Early rheumatoid arthritis is associated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood.

OBJECTIVE: Our aim was to test the hypothesis that there is a deficit in the CD4+CD25high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. METHODS: Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy (n = 43), from individuals with self-limiting reactive arthritis (n = 14), from subjects with stable, well-controlled RA (n = 82) and from healthy controls (n = 72). The frequencies of CD4+CD25high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4+CD25- T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. RESULTS: There was a smaller proportion of CD4+CD25high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4+CD25high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. CONCLUSIONS: These data demonstrate a smaller CD4+CD25high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.

Analysis of the insertion/deletion related polymorphism within T cell antigen receptor beta variable genes in primary Sjögren's syndrome.

OBJECTIVE: To analyse T cell receptor beta variable (TCRBV) gene polymorphisms (insertion/deletion related polymorphism (IDRP) and BV6S7) in primary Sjögren's syndrome (PSS). METHODS: Genomic DNA was extracted from blood samples from patients fulfilling the modified European criteria for PSS (n = 61). Healthy control blood samples were obtained from the Blood Transfusion Service (n = 121). As a disease control group, samples from patients with systemic lupus erythematosus (n = 42) were analysed. BV6S7 was genotyped using an established PCR/RFLP method. The IDRP was determined by comparison of the intensity of PCR product bands from within BV9S2 and an internal control region (BV9S1), to ascertain whether 0, 1, or 2 copies of the insertion were present. RESULTS: There was a decrease (p = 0.018) in the proportion of PSS patients with the deleted/deleted genotype. There was no association with specific BV6S7 alleles or genotypes with either the PSS group or the hypergammaglobulinaemic subgroup. There were no significant differences in haplotype frequencies after Bonferroni correction. CONCLUSIONS: A reduced proportion of patients with PSS have the deleted/deleted genotype. Eighty nine per cent of PSS patients have at least one extra germline copy of BV13S2*1. This may relate to previous observations of increased BV13 specific T cells and mRNA in the salivary glands.

T cell receptor usage in patients with non-progressing HIV infection.

It is still unclear why some patients with HIV progress more slowly than others to developing full blown AIDS. In this study using flow cytometry we have investigated the TCRBV repertoire of peripheral blood T lymphocytes in 17 long-term non-progressing HIV patients (LTNP) to determine if there is a biased usage of T cell receptor V gene products. Patients were identified from hospital records and entered into the study. Three colour flow cytometry was used to determine the expression of the TCRBV3S5, BV5S1, BV5S2, BV5S3, BV6S1, BV7S1, BV9, BV11, BV12, BV13, BV14, BV16, BV17, BV18, BV20, BV21S3, BV22, and BV23 by CD8 and CD4 positive cells isolated from the peripheral blood of patients and controls. Increases in the absolute numbers of CD8+ T cells expressing TCRBV2 and 8 were observed in the HIV-LTNP population (P < 0.05 in both cases). No differences were seen in numbers of CD8+ T cells expressing other TCRBV or in any TCRBV within the CD4+ T cell popu-lation. At follow up (1-2 years later), those patients in which CD4 levels were below 500 x 106/l were those initially found to have lower levels of TCRBV8 +ve CD8 cells. A significant increase in the absolute numbers of T cells coexpressing the gamma delta (gammadelta) T cell receptor and CD8 were also seen in the HIV-LTNP patients compared with controls (P = 0.002). The increase in CD8+ T cells in the HIV-LTNP patients may be interpreted as either an antigen specific, or group of antigen specific responses to viral antigen, or less likely a viral superantigen. A low level of TCRBV8, CD8+ T cells might be predictive of a more rapid disease progression and might indicate a protective role for this population in HIV infected patients. The increase in gammadeltaT cells bearing the CD8 coreceptor suggests a role for this cell type in the response to HIV infection.

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.

Persistence of clonal T-cell expansions following high-dose chemotherapy and autologous peripheral blood progenitor cell rescue.

