.6%) and AN patients under 65 (4%; p<0.001). Older AN patients had a lower incidence of heart failure (24% vs 16%; p=0.04), and a trend towards less arrhythmias (30% vs 21%; p=0.08). CONCLUSIONS: Our data suggests that male AN patients experienced more cardiac arrests, arrhythmias, and heart failure than female AN patients. Additionally, older AN patients have a decreased incidence of CAD and heart failure compared to the general population>65years old.

Primary hyperparathyroidism predicts hypertension: Results from the National Inpatient Sample.

INTRODUCTION: Primary hyperparathyroidism (pHPT), most commonly caused by solitary parathyroid adenomas, leads to mobilization of calcium and is known to result in nephrolithiasis and osteoporosis. To date, studies of pHPT and cardiovascular risk factors and events have produced discrepant findings, likely due to small sample sizes and enrolling populations with varying disease severity. HYPOTHESIS: We utilized a national registry, hypothesizing an association between pHPT and cardiovascular risk factors and events. METHODS: Patients >18years with a diagnosis of pHPT were identified in the Nationwide Inpatient Sample 2009-2010 database using the Ninth Revision of International Classification of Diseases code 252.01. Demographics, risk factors, and cardiovascular event rates were collected and compared to general population data. RESULTS: pHPT was present in 0.1% (n=37,922) of hospital admissions. There was a significant increase in the prevalence of most cardiac risk factors including hypertension (HTN), diabetes mellitus, hyperlipidemia, obesity, and chronic kidney disease. The rates of heart failure (HF) and coronary artery disease (CAD) were higher in the pHPT population. However, after performing multivariate regression for age and cardiac risk factors, pHPT did not independently predict HF or CAD. The risk of HTN, however, was independently predicted by pHPT (OR 1.3; p<0.001). CONCLUSIONS: Primary hyperparathyroidism independently predicted the risk of hypertension in a patient population from a large national database. Despite significant differences in univariate analysis of cardiac risk factors and events, pHPT did not independently predict risk of HF or CAD after multivariate regression analysis. Future studies should explore potential mechanisms relating hypertension to pHPT.

Successful haploidentical donor hematopoietic stem cell transplant and restoration of STAT3 function in an adolescent with autosomal dominant hyper-IgE syndrome.

PURPOSE: Autosomal dominant hyper-IgE syndrome (AD-HIES), caused by mutations in Signal Transducer and Activator of Transcription 3 (STAT3) is associated with defective STAT3 signaling and Th17 differentiation and recurrent bacterial and fungal infections. Most patients suffer significant morbidity and premature mortality. Hematopoietic stem cell transplantation (HSCT) has been reported in a small number of cases, with mixed outcomes. We report successful haploidentical donor HSCT in a patient with AD-HIES. METHODS: Evaluation of lymphocyte subsets, STAT3 signaling, and Th17 cells was performed pre- and post-HSCT. RESULTS: A 14-year old female with AD-HIES developed recurrent methicillin-resistant Staphylococcus aureus (MRSA) abscesses. Immunologic analysis showed elevated IgE (4331 kU/L), absent Th17 cells, and markedly decreased STAT3 phosphorylation in cytokine stimulated peripheral blood mononuclear cells. She had breakthrough abscesses despite clindamycin and trimethoprim-sulfamethoxazole prophylaxis, and developed steroid refractory autoimmune hemolytic anemia. She underwent T-cell depleted haploidentical HSCT from her father following reduced intensity conditioning. She developed one MRSA hand abscess after transplant. Twenty-four months post transplant, she had complete donor chimerism (>95 % donor), normal absolute T cell numbers, and a normal percentage of Th17 cells. IgE was normal at 25 kU/L. She remains well 42 months after transplantation off all antibacterial prophylaxis. CONCLUSIONS: Haploidentical HSCT led to successful bone marrow engraftment, normalization of STAT3 signaling in hematopoietic cells, normalization of IgE, and restoration of immune function in this patient with AD-HIES.

Laser fluorescence angiography reveals perfusion defects in retrograde cardioplegia.

