Antimitochondrial autoantibodies in anti-glomerular basement membrane disease

Anti-glomerular basement membrane (GBM) disease is characterized by the production of an autoantibody with very restricted specificity, with no evidence of polyclonal B cell activation. It was therefore surprising to find that in a solid-phase ELISA a proportion of anti-GBM sera showed significant binding to pyruvate dehydrogenase (PDH), a reactivity usually associated with the antimitochondrial autoantibodies (AMA) found in primary biliary cirrhosis (PBC). The specificity of this reactivity was confirmed by inhibition and competition experiments. The AMA found in anti-GBM sera were of much lower affinity than those found in PBC sera, and recognized a more restricted set of species (mainly the 55-kD and occasionally the 74-kD component of PDH). However, it was possible to block the binding in a Western blot of an anti-GBM serum to both the 55-kD and 74-kD species with F(ab')2 fragments prepared from a PBC serum. Although AMA have been found in diseases other than PBC, such diseases have usually been characterized by polyclonal B cell activation. The stimulus to the production of AMA in anti-GBM disease, and their significance in pathogenesis (if any), are unknown.     

Goodpasture syndrome involving overlap with Wegener's granulomatosis and anti-glomerular basement membrane disease

A 68-year-old Caucasian woman presented to the hospital with nodular pulmonary infiltrates and acute renal failure. Wegener's granulomatosis was initially considered to be most likely because of the presence of increased serum levels of c-anti-neutrophil cytoplasmic antibodies (c-ANCA). A consultation through the Internet after a renal biopsy demonstrated crescentic, necrotizing glomerulonephritis and linear deposits of immunoglobulin G (IgG) and complement C3, typical of anti-glomerular basement membrane (GBM) disease. Hemodialysis was instituted; however, the patient suddenly developed a massive cerebral hemorrhage and died before full therapy could take effect. Postmortem analysis of the patient's sera revealed high titers of IgG against the alpha 3 NC1 domain of type IV collagen. Serologic evidence of both p-ANCA and anti-GBM antibodies are becoming more frequently recognized in the setting of rapidly progressive glomerulonephritis. The patient reported here had the unusual combination of c-ANCA antibodies with anti-GBM disease, and this association raises complex questions regarding the pathogenesis of this type of renal injury.     

Targets of alloantibodies in Alport anti-glomerular basement membrane disease after renal transplantation

A minority of patients with Alport syndrome develop anti-GBM disease in their allografts after renal transplantation. Clinically, the renal disease appears indistinguishable from Goodpasture's disease of native kidneys, in which the target of autoantibodies had been identified as the NC1 domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1. However, in the majority of cases, Alport syndrome is due to mutations in the gene encoding the alpha 5 chain of type IV collagen, located on the X chromosome. Neither chain is detectable in the glomerular basement membrane (GBM) of most patients with Alport syndrome. We investigated the targets of the alloantibodies of 12 Alport patients who developed post-transplant anti-GBM disease by Western blotting onto recombinant NC1 domains made in insect cells. Binding to these antigens, for both typical Goodpasture and Alport anti-GBM antibodies, was strong and conformation-sensitive. Nine antibodies showed selective binding to alpha 5(IV)NC1. This specificity was confirmed by the demonstration of binding to a 26 kDa band of collagenase-solubilized human GBM, and/or binding to normal epidermal as well as renal basement membranes by indirect immunofluorescence. One antibody showed binding to alpha 5 and alpha 3(IV)NC1, while two showed predominant binding to alpha 3(IV)NC1. All seven patients whose pedigree or mutation analysis showed X-linked inheritance had predominant anti-alpha 5 reactivity. One with predominant anti-alpha 3 reactivity had a COL4A3 mutation. These findings show that human anti-GBM disease can be associated with antibodies directed towards different molecular targets. Alpha 5(IV)NC1 is the primary target in most patients with X-linked Alport syndrome who develop post-transplant anti-GBM disease.     

