Autoantigens PLA2R and THSD7A in membranous nephropathy share a common epitope motif in the N-terminal domain.

Patients with membranous nephropathy have autoantibodies against PLA2R (up to 80%), or THSD7A (up to 2%). We previously described the immunodominant epitope within PLA2R but epitopes in THSD7A are still unknown. To find anti-THSD7A sera for this study, we screened 1843 sera from biopsy-proven MN patients by ELISA and identified 22 sera as anti-THSD7A positive representing 1.2% of MN cases. Anti-THSD7A positive sera were further characterized by western blotting and slot blotting on THSD7A protein fragments and peptides. Real time interaction analyses and antibodies off-rate could be reliably determined using bio-layer interferometry. A signature motif in the N-terminal domain of THSD7A (T28mer) with sequence homology to the major PLA2R epitope (P28mer) was identified. B-cell epitope prediction analysis and homology modelling revealed this sequence to be antigenic and surface available suggesting it is accessible for the antibody to bind. All ten selected sera bound to the T28mer confirming this sequence as a dominant epitope in THSD7A. Reactivity to this sequence was lost following kallikrein protease cleavage within the predicted epitope. Importantly, cross-reactivity of both PLA2R and THSD7A autoantibodies was observed at the peptide but not the protein level. We propose that this common motif shared by both autoantigens could be an epitope involved in the initial B-cell triggering event in MN.

Glomerular basement membrane proteoglycans are derived from a large precursor.

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.

Trophoblast-derived heparanase is not required for invasion.

To invade the decidua and myometrium, extravillous trophoblast must degrade an assortment of extracellular matrix (ECM) components. The uterine wall is rich in heparan sulphate proteoglycans (HSPG), which interact with collagen, laminin and fibronectin, and bind a variety of growth factors. HSPG are catabolised by heparanase, an enzyme that is highly expressed in the placenta. The aim of this study was to investigate the role of heparanase in first trimester trophoblast invasion. First trimester cytotrophoblasts (CTB) were isolated by trypsin digestion followed by centrifugation on a Percoll gradient. Cells were cultured on Matrigel to promote an extravillous phenotype. Heparanase expression was studied by immunohistochemistry and confocal microscopy. Trophoblast invasion was assessed using an in vitro transwell assay. A high level of heparanase was observed in isolated first trimester trophoblast; however, a function-blocking antibody did not inhibit invasion of primary CTB or the extravillous trophoblast cell line SGHPL-4 at 21% oxygen. In contrast to cancer cells, heparanase expression was not increased following culture at 3% oxygen, and trophoblast invasion was not retarded by the blocking antibody under these conditions. Heparanase expression was observed in stromal cells and vascular endothelium in first trimester parietal decidua. Expression was evident on the cell surface and in the nucleus of trophoblast and decidual cells. In conclusion, trophoblast heparanase is not required for invasion in vitro. Its abundant expression suggests another role during pregnancy, perhaps in controlling the availability of ECM-bound growth factors or acting as a transcription factor.

Plasmodium falciparum malaria in south-west Nigerian children: is the polymorphism of ICAM-1 and E-selectin genes contributing to the clinical severity of malaria?

Plasmodium falciparum malaria remains a major public health hazard in sub-Saharan African children. While the factors that determine the variations in clinical outcome of a malaria have not been completely defined, both host and parasite factors, as well as the complex molecular interactions between them have been implicated. The cyto-adherent properties of the P. falciparum-infected red blood cells are considered as key properties in the pathogenesis of malaria and the polymorphisms of the host adhesion molecules could contribute to the severity of malaria. Clinical information and blood samples were collected from 223 children from Ibadan (south-west Nigeria), median age of 34.5 months, presenting with different clinical manifestations of malaria--clinically asymptomatic parasitism (ACP), acute uncomplicated malaria (UM) and severe malaria (SM)--as defined by WHO criteria. The polymorphisms of genes coding for four human adhesion molecules at six different loci (ICAM-1 exons 2, 4 and 6, E-selectin exon 2, CD36 exon 10, and PECAM exon 3) were studied. DNA samples were prepared for further genotyping of the six exons mentioned above by PCR-RFLPs using the appropriate restriction digests for each loci. The ICAM-1 exon 4 locus was monomorphic. All the other loci were at Hardy-Weinberg equilibrium (HWE). The E-selectin locus had very low heterozygosity (approximately 0.06) in contrast to the other loci under study (0.23-0.44). Once the data was further processed for covariates (age and parasite density) and taking as the reference category the ACP group, results show that in the presence of the G allele at the ICAM-1 exon 6 there is an increased risk (3.6 times) of severe malaria. As far as the T allele in the E-selectin exon is concerned, the number of sampled DNAs with the T allele within both the UM and SM categories is too low for drawing any relevant conclusion at this stage. In conclusion, these results suggest that genetic polymorphisms at host adhesion molecules loci are an important variable in the susceptibility to severe malaria. Further studies of host loci are needed to further delineate which polymorphisms are associated with severe malaria and increase our knowledge of the biology of host-parasite interactions.

