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Background: Regulation of renal hemodynamics -- especially intraglomerular hemodynamics -- is closely related to the L-arginine (L-Arg)/nitric oxide (NO) pathway, both under basal conditions and in acute renal failure (ARF). Also, superoxide anions -- which may react with NO -- play a role in ischemic ARF. L-Arg not only has beneficial effects on glomerular filtration rate (GFR) but also reduces O2(-) production and prevents NO synthase isoform I up-regulation. Thus, it is of interest to elucidate whether the potential beneficial effects of L-Arg in reperfusion can be augmented by additional treatment with superoxide dismutase (SOD)., Methods: ARF was induced by renal artery clamping for 40 minutes. Animals were treated with either L-Arg, SOD, a combination of both, or saline. GFR, renal plasma flow (RPF), filtration fraction (FF) and blood pressure were recorded at baseline, after induction of ARF, during drug infusion and thereafter., Results: Renal artery clamping induces a severe drop of GFR, RPF and FF, which all are improved by L-Arg and SOD. Increasing GFR is mainly due to better renal perfusion. FF fell after reperfusion and increased with L-Arg and SOD, indicating improvement of disturbed intrarenal hemodynamics. Combined administration of L-Arg and SOD showed similar effects in comparison with each substance alone, but did not induce additional effects on GFR and RPF., Conclusions: L-Arg and SOD exert beneficial effects in ischemic ARF. Probably, improvements in reducing NO availability and in enhancing O2(-) formation are both playing a mediating role. The underlying mechanisms regulating the interplay between NO availability and O2(-) formation need to be elucidated in further studies using -- aside from other means -- selective NOS inhibitors, intervention in different experimental phases and treatment for a longer period.

The oculo-cerebro-renal syndrome of Lowe (OCRL) is a rare X-chromosomal disorder characterised by the triad of congenital cataracts, renal tubular dysfunction, and mental retardation. Typically complete opacification and discoid deformation of the lenses are seen, indicating a developmental defect in early embryogenesis. We report on a 35-year-old patient with a mild Lowe syndrome phenotype including incomplete lenticular opacities. Clinical findings suggest that the gene product of the mutated allele (IVS19 + 1G > A) identified in the patient exhibits some residual function.

Background: The regulation of renal hemodynamics is closely related to the L-arginine (L-Arg)/nitric oxide (NO) pathway. NO - metabolized from L-Arg - is capable of improving renal function in ischemic and toxic acute renal failure (ARF), while NO synthase (NOS) inhibition induces deterioration in renal function. The mortality rate in patients with septic shock is increased when treated with a non-selective NOS inhibitor, while the incidence of ARF requiring renal replacement therapy is unaffected. To date, there are no studies on the impact of NOS substrate (L-Arg) and inhibitor (L-NMMA) on renal function in early lipopolysaccharide (LPS)-induced ARF., Methods: ARF was induced by intravenous (i.v.) LPS. Animals were treated with L-Arg, L-NMMA (NOS substrate and inhibitor), a combination of both or saline. Glomerular filtration rate (GFR), urine flow, fractional sodium excretion, excretion of NO metabolism stable end products and blood pressure (BP) were recorded at baseline, after ARF induction, during drug infusion and thereafter., Results: L-Arg induced better GFR during infusion. Excretion of the NO metabolism end products was highest in the L-Arg group and lowest in the NOS inhibitor group. L-Arg administration had no influence on BP, while L-NMMA induced a slight elevation., Conclusions: We conclude that exogenous L-Arg exerts beneficial effects in early LPS-induced ARF in rats during drug infusion, while NOS inhibition has no influence on GFR. Subcellular compartmentalization of the L-Arg pool in cytoplasma and the rapid utilization of exogenous L-Arg in such a micro-environment could explain this effect, which has been observed in other ARF models and was called the "L-Arg paradox". In further studies the effects of early and prolonged administration of L-Arg in endotoxinemia should be investigated.

