Bacterial DNA in synovial fluid cells of patients with juvenile onset spondyloarthropathies.

OBJECTIVE: To identify bacterial DNA in synovial fluid cells of patients with active juvenile onset spondyloarthropathy (SpA). METHODS: The main group of study constituted 22 patients with juvenile onset SpA. In addition, five patients with adult onset SpA and nine with rheumatoid arthritis (RA) were studied. Polymerase chain reaction (PCR) with either genus- or species-specific primers was performed on synovial fluid cells to detect DNA sequences of Chlamydia trachomatis, Yersinia enterocolitica, Salmonella sp., Shigella sp., Campylobacter sp. and Mycobacterium tuberculosis. The presence of antibacterial antibodies in sera and synovial fluid was also determined by enzyme-linked immunoassay. RESULTS: The synovial fluid of nine patients with juvenile onset SpA, three with adult onset SpA and one with RA contained bacterial DNA. Five juvenile onset SpA samples had DNA of one single bacterium; two juvenile onset SpA and three adult onset SpA had DNA of two bacteria and two juvenile onset SpA had DNA of three bacteria. Overall, Salmonella sp. DNA was detected in seven synovial fluid samples, Shigella sp., Campylobacter sp. and M. tuberculosis were found in four samples each, and C. trachomatis was found in two. The bacterial DNA findings correlated with neither diagnosis nor disease duration. One RA synovial fluid had DNA of Campylobacter sp. Neither serum nor synovial fluid antibacterial antibodies correlated with DNA findings or clinical diagnosis. CONCLUSION: In this study, single and several combinations of bacterial DNA were identified in the synovial fluid of patients with long-term undifferentiated and definite juvenile onset SpA and adult onset SpA. Of relevance is that bacterial DNA corresponds to bacteria producing endemic disease in our population.

Innate immune response mechanisms in non-insulin dependent diabetes mellitus patients assessed by flow cytoenzymology.

It is well known that infections in patients with diabetes mellitus are more severe, although there is controversy for increased susceptibility to them. Non-specific immune response mechanisms could be related to defense and/or susceptibility to pathogens. The aim of this study was to investigate the activity of several enzymes involved in the primary host defense mechanisms in non-insulin dependent diabetes mellitus (NIDDM). Twenty NIDDM females with a mean HbA(1c) level of 8.19% were included. No patient had clinical evidence of infection. As controls 20 healthy females were studied. The enzymes tested were dipeptidyl-peptidase I (DPP-I), cathepsin B and D, NADPH oxidase and superoxide dismutase (oxidative burst) and collagenase. Isolated leukocytes were incubated with the specific substrates in pyrogen free conditions. The intracellular enzyme activity was analyzed by flow cytometry. Collagenase enzymatic activity was similar in the three leukocyte subpopulations studied. Oxidative burst induction in monocytes was comparable between both groups. Enzyme activity of cathepsin B and D in all cell subsets, oxidative burst in PMN cells, and DPP-I in lymphocytes and monocytes from patients, was higher than those from healthy females (P<0.05). Overall, our findings demonstrate an enhanced functional status of several intracellular leukocyte enzymes in NIDDM. Furthermore, the increased oxidative burst induction and the consequent production of free radicals, may contribute to vascular complications. Other mechanisms - either from the non-specific or specific immune response - deserve investigation to establish if diabetic patients are more susceptible to infectious diseases.

Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders. Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid arthritis patients might influence disease outcome.

BACKGROUND: Multidrug resistance (MDR) is characterized by overexpression of P-glycoprotein, a pump molecule that decreases intracellular drug concentrations by increasing drug efflux from cells. OBJECTIVE: To look for correlations between clinical status and P-glycoprotein activity and/or TNF-alpha mRNA levels in patients with rheumatoid arthritis. METHODS: Sixteen patients were studied. Based on response to therapy, eight were refractory and eight nonrefractory to treatment. Findings were compared to those in 24 healthy controls. Flow cytometry was used to evaluate P-glycoprotein activity in peripheral blood mononuclear cells isolated by gradient centrifugation and incubated with the P-glycoprotein substrate daunorubicin. TNF-alpha mRNA levels were determined using quantitative PCR. RESULTS: Patients with rheumatoid arthritis showed an increased number of lymphocytes with high P-glycoprotein activity (p = 0.0001) as compared to the normal controls. P-glycoprotein activity was higher in the refractory than in the non-refractory patient subgroup (p = 0.006). Also, TNF-alpha mRNA levels were markedly higher in the refractory subgroup than in the nonrefractory subgroup, and were undetectable in the normal controls. CONCLUSIONS: Enhanced P-glycoprotein activity may be closely related to an unfavorable clinical course and a poor response to treatment. Increased TNF-alpha expression and chronic exposure to various drugs, including glucocorticoids, may contribute to increase P-glycoprotein activity. Both high P-glycoprotein activity and excessive amounts of TNF-alpha seem associated with poor outcome in rheumatoid arthritis.

Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders. Part II: Increased P-glycoprotein activity in lymphocytes from systemic lupus erythematosus patients might affect steroid requirements for disease control.

BACKGROUND: Over-expression of the membrane glycoprotein called P-glycoprotein has been widely observed in a variety of both normal and neoplastic cells. P-glycoprotein is a pump molecule that transports hydrophobic drugs (including steroids) and toxins outside the cells, thus inhibiting their therapeutic or toxic effects. The gene encoding P-glycoprotein is named multidrug resistance-1 (MDR-1). OBJECTIVE: To evaluate the functional activity of P-glycoprotein in lymphocytes and monocytes from patients with systemic lupus erythematosus. METHODS: 30 systemic lupus erythematosus patients and 20 healthy controls were studied. Peripheral blood mononuclear cells isolated by gradient centrifugation were incubated in the presence of daunorubicin (a fluorescent drug extruded by P-glycoprotein) at 37 degrees C or 4 degrees C for 30 min. P-glycoprotein activity was then analyzed using flow cytometry. Results were expressed as the percentage of lymphocytes or monocytes with high P-glycoprotein activity (i.e., low fluorescence). RESULTS: Mean fluorescence values for lymphocytes and monocytes were comparable between patients and healthy controls. However, because our method allowed to measure P-glycoprotein function at the single-cell level, we were able to show that the mean percentage of lymphocytes with high P-glycoprotein activity was increased in the patients (11.51% +/- 14.3%) as compared to the healthy controls (0.71% +/- 0.57%) (P < 0.05). Moreover, P-glycoprotein activity was lower in the patients in clinical remission than in those with active disease. CONCLUSIONS: Our results suggest that P-glycoprotein function might affect glucocorticoid requirements in systemic lupus erythematosus.

HLA class II genotypes in Mexican Mestizos with familial and nonfamilial multiple sclerosis.

Predisposition to MS is associated with the HLA-DR2 antigen in white patients. The authors investigated the genetic factors behind the increasing frequency of MS in the Mexican population. HLA-DR and DQ were analyzed in 17 patients with MS, 15 of their first-degree relatives, and 99 healthy ethnically matched controls. DR2 or DR3 was found in 15 of 17 patients. In controls, both alleles had frequencies less than 0.05. MS in Mexican patients was associated with HLA-DR2 and DR3.

Increased collagenase and dipeptidyl peptidase I activity in leucocytes from healthy elderly people.

The incidence of infectious diseases increases with ageing. The enzymatic activity of leucocytes may have a relevant role in the morbidity and mortality due to infections in the elderly. In this study we have compared the activity of enzymes involved in the inflammatory response in leucocytes from young and elderly women. A total of 35 healthy females was studied, 20 volunteers aged 78-98 years (mean 89.1 years) and 15 young controls aged 19-34 years (mean 26 years). All of them were in good clinical condition, without any acute or chronic disease. Intracellular enzyme activity was analysed by flow cytometry in leucocytes from young and elderly women. The enzyme substrates employed were for oxidative burst, L-aminopeptidase, collagenase, cathepsin B, C, D and, G and dipeptidyl peptidase I. The intracellular enzyme activity assessed by flow cytometry in leucocytes from young and elderly women was similar, as far as oxidative burst, L-aminopeptidase, cathepsin B, C, D and G are concerned. An increased collagenase activity was detected in granulocytes from elders. The mean fluorescence channels for this enzyme corresponded to 86 +/- 23 and 60 +/- 15 in cells from elders and controls, respectively (P = 0.01224). An increased dipeptidyl peptidase I activity was detected in lymphocytes from elderly women. The corresponding values for this enzyme in elders and the young were 65.9 +/- 43.3 and 17.3 +/- 5, respectively (P = 0. 0036). The proper functional activity of intracellular enzymes involved in inflammatory responses is likely to be determinant for successful ageing.

