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Biomedical subjects

C M Lockwood

Publications and source records attributed to C M Lockwood.

At least 55 records · Page 3Linked to original sources
PubMed1995

Antineutrophil cytoplasmic antibodies and their relevance to the dermatologist.

The term vasculitis embraces a heterogeneous group of conditions which may occur as primary phenomena or secondary to disorders such as rheumatoid arthritis or systemic lupus erythematosus. Classification of vasculitis is often difficult in the absence of identifiable aetiological factors or specific serological markers. However, the primary systemic vasculitides can be segregated morphologically, according to the size of blood vessels involved and the presence of granulomata (Table 1). Recently, the discovery of circulating antineutrophil cytoplasmic antibodies (ANCA) in many patients with these disorders, whose differing specificities substantiate the histological classification, has led to interest being focused on their relevance in the development of vasculitis. This paper reviews the historical aspects of the detection of ANCA, and the value of these antibodies in the diagnosis and management of the primary systemic vasculitides likely to be encountered by the dermatologist. The clinical features in patients with these vasculitides are also outlined.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1995

Bactericidal/permeability-increasing protein (BPI) is an important antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in vasculitis.

Indirect immunofluorescence (IIF) techniques have shown that ANCA are useful serological markers for some small vessel vasculitides, and ELISA assays, using purified molecules as solid-phase ligand, have helped to identify proteinase 3 (PR3) and myeloperoxidase (MPO) as two of the major ANCA antigens. There remain a substantial number of serum samples, which are positive by IIF, yet recognize neither PR3 nor MPO (double-negative samples). We found, by Western blot analysis of soluble neutrophil granule proteins, that certain of these double-negative samples recognized a 55-kD doublet of which the first eight residues shared N-terminal amino acid sequence homology with BPI, a potent antibiotic towards Gram-negative bacteria. We developed a simple, quick and robust two-step immunobiochemical method to purify BPI. This was then employed to detect anti-BPI autoantibodies by ELISA and Western blot analysis. We tested 100 double-negative samples and 400 consecutive new samples sent for routine ANCA testing in the anti-BPI ELISA. We found that 45 of the 100 double-negative and 44 of the 400 new routine samples recognized BPI. By Western blot analysis 20/20 positive anti-BPI samples blotted the 55-kD protein. Inhibition assays confirmed the specificity of binding. Review of the 89 anti-BPI-positive patients showed a male dominance (M:F ratio 55:34), a mean age of 60.4 years and clinical diagnoses ranging from organ limited vasculitis to widespread systemic vasculitis.

Adolescent↗
PubMed1995

The immunosuppressive drug thalidomide induces T helper cell type 2 (Th2) and concomitantly inhibits Th1 cytokine production in mitogen- and antigen-stimulated human peripheral blood mononuclear cell cultures.

Thalidomide is an effective immunomodulatory drug in man, but its mechanism of action remains unclear. We hypothesized that, in addition to its reported inhibitory effects on production of monocyte-derived tumour necrosis factor-alpha (TNF-alpha), thalidomide might be effective at the level of Th immunoregulation. In a comparative study with the immunosuppressant cyclosporin A, we have demonstrated a potent and specific effect of thalidomide on cytokine production relating to the distinct Th1 and Th2 subsets. It induced and enhanced the production of IL-4 and IL-5 and, at the same dose (1000 ng/ml), significantly inhibited interferon-gamma (IFN-gamma) production in phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell (PBMC) cultures. Stimulation of PBMC with recall antigen (streptokinase:streptodornase (SKSD)) at 144 h in the absence of thalidomide resulted in a predominantly Th1 response, with the production of IFN-gamma and IL-2. Thalidomide switched this response from a Th1 to a Th2 type. The effect was most pronounced at 1000 ng/ml thalidomide, where inhibition of IFN-gamma and enhancement of IL-4 production was maximal. In unstimulated cultures thalidomide alone induced IL-4 production. Cyclosporin A, in contrast, inhibited both Th1 and Th2 cytokine production by PHA-stimulated PBMC. Time course data from thalidomide-treated cultures revealed that the augmented IL-4 production diminished as the culture time increased, whereas IFN-gamma production was significantly increased. This response might be due to activation-induced apoptosis of Th2 cells or the induction of Th2 cell anergy, in the continued presence of stimulating agents, with the emergence of IFN-gamma-secreting Th1 cells when Th2 antagonism declines. The effects of thalidomide and related compounds may enhance our understanding of the mechanisms of T helper cell selection, offer the possibility of controlled therapeutic switching between Th1 and Th2 responses, and may lead to a rational approach for the treatment of some T cell-mediated immunological disorders.