Lowe syndrome, or oculocerebrorenal syndrome of Lowe (OCRL), is a rare X-chromosomal disorder characterized by renal dysfunction, congenital cataract, and, in the majority of cases, mental retardation. Although gradual loss of renal function has been seen in most patients, age of onset of deterioration in renal function and its severity and course over time in adult patients have not been documented in detail. We report a 34-year-old man with OCRL without histological changes in renal tissue at the ages of 5 and 8 years, whereas at the age of 29 years, focal and segmental glomerulosclerosis and tubular atrophy were found. During subsequent follow-up of 5 years, progressive loss of renal function occurred, and end-stage renal failure can be expected in a few years. Clinical diagnosis was strongly supported by detecting a nucleotide substitution (IVS19+1g-->a) in the evolutionarily strictly conserved splice consensus sequence of intron 19 of the OCRL1 gene, which may interfere with normal splicing. Clinical course, possible molecular consequences of this novel mutation, and correlation between genotype and phenotype are discussed.

Alport syndrome (AS) is a type IV collagen hereditary disease characterized by progressive hematuric nephritis, hearing loss, and ocular changes. Mutations in the COL4A5 collagen gene are responsible for the more common X-linked dominant form of the disease characterized by much less severe disease in girls and women. A "European Community Alport Syndrome Concerted Action" (ECASCA) group was established to delineate the Alport syndrome phenotype in each gender and to determine genotype-phenotype correlations in a large number of families. Data concerning 329 families, 250 of them with an X-linked transmission, were collected. Characteristics of heterozygous girls and women belonging to the 195 families with proven COL4A5 mutation are compared with those of hemizygous boys and men. Hematuria was observed in 95% of carriers and consistently absent in the others. Proteinuria, hearing loss, and ocular defects developed in 75%, 28%, and 15%, respectively. The probability of developing end-stage renal disease or deafness before the age of 40 yr was 12% and 10%, respectively, in girls and women versus 90 and 80%, respectively, in boys and men. The risk of progression to end-stage renal disease appears to increase after the age of 60 yr in women. Because of the absence of genotype-phenotype correlation and the large intrafamilial phenotypic heterogeneity, early prognosis of the disease in X-linked Alport syndrome carriers remains moot. Risk factors for developing renal failure have been identified: the occurrence and progressive increase in proteinuria, and the development of a hearing defect.

Background: Alport syndrome (AS) is a common hereditary cause for end-stage renal failure due to a defect in type IV collagen genes. The molecular pathogenesis of benign familial haematuria (BFH) is not fully understood. Evidence from linkage analyses and mutation studies point to a role of the COL4A3/COL4A4 genes. The present study describes molecular changes of the COL4A4 gene that cause both diseases: autosomal recessive AS and BFH in a consanguine family with a 400-year-old history of haematuria., Methods: RNA and DNA were isolated and analysed by RT-PCR, PCR, DNA and cDNA sequencing, and Southern blotting. Evaluation of family members comprised creatinine clearence, urine analysis, audiometry and past medical history., Results: Forefathers of this family moved to a German village in the 17th century. Sporadic episodes of macrohaematuria have been reported ever since. Numerous family members with haematuria including the parents of the index family were heterozygous for a splice defect eliminating exon 25 from the alpha4(IV) cDNA. The daughter (15 years old, creatinine clearence 27 ml/min, proteinuria 5 g/day, hearing loss) was homozygous for the mutation, while the son (22 years old, creatinine clearance 68 ml/min, proteinuria 11 g/day, hearing loss, splitted and thickened glomerular basement membrane) was heterozygous. Further analysis showed a second mutation, an 18 bp in-frame deletion in exon 25, for which numerous family members were heterozygous, and both children were homozygous., Conclusions: The COL4A4 splice defect causes BFH-phenotype in heterozygous, and AS in homozygous state. The clinical spectrum of heterozygous individuals reaches from macrohaematuria, intermittent microhaematuria to isolated deafness. The 18 bp in-frame deletion aggravates the phenotype in the compound heterozygous son. These results give further evidence that BFH and autosomal AS are in fact both type IV collagen diseases.

