Modulation of costimulatory molecules CD80/CD86 on B cells and macrophages by stress proteins GroEL, GroES and DnaK.

The aim of this study was to evaluate the effect of the Heat Shock Proteins GroES, GroEL and DnaK on the expression of the costimulatory molecules CD80/CD86 in B cells and macrophages. The interactions among these molecules are able to highly influence the immune response through the regulation of cytokine liberation which, on their own, are able to regulate the immunological response by a feedback mechanism. Our results showed that, on B cells, GroES and GroEL stimulated the expression of CD86 but did not induce the increase of the CD80 expression. CD86 peak expression showed a peak after 24-48 h of culture and decreased 60h after the stimulation. GroES and GroEL also stimulated the expression of CD80 and CD86 on macrophages. The same HSPs did not modify the expression of CD80 and CD86 on cells having characteristics of activated macrophages, the A-THP-1 cell line. DnaK did not induce any increase in the expression of CD80 and CD86 on lymphocytes or macrophages.

Defense mechanisms of IFN-gamma and LPS-primed murine microglia against Acanthamoeba castellanii infection.

In the central nervous system (CNS), cytokine-primed microglia play a central role in host's defense against Acanthamoeba castellanii infection. In this study, the effect of recombinant interferon (rIFN)-gamma and Salmonella enterica serovar enteritidis lipopolysaccharide (LPS), both inflammatory stimuli, on A. castellanii infection in murine microglia was examined. Priming of microglia with rIFN-gamma and LPS synergistically triggered, in a dose-dependent manner, amebastatic activity in these cells. More than 52%, 88% or 95% of this function was then abrogated by anti-IL-1beta (but not anti-IL-1alpha), IL-6 or TNF-alpha neutralizing antibodies, suggesting that these endogenously produced cytokines may participate in the antimicrobial capacity. Consistent with these findings, the priming of microglia with rIFN-gamma and LPS elicited the release of proinflammatory interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. Since L-canavanine affected amebastatic activity only during the priming process but not during the infection process, NO-dependent pathway appears to be not the sole antiparasitic mechanism involved in this function. These data suggest that rIFN-gamma and LPS, likely through a proinflammatory network, up-regulate the release of IL-beta, IL-6 and TNF-alpha, which could trigger antimicrobial activity against A. castellanii infection in the brain.

Histopathological features and modulation of type IV collagen expression induced by Pseudomonas aeruginosa lipopolysaccharide (LPS) and porins on mouse skin.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is responsible for serious infections in the immunocompromised host. Many skin lesions induced by P. aeruginosa have been described. Few investigations have been performed on the local action of P. aeruginosa components. OBJECTIVES: To shed light on the "in vivo" activity of lipopolysaccharide (LPS) and porins extracted from P. aeruginosa, by verifying their effects after inoculation in mouse skin through the observation of histological changes and immunohistochemical expression of collagen IV. RESULTS: Both substances were able to induce a similar inflammatory process and a characteristic reversible change in collagen IV distribution. Interestingly, a fibroblast increase was observed at 24 h in the skin treated with porins, while it appeared later in the skin treated with LPS. Besides these changes, porins particularly increased collagen edema, together with disgregation of hypodermal structures. Moreover "in vitro", porins were able to stimulate fibloblasts 3T3 to convert 72 kDa type IV collagenase into the activated 62 kDa form and to release the 92 kDa collagenase. CONCLUSION: LPS and porins, released by gram-negative bacteria during cell growth and lysis, interact with the host at target cells, such as keratinocytes, fibroblasts and immunocompetent cells, thus contributing significantly to the pathogenesis of P aeruginosa skin infections.

Transforming growth factor beta-1 and interferon-alpha in the AIDS dementia complex (ADC): possible relationship with cerebral viral load?

Mechanisms involved in the pathogenesis of the AIDS-dementia complex are still unclear. The dichotomy between a small number of HIV-infected cells in the brain and their marked dysfunction could be related to a cellular amplification and/or activation of cerebral viral load by several cytokines. This link between cytokines and viral load could play a role in the generation of the clinical dementia syndrome. We have studied cerebral levels of transforming growth factor-beta1 and interferon-alpha, both in the mild and severe AIDS-dementia complex and also compared these cytokines with HIV RNA load in patients with different degrees of dementia. Our data indicate that production of different cytokines characterized the expression of clinical dementia. In the mild AIDS-dementia complex, there was a significant inverse correlation between interferon-alpha and transforming growth factor-beta1 (r = - 0.743; p < 0.001), and HIV-RNA was present in inverse proportion to transforming growth factor beta1 (r = - 0.751; p < 0.001). In patients with severe AIDS-dementia, transforming growth factor-beta1 was undetectable, while interferon-alpha level were higher than in mild AIDS dementia and correlated positively to cerebral HIV-RNA. No significant difference was evident between these cytokines in the serum of ADC patients and in the control samples. Our study suggests that a relationship is possible between productive HIV infection in the cerebral nervous system and a heterogenous and different expression of the immune response via a complex interaction of cytokines with a differential modulation of the dementia phenotype.

