Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels.

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80-100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36-/- mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

Correlation of Pretransplant Donor-specific Antibody Assay Using Luminex Crossmatch with Graft Outcome in Renal Transplant Patients.

The significance of pretransplant anti-human leukocyte antigen antibody levels that are detectable by more sensitive platforms (including the Luminex platform) yet undetected by complement-dependent cytotoxicity (CDC) assay remains unclear. The aim of this study was to determine the clinical significance of the donor-specific antibody (DSA) assay Luminex crossmatch and its impact on short-term renal graft outcome such as acute rejections, graft survival, and graft function. The results of pretransplant DSA-lymphocyte crossmatching (LCXM) assay in 126 renal allograft recipients whose CDCs crossmatches were negative were retrospectively analyzed for correlation with posttransplant outcomes. Of the 126 recipients, 32 (25.4%) had pretransplant DSA positive. Statistically significant association was found between DSA-LCXM positivity with 14th day estimated glomerular filtration rate (eGFR) (P = 0.05), DSA Class I with 3rd (P = 0.014) and 6th month (P = 0.02) eGFR, DSA Class II with 14th day (P = 0.06) and 1st month (P = 0.10) eGFR, mean fluorescent intensity (MFI) DSA with 7th day (P = 0.08) and 14th day (P = 0.09) eGFR, and maximum MFI DSA with 7th day eGFR (P = 0.09). The posttransplant eGFR was higher at various time intervals in DSA-LCXM-negative patients as compared to DSA-positive patients. However, pretransplant DSA-LCXM results did not predict the rejection episodes, graft loss, and 1-year posttransplant 24 h urine protein. Pretransplant DSA detected by LCXM in patients with a negative CDC does not predict adverse short-term outcomes. However, the difference in posttransplant eGFR supports further investigation in long-term effects.

Adefovir nephrotoxicity in a renal allograft recipient.

Adefovir dipivoxil, an oral prodrug of adefovir, is used in the treatment of lamivudine-resistant hepatitis B virus (HBV) infection. Nephrotoxicity manifesting as proximal renal tubular dysfunction and acute tubular necrosis (ATN) were commonly reported in the past, when higher doses were used for the treatment of human immunodeficiency virus infection. However, nephrotoxicity is rare at lower doses that are currently recommended for the treatment of HBV infection. A 31-year-old female was detected to be hepatitis B surface antigen positive months after a kidney transplant. The patient was initiated on lamivudine, but developed resistance after 1 year of treatment, at which time low-dose adefovir was added. The patient developed renal allograft dysfunction after 10 months of starting adefovir. Serum creatinine increased from 1.1 mg/dl to 1.9 mg/dl, along with progressively increasing sub-nephrotic proteinuria. Renal allograft biopsy revealed features of ATN. After discontinuation of adefovir, proteinuria resolved and renal dysfunction improved slowly over the next 2 years. Adefovir-induced nephrotoxicity, although uncommon at lower doses, needs to be considered in the differential diagnosis of renal dysfunction and sub-nephrotic proteinuria occurring in patients receiving adefovir for prolonged periods.

Collapsing glomerulopathy following anabolic steroid use in a 16-year-old boy with IgA nephropathy.

Collapsing glomerulopathy (CG) is a proliferative podocytopathy, increasingly recognized in a variety of disease conditions. We report a case of CG in a 16-year-old boy with IgA nephropathy (IgAN) who presented with acute kidney injury, marked proteinuria and hypertension following a short period of anabolic steroid use. Although CG has been associated with long-term anabolic steroid use among body builders, there is no data on the effect of anabolic steroid use in persons with underlying renal disease like IgAN. We postulate that development of CG in our patient could be temporally linked to intake of body-building steroids along with a predisposing background renal disease of IgAN.

Profile of incident chronic kidney disease related-mineral bone disorders in chronic kidney disease Stage 4 and 5: A hospital based cross-sectional survey.

