Paul A. Gatenby

Autoantibodies to retinal astrocytes associated with age-related macular degeneration*

Sera from 128 patients with age-related macular degeneration (AMD) were examined and profiles of a variety of serum constituents, including immunoglobulins, alpha and beta globulins and autoantibodies, were tabulated. A similar series of tests were carried out on 20 control sera. The results indicate a higher incidence of serum abnormalities, particularly involving alpha-2 globulin, in patients with disturbance of pigmentation of the retinal pigment epithelium (RPE). The sera were further tested for the presence of autoantibodies with specificity for retinal tissue, and five major staining patterns were observed. Many sera produced patterns of labelling on human retina identical to that observed using labelled monoclonal anti-glial fibrillary acid protein (GFAP) antibodies, which are an established marker of retinal astrocytes. Although anti-retinal autoantibodies have been reported in association with a number of ocular pathologies, the observation of anti-astrocyte autoantibodies is new. Astrocytes are involved in the maintenance of the blood-retinal barrier (BRB) and also appear to be the facultative antigen-presenting cells of neural tissue. The present results indicate that the formation of anti-astrocyte autoantibodies may be an early feature of the pathogenesis of AMD.

Treatment of Recurrent Spontaneous Abortion by Immunization With Paternal Lymphocytes: Results of a Controlled Trial

PROBLEM: It remains unclear whether maternal immunization with paternal lymphocytes prior to conception improves the reproductive outcome in women with recurrent abortion in whom all secondary causes have been excluded.

Variable gene usage of T cell receptor γ‐ and δ‐chain transcripts expressed in synovia and peripheral blood of patients with rheumatoid arthritis

The synovial tissue and fluid of patients with rheumatoid arthritis (RA) contain activated T cells that probably have a central role in the disease process which leads to joint destruction. A subset of T cells, γδ T cells detected at the site of inflammation, may be important in the pathogenesis of the disease. This study investigated variable (V) gene usage of γδ T cell receptors (TcRs) expressed in synovia and peripheral blood of patients with RA by using the polymerase chain reaction (PCR) to amplify TcR γ‐and γ‐chain transcripts. Most patients showed no restriction in Vγ gene usage since synovial mononuclear cells (SMC) expressed TcR γ‐chain transcripts which used the same set of Vγ genes as peripheral blood mononuclear cells (PBMC). In contrast, the majority of patients expressed a restricted SMC Vδ‐chain repertoire biased towards Vδ1, but Vδ2 mRNA transcripts were also detected, albeit at low levels in some patients. The TcR δ‐chain repertoires of PBMC from healthy control subjects were also characterized. There was variation in the TcR δ‐chain repertoires of PBMC from patients when compared with controls, particularly with respect to expression of Vδ4. Vδ4 mRNA transcripts were expressed in PBMC of only two of seven RA patients in contrast with eight of the nine controls (P=0.03). These findings are compatible with reports that γδ T cells in the rheumatoid synovium are reactive to Mycobacterium tuberculosis and that response to M. tuberculosis is restricted to VγL9/Vδ2‐bearing T cells, if a superantigen is involved in the pathogenesis of RA.

Is C‐reactive protein really useful in preterm premature rupture of the membranes?

Summary. In a prospective blind study 380 daily serum samples from 55 women with preterm premature rupture of the membranes were analysed for C‐reactive protein (CRP). Although the last CRP before delivery was higher in patients with histological chorioamnionitis (P= 0.007), considerable overlap between infected and non‐infected pregnancies occurred, precluding the use of CRP as a diagnostic test if published normal levels were used. When upper limits were set at 30, 35, or 40 mg/1, the last CRP before delivery proved 90, 95 and 100% specific and 88, 92 and 100% positively predictive of infection in singleton pregnancies. Such high specificities are needed to prevent inappropriate intervention based on false positive results. We therefore propose upper limits for single estimations of 30, 35, or 40 mg/1 depending on the relative risks of preterm delivery versus infection at various gestational ages. In addition, consecutive values <20 mg/1 appeared highly predictive of infection.

