Concomitant in vivo production of 19 different cytokines in human tonsils.

Accumulating data indicate that cytokines, peptides involved in regulation of both physiological and pathological immune responses, are produced predominantly at the site of local antigen stimulation. Cytokine-producing cells were detected at the protein level in human tonsil tissue obtained from children with recurrent tonsillitis or infectious mononucleosis (IM). Concomitant production of 19 different human cytokines, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta 1-3 (TGF-beta 1-3), was identified at a single-cell level by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific antibodies (Ab). Fresh frozen sections were fixed with 4% paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the cell-surface membrane and the Golgi complex. The intracellular localization of all cytokines, except IL-1 and IL-1ra, was demonstrated by a characteristic local cytoplasmic perinuclear configuration in producer cells. In addition, the immunoreactivity for certain cytokines (IL-2, IL-4, IL-5, G-CSF and GM-CSF) was expressed on the cell membranes and extended over a large extracellular area encompassing the producer cell. Localization of the cytokine to the Golgi organelle was established by co-staining with a monoclonal antibody (mAb) specific to the Golgi complex. Both the extra- and intracellular cytokine staining reactions could be blocked by preincubation of the cytokine-specific Ab with the corresponding purified natural or recombinant cytokine. A complex cytokine pattern was established in both groups studied, where most T-helper type 1 (Th1) and Th2 lymphokines were expressed in the tonsils but at different frequencies and localizations. Cells expressing IL-4, IL-5, IL-10 and IL-13, (Th2 response) were evident at higher frequencies in recurrent tonsillitis compared to sections from IM, which were associated with a more pronounced IL-2, IFN-gamma and TNF-beta expression.     

Soluble CD14 from urine copurifies with a potent inducer of cytokines

Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine-inducing activity appeared to co-migrate with the 48-kDa polypeptide which upon sequencing had the same N-terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin-6, respectively. The cytokine-inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine-inducing activity was not affected by LPS-binding protein (LBP) or polyclonal rabbit antibodies against LBP The TNF-inducing activity of uCD14 was also heat labile in contrast to the cytokine-inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.     

T-cell turnover in vivo and the role of cytokines

In addition to responding to specific antigen, CD8+ T-cells with a memory (CD44hi) phenotype undergo bystander proliferation when exposed to certain cytokines, notably type I interferons (IFN I), in vivo; such proliferation does not require T-cell receptor ligation. Since IFN I is unable to induce proliferation of purified CD44hi CD8+ cells in vitro, stimulation of these cells in vivo may reflect IFN I-dependent release of other cytokines. Evidence is presented that IFN I induces macrophages to synthesize IL-15 mRNA and that, at the protein level, IL-15 causes selective stimulation of CD44hi CD8+ (but not CD4+) cells, both in vitro and in vivo. This finding raises the possibility that synthesis of IL-15 during infection may induce bystander proliferation of memory-phenotype CD8+ cells.     

Review article Microsatellites in the HLA region: an overview

Microsatellites are repeats of a DNA base motif (1–6 bp, mostly CA repeats) up to 100 times; they are distributed regularly all over the genome. Many of them are polymorphic and their high polymorphism is based upon a variable number of repeats. They are widely used for genetic mapping, linkage analysis, population genetics, evolutionary studies and in forensic medicine. Such markers have also been described in the HLA region since 1991, and a growing interest in their potential applications is being expressed. The aims of this review are: 1) to outline the presently available information from literature and molecular databases concerning 53 microsatellites in the HLA region (localization, type of repeat, number of alleles, heterozygosity, primers used for amplification); 2) to address the question of technical pitfalls when using such markers; 3) to discuss specific features such as their mutation rate (10^-3 to 10^-6), which is higher than that reported for HLA genes, and their linkage disequilibrium with HLA alleles; 4) to present an integrated map of microsatellites and genes of this region; and 5) to provide a synopsis of their different applications in HLA-related fields (disease studies, population genetics, recombination point studies, HLA region mapping, transplantation) along with perspectives for future use. Although some HLA region microsatellites have already been applied to the analysis of more than 10 diseases, it is now evident that their use in population genetics and the determination of genomic compatibility in bone marrow transplantation represent growing areas of application.     

Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.