Chronic kidney disease related-mineral bone disorder (CKD-MBD) has been poorly studied in pre-dialysis Indian CKD patients. We aimed to study the clinical, biochemical and extra skeletal manifestations of untreated CKD-MBD in pre-dialysis Stage 4 and 5 CKD patients attending nephrology out-patient clinic at a tertiary care hospital in South India. A hospital based cross-sectional survey including, demographic profile, history of CKD-MBD symptoms, measurement of serum calcium, phosphate, parathyroid hormone, 25 hydroxy vitamin D (25(OH) D) and alkaline phosphatase; lateral abdominal X-rays for abdominal aortic calcification (AAC) and echocardiography for valvular calcification (VC) was carried out. Of the 710 patients surveyed, 45% had no CKD-MBD related symptom. Prevalence of hypocalcemia, hyperphosphatemia, hyperparathyroidism (>150 pg/mL) and 25(OH) D levels <30 ng/mL was 66.3%, 59%, 89.3% and 74.7% respectively. Echocardiography was carried out in 471 patients; 96% of whom had VC (calcification score ≥1). Patients with VC were older and had lower 25(OH) D levels than those without. Lateral abdominal X-rays were obtained in 558 patients, 6.8% of whom were found to have AAC, which was associated with older age. Indian patients with incident CKD-MBD have a high prevalence of hypocalcemia, 25(OH) D deficiency and VC even prior to initiating dialysis while AAC does not appear to be common. The association between 25(OH) D deficiency and VC needs further exploration.

Significance of pre-transplant anti-HLA antibodies detected on an ELISA mixed antigen tray platform.

This study aims at examining the clinical impact, of antibodies detected on an ELISA mixed antigen tray format (LATM, One Lambda) in the absence of complement dependent cytotoxicity (CDC) positivity. All patients who underwent renal transplantation in 2007 and 2008 had their final pre-transplant sera retrospectively analyzed by the LATM assay. These patients were then followed-up with clinical, biochemical, and histopathological end points defined by elevation of serum creatinine and/or histopathological criteria. Among 164 patients who were studied, 149 received grafts from live related donors and 15, from deceased donors. 31 (19%) of the transplanted patients demonstrated pre-transplant anti-HLA IgG antibodies on the assay. Totally, 15 were positive for class I antibodies, 4 for class II antibodies, and 12 for both class I and class II antibodies. 44 patients (36%) experienced rejection. 8 out of 31 (26%) ELISA positive patients and 36 out of 133 (27%) ELISA negative patients experienced rejection. Among 15 patients who received deceased donor transplants, 4 were positive for ELISA, and 11 were negative. All 4 (100%) of the ELISA positive patients experienced rejection as compared to 3 out of 11 (27%) ELISA negative patients (P = 0.01). The ELISA LATM assay did not show any predictive value for rejection in our overall patient population; however, results in the specific setting of deceased donor transplants merit further exploration.

Non-O1, non-O139 Vibrio cholerae sepsis in a patient with nephrotic syndrome.

Non-O1, non-O139 Vibrio cholerae is an encapsulated bacterium, ubiquitous in the marine environment and generally considered to be non-pathogenic. However, it is known to cause diarrheal illness, wound infection, and bacteremia in immunocompromised hosts. Here we have describe non-O1, non-O139 V. cholerae sepsis in a patient with nephrotic syndrome following exposure to sea-water. Interestingly, the exposure occurred remotely 4 months prior to the onset of nephrotic syndrome. The occurrence of florid sepsis after a prolonged interval from the time of exposure is peculiar and raises the possibility of an association between occult Vibrio sepsis and nephrotic syndrome.

Combined approach with therapeutic drug monitoring and pharmacogenomics in renal transplant recipients.

In patients undergoing renal transplantation, dose individualization for tacrolimus is routinely achieved with therapeutic drug monitoring (TDM). The patient started on 5.5 mg/day of tacrolimus had a significantly elevated tacrolimus trough concentration. The tacrolimus dose was regularly reduced following TDM at many time periods in the post transplant period but the tacrolimus concentration was consistently elevated. Genomic analysis done after four years revealed mutations in the genes encoding for CYP3A5 and MDR1 (2677G > T). Pharmacogenomics alongside TDM, will soon emerge as the backbone of dose individualization. But for genomics to be beneficial, it should be advocated in the pre-transplant or early post transplant period.

