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.

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.

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.

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.

Renovascular disease is associated with low producer genotypes of the anti-inflammatory cytokine interleukin-10.

Cytokines are important mediators of inflammatory and proliferative responses in disease states including atherosclerosis. Genetic variations in cytokine production could potentially influence the outcome of these responses. The aim of this study was to determine whether cytokine gene polymorphism might influence the development of atherosclerotic renal artery stenosis. Sixty-six patients with atherosclerotic renal artery stenosis and 100 normal healthy individuals were genotyped for interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-2 promoter region polymorphism. TNF-a, TNF-d, and IL-10 microsatellite polymorphisms were also analyzed. The frequency of the anti-inflammatory cytokine IL-10 promoter (-1082 A positive) GA and AA genotypes which are associated with low production were higher in the patient group when compared to the control group. The AA-TT-AA homozygous genotype combination of three single-nucleotide polymorphisms at -1082, -819, and -592 in the IL-10 gene was also observed at a higher frequency in the patient group compared to the controls. The frequency of TNF-alpha, IL-6, and IL-2 polymorphisms did not show any significant difference between the patient and control groups. To correlate IL-10 genotypes with differences in IL-10 protein expression, in vitro mRNA and protein levels were analyzed in lipopolysaccharide-stimulated peripheral blood mononuclear cells from 22 patients with renal artery stenosis and 33 controls. Individuals genotyped as A positive at position -1082 produced lower levels of IL-10 protein and had lower copy numbers of mRNA when compared to individuals genotyped as A negative in both patient and control groups. The increased frequency of the low producer IL-10 promoter, -1082 A-positive genotype in patients with renal artery stenosis, suggests that IL-10 may protect against the development of atherosclerotic renovascular disease.

Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors.

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1beta (interleukin-1beta; 5 ng/ml) and unmodified LDL (200 microg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor alpha and IL-1beta increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1beta inhibited [(3)H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.

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.

Calculated glomerular filtration rate is a useful screening tool to identify scleroderma patients with renal impairment.

OBJECTIVES: Although it only occurs in a minority of patients, renal involvement is a life-threatening complication of scleroderma (SSc). We have investigated the utility of two formulae to calculate glomerular filtration rate (GFR) in a population of SSc patients. METHODS: Twenty-six patients (20 female, 6 male, median age 58 yr, age range 12-80 yr) satisfied our criteria for inclusion in a retrospective comparison of measured and calculated GFR. GFR was measured using (51)Cr-EDTA. The modified Cockcroft and Gault formula and equation 7 from the Modification of Diet in Renal Disease (MDRD) were used to calculate GFR. RESULTS: Eighteen out of 19 patients analysed with a serum creatinine concentration less than the upper limit of the normal range had a measured GFR outside the normal range. Three patients with a normal creatinine concentration had a measured GFR <60 ml/min and in each of these the calculated GFR was also abnormal. All patients with a measured GFR <60 ml/min were identified using both the MDRD and the modified Cockcroft and Gault formula to calculate GFR. The greatest correlation between measured and calculated GFR was seen when the MDRD formula, which employs demographic and serum variables, was used in patients with body surface area (BSA) >1.4 m(2) who were not taking Iloprost (r=0.91). Use of the Cockcroft and Gault formula to calculate creatinine clearance with a correction factor for GFR, the inclusion of patients taking Iloprost and the inclusion of patients with BSA <1.4 m(2) were all associated with a lower degree of correlation. CONCLUSION: Serum creatinine is a poor marker of renal function in SSc patients. Calculating GFR from demographic and serum variables is a simple technique to identify SSc patients who have abnormal renal function. The authors recommend the use of the MDRD formula.

Dysregulation of LDL receptor under the influence of inflammatory cytokines: a new pathway for foam cell formation.

BACKGROUND: Lipid-mediated renal injury is an important component of glomerulosclerosis and its similarity to atherosclerosis is well described. This study focused on the relationship between lipid-mediated injury and inflammation by examining the role of inflammatory cytokines in the regulation of human mesangial cell low-density lipoprotein (LDL) receptors. METHODS: A human mesangial cell line (HMCL) was used to study the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on the regulation of LDL receptor mRNA and protein in the presence of a high concentration of native LDL (250 microg/mL). RESULTS: Native LDL caused foam cell formation in HMCL in the presence of antioxidants, TNF-alpha and IL-1beta. Both cytokines overrode LDL receptor suppression induced by a high concentration of LDL and increased LDL uptake by enhancing receptor expression. These cytokines also caused increased expression of SCAP [sterol responsive element binding protein (SREBP) cleavage activation protein], and an increase in the nuclear translocation of SREBP, which induces LDL receptor expression. CONCLUSION: These observations demonstrate that inflammatory cytokines can modify cholesterol-mediated LDL receptor regulation in mesangial cells, permitting unregulated intracellular accumulation of unmodified LDL and causing foam cell formation. These findings suggest that inflammatory cytokines contribute to lipid-mediated renal damage, and also may have wider implications for the study of inflammation in the atherosclerotic process.

The safety and efficacy of MMF in lupus nephritis: a pilot study.

Inducing and maintaining remission in patients with lupus nephritis may be difficult. Current treatments have significant toxicity. Mycophenolate mofetil (MMF) limits damage in murine models of lupus nephritis. We have assessed the efficacy and tolerability of MMF in the treatment of patients with long-standing or resistant lupus nephritis. We have treated 13 patients with biopsy proven lupus nephritis (two membranous nephropathy, four membranous nephropathy with superimposed proliferative changes, seven with proliferative glomerulonephritis). All patients had relapsed on conventional treatment or there were pressing indications to minimise steroid dosage or avoid alkylating agents. Nine out of 13 were treated with MMF and prednisolone, 3/10 with MMF alone and 1/10 with MMF, prednisolone and cyclosporine. Thirteen patients were treated with MMF for up to 37 months (median 25 months). Three patients were withdrawn from MMF during the first 8 months of treatment. The remainder tolerated MMF (median dose 1 g/day). Serological improvements were observed in 9/13 and steroid dosage was reduced in 8/10 patients. Infections occurred in 3/13. One patient relapsed. MMF significantly reduced the rate of decline of renal function. MMF should be considered in the treatment of long-standing or resistant lupus nephritis. Controlled clinical trials are required to confirm these findings.

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.

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.

Sample sequencing of the human major histocompatibility complex class I region identifies further new genes.

We used sample sequencing, a technique which generates random genomic sequence from cosmid clones and compares them with sequences deposited in the GenBank databases, to identify new genes in the class I region of the human major histocompatibility region. We isolated and ordered cosmid clones from a flow-sorted chromosome (Chr) 6 cosmid library, generating cosmid contigs covering approximately one third of the HLA class I region. Fifteen of these cosmids were then sample sequenced. A total of 216,694 bp of genomic sequence was generated and compared with sequences deposited in GenBank databases. In addition to identifying established class I region genes, a number of potential new genes were identified, including several which were not included in the recent major histocompatibility complex (MHC) consensus sequence map. Of particular interest are several new transcripts in the psoriasis susceptibility region.

Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei.

The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA-F is a human non-classical MHC molecule. Recombinant HLA-F heavy chain was refolded with 2-microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high-affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA-F. HLA-F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines and from HUT-78, a T cell line. HLA-F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA-F tetramers stain peripheral blood monocytes and B cells. HLA-F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA-F, suggest that HLA-F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.