Treatment of Recurrent Spontaneous Abortion by Immunization With Paternal Lymphocytes: Correlates With Outcome

ABSTRACT: Previous observations have suggested that defective recognition of fetal alloantigens by the maternal immune system is associated with recurrent pregnancy failure and that this may be prevented by boosting the maternal immune system with paternal or pooled third‐party leukocytes. The mechanism whereby this process achieves success is not clear, and accordingly to explore this we immunized 28 couples with recurrent fetal loss with 80 × 106 paternal peripheral blood mononuclear leukocytes (PBML) and followed various immunological parameters. The couples studied, in whom 55% achieved a successful pregnancy, showed no increase in sharing of human lymphocyte antigen (HLA)‐A, ‐B, or ‐DR antigens and no consistent evidence of a decreased mixed leukocyte reaction (MLR) or MLR plasma‐blocking factors compared with control couples. Immunization did not alter these parameters but did induce antipaternal lymphocytotoxins, although the presence of the latter did not correlate with pregnancy outcome. There was a correlation between rapid conception after immunization and a subsequent successful pregnancy. A successful pregnancy also correlated with sustained postimmunization, postconception maternal antipaternal allospecific CD‐8+ suppressor T cells. Although these findings provide overall evidence that immunization produces changes in the way in which the maternal immune system interacts with the fetus, larger numbers of couples and a higher dose of paternal lymphocytes will be needed to establish clearly whether this therapy works and its mechanism of action.

Restricted junctional diversity of T cell receptor δ gene rearrangements expressed in systemic lupus erythematosus (SLE) patients

SLE is an autoimmune connective tissue disorder affecting multiple organs, in which T cells may play a central role. This study investigated T cell receptor (TCR) γ/δ repertoire expression in peripheral blood mononuclear cells (PBMC) of SLE patients and healthy individuals using variable (V) gene family‐specific polymerase chain reaction (PCR) amplification of TCR cDNA. The expressed Vγ repertoires were diverse in SLE and control PBMC, although VγIV gene rearrangements were barely detectable or not expressed in some patients. In contrast, δ chain expression was limited in all SLE patients, with Vδ transcripts rearranged primarily to the Vδ1 and Vδ2 genes, as opposed to control PBMC, in which all six Vδ genes were detected. To assess the clonality of TCR populations. cDNA clones containing rearranged Vδ1, Vδ2 and Vγ9 transcripts were sequenced from PBMC of both patients and controls. For controls, δ chain junctional region sequences showed extensive molecular heterogeneity, since virtually all 34 Vδ1 and 32 Vδ2 cDNA clones analysed were unique. A few Vδ9 cDNA clones (321) had the same junctional region sequence motif (EVQEL) encoded largely by the Vγ9 and joining(J)γP gene segments. Identical Vγ9 junctional sequences were found in SLE patients that did not contain the EVQEL motif present in normal peripheral blood γ/δ lymphocytes. Moreover, the predominant Vδ1‐Jδ‐consiant (C) δ and Vδ2‐Jδ‐Cδ gene rearrangements expressed in SLE PBMC showed restricted junctional diversity, but the oligoclonal δ transcripts were different in each patient. These findings suggest in vivo oligoclonal expansion of γ/δ T cells in the periphery of SLE patients in response to a limited number of nominal ligands. Whether γ/δ T cells contribute to the development of systemic autoimmunity remains to be investigated.

Anti-neutrophil cytoplasmic antibodies (ANCA): Their detection and significance: Report from workshops

&NA; Anti‐neutrophil cytoplasmic antibodies (ANCA) are antibodies directed against enzymes that are found mainly within the azurophil or primary granules of neutrophils. There are 3 types of ANCA that can be distinguished by the patterns they produce by indirect immunofluorescence when tested on normal ethanolfixed neutrophils. Diffuse fine granular cytoplasmic fluoresence (cANCA) is typically found in Wegeners granulomatosis, in some cases of microscopic polyarteritis and Churg Strauss syndrome, and in some cases of crescentic and segmental necrotising glomerulonephritis, but it is rare in other conditions. The target antigen is usually proteinase 3. Perinuclear fluorescence (pANCA) is found in many cases of microscopic polyarteritis and in other cases of crescentic and segmental necrotising glomerulonephritis. These antibodies are often directed against myeloperoxidase but other targets include elastase, cathepsin G, lactoferrin, lysozyme and beta‐glucuronidase. The third group designated “atypical” ANCA includes neutrophil nuclear fluorescence and some unusual cytoplasmic patterns, and while a few of the target antigens are shared with pANCA, the others have not been identified. Sera that produce a pANCA or atypical ANCA pattern on alcohol‐fixed neutrophils result in cytoplasmic fluorescence when formalin acetone fixation is used. pANCA or atypical ANCA occur in about 2/3 of all individuals with ulcerative colitis or primary sclerosing cholangitis, and they are found in a third of patients with Crohns disease. The reported incidence of ANCA in rheumatoid arthritis and SLE varies considerably but the patterns are predominantly pANCA and atypical ANCA. Where ANCA are associated with a small vessel vasculitis, such as Wegeners granulomatosis and microscopic polyarteritis, antibody titres may parallel disease activity and have been used to monitor the response to treatment. In other non‐vasculitic conditions ANCA levels do not necessarily reflect disease activity or the presence of a vasculitis. ANCA are also common in HIV infections where all fluorescence patterns are found, but the presence of these antibodies has no clinical significance. There have been many reports of ANCA occurring occasionally in other diseases. ANCA have not yet been demonstrated to be pathogenetic in the small vessel vasculitides. However there is in vitro evidence for 2 mechanisms, one of which focuses on neutrophil activation as the primary event and the other on antibody binding to vascular endothelium. Both mechanisms assume increased levels of circulating tumor necrosis factor or another cytokine that might result from the infections that often precede the onset of Wegeners granulomatosis and microscopic polyarteritis. Successful new regimens in the treatment of Wegeners granulomatosis and microscopic polyarteritis include the use of pooled immunoglobulin, and cyclosporin in combination with low dose prednisolone, but one report of 3‐weekly pulse cyclophosphamide as a single agent indicated that it was effective in only 42% of patients.