Tuberculosis in a renal allograft recipient presenting with intussusception.

Extra-pulmonary tuberculosis (TB) is more common in renal allograft recipients and may present with dissemination or an atypical features. We report a renal allograft recipient with intestinal TB presenting 3 years after transplantation with persistent fever, weight loss, diarrhea, abdominal pain and mass in the abdomen with intestinal obstruction. He was diagnosed to be having an ileocolic intussusception which on resection showed a granulomatous inflammation with presence of acid-fast bacilli (AFB) typical of Mycobacterium tuberculosis. In addition, AFB was detected in the tracheal aspirate, indicating dissemination. He received anti-TB therapy (ATT) from the fourth postoperative day. However, he developed a probable immune reconstitution inflammatory syndrome (IRIS) with multiorgan failure and died on 11(th) postoperative day. This is the first report of intestinal TB presenting as intussusception in a renal allograft recipient. The development of IRIS after starting ATT is rare in renal allograft recipients. This report highlights the need for a high index of suspicion for diagnosing TB early among renal transplant recipients and the therapeutic dilemma with overwhelming infection and development of IRIS upon reduction of immunosuppression and starting ATT.

An unusual case of abdominal wall bleeding after renal allograft biopsy.

We report an unusual case of a enlarging anterior abdominal wall hematoma after percutaneous biopsy of a renal allograft. Angiography-directed embolization of the vessels filling the pseudoaneurysm was done and followed up with surgical exploration of the hematoma. In order to avoid this complication, Color Doppler evaluation of the overlying abdominal wall is suggested to look for significant vessels before the biopsy procedure.

MUC1-specific immune therapy generates a strong anti-tumor response in a MUC1-tolerant colon cancer model.

A MUC1-based vaccine was used in a preclinical model of colon cancer. The trial was conducted in a MUC1-tolerant immune competent host injected with MC38 colon cancer cells expressing MUC1. The vaccine included: MHC class I-restricted MUC1 peptides, MHC class II-restricted pan-helper-peptide, unmethylated CpG oligodeoxynucleotide, and granulocyte macrophage-colony stimulating factor. Immunization was successful in breaking MUC1 self-tolerance, and in eliciting a robust anti-tumor response. The vaccine stimulated IFN-gamma-producing CD4(+) helper and CD8(+) cytotoxic T cells against MUC1 and other undefined MC38 tumor antigens. In the prophylactic setting, immunization caused complete rejection of tumor cells, while in the therapeutic regimen, tumor burden was significantly reduced.

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

Prevalence of rotavirus, adenovirus and astrovirus infection in young children with gastroenteritis in Gaborone, Botswana.

OBJECTIVE: To determine the prevalence of three enteric viruses, namely rotavirus, adenovirus and astrovirus, as agents of diarrhoea in and around Gaborone, Botswana. DESIGN: The sample were categorised into four groups according to the age of the patient: 0-3 months, 4-6 months, 7-12 months and 25-60 months. Total monthly samples across age groups formed basis for calcultating seasonal prevalence of rotavirus infection. SETTING: Stool samples were collected from three medical laboratories in Gaborone and one in the town of Mochudi. These were collected from children under the age of five years with gastroenteritis. SUBJECTS: Stool samples were collected between March 2001 and February 2002 from 346 children less than five years of age suffering from gastroenteritis. These samples had been sent to medical laboratories for microbiological examination. METHODS: The samples were screened for rotavirus (RV), adenovirus (Ad) and astrovirus (AsV) antigens using commercially available ELISA kits. The Ad positive samples were further analysed by commercially available group specific Ad type 40/41 Enzyme Immuno Assays (EIA). RESULTS: Shedding of RV was detected in 9.2%, Ad in 7.8% and AsV in 2.7% of the samples analysed. The enteric Ad (types 40 and 41) were detected in 2% of the samples and the remaining 5.8% of Ad positive samples were non-enteric Ad. An increase of RV was noted in the autumn-winter season but no seasonal pattern was observed in Ad shedding. Seasonal prevalence of AsV could not be determined. The average age of children infected with these agents was less than one year. CONCLUSION: The incidence of rotavirus infection amongst children in Botswana appears to be relatively low. The prevalence rate of adenovirus and astrovirus is similar to other studies in parts of Southern Africa. However, continued enteric virus surveillance and epidemiology amongst this group is required.

