Compensation of Cyclophosphamide Immunosuppression by a Bacterial Immunostimulant (Broncho-Vaxom) in Mice

Abstract

The compensatory effect of a bacterial lysate, Broncho-Vaxom (BV) on the immunosuppressive action of cyclophosphamide (CY) was investigated. In CY immunosuppressed mice, BV treated animals recovered to normal levels of IgM and IgG in serum as well of IgA and IgG in gut secretions significantly earlier than controls. Furthermore, normal cell proliferation in thymus, as estimated by measuring the relative size of this organ was achieved earlier in BV treated mice than in control mice. Oral treatment with BV restores the number of IgM anti SRBC producing cells in spleen, in CY immunosuppressed mice. Since immunosuppression induced by CY increases the susceptibility to various infections, we tested in immunosuppressed animals the protective effect of BV towards IP challenge infections with Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae var ozaenae, Pseudomonas aeruginosa and Candida albicans. BV led to an enhanced resistance towards both pneumococci and staphylococci challenge infections but not to the other challenge microorganisms.     

Compensation of Cyclophosphamide Immunosuppression by a Bacterial Immunostimulant (Broncho-Vaxom) in Mice

The compensatory effect of a bacterial lysate, Broncho-Vaxom (BV) on the immunosuppressive action of cyclophosphamide (CY) was investigated. In CY immunosuppressed mice, BV treated animals recovered to normal levels of IgM and IgG in serum as well of IgA and IgG in gut secretions significantly earlier than controls. Furthermore, normal cell proliferation in thymus, as estimated by measuring the relative size of this organ was achieved earlier in BV treated mice than in control mice. Oral treatment with BV restores the number of IgM anti SRBC producing cells in spleen, in CY immunosuppressed mice. Since immunosuppression induced by CY increases the susceptibility to various infections, we tested in immunosuppressed animals the protective effect of BV towards IP challenge infections with Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae var ozaenae, Pseudomonas aeruginosa and Candida albicans. BV led to an enhanced resistance towards both pneumococci and staphylococci challenge infections but not to the other challenge microorganisms.     

Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues

Lipoprotein from the outer membrane of Escherichia coli and its synthetically prepared N-terminal lipopeptide segments Pam3Cys-Ser-Ser-Asn-Ala and Pam3Cys-Ser, as well as lipoprotein from other Enterobacteriaceae, constitute potent polyclonal B lymphocyte activators. Here, we demonstrate that these compounds were also able to stimulate human and murine leukocytes: in murine macrophages, we could show the induction of interleukin 1 release by the mitogens, as measured in the thymocyte proliferation assay. Moreover, murine peritoneal exudate cells were stimulated to secrete prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). The effect of Pam3Cys-Ser on the murine macrophage cell line P388D1 was also tested: the compound induced an increase in proliferation, as measured by a thymidine incorporation assay. In addition, the cell line could be induced to release IL 1 into the supernatant. Correspondingly, induction of IL 1 release could also be demonstrated in human mononuclear cells. Our results demonstrate that the two novel synthetic lipopeptides are potent stimulators for human monocytes and murine macrophages. These findings may be important for the elucidation of the role of these bacterial surface components in the course of bacterial infections.     

The actions of bacterial DNA on murine macrophages.

Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides ("CpG" motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor alpha. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colony-stimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism.     

p-Cresol inhibits IL-12 production by murine macrophages stimulated with bacterial immunostimulant

p-Cresol, an end product of aromatic amino acids, is produced from food proteins by intestinal bacteria, and is detectable in blood and feces. Especially, blood and fecal levels of p-cresol are high in chronic renal failure (CRF) patients. Although it has been suggested that p-cresol is toxic in the body, the effect of p-cresol on immune responses has not yet been clarified. In this study, we investigated the effect of p-cresol on IL-12 production of macrophages stimulated with Lactobacillus casei strain Shirota (LcS) in vitro. Pre-incubation with p-cresol inhibited IL-12 p40 production of LcS-stimulated J774.1 cells, a murine macrophage-like cell line, in a dose-dependent manner. IL-12 p40 and p70 production of LcS-stimulated murine peritoneal macrophages was also inhibited by p-cresol. The inhibitory effect was not dependent on the cytotoxicity of p-cresol. These results indicate that blood and fecal p-cresol may have adverse effects on the host defense system in CRF patients.     

Immunomodulation by Blastomyces dermatitidis: functional activity of murine peritoneal macrophages.

