Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

An ELISA method for detecting antibodies to the T cell antigen receptor.

An ELISA method for the detection of monoclonal antibodies (MAb) to the T3-T cell antigen receptor (TCR) complex was devised. The T3-TCR complex was solubilised using digitonin and a rat anti-T3 MAb (Campath 3) was used to bind it to an ELISA plate. Normal rat serum was used to block cross-reactivity between the rat MAb and peroxidase-conjugated rabbit anti-mouse immunoglobulins. The assay was tested on four T cell tumour lines and successfully detected MAbs to TCR beta chain variable regions, as well as the anti-T3 MAb UCHT1. Other anti-T3 MAbs were not detected because Campath 3 blocked their binding. None of a panel of MAbs reacting with other T cell surface antigens reacted in the assay.

Continuous in vitro culture of human T lymphocytes.

A method for continuous culture of human T lymphocytes by stimulation with irradiated lymphocytes, lymphoblastoid cells, and phytohaemagglutinin is described. Cultures can be maintained for at least 9 months. Growth appears to be dependent on a soluble factor released by lymphocytes when they are stimulated with lymphoblastoid cells and phytohaemagglutinin.

Inhibition of human antigen-specific T cell proliferation by anti-T blast sera.

Two antisera, one raised against activated human T lymphoblasts and one raised against the human T cell line HPB-ALL, have the ability to inhibit antigen-induced T cell proliferation. Antiproliferative activity is absorbed by T blasts and HPB-ALL cells, but not by B lymphoblastoid cell lines, alpha-ethylisothiouronium bromide-treated sheep red blood cell (AET-SRBC) receptor-negative lymphoid cells, or the T cell lines MOLT-4 and HSB. The effects of these antisera do not require the presence of complement and are not attributable to toxicity during culture. The F(ab')2 fragment of anti-T blast antiserum is also active.

Identification of the epitope recognized by the human V beta 5-specific monoclonal antibody 42/1C1. Potential implications for disease therapy.

A panel of T-cell clones was generated that was specific for amino acid residues 4-13 of the mycobacterial 65-kd stress protein. All the clones were found to express a member of the V beta 5 family, as defined by PCR. However, the clones could be differentiated on the basis of different staining characteristics with the mAb 42/1C1. This antibody is known to recognize both V beta 5.2 and V beta 5.3, as was the PCR primer pair used in the analysis. Sequencing of the TCRs revealed that those clones which were not stained by 42/1C1 expressed a previously unidentified member of the V beta 5 family. By comparing the sequences of the V beta 5 family members that are recognized by 42/1C1 with those that are not, we were able to identify a probable epitope for the antibody. It is also clear from our data that the TCRs of T cells recognizing identical MHC-peptide combinations, although very similar, may be differentiated by mAbs, thereby posing potential problems in any proposed disease therapy involving treatment with monoclonals.

Individuals from multiplex insulin-dependent diabetes mellitus (IDDM) families express higher levels of TCRBV2S1 than controls.

T lymphocytes recognise peptide antigens through the T cell antigen receptor, which is composed of variable alpha and beta chains. There are forty-six functional variable regions on the beta chain. In this study the expression of the T cell receptor beta-chain variable regions 2S1 and 3S1, in a large cohort of multiplex insulin-dependent diabetes mellitus families, have been determined by use of monoclonal antibodies and flow cytometry. Peripheral blood was collected from these multiplex families and three control groups, healthy individuals, sporadic insulin-dependent diabetes mellitus patients and non-insulin-dependent diabetes mellitus patients. The level of TCRBV2S1 expression in the multiplex families was significantly higher than all the control groups for both the CD4+ and CD8+ T lymphocyte subsets. Detailed analysis of the family data showed that this increased expression was not associated with age, sex, HLA type or the diabetic phenotype. The TCRBV3S1 expression in all the diabetic cohorts was significantly lower than the healthy controls, in the CD4 subset only. Detailed analysis of the family data showed only the fathers TCRBV3S1 expression was lower than the healthy controls. This study gives further insight into TCRBV usage which could reflect the mechanism of the autoimmune response in IDDM multiplex families.

Demonstration of the oligoclonality of an enteropathy associated T-cell lymphoma by monoclonal antibodies and PCR analysis of the T-cell receptor V-beta repertoire on fixed tissue.

