In vitro treatment with killed Helicobacter pylori downregulates the production of RANTES by peripheral blood mononuclear cells.

The mechanisms by which Helicobacter pylori colonizes and persists within the gastric mucosa are poorly understood. The gastric immune response observed in vivo during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori cagA(+)/vacA(+ )(live and/or gentamicin-killed) on human peripheral blood mononuclear cells (PBMCs) in order to evaluate the production of regulated activation normal T cell expressed and secreted (RANTES) in vitro. We also evaluated the possible relationship between RANTES release and the presence of IL-12 and IFN-gamma in supernatants of the same cells. In the present study, we show for the first time that the low amount of RANTES in supernatants of PBMC incubated with killed H. pylori is linked, at least in part, to the inhibition of IL-12 and IFN-gamma release.

Differential induction of TNFalpha and IL-18 in human peripheral blood mononuclear cells infected with Leishmania major or Leishmania donovani.

Several cytokines are involved in the host response to Leishmania. However, the role played by cytokines during infection with different species of Leishmania is not univocal. In this work, the production of tumor necrosis factor alpha (TNFalpha) and interleukin 18 (IL-18) during interaction of human phagocytes with Leishmania major or L. donovani was comparatively investigated. Peripheral blood mononuclear cells (PBMC) and monocytes from healthy donors were used. The release of TNFalpha and IL-18 during infection of cells with different species of Leishmania "in vitro" was evaluated. L. donovani induced in both PBMC and monocytes significantly more TNFalpha and IL-18 with respect to L. major. The amounts of TNFalpha released by PBMC were always significantly higher than those released by monocytes of the same donors.

Treatment of PBMC with killed Helicobacter pylori subverts the environment of inflammatory cytokines.

It is well known that inflammation induced by Helicobacter pylori is characterized by the local production of cytokines and chemokines. In the present study, we analyse the kinetics of MCP-1, IL-12 and IL-4 induction during the interaction of peripheral blood mononuclear cells with killed and/or live H. pylori. Our results demonstrate that live H. pylori does not induce IL-4 release whereas it stimulates MCP-1 and IL-12 production. In addition, the neutralization of IL-12 with monoclonal antibodies determines a lower MCP-1 release. These data demonstrate that MCP-1 production is in part supported by IL-12 induced by live H. pylori. On the contrary, killed H. pylori stimulates the IL-4 but not MCP-1 and IL-12 production. The combined treatment with killed and live H. pylori upregulates the IL-4 production and at the same time downregulates IL-12 and MCP-1 production.

Modulatory effect of HHV-6 on MCP-1 production by human monocytes.

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells.

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.

Role of interleukin-18 in peripheral blood mononuclear cells infected with human herpes virus type 6.

OBJECTIVE: Interleukin 18 (IL-18) production represents a critical step in the polarization of the Th1 immune response. Human herpes virus type 6 (HHV-6) possesses a peculiar tropism for immunocompetent cells. To understand the relationships among immunocompetent cells, HHV-6 and cytokines, the role of IL-18 during infection of peripheral blood mononuclear cells (PBMC) with HHV-6 was evaluated. METHODS: PBMC were obtained from healthy HHV-6-seronegative donors, after centrifugation of heparinized venous blood over a Ficoll-Hypaque gradient. Supernatants from PBMC were analyzed for the presence of cytokines. To study the effects of exogenous recombinant human (rh) IL-18 on HHV-6 replication, the number of cells expressing viral antigens and the amount of extracellular virus were analysed. RESULTS: No basal production of IL-18 was found in supernatants of unstimulated PBMC. Appreciable amounts of the cytokine were produced by lipopolysaccharide (LPS)-stimulated PBMC. HHV-6 infection of LPS-treated PBMC downregulated IL-18 production. It was found that the addition of rhIL-18 to HHV-6-infected PBMC downregulated the percentage of antigen-positive cells and the release of extracellular virus. CONCLUSION: Impairment of IL-18 release, which is involved in the induction of antiviral cytokines, such as interferon-gamma, could represent a strategy of the virus to evade the immune response of the host.