Transplantation of metanephroi to sites within the abdominal cavity.

A novel approach to circumventing the shortage in transplantable donor organs is the use of embryonic primordia that develop inside the host. Previously published work has shown that transplantation of rat fetal kidney primordia (metanephroi) onto the omentum of adult rat hosts results in growth and development of the metanephroi into functioning kidney units capable of providing a measurable renal function. However, for anatomical and physiological reasons the omentum may not provide the ideal site for transplantation and may limit the maximum renal function that the transplants can achieve. We postulate that it may be possible to increase the renal function of the transplants by transplantation to sites with increased blood flow. To test this we transplanted rat embryonic day 15 metanephroi into the retroperitoneal fat adjacent to major blood vessels in the peritoneum of unilaterally nephrectomized rats; 21 days later the transplants were examined and suitable transplants connected to the host urinary system. Approximately 130 days later the glomerular filtration rate of the connected transplants was analyzed. Our results show that transplantation of metanephroi to the regions highlighted in this study results in an increased presence of urinary cysts, suggesting increased early renal function in the transplants compared to metanephroi transplanted onto the omentum, but most importantly we show that we can increase the renal function of the transplants to a level comparable with other renal therapies such as dialysis. This work suggests life-sustaining renal function could be achieved through transplantation of renal primordia.

A peroxidase-linked enzyme immunoassay for tumour necrosis factor alpha utilising alternative colorimetric or chemilumimetric substrates.

We present a double antibody immunoassay for tumour necrosis factor alpha (TNF alpha) with a peroxidase dependent endpoint which can be detected by absorbance or chemiluminescence depending on the choice of substrate. The chemilumimetric and colorimetric assays have a detection threshold in human serum of 3.9 pg/ml and 7.8 pg/ml respectively and are able to recognise both rTNF alpha and natural TNF alpha. Concentrations of TNF beta, interleukin-1 alpha (IL-1 alpha), IL-beta, IL-2, IL-3, IL-6 or interferon-gamma (IFN-gamma) up to 5 ng/ml failed to show any cross-reactivity. The monoclonal antibody clone 5-2, used in the assays, did not neutralise rTNF alpha in the L929 bioassay. The assay was able to detect rTNF alpha in the presence of excess concentrations of both TNF alpha receptors (p55 and p75). Removal of interference by rheumatoid factor was achieved by the absorbance of the polyclonal antiserum with mouse serum and the inclusion of 10(-2) M dithiothreitol in the buffer containing the TNF alpha polyclonal antiserum. The assay will be useful for the quantitation of endogenous human TNF alpha in serum, other body fluids and culture supernatants, and can also be used to monitor levels of rTNF alpha in clinical trials.

Investigation of IgG4 levels in atopic patients using a competitive inhibition assay employing biotinylated IgG4 myeloma and avidin peroxidase.

