A study on parasites in Mexican rheumatic disease patients.

Blastocystis hominis is a common human parasite with infection rates up to 50% in developing countries, and giardiasis is the commonest intestinal one in Mexico. No doubt, various parasites as Giardia lamblia and Entamoeba histolytica can cause rheumatic diseases. This study coproparasitoscopic analysis evaluated the cysts by B. hominis, G. lamblia, E. hartmani, E. coli and E. histolytica in Mexican rheumatic disease patients. Also, ELISA was used to detect E. histolytica, Ascaris lumbricoides, Toxocara canis, and Trichinella spiralis in Mexican patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Thirty-six patients (24 with AS and 12 with RA) and 77 healthy control individuals were enrolled in this study. The frequencies of protozoan cysts were comparable in rheumatic disease patients (AS and RA) and healthy control donors (33 and 25 vs. 26%, respectively; p > 0.05). The frequency of antibodies to T. canis was significantly higher in AS patients than in healthy control donors (16 vs. 2.6%, respectively; p = 0.027), whereas no differences were observed for the prevalence of antibodies for the other parasites (E. histolytica, A. lumbricoides and T. spiralis) (p > 0.05). This information indicates the need to intensify educational efforts for the prevention of parasite infections associated with AS disease that cannot be controlled only by drugs.

Morphometric analysis of nodules in human onchocerciasis collected in communities of the southern Chiapas focus, México.

Human onchocerciasis is a disease that remains as an important public health problem. The morphometric and physical characteristics of 363 Onchocerca volvulus nodules collected in the major endemic focus of onchocerciasis in Southern Chiapas (Soconusco), was assessed. In the present work we found that treatment the morphometry of 363 onchocercal nodules preserved in a 67% glycerol solution was determined by measuring the length, width and thick of each nodule with a Vernier caliper. The mass was determined with an analytical balance and the volume by measuring the water displacement, while the specific gravity was calculated by dividing mass over the volume. Statistical analysis was calculated for each parameter. The results showed that the nodules were rather longer than wider or thicker. Morphometric characteristics were 9.87 +/-3.70 (mean +/- standard deviation), 7.52 +/- 2.81, and 4.62 +/-+/- 2.06 mm for length, width and thick respectively. In regard to the shape, 62.81% of the nodules showed a lenticular shape, while 18.18% were spherical and 19.01% were ovoid. Based on the distribution of frequencies of the length, the nodules were classified in three groups: the "small" (5.77 +/- 0.73 mm; n = 104, 28.65%), the "medium" group (9.86 +/- 2.05 mm; n = 203 nodules, 55.92%), and the group of the "big" ones (16.03 +/- 1.91 mm; n = 56, 15.43%). Moreover, the physical characteristics were: for the mass 0.33 +/- 0.24 g, the volume of displaced water was 0.28 +/- 0.26 ml, and the specific gravity was 1.10 +/- 0.55 g/ml. The results indicated that most of the Mexican Onchocerca nodules have a lenticular shape with average size of 10x7x5 mm, which is useful in the knowledge of the genus biodiversity and can be taken as a parameter in clinical or epidemiological trials, where onchocerciasis remains as a public health problem.

Differential induction of TNF-alpha and NOS2 by mitogen-activated protein kinase signaling pathways during Mycobacterium bovis infection.

The role of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of TNF-alpha and NOS2 production by human monocytes infected with Mycobacterium bovis BCG was examined. Inhibition studies showed that ERK1/2 and p38 MAPK activation were necessary for the monocyte response to M. bovis infection. Analysis of MAPK activation showed rapid phosphorylation of ERK1/2 and p38 in response to M. bovis BCG. Phosphorylation was not due to an autocrine effect of TNF-alpha secretion, since an anti-TNF-alpha antibody had no significant effect on the levels of p38 phosphorylation. The inhibitor PD98059 significantly reduced M. bovis BCG-induced TNF-alpha production and almost completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphorylation. In contrast, studies using inhibitors selective for ERK1/2 and p38 showed that p38 plays an essential role in the induction of NOS2, whereas the role of ERK1/2 was minor. These results suggest that ERK1/2 and p38 kinases differentially regulate the M. bovis BCG-mediated induction of TNF-alpha and NOS2 in human monocytes.

