Biomedical subjects

D A Power

Publications and source records attributed to D A Power.

At least 37 records · Page 2Linked to original sources

PubMed1999

AMP-activated protein kinase phosphorylation of endothelial NO synthase.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

AMP-Activated Protein Kinases↗

PubMed1999

Heparin-binding epidermal growth factor-like growth factor is expressed in the adhesive lesions of experimental focal glomerular sclerosis.

BACKGROUND: In this study, we attempted to determine whether heparin-binding epidermal growth factor-like growth factor (HB-EGF) was up-regulated in two chronic models of proteinuria. METHODS: Chronic passive Heymann nephritis (PHN) and puromycin aminonucleoside (PAN) models were induced in Sprague-Dawley rats. HB-EGF expression was studied by Northern blotting, in situ hybridization, and immunohistochemistry. RESULTS: The chronic PAN model was associated with the development of glomerular lesions of focal glomerular sclerosis (FGS), severe interstitial fibrosis, and renal failure. Lesions of FGS were seen in approximately 80% of glomeruli at all time points, with a slight increase in the number of glomeruli showing extensive adhesion between 40 and 90 days. Northern blots of whole kidney tissue showed a 3- to 5.8-fold increased expression of HB-EGF mRNA in the chronic PAN group. Increased mRNA and protein were localized by in situ hybridization and immunohistochemistry to tubules, glomerular epithelial cells (GECs), and cells of Bowman's capsule. HB-EGF mRNA and protein were strongly expressed by epithelial cells involved in the formation of the lesions of FGS. By contrast, in chronic PHN, there was a small increase in HB-EGF, and the extensive lesions of FGS did not develop despite continued, heavy proteinuria. CONCLUSIONS: These data suggest that HB-EGF may contribute to formation of the lesions of FGS, perhaps through stimulation of abortive mitogenesis in GECs or an adhesive interaction between transmembrane HB-EGF and the exposed glomerular basement membrane.

Actins↗

PubMed1999

Renal-dose (low-dose) dopamine for the treatment of sepsis-related and other forms of acute renal failure: ineffective and probably dangerous.

1. Low-dose ('renal-dose') dopamine (i.e. 1-3 micrograms/kg per min) is used widely for the treatment of acute renal failure induced by ischaemia, toxins and/or sepsis. Here we review the scientific rationale, experimental studies and clinical trials evaluating its use in these settings. 2. Renal-dose dopamine augments renal blood flow, sodium excretion and probably glomerular filtration rate in healthy humans and experimental animals and limits ATP utilization and oxygen requirements in nephron segments at risk of ischaemic injury. Renal-dose dopamine is renoprotective in several ischaemic and nephrotoxic models of acute renal failure. 3. However, most studies in humans have not demonstrated prevention of acute renal failure in high-risk patients or improved outcome in those with established acute renal failure. While the safety profile of dopamine in these settings has not been extensively defined, it is known the drug may precipitate serious cardiovascular and metabolic complications in the critically ill. Therefore, we suggest that renal-dose dopamine should not be used for selective renal vasodilatory and natriuretic actions in those patients with acute renal failure until its efficacy is established in randomized control trials. 4. Renal-dose dopamine may be most valuable when combined with agents targeting other events in acute renal failure, such as cast formation, epithelial cell injury and tubule regeneration. These recommendations should not preclude the use of dopamine for its systemic effects in heart failure and septic shock.