Cells, Cultured↗
PubMed1995

Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis.

Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti-neutrophil cytoplasm antibodies. The association of this antigen with specific disease entities requires that there is a source of pure antigen present in large quantities. Further delineation of the molecular mechanisms involved in the antigen-antibody interaction requires the ability to manipulate the molecule. The expression of recombinant MPO in Chinese hamster ovary cells has produced a source of pure protein, suitable for molecular studies. We have shown that this protein is an antigen recognized by 95% of anti-MPO antibodies from patients with systemic vasculitis. This recombinant molecule will be of use in providing an additional specific solid-phase assay for these antibodies and further forms of this protein which mirror the antigenicity of native MPO more exactly may replace chemically purified antigen. It will also be of great value in studies examining the epitopes recognized by anti-MPO antibodies and in studies of immunoregulation and T cell activation.

Animals↗
PubMed1995

Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor V beta 2 gene usage in microscopic polyarteritis.

Antigen recognition by T lymphocytes is mediated by cell surface receptors T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the alpha and beta polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the beta chain (V beta) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR V beta genes. To determine if the dominant usage of a V beta gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific V beta gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (D beta) J beta region. A significant increase in the mean percentage expression of the V beta 2.1 gene was seen in vasculitis patients (11.4 + 1.0% (mean + s.e.m.)) compared with controls (6.6 + 0.6%; P < 0.003). The most marked increase was seen in microscopic polyarteritis (13.9 + 1.7%; P < 0.0001). There were also increases in the expression of V beta 3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of V beta 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR V beta 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR V beta 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding V beta 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis.

Adult↗
PubMed1995

Management of systemic vasculitis: contribution of scintigraphic imaging to evaluation of disease activity and classification.

The use of radio-isotope-labelled leucocyte scans has become established as a non-invasive and accurate means of diagnosing a variety of inflammatory conditions. We report a retrospective study on leucocyte imaging in the management of 50 patients with systemic vasculitis. Leucocyte imaging was useful for detecting unsuspected sites of disease and monitoring disease activity. Scintigraphy was superior to conventional radiography or CT scanning for detecting and monitoring vasculitic involvement of the respiratory tract. The scans were useful for differentiating between Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). There was a close and statistically significant relationship between the clinical diagnosis of WG and nasal uptake on leucocyte scans (p < 0.01), whereas in patients with MPA it was rare. Anti-proteinase 3 autoantibody specificity correlated significantly with nasal uptake of labelled leucocytes (p < 0.03). Leucocyte imaging is a useful non-invasive investigation in patients with systemic vasculitis.

Autoantibodies↗
PubMed1995

ANCA and predicting relapse in systemic vasculitis.

We studied 60 patients with ANCA-positive systemic vasculitis (SV) to assess the prognostic significance of clinical and serological features at presentation, and the value of sequential monitoring of ANCA, C-reactive protein (CRP) and ESR levels as predictors of disease relapse. Patients were recruited at the time of diagnosis, treated with a standard protocol, and assessed monthly for one year. Clinical remission was achieved in 56/60 (93%), and ANCA became undetectable in 50/60 (83%) after treatment. During the one year follow-up period, disease relapses were seen in 23 (38%) patients. No specific associations were observed between initial disease presentation, initial ANCA level or ANCA antigenic specificity and relapse. However, 13/23 (57%) of relapses were preceded by a rise in ANCA a mean of 7.8 weeks earlier, while at the time of relapse 19/23 (83%) were ANCA-positive. Rises in CRP and ESR occurred in 23/60 (38%) and 14/43 (33%), respectively, but were less closely associated with relapse than ANCA. A sustained rise in ANCA was seen in six patients without relapse while clinical relapse occurred with a negative ANCA in four. Sequential ANCA monitoring at monthly intervals during disease remission is of value, at least during the first year, in the prediction and diagnosis of relapse in SV, and is superior to measurement of CRP or ESR.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1995

Antigen specificity in hydralazine associated ANCA positive systemic vasculitis.