Background: Alport syndrome (AS) is a hereditary nephropathy characterized by progressive renal failure, hearing loss and ocular lesions. Numerous mutations of the COL4A5 gene encoding the alpha 5-chain of type IV collagen have been described, establishing the molecular cause of AS. The goal of the present study was to identify the genotype-phenotype correlations that are helpful in clinical counseling. COL4A5-mutations (n=267) in males were analysed including 23 German Alport families., Methods: Exons of the COL4A5 gene were PCR-amplified and screened by Southern blot, direct sequencing or denaturing gradient gel electrophoresis. Phenotypes were obtained by questionnaires or extracted from 44 publications in the literature. Data were analysed by Kaplan-Meier statistics, chi(2) and Kruskal-Wallis tests., Results: Genotype-phenotype data for 23 German Alport families are reported. Analysis of these data and of mutations published in the literature showed the type of mutation being a significant predictor of end-stage renal failure (ESRF) age. The patients' renal phenotypes could be grouped into three cohorts: (1) large rearrangements, frame shift, nonsense, and splice donor mutations had a mean ESRF age of 19.8+/-5.7 years; (2) non-glycine- or 3' glycine-missense mutations, in-frame deletions/insertions and splice acceptor mutations had a mean ESRF age of 25.7+/-7.2 years and fewer extrarenal symptoms; (3) 5' glycine substitutions had an even later onset of ESRF at 30.1+/-7.2 years. Glycine-substitutions occurred less commonly de novo than all other mutations (5.5% vs 13.9%). However, due to the evolutionary advantage of their moderate phenotype, they were the most common mutations. The intrafamilial phenotype of an individual mutation was found to be very consistent with regards to the manifestation of deafness, lenticonus and the time point of onset of ESRF., Conclusions: Knowledge of the mutation adds significant information about the progress of renal and extrarenal disease in males with X-linked AS. We suggest that the considerable prognostic relevance of a patient's genotype should be included in the classification of the Alport phenotype.

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.alpha4.alpha5(IV) network. In Alport syndrome, mutations in any of the genes encoding the alpha3(IV), alpha4(IV), and alpha5(IV) chains cause the absence of the alpha3. alpha4.alpha5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the alpha5 chain was shown to be linked to the alpha3 and alpha4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified alpha1-alpha5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an alpha1.alpha2 and alpha3.alpha4.alpha5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the alpha1.alpha2 and alpha3.alpha4.alpha5 networks of GBM. Moreover, hexamer formation from the alpha3, alpha4, and alpha5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the alpha3.alpha4.alpha5 network by interfering with the assembly of the alpha3.alpha4.alpha5 NC1 hexamer.

Alport syndrome (AS) is a genetic disorder of basement membranes caused by mutations in type IV collagen genes that is characterized by chronic hematuria and progressive nephropathy leading to renal failure. The main extrarenal features include sensorineural hearing loss and ocular lesions. The mode of inheritance is X-linked dominant in about 80%-85% of the affected families, whereas autosomal transmission is rarely encountered. We report a male patient originating from a healthy consanguineous Lebanese family who presented with an unusual association of obstructive uropathy and AS. Hematuria and proteinuria were initially attributed to a suspected poststreptococcal glomerulonephritis (GN) and high-grade subpelvic ureteral stenosis. Persistence of symptoms after medical treatment of poststreptococcal GN and surgical correction of obstructive uropathy finally led to renal biopsy. The observed ultrastructural changes of the glomerular basement membrane were typical for AS. Molecular genetic studies revealed a previously undescribed de novo mutation in the COL4A5 gene, excluding maternal heterozygotic carrier status. This case report emphasizes the importance of hereditary nephritis in the differential diagnosis of chronic hematuria, and demonstrates the value of molecular studies for genetic counselling in AS.