Acute Kidney Injury↗

PubMed1998

Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

Animals↗

PubMed1998

Heparin-binding epidermal growth factor-like growth factor in experimental models of membranous and minimal change nephropathy.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the epidermal growth factor (EGF) family. It binds to heparan sulfate proteoglycans via a cationic domain and is a potent mitogen for epithelial cells, fibroblasts and vascular smooth muscle cells. In the present study we have attempted to identify changes in quantity and distribution of HB-EGF in two models of acute glomerular epithelial cell injury, using Western blotting, immunohistochemistry and in situ hybridization. Prior to disease induction, Western blots showed some expression of HB-EGF protein within glomeruli. Within the first three days in the acute puromycin aminonucleoside (PAN) and passive Heymann nephritis (PHN) models, immunohistochemistry and in situ hybridization demonstrated an up-regulation of HB-EGF mRNA and protein in glomerular epithelial cells (GEC). In both cases, increased protein and mRNA was found prior to the onset of proteinuria and continued until day 21 post-induction, the last time point studied. Early in the course of the models, HB-EGF was localized to the cytoplasm of glomerular epithelial cells. At day 21, however, HB-EGF protein was distributed in a nodular pattern within GEC and along the glomerular basement membrane (GBM) in both models, suggesting that the secreted form might bind to the membrane. The increase in HB-EGF protein within glomeruli was confirmed by Western blots of glomerular membrane protein which, however, demonstrated a single 29 kDa species, consistent with the transmembrane form. These data are not consistent with binding of the secreted form of HB-EGF to the GBM. The transmembrane form of HB-EGF is able to signal in a juxtracrine fashion, so increased expression of HB-EGF mRNA and protein by GEC might contribute to the genesis of proteinuria through the initiation of abortive GEC mitogenesis.

Amino Acid Sequence↗

PubMed1998

Heparin-binding EGF-like growth factor mRNA is upregulated in the peri-infarct region of the remnant kidney model: in vitro evidence suggests a regulatory role in myofibroblast transformation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent fibroblast and epithelial cell mitogen that may be important in wound healing. The aim of this study was to determine its distribution and possible function in segmental renal infarction. At day 1 postinfarction, in situ hybridization showed that HB-EGF mRNA was markedly increased by tubular epithelial cells bordering the infarcted zone. At day 3, typical myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA) were present in large numbers at the peri-ischemic border and, over succeeding days, were also seen within the infarcted area. Some of these cells expressed HB-EGF mRNA by in situ hybridization suggesting possible autocrine stimulation. Endothelial cells appeared to be more resistant to ischemia than tubules because some capillaries at the periphery of the infarct, surrounded by infarcted tubules, also expressed HB-EGF mRNA. The staining intensity of HB-EGF mRNA in individual tubules and endothelial cells was maximal at day 5 after infarction, although Northern blots of tissue from the peri-infarct area only showed significantly increased expression of HB-EGF mRNA at days 1 and 3, perhaps reflecting a smaller area of greater intensity of expression at day 5. Because tubular cells expressing high levels of HB-EGF mRNA were directly apposed to myofibroblasts, an attempt was made to determine whether HB-EGF contributed to upregulation of alpha-SMA by human fibroblasts. Although stimulation of the fibroblast cell line MRC-5 with transforming growth factor-beta1 (TGF-beta1) increased alpha-SMA, HB-EGF reduced expression. HB-EGF also strongly inhibited the increased expression of alpha-SMA due to TGF-beta1. Because HB-EGF is a potent fibroblast mitogen and TGF-beta is usually antiproliferative, this study suggests that HB-EGF may contribute to a local balance between fibroblast proliferation and differentiation into myofibroblasts during scarring.

Actins↗

PubMed1998

Regulation of nuclear factor kappaB by corticosteroids in rat mesangial cells.

Nuclear factor kappaB (NF-kappaB) is one of the most important proinflammatory transcription factors. The anti-inflammatory activity of steroids in leukocytes is partly due to inhibition of signaling by NF-kappaB, but it is not known whether steroids inhibit NF-kappaB in kidney cells. Since the mesangial cell is important in several kidney diseases, especially mesangial proliferative glomerulonephritis, the aims of this study were: (1) to define the mechanism of NF-kappaB activation in rat glomerular mesangial cells; and (2) to determine whether steroids inhibit activation of NF-kappaB in these cells. Electrophoretic mobility shift assays (EMSA) showed that interleukin-1beta and tumor necrosis factor-alpha activated NF-kappaB from 15 min to 48 h after stimulation. Supershift EMSA demonstrated that p65 and p50 were the predominant subunits involved. Degradation of the inhibitory subunit IkappaB-alpha was first observed 15 min after stimulation by Western blot, was maximal at 15 to 30 min (>90% by densitometry), and had returned to near normal levels at 90 min. In contrast, IkappaB-beta was maximally degraded at 60 to 120 min and was still reduced at 48 h (<50% of the untreated level). Although treatment of mesangial cells with dexamethasone increased IkappaB-alpha mRNA by 1.92x and protein by 1.45x over controls, pretreatment did not inhibit degradation of IkappaB-alpha or -beta in response to stimulation, or prevent the increase in NF-kappaB binding activity shown by EMSA. However, dexamethasone significantly inhibited the increase in monocyte chemoattractant protein-1 mRNA seen after stimulation with interleukin 1beta, although this was not complete. It did not reduce transcription of an NF-kappaB reporter. In comparison, the pyrrolidine derivative of dithiocarnamate (PDTC), a known inhibitor of NF-kappaB, prevented the increase in NF-kappaB binding activity and significantly reduced transcription of the NF-kappaB reporter. These studies suggest that steroids can partially inhibit transcriptional activation by NF-kappaB in mesangial cells but not through an increase in IkappaB-alpha protein alone. Their effect must occur at the promoter and may be restricted to some NF-kappaB-responsive genes. Therapies that block NF-kappaB more effectively than steroids in mesangial cells, therefore, may be useful in the treatment of mesangial proliferative glomerulonephritis.

