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.

Effects of cytokines and prolactin on the replication of Toxoplasma gondii in murine microglia.

In the central nervous system, cytokine-activated microglia play a crucial role in host defence against Toxoplasma gondii infections. In this study, the effect of recombinant tumor necrosis factor (rTNF)-alpha and prolactin (PRL) on T. gondii infection in microglia was examined. Pretreatment of microglia with rTNF-alpha and PRL induced toxoplasmastatic activity, the intracellular killing of T. gondii and the release of interleukin (IL)-1 beta IL-3 and IL-6: 50% of the intracellular killing was abrogated by anti-ICAM-1 monoclonal antibodies, whereas more than 54 or 87% of toxoplasmastatic activity was reversed by anti-IL-3 or IL-6 monoclonal antibodies. In addition, the treatment of microglia with either rIL-3 or rIL-6, in the absence or presence of rTNF-alpha significantly limited T. gondii replication. Inasmuch as either NMA or S-M-ITU affected cytokine-activated toxoplasmastatic activity during the infection phase, the NO-dependent pathway itself appears not to be directly involved in the parasitostatic activity. These findings suggest that TNF-alpha and PRL up-regulate the expression of ICAM-1 and the production of endogenous IL-6 and IL-3 by microglia, which could induce anti-parasitic functions against T. gondii infection in the brain.

Neutrophil adhesion and transmigration through bovine endothelial cells in vitro by protein H and LPS of Pasteurella multocida.

This study describes an in vitro investigation on the role of Pasteurella multocida cells and its isolated protein H and LPS on neutrophil adhesion and migration through bovine endothelial cell monolayers. P. multicoda cells, protein H and LPS increased the adhesion and transmigration of neutrophils through BAEC. The bacteria/cell ratio of 100 for P. multocida, protein H concentration 0.05-0.2 microM and LPS concentration 0.5-1.0 microM respectively, induced the maximum adhesion and transmigration of neutrophils through BAEC. The optimal time of incubation with bacteria or bacterial products was 4-6 h. Our results confirm the role of Gram-negative bacteria and of components of the outer membrane such as protein H or LPS in activating the neutrophils and in promoting the adhesion and cells transmigration from the vessels to the site of inflammation.

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.

Apoptosis of human keratinocytes after bacterial invasion.

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.

Porins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocyte transmigration through human endothelial cells in vitro.

Bacteria or bacterial products may constitute important inducers of surface molecule expression on endothelial cells and leucocytes. This study was undertaken to determine the effects of the Salmonella typhimurium porins, LPS-S and LPS-R on the transendothelial migration of leucocytes through human umbilical vein endothelial cells (HUVEC). Treatment of the HUVEC with either porins or LPS-S or LPS-R increased the transmigration of different leucocyte populations, in particular that of neutrophils. The maximal increase occurred using LPS-S treatment, whereas porin stimulation fell between LPS-S and LPS-R. The transmigration increase was dose-dependent and reached its maximum at about 100-1000 ng/ml of stimulus. Optimal endothelial activation occurred after 2-4 h and 4-6 h using LPS and porin, respectively. Stimulation of leucocytes with either porins or LPS slightly increased their transmigration through non-activated endothelial cells. Transmigration increased remarkably during the simultaneous stimulation of endothelial cells by IL-1ss together with either porins or LPS. To assess participation of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and leucocyte adhesion complex (CD11/18) in porin- or LPS-mediated leucocyte migration, blocking MoAbs were used. Each blocking MoAb partially and selectively decreased leucocyte transmigration. The obtained results contribute to clarify some aspects of the inflammatory process at sites of infection.

TNF-alpha expression and herpes simplex infection in human monocytes treated with growth hormone, prolactin and insulin.

We evaluated the in vitro effect of growth hormone (GH), prolactin (PRL) and insulin treatment of human monocytes on Herpes simplex virus type 1 (HSV-1) infection. GH and PRL increased cell susceptibility to infection which was related to a slight TNF-alpha expression and release. Insulin had no significant effect. Cells activated with lipopolysaccharide (LPS) and then treated with PRL showed a lower susceptibility to HSV infection related to a significant increase in TNF-alpha expression and release. On the contrary, GH and insulin increased the susceptibility to infection of activated cells but did not modify TNF-alpha expression with respect to cells treated only with hormones.

