Stimulation with type I collagen induces changes in gene expression in peripheral blood mononuclear cells from patients with diffuse cutaneous systemic sclerosis (scleroderma).

An autoantigenic role for collagen type I (CI) has been suggested previously in diffuse cutaneous systemic sclerosis (dcSSc). Whether CI is indeed capable of affecting the immune system in dcSSc is not known. Patients with early (3 years or less) or late (>3 years) dcSSc and healthy controls donated blood. Peripheral blood mononuclear cells (PBMC) were cultured with or without CI, and expression of genes known for their involvement in autoimmune and inflammatory processes was assessed using cDNA arrays; results were confirmed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay for selected genes. Patients with early and late dcSSc were similarly different from healthy controls in basal gene expression. When cultured with CI, PBMC from patients with early dcSSc differed from healthy controls in expression of 34 genes, whereas PBMC from patients with late dcSSc differed from healthy controls in expression of only 29 genes. Direct comparisons of matched PBMC samples cultured with and without CI revealed differences in expression of eight genes in healthy controls, of five genes in patients with early dcSSc, and no differences in patients with late dcSSc. Thus, PBMC from patients with dcSSc respond differently than do PBMC from healthy controls when cultured with CI. Exposure to CI in culture of PBMC from patients in the early stage of dcSSc in contrast to PBMC from patients with late-stage dcSSc evokes a greater degree of activation of immune-related genes, suggesting that CI is more dominant as an autoantigen in early versus late dcSSc.

Spontaneous formation of germinal centers in autoimmune mice.

The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1-2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220(+), PNA(+), and GL-7(+) B cells, and FDC-M1(+) follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.

Lessons from animal models of vasculitis.

Vasculitis can occur as a primary disease or as a secondary manifestation of either another illness or a type-III hypersensitivity response to a foreign antigen. Over the past four decades, a number of animal models of vasculitis have been described. These models have served as important tools for enhancing our understanding of the basic mechanisms underlying the pathogenesis of vasculitis. In addition, animal models have made possible the preclinical testing of new therapeutic agents. Animal models of vasculitis can be broadly classified into two types--those that are experimentally induced and those that occur spontaneously. Vasculitis can be experimentally induced in animals through the stimulation of a type-III hypersensitivity response to a variety of foreign antigens, by viral or bacterial infection of vascular cells and the immune response to that infection, or by the in-vivo administration of antineutrophil cytoplasmic antibodies, estrogen, or mercuric chloride (HgCl(2)). Systemic vasculitis spontaneously develops in several strains of mice and rats. This paper reviews the current state of knowledge of several animal models of vasculitis and the lessons that have been learned from them.

Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity.

To determine the role of perforin-mediated cytotoxic T lymphocyte (CTL) effector function in immune regulation, we studied a well-characterized mouse model of graft-versus-host disease (GVHD). Induction of acute GVHD using perforin-deficient donor T cells (pfp-->F1) initially resulted in features of acute GVHD, e.g., engraftment of both donor CD4(+) and CD8(+) T cells, upregulation of Fas and FasL, production of antihost CTL, and secretion of both Th1 and Th2 cytokines. Despite fully functional FasL activity, pfp donor cells failed to totally eliminate host B cells, and, by 4 weeks of disease, cytokine production in pfp-->F1 mice had polarized to a Th2 response. Pfp-->F1 mice eventually developed features of chronic GVHD, such as increased numbers of B cells, persistence of donor CD4 T cells, autoantibody production, and lupuslike renal disease. We conclude that in the setting of B- and T-cell activation, perforin plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and ag-specific T cells. Moreover, an ineffective initial CTL response can evolve into a persistent antibody-mediated response and, with it, the potential for sustained humoral autoimmunity.

Diversity and plasticity of the anti-DNA topoisomerase I autoantibody response in scleroderma.

