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Biomedical subjects

J Savige

Publications and source records attributed to J Savige.

At least 19 recordsLinked to original sources
PubMed2004

The binding of proteinase 3 antineutrophil cytoplasmic antibodies (PR3-ANCA) varies in different ELISAs.

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). However, external quality assessment programmes suggest that PR3-ANCA binding varies in different assays. AIM: To demonstrate variations in PR3-ANCA binding in different commercial and in house enzyme linked immunosorbent assays (ELISAs). METHOD: Binding of a PR3-ANCA standard and 19 sera from patients with WG was compared in eight commercial and in house assays. Binding was expressed in different units depending on the kit. RESULTS: One commercial assay performed unsatisfactorily. Three commercial kits produced PR3-ANCA binding (70, 102, and 84 U/ml) close to the expected value for the standard (100 U/ml). Serial dilutions of this standard were linear in only one commercial assay and the in house assay. Sera from patients with WG with borderline binding in the in house assay bound in the eight commercial kits at 0-148 kit units; low binding sera ranged from 0 to 273 units; moderately strong sera bound at 7-260 units; and strongly binding sera bound at 13-336 units. In four assays, at least one strongly positive serum bound at levels greater than the provided range. CONCLUSIONS: Levels of antibody binding and units of binding have not been standardised in commercially available PR3-ANCA ELISAs. This may affect the diagnosis and management of patients with WG, in addition to the implementation of international guidelines for treatment.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2002

Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate.

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 IIF patterns for a single serum. AIM: To determine whether the variation in IIF patterns can be attributed solely to errors in interpretation. METHODS: This study compared the IIF patterns produced by four sera (two with cytoplasmic or C-ANCA; one with perinuclear or P-ANCA with myeloperoxidase (MPO) specificity; and one P-ANCA without MPO specificity) that were tested in 11 different laboratories. The sera were examined according to individual laboratory protocols at dilutions of 1/10 to 1/40 using P1 (n = 4), P2 (n = 2), P3 (n = 2), or in house (n=3) neutrophil preparations and conjugates from manufacturers C1 (n = 3), C2 (n = 1), C3 (n = 2), C4 (n = 1), C5 (n = 2), and C6 (n = 2). The IIF patterns were noted in each laboratory, the testing repeated, and the fluorescent patterns photographed and subsequently discussed at a meeting of the Australian ANCA study group. RESULTS: All IIF patterns described in individual laboratories were confirmed on retesting and by the ANCA study group. Neutrophil substrates produced commercially or in house varied in their ability to demonstrate cytoplasmic granularity and interlobular accentuation, which distinguish between "C-ANCA" and "C-ANCA (atypical)". All commercial and in house neutrophil substrates demonstrated neutrophil nuclear extension of P-ANCA fluorescence, which correlates with MPO specificity. However, eight assays (eight of 43) from eight laboratories resulted in IIF patterns different from those usually seen. One of these produced a C-ANCA (atypical) rather than a C-ANCA pattern. The other seven resulted in at least some cytoplasmic fluorescence when the consensus pattern was P-ANCA with (n = 4) or without (n = 3) MPO specificity. These assays used three different commercial and one in house neutrophil substrate, and six different conjugates, with anti-IgG, anti-(Fab)'(2), anti-Ig (heavy and light chain), and anti-G, A, and M activity. Four of the seven assays tested on commercial substrates had used the manufacturer's conjugates. CONCLUSIONS: This study indicates that the variation in IIF patterns seen with ANCA positive sera tested in different laboratories does not necessarily result from errors in the interpretation of patterns and cannot be attributed solely to the use of a particular neutrophil substrate or conjugate, or to the use of substrate from one manufacturer and conjugate from another.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2001

Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes.

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.

Amino Acid Sequence↗
PubMed2001

Segregation of hematuria in thin basement membrane disease with haplotypes at the loci for Alport syndrome.