Effects of alpha-adrenergic agonists on Toxoplasma gondii replication in human umbilical vein endothelial cells.

We investigated the effects of alpha-adrenergic on the capacity of Toxoplasma gondii to invade and proliferate in cultured human umbilical vein endothelial cells. Pretreatment of human umbilical vein endothelial cells (HUVEC) with alpha 2-adrenergic led to a high degree of intracellular killing of T. gondii in these cells. Moreover alpha 2-adrenergics activated HUVEC, induced a marked and dose-dependent toxoplasmastatic activity, whereas a pretreatment of HUVEC with alpha 1-adrenergics had no antiparasitic effects. These data suggested that antitoxoplasmal effects could involve alpha 2-adrenoreceptors. This hypothesis is supported by the abolishment of the antitoxoplasma capacities by yohimbine (an alpha 2 adreno-receptor blocker) but not by prazosin (which binds alpha 1-adrenergic receptors). Because it has been reported recently that reactive nitrogen intermediates (RNI) are essential for the inhibition of T. gondii in macrophages, we investigated whether these molecules are also involved in the alpha 2-adrenergic- dependent induction of toxoplasmastatic activity. We observed, from the incubation of HUVEC with analogs of arginine (e.g. NG-monomethyl-L-arginine) or arginase that deplete arginine, that a good correlation was found between toxoplasmastatic activity and release of NO2- during the activation phase before infection with T. gondii. No correlation was found between NO2-production during the whole infection phase of the HUVEC and toxoplasmastatic activity. These results raise the interesting possibility that alpha 2 and beta-adrenergics agonists, which naturally occur in body fluids, may regulate the transplacental transmission of T. gondii from mother to foetus.

Susceptibility to toxoplasmosis: correlation between macrophage function, brain cyst formation and mortality in rats.

Rats are resistant to Toxoplasma infection, and in contrast to mice do not form cysts in their tissues. Because rats treated with beta adrenergics, corticosteroids or 60cobalt are more susceptible to toxoplasmosis, we conducted experiments to investigate if the impaired resistance of drug-treated rats is related to macrophage function or induction of cystogenic capacity. Our experiments in 0.7 or 1.2 mg/kg-corticosteroid or 12 Gy-60cobalt-treated rats indicated that the decreased survival rate (P < 0.001 to P < 0.0001, compared to infected-untreated or infected-unirradiated animals) was associated with a decrease of both macrophage toxoplasmastatic activity and intracellular killing (P < 0.05 each group), compared to infected-untreated or infected-unirradiated rats. However in 9 Gy-60cobalt-treated animals the decreased survival rate (P < 0.001, compared with control rats) was accompanied only by a decrease of the toxoplasmastatic activity in comparison to macrophages of the control animals. Moreover in these animals, the release of NO2- by these macrophages was poorly detectable (P < 0.05) or completely inhibited (P < 0.01) in comparison with infected-untreated or infected-unirradiated rats. In contrast, in all groups of rats treated with high doses of beta adrenergic, the decreased survival (P < 0.001 to P < 0.0001, compared with untreated rats) was accompanied by values of intracellular killing and intracellular proliferation of Toxoplasma parasites that did not significantly (P = ns each group) differ from macrophages of infected-untreated rats. Furthermore in the high beta adrenergic treated groups only small amounts of NO2- were detectable (P < 0.05) in comparison with control animals. In addition, our data in rats treated with 0.7 or 1.2 mg/kg of corticosteroid or 12 Gy of 60cobalt indicated that the increased mortality was correlated to the presence of a small number of cysts in their brains (P < 0.05; P = ns; P < 0.01 respectively) in comparison to infected-untreated or infected-unirradiated rats. These results suggest that the susceptibility of drug-immunosuppressed rats is not due exclusively to a deficient macrophage function, but is probably also linked to immune mechanisms involved in the process of cystogenesis.