Role of IL-15 on monocytic resistance to human herpesvirus 6 infection.

Interleukin-15 (IL-15) is a cytokine that possesses a variety of biological functions, including stimulation and maintenance of cellular immune responses. Recently, it has been demonstrated that Human Herpes virus type 6 (HHV-6) enhances NK activity of human PBMC by inducing IL-15. HHV-6 is a typical immunosuppressive agent, as suggested by its tropism for both CD4+ and CD8+ T cells, B cells, monocytes/macrophages, megakaryocytes and NK cells. Moreover, several studies have indicated that mononuclear phagocyte resistance to virus infection is influenced by the cellular differentiation state. This paper describes the effect of pretreatment "in vitro" with IL-15 on the resistance of human monocytes (HM) to HHV-6 infection. Our results demonstrate that undifferentiated HM were highly resistant to HHV-6 infection, whereas HM pretreated with human recombinant IL-15 showed an increased permissiveness for HHV-6 infection. This permissiveness was characterised by higher release of extracellular virus as well as an increased percentage of antigen positive cells. Moreover, we evaluated IL-15 production after the addition of HHV-6 to monocytes precultured in different experimental conditions. Our data indicate that HHV-6-induced IL-15 production by human monocytes is not affected by the condition of "in vitro" precultivation/differentiation. Furthermore, the neutralization of IL-15 induced by HHV-6 in differentiated monocytes did not affect viral replication. These findings suggest that IL-15 acts only on the mechanisms of cellular differentiation, rendering HM more susceptible to HHV-6 infection, without interfering with virus replication.

Altered cytokine production after human herpes virus type 6 infection.

Several strategies allow viruses to elude the surveillance of the immune system and to establish persistent infection in the host. One of such mechanisms is the immunosuppression caused by the direct infection and functional impairment of immune cells. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism for both CD4+ and CD8+ T cells, B cells, monocytes/macrophages, megakaryocytes and NK cells. In this study the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMC) was evaluated during HHV-6 infection "in vitro". Our results demonstrate that HHV-6 up-regulates IL-10 production by PBMC. Furthermore, our data suggest that rhIFN gamma addition counteracts the effect of HHV-6 in promoting IL-10 release. To gain more insight into the role of IFN gamma, anti-IFN gamma monoclonal antibodies were added to PBMC stimulated with LPS. Neutralization of endogenous IFN gamma upregulated IL-10 release. Furthermore, HHV-6 infection inhibited IFN gamma release induced by LPS in PBMC. No basal production of IL-12 was found in PBMC. Moreover, HHV-6 infection did not induce IL-12 release by PBMC. On the contrary, IL-12 was detected in the supernatants of PBMC treated with LPS with or without rhIFN gamma. In these experimental conditions the further addition of HHV-6 markedly impaired IL-12 production. Moreover, the neutralization of IL-10 resulted in a significant up-regulation of IL-12. Finally our data suggest that the immunodysregulation induced by HHV-6 could be accounted for by a shift from a Th-1 to a Th-2 type cytokine profile.

Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major.

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.

Production of TNF alpha and interleukin 6 by differentiated U937 cells infected with Leishmania major.

Several cytokines play a crucial role in the defense of the host against protozoa belonging to the genus Leishmania. However, the role of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) in human leishmaniasis is still controversial. The aim of this work was to study, in an "in vitro" model, the interactions of human phagocytes with L. major. The U937 human monocytic cell line, differentiated with phorbol myristate acetate (PMA) or a combination of 1 alpha,25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), was used in all the experiments. The rate of infection, as well as the production of TNF alpha and IL-6 by cells upon infection with promastigotes, were studied. It was found that, depending on the agent used for differentiation, U937 cells produced different patterns of cytokines. PMA differentiated cells produced significantly more TNF alpha, but less IL-6 than cells differentiated with VD-RA. No direct relationship was found between the ability of differentiated U937 cells to release TNF alpha or IL-6 and their leishmanicidal activity.

Induction of interleukin 1 alpha in murine macrophages infected in vitro with different species and strains of Leishmania.