The apparent clonality of T cells present in enteropathy associated T cell lymphomas (EATCLs) has been previously reported by showing T cell receptor (TCR) gene rearrangement in fresh tumor tissue. The EATCL presented here exhibits the novel phenotype CD3+, HML-1+, CD4+, CD8+, and TCR Vbeta 8+. The oligoclonality of the tumor cells is shown using the polymerase chain reaction (PCR) on cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue. The T cells present in the lymphoma were predominantly TCR Vbeta 8+.

IgA deposition in alcoholic liver disease.

Immunoglobulin deposition in alcoholic and non-alcoholic liver disease was studied using an indirect immunoperoxidase technique. A continuous pattern of IgA deposition, with IgA outlining the sinusoids, was shown to be a specific and sensitive marker for liver disease caused by alcohol in both cirrhotic and non-cirrhotic livers. The sensitivity was lowest in cases of alcoholic disease showing fatty change alone. In one case it was possible to show the absence of IgA in liver disease caused by a drug, which was histologically indistinguishable from alcoholic hepatitis.

Definitive identification of human T cells in formalin fixed paraffin wax embedded tissue.

The use of a new monoclonal antibody, BF1, which reacts in routinely fixed tissues, directed against the beta chain of the T cell antigen receptor was assessed to determine its reactivity in a variety of normal tissues as well as in a number of B and T cell tumours and other lymphoid disorders. It reacted with all five of the unequivocal T cell tumours tested. BF1 will have widespread use in the assessment of lymphoid tissues in patients with the acquired immune deficiency syndrome (AIDS) and the differential diagnosis of tumours.

Kaposi's sarcoma in acquired immune deficiency syndrome (AIDS).

Of 22 patients with Kaposi's sarcoma, 16 had the acquired immune deficiency syndrome (AIDS). The histological pattern in AIDS differs from the more familiar classical Kaposi's sarcoma. The features most useful in making the diagnosis are: dissection of collagen; lymphatic vessel like spaces; angiomatoid lesions; premonitory sign; and spindle cell proliferation. It is important to examine multiple levels of small biopsy specimens and to be cautious in making the diagnosis of patch Kaposi's sarcoma in the presence of recent or healed ulceration and at sites of previous trauma. Only four of 16 patients with AIDS had evidence of systemic Kaposi's sarcoma, supporting the view that Kaposi's sarcoma in AIDS does not necessarily have an aggressive clinical course.

Biopsy pathology of acquired immune deficiency syndrome (AIDS).

Between January 1982 and May 1986 279 biopsy specimens from 82 patients with acquired immune deficiency syndrome (AIDS) were examined. A wide variety of infectious conditions were diagnosed, the commonest being Pneumocystis pneumonia (n = 36), cytomegalovirus (n = 21), a variety of fungi (n = 8), mycobacteria (n = 7). Kaposi's sarcoma was the commonest tumour (n = 40), and there were two cases of extranodal lymphoma. Striking features were the unusual sites of disease and the occasional paucity of organisms.

Cytomegalovirus infection in gastrointestinal tracts of patients infected with HIV-1 or AIDS.

All gastrointestinal tract biopsy specimens from 190 patients positive for HIV-1 or with AIDS were reviewed to assess the prevalence of cytomegalovirus (CMV) infection, morphology of infected cells, and the associated histopathological features. Eighteen patients (10 (7.7%) of 129 HIV antibody positive and eight (13.1%) of 61 with AIDS) had CMV identified in 35 biopsy specimens from the following sites: oesophagus (n = 3); stomach (n = 6); small intestine (n = 4); colorectum (n = 18) and perianal area (n = 4). Eleven patients had CMV alone as the potential cause of symptoms and in seven there were coexistent pathogens or Kaposi's sarcoma. The appearance and type of infected cells at different sites was highly variable. Immunocytochemical techniques and electron microscopic examination were performed to confirm the presence of CMV antigen and CMV virus particles and to exclude the possibility of an adenovirus producing similar cytopathic changes. It is important to recognise the different morphological forms of infected cells, and the use of immunocytochemical techniques is recommended in patients at risk for CMV or in whom CMV infection is suspected.

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

Diagnosis of lung disease in acquired immune deficiency syndrome: biopsy or cytology and implications for management.

