Search

Background: The demonstration of proteinase 3 specific antineutrophil cytoplasmic antibodies (PR3-ANCA), and the estimation of antibody values are useful in the diagnosis and management of patients with Wegener's granulomatosis (WG). [...]

Background: The 'International consensus statement on testing and reporting antineutrophil cytoplasmic antibodies (ANCA)' advocates screening by indirect immunofluorescence (IIF), but external quality assessment programmes often demonstrate different IF patterns for [...]

Background: Dense deposit disease and atypical hemolytic uremic syndrome are often caused by Complement Factor H (CFH) mutations. This study describes the retinal abnormalities in dense deposit disease and, for the first time, atypical haemolytic uremic syndrome. It also reviews our understanding of drusen pathogenesis and their relevance for glomerular disease.Methods: Six individuals with dense deposit disease and one with atypical haemolytic uremic syndrome were studied from 2 to 40 years after presentation. Five had renal transplants. All four who had genetic testing hadCFHmutations. Individuals underwent ophthalmological review and retinal photography, and in some cases, optical coherence tomography, and further tests of retinal function.Results: All subjects with dense deposit disease had impaired night vision and retinal drusen or whitish-yellow deposits. Retinal atrophy, pigmentation, and hemorrhage were common. In late disease, peripheral vision was restricted, central vision was distorted, and there were scotoma from sub-retinal choroidal neovascular membranes and atypical serous retinopathy. Drusen were present but less prominent in the young person with atypical uremic syndrome due to a heterozygousCFHmutation.Conclusions: Drusen are common in forms of C3 glomerulopathy caused by compound heterozygous or heterozygousCFHmutations. They are useful diagnostically but also impair vision. Drusen have an identical composition to glomerular deposits. They are also identical to the drusen of age-related macular degeneration, and may respond to the same treatments. Individuals with a C3 glomerulopathy should be assessed ophthalmologically at diagnosis, and monitored regularly for vision-threatening complications. [ABSTRACT FROM AUTHOR]

Most patients with Alport syndrome have X-linked or autosomal recessive disease that is characterised by renal failure, hearing loss, and, in nearly 75% of the cases, a dot-and-fleck retinopathy and anterior lenticonus. There are only case reports of individuals with the rare autosomal dominant form, who can have haematuria or renal failure, deafness, and, in addition, low platelet counts and neutrophil inclusions. The ocular features of autosomal dominant inheritance have not been described. We have examined the eyes in the members of two families where Alport syndrome was diagnosed on the basis of the clinical features and family history, and where autosomal dominant inheritance was confirmed by father-to-son disease transmission, the associated haematological abnormalities, and haplotypes that segregated with the recently described locus at chromosome 22q. In Family A, the eyes of two individuals with haematuria, hearing loss, and haematological abnormalities and of nine unaffected family members were examined. In Family B, the eyes of two individuals with renal failure, normal hearing, and haematological abnormalities were examined. None of the affected or unaffected members in either family had a dot-and-fleck retinopathy, anterior lenticonus, a history suggesting recurrent corneal erosions, or corneal dystrophy. These results indicate that the protein abnormality in autosomal dominant Alport syndrome does not produce the retinopathy and lenticonus typical of X-linked and autosomal recessive disease. This may be because the abnormal protein is not present or is less important in the ocular basement membranes than elsewhere, or because the presence of a normal allele in autosomal dominant disease compensates for the defective allele. [ABSTRACT FROM AUTHOR]

α1-antitrypsin (α1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of α1-AT phenotypes associated with dysfunctional α1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of α1-AT. Phenotypes usually associated with a moderate or severe reduction in α1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding α1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of α1-AT. No abnormal α1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of α1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum α1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal α1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal α1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have α1-AT phenotypes that are usually associated with low serum levels of α1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal α1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal α1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal α1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production. [ABSTRACT FROM AUTHOR]

Anti-neutrophil cytoplasm antibodies (ANCA) occur occasionally in rheumatoid arthritis (RA), but their incidence and clinical significance have been unclear. In this study we have investigated 58 patients with RA. In 22 patients the disease was inactive and the remaining 36 with active disease were further subdivided into those without clinical evidence of vasculitis (26), those with cutaneous vasculitis (8) and those with systemic vasculitis (2). ANCA were demonstrated by indirect immunofluorescence in 10 of the 58 patients (17%). While both perinuclear (pANCA) and cytoplasmic (cANCA) staining were detected. pANCA were more common (70%). Neutrophil-specific anti-nuclear antibodies (ANNA) were demonstrated in a further eight sera (14%) and ANA were detected on Hep-2 cells in 30 of the 58 sera (52%). ELISAs for the detection of anti-myeloperoxidase and anti-elastase antibodies were then established. Five sera with pANCA and five that contained ANNA were negative for both anti-myeloperoxidase and anti-elastase antibodies, suggesting other as yet unidentified cytoplasmic antigens as the target molecules. However, anti-myeloperoxidase or anti-elastase antibodies were found in four sera that had homogeneous or speckled ANA on both Hcp-2 cells and neutrophils. One serum contained both antibodies. The presence of ANCA detected by indirect immunofluorescence or of anti-myeloperoxidase or anti-elastase antibodies in these patients with RA was not associated with disease activity nor with the demonstration of cutaneous vasculitis or renal disease (P NS). A possible association with systemic vasculitis remains to be confirmed. There is an incomplete correlation between indirect immunofluorescence patterns and antibody specificity in ELISA systems. [ABSTRACT FROM AUTHOR]

Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal Failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes, These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to he important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n=4). the presence of sensori-neural deafness (n=4), a family history of Alport's syndrome (n=5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n=2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome. [ABSTRACT FROM AUTHOR]

The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all formal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3), Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding lo M2 and M3 were found within the kidney and that the published sequences were correct. When hetrologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3, While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs. there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within the collagenase-resistant part of the GBM, suggesting that these do represent parts of novel type IV collagen chains. However, sera containing anti-GBM antibodies did not bind to either peptide in solid-phase ELISAs, and these antibodies may recognize a different peptide sequence, features of the tertiary structure of these peptides or interactions between collagen chains. [ABSTRACT FROM AUTHOR]

Autoantibodies that produce a perinuclear pattern on indirect immunofluorescent examination of ethanol-fixed neutrophils (pANCA) are found in about half of all cases of microscopic polyarteritis. These antibodies are often directed against myeloperoxidase or elastase and we have developed sensitive reproducible ELISAs for their detection and study. Seven sera from 19 patients with microscopic polyarteritis or segmental necrotizing glomerulonephritis contained anti-myeloperoxidase or anti-elastase antibodies or both. In contrast, only one of 18 sera from patients with Wegener's granulomatosis, where the pattern of immunofluorescence is predominantly cytoplasmic, had anti-myeloperoxidase antibodies and no anti-elastase antibodies were detected. Using sera from patients with microscopic polyarteritis, both anti-myeloperoxidase and anti-elastase antibodies were demonstrated to be of high affinity. There was no immunoglobulin class, subclass or light chain restriction noted. Anti-myeloperoxidase and anti-elastase antibodies were also found occasionally in anti-glomerular basement membrane disease, mixed connective tissue disease, systemic lupus erythematosus, post-streptococcal glomerulonephritis and atypical pneumonia. In further studies these antibodies were not associated with other lung infections, although anti-elastase antibodies were noted in one of 14 sera positive for ASOT that were tested. Anti-myeloperoxidase antibodies were found more frequently than anti-elastase antibodies and these antibodies were occasionally present together. In addition some sera with pANCA had neither anti-myeloperoxidase nor antielastase antibodies. The target molecules in these cases remain unclear. [ABSTRACT FROM AUTHOR]

Antibodies that are directed against cytoplasmic constituents of neutrophils and monocytes (antineutrophil cytoplasm antibodies, ANCA) have been described in Wegener's granulomatosis, microscopic polyarteritis (MPA) and some cases of segmental necrotizing glomerulonephrilis (SNGN). Other antibodies occasionally described in Wegener's granulomatosis and MPA include anti-nuclear antibodies (ANA) and anti-glomerular basement membrane (GBM) antibodies. We have studied the diversity of the corresponding antigens in ANCA-associated renal diseases. Sera from 46 patients with active histologically proven Wegener's granulomalosis, MPA and SNGN were tested for ANCA by indirect immunofluorescent examination of normal peripheral blood neutrophils. Thirty-four sera (74%) were positive; 16 were associated with diffuse cytoplasmic staining (cANCA) and 18 with perinuclear staining (pANCA). In addition, five demonstrated antineutrophil- specific nuclear staining (ANNA), On Western blotting of the neutrophil extract, five sera recognized a 29-kD molecule recently identified as neutrophil proteinase 3, Two sera with typical cANCA bound to molecules of 36, 38 and 116 kD and another to a molecule of 22 kD. The final serum associated with pANCA bound lo a molecule of about 12 kD. Thirteen sera out of 46 (28%) tested in an ELISA contained anti-myeloperoxidase antibodies; 10 of these were associated with pANCA and two others with ANNA. Three sera of 17 (18%) tested contained anti-elastase antibodies; these also contained anli-mycloperoxidase antibodies and were associated with pANCA. However, eight sera with pANCA were negative for anti-mycloperoidase antibodies and three of these were also negative for anti-elastase antibodies, suggesting further unidentified target antigen or antigens associated with the pANCA. Fifteen of the 34 sera positive for ANCA also demonstrated anti-nuclear staining on Hep-2 cells (53%) in a speckled, homogeneous, or nucleolar pattern. ANA were significantly associated with the presence of pANCA (P<0.01), and levels of ANA and ANCA fell in parallel after treatment. One scrum with a pANCA was also positive for anti-GBM antibodies. Inhibition studies using ELISAs for anti-GBM antibodies indicated that there was no cross-reactivity between target molecules recognized by these antibodies. The diversity of target molecules recognized by ANCA suggests that cross-reactivity with bacterial structures is less likely as the primary antiological event in the development of these antibodies than tissue destruction; and that crossreactivity with vascular endothelium is also unlikely as the pathogenetic basis of vessel disease. [ABSTRACT FROM AUTHOR]