Adrenal Cortex Hormones↗

PubMed1997

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.

AMP-Activated Protein Kinases↗

PubMed1997

Importance of the tubulointerstitium in human glomerulonephritis. II. Distribution of integrin chains beta 1, alpha 1 to 6 and alpha V.

Accumulation of extracellular matrix is important in the progression of glomerulonephritis. Since adherent cell types utilize integrins to bind and organize extracellular matrix proteins, we have assessed expression of the beta 1 integrins in sequential sections from 85 human renal biopsies and 4 normal kidneys by immunohistochemical staining. Our results demonstrate strong correlations between expression of the alpha 5 chain within the interstitium, the alpha V chain on proximal and distal tubular epithelium and the presence of chronic histological damage. Moreover, staining for interstitial alpha 5 and proximal and distal tubular alpha V were also strongly associated with expression of certain adhesion molecules (ICAM-1, VCAM-1, E-selectin and L-selectin) and the presence of macrophages within the interstitium, which have been linked, in an earlier study, with the degree of chronic histological damage and disease progression. However, in contrast to our earlier study of adhesion molecules, there were also associations between expression of integrin chains within the glomerulus and tubulointerstitium. For example, there were strong positive associations between staining for alpha 5 on glomerular endothelium and its expression on extraglomerular vascular endothelium and between both mesangial alpha 1 and podocyte alpha 3 and tubular staining for the common beta 1 subunit. While the functional significance of these associations is obscure, they suggest some kind of communication between cells in different sites in the kidney. There were also positive associations between staining for different integrins within the glomerulus, notably mesangial cell staining for alpha 2, glomerular endothelial cells staining for alpha 5 and glomerular epithelial cell alpha 3. These results suggest that there is a coordinated upregulation of integrin expression both within the tubulointerstitium and the glomerulus and that at least some of these integrins (interstitial alpha 5 and distal tubular alpha V) are associated with the expression of other adhesion molecules, macrophage infiltration and the presence of markers of disease progression (interstitial fibrosis and tubular atrophy).

Antigens, CD↗

PubMed1996

Adhesion molecule interactions in human glomerulonephritis: importance of the tubulointerstitium.

Infiltration of leukocytes into glomerular and interstitial regions of the kidney is a key event in the pathogenesis of human glomerulonephritis. This process is mediated by specific adhesion molecules, some of which are expressed in a coordinated fashion following endothelial cell activation. We have assessed the pattern of expression of the selectins (E, P and L), and the counter-receptors (LFA-1 and ICAM-1, and VLA-4 and VCAM-1 in 119 renal biopsies using sequential sections, and have correlated this with the degree of histological damage (tubular atrophy and interstitial fibrosis) and the intensity of the macrophage infiltrate. Sections were stained with the monoclonal antibodies using a standard alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. There were strong correlations between the following: (1) expression of LFA-1, VLA-4, and L-selectin in the periglomerular region, interstitium and in focal interstitial infiltrates and the presence of macrophages in these regions; (2) de novo tubular expression of ICAM-1 and VCAM-1; (3) staining for ICAM-1 and VCAM-1 on focal cellular infiltrates within the interstitium; and (4) staining for E- and P-selectin on extraglomerular endothelium. These are also strongly correlated with the degree of chronic histological damage. There was, however, no correlation between glomerular expression of adhesion molecules or glomerular macrophage infiltration and chronic histological damage. Although expression of VCAM-1 by the glomerular mesangium was strongly correlated with the presence of cells staining for VLA-4 within the glomerulus, glomerular expression of adhesion molecules correlated poorly with their expression in other sites. These results show that coordinated up-regulation of adhesion molecule expression in the tubulointerstitium is associated with interstitial fibrosis and tubular atrophy and may contribute, therefore, to the progression of renal disease.