Recombinant murine tumor necrosis factor-alpha (TNF-alpha) was shown to be a strong, systemic stimulus in vivo for members of the chemoattractant cytokine gene families (JE, KC, IP-10). The three genes showed differential sensitivity to TNF-alpha, and their expression demonstrated differential tissue specificity. IP-10 was the most strongly induced messenger RNA and was seen in the liver, kidney, and spleen but very poorly in the lung or skin. JE exhibited a similar pattern, though the magnitude of expression was markedly lower. KC expression was seen only in the liver of TNF-alpha-treated mice. The time course of expression for IP-10 was rapid and transient and showed strong dose dependence. In mice treated with TNF-alpha intravenously, messenger RNA was localized in the splenic stroma but not in adherent macrophages or nonadherent lymphocytes. In situ hybridization found the majority of intercrime expression in the splenic red pulp with little or no expression seen in the white pulp. In vitro, TNF-alpha was a potent stimulus of chemoattractant messenger RNA expression in fibroblasts but not in inflammatory peritoneal macrophages. These results indicate that TNF-alpha may be an important stimulus for chemoattractant cytokine gene expression in vivo, and the primary cell types responsible may be either stromal fibroblasts, microvascular endothelium, and/or a subset of anchored mononuclear phagocytes.     

In vivo and in vitro stimulation of mouse spleen leukocytes by BL-20803, a low-molecular-weight interferon inducer.

BL-20803, a low-molecular-weight compound, although able to elicit circulating interferon in the mouse, failed to protect cultured cell lines in vitro from infection by interferon-sensitive viruses. Of the tissues analyzed for interferon content after oral administration of the drug to mice, spleen and lung contained the largest amounts of the virus inhibitor. Spleen cells from such dosed animals when isolated into in vitro cultures elaborated small amounts of interferon into the culture medium. The time sequence of acquisition by spleen cells of the ability to produce interferon closely correlated with the kinetics of development of the circulating interferon response in the intact mouse. When spleen cells were separated on the basis of adherence or nonadherence to a plastic surface, the bulk of the interferon activity was found to be associated with the adherent cells. Upon exposure to BL-20803 in cell culture, adherent cells and, to a lesser extent, nonadherent cells from untreated mice were stimulated to produce interferon-like activity. The biological behavior of BL-20803 is shown to have striking similarities with that of the structurally different low-molecular-weight inducer tilorone hydrochloride.     

In vivo clearance by the mononuclear phagocyte system in humans: an overview of methods and their interpretation.

The mononuclear phagocyte system (MPS) is responsible for the elimination of foreign material, effete autologous material and immune complexes. To study the relationship between MPS function and human disease, several test substances have been developed, and used to determine the clearance capacity of the MPS in human subjects in vivo. These test substances and the multitude of factors that influence the elimination of these substances (and complicate the interpretation of the test results) are discussed. Use of these probes has provided important new insights, that may lead to the development of treatment modalities by which MPS function is modified in order to influence disease processes more effectively.     

In vivo activation of macrophage oxidative burst activity by cytokines and amphotericin B.

Alterations in macrophage oxidative burst activity following in vivo administration of recombinant murine gamma interferon (IFN-gamma), recombinant murine tumor necrosis factor alpha, and the antifungal antibiotic amphotericin B were investigated. Mice were given intraperitoneal injections of these agents alone and in combination, and the oxidative responses of their resident peritoneal macrophages to challenge with Histoplasma capsulatum or zymosan particles were measured 1 to 5 days later. Various degrees of enhanced oxidative burst activity were achieved following treatment with each agent. However, a synergistic response was observed only when mice were treated with the combination of recombinant murine IFN-gamma and amphotericin B. These results not only confirm the dual role of amphotericin as an antifungal agent and as an immunomodulator but also suggest that IFN-gamma may serve as a useful adjunct in the treatment of intracellular fungal infections.     

Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo

Single-stranded RNA stimulates immune cells and induces the secretion of pro-inflammatory cytokines and type I IFN. As adjuvant RNA can induce a T(h)2 type of humoral response, however, its potency in the induction of cytotoxic T cells in vivo has not been analyzed. Here we show that immunization with the antigen ovalbumin (OVA) and the adjuvant phosphodiester RNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) induced a Toll-like receptor-7-dependent cytotoxic T cell and humoral response. Staining with SIINFEKL-K(b) tetramers demonstrated the induction of antigen-specific T cells that were functional in in vivo cytotoxic T cell assays against SIINFEKL-loaded target cells. In infection experiments with OVA-secreting Listeria monocytogenes, the cytotoxic T cell response strongly reduced the bacterial load in liver and spleen. The RNA-driven humoral response was characterized by OVA-specific antibodies of the IgG1 isotype whereas CpG-DNA induced antigen-specific antibodies of the IgG2a (BALB/c) or IgG2c (C57BL/6) isotype. Furthermore, stimulation with RNA did not induce splenomegaly, a common feature of CpG-DNA-driven immune activation in mice. Taken together, our data confirm that RNA can be used as a safe adjuvant and induces a strong antibody response of the IgG1 isotype. Additionally, we demonstrate that RNA induces an antigen-specific immunity characterized by a potent cytotoxic T cell response to infection.     

Prevalence of stroke in rural community--an overview of Indian experience

Various prevalence studies of stroke conducted in different regions of rural India have been analyzed and compared with prevalence study of stroke in 51,165 rural population of Haryana. The prevalence of stoke varies in different regions of country and ranges from 40 to 270/100,000 rural population. The prevalence rates correspond to that in urban areas in same region but is much lower than stroke prevalence in metropolitan cities in India and from reported prevalence of 400-800/100,000 in Western countries. Ethnic, socio-economic and dietary factors may be responsible for this variance.     

The role of cytokines in arthritic diseases: in vitro and in vivo measurements of cartilage degradation

The injection of partially purified porcine synovial catabolin/IL-1 alpha intra-articularly in rabbits resulted in a 50% loss of glycosaminoglycan (GAG) after 3 days. An increase in the synovial fluid content of GAG was found, and 35SO4 incorporation into proteoglycan was inhibited. Measurements were also made of the GAG loss from articular cartilage into the synovial fluid in human rheumatoid (RA) and osteoarthritic (OA) patients. Very high levels of GAG content in the synovial fluid was found, and calculations were made of the half-life of the cartilage proteoglycan during the active phases of the disease. Estimation of the synovial fluid GAG is believed to be a simple and quantitative method for monitoring the effectiveness of cartilage-"sparing" anti-arthritic drugs.     

An overview of young CLL patients: a single-centre experience from Turkey

Chronic lymphocytic leukaemia (CLL) is generally considered to be a disease of the older population. The aim of this retrospective study was to determine whether younger subjects with CLL (< or = 55 years of age) were different from older patients in their clinical features and prognoses. A total of 198 CLL patients registered to the centre were analyzed: 47 (24%) were 55 years of age or younger and 37 who were followed up regularly were included in the study. The male/female ratio was significantly higher in young patients (3.7 vs. 1.51; p = 0.02). More young patients were asymptomatic than old patients at initial presentation (38.3% vs. 28.5%; p > 0.05). The clinical features, laboratory findings, the distribution of both age groups into clinical stages, and the overall response rate to treatment were similar. The median time to follow-up was 62 months. During this period, 14 of the young patients died (48.3%); all were males. The median survival was longer in the young (64.5 vs. 47 months, p > 0.05). The 5-year survival rate of young patients was more than the old (57% vs. 31%), but the 10-year survival rates did not differ between the two groups (7% vs. 8%). The rate of CLL-related death was higher in young patients (71% vs. 59%; p > 0.05). Univariate analysis revealed no prognostic factor which could influence the survival probability of young patients. In this study, the prognostic values of some variables could not be assessed accurately as the number of regularly followed young patients was low and some data were missing. However, it is expected that survival will be longer in young CLL patients, so the search for different curative treatment strategies will continue.     