Cell-mediated immunity plays the dominant role in the immune response of mice to Blastomyces dermatitidis infections. Since macrophages play an important role in cell-mediated immunity, the interactions between sensitized murine peritoneal macrophages and the yeast phase of B. dermatitidis were investigated. Scanning electron microscopy showed that the sensitized macrophages readily phagocytized B. dermatitidis yeast cells. In addition, there appeared to be activation of metabolic pathways within the sensitized macrophages, as indicated by increased chemiluminescence activity during phagocytosis. Sensitized macrophages were significantly better at controlling intracellular proliferation of the yeast cells when compared to nonsensitized cells. This was determined by disruption of macrophages and plating for viable yeasts. Scanning electron microscope observations offered further substantiation. Experiments with Candida albicans indicated that B. dermatitidis non-specifically activated macrophages. At 2 h postphagocytosis, 30% fewer C. albicans in B. dermatitidis-activated macrophages were able to form germ tubes. These studies demonstrated the multiple potential of activated macrophages with regard to their functional activity.     

低剂量纳曲酮对小鼠巨噬细胞的免疫调节作用及机理研究

目的:探讨纳曲酮(Naltrexone,NTX)对小鼠腹膜巨噬细胞表型,分泌细胞因子及吞噬功能的影响。方法:本实验利用新型四唑化合物比色法测得NTX 对RAW264郾7 细胞作用的最佳剂量;再将培养的腹膜巨噬细胞分为3 组,RPMI1640 空白对照组、脂多糖(LPS)阳性对照组和NTX 处理组,采用流式细胞术检测CD206、CD64 的阳性表达率及对葡聚糖的吞噬能力;ELISA 法检测白介素10(IL鄄10)、白介素6(IL-6)、白介素1茁(IL-1)、肿瘤坏死因子 (TNF )的分泌表达。结果:10-11 mol/ L 剂量可显著促进RAW264-7 细胞的增殖;NTX(10-11 mol/ L)处理可明显提高腹膜巨噬细胞表面CD64 的阳性表达,减少CD206 的表达;提高对葡聚糖的吞噬能力;TNF、IL-6、IL1的表达显著增加,IL-10 的表达无明显差异。结论:低剂量纳曲酮可改变巨噬细胞表型及功能,起免疫调节作用。     

Morphological and functional changes during cytomegalovirus replication in murine macrophages

Structural and functional changes were studied in murine peritoneal macrophages infected with murine cytomegalovirus by using centrifugal enhancement to achieve a high-level (greater than 90%) pulsed infection. During 3 d of culture the infected cells became enlarged and rounded with smooth surface contours. Transmission electron microscopy demonstrated various stages of viral maturation in the nucleus and cytoplasm. Intracellular organization was generally retained, apart from the development of large, irregular, intracytoplasmic vacuoles, in which enveloped virions and cell debris accumulated. The infected macrophages lost most surface markers tested (F4/80, Mac-1, FcR, and the receptor for gluteraldehyde-fixed sheep red blood cells), but H-2 expression was increased. Moreover, ingestion of colloidal gold or horseradish peroxidase was depressed, and the levels of acid phosphatase activity, lymphocytostatic activity, and interleukin 1 production were also decreased. The latter may explain the observed loss of accessory cell function.     

Induction of interleukin 1 secretion by murine macrophages and human monocytes after stimulation by RU 41740, a bacterial immunomodulator

RU 41740, a glycoprotein extract from K. pneumoniae K2O1 strain, is an immunomodulating compound which has been shown to reduce infectious episodes in patients prone to recurrent infections. Data from preliminary experiments suggest that RU 41740 may affect several target cells, including T cells, B cells or macrophages. In the present report we show that RU 41740 can trigger mouse macrophages and human adherent mononuclear cells to produce interleukin 1 activity. Indeed, supernatants from mouse peritoneal adherent cells and human monocytes incubated in presence of RU 41740, can stimulate blastogenesis in thymocytes from C3H/HeJ mice. The data suggest that the immunomodulating effect of RU 41740 could be related to its ability to induce interleukin 1 production.     

Stimulation of the ceramide pathway partially mimics lipopolysaccharide-induced responses in murine peritoneal macrophages.