A rapid ELISA for measurement of anti-glomerular basement membrane antibodies using microwaves

The presence of anti-glomerular basement membrane antibodies is one of the features of Goodpasture's syndrome. Since the disease has a rapidly progressive course, an early diagnosis is essential. As was already demonstrated in other ELISA methods, 2.45-GHz microwave irradiation can accelerate all kinds of time consuming processes in several laboratory techniques. The application of microwaves in an ELISA for the measurement of anti-GBM antibodies in serum indicated that a considerable time reduction of 75% can be achieved, resulting in a rapid and reliable assay. In addition, microwaves can also have a positive effect on the resolution of that particular ELISA as shown in this study.     

Experimental induction of glomerulonephritis mediated by anti-glomerular basement membrane and anti-brush border antibodies in a single rat

This report describes induction of nephritis, which was concurrently mediated by [anti-glomerular basement membrane antibody (anti-GBM) and anti-brush border antibody, in the Wistar rat immunized with a solubilized renal antigen (S-RA). The antigen was prepared by digestion of rat cortical tissue with trypsin and pronase. Ouchterlony test using antisera to the rat GBM and brush border showed that the S-RA contained both antigens. From the S-RA the brush border antigen was isolated by affinity chromatography. At the 8th week rats injected with the S-RA showed a linear or combined linear and granular distribution of rat IgG and C3 along the GBM in immunofluorescence. The capillary granular pattern was only observed at the 16th week. In contrast rats injected with the brush border antigen remained in a capillary granular pattern throughout the experimental course. It was suggested that the rat nephritis injected with the S-RA was mediated by the antibodies capable of reacting with at least two different antigens, namely the GBM and the brush border. The possibility was confirmed by demonstrating the coexistence of these two kinds of antibodies in the serum and kidney eluate from the nephritic rats.     

Cytokine and adhesion molecule requirements for lung injury induced by anti-glomerular basement membrane antibody

Acute hemorrhagic lung injury occurs in humans with anti-GBM antibody (Goodpasture's syndrome), however, the mechanism of this injury is still largely unknown. To date, treatment has been confined to steroids and plasmaphoresis. Infusion of anti-GBM antibody into rats caused lung injury with intra-alveolar hemorrhage and intrapulmonary accumulation of neutrophils. Lung injury was dependent on the presence of neutrophils and complement and required both TNF alpha and IL-1. Experiments employing blocking antibodies to adhesion molecules demonstrated requirements for the beta 1 integrin VLA-4, beta 2 integrins LFA-1 and Mac-1, and L-selection. The endothelial cell adhesion molecules, E-selectin and ICAM-1, were also required for the full development of lung injury. Inhibition of TNF alpha or IL-1 or adhesion molecules reduced both lung injury and tissue neutrophil accumulation. Thus, this study underscores cytokine and adhesion molecule requirements for neutrophil mediated injury in lung and kidney caused by anti-GBM, suggesting potential targets for the treatment of Goodpasture's syndrome in humans.     

Th2 responses induce humorally mediated injury in experimental anti-glomerular basement membrane glomerulonephritis

Clinical and endocrinological effects of exemestane (6-methylenandrosta-1,4-diene-3,17-dione; PNU 155971) were evaluated in an open Phase I study. Thirteen postmenopausal women suffering from advanced breast cancer received exemestane in escalating doses over a 12-week period. Starting on 5 mg once daily (o.d.), exemestane was subsequently escalated at 2-week intervals to 10, 25, 50, 100, and 200 mg o.d. Each patient subsequently continued treatment on the highest tolerated dose until time of progression. One patient terminated treatment after 6 days due to diarrhea that was probably not related to drug therapy, although a relationship could not be excluded. Apart from this, no serious side effects were seen during the dose escalation period. Exemestane (10 mg o.d.) caused maximal suppression of plasma estradiol (E2) and estrone (E1) to a mean of 14.6 and 5.8% of pretreatment levels, respectively, whereas 25 mg of exemestane o.d. suppressed estrone sulfate (E1S) to 8.9% of pretreatment levels. No fall in adrenal steroid levels was recorded. Exemestane (5 mg o.d.) suppressed urinary E2 and E1 to a mean of 11.9 and 12.2% of pretreatment levels, respectively. Administering exemestane at doses of 50-200 mg o.d. caused no further suppression of urinary E1, whereas urinary E2 fell to 6-7% of pretreatment levels. Median time to progression was 63 weeks. We conclude that exemestane is a well-tolerated aromatase inhibitor that effectively suppresses plasma and urinary estrogens in postmenopausal patients with breast cancer.     

