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.

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.

Association between ICAM-1 Gly-Arg polymorphism and renal parenchymal scarring following childhood urinary tract infection.

Renal parenchymal scarring (RPS) following urinary tract infection (UTI) is an important cause of renal morbidity in children. Studies have shown that the intensity of the inflammatory response following infection is related to the risk of RPS. However, genetic variability in this response has not been studied. Adhesion molecules play a crucial role in leucocyte recruitment following infection, and polymorphisms have been reported in the genes for key cell adhesion molecules. We have investigated the possibility that children who develop RPS following UTI may exhibit altered genotype or allele frequencies for polymorphisms of the intercellular adhesion molecule-1 (ICAM-1) (exons 4 and 6), E-selectin (exons 2 and 4), platelet endothelial cell adhesion molecule-1 (PECAM-1) (exon 3) and CD11b (3'UTR) genes, which may predict outcome of UTI. DNA was isolated from 99 children shown to have developed RPS, 43 children with no evidence of scarring (NS) following UTI and 170 healthy controls. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis. When the RPS group was compared with the NS group, there was a significant reduction in the frequency of the ICAM-1 exon 4 A allele (10.6 vs. 21.3%, respectively, chi2 = 6.01, P = 0.014). There was no significant difference in either allele or genotype frequency for any of the other polymorphisms studied. These data suggest that the A allele of the ICAM-1 exon 4 polymorphism may protect against the risk of RPS following UTI and may participate in the regulation of the inflammatory response following UTI.

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.

Angiogenic cytokines in patients with idiopathic interstitial pneumonia.

BACKGROUND: Angiogenesis has been implicated in the pathogenesis of idiopathic interstitial pneumonia (IIP). The aim of this study was to examine the relationship between plasma concentrations of the angiogenic cytokines interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), and endothelin-1 (ET-1) and clinical parameters of disease progression over a 6 month period to identify potential aetiological mediators and prognostic markers of disease activity in patients with IIP. METHODS: Forty nine patients with IIP (40 men) were recruited to the study. Plasma cytokine measurements, pulmonary function tests, and high resolution computed tomography (HRCT) scans were performed on recruitment and after 6 months. Plasma cytokine measurements were also performed in 15 healthy volunteers for control purposes. RESULTS: Patients with IIP had significantly higher median (IQR) baseline concentrations of IL-8 and ET-1 than controls (155 (77-303) pg/ml v 31 (0-100) pg/ml, p<0.001) and (1.21 (0.91-1.88) pg/ml v 0.84 (0.67-1.13) pg/ml, p<0.01), respectively. Baseline concentrations of IL-8, ET-1, and VEGF were significantly related to the baseline HRCT fibrosis score (r = 0.42, p<0.005; r = 0.39, p<0.01; and r = 0.42, p<0.005, respectively). Patients with IIP who developed progressive disease had significantly higher baseline levels of IL-8 (345 (270-497) pg/ml v 121 (73-266) pg/ml, p = 0.001) and VEGF (1048 (666-2149) pg/ml v 658 (438-837) pg/ml, p = 0.019). Over 6 months the change in VEGF was significantly related to the change in HRCT fibrosis score (r = 0.565, p = 0.035) and negatively related to the change in forced vital capacity (r = -0.353, p = 0.035).

Steroid-sensitive nephrotic syndrome and vascular endothelial growth factor gene polymorphisms.

Genetic polymorphisms have been recognized as important determinants of gene expression. Three common single nucleotide polymorphisms have been identified in the promoter and 5' untranslated region of the vascular endothelial growth factor (VEGF) gene: -460 C --> T, -141 A --> C and +405 G --> C. As VEGF has been postulated to play a role in the pathogenesis of childhood steroid-sensitive nephrotic syndrome (SSNS), this study tested the hypothesis that VEGF genotype may be associated with susceptibility to SSNS. We examined the genotype frequencies of these polymorphisms in a total of 116 children with SSNS and 150 control subjects, using polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). There were no statistically significant differences in any of the genotype frequencies between SSNS patients and controls. We conclude that VEGF -460, -141 and +405 genotypes are not associated with susceptibility to childhood SSNS.

Recent advances in cutaneous angiogenesis.