Modification of a 'sandwich' ELISA assay developed for the determination of serum IgE levels proved to be unsatisfactory for the measurement of IgG4. This was attributed to the limited capacity of the microtitre plate solid phase which required high serum dilutions in order to measure IgG4 levels. To overcome this problem a competitive inhibition assay was developed with monoclonal anti-IgG4 attached to the plate. In this system biotinylated IgG4 myeloma and sample IgG4 compete for the limited antibody binding sites present on the solid phase. The attached biotinylated myeloma is detected by addition of avidin conjugated with peroxidase and following development with substrate, IgG4 levels are calculated by reference to a calibrated inhibition curve. The inhibition ELISA assay has been used clinically to measure IgG4 levels in atopic and normal individuals and the values obtained correlated closely (r = 0.99) with the IgG4 levels determined by radial immunodiffusion. For 43 atopic dermatitis patients investigated the median IgG4 level was 1.1 g/l which was significantly elevated when compared to a median of 0.385 g/l for 60 blood donors (P less than 0.0001, Mann-Whitney U). Among the 47 hay fever patients investigated the median was 0.6 g/l which, although lower than in atopic dermatitis, was again significantly increased (P less than 0.025). Within this latter group, 25 patients were investigated for the effects of desensitization with commercial grass pollen injections. The total IgG4 showed a variable but significant rise between the start and finish of treatment (P less than 0.01 Wilcoxon signed ranks test).

Antithymocyte globulin preparations after heart transplantation. Cytokine responses in vivo and in vitro.

It is accepted that antithymocyte globulin (ATG) preparations vary in their bioactivity and side effects. However, this is poorly documented in the literature. We compared the clinical course and cytokine response of heart transplant patients who had received either Merieux or Stanford ATG preparations. The serum cytokine response (interleukin [IL]-6, tumor necrosis factor [TNF]-alpha, IL-4, and IL-10) of 28 consecutive heart transplant recipients was measured for 14 days after surgery using ELISAs. The effect of various ATG preparations on cytokine stimulation of whole blood in vitro was also evaluated. There was a much greater in vivo IL-6 and TNF-alpha response to Merieux than to Stanford ATG (P < 0.0005). There was little IL-4 or IL-10 response with either preparation. No side effects could be attributed to either treatment. No significant difference was seen in the frequency of rejection at 30, 90, or 365 days. More infection episodes occurred in the group treated with Stanford ATG at 30 days (0.5 compared with 0.2 episodes/patient; P = 0.097), 90 days (1.2 compared with 0.5 episodes/patient; P = 0.17), and 365 days (2.8 compared with 1.8; P = 0.59), although none of these differences were statistically significant. When tested in vitro for cytokine stimulation, the in vivo pattern was confirmed, with Merieux ATG producing greater levels of TNF-alpha and IL-6 than Stanford ATG. The differences in cytokine stimulation may be reflected in different immunosuppressive activities. Further research to elucidate the important components of immunosuppressive activity while excluding potentially detrimental effects is important.

Transforming growth factor beta 1 in renal allograft recipients.

Transforming growth factor beta (TGF beta 1) is a prosclerotic cytokine implicated in several disease processes. Recent reports have demonstrated a role for TGF beta 1 in experimental models of glomerulonephritis, focusing attention on the relevance of TGF beta to renal fibrogenesis in human disease. The study reported here was designed to investigate whether circulating, active TGF beta 1 could be detected in renal allograft recipients, and whether plasma levels correlated with episodes of rejection, a process involving both inflammation and fibrosis. We have developed an ELISA assay for active TGF beta 1 using commercially available antibodies, and measured plasma levels in 43 healthy controls, 11 patients with membranous nephropathy (MN) and impaired renal function, 17 transplant recipients with stable renal function, 27 patients with acute cellular rejection, 7 patients with chronic vascular rejection, and 10 patients with acute tubular necrosis and/or cyclosporine toxicity. In the last three groups diagnoses were biopsy-proved, and all samples were collected at the time of biopsy. TGF beta 1 was also measured in urine samples from 8, 11, 0, 9, 4, and 7 individuals, respectively, from each group. TGF beta 1 was not detected in plasma from any of the healthy controls or any of the MN patients, (detection limit of assay 0.1 ng/ml). By comparison, it was significantly increased in all groups of transplant recipients (unpaired t test, P < 0.01), but there were no significant differences between the transplant groups. Plasma TGF beta 1 level did not correlate with renal function (estimated by either serum creatinine or reciprocal creatinine), kidney donor age, recipient age, time since transplantation, or cyclosporine plasma trough level. TGF beta 1 was found in every urine sample tested from healthy controls, with a range from 1 ng/ml to 35 ng/ml. Among the 20 transplant patient urines tested, 2 were negative, 18 were positive but within the range determined for the healthy controls. There were no significant differences between the groups.