Susceptibility to anti-glomerular basement membrane disease and Goodpasture syndrome is linked to MHC class II genes and the emergence of T cell-mediated immunity in mice

We developed a new mouse model of human anti-glomerular basement membrane (GBM) disease to better characterize the genetic determinants of cell-mediated injury. While all major histocompatibility complex (MHC) haplotypes (H-2a, k, s, b, and d) immunized with alpha3 NC1 domains of type IV collagen produce anti-alpha3(IV) NC1 antibodies that cross-react with human Goodpasture [anti-GBM/anti-alpha3(IV) NC1] autoantibodies, only a few strains developed nephritis and lung hemorrhage associated with Goodpasture syndrome. Crescentic glomerulonephritis and lung hemorrhage were MHC-restricted in haplotypes H-2s, b, and d (A beta/A alpha region in H-2s) and associated with the emergence of an IL-12/Th1-like T cell phenotype. Lymphocytes or anti-alpha3(IV) NC1 antibodies from nephritogenic strains transfer disease to syngeneic recipients. However, passive transfer of isogenic alpha3(IV) NC1 antibodies into -/- T cell receptor-deficient mice failed to produce nephritis. Finally, nephritis and its associated IL-12/Th1-like T cell response attenuate in disease-susceptible mice tolerized orally to alpha3(IV) collagen before immunization. Our findings suggest collectively, as a hypothesis, that anti-GBM antibodies in mice only facilitate disease in MHC haplotypes capable of generating nephritogenic lymphocytes with special T cell repertoires.     

Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity. 2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity. 3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells. 4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.     

Polyarteritis nodosa and antiglomerular basement membrane disease without antineutrophil cytoplasm antibodies

Antiglomerular basement membrane (GBM) antibodies have been described previously in patients with microscopic polyarteritis but not in patients with polyarteritis nodosa alone. Where anti-GBM antibodies occur in microscopic polyarteritis, antineutrophil cytoplasm antibodies (ANCA) are usually present. We describe here a patient with polyarteritis nodosa and anti-GBM antibodies in whom ANCA could not be demonstrated. A 72-year-old woman presented with abdominal pain, diarrhoea and acute renal failure. A renal biopsy showed crescentic glomerulonephritis and linear immunofluorescence of the GBM consistent with anti-GBM disease. In addition, there was evidence of large-and medium-sized vessel vasculitis on abdominal angiography, performed because of persisting abdominal pain. There was no small vessel vasculitis on histological examination of the renal biopsy and ANCA could not be demonstrated by indirect immunofluorescence or ELISA.     

Lamina lucida of basement membrane: an artefact

In tissues prepared with chemical fixation followed by conventional dehydration, basement membranes have been observed to be laminated structures composed of a lamina lucida and lamina densa as well as a poorly limited transitional zone referred to as the pars fibroreticularis. Scattered attempts in the application of new techniques of tissue preparation such as cryofixation or freeze substitution for the study of the basement membrane structure have been made in recent years. From these studies, the possibility has arisen in which basement membranes are composed of only the lamina densa without a lamina lucida. In recent studies in this laboratory, the attempt was made to determine whether or not this lamina lucida is an artefact, and if so, which step in the conventional method of tissue preparation is responsible for its formation. Basement membranes from diverse sources in the mouse and rat including the testis, ductus epididymis, eye, thyroid, kidney, and skin, were observed after either cryofixation by slam freezing followed by freeze substitution, or aldehyde fixation followed by freeze substitution. The basement membranes after preservation with either of these two methods were composed of only the lamina densa with no lamina lucida. It indicates that an artefactual formation of the lamina lucida occurs during dehydration in conventional tissue preparation rather than during chemical fixation. In view of the well known superiority of freeze substitution over conventional dehydration, the lamina lucida of the basement membrane is likely to be an artefact. Therefore, it is concluded that the lamina lucida is an artefact formed during conventional tissue preparation, and in its original condition in the living state, the basement membrane is composed of a single layer made up of lamina densa material.     

