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Biomedical subjects

Charles D Pusey

Publications and source records attributed to Charles D Pusey.

At least 37 records · Page 2Linked to original sources
PubMed2004

DNA adducts and p53 mutations in a patient with aristolochic acid-associated nephropathy.

BACKGROUND: Aristolochic acid-associated nephropathy (AAN) is a specific type of renal disease that predisposes patients to a high risk of urothelial carcinoma. The authors have analyzed DNA from a patient who had urothelial malignancy 6 years after presenting with AAN and later had a breast carcinoma that metastasized to the liver. METHODS AND RESULTS: DNA was isolated from the primary breast tumor, the liver tumor, and the original urothelial malignancy and assayed for aristolochic acid (AA)-DNA adducts and mutations in the p53 gene. The adduct detected was the adenosine adduct of aristolochic acid I 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In DNA from the breast and liver tumors the authors showed the same missense mutation in codon 245 (GGC-->GAC; Gly-->Asp) of exon 7 of p53. In contrast, DNA extracted from the urothelial tumor showed an AAG to TAG mutation in codon 139 (Lys-->Stop) of exon 5. CONCLUSION: A to T transversions, as observed here, are the typical mutations observed in the H-ras gene of tumors induced when rodents are treated with AA and correspond with DNA adduct formation at adenosine residues. These data indicate the probable molecular mechanism whereby AA causes urothelial malignancy.

Aristolochia↗
PubMed2004

Microscopic polyangiitis.

Microscopic polyangiitis is a systemic vasculitis affecting smal-l and medium-sized vessels and is characteristically associated with a focal and segmental necrotizing glomerulonephritis. It may present as a pulmonary-renal syndrome with rapidly progressive glomerulonephritis and alveolar hemorrhage, but the pattern of disease will vary according to the organ systems involved. Granulomatous disease of the upper or lower respiratory tract is not a feature, and its presence suggests the diagnosis of Wegener's granulomatosis. The etiology of the condition is unclear, but most patients have antineutrophil cytoplasm antibodies (ANCA) with specificity for either myeloperoxidase (MPO) or proteinase 3 (PR3), and there is increasing evidence that these may be pathogenic. Current treatment includes an induction phase using cyclophosphamide and steroids to attain remission, followed by a maintenance phase in which the levels of immunosuppression are gradually reduced. Azathioprine may be substituted for cyclophosphamide at 3 months. Adjunctive plasma exchange or intravenous methylprednisolone is used in the management of either or both severe renal disease and alveolar hemorrhage, and new evidence suggests that plasma exchange is more effective in recovery of renal function. Overall, 1-year survival in systemic vasculitis is around 85%, and up to 50% of patients relapse, although relapse is less common in those with MPO-ANCA. Newer therapies are being explored in an attempt to increase the efficacy and reduce the toxicity of treatment.

Journal Article↗
PubMed2004

Retransplantation in Alport post-transplant anti-GBM disease.

BACKGROUND: Post transplant anti-glomerular basement membrane (GBM) disease affects up to 5% of patients with Alport's syndrome. Defects in the COL4A5 gene are responsible for most cases, and alpha 5(IV)NC1 is the usual target for alloantibodies. Gene deletions are more commonly associated with this complication than are point mutations. The disease is severe in renal allografts and nearly always results in graft loss. METHODS: Three cases of retransplantation in Alport's syndrome are described here in detail. All cases were started on immunosuppressive therapy early in the course of their disease and one patient (case 2) received pre-emptive anti-T-cell therapy (Campath IH). Anti-GBM antibodies in these cases were investigated by standard anti-GBM enzyme-linked immunosorbent assay (ELISA), by indirect immunofluorescence, and by Western blotting using collagenase-digested human GBM and recombinant type IV collagen NC1 domains made in insect cells. RESULTS: All cases showed early antibody and complement fixation to human GBM. Target alloantibodies were to alpha 5(IV)NC1 domain predominantly. Cases two and three gave negative results on standard ELISA for anti-GBM antibodies. Pathologic examination revealed crescentic glomerulonephritis, which was rapid in onset in case 1, blunted and less aggressive in case 3, and case 2 developed segmental necrosis without crescent formation. Neutrophilic infiltrates were an early feature in all 3 cases. All cases are compared with a review of all retransplanted cases in the literature. CONCLUSION: Alport anti-GBM disease is a severe disease in retransplanted patients. Anti-T-cell therapy seemed to modify the pathologic findings but did not prevent graft loss. Longer term plasma exchange and mycophenolate mofetil may attenuate the illness, but in these cases did not prevent graft loss. Western blotting detected alloantibodies to alpha 5(IV) NC1 domain and is more sensitive and specific for this disease than standard ELISAs.