BACKGROUND: Inherited hematuria is common and is usually attributed to thin basement membrane disease (TBMD). The aim of this study was to determine how often hematuria in families with TBMD segregated with haplotypes at the chromosomal loci for autosomal recessive and X-linked Alport syndrome (COL4A3/COL4A4 and COL4A5, respectively). METHODS: The families of 22 individuals with TBMD on renal biopsy and with urinary glomerular red blood cell (RBC) counts of more than 50,000/mL were studied using phase-contrast microscopy of the urine and DNA microsatellite markers. Eighteen families had at least two members with hematuria. RESULTS: Hematuria segregated with or was consistent with segregation at the COL4A3/COL4A4 locus in eight (36%) families (P < 0.05 in 5 of these) and at the COL4A5 locus in four (18%) families (P < 0.05 in 2). The lack of segregation in the other 10 (45%) families may have occurred because of incomplete penetrance of the hematuria, de novo mutations, coincidental hematuria in other family members, or the presence of a novel gene locus. In four different families, three of which had hematuria that segregated with the COL4A3/COL4A4 locus, four family members with the hematuria haplotype had spouses with coincidental hematuria (4 of 29, 14%). However, none of their four offspring who had also inherited the hematuria haplotype had the clinical features of autosomal recessive Alport syndrome. CONCLUSIONS: Hematuria in families with TBMD commonly segregates with the COL4A3/COL4A4 locus and thus results from mutations in the same genes as autosomal recessive Alport syndrome. Sometimes TBMD may be confused with the carrier state for X-linked Alport syndrome. However, nearly half of the families in this study had hematuria that did not segregate with the loci for either autosomal recessive or X-linked Alport syndrome.

Adolescent↗
PubMed2001

COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome.

BACKGROUND: Carriers of autosomal-recessive and X-linked Alport syndrome often have a thinned glomerular basement membrane (GBM) and have mutations in the COL4A3/COL4A4 and COL4A5 genes respectively. Recently, we have shown that many individuals with thin basement membrane disease (TBMD) are also from families where hematuria segregates with the COL4A3/COL4A4 locus. This study describes the first COL4A4 mutation in an individual with biopsy-proven TBMD who did not have a family member with autosomal-recessive or X-linked Alport syndrome, inherited renal failure, or deafness. METHODS: The index case and all available family members were examined for dysmorphic hematuria> 50,000/mL using phase contrast microscopy and for segregation of hematuria with the COL4A3/COL4A4 and COL4A5 loci using DNA satellite markers. COL4A4 exons from the index case were then studied using the enzyme mismatch cleavage method, and exons that demonstrated abnormal cleavage products were sequenced. RESULTS: Hematuria in this family segregated with a haplotype at the COL4A3/COL4A4 locus (P = 0.031) but not with haplotypes at the COL4A5 locus. A mutation in COL4A4 that changed C to T resulting in an arginine residue being replaced by a stop codon (R1377X) was demonstrated in exon 44, which encodes part of the alpha 4(IV) collagen sequence close to the junction with the noncollagenous domain. This mutation was present in all five family members with hematuria, but not in the four unaffected family members, 33 unrelated individuals with TBMD, or 22 nonhematuric normals. CONCLUSIONS: R1377X has been described previously in a compound heterozygous form of autosomal-recessive Alport syndrome. Our observation is evidence that TBMD can represent a carrier state for autosomal-recessive Alport syndrome in at least some individuals.

Basement Membrane↗
PubMed2001

A comparison of the clinical, histopathologic, and ultrastructural phenotypes in carriers of X-linked and autosomal recessive Alport's syndrome.

Previous series that described phenotypes in carriers of Alport's syndrome did not distinguish genetically between carriers of X-linked and autosomal recessive disease. In this study, modes of inheritance in unselected families with Alport's syndrome associated with two city and two provincial hospitals were determined using microsatellite markers, and carriers of disease haplotypes were identified within these families. All 47 carriers (100%) from 18 families with X-linked Alport's syndrome had dysmorphic hematuria on phase-contrast microscopy, but few developed renal failure (3 of 40 carriers; 8%), clinical hearing loss (2 of 45 carriers; 4%), retinopathy (1 of 30 carriers; 3%), or lenticonus (0 of 30 carriers; 0%). Eleven of the 14 carriers (79%) from 2 families with autosomal recessive disease had dysmorphic hematuria, but none had renal failure, clinical hearing loss, retinopathy, or lenticonus. Urinary red blood cell counts in carriers of X-linked Alport's syndrome were greater than those in carriers of autosomal recessive disease (P < 0.0001), but the frequency of proteinuria and hypertension and levels of proteinuria were not different. There was more tubulointerstitial damage in carriers of X-linked disease (P = 0.012); however, carriers of autosomal recessive disease had more widespread and more uniform thinning of the glomerular basement membrane (P < 0.0001) and less lamellation (P < 0.04).

