Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

The MHC: relationship between linkage and function.

It is intriguing that several genes with associated functions, including all of class I and class II genes, as well as some genes affecting antigen presentation of both class I and class II pathways, are linked in the MHC. Recent observations have led to speculation that there may be a functional explanation for keeping these related genes together.

Interpreting regulatory authority guidance on immunosuppressive therapy for renal transplantation: a response to the UK's National Institute for Clinical Excellence (NICE).

AIMS: A group of UK consultant transplant physicians and surgeons (the Consensus Group) met to consider the implications and interpretation of the National Institute for Clinical Excellence's (NICE) Technology Appraisal No. 85 on the use of immunosuppressive therapy for renal transplantation in adults. METHODS: This group considered what the implications of these guidelines might be for clinical practice and consensus was developed on those areas which were potentially open to different interpretations. A wider survey of nephrologists and transplant surgeons throughout the UK was also performed to gauge the impact of the NICE recommendations. RESULTS AND CONCLUSIONS: The outcome of the discussions of the Consensus Group are presented with particular reference to the recommendations of how to respond to calcineurin inhibitor (CNI) intolerance. The survey suggested that the publication of this NICE guidance has resulted in relatively few changes in prescribing practice: UK transplant centers continue to use a wide range of locally developed protocols for immunosuppressive therapy. These include the use of agents such as mycophenolate mofetil (MMF) and sirolimus, despite the fact that both drugs appeared to receive only conditional acceptance in the NICE Guidelines.

Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci.

The pathogenesis of all forms of psoriasis remains obscure. Segregation analysis and twin studies together with ethnic differences in disease frequency all point to an underlying genetic susceptibility to psoriasis, which is both complex and likely to reflect the action of a number of genes. We performed a genome wide analysis using a total of 271 polymorphic autosomal markers on 284 sib relative pairs identified within 158 independent families. We detected evidence for linkage at 6p21 (PSORS1) with a non-parametric linkage score (NPL)=4.7, p=2 x 10(-6) and at chromosome 1p (NPL=3.6, p=1.9 x 10(-4)) in all families studied. Significant excess (p=0. 004) paternal allele sharing was detected for markers spanning the PSORS1 locus. A further three regions reached NPL scores of 2 or greater, including a region at chromosome 7 (NPL 2.1), for which linkage for a number of autoimmune disorders has been reported. Partitioning of the data set according to allele sharing at 6p21 (PSORS1) favoured linkage to chromosomes 2p (NPL 2.09) and 14q (NPL 2.0), both regions implicated in previous independent genome scans, and suggests evidence for epistasis between PSORS1 and genes at other genomic locations. This study has provided linkage evidence in favour of a novel susceptibility locus for psoriasis and provides evidence of the complex mechanisms underlying the genetic predisposition to this common skin disease.

An improved method of fluorescent dual insulin and endothelial staining allows visualisation of the revascularisation of intraportally transplanted islets.

BACKGROUND: Intraportally transplanted islets are avascular at the time of transplantation and take up to 14 days to fully revascularize, during which time up to 60% of islet mass may be lost. To investigate and improve islet revascularization, a robust method for the visualization and quantification of this process is required. METHODS: Islets isolated from Lewis rats were transplanted intraportally into the liver of diabetic syngeneic Lewis recipients. The animals were humanely killed either on the day of transplant or at 3, 5, 7, or 14 days posttransplant. The harvested livers were sectioned and stained with Bandeiraea simplicifolia lectin (for endothelial cells) and anti-insulin antibody and counterstained with DAPI. The slides were visualized with a fluorescent microscope. RESULTS: Islets were visualized over the whole time course. Insulin and endothelial staining was clearly visualized on the day of transplantation, but by day 3 endothelial staining was scarce within the islet. By day 5, early vessel formation could be seen within the islet, but insulin staining was patchy and associated with apoptotic nuclei. By day 7, vessels could be seen throughout the islet and insulin staining had returned. Day 14 sections showed a fully revascularized islet. CONCLUSIONS: The staining provided good delineation of islet endothelium and beta-cell location, with clear observation of the revascularization process. This technique also suggests that days 3 through 5 may be a critical period for islet survival and provides a good model for studying the effects of manipulating the revascularization process.