Infection of human monocytes with Mycobacterium bovis BCG induces production of CC-chemokines.

DESIGN: CC-chemokines are potent leukocyte activators and chemoattractants, which have an important role in granuloma formation, function critical for the immune responses to mycobacterial infection. This study investigated whether infection of human monocytes with Mycobacterium bovis bacillus Calmette-Guérin (BCG) elicits secretion of RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. METHODS: RANTES, MIP-1alpha and MIP-1beta synthesis was measured by the presence of protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay. To investigate the mechanism of M. bovis BCG stimulation of RANTES, we carried out inhibition assays with antibodies to CD40 and we used an intracellular calcium chelator BAPTA-AM. RESULTS: Infection of human monocytes with M. bovis BCG induced RANTES, MIP-1alpha and MIP-1beta secretion in a dose-dependent manner. This stimulation of CC-chemokines production was not attributed to LPS contamination. M. bovis-induced RANTES secretion was dependent upon bacterial uptake and on tumor necrosis factor (TNF)-alpha. Interestingly, the production of RANTES by M. bovis BCG-infected monocytes occurs through a mechanism that requires intracellular calcium and was significantly inhibited (P<0.05) with antibodies to CD40. CONCLUSIONS: These results suggest that the ability of M. bovis BCG to produce CC-chemokines might lead to protection in the acquired immune response of mycobacterial infection and at the same time indicate that M. bovis BCG-induced RANTES secretion is mediated by CD40 and dependent on the intracellular calcium influx.

CD40 ligand expression in mycobacterium bovis BCG infection and its regulation by cytokines: a direct role of interleukin 12.

BACKGROUND: Activation and clonal expansion of T cells require not only the recognition of processed antigen on the surface of the antigen-presenting cell (APC) by T-cell receptor (TCR), but also involve co-stimulatory signals that are provided by the simultaneous engagement of cell surface molecules expressed by both the APC and the T cell. Interaction between CD40 and its ligand (CD40L) is known to mediate host immune response and T-cell-mediated effector functions in mycobacterial infections in mice. In this work, we investigated the capacity of Mycobacterium bovis (M. bovis) BCG to induce the expression of CD40L on human T cells. METHODS: Human cells were obtained from healthy adults by centrifugation using Ficoll/Hypaque. Cells (1 x 10(6)) were incubated in RPMI medium with BCG. After incubation at 37 degrees C in 5% CO(2) atmosphere for 40 h, cells were collected and double-stained with anti-CD40L-PE and anti-CD4-FITC or anti-CD8-FITC mAb. The quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: M. bovis BCG stimulation induced a significant amount of CD40L expression on CD4+ T cells, while CD40L was not significantly detected on human CD8+ T cells. The highest CD40L expression on BCG-activated T cells in synergism with interleukin-12 endogenous occurred after a 40-h cell culture with a dose of 10 microg/mL of BCG (84.86 +/- 11.77; mean +/- standard deviation [SD]). This CD40L expression on BCG-activated human T cells was significantly inhibited by anti-IL-12 mAb (5.08 +/- 1.7; mean +/- SD). In contrast, anti-IFN-gamma and anti-IL-2 mAb did not have an important role in this expression. CONCLUSIONS: These results indicate that the capacity of BCG to induce CD40L expression on human cells represents a novel mechanism underlying the regulation of cellular responses against tuberculosis. Furthermore, the results showed a direct role of IL-12 to enhance the expression of CD40L on BCG-activated human cells.

Down-modulation of mycobacterial-induced IL-1 beta production in human mononuclear cells by IL-4.