Reconstitution experiments in the heterologous phase of nephroioxic nephritis (HNTN) have shown that tissue injury is directly related to the number of polymorphonuclear neutrophils (PMN) infiltrating the glomeruli. Recent studies have indicated that endotoxin pretreatment in this model enhances tissue injury under certain conditions. The aim of this project was to investigate the relationship between glomerular PMN infiltration and tissue injury after endotoxin in HNTN. A dose of nephrotoxic globulin (NTG) that resulted in low levels of albuminuria was chosen. The administration of E. coli lipopolysaccharide (LPS) to Sprague-Dawley rats 24 h before the induction of HNTN resulted in the earlier appearance of larger numbers of glomerular PMN than in animals injected with nephrotoxic globulin alone (P < 0.05 at 1/2 and 1 h). However there was no significant increase in albuminuria nor in renal histological damage. There was no difference in the amount or rate of glomerular deposition of the NTG or rat complement between the two groups of rats. The more rapid accumulation of glomerular PMN can, however, be accounted for by the LPS-induced activation of endothelium, since endotoxin was detected in the kidneys 24 h after the injection of LPS. No endotoxin was present in the serum at the time of injection of NTG. Thus we have demonstrated in a model of PMN-mediated tissue injury, that an increase in PMN influx is not necessarily associated with exaggerated injury. [ABSTRACT FROM AUTHOR]

A functional comparison between elicited peripheral blood neutrophils has been made in vivo and in vitro. Preliminary experiments showed that separation of peripheral blood cells on a metrizamide gradient yielded too few neutrophils for efficient radiolabelling with indium (In): hence a mixed cell preparation comprising 80%, neutrophils was elicited in the peripheral blood of adult male rats by the administration of endotoxin (0.25 mg i.a.) and cobra venom factor (200 μl i.p.) 20 h before. Peritoneal neutrophils were collected 4 h after the i.p. injection of 6 ml thioglycollate. Both populations differed markedly from normal peripheral neutrophils on the in vitro testing of random locomotion, chemotaxis and phagocytosis of Candida. After labelling with IIIIn-tropolonate, a greater proportion (mean = 8%) of peripheral blood cells localized to an E. coli/Freund's complete adjuvant-induced abscess compared with peritoneal neutrophils (mean = 3%). The abscess could be visualized externally by scanning with both cell preparations, but the distribution of activity differed markedly. The greater hepatic sequestration of peritoneal neutrophils suggested cell damage or activation. To overcome the difficulty of harvesting normal peripheral blood neutrophils in the rat, either of these populations can be used to follow the kinetics of inflammation. However, elicited peripheral blood cells yield a higher proportion of responding cells. [ABSTRACT FROM AUTHOR]

Background: This study compared the clinical and laboratory characteristics of patients with antiglomerular basement membrane (GBM) disease and normal renal function, with those of patients with anti-GBM disease where there was renal impairment. [ABSTRACT FROM PUBLISHER]

Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (β-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, β-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, β-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C-digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system. [ABSTRACT FROM AUTHOR]

Anti-proteinase 3 antibodies are a subgroup of anti-neutrophil cytoplasmic antibodies (ANCA), and we have established an ELISA for their detection using high performance liquid chromatography (HPLC)-purified protein. This assay is sensitive and specific: inhibition studies have shown that despite the homology between proteinase 3 and elastase there is no cross-reactivity between the corresponding antibodies for their targets. Anti-proteinase 3 antibodies were associated most often with cytoplasmic fluorescence (17/22, 77%), but occasionally with a perinuclear (3/22, 14%) or atypical pattern (1/2). These antibodies were found in 23 out of 76 sera (30%) that were positive in an ELISA based on a crude neutrophil cytoplasmic extract, and they were associated with both 29 and 55 kD bands on Western blots, Anti-proteinase 3 antibodies were found in most individuals with active Wegener's granulomatosis (10/13, 77%), but less often in individuals with microscopic polyarteritis (2/10, 20%) or segmental necrotizing glomerulonephritis (3/6, 50%). However, antiproteinase 3 antibodies were not detected in any of 32 sera from individuals with rheumatoid arthritis or systemic lupus erythematosus (SLE). Occasionally anti-proteinase 3 antibodies were associated with anti-glomerular basement membrane antibodies (1/11, 9%) or with anti-mycloperoxidase antibodies(1/11, 9%). IgM anti-proteinase 3 antibodies were uncommon (2/22 sera, 9%), and no IgA antibodies were demonstrated in any of 22 sera from patients with active systemic vasculitis. Significantly more individuals presented with anti-proteinase 3 antibodies in April-May-June, suggesting that an infective agent prevalent in Autumn might have a causative role in the associated diseases, Anti-proteinase 3 antibodies are the most common target antigen associated with Wegener's granulomatosis and cytoplasmic fluorescence. [ABSTRACT FROM AUTHOR]