Characterization of the major susceptibility region for psoriasis at chromosome 6p21.3.

Psoriasis is a common inflammatory skin condition caused by genetic and environmental factors. Recent genome-wide linkage analyses have identified a locus encoding susceptibility to psoriasis and placed this gene in the 12 cM interval between markers D6S426 and D6S276 on chromosome 6p21.3. This is a broad region and encompasses the human major histocompatibility complex. We have sought to localize the susceptibility gene more precisely by exploiting the linkage, haplotype, and linkage disequilibrium information available through genotyping 118 affected sib pairs, their parents and other affected family members. A total of 14 highly polymorphic markers were genotyped, combining anonymous loci with the class I genes HLA-B and -C distributed across a genetic interval of approximately 14 cM including the entire major histocompatibility complex. Through the application of higher density mapping within the major histocompatibility complex, we identified those regions most commonly shared identical by descent in patients with psoriasis. Using the transmission-disequilibrium test, we found significant evidence of linkage and allelic association across an interval defined by the markers tn62 (p = 1.0 x 10(-7)), HLA-B (p = 4.0 x 10(-7)), and HLA-C (p = 2.7 x 10(-9)), a region encompassed within a 285 kb genomic DNA fragment. Hence these studies contribute to the refinement of the localization of a major psoriasis susceptibility gene and place the critical region near to HLA-C.

Surviving native beta-cells determine outcome of syngeneic intraportal islet transplantation.

In moderately diabetic rats (plasma glucose 20-30 mmol/L), where there is some residual pancreatic islet function, normoglycemia can be restored by transplantation of pancreatic islets into the liver via the portal vein. To examine whether normoglycemia can also be achieved in more severely diabetic animals (which more closely resemble human type I diabetes), we have compared the effect of transplanting 1000 islets intraportally in Lewis rats made moderately diabetic (55 mg/kg streptozotocin injected IP while nonfasting) or severely diabetic (65 mg/kg streptozotocin injected IP while fasting). In the moderately diabetic rats in which residual pancreatic insulin was 128 +/- 40 mU insulin (2.0% of control), plasma glucose stabilized (32 +/- 2.8 mmol/L at 1 week, 34 +/- 2 mmol/L at 3 weeks) as did body weight (falling from 290 +/- 5 to 265 +/- 5 g at 1 week and 253 +/- 6 g at 3 weeks). In contrast, in severely diabetic rats in which residual pancreatic insulin was only 13.5 +/- 4.2 mU insulin (0.21% of control), there was a progressive rise in plasma glucose (30 +/- 1.3 mmol/L at 1 week, 49 +/- 4 mmol/L at 2 weeks, and 67 +/- 7 mmol/L at 3 weeks) and a progressive fall in body weight (from 304 +/- 10 to 260 +/- 5 g by week 1 and to 209 +/- 6 g by week 3). Following islet transplantation, nonfasting plasma glucose normalized in moderately diabetic rats (10.5 +/- 0.6 vs. 9.1 +/- 0.6 mmol/L in nondiabetic controls, NS) after 23 +/- 5 days. In contrast, in the severely diabetic rats plasma glucose stabilized at 32 +/- 5 mmol/L (p < 0.05 compared to moderately diabetic group) but did not normalize. This difference was not attributable to different plasma glucose levels at the time of transplantation (35.1 +/- 1.8 in moderately diabetic vs. 32.5 +/- 2.5 mmol/L in severely diabetic rats). These observations demonstrate that residual native beta-cells (equivalent to only 60-80 islets) contribute to the survival or function of intraportally transplanted islets.

Human mesangial cells express inducible macrophage scavenger receptor: an Ap-1 and ets mediated response.