Adolescent↗
PubMed2004

Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis.

BACKGROUND: Experimental autoimmune glomerulonephritis (EAG) was induced in Wistar-Kyoto (WKY) rats by immunization with rat glomerular basement membrane (GBM) in adjuvant. This model is characterized by anti-GBM antibody production, accompanied by focal necrotizing glomerulonephritis with crescent formation. There is also glomerular infiltration by T cells and macrophages. Our hypothesis was that blocking the interaction between CD154 (CD40L) on Th cells and CD40 on antigen-presenting cells should inhibit T-cell activation, and thus the development of EAG. METHODS: The in vivo effects of a hamster anti-rat monoclonal antibody to CD154 (AH.F5) were examined in EAG starting at day -1 prior to immunization, day +7 after immunization, or day +14 after immunization. RESULTS: When administered from day -1 at a dose of 10 mg/kg intraperitoneally three times per week for the duration of the study (4 weeks), AH.F5 resulted in a marked reduction in circulating anti-alpha3(IV)NC1 antibodies, deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of glomerular T cells and macrophages. When administered from day +7 at the same dose, AH.F5 resulted in a moderate reduction in the severity of disease, while administration from day +14 had no significant effect. CONCLUSION: These studies demonstrate for the first time that early blockade of the CD154-CD40 T-cell costimulatory pathway can prevent the development of crescentic nephritis, and that delayed treatment can reduce the severity of disease. This confirms the importance of T cell mediated immunity in the pathogenesis of EAG, and suggests that strategies targeting T-cell costimulation may provide a novel approach in the treatment of human glomerulonephritis.

Albuminuria↗
PubMed2004

Clinical features and outcome of patients with both ANCA and anti-GBM antibodies.

BACKGROUND: Patients have been described who have both anti-neutrophil cytoplasm antibodies (ANCA) and anti-glomerular basement membrane (GBM) antibodies. We have attempted to define the true prevalence of such "double positive" patients, and describe in detail their clinical features and outcome. METHODS: We have reviewed all serologic assays performed between 1990 and 2000 in a single institution, and the case notes of patients having sera positive for both ANCA and anti-GBM antibodies. During this time 20,392 sera were initially tested for ANCA, and 4808 sera tested for anti-GBM antibodies. RESULTS: Five percent of all ANCA-positive serum samples were also positive for anti-GBM antibodies, and 32% of all anti-GBM positive samples had detectable ANCA. Of 27 patients with both antibodies, 82% had anti-myeloperoxidase specific P-ANCA. Pulmonary hemorrhage occurred in 44%. Renal biopsy showed extensive glomerular cellular crescents in most patients. Patient and renal survival rates were 52% and 26%, respectively, at one year. Sixty-eight percent of patients were dialysis-dependent at presentation, and none of these recovered renal function, despite immunosuppression with or without plasma exchange. CONCLUSION: Serologic evidence of double positivity for both ANCA and anti-GBM antibodies is common in patients with either antibody. In our study these patients have a poor prognosis when presenting with severe disease and initially behave more like anti-GBM disease than vasculitis. Recovery from severe renal failure is rare.