Possible role of interleukin-10 in autoantibody production and in the fate of human cord blood CD5+ B lymphocytes.

Mononuclear cell subsets in 25 human umbilical cord blood samples and 10 healthy adults were studied. We found a decreased percentage of CD3+ cells, CD8+ cells and gammadelta T cells in cord blood compared with blood from healthy adults. The CD16+56+ and CD19+CD5+ phenotypes were overexpressed in cord blood. We then measured spontaneous gene expression and the production of interleukin-10 in mononuclear cells from cord blood and adult subjects. Although we found no difference between cord blood cells and those from healthy adults, a tendency towards spontaneous interleukin-10 production was observed in cord blood. Phorbol 12-myristate 13-acetate stimulation revealed that, among lymphocytes, cord blood B cells are the main cellular source of interleukin-10. Finally, we found no evidence of augmented spontaneous apoptosis but an increased bcl-2 gene expression in non-T cells from cord blood. Interleukin-10 might protect CD19+CD5+ B cells from apoptosis by inducing bcl-2 and promoting autoantibody production in this B-cell subpopulation.

Differential oncogene and TNF-alpha mRNA expression in bone marrow cells from systemic lupus erythematosus patients.

The aim of the study was to investigate the bone marrow expression of genes involved in cell growth and apoptosis in patients with systemic lupus erythematosus (SLE). Spontaneous expression of bcl-2, bax, c-myc. c-fos, c-jun, p53, Fas and tumour necrosis factor (TNF)-alpha by bone marrow cells was measured using either semiquantitative or quantitative reverse transcription polymerase chain reaction in SLE patients (n = 8) and in eight normal control subjects. The expression of bcl-2 was found to be higher in SLE patients than in controls. Bone marrow cells from SLE patients showed significant down-regulation of bax, c-myc, c-fos and p53 (P < or = 0.05), as compared to normal controls. In both SLE patients and controls the expression of c-jun and Fas was very low or undetectable. Finally, TNF-alpha gene expression was higher in bone marrow samples from SLE patients than in those of controls (P= 0.01). The abnormal expression of genes regulating cell growth and apoptosis in bone marrow cells from SLE patients may help explain the presence of autoreactive cells in secondary lymphoid organs and peripheral blood of SLE patients.

[Anaplastic carcinoma of the thyroid at the Instituto Nacional de la Nutrición Salvador Zubirán]. / Carcinoma anaplásico de tiroides en el Instituto Nacional de la Nutrición Salvador Zubirán.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is a highly aggressive tumor with a median survival rate of 6 months. AIM: To analyze presentation, treatment, morphology, immunohistochemistry, and nuclear DNA analysis of a cohort of patients with ATC. PATIENTS AND METHODS: Twelve patients with ATC (11 female) with a mean age of 65 years were seen at our hospital from 1970-1995. The data were obtained from the clinical records and the morphology, immunohistochemic studies and DNA pattern were performed in slides obtained from archival specimens. RESULTS: Previous or coexisting thyroide disease was documented in 10 patients (9 multinodular goiters and one Grave's). The most frequent presentation was a rapidly growing tumor associated with dysphagia, cervical pain, hoarseness and dyspnea. A cold thyroid nodule was detected by thyroid scan in 10 patients. The most frequent subtype was the spindle cell variety. Papillary thyroid carcinoma coexisted in eight cases, two of them corresponded to the tall cell variant. Reactivity for S-100 protein and vimentin was studied in six patients: all were positive for S-100 protein and vimentin, 5/6 for epithelial membrane antigen, half for carcinoembriogenic antigen, 2/6 for thyroglobulin and calcitonin, and one for neuronal specific enolase. These six tumors showed a diploid DNA pattern. Tumor resection was achieved in 2/11 and none survived six years after diagnosis. CONCLUSIONS: ATC is a highly aggressive tumor coexisting with thyroid pathologies. Spindle cell variant is the most frequent with positive reactivity for S-100 protein, vimentin and epithelial membrane antigen. Most tumors have a diploid DNA content.