BACKGROUND: Type A scavenger (SR) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipid via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMC), which can be converted to foam cells in vivo, have not previously been shown to express SR in normal culture. We investigated whether or not there was an inducible form of SR in a human mesangial cell line (HMCL). METHODS: SR activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using a flow cytometer. SR mRNA expression was examined using RT-PCR followed by Southern blotting. To investigate the molecular mechanism of SR expression, several reporter gene constructs were designed. The first contained a full SR promoter, the second a part of the SR promoter that has both activated protein-1 (AP-1) and ets transcriptional factor binding sites. Other constructs were identical to the second except they contained either AP-1 or ets motif mutations. RESULTS: Phorbol 12-myristate 13-acetate (PMA) increased both the percentage of SR positive cells and SR mean fluorescence intensity. PMA also increased SR mRNA and promoter activity in a time and dose responsive manner. Function analysis showed that both AP-1 and ets motifs were specific response elements to PMA stimulation in HMCL. CONCLUSIONS: The present study suggests that the combination of interaction between AP-1 and ets transcriptional factors may mediate the inducible expression of the SR gene in HMCL, which may contribute to foam cell formation.

Calcineurin inhibitors enhance low-density lipoprotein oxidation in transplant patients.

BACKGROUND: Our objective was to assess the pro-oxidant status of neoral and tacrolimus in renal transplant patients and monitor the protection provided by vitamin C and vitamin E in normalizing low density lipoprotein (LDL) oxidation lag time of tacrolimus-treated patients. METHODS: Plasma LDL was isolated by density gradient ultracentrifugation from renal transplant patients receiving neoral, tacrolimus and tacrolimus with vitamin C and vitamin E. Oxidation was initiated by the addition of CuCl2 at 37 degrees C and monitored at 234 nm over 480 minutes and oxidation lag time was computed. Total antioxidant capacity of serum was measured using the enhanced chemiluminescent method. RESULTS: LDL from tacrolimus-treated patients had significantly lower oxidation lag time and serum antioxidant activity in comparison with neoral-treated patients, and this was particularly significant during the first four months after transplantation. Vitamin C and E supplementation in tacrolimus treated patients provided protection against oxidation and normalized their oxidation lag time. CONCLUSION: Calcineurin-inhibiting drugs, CsA and tacrolimus, have pro-oxidant activity and they increase the susceptibility of LDL to oxidation. Neoral formulation is fortified with DL-alpha tocopherol and therefore provides protection against oxidation. The present study clearly demonstrates the benefit of giving vitamin C and E supplements to patients taking tacrolimus and this seems to be particularly important during the early period after transplantation.

Effect of donor treatment with heparinoids on graft function after intraportal transplantation of a marginal islet mass: an experimental study.

Heparinoids interact with factors that are involved in ischemia-reperfusion injury and thus may prevent organ injury. We therefore studied the effects on subsequent intraportal islet transplantation of systemic administration of unfractionated and N-desulphated heparin to donors prior to pancreatectomy. Donor rats were given an intravenous injection of either heparin (1.3 mg/kg or 13.3 mg/kg; 200 U/kg or 2000 U/kg, respectively) or N-desulphated heparin (50 mg/kg; approximately 5 U/kg) at 5 to 10 minutes prior to pancreas procurement. Five hundred freshly isolated islets were injected intraportally into syngeneic male Lewis recipients that had developed streptozotocin-induced diabetes. Blood glucose and body weight were monitored for 5 weeks thereafter. Rats transplanted with islets from donors given high dose heparin showed a fall in blood glucose from 25.1 +/- 1.4 to 11.0 +/- 2.7 mmol/L (P <.01) with 60% of animals euglycemic within the first week. In contrast, the controls, did not show a fall in glucose levels at 1 week and none had become euglycaemic. Normalization of glycemia was slower in recipients of islets from animals treated with the lower dose of heparin. Results were intermediate with islets from donors given N-desulphated heparin. Nevertheless, all heparinoids used in this study caused more than a doubling of the number of animals achieving normoglycemia by 3 to 4 weeks. We hypothesize that pretreatment of the donor with heparin protects islet integrity during procurement and isolation and hence accelerates islet engraftment and remodelling. Since the effect was seen with N-desulphated heparin, which has negligible anticoagulant properties, we believe the mechanism to be independent of the anticoagulant activity.