Anti-Glomerular Basement Membrane Disease↗
PubMed2004

Glomerular T cells are of restricted clonality and express multiple CDR3 motifs across different Vbeta T-cell receptor families in experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis (EAG) is an animal model of Goodpasture's disease which can be induced in Wistar-Kyoto (WKY) rats by a single intramuscular injection of collagenase-digested rat glomerular basement membrane (GBM) in adjuvant. This model is characterised by anti-GBM antibody production, accompanied by focal necrotising glomerulonephritis with crescent formation and glomerular infiltration by T cells and macrophages. Previous work has shown that EAG is a T-cell-dependent disease. We proposed that intraglomerular T cells might be directly involved in pathogenesis and would be oligoclonal. In this study, EAG was induced by standard methods, the kidneys perfused with saline at week 2 and week 4, and the glomeruli separated by a sieving method. Glomerular RNA was extracted and reverse transcribed. RT-PCR showed overexpression of an average of two Vbeta families in each kidney analysed. However, no predominant single Vbeta family was overexpressed in any of the experimental animals. CDR3 spectratyping of Fam-labelled PCR products showed a marked restriction involving different Vbeta families. Sequencing demonstrated multiple CDR3 motifs, each expressed in association with different Vbeta gene segments. Our results show that glomerular T cells are of restricted clonality and suggest a role for antigen-specific effector T cells in the pathogenesis of EAG.

Animals↗
PubMed2003

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease.

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.

Animals↗
PubMed2003

Outcome of ANCA-associated renal vasculitis: a 5-year retrospective study.

BACKGROUND: Renal involvement is frequently present in antineutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitis and is an important cause of end-stage renal failure (ESRF). METHODS: This retrospective, multicenter, sequential cohort study reports presenting features and outcome of 246 new patients diagnosed in London, UK, between 1995 and 2000. RESULTS: Diagnostic subgroups were microscopic polyangiitis, 120 patients (49%); Wegener's granulomatosis (WG), 82 patients (33%); renal-limited vasculitis, 33 patients (13.5%); and Churg-Strauss angiitis, 11 patients (4.5%). Median age was 66 years, 57% were men, and median creatinine level at presentation was 3.87 mg/dL (342 micromol/L). ANCA was present in 92%. Cumulative patient survival at 1 and 5 years was 82% and 76%, respectively. Mortality was associated with age older than 60 years (P < 0.001), development of ESRF (P < 0.001), initial creatinine level greater than 2.26 mg/dL (200 micromol/L; P = 0.01), and sepsis (P < 0.048). ESRF occurred in 68 patients (28%), of whom 47% died. Fifty-six patients who presented with a creatinine level greater than 5.65 mg/dL (500 micromol/L) survived, and 31 patients (55%) achieved dialysis independence. Relapse occurred in 34% after a median of 13 months and was more common in patients with WG (P = 0.048) and proteinase 3-ANCA (P = 0.034). Leukopenia occurred in 41% and was associated with sepsis (P < 0.001). CONCLUSION: Mortality and morbidity of ANCA-associated systemic vasculitis are improving compared with previous series, but remain high. Renal vasculitis often affects older patients, who have a particularly poor outcome. Early diagnosis improves outcome. Leukopenia, caused by immunosuppressive therapy, should be avoided because of the close association with sepsis and death.

Adolescent↗
PubMed2003

Regulation by CD25+ lymphocytes of autoantigen-specific T-cell responses in Goodpasture's (anti-GBM) disease.

BACKGROUND: Goodpasture's, or anti-glomerular basement membrane (GBM), disease is unusual among autoimmune diseases in that it rarely follows a relapsing-remitting course. Moreover, untreated, autoantibodies disappear spontaneously after 1 to 3 years and, following treatment, autoreactive T cells diminish in frequency. This suggests that operational tolerance toward the autoantigen is reestablished. However, the mechanisms underlying this have remained unclear. Recent data have suggested that a population of regulatory T lymphocytes can suppress both autoimmune and alloimmune responses in animal models and are present in normal individuals. However, to date, they have not been demonstrated to play a role in human renal autoimmune disease. METHODS: We studied the role of regulatory CD25+ cells in suppressing T-cell responses to the Goodpasture autoantigen in nine patients with Goodpasture's disease. RESULTS: At the time of acute presentation, there was no evidence of a regulatory cell population. However, from 3 months onward a population emerged, capable of suppressing the response to the Goodpasture autoantigen. Following depletion of CD25+ cells, the frequencies of autoreactive-, GBM-, or collagen alpha 3(IV)NC1-specific T cells were significantly increased (P = 0.031 by paired t test), with five of seven (71%) convalescent patients and no acute patients demonstrating regulation. CONCLUSION: These data demonstrate that, in Goodpasture's disease, regulatory CD25+ T cells play a role in inhibiting the autoimmune response. Their emergence and persistence may underlie the "single hit" nature of this condition. Understanding the conditions required for the development and propagation of these cells would allow development of novel therapeutic strategies for inducing hyporesponsiveness in autoimmune disease.

