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Biomedical subjects

S J Harper

Publications and source records attributed to S J Harper.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Diabetic Nephropathies↗
PubMed1999

Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant.

Vascular endothelial growth factor (VEGF) mediates increased vascular permeability and endothelial mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(121) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(145), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

Aged↗
PubMed1998

Quantification of Fos immunoreactivity in cortical cultures treated with growth factors.

Among the effects mediated by growth factors in vivo are the switch to growth arrest and differentiation during normal development of the nervous system, survival during the period of naturally occurring cell death, and plasticity and repair of neurons in the adult brain. Much interest has focused on the signalling pathways utilised by growth factors with a large proportion of experiments carried out using the phaeochromocytoma cell line. Here we have quantified Fos immunoreactivity following stimulation of primary cultures of rat cortical neurons with a variety of growth factors including neurotrophins and cytokines. Expression of Fos has been quantified in these cultures using an ELISA technique, and immunocytochemistry followed by digital stereology. Treatment of cultures with brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4) causes a dose-dependent increase in Fos expression, while neurotrophin-3 (NT-3) causes an increase but at high concentrations only. A sub-population of cortical neurons within the cultures express Fos in response to fibroblast growth factor-1 or fibroblast growth factor-2 but no cells respond with Fos expression on treatment with insulin-like growth factor-1. We conclude that BDNF and NT-4 cause dose-dependent increases in the number of Fos immunoreactive cells.

Animals↗
PubMed1998

Mouse cortical neurones lacking APP show normal neurite outgrowth and survival responses in vitro.

We have cultured neurones from the developing cortex of mice that have had the amyloid precursor protein gene deleted (APP-null). Neurones cultured for a period of 24 h show similar neurite outgrowth and survival responses to wild-type neurones. Similar neurite outgrowth responses were also seen when neurones from APP-null mice were treated with a neurotrophic peptide derived from the APP sequence and compared with wild-type neurones. Finally, cortical cultures derived from APP-null mice showed similar survival responses to the toxic amyloid-beta peptide.

Amino Acid Sequence↗
PubMed1998

The nucleotide sequence of the IgA1 hinge region in IgA nephropathy.

BACKGROUND: Mesangial IgA1 deposition is characteristic of IgA nephropathy (IgAN). Structural abnormalities of the IgA1 glycoprotein may play a key role in its mesangial deposition, particularly the recently described abnormalities of O-glycosylation of the IgA1 hinge region. The mechanism of abnormal O-glycosylation has not yet been elucidated; it is not clear whether there is an alteration in the amino acid sequence of the hinge region, modifying the number of O-glycosylation sites available, or whether there is a post-translational defect in the glycosylation process. METHODS: The O-glycosylation of serum IgA1 from a series of patients with IgAN and matched controls was assessed by lectin binding assay. We then used dideoxy-sequencing of the PCR-amplified hinge region of the alpha1 heavy chain gene to compare the hinge region nucleotide sequence in IgAN and controls. We also compared cDNA transcripts of alpha1 hinge region mRNA to look for evidence for a transcriptional abnormality in IgAN. RESULTS: Lectin binding assays confirmed that the IgAN subjects used in this study did indeed display the previously reported abnormality of IgA1 O-glycosylation. However, the hinge region nucleotide sequence of the alpha1 gene was identical in IgAN and controls. There was also no difference in the sizes of cDNA transcripts of hinge region mRNA from patients with IgAN and controls. CONCLUSIONS: We found no evidence for any nucleotide sequence alteration or transcriptional abnormality of the alpha1 hinge region in IgAN, and we conclude that the O-glycosylation defect is post-translational.

Adolescent↗
PubMed1997

Expression of the mucosal gamma delta T cell receptor V region repertoire in patients with IgA nephropathy.