Anti-glomerular basement membrane (GBM)-antibody-mediated disease with normal renal function

The purpose of this study was to compare the objective response rate, duration of remission, and survival of 5-fluorouracil (5-FU) versus those of 5-FU plus levamisole in metastatic colorectal cancer using the same dose and schedule of these agents as in the North Central Cancer Treatment Group and intergroup studies of adjuvant therapy. Patients with no prior history of chemotherapy for metastatic disease were entered on this Hoosier Oncology Group randomized Phase III trial. Patients were stratified by Karnofsky performance status and presence or absence of liver metastases. They were randomized to receive 450 mg/m2 5-FU i.v. for 5 days followed by 15 mg/kg i.v. weekly (arm 1) or the same dose of 5-FU plus levamisole 50 mg p.o. every 8 h for 3 days every 2 weeks (arm 2). The duration of treatment for both arms was 26 weeks. From April 1990 to March 1995, 199 patients were entered. One hundred eighty-two patients, 91 in each arm, were fully evaluable. The response rates were 12% on arm 1 and 13% on arm 2. The median duration of response was 18 weeks on both arms. The median survival was 48 weeks on arm 1 and 41 weeks on arm 2 (P = 0.20). This study failed to show any improvement in survival, response, or duration of remission with the addition of levamisole to 5-FU in the treatment of metastatic colorectal cancer.     

Vascular adventitial cell expression of collagen I messenger ribonucleic acid in anti-glomerular basement membrane antibody-induced crescentic nephritis in the rabbit. A cellular source for interstitial collagen synthesis in inflammatory renal disease

BACKGROUND: Scarring in the interstitial compartment of the renal cortex heralds a poor prognosis in many forms of renal injury, however, the mechanism through which glomerular inflammation leads to interstitial scarring is not understood. In a model of anti-GBM disease in the rabbit, development of crescentic glomerulonephritis is associated with marked interstitial fibrosis and decreased renal function. We previously demonstrated that collagen accumulation in the model was preceded by increases in collagen I and IV mRNA and that these changes were primarily extraglomerular at early time points when inflammation was predominantly intraglomerular. In order to identify the cellular origins of extraglomerular collagen synthesis in this model, in situ hybridization using an alpha 2(I) procollagen probe was performed. EXPERIMENTAL DESIGN: A 602 bp rabbit alpha 2(I) procollagen cDNA was cloned using a PCR strategy and sequenced. The nucleotide sequence of the coding region was 94% identical with the human alpha 2(I) procollagen sequence. Northern blots were performed to define conditions of specific hybridization of the anti-sense riboprobe. Tissue sections from normal rabbit kidneys and from kidneys 4, 5, 7, 10 and 14 days after injection of anti-GBM antibody were hybridized with 35S-labeled sense and anti-sense riboprobes. Cells containing alpha 2(I) mRNA were identified by autoradiography and mRNA abundance was quantitated by grain density. RESULTS: No specific hybridization was detected with the sense probe at any time. alpha 2(I) mRNA was undetectable with the anti-sense probe in normal kidney sections. In contrast, the anti-sense probe hybridized specifically at all time points after induction of anti-GBM disease. In agreement with previous filter hybridization studies, on day 4, when inflammation was predominantly intraglomerular, cells in the periarterial adventitial compartment of renal cortex hybridized strongly. At later time points, labeling was also present in the interstitial spaces, the periglomerular region, in Bowman's space and in the glomerular tuft itself. CONCLUSIONS: We conclude that perivascular adventitial cells are among the first to respond to glomerular inflammation and represent a pool of cells that subsequently contribute to interstitial and glomerular scarring.     