Conformational properties of alpha-tubulin tail peptide: implications for tail-body interaction.

The carboxy-terminal segments of the alpha/beta-tubulins are flexible regions rich in acidic amino acid residues. It is generally believed that these regions play crucial roles in tubulin polymerization and interaction with many ligands, including colchicine. Exactly how these effects are exerted are not known at present. One such interesting aspect is the pH dependence of colchicine-tubulin interaction and the influence of the alpha-tail on the binding interaction. We have investigated the location of the colchicine-binding site on tubulin by docking. It has been located on the alpha/beta interface on the N-terminal side, which is also supported by much of the solution data. This location is too far from the tail regions, suggesting that influence of the tail region is transmitted by a pH-dependent conformational change. Two-dimensional NMR studies indicate that at pH 7 a 13-residue peptide corresponding to alpha-tubulin tail shows little NOE constraints, suggesting extended conformation. On the contrary, at pH 5, a relatively compact structure was deduced from the interproton NOE constraints. Pulsed field gradient measurement of diffusion constant indicates that the peptide at pH 5 is substantially faster diffusing than at pH 7. The Perrin factors calculated from diffusion data indicates that the peptide structure at pH is significantly more compact than at pH 7. Temperature coefficients of several amide protons at pH 5 fall below 5 ppb/(o)K, indicating a degree of protection. A difference is also seen in the CD spectra obtained at different pHs, consistent with the NMR data. We have investigated the probable spatial organization of the tail of the alpha-subunit of tubulin, in the high pH extended form and the low pH compact form. On the basis of correlation of pH dependence of many properties of tubulin and the conformation of the alpha-tail peptide, we propose that the intrinsic conformational preference of the tail-region modulate the tail-body interaction, which in turn has important bearing on colchicine binding properties.

Dielectric relaxation in a single tryptophan protein.

Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single-tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift ( approximately 5 nm at 50% quenching by acrylamide) is striking in a single-tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady-state polarization and time-resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.

Neural network prediction of 3(10)-helices in proteins.

Secondary structure prediction from the primary sequence of a protein is fundamental to understanding its structure and folding properties. Although several prediction methodologies are in vogue, their performances are far from being completely satisfactory. Among these, non-linear neural networks have been shown to be relatively effective, especially for predicting beta-turns, where dominant interactions are local, arising from four sequence-contiguous residues. Most 3(10)-helices in proteins are also short, comprising of three sequence-contiguous residues and two capping residues. In order to understand the extent of local interactions in these 3(10)-helices, we have applied a neural network model with varying window size to predict 3(10)-helices in proteins. We found the prediction accuracy of 3(10)-helices (approximately 14%), as judged by the Matthew's Correlation Coefficient, to be less than that of beta-turns (approximately 20%). The optimal window size for the prediction of 3(10)-helices was about 9 residues. The significance and implications of these results in understanding the occurrence of 3(10)-helices and preferences of amino acid residues in 3(10)-helices are discussed.

Solution structure of an immunoactive peptide fragment of Staphylococcal protein-A.