Alport syndrome (AS) is a type IV collagen hereditary disease characterized by the association of progressive hematuric nephritis, hearing loss, and, frequently, ocular changes. Mutations in the COL4A5 collagen gene are responsible for the more common X-linked dominant form of the disease. Considerable allelic heterogeneity has been observed. A "European Community Alport Syndrome Concerted Action" has been established to delineate accurately the AS phenotype and to determine genotype-phenotype correlations in a large number of families. Data concerning 329 families, 250 of them with an X-linked transmission, were collected. Characteristics of the 401 male patients belonging to the 195 families with COL4A5 mutation are presented. All male patients were hematuric, and the rate of progression to end-stage renal failure and deafness was mutation-dependent. Large deletions, non-sense mutations, or small mutations changing the reading frame conferred to affected male patients a 90% probability of developing end-stage renal failure before 30 yr of age, whereas the same risk was of 50 and 70%, respectively, in patients with missense or splice site mutation. The risk of developing hearing loss before 30 yr of age was approximately 60% in patients with missense mutations, contrary to 90% for the other types of mutations. The natural history of X-linked AS and correlations with COL4A5 mutations have been established in a large cohort of male patients. These data could be used for further evaluation of therapeutic approaches.

Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (Netzer, K.-O. et al. (1999) J. Biol. Chem. 274, 11267-11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract alpha3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease.

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

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.

Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.

Systemic lupus erythematosus is a chronic disease with many clinical features, while Goodpasture's syndrome usually becomes manifest with progressive glomerulonephritis and pulmonary hemorrhage. Rapidly declining renal function and even pulmonary hemorrhage may be the common feature. Early and precise diagnosis is most important as it may provide general prognostic information and serve as a guideline for initial therapy. Immunosuppression with oral cyclophosphamide and high dose corticosteroids together with plasmapheresis is used in Goodpasture's syndrome. Progressive lupus nephritis requires high dose corticosteroids together with i.v. pulses of cyclophosphamide for at least six months, followed by maintenance immunosuppression. The benefits of therapy must always be weighed against the risks. Nevertheless, current therapy remains less than optimal. A better understanding of the pathogenesis of systemic lupus erythrematosis (SLE) and Goodpasture's syndrome may provide more specific information about the nature and the role of the immune response and thus lead to new treatment strategies.

Conditions for polymerase chain-reaction amplification of ten exon regions (Exons 3, 7, 11 through 13, and 15 through 19) of the collagen COL4A5 gene and four exon regions (Exons 2, and 12 through 14) of the COL4A6 gene were sequenced and established in this study. These Type IV collagen genes contain 51 and 48 exons, respectively. The sequences of these exons were determined in the two genes in 250 male patients with hematuria and suspected Alport syndrome. Seventeen mutations were found in nine of the ten exons studied in the COL4A5 gene in 17 patients, whereas no mutations were identified in COL4A6. One mutation was identical in two patients known to be unrelated. The results indicate that mutations in COL4A5 that leading to renal failure are more frequent than those involved in classic Alport syndrome, and also that mutations in COL4A6 are not likely to cause this disease. Furthermore, mutations in COL4A5 are distributed quite randomly and no "hot spots" were found.

The mechanisms of glomerular injury are multiple: one aspect is cell-mediated immunity in inflammatory kidney disease. Its fundamental role has been established within the last decade. T cells seem to play a regulatory role in the course of inflammation not only in the proliferative and crescentic disease, but also in nonproliferative glomerulonephritis. T cells become increasingly important in the pathogenesis of both glomerular and interstitial kidney diseases and seem to determine course and prognosis. The recent advances in understanding the mechanisms of T cell-mediated inflammation in kidney disease offer new approaches in therapy.