BACKGROUND: Adequate perfusion of the right ventricle with retrograde cardioplegia has always been questioned. However, clinical studies suggested sufficient protection and, up to now, intraoperative assessment of cardioplegia distribution has been difficult. METHODS: As a pilot study in 14 patients, we used indocyanine green laser fluorescence angiography (ICGLA) to assess vascular and myocardial perfusion of different areas of the right anterior ventricular wall. Regions of interest were analyzed quantitatively using a new software package. RESULTS: ICGLA allowed rapid and reliable visualization of cardioplegic flow and distribution. Retrograde cardioplegia revealed perfusion defects in the territory of the right anterior cardiac veins when compared to antegrade delivery and to areas close to the left anterior descending vein(s), confirmed by quantitative analyses of maximal fluorescence intensity. Five patients were excluded from quantitative analyses. The learning curve, pitfalls, limitations and special image details are described. CONCLUSION: A larger study is necessary to examine the relevance of perfusion defects to metabolic changes in affected myocytes and to global right ventricular function.

Development of tics in a thirteen-year-old male following atomoxetine use.

Tics and Tourette syndrome are common comorbidities of patients diagnosed with attention-deficit/hyperactivity disorder (ADHD). One of the mainstay pharmacologic therapies for ADHD has been stimulants. However, this class of drugs has been associated with tic exacerbations, thus limiting their utility in this patients subgroup. Atomoxetine has been explored as an alternative treatment as one of the few non-stimulants available to treat ADHD. Early data identifies atomoxetine's influence on Tourette symptomatology to be not merely equivocal but potentially suppressive in the manifestation of tics. There are, however, case studies describing patients experiencing recurrences of tics following treatment with atomoxetine. We present a unique case of a patient, without any prior history of a movement disorder, who developed tics following a single dose of atomoxetine that did not improve until interventional therapy was initiated.

Comparison of vasa vasorum after intravascular stent placement with sirolimis drug-eluting and bare metal stents.

The cytostatic drug, sirolimis has shown prevention in neointimal hyperplasia after stent placement. Recent studies have shown persistent inflammation seen with drug-eluting stents (DES) may result in late stent thrombosis. The aim of this study is to compare effects of bare metal stents (BMS) and sirolimis DES on the neointima and vasa vasorum in stented rabbit aortas. Stents were implanted in eight New Zealand rabbits for 9 weeks. Group I rabbits received BMS. Group II rabbits received sirolimis DES. A balloon-mounted BMS or DES was placed in the infrarenal aorta. Following euthanasia, aortas were perfused with barium sulfate and sectioned for histology. After 9 weeks the qualitative intrastent luminal diameter was fairly uniform in both the DES and the BMS. The thickness of neointima was similar in both groups. The number of vasa vasorum in the sirolimis DES increased compared with the BMS (P < 0.05). An increased number of vasa vasorum produced by the DES when compared with the BMS shows a difference in response to local vessel injury in rabbits. This result suggests that vasa vasorum may play a role in the persistent inflammation generated by sirolimis-coated stents.

The effect off-pump coronary artery bypass surgery on in-hospital mortality and morbidity.

OBJECTIVE: Off-pump coronary artery bypass (OPCAB) surgery is being increasingly reported to show better outcomes compared to conventional on bypass grafting. We examined the effect of OPCAB on in-hospital mortality and morbidity, while adjusting for patient and disease characteristics, in four institutions in the North West of England. METHODS: Between April 1997 and March 2001, 10,941 consecutive patients underwent isolated coronary artery bypass surgery at these four institutions. Of these, 7.7% were performed off-pump. We used logistic regression to examine the effect of OPCAB on in-hospital mortality and morbidity after adjusting for potentially confounding variables. RESULTS: The crude odds ratio (OR) for death (off-pump versus on-pump coronary bypass grafting) was 0.48 (95% confidence interval, CI 0.26-0.92; P=0.023). After adjustment for all major risk factors, the OR for death was 0.59 (95% CI 0.31-1.12; P=0.105). Off-pump patients had a substantially reduced risk of post-operative stroke (0.6 versus 2.3%, respectively; adjusted OR 0.26 (95% CI 0.09-0.70; P=0.008) and a significant reduction in post-operative hospital stay. Other morbidity outcomes were similar in both groups. CONCLUSIONS: Off-pump coronary artery bypass incurs no increased risk of in-hospital mortality. In contrast, there is a significant reduction in morbidity in patients undergoing off-pump coronary bypass grafting when compared to that performed on cardiopulmonary bypass.