Effect of prolactin, rIFN-gamma or rTNF-alpha in murine toxoplasmosis.

Mice lethally infected with T. gondii and treated with prolactin (PRL), recombinant interferon gamma (rIFN-gamma) or recombinant tumour necrosis factor (rTNF-alpha) were protected against death, as compared to untreated controls. The protective effect of PRL (0.5-2 mg/kg/twice daily for 12 days) was dose dependent and statistically significant (P < 0.001). The survival was 50% or 40% in mice that received doses of 1 x 10(4) U of rIFN-gamma or 4 x 10(4) U of rTNF-alpha at -2, 0, +2 days before and after infection (P < 0.0001). An increase of time to death, up to 60 days after challenge, and of survival rate (50% up to 70%) were observed in animals treated with PRL in combination with either rTNF-alpha or rIFN-gamma, compared to those that received treatments with the same therapeutic agents alone; however the differences were not statistically significant. In addition, a slight synergistic effect on brain cyst formation, with lower number of Toxoplasma cysts, was observed in mice treated with PRL plus TNF-alpha (P < 0.01), compared with animals that received rTNF-alpha alone (P < 0.05). These data suggest that PRL can regulate in vivo endogenous TNF-alpha production in the cytokine cascade. We conclude that prolactin may play an important role in modulating the host's immune defence against T. gondii opportunistic infection.

Enhanced cellular response in mice treated with a Brucella antigen-liposome mixture.

The capacity of liposomes constituted by dycetyl-phosphate (0.009 mM), cholesterol (0.017 mM), lecithin (0.003 mM), and myristic (0.1 mM), stearic (0.1 mM), or oleic acid (0.1 mM) to modify the lymphocyte response to Brucella melitensis antigens in mice was studied. Mice treated with antigens mixed with liposomes containing myristic, stearic or oleic acid had higher antibody titres than mice given antigen suspended in a saline solution. Liposomes alone, without Brucella antigens, resulted in increased 3H-thymidine incorporation by lymphocytes both in vivo and in vitro. The addition of polyclonal activators (LPS and ConA) caused a further increase of 3H-thymidine uptake. Moreover, spleen lymphocytes from mice inoculated with Brucella antigens mixed with the liposomes had a significantly lower population of B lymphocytes (10%), and a notable increase in the Tc lymphocytes (20%). Autoradiography of sections of popliteal ganglia of treated mice showed that the radioactivity was concentrated mainly in the membrane structures of the cell.

Effects of irradiation doses on alterations in cytokine release by monocytes and lymphocytes.

We evaluated the effect of increasing doses of gamma-irradiation on the release of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) from lymphocytes, and interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from monocytes. The most radiosensitive populations were the T lymphocytes which release IL-4 and IFN-gamma after irradiation alone. This release increased when the cells were activated by polyclonal activators such as Concanavalin A (Con A). Monocytes irradiated and stimulated with lipopolysaccharide (LPS) released TNF-alpha with values near those of the control cells. Con A produced no effect. The release of IL-1 beta decreased as the irradiation dose was increased, while stimulation with LPS and Con A caused a greater IL-1 beta increase after small irradiation doses. The results obtained show that minimum doses of irradiation can induce significant alterations in an amplified and unbalanced immunologic response through release of cytokines.

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.

Impairment of natural resistance to Toxoplasma gondii infection in rats treated with beta adrenergics, beta blockers, corticosteroids or total body irradiation.

The data shows that Fisher rats treated with propranolol, betamethasone or exposed to cobalt60-treatment before challenge with T. gondii exhibit an impaired resistance to this opportunistic parasite infection. In fact, in rats propranolol-treatment induces an increase mortality rate up to 66.6% (P < 0.01, with respect to controls). Whereas betamethasone or cobalt60-treatment induces an increased of mortality rate about 33.3% (P < 0.05 respectively, with respect to unirradiated rats). In contrast to beta blocker, corticosteroid-treated or irradiated rats, beta adrenergic-treated rats do not significantly differ from untreated rats in their time of survival. These data lead the authors to hypothesise that in rats the natural resistance to T. gondii can be modulate beta adrenergics or corticosteroids.