Antibodies to 65 kDa and 70 kDa heat shock proteins in rheumatoid arthritis and systemic lupus erythematosus

To test the potential role of autoimmunity to the highly conserved heat shock proteins (HSP) in immune arthritides, the sera from 99 patients with rheumatoid arthritis (RA), 48 patients with systemic lupus erythematosus (SLE) and 65 normal controls were examined by ELISA for IgG and IgM antibodies to the 65 kDa and 70 kDa heat shock proteins from Mycobacterium bovis (Bacille Calmette‐Guerin; BCG). In RA sera there are significant numbers of individuals with increased IgM anti‐65 kDa and anti‐BCG reactivity as well as IgG anti‐70 kDa when compared with controls. In SLE both IgM and IgG anti‐BCG, together with IgM anti‐65 kDa, differed significantly from controls. The results were compared with previous reports in similar groups of patients, and it is clear that no consistent pattern of reactivity emerges. While further work may be justified looking carefully at the disease duration and other subsets of both RA and SLE, it is difficult at this stage to conclude that antibodies to autologous HSP that cross‐react with mycobacterial HSP play a major role in disease pathogenesis.

THE ROLE OF COMPLEMENT IN THE AETIOPATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

The role of classical pathway complement components in systemic lupus erythematosus (SLE) is reviewed. Their importance in maintaining immune complexes (IC) in soluble form and in enhancing clearance of IC through binding to red cell CR1 is such that deficiency, complete or partial, of these components or some of their controlling enzymes can lead to IC mediated disease like SLE. C2 and C4 are encoded within the class III region of the major histocompatibility complex (MHC). There are certain well described associations between class II MHC genes and the occurrence of SLE and the relative importance of the two sets of gene products and their potential interactions are discussed. Complement C4 plays a role in drug induced lupus as many of the lupus associated drugs bind to C4 and interfere with its protective functions. Classical genetic studies provide clear evidence that non MHC genes are important in the aetiopathogenesis of SLE. Non MHC encoded complement deficiencies and functional deficits may well represent some of these other genetic factors and is clearly a fertile area for future research.

Analysis of T cell receptor Vα and Vβ gene usage in synovia of patients with rheumatoid arthritis

T cells are thought to play a fundamental role in the pathogenesis of rheumatoid arthritis (RA), Activated T cells expressing α/β T cell receptor (Tcr) infiltrate the rheumatoid synovium and could potentially initiate a local inflammatory response directed against joint constituents, A Tcr repertoire with restricted heterogeneity may reflect a selective expansion of T cells reactive to a few antigenic determinants within the synovium. To determine whether predominant Vα and/or Vβ gene usage of the expressed α/β Tcr repertoire is a feature of synovial T cells in patients with RA, the polymerase chain reaction (PCR) was used to amplify Tcr‐α and Tcr‐β chain transcripts. The peripheral blood and synovia of five patients with adult RA were examined and no evidence of preferential use of 19 Tcr Vα gene families was found. Similarly, most of the 18 Tcr Vβ gene families could be detected in RA synovia although there were quantitative differences in Tcr Vβ gene expression when compared to peripheral blood. This report shows that when the extremely sensitive assay of oligonucleotide hybridization of PCR amplified Tcr transcripts is used, permitting identification of specific V gene families, the α/β Tcr repertoire in the rheumatoid synovium is more diverse than was previously thought. Therefore, in patients with RA of long duration, the synovial T cell response is most likely to be polyclonal.