One hundred and twenty consecutive bronchoscopic examinations were carried out on 80 patients with acquired immune deficiency syndrome (AIDS) between January 1982 and December 1986. Ninety one paired biopsy and cytology specimens from 72 of these patients were analysed. There was no significant difference between biopsy and cytology in diagnosing Pneumocystis carinii pneumonia (0.95 greater than p greater than 0.1). In 10 cases P carinii pneumonia was diagnosed by biopsy but not cytology and in seven cases by cytology but not biopsy. Nineteen patients had multiple infections or Kaposi's sarcoma. Biopsy was more useful than cytology in the diagnosis of other infections (n = 20) and Kaposi's sarcoma (n = 2) with positive cytological correlation in only three of the infections. Biopsy and cytology together have a diagnostic yield of 78.3%. We conclude that all patients presenting with respiratory disease who have, or are in a high risk group for, AIDS should be examined by bronchoscopy at an early stage with both cytology and biopsy.

Campylobacter pylori in the upper gastrointestinal tract of patients with HIV-1 infection.

Fifty one patients with human immuno-deficiency virus (HIV-1) infection who had been consecutively endoscoped for upper gastrointestinal symptoms were biopsied (stomach or duodenum, or both) and compared with 59 age and sex matched controls for the presence of Campylobacter pylori. In 28 (47%) of the control group but in only seven (14%) of the HIV seropositive patients were C pylori seen on histological examination (p less than 0.001, odds ratio 5.6, 95% confidence interval 2.2-14.5). Sixteen patients who were HIV antibody positive had other index diseases for the diagnosis of AIDS in the biopsy material and, when these were excluded, comparison with the control group still showed a significant difference; p less than 0.01, odds ratio 3.6, 95%, confidence interval 1.4-9.6. In this series, therefore, C pylori were far less common in HIV antibody positive patients than in controls. Among the HIV positive patients, a higher proportion of C pylori negative cases had AIDS but this trend was not significant. The findings of this study indicate that whatever abnormalities of cell mediated mucosal immunoregulation are caused by HIV infection, they do not seem to be important in the response to infection by C pylori.

Interleukin-10. A role in the development of postoperative immunosuppression.

BACKGROUND: The cause of diminished monocyte major histocompatibility complex class II antigen expression after surgery or trauma is unclear. Interleukin-10 (IL-10) regulates inflammatory cytokine production and major histocompatibility complex class II (HLA-DR) expression in vitro. OBJECTIVES: To quantify in vivo IL-10 messenger RNA (mRNA) and protein and monocyte HLA-DR expression after major surgery and to investigate the effects of IL-10 neutralizing blockade on monocyte HLA-DR expression in vitro. DESIGN: Inception cohort study of 48 surgical patients from preoperative status to postoperative day 7 and 9 healthy volunteers (controls). SETTING: Large teaching hospital, Northern England. PATIENTS: Monocyte HLA-DR and cytokine mRNA expression was determined in 32 of 48 consecutive patients undergoing elective major resectional surgery. Mononuclear cells for in vitro studies and serum samples for IL-10 measurement were obtained from the remaining 16 patients. MAIN OUTCOME MEASURES: Monocyte HLA-DR expression determined by flow cytometry, IL-10, and tumor necrosis factor mRNA in peripheral blood mononuclear cells assayed by multiplex reverse transcriptase polymerase chain reaction, and serum IL-10 determined by enzyme-linked immunosorbent assay. RESULTS: Monocyte HLA-DR expression (in mean channel fluorescence units [MCF]) was significantly reduced 24 hours after surgery (MCF [+/- SEM], 32.6 +/- 2.3 vs 16.3 +/- 1.2; P < .001) and remained low during the first postoperative week. A relative increase in IL-10 to G3PDH mRNA ratio (mean [+/- SEM], 0.95 +/- 0.08 vs 0.59 +/- 0.06; P < .01) and serum IL-10 (mean [+/- SEM], 18.1 +/- 4.1 vs 5.4 +/- 0.8 pg/mL; P < .01) was noted on the first postoperative day. A significant correlation existed between HLA-DR antigen expression and the presence of IL-10 mRNA transcript on the first postoperative day (P < .01). Lipopolysaccharide-induced up-regulation of monocyte HLA-DR expression was significantly impaired on the first postoperative day (mean [+/- SEM], 151% +/- 24.4% vs 60% +/- 10.1%; P < .01), but this was partially reversed by IL-10 neutralizing antibody (mean [+/- SEM], 60% +/- 10.1% vs 115% +/- 11.6%; P < .01). CONCLUSIONS: Interleukin-10 gene expression correlates with the fall in monocyte HLA-DR antigen expression in patients undergoing major abdominal surgery and may account for the immunosuppression associated with surgical injury.