IgA nephropathy (IgAN) is characterized by the deposition of IgA in the glomerular mesangium and often leads to progressive renal dysfunction and kidney failure. We have previously shown that the mesangial IgA is likely to derive from the bone marrow plasma cells, and suggested that a primary abnormality within the mucosal immune system may underly the pathogenesis of IgAN. This study has analyzed the T cell receptor (TCR) variable (V) region expression by gamma delta T cells in the intestinal mucosa of patients with IgAN. The V gamma and V delta usage of TCR transcripts was determined using a semiquantitative reverse transcriptase-PCR protocol. Primers specific for the four human V gamma and six V delta subfamilies were used each with a constant (C) gamma or C delta specific primer, and the PCR-amplified TCR transcripts were detected by Southern blotting and oligonucleotide hybridization. gamma delta TCR V region expression was determined in gut biopsies and peripheral blood of 11 patients with IgAN, and the TCR V gamma and V delta repertoires were compared to those in gut and peripheral blood of 11 control individuals. gamma delta T cells in normal blood predominantly expressed V gamma 2 (V gamma 9 gene) and V delta 2 gene segments whereas those in normal gut mainly expressed V gamma 3 and V delta 3. In IgAN patients, V delta 2 was also the predominant V delta gene utilized by peripheral blood gamma delta T cells, however, we observed a predominance of V gamma 3 and reduced V gamma 2 usage by these cells. gamma delta T cells in the gut of IgAN patients mainly used V gamma 3 and V delta 1. While the gamma and delta TCR V region repertoires did not differ significantly between the peripheral blood of patients and controls, there were significant differences in V gamma and V delta repertoire expression between IgAN and control gut biopsies. V gamma 3 gene expression was significantly decreased in IgAN gut compared to control gut (P = 0.023). In addition, there was a significant decrease in V delta 3 gene expression in IgAN gut compared to control gut (P = 0.043). These findings indicate that a subpopulation of gamma delta T cells, which represent the majority of gamma delta T cells in normal gut mucosa, are significantly diminished in the gut of patients with IgAN. This suggests that a "hole" in the mucosal gamma delta T cell repertoire may play a fundamental role in contributing to the pathogenesis of IgAN.

Adult↗
PubMed1997

Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy.

BACKGROUND: Reduced galactosylation of the O-linked glycans of the IgA1 hinge region in IgAN has recently been described. To investigate the underlying defect resulting in this abnormality, we have measured the activity of beta 1,3 galactosyltransferase, the enzyme responsible for galactosylation of O-linked sugars. METHODS: A galactose-acceptor substrate was prepared from degalactosylated hinge region fragments of normal IgA1, and incubated with the T cell, B cell, and monocyte lysates from patients with IgAN and controls for acceptor regalactosylation. The extent of acceptor galactosylation was then measured with biotinylated Vicia villosa lectin (VV), which is specific for ungalactosylated moieties. Lectin binding of serum IgA from the same subjects was also measured. RESULTS: T cell and monocyte beta 1,3 galactosyltransferase activities did not differ between IgAN and control, but B cell lysates in IgAN showed significantly lower beta 1,3 galactosyltransferase activity than control (6.2 +/- 0.71 vs. 9.5 +/- 1.03 AU/microgram, P = 0.018). Furthermore, B cell beta 1,3 galactosyltransferase activity showed a negative correlation (r = -0.87, P = 0.002) with VV lectin binding of serum IgA in IgAN, but not controls. CONCLUSIONS: These data indicate that altered IgA1 O-galactosylation in IgAN results from a B cell-restricted reduction of beta 1,3 galactosyltransferase activity. This enzyme defect may be a fundamental pathogenic abnormality in IgAN.

Adult↗
PubMed1997

A comparative study of digoxigenin, 2,4-dinitrophenyl, and alkaline phosphatase as deoxyoligonucleotide labels in non-radioisotopic in situ hybridisation.

AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in non-radioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. RESULTS: The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. CONCLUSIONS: 3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails.