Practitioner versus medication-expert opinion on psychiatric pharmacotherapy of mentally retarded patients with mental disorders.

The agreement of practicing psychiatrists with medication experts regarding how psychotropic drugs should be used to treat behavioral and psychiatric problems in patients with mental retardation was studied. The medication survey used in developing guidelines on the treatment of behavioral and psychiatric problems in mental retardation was sent to 85 psychiatrists who had been identified as caring for the mentally retarded in the Texas public mental health system. The comparison of these practitioners with the medication experts included first-line and second-line treatment choices. Survey analysis was based on using 95% confidence intervals (CIs) to determine the type of rating. If the 95% CIs for the practitioners' responses overlapped the 95% CIs for experts, the two groups were judged to be in agreement. Thirty-seven practitioners (43.5%) completed and returned the survey. Few differences between the practitioners and the medication experts were found with respect to treatments for specific mental illness diagnoses. However, the practitioners rated venlafaxine and mirtazapine higher than the medication experts. Lithium augmentation of therapy with selective serotonin-reuptake inhibitors for nonpsychotic depression was rated first-line by the practitioners and second-line by the medication experts. Practicing psychiatrists and medication experts generally agreed about the use of psychotropic drugs for mental illness in patients with mental retardation.

Post-cardiotomy mechanical circulatory support using a conventional bypass circuit in children.

OBJECTIVE: Mechanical circulatory support (MCS) is increasingly used after cardiotomy in children when conventional medical treatment fails. Poor overall survival and long-term outcome have been reported. We report our experience of post-cardiotomy MCS using a conventional bypass circuit. METHODS: Over a 4 year and 6 month period 10 patients, with a median age of 16 days (range 5 days to 16 years) required MCS. Eight patients required support for failure to wean from cardiopulmonary bypass during primary correction. Two patients required support for cardiac arrest or poor cardiac output in the postoperative period. RESULTS: The median duration of support was 43 h (range 26-146 h). Seven hospital survivors were alive and well at median follow-up of 18 months (range 4-36 months). One patient could not be weaned off support. Two more patients died after successful weaning from support. Complications in nine patients who could be weaned off support included renal failure (n=6), cerebrovascular events (n=3) and mediastinitis (n=2). CONCLUSIONS: Overall long-term survival (70%) and quality of recovery is usually good even though initial mortality and complication rates may be high. We think that post cardiotomy mechanical circulatory bypass using a conventional bypass circuit can offer a favourable outcome to selected patients.

Central hyperventilation related to administration of topiramate.

Topiramate is a recently released antiepileptic agent used in the treatment of patients with refractory seizure disorders. In addition to its antiepileptogenic activities, it results in inhibition of carbonic anhydrase isoenzymes II and IV, which are present in the central nervous system. A 15-year-old female who presented with hyperpnea and primary respiratory alkalosis is reported. Other possible etiologies of the central hyperventilation syndrome were excluded. The problem resolved within 24 hours after discontinuing topiramate.

Two types of interneurons in the cat visual thalamus are distinguished by morphology, synaptic connections, and nitric oxide synthase content.

The distribution of the neuronal form of the nitric oxide-synthesizing enzyme, brain nitric oxide synthase (BNOS), was examined in the cat thalamus by using immunocytochemical techniques. BNOS was found in both cells and fibers throughout the visual thalamus. BNOS-stained cells were found consistently in the C laminae of the lateral geniculate nucleus (LGN), the pulvinar nucleus, and the lateral posterior nucleus (LP). In the A laminae of the LGN, variable numbers of BNOS-stained cells also could be detected. BNOS-stained cells were identified as a subset of interneurons because they all stained for glutamic acid decarboxylase (GAD), but not all GAD-stained cells contained BNOS. The average soma area of BNOS-stained cells was slightly greater than the average soma area of GAD-stained cells. BNOS-stained cells display a distinctive dendritic morphology, which is consistent with previous descriptions of class V neurons (Updyke [1979] J. Comp. Neurol. 186:603-619); they have widespread but fairly sparse arbors of thin, somewhat beaded dendrites. BNOS-stained cells participate in a distinct synaptic circuitry. Although many GAD-stained profiles are filled with vesicles and participate in complex synaptic arrangements, known as glomeruli, BNOS-stained dendrites contain small clusters of vesicles and form dendrodendritic contacts in the extraglomerular neuropil. Thus, there appear to be at least two types of gamma-aminobutyric acidergic interneurons in the visual thalamus of the cat. Interneurons that do not contain BNOS (class III morphology) may exert their effects primarily within synaptic glomeruli (Hamos et al. [1985] Nature 317:618-621), whereas interneurons that contain BNOS (class V morphology) contribute primarily to the extraglomerular neuropil.