It is now generally agreed that several cytokines released by immunocompetent cells such as macrophages play a crucial role in the outcome of infections caused by protozoa belonging to the genus Leishmania. In particular, tumor necrosis factor (TNF) induction during the course of cutaneous leishmaniasis has been related to resistance to L. major infection in mice. However, the role played by interleukin 1 (IL-1) in the host response to leishmaniasis has yet to be completely elucidated. The aim of this work was to study whether different species and strains of Leishmania could induce IL-1 alpha in murine macrophages in vitro. Resident peritoneal macrophages of BALB/c and C3H/HeN mice were infected with L. donovani, L. major, or different strains of L. infantum. It was found that L. donovani did not induce IL-1 alpha in macrophages from either mice strain. Infection with L. major or with three out of six strains of L. infantum induced consistent amounts of IL-1 alpha, but only in macrophages from genetically resistant C3H/HeN mice. No relationship was found between the rate of infection of macrophages and the amount of IL-1 alpha detected in the supernatants of infected macrophages. Data obtained confirm that the release of IL-1 alpha by murine macrophages infected in vitro with Leishmania is influenced by the genetic background of the cells as well as by the parasite species.

Induction of tumor necrosis factor alpha by Leishmania infantum in murine macrophages from different inbred mice strains.

The present study was undertaken to determine whether the viscerotropic species, Leishmania infantum, endemic in Italy, could induce tumor necrosis factor alpha (TNF alpha) in murine macrophages. Genetically susceptible (Lshs) and resistant (Lshr) mice were used in the attempt to correlate TNF alpha production with the ability to control parasite growth and replication. Resident peritoneal macrophages of C3H/HeN, DBA/2, CBA (Lshr), C57BL/10 and BALB/c (Lshs) mice were infected in vitro with promastigotes at a parasite to cell ratio of 8:1. No significant differences in the percentages of infected peritoneal cells of Lshs versus Lshr mice were observed until 72 h of in vitro culture. On the contrary, Kupffer cells from Lshr mice inhibited Leishmania replication. Peritoneal macrophages of resistant mice produced significantly higher amounts of TNF alpha as compared to susceptible mice. TNF alpha production of both resistant and susceptible mice peaked at about 5 h after the challenge with the parasite. No TNF alpha was found in supernatants of infected Kupffer cells from all the strains tested. The ability of macrophages from susceptible or resistant mice strains to produce TNF alpha after challenge with Leishmania infantum does not seem related to their capacity to control parasite replication in vitro.

Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenomenon induced by Salmonella minnesota R595 lipopolysaccharide.

Endotoxin-neutralizing activity may be an important property for antibiotics to be used in severe sepsis. Several antibiotics, belonging to different classes, were evaluated as to their endotoxin-neutralizing ability, using the inhibition of an in vitro metachromatic assay for lipopolysaccharides and a murine generalized Shwartzman reaction model. Gentamicin, amikacin, and sisomicin have been found to share significant in vitro antiendotoxin activity at an antibiotic/endotoxin ratio as low as 1.0/5 (by weight) and to reduce the murine generalized Shwartzman reaction at an antibiotic/endotoxin ratio of 3.3/5.

Role of exogenous interferons on intrinsic antiviral activity of macrophages from patients affected by neoplasia.

Macrophages derived from in vitro cultured monocytes were infected with herpes simplex virus type 2. A marked impairment in the intrinsic antiviral activity was found in macrophages obtained from patients with breast cancer or melanoma. Moreover, the antiviral activity of macrophages from healthy donors, differentiated in serum from patients with neoplasia, was also impaired. The aim of this work was the evaluation of alpha, beta, gamma exogenous interferon in restoring the intrinsic antiviral activity of macrophages from patients affected by breast cancer or melanoma under different conditions. Pretreatment of macrophages with alpha, beta interferons, but not gamma interferon, restored their impaired intrinsic antiviral activity.

Different effects of bacterial lipopolysaccharide on superoxide anion production by macrophages from normal and tumor-bearing rats.