Analysing the regeneration of T lymphocytes after high-dose chemotherapy with autologous peripheral blood progenitor cell rescue (PBPCR) may help elucidate the mechanisms of immune recovery. The T-cell receptor variable beta chain (TCRBV) repertoire of adult patients undergoing high-dose chemotherapy was analysed by flow cytometry, before and after treatment. Four patients were found to have a stable expansion present (TCRBV3, 17, 21 and 22) ranging from 8% to 42% of the CD4(+) or CD8(+) repertoire. We demonstrated that, in these patients, following high-dose chemotherapy and autologous stem cell transplantation, the clonal expansions reappeared in peripheral blood and returned to pretransplant levels. Three expansions (CD3(+)CD8(+)TCRBV3(+), CD3(+)CD4(+)TCRBV21(+) and CD3(+)CD8(+)TCRBV22(+)) were further defined by sequence analysis of the complementarity-determining region (CDR)3 portion within the TCR rearrangements. These were shown to be predominantly clonal, with the same sequences being identified in peripheral blood before and after PBPCR, providing evidence that the overwhelming majority of T cells in these expansions arise from mature lymphocytes. This study demonstrated that patients undergoing autologous PBPCR for high-dose chemotherapy regenerate clonal expansions, consistent with pretreatment levels. They also regenerate T-cell repertoires with each TCRBV family represented to a similar level as that prior to high-dose chemotherapy.

Predominance of one T-cell antigen receptor BV haplotype in African populations.

The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable alpha and beta chains. We investigated allele frequencies for four variable-region gene segments of the beta chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes.

Intra-articular primatised anti-CD4: efficacy in resistant rheumatoid knees. A study of combined arthroscopy, magnetic resonance imaging, and histology.

OBJECTIVES: CD4+ T cells sustain the chronic synovial inflammatory response in rheumatoid arthritis (RA). SB-210396/CE 9.1 is an anti-CD4 monoclonal antibody that has documented efficacy in RA when given intravenously. This study aimed to establish the safety and efficacy of the intra-articular administration of SB-210396/CE 9.1 compared with placebo, examining its mode of action using a combined imaging approach of arthroscopy, magnetic resonance imaging (MRI), and histology. METHODS: Thirteen RA patients with active, resistant knee synovitis, were randomised to intra-articular injection of placebo (n=3), 0.4 mg (n=3) or 40 mg (n=7) of anti-CD4 after sequential dynamic gadolinium enhanced MRI, followed by same day arthroscopy and synovial membrane biopsy. Imaging and arthroscopic synovial membrane sampling were repeated at six weeks. This study used a unique region of interest (ROI) analysis mapping the MRI area analysed to the specific biopsy site identified arthroscopically, thus providing data for all three modalities at the same synovial membrane site. RESULTS: 12 patients completed the study (one placebo treated patient refused further MRI). Arthroscopic improvement was observed in 0 of 2 placebo patients but in 10 of 10 patients receiving active drug (>20% in 6 of 10). Improvement in MRI was consistently observed in all patients of the 40 mg group but not in the other two groups. A reduction in SM CD4+ score was noted in the 40 mg group and in the 0.4 mg group. Strong correlations both before and after treatment, were identified between the three imaging modalities. Intra-articular delivery of SB-210396/CE 9.1 was well tolerated. CONCLUSIONS: SB-210396/CE 9.1 is safe when administered by intra-articular injection. A trend toward efficacy was found by coordinated MRI, arthroscopic, and histological imaging, not seen in the placebo group. The value of ROI analysis was demonstrated.

T-cell receptor gene usage in patients with fibrosing alveolitis and control subjects.