Synaptic targets of cholinergic terminals in the cat lateral posterior nucleus.

We examined profiles in the neuropil of the lateral division of the lateral posterior (LP) nucleus of the cat stained with antibodies against choline acetyl transferase (ChAT) or gamma-aminobutyric acid (GABA), and several differences in the synaptic circuitry of the lateral LP nucleus compared with the pulvinar nucleus and lateral geniculate nucleus (LGN) were identified. In the lateral LP nucleus, there are fewer glomerular arrangements, fewer GABAergic terminals, and fewer cholinergic terminals. Correspondingly, the neuropil of the lateral LP nucleus appears to be composed of a higher percentage of small type I cortical terminals (RS profiles). Similar to the pulvinar nucleus and the LGN, the cholinergic terminals present in the lateral LP nucleus contact both GABA-negative profiles (thalamocortical cells; 74%) and GABA-positive profiles (interneurons; 26%). However, in contrast to the pulvinar nucleus and the LGN, the majority of cholinergic terminals in the lateral LP nucleus contact small-caliber dendritic shafts outside of glomeruli (60 of 82; 73%). Consequently, most cholinergic terminals are in close proximity to RS profiles. Therefore, whereas the cholinergic input to the LGN and pulvinar nucleus appears to be positioned to selectively influence the response of thalamocortical cells to terminals that innervate glomeruli (retinal terminals or large type II cortical terminals), the cholinergic input to the lateral LP nucleus may function primarily in the modulation of responses to terminals that innervate distal dendrites (small type I cortical terminals).

Calibration of the Periotron 8000 and 6000 by polynomial regression.

This paper reports the detailed calibration of the new Periotron 8000 with different fluids and uses the method of least squares to derive polynomial regression equations up to the 6th order, to investigate the most accurate descriptor of the resulting calibration lines. The use of a 4th order polynomial regression equation (recommended by the manufacturer) provided better coefficients of determination (R2: 0.999) and root mean square errors (RMSE = 1.6) than either linear regression (R2: 0.986, RMSE = 10.9) or quadratic models (R2: 0.998, RMSE = 3.2). Data derived using the manufacturer's MLCONVERT software program lacked accuracy and incurred large errors for volumes > 0.5 microliter. Calibrations performed on one day could be used with accuracy to derive volumes > 0.1 microliter collected on subsequent days, when using the same machine (s.d. for residuals plot = 2.49 Periotron units), but this was not the case for different machines (s.d. = 9.57 Periotron units). Varying serum protein concentration by up to 500% had a negligible effect on calculated volumes above 0.1 microliter. We conclude that the Periotron 8000 is at least as reliable a machine as the Periotron 6000, and that the calibration lines for both machines are best described using 4th order polynomial regression equations and "look-up" tables, rather than quadratic (Periotron 6000) or the manufacturer's software (Periotron 8000). Serum seems to be an acceptable GCF substitute for calibrations, which can be performed 1 day, and used on subsequent days for a given machine and for volumes above 0.1 microliter.

Cardiovascular, adrenocorticotropin, and cortisol responses to hypertonic saline in euvolemic sheep are altered by prostaglandin synthase inhibition.