Biopsy↗

PubMed1996

CD44 in glomerulonephritis: expression in human renal biopsies, the Thy 1.1 model, and by cultured mesangial cells.

CD44 is a transmembrane proteoglycan that serves as a cell adhesion receptor and is involved in cell-cell and cell-matrix interactions, both key events in the pathogenesis of clinical and experimental glomerulonephritis. In addition, recent evidence suggests that the binding of cytokines to proteoglycans could regulate cytokine function. We have, therefore, studied the expression of CD44 by mesangial cells in culture and in experimental (Thy 1.1 model) and human glomerulonephritis. Mesangial expression of CD44 detected by immunohistochemistry was markedly increased four days after induction of the Thy 1.1 model, coinciding with the peak of mesangial cell proliferation and macrophage infiltration. Analysis of 92 human renal biopsies by immunohistochemistry showed that CD44 expression by infiltrating cells within the glomerulus, in focal interstitial infiltrates and within the interstitium (interstitial fibroblasts, and extracellular matrix), was significantly increased in biopsies with a greater degree of histological damage. There was, however, no increase in mesangial staining in diseased kidneys as compared with control sections. In contrast, cultured human mesangial cells expressed CD44 strongly when assayed by immunohistochemistry, immunoprecipitation and Northern blotting. CD44, therefore, is an example of a protein strongly expressed by mesangial cells in vitro and weakly or not at all in vivo, but which is up-regulated in a disease model. In human disease, however, little expression was detected within the glomerular mesangium, which may be related to the greater proliferation and more profound disruption of mesangial architecture seen in the Thy 1.1 model. CD44 expression by infiltrating cells and by components of the interstitium could, however, play an important role in the pathogenesis of chronic progressive renal disease in humans.

Animals↗

PubMed1995

Phenotypic analysis of the glomerular and periglomerular mononuclear cell infiltrates in the Thy 1.1 model of glomerulonephritis.

BACKGROUND: The phenotype of macrophages invading the mesangium and periglomerular region has not been described in experimental mesangial proliferative glomerulonephritis, although it has implications for the mechanism of entry of these cells into these locations and their function once there. We have, therefore, determined the phenotype of the periglomerular leukocytic infiltrate in the Thy 1.1 model of mesangial proliferative glomerulonephritis and compared it with that of cells invading the mesangium. EXPERIMENTAL DESIGN: The Thy 1.1 model was induced in Lewis rats, and sections were taken at 1 hour and at 1, 4, 9, 30, and 90 days postinduction. Immunohistochemistry was performed using monoclonal antibodies against macrophage markers (ED-1, CD4, RT1-B, and ED-2), chains of the beta 2 integrins (CD18, CD11a, and CD11b), T and B cell markers (CD8, T cell receptor, interleukin-2 receptor, and MRC OX33), and markers of mesangial cell proliferation (proliferating cell nuclear antigen, alpha-smooth muscle actin). Sections were compared with those obtained from control animals. RESULTS: In untreated rats, a striking resident periglomerular macrophage population (phenotype ED-1-ve ED-2-ve CD4+ve RT1-B+ve CD18+ve CD11a+ve CD11b+ve) was found, confirming a previous report. From 24 hours postinduction, this resident macrophage population was supplemented by a population whose predominant phenotype (ED-1+ve ED-2-ve CD4+ve RT1-B+ve CD18+ve CD11a+ve CD11b+ve) was identical to that of macrophages infiltrating the mesangium. Both infiltrates peaked at 4 days and returned to base-line levels by 1 to 3 months. There was no significant lymphocyte infiltrate within the glomerulus and only a minimal periglomerular T cell infiltrate. CONCLUSIONS: These data show, first, that disease limited to the mesangium can lead directly to periglomerular macrophage infiltration. Second, the presence of CR3 (i.e., CD11b and CD18) and LFA-1 (i.e., CD11a and CD18) on the macrophage infiltrate indicates that both ligands are important for cells to enter the mesangium and periglomerular areas. Third, the marked phenotypic and temporal similarities between the mesangial and periglomerular macrophage infiltrates suggests that a common factor(s) is involved in their pathogenesis. Finally, expression of RT1-B (Ia) but not ED-2 is reported to be typical of interstitial dendritic cells rather than tissue macrophages, suggesting a unique function for the glomerular and periglomerular macrophage infiltrate in this model.