Staphylococcal protein-A (SpA) is known to bind the Fc fragment of immunoglobin G in vitro and induce a myriad of immunogenic responses in vivo. The latter is ascribed to be due to the interaction of Fc and SpA. It has also been proposed that in vivo proteolytically cleaved fragments of SpA may be functioning in the same manner. One such fragment (EQQNAFYEILHLPNLNEEQR), fragment 8-27 of the B-domain (SpA-B), was recently shown to exhibit in vivo immunogenic response [Sinha, P., Sengupta, J., and Ray, P. K. (1999) Biochem. Biophys. Res. Commun. 258, 141-147]. As a first step towards understanding the mode of interaction of this peptide with the Fc fragment, we have studied the solution conformation of this isolated peptide by CD and NMR. The peptide, with 7 contact residues in the crystal structure of the SpA-B/Fc complex and comprising of mostly helixI and part of helixII of the 3-helix bundle of SpA-B, was found to be present predominantly in extended structure. However it showed nascent turn/helix like conformations around F14 & Y15. These two residues are known to play a vital role in SpA-B/Fc interaction as deciphered from crystal structure and NMR studies of SpA-B/Fc complex and mutational studies. The implications of our results, especially the nascent conformations found around F14 & Y15, in design of SpA-B mimetic small molecules are discussed.

Chronic arsenic toxicity in Bangladesh and West Bengal, India--a review and commentary.

Fifty districts of Bangladesh and 9 districts in West Bengal, India have arsenic levels in groundwater above the World Health Organization's maximum permissible limit of 50 microg/L. The area and population of 50 districts of Bangladesh and 9 districts in West Bengal are 118,849 km2 and 104.9 million and 38,865 km2 and 42.7 million, respectively. Our current data show arsenic levels above 50 microg/ L in 2000 villages, 178 police stations of 50 affected districts in Bangladesh and 2600 villages, 74 police stations/blocks of 9 affected districts in West Bengal. We have so far analyzed 34,000 and 101,934 hand tube-well water samples from Bangladesh and West Bengal respectively by FI-HG-AAS of which 56% and 52%, respectively, contained arsenic above 10 microg/L and 37% and 25% arsenic above 50 microg/L. In our preliminary study 18,000 persons in Bangladesh and 86,000 persons in West Bengal were clinically examined in arsenic-affected districts. Of them, 3695 (20.6% including 6.11% children) in Bangladesh and 8500 (9.8% including 1.7% children) in West Bengal had arsenical dermatological features. Symptoms of chronic arsenic toxicity developed insidiously after 6 months to 2 years or more of exposure. The time of onset depends on the concentration of arsenic in the drinking water, volume of intake, and the health and nutritional status of individuals. Major dermatological signs are diffuse or spotted melanosis, leucomelanosis, and keratosis. Chronic arsenicosis is a multisystem disorder. Apart from generalized weakness, appetite and weight loss, and anemia, our patients had symptoms relating to involvement of the lungs, gastrointestinal system, liver, spleen, genitourinary system, hemopoietic system, eyes, nervous system, and cardiovascular system. We found evidence of arsenic neuropathy in 37.3% (154 of 413 cases) in one group and 86.8% (33 of 38 cases) in another. Most of these cases had mild and predominantly sensory neuropathy. Central nervous system involvement was evident with and without neuropathy. Electrodiagnostic studies proved helpful for the diagnosis of neurological involvement. Advanced neglected cases with many years of exposure presented with cancer of skin and of the lung, liver, kidney, and bladder. The diagnosis of subclinical arsenicosis was made in 83%, 93%, and 95% of hair, nail and urine samples, respectively, in Bangladesh; and 57%, 83%, and 89% of hair, nail, and urine samples, respectively in West Bengal. Approximately 90% of children below 11 years of age living in the affected areas show hair and nail arsenic above the normal level. Children appear to have a higher body burden than adults despite fewer dermatological manifestations. Limited trials of 4 arsenic chelators in the treatment of chronic arsenic toxicity in West Bengal over the last 2 decades do not provide any clinical, biochemical, or histopathological benefit except for the accompanying preliminary report of clinical benefit with dimercaptopropanesulfonate therapy. Extensive efforts are needed in both countries to combat the arsenic crisis including control of tube-wells, watershed management with effective use of the prodigious supplies of surface water, traditional water management, public awareness programs, and education concerning the apparent benefits of optimal nutrition.