Acute Disease↗
PubMed2003

Membranous glomerulonephritis associated with follicular B-cell lymphoma and subepithelial deposition of IgG1-kappa paraprotein.

A case is described of B-cell lymphoma/chronic lymphocytic leukaemia associated with membranous glomerulonephritis in which the subepithelial deposits of immunoglobulin showed kappa light chain restriction by immunofluorescence. Surface IgG-kappa immunoglobulin was demonstrated on the malignant B cells, and a monoclonal protein of the same type was eluted from kidney. The mechanism of membranous glomerulonephritis in this type of lymphoid malignancy is clearly different from that in epithelial malignancies.

Aged↗
PubMed2003

Blocking VLA-4 prevents progression of experimental crescentic glomerulonephritis.

BACKGROUND/AIMS: Integrins are adhesion molecules of fundamental importance to the recruitment of leucocytes in inflammation. The alpha4beta1 integrin (VLA-4) is a leucocyte ligand for endothelial vascular cell adhesion molecule-1 (VCAM-1), fibronectin and osteopontin. We addressed the role of VLA-4 in mediating progressive renal injury in vivo using a blocking monoclonal antibody (mAb) in a rat model of crescentic glomerulonephritis. METHODS: WKY rats with nephrotoxic nephritis were given anti-VLA-4 or control mAb at 2.5 mg/kg by i.p. injection on alternate days. In separate experiments, antibodies were given from days 5-13, from days 13-21 or from days 14-28. RESULTS: Early treatment with anti-VLA-4 mAb from days 5-13 showed a significant effect on renal function, with a reduction in albuminuria (p < 0.01) and a higher creatinine clearance (p < 0.05). Delayed treatment from days 13-21 also showed a reduction in albuminuria (p < 0.05) and serum creatinine (p < 0.05). However, there was no significant effect on glomerular or interstitial scarring in these two experiments. In the late treatment study, in which anti-VLA-4 mAb was administered from days 14-28, serum creatinine was reduced (p < 0.05), creatinine clearance was improved (p < 0.05), and renal survival was significantly prolonged (p < 0.05). Interstitial scarring was significantly less in treated rats (p < 0.05). Glomerular macrophage and CD8+ cell counts were higher in anti-VLA-4 mAb treated rats (p < 0.05), possibly reflecting greater glomerular scarring in control animals. CONCLUSION: Leucocyte VLA-4 mediates pro-inflammatory and pro-fibrotic effects within the kidney, independent of any role in recruitment of leucocytes into the kidney. Blocking VLA-4 is a promising therapeutic approach in human glomerulonephritis.

Albuminuria↗
PubMed2002

Elevated Akt phosphorylation as an indicator of renal tubular epithelial cell stress.