2,4-Dinitrophenol↗
PubMed1996

The effects of insulin-like growth factor analogues on survival of cultured cerebral cortex and cerebellar granule neurones.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: (Gln3, Ala4, Tyr15, (Leu16)IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

Animals↗
PubMed1996

Early measurement of interstitial fibrosis predicts long-term renal function and graft survival in renal transplantation.

This study investigated the relationships between renal allograft interstitial fibrosis, renal function and graft survival. A total of 107 consecutive renal transplant recipients immunosuppressed with cyclosporin were studied. Needle core transplant biopsies were performed before operation and at 1, 6 and 12 months after transplantation. Allograft fibrosis was assessed by histomorphometric analysis of graft interstitial volume fraction. Renal function was measured by isotopic glomerular filtration rate (GFR) measurement at the same time points. Interstitial volume fraction was already high in preperfusion biopsies, significantly increased with time but stabilized at 6 months after transplantation. GFR correlated negatively with interstitial volume fraction at 6 months (P = 0.05). Interstitial volume fraction at 1 month was not a useful predictor of subsequent graft survival but for allografts surviving to 6 months an interstitial volume fraction above 25 per cent predicted significantly poorer survival (P = 0.04). It provides an objective measure of chronic allograft damage and may prove to be a useful surrogate endpoint in the study of therapeutic intervention.

Biopsy, Needle↗
PubMed1996

The beneficial effects of oral nifedipine on cyclosporin-treated renal transplant recipients--a randomised prospective study.

The aim of this study was to test the hypothesis that nifedipine will improve graft survival in cyclosporin A (CyA)-treated renal transplant recipients. One hundred and forty-seven patients were randomised to one of three regimens. Group A received CyA, 7 mg/kg per day, and prednisolone; group B followed the same regimen as group A plus oral nifedipine and group C received CyA, 4 mg/kg per day, prednisolone and azathioprine. Calcium channel blockers were avoided in groups A and C. The crude 2-year (P = 0.0223) and 4-year (P = 0.0181) graft survival was significantly better in group B (86% and 81%, respectively) than in group A (75% and 63%, respectively). Delayed initial function was seen least frequently in group B (10.2%) compared to groups A (31%) and C (28%; P < 0.01). Group B also experienced fewer rejection episodes than groups A and C (P < 0.05). We conclude that the combination of oral nifedipine and CyA significantly improves initial graft function, rejection frequency and long term graft survival.

Administration, Oral↗
PubMed1996

The effect of epidural blockade on gastric intramucosal pH in the peri-operative period.

Patients undergoing major surgery are at risk of developing gut ischaemia and multiple organ failure. The gastric tonometer provides a relatively non-invasive method of assessing the adequacy of gut blood flow. Patients who develop repeated episodes of splanchnic ischaemia in the postoperative period, as evidenced by a low gastric intramucosal pH, have a higher mortality than those who do not. This randomised, controlled study was conducted to assess the effect of epidural blockade with bupivacaine on gastric intramucosal pH measurements in patients undergoing major surgery. A significantly lower proportion of patients with epidural blockade developed gastric intramucosal pH values < 7.32 postoperatively compared to controls (3/15 versus 13/16, p < 0.001). The significance of these results is discussed.

Anesthesia, Epidural↗
PubMed1996

Increased dimeric IgA producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA.

AIM: To investigate the possible role of the systemic IgA immune system in the pathogenesis of IgA nephropathy METHODS: J chain mRNA expression in the IgA cells of the bone marrow was studied. Bone marrow trephine biopsy specimens from seven patients with IgA nephropathy and seven matched controls were examined by (1) non-isotopic in situ hybridisation (ISH) and (2) combined immunofluorescence and non-isotopic ISH to identify the plasma cell type. Serum polymeric IgA was also determined using standard high pressure liquid chromatography and sandwich enzyme linked immunosorbent assay. RESULTS: Non-isotopic ISH revealed a similar number of J chain mRNA positive cells/unit length in biopsy specimens from patients (16.5 +/- 2.7 cells/mm) and controls (17.7 +/- 2.4 cells/mm). Combined immunofluorescence and ISH revealed a greater proportion of J chain mRNA positive IgA cells in patients (7.6 +/- 1.45%) compared with controls (3 +/- 0.8%). Serum polymeric IgA was similar in both patients (91 +/- 22 mg/l) and controls (77 +/- 24 mg/l). CONCLUSION: These data suggest that excess production of dimeric IgA occurs in the bone marrow in IgA nephropathy.

