Goodpasture antigen: expression of the full-length alpha3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain

BACKGROUND: Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the alpha3(IV) chain of type IV collagen, one of five alpha(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the alpha3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain. A knowledge of GP epitopes is important for the understanding of the etiology and pathogenesis of the disease and for the development of therapeutic strategies. METHODS: A cDNA construct was prepared for the full-length human alpha3(IV) chain. The construct was stably transfected into human embryonic kidney 293 cells. The purified full-length r-alpha3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for binding of GP antibodies from five patients was compared with that of the human r-alpha3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay. RESULTS: The r-alpha3(IV) chain was secreted from 293 cells as a single polypeptide chain that did not spontaneously undergo assembly into a triple-helical molecule. An analysis of GP-antibody binding to the full-length r-alpha3(IV) chain showed binding exclusively to the globular NC1 domain. CONCLUSION: The full-length human alpha3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontriple-helical NC1 domain of the alpha3(IV) chain, indicating the presence of specific immunogenic properties. The alpha3(IV) chain alone does not spontaneously undergo assembly into a triple-helical homotrimeric molecule, suggesting that coassembly with either the alpha4(IV) and/or the alpha5(IV) chain may be required for triple-helix formation.     

High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. METHODS: Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. RESULTS: All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. CONCLUSIONS: The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation, despite removal of circulating Abs and adequate immunosuppression.     

Antibiotic modulation in a clinically relevant model of chronic intraabdominal infection

Continuous and twice-daily cefoxitin dosing was used in a highly lethal model of acute peritonitis in mice using intraperitoneal (IP) Klebsiella pneumoniae (Kpn). The purpose was to use antibiotics to create a model of chronic infection. Male Balb/c mice (averaging 20 g body weight) were inoculated IP with 10(3) colony-forming units (CFU) Kpn serotype 2. Controls received subcutaneous saline either twice daily or continuously. Antibiotic groups received 300 mg/kg per day of cefoxitin either twice daily or continuously. Survival and daily weight losses were determined. Another group was inoculated with 10(3) Kpn given twice daily saline or cefoxitin and harvested at 24 hours. Leukocyte counts were performed on peritoneal exudate cells (PEC) and peripheral blood. Cultures determined Kpn counts in blood, lung, and PEC. By 24 hours, saline-treated animals had lost more weight than cefoxitin mice (1 g vs. 2 g, P < 0.05). Continuous cefoxitin showed significant advantage with 50 per cent mortality at 5 days. Kpn levels were not significantly altered by cefoxitin. Cefoxitin treatment extended chronicity by preventing weight loss and increasing survival in a highly lethal, monomicrobial peritonitis model. This model will allow future study of specific host defense mechanisms over a prolonged time period.     

Ulcerative colitis in a renal transplant patient with previous Goodpasture disease

A renal transplant was performed in a 6-year-old boy who developed end stage renal disease (ESRD) after presenting with antiglomerular basement membrane (anti-GBM) disease. At 10 years of age he developed ulcerative colitis while being immunosuppressed with cyclosporin, prednisone, and azothioprine. He had a pancolectomy, and at 14 years has no symptoms of ulcerative colitis or anti-GBM disease. HLA typing revealed that he was homozygous for HLA DR2. The co-occurrence of anti-GBM disease and ulcerative colitis has not previously been described. Although there is no known common etiology for these two autoimmune diseases, we propose that the patient’s homozygosity at HLA DR2 may have predisposed him to both. Received: 21 September 2000 / Revised: 26 January 2001 / Accepted: 1 February 2001     

Are antiphospholipid antibodies clinically relevant in dialysis patients?

A cohort of 60 stable dialysis patients (56 haemodialysis, 4 continuous ambulatory peritoneal dialysis) was followed for 1 year to determine the relationship between anticardiolipin (aCL) antibodies and lupus anticoagulant (LA), and clinical events. The outcome measures were death, arterial, venous, and fistula thromboses, and fistula repairs. At baseline 15% had antibodies (5/60 IgG aCL, 3/60 LA, and 1/60 had both); 15 patients had a history of arterial thrombosis (1 patient was aCL positive), seven venous thrombosis (1 patient LA positive), 10 fistula thromboses (1 aCL positive); and 21 had a history of fistula repairs (4 aCL positive). Renal diagnosis, age (66.78 versus 59.67 years) and duration of dialysis (38.11 versus 45.25 months) were similar in patients with and without aCL antibodies or LA. Only the sex ratio showed a female predominance in the aCL- or LA-positive patients compared to the negative patients (3M:6F versus 36M:15F), but this was not significant (P = 0.07). After 1 year there were 10 deaths (1 LA positive), 12 thrombotic events in eight patients (none aCL or LA positive), and nine fistula repairs in seven patients (1 aCL-positive patient). We are unable to show higher rates of death, thrombotic event, or fistula repair in dialysis patients with aCL antibodies or LA followed up for 1 year in one centre. The clinical importance of antiphospholipid antibodies in dialysis patients is uncertain.     

Burn injury and pulmonary sepsis: development of a clinically relevant model

BACKGROUND: Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail. Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure. Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality. METHODS: Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S). Burned animals had a full-thickness 15% dorsal scald burn. BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units). S and B animals received saline intratracheally. All animals were resuscitated with 2 mL of intraperitoneal saline. Mortality was recorded at 24, 48, and 72 hours. Bacterial sepsis was confirmed by tissue Gram's stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa. Femoral bone marrow cells were collected at 72 hours from surviving animals. Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur. Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6. RESULTS: Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups. Pneumonia was documented in all infected animals at 24 hours by Gram's stain and positive tissue cultures for Pseudomonas aeruginosa. Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only. Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05). Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur). Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus. CONCLUSION: Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis. Only the combined injury resulted in systemic sepsis and significantly increased mortality. We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.     