Porins and lipopolysaccharide induce apoptosis in human spermatozoa.

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.

Haemophilus influenzae porin contributes to signaling of the inflammatory cascade in rat brain.

In the present study we observed that the Haemophilus influenzae type b (Hib) porin, among the different surface bacterial components, is involved in the pathophysiology of bacterial meningitis. This study demonstrates that inoculation of Hib porin into the fourth cerebral ventricle causes the simultaneous expression of interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), and macrophage inflammatory protein 2 (MIP-2) at 6 h after inoculation. At 24 h, the expression of MIP-2 decreases while the expression of IL-1alpha and TNF-alpha increases. The mRNA expression of IL-1alpha, TNF-alpha, and MIP-2 is correlated with injury to the blood-brain barrier as demonstrated by the appearance of serum proteins and leukocytes in cerebrospinal fluid and by the increase in brain water content.

Interactions between cytokines and growth hormone for resistance to experimental.

Cytokine-activated human vein endothelial cells (HUVEC) may play an important role in resistance to Toxoplasma gondii infection. In this study, it was investigated the role of rTNF-alpha and GH in the induction of antitoxoplasmal activities in HUVEC. Co-treatment of HUVEC with rTNF-alpha plus GH induced both toxoplasmastatic activity and the intracellular killing of T. gondii (p <0.01 each vs untreated cells). Thus, these functions were inhibited by both neutralizing antibodies to IL-6 and GM-CSF (but not to IL-3) suggesting that these cytokines participate in the inhibitory process. Consistent with this hypothesis, the treatment of HUVEC with rIL-6 or rGM-CSF in the presence of rTNF-alpha, limited T. gondii multiplication in a dose-dependent manner (p <0.01 each vs untreated cells). In order to elucidate the inhibitory mechanism of HUVEC, it was assessed by L-arginine analogs (e.g., NG-monomethyl-arginine) whether NO2 molecules originating from HUVEC were directly or indirectly involved in the rTNF-alpha/GH-dependent induction of toxoplasmastatic activity. A good correlation was found between toxoplasmastatic activity and NO2 release during the activation phase, before infection of the HUVEC with T. gondii, but no correlation was found between the parasitostatic activity and NO2 release during the infection phase. These data indicate that NO2- itself does not directly affect toxoplasmastatic activity. Besides, the reduction of intracellular killing by monoclonal antibodies to ICAM-1 suggest that this adhesin plays a role in controlling T. gondii entry into cells.

Profile of cerebrospinal fluid and serum cytokines in patients with relapsing-remitting multiple sclerosis: a correlation with clinical activity.

Levels of tumor necrosis factor (TNF)-alpha, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-10 and transforming growth factor (TGF)-beta in cerebrospinal fluid (CSF) and serum of 29 patients with multiple sclerosis (MS) of the relapsing-remitting type and of 20 controls with other non inflammatory neurological diseases were studied. Sixteen patients were in the active phase of disease and 13 in remission. In CSF, higher IL-10 and TGF-beta concentrations were found in patients with a stable phase of MS, while in the active phase there were elevated levels of TNF-alpha and GM-CSF. These results suggest that a different cytokine pattern could be probably involved in the pathogenesis of relapsing-remitting MS.

Insulin regulates IL-1alpha, Ifn-y and IL-4 release from murine splenocytes stimulated with staphylococcal protein A, toxic shock syndrome toxin-1 and streptococcal lysin S.

In this study, changes were investigated in release of IL-1alpha, IFN-gamma and IL-4 from mouse splenocytes stimulated with staphylococcal protein A (SpA), toxic shock syndrome toxin-1 (TSST-1) or streptococcal lysin S (SLS) in the presence of insulin. The results show that insulin-treated splenocytes stimulated by SpA had a 25% increase in IFN-gamma release and a 50% decrease in IL-4 compared with splenocytes treated with SpA alone. IL-1alpha release was unchanged compared with controls. Insulintreated splenocytes stimulated with TSST-1 had a 30% fall in IL-1alpha and IFN-gamma release compared with controls. There were no changes in IL-4 release. Splenocytes stimulated with SLS after insulin treatment increased their release of IL-1alpha and IFN-gamma by 50%, whereas IL-4 release was unchanged. The data suggest that the insulin may have important functional implications in immunoregulation.