Foamy viruses (FVs) persist in healthy individuals of various mammalian species, including nonhuman primates. Laboratory markers of FV infection are (1) virus in throat epithelium or peripheral blood lymphocytes (PBLs), (2) proviral DNA sequences in PBLs and various solid organs, and (3) antibodies reactive to viral antigens on Western blots, in radioimmunoprecipitation tests, and in immunofluorescence assays. Using PCR and serological tests, we readily detected FV markers in naturally infected African green monkeys, rhesus monkeys, and chimpanzees, as well as in accidentally infected humans. Transmission of simian foamy viruses to humans (by bite or inadvertent laboratory infection) leads to viral markers, without affecting the recipient. Reports on FV-associated clinical disorders (e.g., thyroid or neurological) have remained controversial. In this study we failed to detect, by PCR, viral sequences in the samples from 223 patients, including 16 HIV-infected Africans, 46 Graves' disease patients, and 28 patients with the de Quervain's thyroiditis. Evaluation of 2688 sera from suspected high-risk areas (e.g., Central and East Africa, or high-risk groups such as HIV-infected individuals and patients with AIDS, thyroid, and neurological disorders) did not reveal FV-specific antibodies in a single case. Previously reported FV seroprevalence in various populations has never been verified by appropriate confirmatory tests. The strain of "human foamy virus" has remained a unique isolate. In conclusion, FVs are unlikely--at present--to circulate in human populations.

Mutations in the COL4A5 gene encoding the alpha 5(IV) chain of type IV collagen have been implicated as the primary defect in X-linked Alport syndrome. Several kinds of mutations have been reported so far, spanning point mutations to complete gene deletions. About 5% of Alport patients, who undergo renal transplantation, develop anti-glomerular basement membrane (GBM) nephritis, causing loss of allograft function. In one such patient, COL4A5 gene deletion was recently identified. In the present study, the GBM constituent, targeted by the anti-GBM alloantibodies from the patient who had complete COL4A5 gene deletion was identified. Its identity was determined on the basis of circulating antibody binding to various GBM constituents, domains of bovine type IV collagen and recombinant NC1 domain of human type IV collagen. These results establish, for the first time, the absence of the alpha 5(IV) chain in Alport GBM and, in the same patient, the production of an alloantibody that is targeted to a different chain of type IV collagen, the alpha 3(IV) chain. These findings provide further support for the hypothesis that: (1) anti-alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in Alport GBM, as reflected by the production of anti-alpha 3(IV) alloantibodies.

Anti-basement membrane antibody (anti-BM Ab) mediated disease is reported to be a rare disorder frequently leading to severe deterioration of renal function. It was our purpose to work out parameters necessary to predict the outcome reliably and to examine, who will benefit most from therapy. Data from 35 patients were evaluated retrospectively. Diagnosis was based on the detection of linear IgG staining (n = 28) along the glomerular basement membrane (GBM) in renal biopsies and/or on the demonstration of anti-BM Ab both by ELISA and immunoblotting (n = 35). Patients were followed up for at least 6 months. Several variables were analysed as to whether they are appropriate prognostic factors. Twenty patients (57%) presented with Goodpasture's syndrome, 13 (37%) had anti-GBM glomerulonephritis alone, whereas two patients suffered solely from pulmonary haemosiderosis. Frequent initial symptoms were haemoptysis (n = 18), haematuria (n = 26), proteinuria (n = 26) and elevated serum creatinine (n = 27). Among all, 10 patients improved, having stable renal function. Twenty-one patients developed end-stage renal failure and four died. Parameters indicating a poor prognosis were a serum(s)-creatinine greater than 600 mumol/l and crescent formation in more than 50% of the glomeruli on renal biopsy. By combining these two parameters the outcome could be reliably predicted. The initial antibody titre and cigarette smoking were without predictive value. In conclusion, the earlier therapy starts, the better will be the result. Patients presenting early with a serum creatinine < 200 mumol/l and without severe glomerular alterations gained the most benefit from therapy, indicating that outcome may be improved by early diagnosis.