Inherited diseases of the glomerular basement membrane

The inherited diseases of the glomerular basement membrane include Alport's syndrome (AS), nail-patella syndrome, and thin basement membrane nephropathy. Classical AS is inherited in an X-linked manner and accounts for approximately 85% of the cases. Its manifestations include hematuria, sensorineural hearing loss, ocular defects, and a progression to renal failure. A defect(s) in the alpha 5 (IV) chain of type IV collagen is believed to be the etiology of classic AS, and alterations in its encoding gene localized to the X-chromosome have been elucidated. Although isolated cases of anti-glomerular basement membrane glomerulonephritis have been reported following renal transplantation in patients with AS, it is considered an effective form of renal replacement therapy. Less is known regarding the genetic basis of the autosomal-dominant form of AS, which apparently accounts for the remaining 15% of the cases. Nail-patella syndrome is characterized by nail dysplasia, patellar hypoplasia or aplasia, and nephropathy. It is inherited in an autosomal-dominant fashion with the gene locus assigned to the long arm of chromosome 9. Possible linkage between the COL5A1 gene and the gene for nail-patella syndrome has been suggested. Approximately 30% of the patients progress to end-stage renal failure. Renal transplantation has been successful in treating patients who progress to end-stage renal failure. Thin basement membrane nephropathy is an autosomal dominant trait that accounts for approximately 30% of the cases presenting as persistent, asymptomatic hematuria. The cause of thin basement membrane nephropathy is unknown at present. No decline in renal function is associated with thin basement membrane nephropathy.     

Short-term results and intermediate-term follow-up of laser wire recanalization of chronic coronary artery occlusions: a single-center experience

OBJECTIVES: This study sought to elucidate the short-term efficacy and intermediate-term outcome of excimer laser recanalization of chronic coronary artery occlusions in patients in whom attempts at mechanical revascularization had failed. BACKGROUND: Recanalization of chronic coronary occlusions with the use of a mechanical guide wire fails in 30% to 50% of cases, mostly because of inability to pass the wire through the lesion. The value of using excimer laser energy in this setting has not yet been determined. METHODS: The study group comprised 66 consecutive patients with 68 chronic coronary occlusions. Patients were eligible for inclusion in the study if a previous attempt at mechanical revascularization had failed and if their angiographic status was such that 1) the vessel segment distal to the occlusion could be visualized by way of collateral vessels, 2) the entry point of the occlusion was clearly outlined, and 3) not more than one anatomic bend was expected within the occlusion. Excimer laser energy was applied to the lesion through a 0.018-in. (0.046 cm) fiber-optic guide wire. Adjunctive balloon angioplasty and stenting were performed in all successfully treated patients but one. RESULTS: Thirty-four occlusions (50%) in 32 patients (48%) could be crossed with the laser wire. Location and age of the occlusion had no adverse influence on the outcome of laser wire recanalization, nor did the presence of bridging collateral vessels, a major side branch at the site of the lesion or a blunt stump of the occlusion. An inverse relation was found between the success rate and the length of the occlusion, such that a 19% reduction of the success rate accompanied each 10-mm increment of the mean occlusion length. Thus, the success rate was 68% for lesions < or = 10 mm but only 25% for lesions > 30 to < or = 40 mm. The presence of a bend in the lesion exceeding 60 degrees was strongly related to procedural failure. During a median angiographic follow-up period of 18 weeks, restenosis > 50% (n = 6) or reocclusion (n = 4) was found in 10 of the 32 successfully treated patients, for an intermediate-term success rate of 33% (22 of 66). Clinical follow-up revealed improved anginal status in 21 patients (66%) after a median of 24 weeks. Major complications (death, myocardial infarction, emergency operation) were not encountered. CONCLUSIONS: Successful recanalization of a chronic coronary occlusion by using currently available laser wires can be expected in 50% of selected patients in whom attempts at mechanical revascularization fail. Restenosis or reocclusion accounts for an overall 6-month success rate of 35%.     

Laparoscopic Nissen fundoplication for gastroesophageal reflux disease: clinical experience and outcome in first 100 patients

Using retrospective chart review, the authors evaluated the results of laparoscopic Nissen fundoplication in their first 100 patients. All patients were diagnosed with gastroesophageal reflux disease. More than 90% of the patients in this series were symptomatically improved, and 92% of those studied endoscopically had healed esophagitis and intact fundoplication. No deaths, esophageal injuries, or splenic injuries occurred. Laparoscopic fundoplication can be performed safely and efficiently. Using a linear stapler enables rapid and safe fundi mobilization. Selective manometrics and ambulatory pH monitoring provide excellent results. Laparoscopic Nissen is safe and as effective as the open procedure. Research centers have noted some differences in postoperative function of the lower esophageal sphincter, but symptomatically patient satisfaction is comparable.