The anti-hypertensive agent hydralazine can cause a lupus-like syndrome characterized by serosal inflammation, arthralgias and rashes. The kidneys however are usually spared. The condition is characterized by circulating immune complexes and antinuclear antibodies, whilst antibodies against double-stranded DNA are rare. Hydralazine can also cause a systemic vasculitis with a pauci-immune rapidly progressive glomerulonephritis, which is associated with autoantibodies directed against components of the neutrophil cytoplasm. In this study, ten patients with hydralazine-induced vasculitis had antibodies with specificities for both myeloperoxidase and lactoferrin. We suggest that this particular pattern of autoantibodies, together with antibodies with reactivity against nuclear components including double-stranded DNA, are characteristic findings in hydralazine-induced vasculitis. In addition, renal involvement appears to be more common in this group of patients with vasculitis than in those with the lupus-like syndrome.

Adult↗
PubMed1994

High immunoreactivity of lactoferrin contaminating commercially purified myeloperoxidase.

Three sets of experiments were performed to investigate the quality of myeloperoxidase (MPO) preparations and anti-MPO reagents. In the first experiment, two groups of three and four mice were immunized with commercially purified MPO (Calbiochem). Immunization was performed in PBS in the first group and in acetate buffer in the second. From the first group, five monoclonals were raised, and their specificities examined by ELISA and immunoblotting. Surprisingly, these antibodies reacted with lactoferrin (LF) and not MPO. In the second group, 13 monoclonals were raised; six of these reacted with MPO and seven reacted with LF. In a second set of experiments, MPO and LF reactivity were tested in different buffer conditions in the ELISA procedure. Slight variations in the detection of contaminating LF were found. In a third experiment, polyclonal reagents directed against MPO and LF were tested in MPO immunoblotting studies. A polyclonal anti-MPO reagent reacted not only with MPO but also with contaminating material including LF. The anti-MPO polyclonal reagent also reacted with LF on immunoblotting. We conclude that: (i) caution should be exercised when defining anti-neutrophil cytoplasm specificities of human sera and monoclonals by ELISA, (ii) the low concentration of contaminating LF in the commercially purified reference MPO preparation should be taken into consideration since it appears to have high immunoreactivity, (iii) changes in MPO immunoreactivity may occur under different buffer and pH conditions.

Animals↗
PubMed1994

Autoantibodies to lactoferrin and histone in systemic vasculitis identified by anti-myeloperoxidase solid phase assays.

We aimed at confirming the antigen specificity recognized by anti-neutrophil cytoplasm antibodies (ANCA) in patients presenting systemic vasculitis with anti-myeloperoxidase (MPO) activity on ELISA. Thirty-five consecutive patients with reactivity in anti-MPO ELISA and systemic microscopic vasculitides were tested in slot and Western blot analyses. Eleven of 35 sera exhibited binding in Western blot studies with the MPO preparation used in the ELISA: five sera bound at the size of MPO, but five sera reacted with a 78 kD species (p78) co-purifying with MPO, and one serum blotted both MPO and p78. Sequence analysis and antigen-specific assays including Western blot studies showed that p78 is lactoferrin. All anti-lactoferrin positive sera, but no anti-MPO positive sera, also exhibited anti-nuclear binding on HEp2 cells with specificity for histone. We concluded that: (a) a subgroup of patients presenting systemic vasculitis with false anti-MPO reactivity on ELISA had anti-lactoferrin antibodies; (b) anti-lactoferrin was associated with anti-nuclear activity with specificity for histone; (c) these patients had systemic vasculitis without histological evidence of immune complex deposition.

Amino Acid Sequence↗
PubMed1994

Does truly 'idiopathic' crescentic glomerulonephritis exist?

Crescentic glomerulonephritis is usually classified into antiglomerular basement membrane (GBM) disease, immune-complex disease, or pauci-immune crescentic nephritis. The last category includes patients with systemic vasculitis as well as 'idiopathic' isolated crescentic nephritis. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in many patients with apparently isolated crescentic nephritis suggests that this represents a renal-limited form of vasculitis, and that truly 'idiopathic' crescentic nephritis is a very rare entity. We reviewed all renal biopsies with extracapillary proliferation seen at our centre since the availability of an ANCA assay (4-year period). There were 89 such biopsies of a total of 1240, of which 82 had sufficient details for further analysis. Of these, 10 had anti-GBM disease, 35 had epithelial proliferation associated with a variety of other diseases, and 36 had ANCA-associated disease. Nine of this last group had no extrarenal features and would previously have been classified as 'idiopathic' crescentic glomerulonephritis. The single remaining patient had an inactive glomerulonephritis with a scarred crescent; the predominant lesion was an interstitial nephritis. We therefore conclude that truly 'idiopathic' crescentic nephritis is very rare, if it exists at all. The ability to provide a practically complete classification of crescentic nephritis has important prognostic and therapeutic consequences.