BACKGROUND: Fibrosing alveolitis is characterized by inflammation, fibrosis and increased numbers of activated CD4+ T-cells in the lower respiratory tract. The aims of this study were to compare the T-cell antigen receptor repertoire in the lungs of subjects with fibrosing alveolitis systemic sclerosis (FASSc) with cryptogenic fibrosing alveolitis (CFA) and normal control subjects, to determine whether FASSc is driven by a specific T-cell trigger and is determined by a T-cell driven immune response, and to assess the clonality of CD4+ and CD8+ TcR usage in subjects with FASSc. MATERIALS AND METHODS: We used reverse transcription polymerase chain reaction with specific V alpha- and V beta-chain primers to identify the TcR gene usage in biopsy material, bronchoalveolar lavage fluid or peripheral blood from our subjects. RESULTS: We found individual-specific restriction of V alpha- and V beta-chain usage in lung biopsies from patients and control subjects. To establish whether this was due to expression bias in the CD4+ or CD8+ T-cells and was restricted to the lung, the alpha beta-T-cell receptor chain usage was assessed in T-cell subsets separated from the lungs of patients with fibrosing alveolitis and was compared with that of the peripheral blood. There was no consistent difference in the expression of any variable family chain among the population studied, although there was a significant difference between lung and peripheral blood lymphocyte V beta-families in CD8+ T-cells (P = 0.0007). CONCLUSION: We conclude that there is individual TcR V alpha- and V beta-expression bias in subjects with fibrosing alveolitis.

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.

Rib lesions and tuberculosis: the palaeopathological evidence.

SETTING: Tuberculosis diagnosis in past populations relies on lesions in the spine and major weight bearing joints of the body. Bone formation on visceral surfaces of ribs has also been suggested to be the result of chronic pulmonary disease. OBJECTIVE: To test whether these lesions are the result of pulmonary infection (most likely tuberculosis), by reviewing past work, and to discuss whether these lesions could be considered another diagnostic criterion for pulmonary tuberculosis. DESIGN: A review of the literature on new bone formation on ribs, and consideration of further evidence from archaeological skeletal material from the UK. RESULTS: Results from modern studies suggest that bone formation on ribs is often the result of pulmonary tuberculosis, that lesions are relatively common in archaeological skeletal material, and that some skeletons have rib lesions plus pathognomonic changes of tuberculosis. CONCLUSION: Evidence suggests that new bone formation on visceral surfaces of ribs should be considered a possible indicator of tuberculosis. If accepted, historical evidence, when correlated with rib data, produces closer approximations to the frequency of the disease in the past. This study indicates the importance of palaeopathology in identifying sometimes subtle lesions that may not be noted by clinicians because of their non-visibility on radiographs.

Monocyte activation after open and laparoscopic surgery.

BACKGROUND: The level of expression of major histocompatibility complex class II antigens, human leucocyte antigen (HLA)-DR, on monocytes correlates with the development of sepsis after surgery or trauma. Normally interferon (IFN) gamma can increase monocyte HLA-DR expression and thus may be a potential biological prophylactic antisepsis agent in patients suffering from accidental or surgical trauma. The purpose of this study was to determine the capacity of monocytes to respond to IFN-gamma after conventional open surgery and laparoscopic surgery of similar magnitude. METHODS: A whole blood flow cytometric assay was used to measure monocyte HLA-DR antigen expression before and after monocyte activation with either IFN-gamma or bacterial lipopolysaccharide (LPS). Blood was obtained before operation and on postoperative day 1 from 43 patients undergoing conventional open surgery and from 15 undergoing laparoscopic surgery of similar magnitude. RESULTS: Whole blood monocyte HLA-DR expression fell significantly after both conventional open and laparoscopic surgery. IFN-gamma caused monocyte HLA-DR expression to rise by a median (interquartile range) of 43 (26-60) per cent and 63 (10-124) per cent before operation in the open and laparoscopic groups respectively. However, after operation the corresponding values were 20 (6-41) per cent and 26 (9-74) per cent (P < 0.003 in all cases). Identical findings but of greater magnitude were observed with LPS stimulation before and after operation in the two surgical groups. CONCLUSION: Monocyte HLA-DR expression is diminished by surgical operations and is relatively refractory to further stimulation by IFN-gamma or LPS after surgery. Laparoscopic surgery is as suppressive as conventional surgery in this regard. The resistance of postoperative monocytes to further activation by IFN-gamma suggests that this agent may be ineffective as a biological response modifier after major surgery or trauma.

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.

Streptococcus pyogenes type 5 M protein is an antigen, not a superantigen, for human T cells.