Animals↗

PubMed1994

Regulation of alternative splicing of the fibronectin IIICS domain by cytokines.

The primary fibronectin gene transcript is alternatively spliced in three regions, designated EIIIA, EIIIB and IIICS. While the functions of the EIIIA and EIIIB domains are still to be established, either end of the IIICS domain contains the cell-binding sites CS1 and CS5 recognised by the integrin VLA-4 which is present on mononuclear cells. We have determined the effects of three cytokines upon fibronectin mRNA and IIICS splice variants in four cultured human cell lines using Northern hybridisation and PCR. In the cell line CAKI-2, interleukin 1 beta, Platelet-derived growth factor BB and transforming growth factor beta 1 all increased mRNA splice variants where the CS1 and CS5 regions were removed. This cytokine-induced change would lead to a decrease in the CS1 and CS5 cell binding sites within the extracellular matrix. All the changes in spliceosome function increase use of downstream 3' splice acceptor sites and may represent use of a single common pathway by three different cytokines.

Alternative Splicing↗

PubMed1994

Urokinase-plasminogen activator is synthesized in vitro by human glomerular epithelial cells but not by mesangial cells.

The plasmin protease system may have a role in maintaining the patency of renal tubules and in regulating matrix degradation within the glomerulus. Urokinase-plasminogen activator (u-PA) is a serine protease which plays an important part in the regulation of plasmin production from plasminogen. The synthesis of u-PA by cultured human glomerular cells, in particular mesangial cells, is controversial. The present study describes the presence of u-PA in supernatants of pure cultures of human glomerular epithelial cells (EC), cocultures of EC and human mesangial cells (MC) and whole glomeruli, but not within pure cultures of MC. To confirm the synthesis of u-PA mRNA in glomerular EC, cocultures of EC and MC were tested by in situ hybridization with u-PA antisense and sense digoxigenin-labeled RNA probes. Cytoplasmic localization of u-PA mRNA was demonstrated only in the EC, thus confirming the absence of synthesis of u-PA by human mesangial cells in culture.

Electrophoresis, Polyacrylamide Gel↗

PubMed1994

Membranous glomerulonephritis associated with hepatitis C virus infection in an adolescent.

The clinical, laboratory and pathological findings are reported in an adolescent patient with hyper-IgM immunodeficiency syndrome (dysgammaglobulinemia) which was complicated by primary sclerosing cholangitis and membranous glomerulonephritis. Hepatitis C virus (HCV) RNA was detected in serum by the polymerase chain reaction. A possible etiological relationship between hepatitis C virus infection and membranous nephropathy is discussed.

Adolescent↗

PubMed1993

The effect of transforming growth factor beta 1 on mesangial cell fibronectin synthesis: increased incorporation into the extracellular matrix and reduced pI but no effect on alternative splicing.