Characterization of the phosphoinositide 3-kinase-signaling pathway in a human renal tubular epithelial cell (TEC) line HKC-8 revealed high levels of Akt phosphorylation in serum-starved cultures. In contrast to Erk1/2, little additional phosphorylation of Akt was observed after cytokine or serum stimulation. Replacement of the conditioned medium attenuated Akt phosphorylation such that 90 min after the addition of warmed serum-free media, Akt phosphorylation had fallen sufficiently to allow an epidermal growth factor-stimulated increase to be detected readily. Although the mechanism by which the phosphoinositide 3-kinase/Akt pathway is activated in serum-starved TEC is unknown, the mediator responsible is secreted from these cells. Thus, conditioned media removed from a dish of quiescent TECs stimulated Akt phosphorylation in washed TEC cultures within 10 min. Biochemical characterization of the bioactive agent identified a heat labile factor of small apparent molecular mass. The basal level of Akt phosphorylation observed in serum-starved cultures was inhibited by wortmannin at concentrations that demonstrated its dependence on 3-phosphoinositide synthesis (IC(50) = 8 nm). Regular removal of conditioned media from TEC cultures and its replacement with serum free media resulted in a sustained attenuation of Akt phosphorylation. Interestingly, after 5 days of this treatment, washed TEC cultures contained a greater number of viable cells than cultures maintained in conditioned media throughout. This observation was not explained by a difference in the rate of DNA synthesis. Instead, the number of cells undergoing apoptosis increased markedly in the unwashed cultures. Consequently, we propose that in HKC-8 cells Akt phosphorylation is up-regulated in an effort to minimize cell death. This stress-activated response is initiated by a factor secreted into the conditioned medium that stimulates the phosphoinositide 3-kinase signaling pathway.

Cell Line↗
PubMed2002

Treatment with an antibody to VLA-1 integrin reduces glomerular and tubulointerstitial scarring in a rat model of crescentic glomerulonephritis.

The alpha 1 beta 1 integrin (VLA-1) is a major collagen/laminin receptor that regulates fibroblast proliferation and mesangial cell migration and cell contraction. We have examined the effect of an antibody to VLA-1 in crescentic glomerulonephritis. Nephrotoxic nephritis was induced in Wistar-Kyoto rats and rats were given monoclonal antibody to VLA-1 (Ha31/8), 2.5 mg/kg, on alternate days. Antibodies were given from day -1 to day 10 or from day 14 to day 28. Treatment from day -1 to day 10, during the early inflammatory phase of nephrotoxic nephritis, had no effect on albuminuria or glomerular crescent formation. In the delayed treatment experiment, all rats developed florid crescentic glomerulonephritis, and control rats showed marked glomerular and tubulointerstitial scarring at day 32. VLA-1 expression, by immunohistochemistry, was increased in glomeruli and around tubules. Proteinuria did not differ between groups. In anti-VLA-1-treated rats, serum creatinine was significantly lower at day 32 (P = 0.002) and renal survival was significantly better (P = 0.045). Both glomerular and interstitial scarring were significantly less at day 32 in rats given anti-VLA-1 (P = 0.002). Deposition of ED(A) fibronectin, a marker of new matrix synthesis, and of type IV collagen, were reduced in glomeruli and interstitium in anti-VLA-1-treated animals (P = 0.0006). Expression of alpha-smooth muscle actin, a marker of myofibroblasts, showed no significant difference. Expression of matrix metalloproteinase-9 was increased in the glomeruli of rats treated with anti-VLA-1. We conclude that VLA-1 mediates both glomerular and interstitial fibrosis in crescentic glomerulonephritis and that neutralization of VLA-1, which enhanced expression of matrix metalloproteinase-9, is a possible therapeutic strategy in progressive renal scarring.

Animals↗
PubMed2002

Goodpasture's disease in the absence of circulating anti-glomerular basement membrane antibodies as detected by standard techniques.

Goodpasture's disease is characterized by rapidly progressive glomerulonephritis, often accompanied by pulmonary hemorrhage, in association with deposition of antibodies in a linear pattern on the glomerular basement membrane (GBM). The diagnosis of Goodpasture's disease in patients with acute renal failure often relies on the use of immunoassays to detect circulating anti-GBM antibodies in serum samples. We describe three cases of Goodpasture's disease in which no circulating anti-GBM antibodies were detectable in serum by well-established enzyme-linked immunosorbent assay or Western blotting techniques. The diagnosis of Goodpasture's disease was confirmed by renal biopsy, with linear deposition of immunoglobulin along the GBM and crescentic glomerulonephritis. In addition, an alternative method of antibody detection using a highly sensitive biosensor system confirmed that circulating antibodies were present in sera from both patients tested. Because this technique is not routinely available for the detection of anti-GBM antibodies, we suggest that diagnosis always be confirmed with a renal biopsy, and despite negative serological test results using immunoassay, the diagnosis of Goodpasture's disease should still be considered in the correct clinical context.

Adolescent↗