Recent studies have suggested that lipolysaccharide (LPS) stimulates cells by mimicking the second-messenger function of ceramide, a lipid generated in the cell by the action of sphingomyelinase (SMase). To examine this possibility further, we compared the abilities of LPS, SMase, and/or ceramide analogs to induce cytokine secretion, modulate gene expression, and induce endotoxin tolerance in macrophages. SMase and LPS induced secretion of tumor necrosis factor alpha (TNF-alpha) to comparable degrees; however, unlike LPS, SMase failed to stimulate detectable interferon activity. Cell-permeable analogs of ceramide induced the expression of many LPS-inducible genes; however, the expression of interferon-inducible protein 10 (IP-10) and interferon consensus sequence-binding protein (ICSBP) mRNAs was significantly lower than that induced by LPS. Both SMase-induced TNF-alpha secretion and LPS-induced TNF-alpha secretion were inhibited by pretreatment with a serine/threonine phosphatase inhibitor, calyculin A. Macrophages preexposed in vitro to LPS to induce a well-characterized state of endotoxin tolerance secreted little or no TNF-alpha upon secondary challenge with either LPS or SMase, whereas macrophages preexposed to SMase secreted high levels of TNF-alpha upon secondary stimulation with LPS or SMase. Collectively, these results suggest that ceramide activates a subset of LPS-induced signaling pathways in murine peritoneal exudate macrophages.     

Differential regulation of type IV collagenases and metalloelastase in murine macrophages by the synthetic bacterial lipopeptide JBT 3002

We determined whether the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) in murine macrophages is regulated by the novel synthetic bacterial lipopeptide JBT 3002. Multilamellar liposomes (MLV) encapsulating JBT 3002 (MLV-JBT 3002) stimulated the production of 72-kDa and 92-kDa (gelatinase A and B) type IV collagenase and inhibited the production of murine metalloelastase (MME) in a dose-dependent manner in murine peritoneal macrophages. MLV-JBT 3002 also induced production of TIMP-1. MLV-JBT 3002 did not induce collagenase production in tumor cells. Priming murine macrophages with interferon-gamma (IFN-gamma) inhibited JBT 3002-stimulated production of both MMP-9 and MMP-2 and further inhibited production of MME by a mechanism involving nitric oxide (NO). This conclusion is based on data showing that IFN-gamma failed to inhibit production of MMP in the presence of L-methyl arginine or in macrophages from inducible nitric oxide synthase knockout mice. These data suggest that JBT 3002 differentially regulates the production of various MMPs and TIMP in macrophages.     

Anticancer and anti-inflammatory activities of shallot (Allium ascalonicum) extract

Introduction

Alliumplants are an important part of the diet of many populations and there is a long-held belief in their health-enhancing properties such as cancer prevention. In this study, the anticancer and anti-inflammatory activities of the aqueous extract of the Allium ascalonicum bulbs have been studied.

Material and methods

The antiproliferative and anti-growth activity of the aqueous extract of A. ascalonicum was examined in vitro on different tumor cell lines. Furthermore, the acetic acid-induced vascular permeability as an in vivo assay was used for studying anti-inflammatory activity of the extract.

Results

The aqueous extract of A. ascalonicum had the most anti-growth activity on the cancer cell lines; Jurkat and K562 against Wehi 164 with lower cytotoxic preference. The extract also showed much less cytotoxicity against the normal cell (HUVEC) line and significant anti-inflammatory activity in vivo.

Conclusions

It is of interest that the extract of this plant has shown much less cytotoxicity against the normal cell line, and, if this also occurs in vivo, the use of this plant clinically for the treatment of cancer patients would have some scientific support. The results of these assays indicated that A. ascalonicum can be a candidate for prevention and treatment of many diseases related to inflammation and malignancy.     

Stimulation of the trypanocidal and endoribonuclease activities by the interferon induced (2'-5') oligoadenylates

Interferon or 2'-5' oligoadenylates (2'-5' An) activated the microbicidal activity of primary cultures of rat glia cells and of the mouse macrophage transformed cell line J774 against infection by Trypanosoma cruzi. Pretreatment with gamma-interferon (gamma-IFN) or 2'-5' A3 of rat glia cells or direct addition of these compounds during the incubation with the parasite enhanced the uptake of metacyclic trypanosomes by the cells. Furthermore, glia cells treated with gamma-IFN or 2'-5' A3 were able to restrict the growth and to eventually destroy intracellular amastigotes. Bacterial lipopolysaccharide (LPS) synergized with gamma-IFN as well as with 2'-5' A3 and 2'-5' A4, but not with dephosphorylated 'core' molecules or ATP, to induce a partial trypanocidal activity in J774 cells. In addition, those treatments with gamma-IFN or 2'-5' A3 activated to a similar extent an endoribonuclease, which degraded ribosomal RNA, in rat glia cells, suggesting a role of this enzyme in the mechanism of the trypanocidal activity of gamma-IFN.     

Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide

RON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.     