An understanding of the molecular basis of angiogenesis is key to the appreciation of many of the advances made in the field of neovascularization over the past two decades. The sequence of events involved in angiogenesis includes: (i) increased vascular permeability and leakage; (ii) degradation of basement membrane; (iii) endothelial cell proliferation and migration through the surrounding extracellular matrix; and (iv) maturation and stabilization of the newly formed vessel bed. This review provides an update on the molecular basis of such pathways in the skin, with particular emphasis on the endothelial cell-specific vascular endothelial growth factor and angiopoietins as modulators of angiogenesis that can be targeted in therapy of cutaneous disease.

A PCR-RFLP typing method for adhesion molecule gene polymorphisms and allele frequencies in a normal UK population.

We report simple and reproducible PCR-RFLP typing methods for the polymorphisms in the ICAM-1, E-selectin and PECAM-1 genes. The genotype and allele frequencies detected in a normal UK population did not deviate significantly from the Hardy-Weinberg equilibrium; neither did they differ from frequencies previously reported using SSP or SSCP methods.

Plasma and urinary soluble adhesion molecule expression is increased during first documented acute pyelonephritis.

BACKGROUND: The degree of inflammatory reaction and leucocyte trafficking during acute pyelonephritis has been related to the risk of developing renal parenchymal scarring. Adhesion molecules play a central role in leucocyte recruitment during inflammation. AIMS: (1) To determine whether circulating and urinary concentrations of E-selectin and intercellular adhesion molecule 1 (ICAM-1) were abnormal during first documented acute pyelonephritis; (2) to investigate whether circulating or urinary concentrations were predictive for the development of abnormalities on DMSA imaging. METHODS: Plasma and urine samples were collected from 40 children with a first episode of acute pyelonephritis within one week of infection (acute sample) and at six weeks (late sample). Control samples were collected from 21 healthy age matched controls and 18 age matched controls with febrile illness not secondary to urinary tract infection. RESULTS: Plasma and urinary sE-selectin were higher in acute samples (median 176.3 ng/ml and 0.12 ng/mmol respectively) compared with late (97.8 ng/ml and 0.029 ng/mmol) and both control (65.6 ng/ml and 0 ng/mmol) and febrile control (urine 0 ng/mmol) samples. Plasma sICAM-1 was higher in acute samples (428 ng/ml) than controls (365.2 ng/ml), and acute sICAM-1 urine concentrations were higher than febrile control concentrations (3.2 v 0.7 ng/mmol). No correlations were detected between sE-selectin or sICAM-1 and acute or late DMSA scan changes. CONCLUSION: Plasma and urinary sE-selectin and sICAM-1 are significantly increased during acute pyelonephritis, though no correlation exists between the presence of high plasma or urine concentrations and DMSA scan changes, both during acute infection and six weeks post-infection.

Vascular endothelial growth factor expression correlates with tumour grade and vascularity in gliomas.

AIMS: Tumour vascularity and vascular endothelial growth factor (VEGF) expression were studied in 41 primary brain tumours of astrocytic and oligodendroglial origin, in order to define the potential role of VEGF in the vascularization and growth of these tumours. METHODS AND RESULTS: Two commercial monoclonal antibodies to the VEGF protein (from R&D Systems and NeoMarkers), raised against different isoforms, were utilized. Each monoclonal antibody consistently detected the expression of VEGF in different cell types. The R&D Systems antibody only produced surface staining of endothelial cells in tumour capillaries, whereas staining with the Neomarkers antibody was largely confined to tumour cell cytoplasm. High levels of staining were seen with the R&D Systems and NeoMarkers antibodies in 13 and 14 of 15 glioblastomas, respectively, four and three of five oligodendrogliomas, four and seven of 10 anaplastic astrocytomas, one and three of six low-grade astrocytomas and none and none of five pilocytic astrocytomas. There was a close correlation between VEGF expression, tumour vascularity and grade. CONCLUSIONS: These findings support a role for VEGF in the angiogenesis of glioblastoma, anaplastic astrocytoma and oligodendroglioma. The distinct immunoreactivities of the two commercial monoclonal antibodies indicate either there is expression of different splice variants of VEGF or that the epitopes are differentially revealed during synthesis, secretion and receptor-binding of the growth factor. This highlights the importance of using more than one antibody in the evaluation of tissue VEGF expression.

Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.

BACKGROUND: In IgA nephropathy (IgAN), circulating IgA1 molecules display an abnormal pattern of O-glycosylation. This abnormality may potentially contribute to mesangial IgA1 deposition, but this is unproven because the O-glycosylation of mesangial IgA1 has not been analyzed. METHODS: IgA1 was eluted from glomeruli isolated from the kidneys of three IgAN patients obtained after nephrectomy or at postmortem. Serum from these patients, other patients with IgAN, and controls was subjected to the same treatment as the glomerular eluates. The O-glycosylation of eluted and serum IgA1 was measured by lectin binding using an enzyme-linked immunosorbent assay-based system. RESULTS: In all three cases, the lectin binding of IgA1 eluted from the glomeruli of IgAN patients was markedly higher than that of the serum IgA1 of the same individual, and also all but one of a series of serum IgA1 samples from other patients and controls. CONCLUSIONS: The higher lectin binding of glomerular compared with serum IgA1 suggests that O-glycosylated IgA1 molecules abnormally and selectively deposit in the kidney. These results provide the first evidence that mesangial IgA1 is abnormally O-glycosylated, and support a direct role for abnormal IgA1 O-glycosylation in the mechanism of mesangial IgA deposition in IgAN.

Plasma transforming growth factor beta(1) and platelet activation: implications for studies in transplant recipients.

BACKGROUND: Evidence from animal models supports the hypothesis that dysregulated transforming growth factor beta(1) (TGF beta(1)) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGF beta(1) levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGF beta(1) is detecting the platelet-released pool of TGF beta(1), artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGF beta(1) pool; and (ii) the effect of different immunosuppressive drugs on apparent TGF beta(1) plasma levels. METHODS: The effect of two different venepuncture procedures on plasma TGF beta(1) was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGF beta(1). Plasma TGF beta(1) and beta thromboglobulin were measured by ELISA. RESULTS: Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGF beta(1) (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGF beta(1) (median 7.70 ng/ml, interquartile range 5.87-13.64 ng/ml) following acid/ urea activation. The median beta TG level (a measure of platelet degranulation) was 0.71 microg/ml (interquartile range 0.53-1.19 microg/ml) in the special collections compared with 3.39 microg/ml (interquartile range 2.27-4.33 microg/ml) in the vacutainer samples (P=0.0029). Among 52 allograft recipients there was a significantly higher mean TGF beta(1) level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28,090+/-26,860 pg/ml vs 7173+/-10 610 pg/ml, respectively; P<0.002). Mean plasma beta TG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14+/-5.54 microg/ml vs 3.66+/-3.32 microg/ml, respectively; P<0.002). However, when TGF beta(1) values were corrected for the degree of platelet activation (by factoring with beta TG) there was no significant difference between TGF beta(1) levels on cyclosporine or tacrolimus (4117+/-2993 pg/microg beta TG vs 2971+/-658 pg/microg beta TG, respectively; P=0.294). CONCLUSIONS: To avoid erroneous hypotheses concerning TGF beta(1) and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGF beta(1) in clinical practice.

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.

Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production.

Dysregulated vascular endothelial growth factor (VEGF) expression has been implicated as a major contributor to the development of a number of common disease pathologies. The aim of this study was to establish the extent of genetic variability within the VEGF gene and to determine whether this genetic variation influenced levels of VEGF protein expression. The promoter region and exon 1 of the VEGF gene were screened for polymorphisms using single-stranded conformation (SSCP) polymorphism analysis and direct PCR-sequencing. We identified 15 novel sequence polymorphisms most of which were rare. Eleven of these polymorphisms were single base substitutions, three were single base insertions and one was a two base deletion. Thirteen of the polymorphisms were located within the promoter and two in the 5' untranslated region (5'UTR) of the gene. We established PCR-RFLP typing systems for ten of the polymorphisms. For the two common polymorphisms at -460 and +405, we developed a combined sequence specific priming (SSP) PCR typing system to determine the cis/trans orientation of each allele and hence, ascertain haplotypes. A significant correlation was observed between lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cell (PBMC) VEGF protein production and genotype for the +405 polymorphism.