Human foamy virus (HFV) is a retrovirus encoding structural genes and, like human immunodeficiency virus and human T cell leukemia virus I, several ancillary reading frames collectively termed the be1 genes. We have previously shown that HFV transgenic mice develop an encephalopathy with neuronal loss in hippocampus and cerebral cortex. We have now raised and characterized rabbit antisera to various recombinant portions of gag, pol, env, and bel-1, the viral trans-activator. Immunoreactivity for gag and bel-1 was observed in nuclei and processes of hippocampal and cortical neurons before the onset of morphological lesions and correlated with the appearance of HFV mRNA. Astrocyte-derived multinucleated giant cells containing HFV proteins were present in the brain of transgenic mice coexpressing full-length HFV genes but not in mice expressing truncated gag and env, suggesting that these genes contain a fusogenic domain. Expression of full-length structural genes decreased the life expectancy of transgenic mice, implying an adjuvant role for these proteins in HFV-induced brain damage.

Mutations affecting the COL4A5 gene encoding the alpha 5 chain of type IV collagen, are involved in the pathogenesis of X-linked Alport syndrome. We used denaturing gradient gel electrophoresis (DGGE) to screen PCR amplified exons of COL4A5 for point mutations in a set of 18 Alport patients previously characterized by Southern blotting. One sequence variant was identified in the exon 38 region of a male Alport patient. Sequence analysis revealed a G to C transversion in the 5' intron splice donor site downstream from exon 38 (GT to CT). To determine the effect of the mutation on mRNA splicing, alpha 5(IV) cDNA was generated from total RNA of peripheral blood lymphocytes. Subsequent cDNA PCR yielded a product 81 base pairs shorter in the affected Alport patient, compared to normal controls. The absence of exon 38 from the alpha 5(IV) cDNA was confirmed by sequence analysis. The results demonstrated that the mutation leads to skipping of exon 38 in the processing of alpha 5(IV) pre-mRNA. The shortened transcript lacked 27 codons encoding a Gly-X-Y-repeat sequence with a preserved reading frame, enabling the translation of codons further downstream. Clinically, the patient presented with juvenile onset Alport syndrome, end-stage renal failure, and deafness. He had no ocular lesions. Typical ultrastructural changes of the glomerular basement membrane (GBM) were shown on electron microscopy. The patient developed anti-GBM antibodies after renal transplantation, however, renal function deteriorated only moderately.

Human foamy virus pol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5' pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. In immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (M(r)) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domains of pol. With concentrated virus as antigen, the RT and RNaseH antibodies recognized a protein of 80K, the IN antiserum recognized a protein of 40K, and the PR antiserum detected a protein of approximately 10K.

Mutations in the COL4A5 gene encoding the alpha 5 chain of type IV collagen have been found in linkage with X-chromosomal Alport syndrome (AS). To identify COL4A5 mutations in patients from Germany with clinically defined AS, DNA from 20 unrelated patients was analyzed by conventional Southern blotting. By using full length alpha 5(IV) cDNA probes, large COL4A5 deletions could be detected in two patients. In one case, a 34 kb deletion affecting the 14 most 3' exons of the gene was observed. The second patient harbored a complete COL4A5 deletion. In both cases, functional alpha 5(IV) mRNA was unlikely to be present. Clinically, both patients developed end-stage renal failure before age 30. Furthermore, they had characteristic retinal flecks, and sensorineural hearing loss with typical changes on the audiogram. The patient with the complete deletion of COL4A5 lost the renal allograft due to an anti-GBM mediated glomerulonephritis.