Endotoxin and cytokines alter contractile protein expression in cardiac myocytes in vivo

Release of bacterial endotoxin and cytokines induce cardiac failure during sepsis. We investigated the direct effects of E. coli endotoxin (lipopolysaccharide, LPS) and cytokines induced by LPS on the cardiac myocyte gene program. For in vivo-experiments adult Wistar rats were given 600 microg/day LPS i.v. for 24 h or 7 days. In addition, cultured adult rat cardiac myocytes were treated with LPS, interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma) or IL-6 for 24 h. mRNA expression was evaluated for cardiac-alpha-actin (cAct), skeletal-alpha-actin (skAct), beta- and alpha-myosin heavy chain (MHC). LPS induced betaMHC-mRNA 3.6-fold and repressed alphaMHC 2.7-fold and cAct 2.5-fold after 24 h in vivo. Up-regulation of betaMHC (3-fold) and repression of cAct (2.5-fold) were still observed after 7 days LPS infusion, whereas alphaMHC-mRNA levels had returned to normal. At the protein level, increased expression of betaMHC by LPS treatment occurred already after 24 h and was maintained thereafter. LPS had no influence on skAct-mRNA. Similar changes in contractile protein mRNA expression were observed in LPS-treated cardiomyocytes in culture, whereas the tested cytokines either activated (IL-1beta, IFNgamma) or repressed (TNFalpha, IL-6) both MHC-isoforms and cAct. In conclusion, LPS and proinflammatory cytokines induce changes in contractile protein expression that may contribute to the acute heart failure observed during endotoxaemia.     

Cytokines: an overview.

Cytokines are a broad, heterogeneous group of proteins and polypeptides that regulate intercellular communication. Examples of cytokines include interleukins, interferons, colony-stimulating factors, and a variety of growth factors. The preparation of large quantities of highly purified recombinant cytokines has provided a basis for their biological and physicochemical characterization. The pleiotropic biological effects of these factors are expressed through binding to specific, high-affinity cell-surface receptors. Although they are different in amino acid sequence, cytokines have a number of biological and physicochemical properties in common.     

Bacterium-dependent induction of cytokines in mononuclear cells and their pathologic consequences in vivo

Viridans streptococci are a heterogeneous group of gram-positive bacteria that are normal inhabitants of the mouth. These organisms are thought to contribute significantly to the etiology of infective endocarditis, although recently they have been implicated in serious infections in other settings. Another group of oral bacteria, gram-negative anaerobes, is associated with chronic dental infections, such as periodontal diseases or endodontic lesion formation. We evaluated the ability of the oral pathogens Streptococcus mutans and Porphyromonas endodontalis to induce a pathogenic response in vivo, with the goal of quantifying the inflammatory response in soft tissue by measuring leukocyte recruitment and hard tissues by measuring osteoclastogenesis. S. mutans induced a strong inflammatory response and was a potent inducer of osteoclast formation, while P. endodontalis was not. To further study the mechanisms by which P. endodontalis and S. mutans elicit significantly different levels of inflammatory responses in vivo, we tested the capacity of each to induce production of cytokines by mononuclear cells in vitro. S. mutans stimulated high levels of interleukin-12 (IL-12), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), all of which are associated with inflammation, enhanced monocyte function, and generation of a Th1 response. In contrast, P. endodontalis stimulated production of IL-10 but not of TNF-alpha, IL-12, or IFN-gamma. These results demonstrate that oral pathogens differ dramatically in their abilities to induce inflammatory and immunoregulatory cytokines. Moreover, there is a high degree of correlation between the cytokine profile induced by these bacteria in vitro and their pathogenic capacity in vivo.     

Immune response and in vivo production of cytokines in patients with liver hydatidosis

Cytokines play an important role in the human immunological response, but the exact role of cytokines in the human immune response against parasites, especially against Echinococcus granulosus, remains unclear. IL-1, IL-2, IL-4 and tumour necrosis factor (TNF) levels in peripheral blood of 21 patients with liver hydatidosis were evaluated before surgical treatment, and the levels of IgA, IgM, IgG, IgE, specific IgE against E. granulosus, C3, C4 and BF complement fractions and CD20, CD3, CD4, CD8 and CD16 cell percentages were also determined, as was the relationship between these variables and cytokine levels. Data from hydatid patients were compared with data obtained from 21 healthy volunteers. Hydatid patients showed increases of IgG, IgE, IgEs and IL-2 (P< 0.01), and decreases of IL-1 and TNF levels (P< 0.001), but these variables (respectively) increased in patients showing cysts in the central area of the liver or with a wide opening of cysts in the biliary tract. The increase of IL-1, IL-2 and IL-4 showed a close relationship with the number, characteristics and above all the location of cysts within the liver itself. IgG and IL-4 levels and also IgG and IgE levels showed a significant correlation (P< 0.05).