Effect of tuftsin on the functional activity and intracellular pH of murine peritoneal macrophages

The effect of tuftsin and its tripeptide analog in various concentrations (from 0.001 to 10.0 μg/ml) on phagocytosis and on the intracellular pH is studied in murine peritoneal macrophages. Tuftsin causes a uniform dose-dependent increase of these two parameters in the cells. This effect is maximally pronounced at concentrations of the peptide close to its physiological level (about 0.3 μg/ml) and gradually decreases as its content in the incubation medium is lowered or raised. On the other hand, the tripeptide analog of tuftsin does not exhibit such an effect on the cells and under the same conditions suppresses phagocytosis and acidifies their intracellular medium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 3, pp. 265–267, March 1994 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Direct activation of human monocyte-derived macrophages by a bacterial glycoprotein extract inhibits the intracellular multiplication of virulent Legionella pneumophila serogroup 1.

Intracellular multiplication of virulent Legionella pneumophila serogroup 1 was inhibited by human monocyte-derived macrophages activated by a glycoprotein extract of Klebsiella pneumoniae, RU 41.740. Macrophage cultures were infected with L. pneumophila in the presence of immune antibodies on day 7 of culture. Extracellular bacteria were removed an hour after infection, and various concentrations of RU 41.740 or an antibiotic, erythromycin, were added. Intracellular multiplication in the presence of RU 41.740 was significantly slowed down compared with that of cultures without RU 41.740. The reduction was, however, significantly less than that effected by erythromycin, which was used as a positive control for inhibition of intracellular multiplication. Cultures incubated with RU 41.740 before infection also demonstrated a significant reduction in the intracellular multiplication of L. pneumophila. In addition, RU 41.740 increased superoxide anion production from human monocytes in suspension in the presence of L. pneumophila. These results show that direct nonspecific activation of macrophages by a bacterial glycoprotein inhibits the intracellular multiplication of L. pneumophila and may suggest a role for activated macrophages in host defense against intracellular pathogens.     

Antimicrobial activities of the methanol extract and compounds from Artocarpus communis (Moraceae)

Background

Artocarpus communis is used traditionally in Cameroon to treat several ailments, including infectious and associated diseases. This work was therefore designed to investigate the antimicrobial activities of the methanol extract (ACB) and compounds isolated from the bark of this plant, namely peruvianursenyl acetate C (1), α-amyrenol or viminalol (2), artonin E (4) and 2-[(3,5-dihydroxy)-(Z)-4-(3-methylbut-1-enyl)phenyl]benzofuran-6-ol (5).

Methods

The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against seven bacterial and one fungal species.

Results

The MIC results indicated that ACB as well as compounds 4 and 5 were able to prevent the growth of all tested microbial species. All other compounds showed selective activities. The lowest MIC value of 64 μg/ml for the crude extract was recorded on Staphylococcus aureus ATCC 25922 and Escherichia coli ATCC 8739. The corresponding value of 32 μg/ml was recorded with compounds 4 and 5 on Pseudomonas aeruginosa PA01 and compound 5 on E. coli ATCC 8739, their inhibition effect on P. aeruginosa PA01 being more than that of chloramphenicol used as reference antibiotic.

Conclusion

The overall results of this study provided supportive data for the use of A. communis as well as some of its constituents for the treatment of infections associated with the studied microorganisms.     

Candida albicans suppresses nitric oxide (NO) production by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages

We examined the in vitro effect of Candida albicans on NO production by macrophages. Candida albicans suppressed not only NO production but also expression of inducible NO synthase (iNOS) mRNA by murine IFN-gamma and bacterial LPS-stimulated peritoneal macrophages. The suppression was not associated with inhibition but rather stimulation of IL-1 beta production. This effect was observed when more than 1 x 10(3)/ml of Candida albicans were added to macrophage cultures (1 x 10(6) cells/ml) and reached a maximal level at 1 x 10(6)/ml. The NO inhibitory effect of Candida albicans was mediated predominantly by as yet unidentified soluble factor(s) and to a lesser extent by direct contact. In addition, heat- or paraformaldehyde-killed Candida albicans did not show this inhibitory activity. Culture supernatant of Candida albicans also inhibited NO production by activated macrophages in a dose-dependent manner, and increased IL-1 beta production. Finally, the inhibitory effect was not mediated by IL-10 and transforming growth factor-beta (TGF-beta), since neutralizing antibodies to these cytokines did not influence Candida albicans-induced reduction in macrophage NO production. Our results suggest that Candida albicans may evade host defence mechanism(s) through a soluble factor-mediated suppression of NO production by stimulated macrophages, and that the effect is independent of production of immunosuppressive cytokines such as IL-10 and TGF-beta.