Increased usage of V beta 2 and V beta 6 in rheumatoid synovial fluid T cells.

OBJECTIVE: To determine if the T cell antigen receptor V beta usage of unstimulated rheumatoid arthritis (RA) synovial fluid (SF) T cells is biased compared with those in peripheral blood (PB). METHODS: Freshly isolated, matched synovial fluid and peripheral blood T cells were analyzed for V beta gene expression using quantitative polymerase chain reaction (PCR) methods. Ten synovial fluid samples from the knees of 7 patients with RA were studied. The PCR assay used 26 V beta primers with a constant region C beta primer, and 2 C alpha primers that co-amplified a product that served as an internal standard. Cycle number and complementary DNA content were controlled to ensure the linear accumulation of PCR products. Labeled products were separated on 10% polyacrylamide gels and counted with a Betascope blot analyzer. RESULTS: There were consistent differences between the V beta gene usage of SF and PB T cells directly isolated from patients with RA, regardless of HLA-DR haplotype. In all synovial specimens, V beta 2 was increased relative to the peripheral blood, while V beta 13.1 and V beta 13.2 were decreased. V beta 6 and V beta 21 were increased in 9 of the 10 synovial samples. Analyses of bilateral SF specimens from 2 subjects and serial specimens from the same knee of 1 subject revealed virtually identical patterns in each patient. The SF V beta bias was not solely due to differences in the proportion of CD4+ and CD8+ cells, because the CD4:CD8 ratios in SF and PB were similar. However, V beta gene usage of separated CD4+ and CD8+ synovial T cells showed that V beta 2 and V beta 6 were more highly expressed on CD4 cells. CONCLUSION: Freshly isolated synovial T cells from inflamed (not end-stage) knees of patients with RA have a remarkably consistent biased V beta gene usage compared with PB T cells. V beta 2 and V beta 6 are uniformly increased, and this increase is primarily in CD4+ T cells. The same V beta bias in the SF T cells of several RA patients suggests that shared antigens may be stimulating the T cell response.

Diversity of rheumatoid synovial tissue T cells by T cell receptor analysis. Oligoclonal expansion in interleukin-2-responsive cells.

The synovitis of rheumatoid arthritis (RA) is characterized by infiltrates of CD4+ T lymphocytes. To determine the clonal diversity of these cells, we cloned T cells with interleukin-2 (IL-2), alone or with phytohemagglutinin (PHA), directly from actively inflamed synovial tissue obtained at synovectomy. A total of 205 clones from 4 specimens was analyzed for T cell receptor (TCR) gene rearrangements using Hind III and Eco RI digests with beta chain and gamma chain complementary DNA probes. A comparison of the TCR rearrangements enabled us to determine if the T cell clones arose from the same or different precursor cells. Most of the T cell clones (92%) had distinct TCR gene rearrangement patterns, indicating a unique clonal origin. However, a few clones (1 quadruplicate and 6 pairs) with identical TCR rearrangements were identified, and these clonal multiples were most commonly found in clones selected with IL-2 alone. Mass cultures were propagated with IL-2, alone or with PHA, and at each passage, cells were removed for TCR analysis. The later passages of the lines selected with IL-2 had oligoclonal TCR rearrangements, whereas no oligoclonal rearrangements were found in the PHA + IL-2-selected cell lines. The TCR rearrangements in the later passages of the IL-2 mass cultures were often identical to the TCR rearrangements that were found in the IL-2-derived clonal multiples. These findings indicate that while the majority of CD4+ T cells within the actively inflamed rheumatoid joint have diverse clonal origins, small numbers of clonal multiples and oligoclonal populations are present, and these cells may be enriched in an IL-2-responsive T cell subset.

Radioimmunoassay for detection of the Snow Mountain Agent of viral gastroenteritis.

The Snow Mountain Agent (SMA) is a Norwalk-like viral agent of acute gastroenteritis that has been detected only by immune electron microscopy (IEM). We established a solid phase microtiter radioimmunoassay (RIA) for SMA antigen employing pre-and post-challenge sera from volunteer studies as capture antibodies. SMA was detected in 12 of 67 stool samples from volunteers who were ill after oral challenge with SMA. All samples in which virus particles were detected by IEM were positive by RIA, but the RIA was 10-80 times more sensitive than IEM. To detect serum antibody to SMA, a blocking test was developed, employing diarrheal stool containing SMA as a standard source of antigen. Serum antibody rises were detected in eight out of nine volunteers with experimentally induced illness following challenge with SMA, as well as in three out of three naturally occurring cases. A preliminary sero-epidemiologic survey suggested that infection with SMA was common in the population surveyed. This RIA should permit large scale seroepidemiologic studies of SMA to be carried out, and should also facilitate characterization of this agent.

Detection by immune electron microscopy of the Snow Mountain agent of acute viral gastroenteritis.

An extensive outbreak of acute gastroenteritis of unknown etiology occurred at Snow Mountain, Colorado, in December 1976. Virus-like particles, 27 nm in diameter, were observed by electron microscopy in two of five stool specimens from individuals in the outbreak. Oral administration of a filtrate from one of the specimens induced disease in nine of 12 normal volunteers. Experimentally induced illness was similar to that observed during the outbreak. Stool specimens examined by immune electron microscopy revealed 27-nm virus-like particles in three of nine ill volunteers at the onset of illness. When the particles were used as a source of antigen, increases in levels of serum antibody were demonstrated in persons with either experimentally induced or naturally occurring illness. Therefore, it is likely that this virus-like particle is the etiologic agent of the Snow Mountain outbreak. The Snow Mountain agent appears to be morphologically similar to, but antigenically distinct from, the Norwalk and Hawaii agents by immune electron microscopy and may represent an additional antigenic type among the agents that resemble the Norwalk particle.

Isolation of reovirus-like agents from patients with Crohn's disease.

220 nm filtrates of intestinal resections from 6 of 10 patients with Crohn's disease produced a cytopathic effect in WI-38 tissue-culture monolayers, whereas controls had no effect. Characterisation of the virus responsible has been completed in 3 of the 6 positive isolates and indicates that it is an R.N.A. virus, 55-60 nm in diameter, heat, ether, and acid stable, and antigenically related to Nebraska calf-diarrhoea virus. It therefore belongs to the Reoviridae family.

Serum and intestinal fluid immunoglobulins and jejunal IgA secretion in Crohn's disease.

Immunoglobulins in serum and proximal intestinal fluids and secretion of IgA by cultured jejunal mucosa were measured in 12 healthy subjects and 36 patients with Crohn's disease. Concentrations of IgA, IgG, IgM, and IgE in serum and intestinal fluids were similar in the two groups, except for increased serum IgA concentrations in the patients. Elevation of IgA and chronicity of disease were correlated, which suggests that the IgA alteration was a response to duration of disease rather than a primary pathogenetic factor. IgA secretion by cultured jejunum was similar in control and patient groups. Thus, no evidence was found that abnormalities of secretory immunoglobulins are pathogenetically involved in Crohn's disease.