Tuberculosis is characterized by a cellular immune response mediated by various cytokines, including IL-1 beta released by stimulated mononuclear cells. It is now well established that IL-I beta plays an important role in local and systemic inflammatory response in tuberculosis. Here we have demonstrated, for the first time, that addition to IL-4 to human mononuclear cells obtained from 11 healthy bacille Calmette-Guérin (BCG)-vaccinated donors reduced BCG-induced production of IL-1 beta by 91.46 +/- 2.2%. This inhibitory effect was found highly significant (P less than 0.001) and was dose-dependent. The effect of IL-4 on the secretion of IL-1 beta was specific, since a complete reversion was obtained with a neutralizing MoAb to human IL-4. In addition, this inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in response to IL-4. Interestingly, the induction of IL-1 beta was regulated by IL-4, at least in part, by direct mechanism mediated through the extracellular domain of the 130-kD IL-4 receptor, as demonstrated by incubation of the mononuclear cells with the neutralizing anti-IL-4 receptor MoAb. Finally, a significant down-regulation of IL-1 beta secretion was observed in hsp70-stimulated mononuclear cells cultured with IL-4. Further experimental work is needed to establish the relevance of IL-4 in human mycobacterial infection in vivo. However, an understanding of the mechanisms that control IL-1 beta secretion in human mycobacterial infections is essential to understand the pathogenesis of tuberculosis.

Activation of T cells to peptides that span the 19 kDa protein of Mycobacterium tuberculosis.

It has been suggested that the cellular immune response to mycobacterial antigens, is implicated in the pathogenicity of inflammatory joint diseases such as rheumatoid arthritis. Therefore, the aim of this study was to identify T-cell epitopes in a series of 20-mer peptides spanning the entire 19-kDa protein of M. tuberculosis. Mononuclear cells obtained from six rheumatoid arthritis (RA) patients, were analyzed for their proliferation to both the 19-kDa containing immunoblot fraction and to the synthetic peptides. Mononuclear cells from three rheumatoid arthritis patients responded in a dose-dependent manner to peptides 1-20, 60-79, 71-90, 82-101, 90-109 and 121-140, whereas the other eight peptides: 11-30, 21-40, 41-60, 50-69, 100-119, 112-131, 131-150 and 140-159 did not stimulate significant proliferative responses in any of the patients tested. These results indicate, for the first time, the presence of dominant epitopes in the cellular immune response to the 19-kDa M. tuberculosis antigen by rheumatoid T cells.

Chemically unrelated mycobacterial peptides as antigens and competitors in antigen recognition by human T cells.

Understanding resistance to mycobacterial infectious diseases requires identification of antigens and epitopes (antigenic determinants) that stimulate cell-mediated immune responses. In this study, a DR1-restricted T cell epitope (residues 1 through 20) of the 19-kD protein of M. tuberculosis was identified. Peripheral blood mononuclear cells from two HLA-DR1 patients with tuberculosis responded not only to the 19-kD immunoblot fraction of M. tuberculosis but also to the peptide 1-20. A M. tuberculosis-reactive T cell clone isolated from one of the patient (whose mononuclear cells showed a stronger proliferative response to the 19-kD protein) recognized the peptide 1-20, while failed to recognize a negative control peptide (residues 65 through 85 of the 65-kD mycobacterial protein: peptide 65-85) or a negative control antigen (candida). The antigen recognition to peptide 1-20 was shown to be DR1 restricted. This MHC restriction was confirmed using a DR1-restricted mycobacterial T cell epitope as competitor. These results demonstrated that this mycobacterial competitor significantly reduced the antigen recognition of peptide 1-20. The reduction observed was dose-dependent.

Modulation for antigen presentation in tuberculosis by using synthetic peptides.

Competition assay technology has been a very useful tool in the study of parasite antigens and has been inferred but never proven that this approach can be applied to select T-cell epitopes by using another microorganisms. In this study, HLA-restricted T-cell clones specific to synthetic peptides derived from the 65 kDa mycobacterial protein were used to investigate whether these peptides are able to compete with each other at the level of MHC-binding sites in tuberculosis. Fixed APCs were pulsed with suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptide. The results showed that two peptides from this protein were able to compete with each other inducing a significant inhibition in the proliferation assays while there was no competition by using a control peptide. The amount of cross-reactivity was influenced by the peptide concentrations. More important was the observation that these peptides were able to bind to the same HLA-class II molecules therefore blocking the binding of each other. The fact that these peptides have not an identical amino acid sequence support the idea that the MHC-peptide interaction must have a broad specificity to be able to bind a large number of peptides. These results demonstrate that it is possible to modulate the antigen presentation by blocking the peptide MHC-class II interaction in tuberculosis and support the idea that this approach facilitates the selection of appropriate T-cell epitopes to be incorporated in a vaccine.