OBJECTIVE: To examine domain recognition by anti-DNA topoisomerase I (anti-DNA topo I, or anti-topo I) antibodies over time in scleroderma patients. METHODS: Serial serum samples from scleroderma patients with known reactivity to Scl-70, a 70-kd topo I breakdown product, were tested by immunoblot for IgM, IgG, IgA, kappa, and lambda reactivity to Scl-70 and 8 overlapping recombinant peptide fragments (F1-F8) that span the human topo I molecule. RESULTS: IgM, IgG, kappa, and lambda anti-topo I antibodies in both early-disease and late-disease serum samples preferentially recognized the Scl-70 molecule rather than the F1-F8 peptides, suggesting preferential recognition of conformational determinants on Scl-70 throughout the disease course. Amounts of both primary and secondary anti-topo I antibodies to Scl-70 varied over time, including increases in primary antibody responses late in the disease course. Striking variability in recognition of the F1-F8 peptides by IgM, IgG, IgA, kappa, and lambda anti-topo I antibodies was seen in serial samples. Most often, the change in FI-F8 recognition from one sample to the next was unpredictable, although occasionally patterns of antibody recognition were reciprocal in serial samples. Of note, in several patients, what could have been interpreted as domain spreading among F1-F8 in 2 successive samples was just a part of changing antibody reactivity to these peptides that again became more restricted in a third sample. CONCLUSION: Titers and immunodominant domains recognized by both primary and secondary anti-topo I antibodies are highly variable over time. This suggests continual antigen presentation and regulation of the anti-topo I antibody response in scleroderma, even late in the disease course.

IL-10 kinetics in thyroiditis-prone BB/Wor rats.

UNLABELLED: Cytokines modulate the course of autoimmunity, but their role in the evolution of spontaneous disease is unclear. This study compared the cytokine kinetics of T cell cultures from thyroiditis (LT)-prone NB line BB/Wor rats with those of Wistar (Wis) rat controls following activation with the thyroid-specific antigen thyroglobulin (Tg) or Concanavalin A (Con A). DESIGN: T cell enhanced splenocytes from 60 day old Wis and NB rats were activated with 0.5 microg/ml rat thyroglobulin (Tg) or Con A in the presence of homologous irradiated splenocytes as antigen presenting cells (APC's). In addition, the effect of APC's was determined in a crisscross experiment which examined NB T cell responses to Con A in the presence of Wis APC's. ELISA and RT-PCR were used to examine IL-2, IL-4, IL-10, TNFalpha, IFNgamma, IL-I0 concentrations and mRNA expression in the supernatant and cells from parallel cultures harvested at specific intervals. Frozen thyroids from 60 day old NB, Wis and Fisher rats were examined for the presence of IL-10 by immunohistochemistry. T cell proliferation was measured by 3H thymidine uptake. RESULTS: Following activation with either Tg or Con A, IL-10 concentrations exceeded IFNgamma in NB rat cultures, but IFNgamma exceeded IL-10 in Wis cultures. Wis splenocytes significantly enhanced NB T cell proliferation and cytokine responses to Con A. Thyroids from 60 day NB rats contained IL-10, but no IFNgamma. There was no IL-10 in thyroids from Wistar or Fisher rats. CONCLUSION: Splenocyte responses in LT-prone BB/Wor rats favor IL-10 production. Future investigations will examine the source of intrathyroidal IL-10 and its role in LT.

[Comparative analysis of existing criteria in the assessment of iodine-deficiency in dwellers of the Far North areas]. / Sravnitel'nyi analiz sushchestvuiushchikh kriteriev otsenki iododefitsitnykh sostoianii u zhitelei Krainego Severa.

New standards of the thyroid volume proposed by F. Delange (1977) are now being introduced. In view of this, we compared incidence of goiter by F. Delange and by R. Gutekunst (1988) criteria. The examination covered 3920 schoolchildren in 12 settlements of the Tyumen region in 1996. Iodine deficiency was mild to moderate. The greatest deficiency was observed in the North. Ultrasonic investigation of the thyroid by standards of R. Gutekunst (1988) was also indicative of the greatest iodine deficiency in the North. In the Far North, Polar Urals there was severe goiter endemia, in the rest areas the endemy was moderate. By F. Delange (1977) criteria, mild and moderate endemy was in the Far North and Polar Urals, the endemy was absent in the rest areas. As shown by comparison of the palpation data to thyroid ultrasonography, iodinuria, R. Gutekunst criteria are more reliable whereas criteria of F. Delange provide overestimated data.

Vasculitis in the Palmerston North mouse model of lupus: phenotype and cytokine production profile of infiltrating cells.