Use of perfluorodecalin for pancreatic islet culture prior to transplantation: a liquid-liquid interface culture system--preliminary report.

Although the issue remains controversial, short-term culture is probably beneficial for islet graft quality. However, significant islet loss is invariably observed. This is related to reduced survival of large islets, which is compromised by hypoxia under standard culture conditions. We aimed to develop a method of culture, which would avoid exposure to relative hypoxia and hence maintain the quality of islets. Isolated rat islets cultured for 48 h in a liquid-liquid interface culture system (LICS) with a perfluorocarbon were compared to islets cultured under standard (C1) and suboptimal conditions (C2). Islets were tested for viability and response to a glucose challenge, and a marginal mass was transplanted into syngeneic diabetic recipients. The viability of islets after 24-h culture in LICS was higher than in C1 and C2 groups (89.0% vs. 77.5% and 64.6%, respectively) and decreased with time to reach 79.0%, 62.9%, and 53.4% after 72-h culture. The stimulation index in LICS-cultured islets was also significantly higher than in C1 and C2 groups (12.3 ± 0.4 vs. 5.8 ± 0.5 and 4.1 ± 0.2, respectively). Following transplantation of LICS-cultured islets 50% of recipients were rendered normoglycemic compared with 14.3% and 31.3% for C2 and fresh islets, respectively. Our liquid-liquid interface culture system using perfluorodecalin provides optimized culture conditions, which preserve both islet viability and their ability to engraft successfully after intraportal transplantation and could be used for islet transportation.

Plasma concentrations of mycophenolic acid acyl glucuronide are not associated with diarrhea in renal transplant recipients.

The aim of this study was to determine whether plasma concentrations of the acyl (AcMPAG) and phenolic (MPAG) glucuronide metabolites of mycophenolic acid (MPA) were related to diarrhoea in renal transplant patients on mycophenolate mofetil (MMF) with cyclosporine (CsA) or tacrolimus (TCL). Blood samples (0, 30, 120 min) were taken at days 3, 10, week 4, months 3, 6 and 12 for determination of MPA, MPAG and AcMPAG. MPA-AUC was estimated using validated algorithms. Two hour AUCs were calculated for MPAG and AcMPAG. Immunosuppressive therapy consisted of CsA/MMF (n= 110) and of TCL/MMF (n= 180). In 70/290 (24%) patients 86 episodes of diarrhoea were recorded during 12 months. Significantly more patients on TCL (31.1%) suffered from diarrhea compared to CsA (12.7%). MMF dose, MPA-AUC and the 2 h AUCs of MPAG and AcMPAG did not differ between patients with and without diarrhoea. Plasma AcMPAG and MPAG concentrations were substantially higher in patients on CsA compared with TCL, while MPA-AUC was lower in the former group. These data support the concept that CsA inhibits the biliary excretion of MPAG and AcMPAG, thereby potentially reducing the risk of intestinal injury through enterohepatic recycling of MPA and its metabolites.

Low plasma protein nitrotyrosine levels distinguish primary Raynaud's phenomenon from scleroderma.

OBJECTIVE: To investigate the hypothesis that increased formation of reactive nitrogen species may contribute to the vascular pathology that develops in patients with connective tissue disease such as scleroderma. PATIENTS AND METHODS: The level of protein-bound nitrotyrosine in plasma was measured by stable isotope dilution gas chromatography/negative ion chemical ionisation mass spectrometry in 11 patients with primary Raynaud's phenomenon, 37 with scleroderma, 13 with chronic renal impairment, and in 23 healthy controls. RESULTS: Plasma protein-bound nitrotyrosine was markedly decreased in patients with primary Raynaud's phenomenon (mean (SEM) 0.60 (0.06) ng/mg dry protein) compared with patients with scleroderma (1.78 (0.21) ng/mg protein), chronic renal impairment (1.42 (0.17) ng/mg protein) or healthy controls (1.63+/-0.15 ng/mg protein, ANOVA p<0.001). CONCLUSION: These data suggest that there is decreased nitration of plasma proteins, or increased degradation of nitrated proteins from the circulation of patients with primary but not secondary Raynaud's phenomenon.