Production of leukocyte migration inhibition factor (LIF) and migration stimulation factor (MStF) by CD4+ and CD8+ human lymphocytes subsets and T-cell clones.

The production of the lymphokines leukocyte migration inhibition factor (LIF) and migration stimulation factor (MStF) at the level of CD4+ and CD8+ human lymphocyte subsets was investigated. In a first series of experiments, anti-CD4 and anti-CD8 monoclonal antibodies capable of inhibiting the activation by concanavalin-A (Con-A) of the respective T-cell subset were used. It was observed that when CD8+ cell activation was blocked, LIF was always produced after Con-A activation. When CD4+ cell activation was blocked, MStF was produced in five out of nine experiments (no activity in the other four). The addition of N-acetyl-D-glucosamine to block LIF in supernatants of anti-CD8 treated cells was unable to show evidence of masked MStF activity. In a second series of experiments, T-cell clones were established from continuous growing T-lymphocyte cell lines developed from cultures of Con-A activated normal human leukocyte cultures. The phenotype of 22 clones was determined and their ability to produce LIF or MStF investigated. Four clones produced MStF after Con-A activation and all of them were CD3+, CD4-, CD8+. Three clones produced LIF after Con-A activation and all of them were CD3+, CD4+, CD8-. We conclude that LIF is produced by CD4+ cells and MStF by CD8+ cells.

Human lymphocytes produce both leukocyte migration inhibition (LIF) and simulation (MStF) factors after mitogen, alloantigens or antigenb activation

Utilizando un sistema desarrollado para producir factor inhibidor de la migración de leucocitos (LIF) en altos títulos, hemos encontrado que cuando activamos linfocitos humanos con concanavalina-A, obtenemos LIF en 55% de los experimentos y factor estimulador de la migración en 38%. Cuando los linfocitos fueron activados por medio de cultivos mixtos de linfocitos encontramos resultados muy parecidos. Se seleccionaron sobrenadantes de linfocitos humanos activados con concanavalina-A que tuvieron una alta actividad de LIF. Al bloquear el LIF con N-acetil-D-glucosamina se pudo observar que estos sobrenadantes contenían también factor estimulador de la migración. En pruebas de LIF directo usando como antígeno a la estreptocinasa-estreptodornasa, encontramos que cuando el ensayo se realiza en presencia de N-acetil-D-glucosamina el valor de índice de migración se incrementa de manera importante. Concluimos que cuando los linfocitos humanos son estimulados ya sea por mitógenos, aloantígenos o antígenos, ellos producen tanto factor inhibidor como estimulador de la migración de leucocitos. Finalmente se discute la importancia de este hallazgo en la determinación de immunidad celular in vitro en humanos por medio de la prueba de LIF

La prueba de LIF en humanos. Sugerencias para la evaluacion de los resultados. / LIF test in humans. Suggestions for evaluation of the results

Se analisa los resultados de 141 pruebas de inhibicion de la migracion de leucocitos humanos (LIF) con un total de 564 capilares. Cuando se usan dos capilares por grupo experimental la aparicion de valores al azar de indice de migracion (IM) iguales o menores a 0.80 es de una de cada veinticinco veces. Cuando se usan cuatro capilares por grupo se usaron pruebas estadisticas (t de student y U de MannWhitney) para evaluar la prueba observandose que el limite de significancia se situa alrededor de IM de 0.84 - 0,85%.Cuando se usan cuatro capilares por grupo el 92% de los valores de migracion mas alejados de la media del grupo no se alejan de la misma mas de un 20% y el 98% no mas de un 30%. Con base en estos analisis se dan una serie de sugerencias para evaluar confiablemente los resultados de la prueba de LIF