Adult↗
PubMed1995

Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy determined by in situ hybridization for J chain mRNA.

The origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed and in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7 +/- 4.6 cells/10(5) micron2) compared with controls (mean 36.9 +/- 3.5 cells/10(5) micron2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positive interfollicular IgA cells in patient tonsils (32 +/- 3.4%) compared with controls (21 +/- 2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain-positive Iga-negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.

Adolescent↗
PubMed1995

Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown. Mesangial IgA is of the IgA1 subclass, and since no consistent antigenic target for the IgA1 has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or IgA2. Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgA1, and the O-linked sugars of IgA1 and C1 inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.

Adult↗
PubMed1994

Increased immunoglobulin A and immunoglobulin A1 cells in bone marrow trephine biopsy specimens in immunoglobulin A nephropathy.

The origin of mesangial immunoglobulin A (IgA) in IgA nephropathy remains unknown. To investigate potential abnormalities within the bone marrow in this condition, bone marrow trephine biopsy specimens from seven patients and matched controls were studied using two-color immunofluorescence. In addition, serum levels of IgA and IgA1 were determined by radial immunodiffusion. Serum levels of IgA and IgA1 were higher in patients than in controls (4.53 +/- 1.38 g/L v 2.56 +/- 1 g/L, P < 0.01 and 3.68 +/- 1.11 g/L v 1.92 +/- 0.7 g/L, P < 0.005, respectively). In addition, patient trephine biopsy specimens contained an increased percentage of IgA plasma cells (61.6% +/- 4.4%) compared with controls (47.3% +/- 2.5%) (P < 0.02). The proportion of IgA plasma cells bearing subclass IgA1 was also greater in the patient biopsy specimens (91.6% +/- 1.9%) compared with controls (81.4% +/- 2.7%) (P < 0.01). In patients a positive correlation between the percentage of marrow IgA plasma cells and serum IgA levels was found (r = 0.94, P < 0.002). However, our studies failed to demonstrate a similar correlation between serum IgA1 levels and IgA1 marrow cells. These findings support the hypothesis that mesangial IgA may derive from the bone marrow.

Adult↗
PubMed1994

Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy.

Glomerular IgA in IgA nephropathy (IgAN) is at least in part polymeric, and is thought to derive from the mucosal IgA system in view of the association between mucosal infection and haematuria in this condition. To investigate this hypothesis, an in situ hybridization (ISH) technique was developed for the detection of J chain mRNA, the expression of which has been correlated with the secretion of high level polymeric immunoglobulin (pIg). Endoscopic duodenal biopsies from ten patients and matched controls were examined by: (i) two color immunofluorescence (IF); (ii) ISH; and (iii) combined ISH and IF, to permit simultaneous identification of plasma cell type. IF revealed a reduction in the percentage of IgA plasma cells (P < 0.02) and increased absolute numbers of IgG cells (P < 0.02) in patient biopsies. ISH demonstrated fewer J chain mRNA expressing plasma cells (P < 0.005) with lower signal intensity (P < 0.002) in patients' biopsies compared with controls. Combined ISH and IF confirmed a reduction in J chain mRNA-positive IgA plasma cells in the patient biopsies (P < 0.02). The reduction in J chain mRNA expression in duodenal IgA plasma cells in IgAN argues against the gastrointestinal lamina propria as the source of glomerular pIgA.

Adult↗