Serum cytokines in human heart transplant recipients. Is there a relationship to rejection?

Cytokines are important in the pathogenesis of allograft rejection. Some studies have suggested a positive relationship between serum levels of cytokines and rejection, so this study was designed to investigate the presence of a range of cytokines in a large cohort of cardiac transplant recipients. We used enzyme linked immunosorbent assays (ELISA) to examine sequential serum samples from 28 consecutive heart transplant recipients; length of follow up varied between 2 and 566 days (median 357 days). Serum levels of IL-2, 4, 6, 10, TNF-alpha, and IFN-gamma were measured. We compared these results with detailed data on patients' clinical courses, including histological rejection, infection, and therapeutic use of antithymocyte globulin (ATG). No significant relationship was found between rejection and serum cytokine levels for samples taken more than 30 days after transplantation. Prior to this cytokine levels were significantly disturbed by the use of cytolytic therapy for induction immunosuppression. Serum cytokine levels sometimes showed peaks that appeared to be related to rejection, or occasionally to infection, but these relationships were not consistent. Serum TNF-alpha and IL-6 were consistently elevated within a few days of administration of ATG. We conclude that there is no systematic relationship between serum cytokine levels and histological rejection or infection in cardiac transplant recipients.

Serum and urine IL-6 and TNF-alpha in renal transplant recipients with graft dysfunction.

The object of the study presented here was to test whether measurement of blood or urine IL-6 or TNF-alpha could discriminate between the most common causes of renal allograft dysfunction, thus avoiding a biopsy. We present data here in which serum and urine IL-6 and TNF-alpha levels were measured at the same time as a diagnostic renal biopsy was performed. TNF-alpha and IL-6 were measured by sandwich ELISA. Thirty patients had acute cellular rejection, 18 had acute tubular necrosis/CsA toxicity, and 9 had chronic vascular rejection. There was no difference in the levels of IL-6 measured in serum and urine among the three categories of graft dysfunction (t < 1.31; P > 0.20). A similar result with considerable overlap between the groups was seen with TNF-alpha (t < 0.78; P > 0.44). Stratifying the results according to the precise immunosuppressive therapy, CsA dose, body weight, CsA level, body temperature, serum creatinine, the number of previous rejection episodes, original cause of renal failure, or the time elapsed since the transplant did not alter the results. The ratio of serum IL-6 divided by trough CsA level was compared among the three groups and there was no significant difference among them (t < 1.79; P > 0.09). In the light of our results, we therefore suggest that previously published reports of the clinical value of serum and or urine IL-6 and or TNF-alpha in relatively small numbers of patients, not all of whom had been biopsied and in whom rigorous clinical and statistical criteria had not been met, should be viewed with caution.

Expression of cytokine messenger RNA after heart transplantation: relationship with rejection and serum cytokines.

Different groups of cytokines may initiate or inhibit the rejection process. We used the polymerase chain reaction to study the expression of cytokine mRNA for interleukin (IL)-2, -4, -6 and -10, tumor necrosis factor-alpha, and interferon-gamma in 187 biopsy specimens from 24 human cardiac transplant recipients 5-555 days after transplantation. Cytokine levels in the serum were also measured. Cytokine mRNA was detected in 38.5% of biopsy specimens. IL-10 mRNA was detected more frequently with mild or absent rejection (11.6% in grades 0 and 1 - vs. 1.4% in grades 2 and 3, P=0.01). Up to 90 days after transplantation, IL-2 mRNA was detected more frequently with moderate rejection (13% in grades 2 and 3 vs. 0% in grades 0 and 1, P=0.076), and IL-4 mRNA was detected more frequently with mild or absent rejection (16% in grades 0 and 1 - vs. 0% in grades 2 and 3, P=0.061). More than 90 days after transplantation, IL-2 mRNA was detected more frequently with mild or absent rejection (10% in grades 0 and 1 vs. 0% in grades 2 and 3, P=0.078). Serum IL-4 levels corresponding to biopsy specimens positive for IL-4 mRNA were higher than those corresponding to specimens negative for IL-4 mRNA (59 pg/ml vs. 32 pg/ml medians, P=0.028). Our results suggest that IL-10 and possibly IL-4 (T helper 2 cytokines) may suppress graft rejection, whereas IL-2 (T helper 1 cytokine) may promote cellular rejection. In addition, cytokine profiles may change with length of time after transplantation. The association of elevated serum levels of IL-4 with increased expression of intragraft IL-4 mRNA may suggest release of this cytokine from the graft into the circulation.