Background Both IgA glomerulonephritis (IgA gn) and thin basement membrane disease (TBMD) are common forms of glomerulonephritis. Patients with these conditions may present with identical clinical features, but higher urinary RBC counts, heavier proteinuria, and impaired renal function are more common in patients with IgA gn. Because IgA gn and TBMD are common, some patients will have both diseases. Subjects We describe the clinical features of two individuals with both IgA gn and TBMD, and compare them with the clinical and laboratory characteristics in patients with TBMD (n=15) or IgA gn (n=32) alone. Results IgA gn was found in two individuals of the 110 with TBMD who were studied. They both had haematuria with >/100 000 RBC/ml and proteinuria >0.2/day (one had more than 1 g/day). These features were more consistent with IgA gn than TBMD alone. However, both individuals had normal serum creatinine and creatinine clearance at presentation. Additional clinical features were macroscopic haematuria in one and hypertension in both. Conclusion IgA deposits are not uncommon in patients with TBMD, and these patients have clinical features that resemble those seen in IgA gn rather than TBMD. Patients with both IgA gn and TBMD do not necessarily have the worse prognosis noted in some patients with IgA gn. [ABSTRACT FROM PUBLISHER]

We have examined 10 families with focal segmental glomerulosclerosis (FSGS) on renal biopsy for evidence of linkage to the 3 known loci, 1q, 11q and 19q using microsatellite markers. Seventy-five family members were examined including 27 affected individuals with proteinuria > 1g/day or renal failure. Inheritance was autosomal dominant in 4 families and autosomal recessive in 6 families. DNA was extracted from blood or buccal smears, and microsatellite markers located near the candidate gene loci were amplified using PCR. Proteinuria segregated at the lq locus in 2 families, at the 11q locus in 4 families (p 1/16 to 1/256) and at none of the candidate loci in 4 families. This is the first time that autosomal dominant inheritance has been described at the lq locus and autosomal recessive disease at the l lq locus. All individuals with autosomal recessive disease were compound heterozygotes except in one consanguinous family where the affected individual was homozygous at the locus. Only one of 13 carriers (7%) of autosomal recessive disease had proteinuria. When family members were studied for the ACE genotype, the DD polymorphism did not correlate with worse proteinuria. Thus FSGS is commonly inherited, with both autosomal recessive and dominant inheritance, and the 11q locus occurring most often. There is at least one further genetic locus for FSGS. Carriers cannot be identified on the basis of the presence of proteinuria. The genes responsible for FSGS at each locus are still unknown. [ABSTRACT FROM AUTHOR]

Background: Cardiac injury after orthopaedic surgery is an increasing problem particularly in an ageing population. The detection of cardiac injury has been aided by the use of cardiac troponins which has also raised questions about the utility of this enzyme in the post-operative setting.Objective: This review evaluates the diagnosis and pathophysiology of myocardial infarction after orthopaedic surgery and examines how myocardial injury is detected, with particular emphasis on the role of troponin testing.Subjects: Eight recent orthopaedic trials evaluating the use of troponin were identified in the literature and included in this review.Results: This review found that the diagnosis of myocardial infarction ismore difficult after surgery since classic symptoms may be atypical or absent. Therefore, there ismore reliance on the typical rise and fall in troponin to diagnose cardiac injury especially because electrocardiograph changes may be hard to detect. The pathophysiology of ischaemia after orthopaedic surgery may be different to ischaemia in the non-surgical setting. The incidence of troponin elevation is between 22 and 52.9% after emergency orthopaedic operations. Of note, patients sustaining a troponin elevation are often asymptomatic. Small studies have found troponin to be a prognostic marker of in-hospital cardiac complications, increased length of stay, increased likelihood of discharge to residential care and death at 1 year. No interventional studies have been published to date.Conclusion: Cardiac injury is an important complication after orthopaedic surgery. Studies have found that troponin testing can detect asymptomatic cardiac injury. These patients are at risk of poorer outcomes and future research should be directed towards treatment of these patients. [ABSTRACT FROM AUTHOR]