We review the recent progress achieved on the understanding of the molecular basis of Alport's syndrome. This inherited disease is defined as progressive nephritis with sensorineural hearing loss. In 80%-85% of the families, inheritance is compatible with X-linked dominant transmission, whereas in the remaining cases autosomal dominant transmission is assumed. Histology studies demonstrated that the main defect is within the glomerular basement membrane (GBM). In addition, evidence for an altered GBM antigenicity came from immunofluorescence studies which showed a reduced or absent binding of anti-GBM autoantibodies or monoclonal antibodies to the "Goodpasture antigen" in some families. Subsequent studies added substantial evidence that Alport's syndrome is a type IV collagen disease. Genetic linkage analyses coherently identified an Alport locus at the X-chromosomal region Xq21.3-22. Recently, a previously unknown alpha 5 chain of type IV collagen was identified, and the corresponding gene was also mapped to Xq22. Subsequent studies on Alport families by various groups identified more than 25 COL4A5 lesions. Segregation in linkage with the Alport phenotype could be shown in large kindreds. Mainly deletions and only a few point mutations were described. Most lesions reported so far are heterogeneous. We were able to identify two deletions and one point mutation involving a 3' splice site in 20 Alport families from Germany. One of the patients with a COL4A5 deletion and the patient with the splice site mutation developed anti-GBM antibodies after renal transplantation. In contrast, no COL4A5 lesions have been found in 2 further patients with posttransplant anti-GBM nephritis.(ABSTRACT TRUNCATED AT 250 WORDS)

We have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelled viral proteins were immunoprecipitated from HFV-infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 31K to 170K. Labelling of proteins with [14C]glucosamine or with [35S]methionine in the presence of tunicamycin, as well as endo-beta-N-acetylglycosaminidase H and F treatment of [35S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.

An infectious molecular clone (pHSRV) of the human Spumaretrovirus (HSRV) was constructed using viral DNA and cDNA clones. The infectivity of pHSRV was proven by transfection of cell cultures and subsequent infection of susceptible cultures with cell free transfection derived virus. pHSRV derived virus produced foamy virus typical cytopathic effects in susceptible cultures. Infected cells could be stained specifically with foamy virus antisera by means of indirect immunofluorescence. Radioimmunoprecipitation revealed the presence of characteristic HSRV structural proteins in pHSRV infected cultures. By cotransfection of pHSRV and an indicator plasmid it was found that pHSRV is able to transactivate the viral LTR. Viral transcripts were found to be approximately 200 bases longer in pHSRV infected cultures compared to wildtype infected cultures. This difference is most likely due to an insertion of DNA of non-viral origin in the U3 region of the 3'LTR of the infectious clone.

Unlabelled: Experiments were carried out on 183 rats to study the effect of a complex therapy for treatment of mesenteric shock. The superior mesenteric artery (SMA) was temporarily ligated for 90 min under ether anesthesia; this was followed by an analgesic treatment. After release of the ligated artery, fluid therapy was instituted by administering the equivalent of 7.5% of body weight of one of three different solutions: ringer lactate (RL), hydroxyethyl starch (HES) in RL, and perfluorochemicals (PFC, 4 g/kg b.wt. in RL with HES), the latter with the aim to improve the oxygen transport to the tissue. The same fluid therapy was carried out on rats pretreated with E. coli endotoxin. Endotoxin pretreatment was chosen to compensate the negative effect of PFC on the reticuloendothelial system (RES) as shown in previous studies. Survival time and survival rate were recorded as well as hematocrit values at different times before and after treatment. Experimental groups were:, Controls: (1) SMA occlusion without release; (2) 90-min occlusion without therapy. Treated animals: (3) RL therapy; (4) therapy with HES in RL; (5) therapy with PFC in RL and HES; (6), (7), and (8) identical therapies as Groups 3, 4, and 5, respectively, but with endotoxin pretreatment. Survival time increased to the same extent if HES or PFC were added to RL. There was a further increase both in survival times and rates with endotoxin pretreatment (Groups 6, 7, and 8 vs. Groups 3, 4, and 5). The highest survival time and rate were obtained in Group 8, which received PFC therapy with endotoxin pretreatment. There was a slight negative correlation between survival time and hematocrit values if all groups were considered together.(ABSTRACT TRUNCATED AT 250 WORDS)