A genetic marker of high TNF-alpha production in heart transplant recipients.

The cytokine TNF-alpha has been implicated in the pathogenesis of both acute and chronic transplant rejection. Levels of the cytokine are known to vary in a normal population, leading to speculation that high responders may be at greater risk of rejection. Particular TNF region polymorphic markers have been associated with increased TNF-alpha levels and a biallelic polymorphism has been identified at position -308 of the TNF-alpha promoter that may contribute significantly to the interindividual variation in healthy persons. We describe here a new association between a polymorphic locus in the TNF gene region and increased production of TNF-alpha in heart transplant recipients. We studied two microsatellite markers that flank the TNFA gene, as well as a biallelic polymorphism at position -308 of the TNFA promoter, and found that the microsatellite allele TNFd3 was significantly associated with the capacity of leukocytes to produce TNF-alpha in vitro. No association was demonstrated for the promoter region polymorphism. Patients were receiving cyclosporine (CsA) and prednisolone (pred) at the time of sampling, which are known to interrupt 5' regulation of TNFA transcription in T cells and macrophages and may therefore negate the influence of the -308 polymorphism. Because of this we suggest that TNFd3 may be a marker for a 3' repressor region polymorphism that is of importance in immunosuppressed individuals.

Serum cytokine levels in patients undergoing bone marrow transplantation.

Daily serum samples collected during 6 autologous and 13 allogeneic BMT were assayed retrospectively for tumour necrosis factor-alpha (TNF), interleukin-6 (IL-6) and C-reactive protein (CRP). In addition, for 6 allogeneic transplant patients soluble TNF receptor (sTNFR) levels were determined. IL-6 levels were regularly raised during febrile episodes and closely mirrored changes in serum CRP but were not predictive for non-infectious major transplant-related complications (TRC). Levels of TNF showed no such close association with infection and in contrast to previously reported data for allogeneic transplants having TRC, TNF levels were consistently detectable in only 3 of 9 patients. Pre-transplant levels were not predictive for the development of TRC and no profile was recognized to be specific for a particular complication. In transplants with only minor complications TNF levels remained consistently undetectable. sTNFR levels increased in a more stable manner in association with TRC, suggesting that they may be a more suitable marker to monitor major TRC.

Mast cells: the forgotten cells of renal fibrosis.

BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.

Vascular endothelial growth factor mRNA expression in minimal change, membranous, and diabetic nephropathy demonstrated by non-isotopic in situ hybridisation.

AIM: To investigate vascular endothelial growth factor (VEGF) mRNA expression in glomerular disease in the context of heavy proteinuria. METHODS: Non-radioisotopic in situ hybridisation was performed using a cocktail of 12 deoxyoligonucleotides complementary to VEGF mRNA labelled during solid phase synthesis with 2,4-dinitrophenyl. Archival renal biopsies were studied from cases of minimal change nephropathy, membranous nephropathy, diabetic nephropathy, and controls, matched for age, sex, race, and storage time. Hybrid detection used NBT/BCIP colorimetric development. RESULTS: More VEGF mRNA positive glomerular cells per unit cross sectional diameter were seen in minimal change nephropathy (mean (SEM), 19.35 (1.5)) compared with controls (12.6 (1.73)), p < 0.01. In contrast, fewer were seen in diabetic nephropathy (5.93 (0.97)) compared with controls (9.97 (1.25)), p < 0.03. Analysis of membranous nephropathy (10 (1.62)) showed no difference from controls (10.98 (1.51)), NS. In addition, in minimal change nephropathy there was a significant correlation between 24 hour protein excretion at the time of biopsy and the number of VEGF mRNA cells per glomerulus (r = 0.08, p = 0.01). CONCLUSIONS: Using non-radioisotopic in situ hybridisation, VEGF mRNA is almost exclusively expressed by visceral glomerular epithelial cells. Abnormal numbers of cells are seen in both minimal change and diabetic nephropathy. As VEGF exists in a number of functionally distinct isoforms, further study of qualitative VEGF isoform expression in diagnostic groups is indicated.

