Analysis of immunoregulatory cytokines in ocular fluid samples from patients with uveitis.

PURPOSE: To investigate the T-helper cell cytokine profiles in two well-defined clinical uveitis entities caused by an infectious mechanism. METHODS: Cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and interferon [IFN]-gamma) were measured in ocular fluid samples obtained from patients with herpes simplex- or varicella-zoster virus-induced acute retinal necrosis (ARN; n = 17) and toxoplasma chorioretinitis (n = 27) using enzyme-linked immunosorbent assay techniques. The data were compared with data for 51 control samples taken during cataract surgery (n = 10), vitrectomy in diabetic retinopathy (n = 10), eye bank eyes (n = 10) and with samples from patients with "autoimmune" uveitis (n = 21). RESULTS: Interleukin-6 was detected in 44 of 51 control samples and 43 of 44 eyes of patients with uveitis. The highest levels in the control samples were detected in 9 of 10 vitreous samples from patients with diabetic retinopathy (mean, 648 pg/ml). In 8 of 10 samples taken from patients during cataract surgery and in 7 of 10 eye bank eyes the amount of IL-6 was significantly lower (mean, 10 pg/ml and 136 pg/ml, respectively). Interleukin-6 levels in patients with ARN (mean, 1436 pg/ml) were significantly higher than in those with toxoplasma chorioretinitis (mean, 272 pg/ml). Interleukin-2 was detected in one of the samples from patients with toxoplasma chorioretinitis (1105 pg/ml) and in three samples from the control subjects suffering from Fuchs' heterochromic anterior uveitis (mean, 752 pg/ml). No IL-4 (<2 pg/ml) was detected either in patient or control samples. Interferon-gamma could be detected in 7 of 17 ARN patients (range, 277-3483 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 12-250 pg/ml), and in 1 of 21 of the samples from control subjects with uveitis (31 pg/ml) but was absent in nonuveitic control samples. Interleukin-10 was detected in 10 of 17 ARN patients (range, 29-3927 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 4-67 pg/ml), and in only 3 of 51 control samples (6 pg/ml, 16 pg/ml, and 20 pg/ml). CONCLUSIONS: Various immunoregulatory cytokines (IL-6, IL-10, and IFN-gamma) were detected in ocular fluid samples from patients with uveitis. A separate role for either a T-helper type 1 or T-helper type 2 response in the pathogenesis of clinical uveitis could not be proven.

Differences between intraocular and serum antibody responses in patients with ocular toxoplasmosis.

PURPOSE: To investigate the antigen specificity of the intraocular anti-Toxoplasma gondii antibody response in patients with ocular toxoplasmosis. METHODS: Paired ocular fluid and serum samples were collected from 13 patients with active ocular toxoplasmosis. Serum IgM anti-T. gondii antibodies were tested to distinguish recently-acquired from chronic infection. Anti-T. gondii IgG specificity was analyzed by immunoblotting using a crude T. gondii extract. RESULTS: Two of the 13 patients tested were IgM positive and considered to have acquired ocular toxoplasmosis. The antibody specificity in ocular fluid and serum of these two patients was similar, whereas in the patients with presumed chronic disease, marked differences could be observed. Most ocular fluid samples contained antibodies that stained a 28-kD antigen more intensely than did antibodies from paired serum samples. Using absorption and elution experiments, we demonstrated that this 28-kD protein was identical to the GRA-2 antigen, which is expressed in both the tachyzoite and the bradyzoite stages of the parasite. CONCLUSIONS: Our results show that the intraocular T. gondii antibody response of patients with recurrent ocular toxoplasmosis differs from the systemic response. This finding may have implications for our understanding of the immunopathogenesis of ocular toxoplasmosis and could be employed to improve diagnosis of the disease.

Heterogeneity of rearranged T cell receptor V alpha and V beta gene transcripts in synovial fluid T cells of HLA-B27 positive reactive arthritis patients.

To analyze the T cell receptor (TCR) V-alpha/beta gene usage by synovial fluid (SF) and peripheral blood (PB) T cells of HLA-B27+ reactive arthritis (ReA) patients. The TCR V-alpha/beta gene usage was determined by the polymerase chain reaction on freshly isolated SF and PB mononuclear cells (MNC) of five HLA-B27+ ReA patients. A total of 30 TCR V alpha and 23 V beta (sub)family specific primers in combination with a C alpha or C beta specific primer, respectively, were used. In five patients most of the TCR V alpha and V beta gene segments expressed by PB T cells were also detected in the paired SF samples. Although one patient showed an increased expression of TCR V alpha2 in SF when compared to PB, the SF samples showed a heterogeneous TCR V-gene repertoire similar to PB. Although this study was limited to a small group of patients, the apparent lack of a restricted TCR V-gene repertoire in SF does not support the involvement of a single or limited number of T cell subsets in the disease process of HLA-B27+ ReA patients.

Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells.

Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages. A well-defined HLA-B27-restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (Th) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E. coli and Salmonella typhimurium. Macrophages infected with both recombinant bacteria presented the Th epitope to the specific CD4+ T cell clone, but failed to present the CTL epitope to the specific CD8+ T cell clone. Presentation of the Th epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II. Presentation of the Th epitope to the CD4+ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation. The efficiency of macrophages to process and present the inserted Th epitope was similar for both the recombinant E. coli and S. typhimurium strains.

Polymorphism of tumour necrosis factor-alpha (TNF-alpha) at position -308 in relation to ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the localization, in the proximity of the HLA-B locus, and the biological activities of TNF-alpha, we investigated the association between AS and a single base polymorphism located at position -308 of the TNF-alpha gene. An allele-specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF-alpha alleles was determined in 66 AS patients and 37 healthy controls. The TNF-alpha allele frequency was not significantly different between AS patients and controls.

Effects of multiplicity of infection, bacterial protein synthesis, and growth phase on adhesion to and invasion of human cell lines by Salmonella typhimurium.

Monolayers of intestine 407 (Int-407) cells were infected with the virulent Salmonella typhimurium strain C52, and the adhesion to and invasion of these cells were studied. The effects of the multiplicity of infection and growth phase of the bacteria (logarithmic versus stationary) on the interaction with eukaryotic cells were investigated. In contrast to other reports, we found no differences in the adhesive and invasive capacities of bacteria derived from logarithmic- or stationary-phase cultures. Invasion by S. typhimurium required bacterial protein synthesis and live Int-407 cells. Bacteria adhered equally well to dead or live Int-407 cells, which indicates that adhesion does not require metabolically active cells. Adhesion of S. typhimurium followed saturation kinetics, with a maximum of 10 adhesive bacteria per cell. This indicates that there is a limited number of bacterial adhesion sites (receptors) available on the surface of the host cell. Killed and live bacteria adhered equally well and competed with each other for cellular adhesion sites. This and adhesion experiments performed in the presence of chloramphenicol showed that bacterial protein synthesis is not required for adhesion. The general validity of the results obtained with S. typhimurium C52 was confirmed by comparing the invasion and adhesion data with those of the frequently used SL1344 and SR11 strains. In addition, we assayed the adhesion and invasion of S. typhimurium C52, SL1344, and SR11 and 27 S. typhimurium field isolates with Int-407, HeLa, and HEp-2 cells.