M proteins are coiled-coil dimers expressed on group A streptococcal cell surfaces. They have an important role in host antistreptococcal immunity and in poststreptococcal autoimmune sequelae. Controversy has arisen regarding whether type 5 M proteins are superantigenic for human T cells. To investigate this, we have produced and tested M5 in the form of two novel recombinant proteins. We found no evidence of superantigenicity using either recombinant whole M5 protein (rM5) or recombinant pep M5 protein (rpepM5) to activate peripheral blood mononuclear cells (PBMC) from healthy adult volunteers. Short-term, rM5-specific T-cell lines from different subjects were uniformly self-APC restricted and showed no consistent pattern of TCR V beta usage. A synthetic peptide of M5 residues 217-237 was found to contain epitope(s) recognized by some rM5-specific human T cells. PBMC responses to rM5 and rpepM5 in 3- and 7-day proliferation assays were characteristic of antigenic rather than superantigenic stimulation. We conclude that type 5 M protein activates human T cells as a conventional antigen.

Brief communication: when Adam delved ... an activity-related lesion in three human skeletal populations.

A rare, activity-related lesion, the clay-shoveller's fracture, was identified during osteological analysis in three human populations dating from the Roman to the later Medieval period in England, circa fourth to 14th centuries A.D. The prevalence of this fracture in these populations suggests an osteological indicator for several possible manual activities, but also one that may be the result of a long-standing human subsistence adaptation requiring digging in the soil. Since males as opposed to females appear to be preferentially affected, the occurrence of such injuries has the potential to provide an insight into the sexual division of labor in earlier human populations.

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+.

The immunomodulatory effect of levamisole is influenced by postoperative changes and type of lymphocyte stimulant.

The results of both laboratory and clinical research into the immunomodulatory activity of levamisole have shown a considerable degree of inconsistency and sometimes contradiction. This is probably a reflection of the lack of understanding of the mechanism(s) of action of levamisole and it is therefore necessary to base conclusions about its immunomodulatory efficacy in the treatment of disease on experimental assays that take into consideration the in vivo conditions. This investigation was designed to compare the immunomodulatory activity of levamisole under clinically achievable and non-achievable conditions as judged by changes in the perioperative proliferative response of lymphocytes from 30 patients with colorectal cancer. The results obtained showed that proliferation in antigen (purified protein derivative, PPD)-stimulated, but not phytohaemagglutinin(PHA)- or staphylococcal-enterotoxin-B(SEB)-stimulated, lymphocyte cultures was consistently and significantly augmented by levamisole in concentrations of 25 ng-25 micrograms/ml. High concentrations of levamisole (25 micrograms/ml and 100 micrograms/ml) were inhibitory to PHA- and SEB-stimulated, but not PPD-stimulated, lymphocyte cultures, especially in the postoperative period. Of particular interest was the observation that, although levamisole temporarily lost its stimulatory activity in the postoperative period (third postoperative day), it did enhance antigen-stimulated lymphocytes at the time of the nadir of the postoperative suppression of lymphocyte proliferation (first postoperative day). Clinically achievable concentrations of levamisole are therefore effective both before and after operation in enhancing the response of lymphocytes to antigens.

Large granular lymphocyte expansions in patients with Felty's syndrome: analysis using anti-T cell receptor V beta-specific monoclonal antibodies.

Felty's syndrome (FS), the association of rheumatoid arthritis (RA) and idiopathic neutropenia, remains an unexplained phenomenon. HLA-DR4 is found in over 90% of cases. Patients with FS may have a T cell lymphocytosis of CD3+CD8+CD57+ large granular lymphocytes (LGL syndrome). In this study of 47 patients with FS, 19% had clear evidence for LGL expansions, while in total 42% had variable evidence for the LGL syndrome using currently available techniques. Of these T cell expansions, 76% were clonal, as demonstrated by Southern blotting and analysis with T cell receptor (TCR) beta chain constant region probes. This technique may fail to detect clonal populations in some patients. Cytofluorographic analysis using antibodies specific for TCR V beta chains identified patients with clonal LGL expansions with results comparable to those obtained with Southern blotting. No evidence for shared V beta usage among expansions from different patients was seen. The role of LGL in RA and FS is currently unclear, but this technique offers a practical and accessible means of identifying patients with LGL expansions, as a starting point for further investigation.