Adolescent↗
PubMed2001

Testing for antineutrophil cytoplasmic antibodies.

The most common reason to request a test for antineutrophil cytoplasmic antibodies (ANCA) is to diagnose Wegener's granulomatosis and microscopic polyangiitis and to monitor inflammatory activity in these diseases. Several retrospective and prospective studies have suggested that the demonstration of ANCA lacks sensitivity and specificity, but these series have detected ANCA with neutrophil-indirect immunofluorescence alone, have used a disease classification that did not describe microscopic polyangiitis and have included patients with inactive disease. The 'International Consensus Statement on Testing and Reporting ANCA' has been developed to optimize the clinical relevance of ANCA testing by the adoption of standardized testing and reporting procedures. International collaborative efforts continue to focus on improving the tests for ANCA.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2000

Clinical associations and characterisation of antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein and azurocidin.

Bactericidal/permeability-increasing protein (BPI) and azurocidin (AZ) are recently described target antigens of antineutrophil cytoplasmic antibodies (ANCA). In this study, BPI-ANCA were demonstrated most often in patients with ulcerative colitis (36/92, 39%), Crohn's disease (17/66, 26%) and cystic fibrosis (11/14, 79%), but also in patients with rheumatoid arthritis (8/40, 20%), systemic lupus erythematosus (SLE) (111/65, 17%) and mixed connective tissue disease (4/18, 22%). BPI-ANCA were also common in sera containing antinuclear (ANA) (9/43, 21%) or antidouble-stranded (ds) DNA (7/28, 25%) antibodies. There was no increased frequency of abnormal alpha1-antitrypsin (alphal1AT) phenotypes in patients with BPI-ANCA, and BPI-ANCA were not more common in individuals with an abnormal phenotype. The predominant IgG subclasses were IgG1 and IgG3; IgA but not IgM was present. Both IgG and IgA BPI-ANCA were high affinity antibodies, and the affinity of IgG antibodies did not change with time in the sera tested. Four of the five sera (80%) containing BPI-ANCA did not bind to denatured, reduced BPI, suggesting that most BPI-ANCA recognised conformational epitopes. AZ-ANCA were demonstrated in 2/11 patients (18%) with Wegener's granulomatosis, 3/12 (25%) with cystic fibrosis and 3/14 (21%) with chronic active hepatitis. AZ-ANCA were present in 5/25 sera (25%) with ANA, but the levels were only marginally elevated. AZ-ANCA were uncommon in patients with inflammatory bowel and rheumatological diseases, and in sera containing other autoantibodies. Again, there was no association with abnormal alpha1-AT phenotypes. BPI represents a major ANCA target antigen in patients with rheumatological as well as inflammatory bowel disease and cystic fibrosis, but AZ-ANCA are uncommon.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2000

Antineutrophil cytoplasmic antibodies and associated diseases: a review of the clinical and laboratory features.

There have been a number of recent advances in this field. First, the "International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)" has been developed to optimize ANCA testing. It requires that all sera are tested by indirect immunofluorescent (IIF) examination of normal peripheral blood neutrophils and, where there is positive fluorescence, in enzyme-linked immunosorbent assays (ELISAs) for antibodies against both proteinase 3 (PR3) and myeloperoxidase (MPO). Testing will be further improved when international standards and common ELISA units are available. Second, new diagnostic criteria for the small vessel vasculitides that take into account ANCA-positivity and target antigen specificity as well as histologic features are currently being produced. Third, we understand that the complications associated with treatment of the ANCA-associated vasculitides are often more hazardous than the underlying disease, and regimens that use effective but less toxic agents are being evaluated. The factors associated with increased risk of relapse, however, remain incompletely understood. Finally, ANCA with specificities other than PR3 and MPO are present in many nonvasculitic autoimmune diseases. Their clinical significance is still largely unclear, and some of the target antigens are present in other cells as well as neutrophils and thus are not strictly "ANCA."