Small volume intravenous infusions of hypertonic saline (HTS) increase blood pressure, heart rate, adrenocorticotropic hormone (ACTH), and cortisol by mechanisms that are not fully understood. We hypothesized that HTS infusions increase prostaglandin biosynthesis and that a prostaglandin synthase metabolite is responsible for mediating actions of HTS. We further hypothesized that thromboxane A2 (TxA2) is the specific metabolite responsible for mediating responses to HTS infusion. Adult female sheep (n=8) were chronically instrumented with vascular catheters and infused intravenously with 7.5% saline at a rate of 4 mL x kg(-1) over 5 min with or without pretreatment with the prostaglandin synthase inhibitor flunixin. Blood pressure, ACTH, and cortisol increased in response to HTS, and these responses were prevented by flunixin. Heart rate increased in response to HTS infusion, and flunixin reduced but did not prevent a heart rate response. Hematocrit decreased significantly in response to HTS but only following flunixin treatment. Arginine vasopressin increased but only modestly in response to HTS, and responses were not different following flunixin. Arterial pH, partial pressure of CO2, and partial pressure of O2 did not change. Circulating concentrations of thromboxane B2, a stable metabolite of TxA2 and an index of TxA2 formation, remained low and did not change in response to HTS. We conclude that heart rate, blood pressure, ACTH, and cortisol responses to HTS are mediated at least in part by a product of prostaglandin synthase metabolism. These responses were not due to increases in circulating concentrations of TxA2 but might involve local formation of TxA2 or some other prostaglandin synthase metabolite.

Synaptic targets of cholinergic terminals in the pulvinar nucleus of the cat.

We compared the cholinergic innervation of the pulvinar nucleus, a thalamic association nucleus, to previous studies of the cholinergic innervation of the dorsal lateral geniculate nucleus (dLGN), a thalamic relay nucleus. Both nuclei receive a dense innervation from cholinergic cells of the brainstem parabrachial region (PBR). In the dLGN, PBR terminals are located in close proximity to retinal terminals. Our goal was to determine whether PBR terminals in the pulvinar nucleus are located in close proximity to corticothalamic terminals. We identified PBR terminals with a monoclonal antibody directed against choline acetyltransferase (ChAT). Cholinergic terminals contacted dendrites (142 of 160, or 89%) or vesicle-filled profiles (18 of 160, or 11%). A subset of 55 terminals was stained for gamma-aminobutyric acid (GABA) to determine whether profiles postsynaptic to cholinergic terminals originate from thalamocortical cells (GABA-) or interneurons (GABA+). The majority (44 of 55, or 80%) of postsynaptic profiles were GABA- dendrites. The minority (11 of 55, or 20%) were GABA+ dendrites with vesicles. This distribution of contacts is very similar to that seen in the dLGN. However, the most significant finding was that most cholinergic contacts (121 of 160, or 76%) were located within complex clusters identified as glomeruli. This is the primary site of contacts made by corticothalamic terminals originating from layer V cells. These results suggest that while the PBR enhances retinal signals in the dLGN, it may also enhance cortical signals in the pulvinar nucleus. Thus, activity in the PBR may stimulate both an increased flow of retinal information to visual cortex, as well as an increased flow of information between different visuomotor areas of cortex.

Prospective study of bone, indium-111-labeled white blood cell, and gallium-67 scanning for the evaluation of osteomyelitis in the diabetic foot.

Twenty-two adult diabetic patients with clinical suspicion of foot and/or ankle infection were prospectively evaluated using radiography, technetium-99m methylene diphosphonate bone scanning (99mTc), indium-111-labeled leukocyte scanning (111In), and gallium-67 scanning (67Ga) to determine the presence of clinically suspected osteomyelitis. Biopsy for culture and histology was performed in 16 patients. The diagnosis of osteomyelitis was confirmed by biopsy in 12 patients. The remaining 10 patients had no evidence of osteomyelitis with long-term follow-up. 99mTc was shown to be of limited valued when used alone in these patients with peripheral neuropathy. 67Ga, either alone or in combination with 99mTc bone scanning, was of little diagnostic value and gave no additional information that was not available from 111In. The combination of three-phase 99mTc and 111In had the highest diagnostic efficacy (100% sensitivity, 80% specificity, and 91% accuracy), followed closely by 111In alone (100% sensitivity, 70% specificity, and 86% accuracy). We conclude that for adult diabetic patients with clinical suspicion of osteomyelitis but no radiographic findings of that disease, 111In alone is an appropriate nuclear medicine evaluation for ruling out infection if it is negative. However, if an area of 111In white blood cell uptake is present, a "simultaneous" 99mTc is often helpful in providing the anatomic correlation to differentiate osteomyelitis from infection that is limited to soft tissue.