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.

Effect of modification of HEp 2 cell membrane lipidic phase on susceptibility to infection from herpes simplex virus.

We have verified the consequences of alterations of the lipidic phase of HEp 2 cell membranes on susceptibility to infection from Herpes simplex virus, and have related these results to modification in membrane fluidity. This was carried out by treating HEp 2 cells with saturated and unsaturated fatty acids with chain lengths from eight to 18 carbon atoms. The results show that HEp 2 cells treated with saturated fatty acids with eight and ten carbon atoms have both increased membrane fluidity and increased susceptibility to Herpes simplex virus infection while both parameters were reduced when HEp 2 cells were treated with saturated fatty acids having 14 and 18 carbon atoms. Fatty acids with 16 carbon atoms, however, show an increase in membrane fluidity which has no significant correlation to infection susceptibility. As to unsaturated acids, those with eight carbon atoms, 2-octenoic acid (trans-8: 1[2]) and 2-octynoic acid (8:::1[2]), increase the susceptibility to infection and fluidity while low concentrations of monounsaturated acids with 14 and 18 carbon atoms, myristoleic acid (cis-14:1[9]) and oleic acid (cis-18:1[9]), reduce both the susceptibility to infection and the fluidity of the membrane.

Comparative study of some biological activities of porins extracted from various microorganisms.

The outer membrane of Gram-negative bacteria contains some major proteins, called porins, with particular chemical and physical characteristics which reflect some specialized functions peculiar to the outer membrane. In recent years the biological properties of Salmonella typhimurium SH5014 porins have been the focus of our experimental studies. The aim of the present research was to make a comparative investigation of the biological activities of porins extracted from other microorganisms classified in the Enterobacteriaceae, together with a taxonomically different one, Eikenella corrodens frequently isolated from gum pockets of patients with periodontal disease. Porins from E. coli K12, Proteus mirabilis and Eikenella corrodens were extracted, purified and separated from LPS by means of phenol extraction. The purity of preparation was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis in slabs. We studied the interactions of these proteins with some cellular and humoral systems within the host. Mouse peritoneal macrophages in the presence of these proteins show morphological and functional modifications, depending upon the type of porins and the concentrations used. The ability of porins to interact with the complement system was investigated in vitro. A pool of sera from 20 blood donors served as a source of human complement. The results obtained with porins of various sources were shown comparatively.

Phagocytosis and resistance to Salmonella typhimurium infection in mice fed with lipidic diet.

The peritoneal macrophages from mice on a lipidic diet have shown an increase of surface hydrophobicity of cytoplasmatic membrane. This fact is correlated with a decrease of the phagocytic index and with an impairment of Salmonella typhimurium.

Cytotoxic antibody dependent cells in mice experimentally infected with Brucella abortus.

In this study we investigated the cytotoxic activity mediated by antibody dependent cells (K cell) in C57BL/6 mice experimentally infected with Brucella abortus. The involvement of the K cells in experimental infection from Brucella was studied by evaluating the bacteriolysis in the presence of specific antibodies. After 3 h of incubation, approximately 30 +/- 3% of the labeled Brucellae undergo lysis, whereas the presence of lymphocytic populations alone, or of a specific decomplemented serum, determines a lysis of 5 +/- 3%. Cytotoxic antibody dependent activity of purified lymphomonocytic populations (ADCC) evaluated against cells labeled with 51Cr sensitized with specific antibodies obtained from mice is considerably increased in mice experimentally infected with Brucellae with respect to that present in non-infected mice. The increased activity begins to manifest itself 5 to 7 days after inoculation of the Brucellae reaching a maximum on the 10th day. The same results were obtained in the lymphocytic populations prepared from normal animals and those infected by Brucellae searching for K cells with the ability to form rosettes (EA-RFC).