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2000

Ultrastructural appearance of renal and other basement membranes in the Bull terrier model of autosomal dominant hereditary nephritis.

Bull terrier hereditary nephritis may represent a model for autosomal dominant Alport's syndrome because affected dogs have the typically lamellated glomerular basement membrane (GBM) and father-to-son disease transmission occurs. This study examined the ultrastructural appearance of the renal and extrarenal basement membranes and their composition in affected Bull terriers. Affected stillborn animals and puppies had subepithelial frilling and vacuolation of the GBM. In adult dogs, lamellation was common, and subepithelial frilling and vacuolation were less prominent. Foot-process effacement and mesangial matrix expansion occurred frequently. Basement membranes in the glomeruli, tubules, and Bowman's capsule were significantly thickened and often mineralized. Immunohistochemical examination showed alpha 1(IV) and alpha 2(IV) collagen chains in all renal basement membranes; alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains in the GBM, distal tubular basement membrane, and Bowman's capsule; and the alpha 6(IV) chain in Bowman's capsule. Conversely, the basement membranes from the affected Bull terrier cornea, lens capsule, retina, skin, lung, and muscle had a normal ultrastructural appearance and were not thickened compared with membranes in normal age-matched dogs. The distribution of basement membrane abnormalities in Bull terrier hereditary nephritis may occur because the defective protein is present exclusively or more abundantly in the kidney and is structurally more important in the kidney or because of local intrarenal stresses.

Age Factors↗
PubMed2000

Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA).

BACKGROUND: The "International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)" requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time. AIMS: To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA). METHODS: Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO). RESULTS: IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears. CONCLUSIONS: Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2000

Absence of ocular manifestations in autosomal dominant Alport syndrome associated with haematological abnormalties.

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.

Adolescent↗
PubMed1999

Pyoderma gangrenosum with secondary pyarthrosis following propylthiouracil.

The association of pyoderma gangrenosum and arthritic symptoms is well documented. We present a rarely reported variant of this in a 44-year-old woman with pyoderma gangrenosum and bilateral large purulent effusions of her knees. She had no evidence of underlying rheumatoid arthritis or a specific seronegative spondyloarthropathy. Of note she had a history of Graves' disease for which she had been treated with propylthiouracil for 3 years and on investigation at this presentation had a markedly elevated perinuclear antineutrophil cytoplasm antibody (P-ANCA) level with specificities for IgM myeloperoxidase, IgG elastase and IgG lactoferrin. We believe this patient had pyoderma gangrenosum with secondary sterile pyarthrosis and a P-ANCA precipitated by propylthiouracil.

Adult↗
PubMed1999

A comparison of commercial and in-house ELISAs for antineutrophil cytoplasmic antibodies directed against proteinase 3 and myeloperoxidase.

This study compares the concordance of results in different ELISAs for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO). Sera were considered "true positives" if they were positive according to the manufacturer's criteria in a least three of the five PR3-ANCA ELISAs, or in at least four of the six MPO-ANCA ELISAs. Of the 26 sera that demonstrated cytoplasmic fluorescence (C-ANCA), 23 (89%) contained PR3-ANCA and three (11%) had MPO-ANCA. Two sera that were negative by indirect immunofluorescence (IIF) contained PR3-ANCA. Of the 26 sera with perinuclear fluorescence (P-ANCA), 19 (73%) contained MPO-ANCA, and one (4%) had PR3-ANCA. Six sera with P-ANCA did not have PR3- or MPO-ANCA. No serum that was negative by IIF contained MPO-ANCA. For the different PR3-ANCA ELISAs, sensitivities ranged from 88 to 100%, and specificities from 91 to 100%. For the MPO-ANCA ELISAs, sensitivities varied from 59 to 100% and specificities from 83 to 100%. The highest sensitivity and specificity for both the PR3- and MPO-ANCA ELISAs were obtained with the IBL and Eurodiagnostica assays. The in-house PR3-ANCA ELISA performed slightly less well than the commercial assays, but the performance of the in-house MPO-ANCA assay was comparable or better.

Antibodies, Antineutrophil Cytoplasmic↗