OBJECTIVE: To define the phenotype of cells in the perivascular and vascular infiltrates of Palmerston North (PN) mice and the cytokines that those cells produce. METHODS: Immunohistologic analysis, flow cytometric analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were performed on tissues and cells from female PN mice and age-matched and sex-matched DBA/2 mice. RESULTS: With aging, PN mice developed a female-predominant, lupus-like disease, with a severe systemic mononuclear cell perivasculitis and vasculitis. The perivasculitis involved arteries and veins in kidney, liver, brain, and lung; the vasculitis predominantly involved veins and venules. The perivascular and vascular infiltrates in female PN mice were composed mainly of an unusual cell type that expressed phenotypic markers characteristic of both T cells (Thy1+, CD3+, CD4+, T cell receptor + [TCR+]) and B cells (B220+). In addition, the infiltrates contained a smaller number of conventional CD4+,B220- T cells and macrophages. Very few CD8+ T cells or surface Ig+ B cells were seen. Unlike the Thy1+,B220+ T cells present in MRL/lpr mice, most of which were CD4-,CD8- and TCRalpha/beta+, the majority of the Thy1+,B220+ T cells in the perivascular/vascular infiltrates of PN mice were CD4+ and expressed either TCRalpha/beta or TCRgamma/delta. By immunohistologic staining, the cells in the perivascular and vascular infiltrates in the kidneys of older PN mice were shown to produce interleukin-4 (IL-4), IL-6, and IL-10, but not IL-2, interferon-gamma, transforming growth factor beta, tumor necrosis factor alpha, or IL-1beta. By RT-PCR, the kidneys of older PN mice were found to express high levels of IL-4, IL-6, and IL-10 messenger RNA. CONCLUSION: The vascular and perivascular infiltrates in PN mice are composed predominantly of an unusual subpopulation of T cells that are Thy1+,B220+,CD4+,CD8-, express either TCRalpha/beta or TCRgamma/delta, and produce mainly type 2 cytokines. The exact role of these cells in the immunopathogenesis of lupus-like disease in PN mice remains to be elucidated.

5'-degenerate 3'-dideoxy-terminated competitors of PCR primers increase specificity of amplification.

Amplification of a product in PCR with specific primers may be viewed as an artificial Darwinian-type "selection of the fittest". In other selective systems, such as general evolution, immune system and probably brain cortex, the stringency of selection is not absolute but rather degenerate, with selection of many highly fit units, not limited, however, to only the fittest. In PCR also, annealing of the primers is not absolutely specific. The subsequent amplification frequently leads to amplification of not only the desired product but also to less-specific sequences. Using theoretical analysis of the degenerate mode of selection, we predict theoretically and prove experimentally that 5'-degenerate, 3'-dideoxy-terminated competitors of PCR primers can be used to dramatically improve the specificity of PCR amplification without affecting the quantitation of the final specific product.

[Endemic goiter in the extreme North of West Siberia]. / Endemicheskii zob na Krainem Severe Zapadnoi Sibiri.

Random examinations covering 8-60-year-old 4345 citizens of 12 settlements of the Yamal-Nenets Autonomic Territory discovered goiter endemia throughout the territory but most evident the endemy manifested in the Far North. The prevalence of endemic goiter among schoolchildren made up 52.8% (enlargement of the goiter of the 1st and 2nd degree), among adults-49.2%. By ultrasound investigation, the above percentages were 29 and 26.4%, respectively. This corresponds to moderate endemia. The median of urinary iodine excretion averaged in the territory 5.1 micrograms%, while overall iodine insufficiency (number of children with urinary iodine < 10 micrograms%) was 81.9%. In the Far North iodine excretion was less but goiter incidence was higher than normal. Thus, in the Far North goiter endemia is rather serious.

[Biosynthesis of bovine growth hormone in Escherichia coli DH1 cells and its purification on a pilot scale]. / Biosintez bych'ego gormona rosta v kletkakh Escherichia coli DH1 i ego vydelenie na pilotnom urovne.

The biosynthesis of the bovine growth hormone (BGH) in E. coli DH1 cells containing the plasmid pbGH(1-119)ptrp was studied. It was found that BGH accumulated in the cytoplasm of bacterial cells as optically dense granules that can be isolated by low-speed centrifugation. A fraction of purified protein granules was used for further purification of BGH. A purified BGH preparation contained two polypeptides with molecular weights of 18,000 and 22,000 D. The state of plasmid was investigated during cultivation and storage of the culture.

[Continuous cultivation of Penicillium brevicompactum that forms extracellular ribonucleases]. / Nepreryvnoe kul'tivirovanie Penicillium brevicompactum, obrazuiushchego vnekletochnye ribonukleazy.

Conditions for continuous cultivation of Penicillium brevi-compactum producing extra-cellular ribonucleases were studied. The two-step process of fermentation in the course of which the flow of the medium in the first fermenter was maintained at 0.054 hr-1, and in the second fermenter at 0.0527 hr-1, made it possible to produce 3--4 times more enzymes as compared to the batch culture.