An association between antibodies specific for endothelial cells and renal transplant failure.

Human leucocyte antigen (HLA)-specific antibodies, present at the time of transplant, cause renal transplant rejection but cases of rejection of HLA-identical renal transplants indicate that antibodies to non-HLA antigens may also be detrimental. There is increasing evidence that antibodies to antigens present on endothelial cells and monocytes, and on endothelial cells alone, are associated with transplant rejection. We investigated 105 patients with failed renal transplants for the presence of endothelial cell reactive antibodies and compared them with 94 successful transplant patients to determine the role of non-HLA antibodies in transplant failure. Patient sera were tested by enzyme-linked immunoabsorbent assay (ELISA) using as a target fixed cells either from the endothelial/epithelial cell line EAHy.926 or primary cultures of human umbilical vein endothelial cells. Antibody binding was detected using an alkaline phosphatase-conjugated anti-human immunoglobulin G (IgG) antibody. Fourteen of the 105 failed transplant patients had endothelial cell-reactive antibodies as compared with only three of the 94 patients with successful transplants (Fisher's exact test, p = 0.02). Antibody-positive sera were absorbed with the epithelial cell line A549 to remove antibodies directed against the epithelial component of EAHy.926 and with a pool of lymphoblastoid cell line cells to remove HLA-specific antibodies. Absorption did not reduce antibody activity showing the antibodies to be directed against endothelial cell determinants. Antibody-positive sera were also tested by flow cytometry against the monocyte cell line THP-1 and 13 of the 14 patients were negative. In conclusion, we have demonstrated the presence of IgG antibodies directed against endothelial cell determinants in renal transplant recipients in association with renal transplant failure.

Serum soluble adhesion molecules and cytokines in cardiac allograft rejection. The Manchester Transplant Cytokine Group.

AIMS: To examine the relationship between soluble adhesion molecules ICAM-1, E-selectin and VCAM-1, serum cytokines TNF alpha, IL6 and IL2, the IL2 soluble receptor p55 and cardiac rejection in cardiac allograft recipients. METHODS: Seventy-six serum samples from 56 patients were examined. Samples were taken on the day of biopsy. No patient was experiencing concurrent infection. All the samples were examined for ICAM-1, TNF alpha, IL6 and IL2R p55. A smaller number were examined for E-selectin, VCAM-1 and IL2. Specific enzyme-linked immunosorbent assays were used. RESULTS: When grade 0 and grade 3a rejection groups were compared a significant difference was seen between IL6 levels (means 32 pg/ml vs 51 pg/ml, medians both 32 pg/ml, p = 0.007), and a significant difference between ICAM-1 levels (medians 207 ng/ml vs 250 ng/ml versus 303 ng/ml, p = 0.045). No patient without rejection had detectable levels of IL6. There was a correlation between ICAM-1 and E-selectin levels (R = 0.6, p = 0.003). There was no correlation between the other parameters and rejection or each other. CONCLUSIONS: Cytokines and adhesion molecules are of great importance in the mechanisms of transplant rejection and this, in some cases, is reflected in the serum. However, this is not sufficiently consistent to be of diagnostic value.

Sequential development of systemic vasculitis with anti-neutrophil cytoplasmic antibodies complicating anti-glomerular basement membrane disease.

Anti-neutrophil cytoplasmic antibodies (cANCA) were detected in 3 patients with anti-glomerular basement membrane (anti-GBM) disease. All 3 cases presented late in their clinical course, with severe renal involvement and alveolar hemorrhage. Anti-neutrophil cytoplasmic antibodies were associated with clinical features outwith the lungs and kidneys; in one case cANCA were initially absent but subsequently developed concurrently with the clinical appearance of systemic vasculitis as the anti-GBM antibody titer was falling. These findings confirm that cANCA can complicate anti-GBM disease and suggest that cANCA may identify a distinct subset of patients with anti-GBM disease, supporting a pathogenic role for cANCA in the development of systemic vasculitis.