Bacterial endotoxins or lipopolysaccharides (LPS) exhibit a wide range of modulatory activities on immunocompetent cells. Among the numerous effects of LPS on macrophages, an enhancement of superoxide anion (O2-) release has been reported. In previous studies carried out on tumor-bearing rats, it was found that several functions of peritoneal macrophages such as phagocytic, microbicidal and antiviral activities were depressed. In this paper we evaluated the spontaneous or phorbol myristate acetate (PMA)-induced production of superoxide anion by macrophages from tumor-bearing rats with respect to controls. Moreover, the effect of in vitro priming with LPS on O2- production by the same cells was studied. It was found that the pattern of superoxide release by macrophages from tumor-bearing rats is significantly different from controls. Preincubation of macrophages from normal rats with LPS enhanced the spontaneous and PMA-induced production of O2-. In contrast, the same concentrations of LPS did not prime macrophages from tumor-bearing rats.

Influence of sera from patients affected by neoplasia on some human macrophage functions.

It has been repeatedly reported that several functions of mononuclear cells are impaired in patients affected by neoplasia. Moreover, inhibitory activity of serum and tumor extracts on macrophages have been described. In a previous study, we found a marked impairment of the intrinsic antiviral activity of macrophages derived from monocytes isolated from peripheral blood of patients with breast carcinoma or melanoma compared with that from blood of normal subjects. The aim of the present work was to study whether this impairment was due to circulating inhibitory factors. Macrophages were differentiated in vitro in sera from patients with neoplasia and in sera from healthy donors and then challenged with herpes simplex virus type 2 (HSV-2). Macrophages from normal subjects, incubated with sera from patients, were significantly impaired in their intrinsic antiviral activity. These results support the possibility that circulating inhibitory factors influence the functionality of mononuclear phagocytes in the tumor-bearing host.

Evaluation of macrophage antiviral activity in patients affected by neoplasia.

The intrinsic antiviral activity of macrophages has been studied in healthy donors and in patients affected by breast cancer and melanoma. In vitro differentiated macrophages from blood-derived monocytes were infected with measles virus, herpes simplex virus type 2 and adenovirus 17. The challenge was carried out with different multiplicities of infection and the synthesis of virus was tested by evaluating the single cycle growth curve in 24 h. The results obtained show that the restriction of virus infectivity by macrophages is strongly influenced by the multiplicity of infection. This was particularly evident with the adenovirus 17. Moreover, macrophages from patients with melanoma and breast cancer showed an impairment of the intrinsic antiviral activity in comparison with normal subjects.

Impairment of macrophage antiviral activity by soluble tumor products. Effects of bacterial immunomodulators.

The antiviral activities of normal rat peritoneal macrophages versus Herpes Simplex Virus type 1 were inhibited by sera from tumor-bearing rats and 3M KCl extracts of tumor mass. The inhibitory activity was demonstrated on the extrinsic as well as on the intrinsic macrophage functions. Sera from Corynebacterium parvum and Listeria monocytogenes treated tumor bearing rats did not inhibit these macrophages functions. Furthermore the 3 M KCl extracts from the tumor mass of the above treated TBR show a decrease in the capability to impair these macrophage functions. On the other hand, the treatment with the oral polyvalent adjuvant "Buccalin" was not able to restore the compromised antiviral activity in tumor bearing rats.

Spontaneous and induced interferon production by peripheral blood leucocytes from control population and patients with herpes genitalis.

The spontaneous and PHA induced levels of interferon were measured in peripheral blood leucocyte cultures of twenty-eight individuals diagnosed as positive for herpes genitalis, and in a group of control subjects. As reported by Cunningham and Merigan [1983] for herpes labialis, leucocytes from individuals with herpes genitalis produced low levels of interferon spontaneously; however, similar results were found for individuals within the control population. No statistically significant difference could be found for PHA induced interferon levels, antigen induced interferon levels, or helper/suppressor cell ratios between the herpes genitalis population and control population. Our results indicate that interferon does not play a major role in the latency or recurrence of herpes genitalis.