Adolescent↗
PubMed1994

Endothelial cell activation in patients with systemic vasculitis.

Vascular endothelial cells respond in vitro to a number of stimuli, and in particular to cytokines, by undergoing functional and morphological alterations which endow them with the capacity to promote inflammatory reactions. We studied this process of endothelial cell activation in 20 skin biopsies from 18 patients with systemic vasculitis. At sites of cutaneous inflammation, blood vessels were lined with swollen endothelial cells which expressed increased levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and were associated with a mononuclear cell inflammatory infiltrate. Neutrophil infiltration was only found in the presence of endothelial leucocyte adhesion molecule-1 (ELAM-1), which was expressed in 15/20 biopsies. ELAM-1 and VCAM-1 were associated with the presence of inflammatory cytokines which induce expression of these molecules in cultured endothelial cells. Endothelial activation in vivo appears to parallel that observed in vitro, and is likely to be important in determining the nature of an inflammatory response.

Adolescent↗
PubMed1994

Antineutrophil cytoplasm antibodies (ANCA) of IgA isotype in adult Henoch-Schönlein purpura.

ANCA are associated with certain forms of systemic vasculitis, and have been reported previously to be of the IgG and IgM isotype. We examined the possible association between IgA ANCA and the IgA-related diseases Henoch-Schönlein purpura (HSP) and IgA nephropathy (IgAN). IgA and IgG ANCA were detected by isotype-specific solid-phase assays with a crude neutrophil extract, and their presence was confirmed by antigen-specific fluid-phase competitive inhibition tests and by indirect immunofluorescence. The possible interference by IgA rheumatoid factor was excluded. IgA ANCA were detected in sera from 11/14 HSP patients (79%), from 1/30 IgAN patients (3%), from 1/40 patients with vasculitides classically associated with IgG ANCA (2.5%), and in none of 60 sera from healthy blood donors. IgG ANCA were present with IgA ANCA in three patients with HSP. Only one HSP serum had anti-myeloperoxidase (MPO) activity by both IgA and IgG isotype-specific ELISA, and none was positive for proteinase 3 (PR3). Western blot analysis performed with neutrophil extract showed that the four strongest IgA ANCA-positive HSP sera reacted with a 51-kD protein; Western blot performed on cellular fractions showed that this protein is primarily membrane-associated, and different from fibronectin. Our study suggests that adult HSP is closely associated with circulating IgA ANCA, which may be directed against a different autoantigen than that recognized by IgG ANCA.

Adolescent↗
PubMed1994

Rheological response of neutrophils to different types of stimulation.

The potential for neutrophils to obstruct microvessels was evaluated by measuring transit of individual neutrophils through 8-microns pores in an automated cell transit analyzer (CTA) or into micropipettes (4-8 microns ID). Stimulation in vitro by the chemotactic agent N-formyl-methionyl-leucyl-phenylalanine. (fMLP), cigarette smoke, or purified antineutrophil cytoplasm antibodies greatly increased flow resistance, but the response varied in its dependence on time and pore diameter. Cigarette smoke or fMLP caused rapid loss of cellular deformability, although observations were complicated by changes in cell shape: progressive bipolar shape formation (after treatment with fMLP) could facilitate entry into larger pores (approximately 8 microns), whereas blebs induced by cigarette smoke caused bridging of these pores with cell immobilization. These processes led to an underestimation of the changes in deformability by the CTA. Neutrophils responded slowly to the antineutrophil cytoplasm antibodies (approximately 30 min), with a greater increase in flow resistance evaluated by a micro-pipette (4-6 microns ID) than by the CTA. We conclude that the effect of neutrophil stimulation on flow through capillary-sized vessels is potentially great (with resistance typically increased 10-fold or even complete blockage) but may depend on the vascular and cellular geometry and may be local or disseminated, depending on the rate of the rheological response.

Adult↗