The effect of hemopure on coagulation in clinically relevant concentrations

Hemopure is a new colloidal blood substitute that may influence coagulation. We designed this study to examine the influence of this product on in vitro coagulation of whole blood by using the thrombelastograph (TEG). Blood samples from 20 volunteers were obtained. Hemopure was added to blood samples to obtain 0.5, 1, and 2 g/dL mixtures of Hemopure in blood. Control consisted of an undiluted sample and, for comparison, two samples diluted with volumes of lactated Ringer's solution (LR) equivalent to the two higher Hemopure dilutions. TEG with Hemopure at a concentration of 2 g/dL showed significantly shorter reaction and clot formation k times and an increased alpha angle compared with control. LR dilution with equivalent volume to 2 g/dL Hemopure solution also resulted in significantly shorter reaction and k times, as well as an increased alpha angle. Coagulation in samples with Hemopure at concentrations of 0.5 and 1 mg/dL did not vary significantly from control. Maximum amplitude did not vary significantly from control in any samples. The effect of Hemopure on TEG measures of coagulation is not significantly different from that of LR at clinically relevant concentrations.     

Sera from patients with anti-GBM nephritis including Goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen {alpha} chains

BACKGROUND.: Goodpasture (GP) syndrome is defined by the clinical associationof pulmonary haemorrhage with rapidly progressive glomerulonephritis.The disease is caused by pathogenic autoantibodies directedagainst type IV collagen, which is a major structural componentof glomerular basement membranes (GBM). METHODS.: The non-collagenous domains (NC1) of all six human type IV collagenchains was produced in E. coli as recombinant fusion proteinswith glutathione-S transferase. Sera from 10 patients with differenttypes of anti-GBM nephritis, including GP syndrome, were testedfor reactivity with the six proteins using immunoblotting ofdenatured and reduced proteins and ELISA without reduction. RESULTS.: All 10 sera reacted with the 3(IV) collagen chain by immunoblottingand ELISA. One serum also recognized the 2(IV) 4(IV), 5(IV)and 6(IV) chains by immunoblotting. ELISA measurements revealedreactivity of several other sera with 2(IV), 4(IV) or 6(IV)but not with 5(IV) collagen chains. No reactivity was observedwith the 1(IV) chain. CONCLUSION.: Autoantibodies in anti-GBM nephritis may not be directed onlyagainst the 3(IV) collagen chain and they frequently recognizeconformational epitopes.     

Effects of aging on behavioral assessment performance: implications for clinically relevant models of neurological disease

Object Despite the role of aging in development of neurological and neurodegenerative diseases, the effects of age are often disregarded in experimental design of preclinical studies. Functional assessment increases the clinical relevance of animal models of neurological disease and adds value beyond traditional histological measures. However, the relationship between age and functional impairment has not been systematically assessed through a battery of functional tests. Methods In this study, various sensorimotor and behavioral tests were used to evaluate effects of aging on functional performance in naive animals. Sensorimotor measures included locomotor activity; Rotarod, inclined plane, and grip-strength testing; and modified Neurological Severity Score. The Morris water maze was used to examine differences in learning and memory, and the elevated plus maze and forced swim test were used to assess anxiety-like and depressive-like behaviors, respectively. Results Older Sprague-Dawley rats (18-20 months) were found to perform significantly worse on the inclined plane tests, and they exhibited alterations in elevated-plus maze and forced swim test compared with young adult rats (3-4 months). Specifically, older rats exhibited reduced exploration of open arms in elevated plus maze and higher immobility time in forced swim test. Spatial acquisition and reference memory were diminished in older rats compared with those in young adult rats. Conclusions This study demonstrates clear differences between naive young adult and older animals, which may have implications in functional assessment for preclinical models of neurological disease.     

Prospects for clinically relevant epigenetic tests in the andrology laboratory

The review published by K1aver and Gromoll is a timely reminder of the ever-changing landscape of the clinical andrology laboratory as well as the limitations of current approaches to properly diagnose and treat male infertility. In a variety of disciplines, the clinical relevance of epigenetic status is becoming increasingly apparent. In this paper, the authors review the various studies that have evaluated the epigenetics （primarily DNA methylation） of sperm in the context of infertility, and they discuss the potential value of sperm epigenetic analysis in the workup of idiopathic male infertility.     

Identification of post-transplant anti-{alpha}5(IV) collagen alloantibodies in X-linked Alport syndrome

X-linked Alport syndrome (AS) is a heritable disorder whichis associated with mutations in the type IV collagen 5(IV) chaingene (COL4AS) located on chromosome X. Following renal transplantation,an average of 6% of male AS patients develop anti-GBM nephritis.We studied the specificity of the antibodies against type IVcollagen in the serum of a patient with COL4A5 partial deletion.The specificity of these alloantibodies was determined againstcollagenasedigested GBM, as well as against recombinant noncollagenous(NCl) domains of the type IV collagen 1(IV)—6(IV) chainsexpressed in Escherichia coli. Immunoblotting and ELISA demonstratedthat these antibodies bound specifically to the NCl domain of5(IV) collagen. There was no binding to the NCl domain of theother chains, including the Goodpasture antigen. CompetitiveELISA confirmed the results obtained by ELISA and immunoblotting.This patient developed alloantibodies directed against antigenspresent in the grafted kidney, but absent from his Alport kidney.The pathogenesis of post-transplantation glomerulonephritisin the Alport patient studied is thus similar to that of Goodpasturesyndrome, with the exception that the pathogenic antibodiesare targeted to another chain of type IV collagen.