Fibronectin is a multidomain glycoprotein which accumulates in mesangial proliferative glomerulonephritis (MPGN). Recent evidence has implicated transforming growth factor beta (TGF-beta) in the pathogenesis of experimental MPGN. We have, therefore, examined the influence of TGF-beta 1 on mesangial cell fibronectin synthesis. Considering, first, levels of mRNA, TGF-beta 1 increased steady-state fibronectin RNA in cultured mesangial cells by 1.9 times 24 hr after treatment of cycling mesangial cells and by 11.8 times in growth-arrested cells. There was, however, no alteration in fibronectin pre-mRNA splicing in either the EIIIA or IIICS regions. Fibronectin protein concentrations in cell culture supernatants, determined by immunoprecipitation of supernatants from cells labeled with [35S]methionine and by ELISA, were not increased by treatment with TGF-beta 1. Western blots and immunoprecipitation of metabolically labeled cells showed that fibronectin was increased, however, in the deoxycholate-insoluble extracellular matrix (ECM) of cells stimulated with TGF-beta 1. TGF-beta 1 altered the physicochemical properties of fibronectin in ECM and supernatant such that the isoelectric point of fibronectin, determined from Western blots of 2D SDS-PAGE gels, was reduced so that both became more acidic. These studies demonstrate, therefore, that in addition to increasing its synthesis, TGF-beta 1 increases incorporation of fibronectin into the ECM. Because fibronectin possesses binding sites for other ECM proteins, greater incorporation of fibronectin following TGF-beta 1 treatment may be an important pathogenetic mechanism in mesangial sclerosis. Moreover, the altered charge of fibronectin may increase localization of serum immunoglobulins to the mesangium.

Alternative Splicing↗

PubMed1993

A human lymphocytotoxic autoantibody is encoded by antibody variable region genes in their germ line configuration.

Lymphocytotoxic autoantibodies are commonly found in sera from kidney dialysis patients, where they interfere with interpretation of the crossmatch test. We have produced a monoclonal lymphocytotoxic autoantibody by EBV transformation of PBLs from a dialysis patient, followed by fusion of the transformed cells with a heteromyeloma. The autoantibody derived, called FWE, was IgM kappa class and its pattern of reactivity against T and B lymphocytes, cells from patients with chronic lymphocytic leukemia and K562, was similar to that described for lymphocytotoxic autoantibodies found in sera. It was absorbed by fetal but not adult human erythrocytes, suggesting the antigenic determinant might be the blood group antigen i. cDNA encoding the variable domain of FWE was amplified by polymerase chain reaction, cloned into the vector M13 mp18, and sequenced. The variable region of the kappa light chain (V kappa) was 98.8% identical over a 260-bp stretch with a known germ line sequence and the junctional (J kappa) region was identical over 16 bp with the germ line sequence J kappa 2. The variable region of the heavy chain (VH) was 99.3% identical over a 268-bp overlap with the germ line gene VH4.21, a member of the VHIV family, and the junctional region of the heavy chain (JH) was identical with the germ line JH gene JH5 over 46 bp, with a truncated 5' end. The diversity region was not identified. These data suggest that the genes required to produce human lymphocytotoxic autoantibodies are encoded within the germ line and, therefore, that all dialysis patients may be able to produce them under certain circumstances.

Amino Acid Sequence↗

PubMed1993

An immunohistological study of epidermal growth factor receptor and neu receptor and neu receptor expression in proliferative glomerulonephritis.

Many forms of glomerulonephritis including IgA nephropathy are characterized by mesangial cellular proliferation. Since epidermal growth factor is a potent mitogen for cultured human mesangial cells, we have attempted to localize and quantify the expression of its receptor in normal and abnormal renal biopsies using immunohistochemistry. Using a particular antibody (Amersham, clone EGFR1), the epidermal growth factor receptor (EGF-R) was shown to be predominantly localized in the mesangium of the glomerulus. Visual estimates of intensity of staining suggested that expression of this receptor may be increased in some IgA disease patients with mesangial proliferative glomerular lesions. The neu receptor which has a 50% homology with EGF-R was, however, absent from the glomerulus and cultured mesangial cells did not express detectable levels. Expression of EGF-R by cultured mesangial cells, as assessed by immunostaining, was weak and it was not possible to induce detectable upregulation using different cytokines. The factors leading to increased expression of EGF-R in glomerulonephritis, therefore, remain unknown. Our findings suggest that signalling via EGF-R may play a role in the pathogenesis of proliferative glomerulonephritis. Despite its homology with EGF-R, the neu receptor is unlikely to have similar importance.

Cells, Cultured↗