The elevation of cellular phosphatidic acid levels caused by polyomavirus transformation can be disassociated from the activation of phospholipase D.

Middle T (mT), the oncogene of murine polyomavirus, causes transformation of rat fibroblasts by activating a number of signal transducing pathways usually used by polypeptide growth factors and their receptors. Here, we report data regarding the activation of signal transducing pathways involving phospholipase D (PL-D). The hydrolysis of phospholipids by PL-D produces phosphatidic acid (PA), a compound with multiple biological effects. The PA content of cells expressing wild-type mT, introduced via a number of different methods, is approximately 50% higher than their untransformed counterparts. This increase in cellular PA content is associated with an approximately 65% increase in PL-D activity in cells expressing wild-type mT. We have also examined the effects of a number of site-directed mutants of mT, on both cellular PA levels and on PL-D activity. Mutants that do not produce mT (Py808A) or that produce a truncated, nonmembrane bound mT (Py1387T) have PA levels similar to that of control cells. Cells expressing the 322YF mutant of mT (which abolishes interaction of mT with phospholipase C gamma1) show increases in both PA levels and PL-D activity that are similar to those seen with wild-type mT. Expression of mutants that abolish the interaction of mT with either shc or with phosphatidylinositol 3-kinase (250YS and 315YF, respectively) cause an increase in PL-D activity comparable to that seen with wild-type mT. However, the PA content of cells expressing these mutants is not elevated. These results suggest that mT causes activation of cellular PL-D, but this activation alone is not sufficient to cause an increase in cellular PA content. Therefore, wild-type mT must affect another, as yet unknown, step in PA metabolism.

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water.

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

Prolactin and insulin regulate the release of IL-1-alpha and IFN-gamma from murine splenocytes activated with porins or LPS of Salmonella typhimurium.

Murine splenocytes treated with prolactin (PRL) or insulin were stimulated in vitro with porins or lipopolysaccharide (LPS) of Salmonella typhimurium. It was seen that PRL inhibits the release of IFN-gamma from splenocytes treated with porins by about 20% while having no effect on the release of IL-1-alpha. Splenocytes porin-stimulated splenocytes exhibited a remarkable increase in IL-1-alpha release (100%) and a diminished release of IFN-gamma (about 50%) in the presence of insulin. The splenocytes stimulated with LPS had a reduced release of IL-1-alpha (75%) and IFN-gamma (about 50%) when insulin was added. The data suggest that classical endocrine system participates in a bioregulatory feedback loop that may prevent unwanted toxicity from cytokine excess. However, some bacterial products sometimes enormously unbalance this regulatory network.

Beneficial effects of myristic, stearic or oleic acid as part of liposomes on experimental infection and antitumor effect in a murine model.

Liposomes consisting of dicetyl-phosphate, cholesterol, lecithin and stearic or myristic or oleic acid, exert a protective effect for mice against experimental infection by Salmonella typhimurium, and delay both the onset and mortality B16 melanoma in these animals. Liposomes labelled with 3H-myristic acid were used as probes in the spleen and liver. We found that the treatment schedule rather than route of administration of liposomes, is important. The results show that in order to induce protection, preventive treatment must start at least three days before. Longer treatments do not increase the degree of protection, and treatments started at the same time as, or following experimental infection or tumor transplantation, have no effect.

Toxic effect on human spermatozoa by Chlamydia trachomatis purified lipopolysaccharide.

We have investigated the effect that lipopolysaccharide extracted from Chlamydia trachomatis has on human spermatozoa. A lipopolysaccharide of 0.1 microgram ml-1 caused a spermatozoa mortality rate of 65 +/- 4% evaluated by eosin exclusion test. The toxic activity occurred rapidly even after brief incubation times, reaching the maximum (100% mortality) within 60 min.

The presence in human spermatozoa of sites binding some proteins of the outer membrane of Escherichia coli.

In previous works, we noted that porins, surface components of Gram negative bacteria, are toxic for human spermatozoa. In this work we see that porins of E. coli labeled with I125 (0.1-1.0 microgram/ml) bind to spermatozoa, showing the presence of saturable molecular configurations of 0.5-0.6 micrograms/ml. In the presence of non labeled porins, the binding of labeled porins to the spermatozoa is greatly reduced. At saturating concentrations of 0.6 micrograms/ml labeled porins, the binding reaches a saturation plateau at about 15 min.

Correlation between modification of membrane phospholipids and some biological activity of lymphocytes, neutrophils and macrophages.

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs. d) The cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at C16 cause a decrease in the formation of phagolisosomes in the macrophages tested.

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.