Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence ...and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guérin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-α and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.
One-third of all Trypanosoma cruzi -infected patients eventually develop chronic Chagas' disease cardiomyopathy (CCC), a particularly lethal inflammatory dilated cardiomyopathy, where parasites are ...scarce and heart-infiltrating mononuclear cells seem to be the effectors of tissue damage. Since T. cruzi is a major inducer of interleukin-12 production, the role of inflammatory cytokines in the pathogenesis of CCC was investigated. We assayed cytokine production by peripheral blood mononuclear cells (PBMC) from CCC and asymptomatic T. cruzi -infected (ASY) individuals, as well as by T cell lines from endomyocardial biopsies from CCC patients. PBMC from CCC and ASY patients produced higher IFN-γ levels than normal (N) individuals in response to B13 protein and phytohaemagglutinin PHA; IFN-γ high responders (≥1ng/ml) were 2–3 fold more frequent among CCC patients than ASY individuals. Conversely, IL-4 production in response to the same stimuli was suppressed among T. cruzi -infected patients. The frequency of PHA-induced IFN γproducing cells on PBMC was significantly higher among CCC than ASY and N individuals. IFN-γ and TNF-α were produced by ten out of ten PHAstimulated T cell lines from CCC patients; IL-2 and IL-10 were produced by four out of ten and one out of ten lines, respectively; IL-4, IL-1α, IL-1β, IL-6 and IL-12 were undetectable. Our results suggest that CCC and ASY patients may respond differentially to the IFN-γ-inducing stimulus provided by T. cruzi infection. Given the T1-type cytokine profile of heart-infiltrating T cell lines from CCC patients, the ability to mount a vigorous IFN-γ response may play a role on the differential susceptibility to CCC development.
The present study investigated the effects of the anthraquinone derivative (O,O′-bis-(3′-iodopropyl)-1,4-dihidroxyanthraquinone — DIPDHAQ), mitoxantrone analog, in an experimental autoimmune ...encephalomyelitis (EAE) model. The results showed that DIPDHAQ treatment improved the clinical signs of the disease (n=10; vehicle: 3.8±0.3; DIPDHAQ: 1.4±0.9). The improvement was associated with a decrease of inflammatory cells, demyelination, IL-17, IFN-γ, IL-12p40, IL-6, TGF-β, CCL5 and CCL20 levels in the spinal cord. DIPDHAQ presented a low cytotoxicity when in vitro assays were performed. Therefore, the findings suggest a major role for DIPDHAQ in multiple sclerosis, disease characterized as an autoimmune inflammatory disorder against myelin proteins of the brain and spinal cord. The attenuation of inflammation and consequently improvement of clinical signs, involving a decrease of pro-inflammatory cytokines and the low cytotoxicity of DIPDHAQ, suggest that this compound could be used as an alternative treatment for autoimmune diseases in the central nervous system.
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►EAE is a murine autoimmune disease used to study multiple sclerosis. ►The use of a mitoxantrone analog (DIPDHAQ) in the treatment of EAE was investigated. ►DIPDHAQ treatment caused reduction of the EAE clinical signs. ►DIPDHAQ reduces infiltrated cells and demyelination in the spinal cords. ►DIPDHAQ reduces CCL5, CCL20, IL-6, IL-17, IL-12 and IFN-γ in the spinal cords.
Abstract Thalidomide is used to treat a variety of diseases including erythema nodosum leprosum, an inflammatory complication of leprosy. However, this drug has severe teratogenic activity and novel ...thalidomide analogues might be used to treat diseases without this severe side effect. A series of diamine compounds containing two hydrolyzed phthalimide units were chosen as analogues of thalidomide and evaluated regarding their capacity to regulate the production of molecules involved in inflammatory responses. TNF-α, IL-12 and IL-10 production, and the expression of CD80 and CD86 were investigated in LPS plus IFN-γ-stimulated J774A.1 cells by ELISA and flow cytometry, respectively. The expression of TNF-α and IL-10 mRNA was analyzed by real time RT-PCR. TNF-α, IL-6, IFN-γ, CXCL9 and CXCL10 production by human peripheral blood mononuclear cells (PBMC) were evaluated by flow cytometry. Compounds 3 , 6 and 9 greatly inhibited TNF-α and IL-12 production while enhancing IL-10. In addition, CD80 expression was inhibited, but not CD86. The compounds inhibited TNF-α production by PBMC more than thalidomide and also had an inhibitory effect on the production of IL-6, IFN-γ, CXCL9 and CXCL10. Levels of mRNA for TNF-α were reduced after treatment with the compounds, suggesting post- transcriptional effects. The compounds had no effect on cell viability. Our results indicate that the novel diamine compounds 3 , 6 and 9 inhibit critical pro-inflammatory cytokines and stimulate IL-10, which make them attractive candidate drugs for the treatment of certain inflammatory conditions and cancer.
IFN-γ responses to
Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of
M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been ...shown to be expressed by IFN-γ stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-γ, measuring MIG may provide an interesting marker to assess downstream IFN-γ induced responses, in contrast to assays that mainly focus on quantifying production of IFN-γ per se. We, therefore, investigated MIG and IFN-γ responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of
M. tuberculosis, in 29 BCG vaccinee controls and 24 TB patients. IFN-γ secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-γ secreting cells (
r
=
0.55,
P
<
0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-γ and MIG following stimulation with ESAT-6/CFP-10 or PPD (
P
<
0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccinees (
P
<
0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-γ production induced by
M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-γ in TB.
In this study, we have identified a secreted 13 kDa lectin from Mtb (Mtb, Mycobacterium tuberculosis; sMTL-13) by homology search of a non-redundant lectin database. Bioinformatic analysis revealed ...that sMTL-13 belongs to the ricin-type β-trefoil family of proteins containing a Sec-type signal peptide present in Mtb complex species, but not in non-tuberculous mycobacteria. Following heterologous expression of sMTL-13 and generation of an mAb (clone 276.B7/IgG1κ), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non-tuberculous mycobacteria-derived culture filtrate proteins. In addition, sMTL-13 leads to an increased IFN-γ production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab against sMTL-13, a response found to be decreased following successful anti-tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin-like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment.
Genistein modulates inflammatory responses in part by reducing the production of the pro‐inflammatory cytokines IL‐12, TNF‐α, and nitric oxide, by activated macrophages in response to ...lipopolysaccharide stimulus. Previous studies have shown that synthetic lipophilic genistein glycosides were significantly more active than hydrophilic glycosides. The aims of this study were to synthesize and to evaluate the effect of novel lipophilic genistein derivatives on IL‐12, TNF‐α, and nitric oxide production by J774A.1 cells. The results show that the modification of genistein enables the generation of non‐cytotoxic compounds with increased IL‐12 inhibition. However, these derivatives failed to inhibit TNF‐α. The nitric oxide production was notably inhibited by the monoester (2, 3) and monoether (6, 7) compounds in a dose‐dependent manner.
This work describes the synthesis and evaluation of the effect of novel lipophilic genistein derivatives on IL‐12, TNF‐α and NO production by J774A.1 cells. The results show that the modification of genistein enables the generation of non‐cytotoxic compounds with increased IL‐12 inhibition. The NO production was notably inhibited by the monoester (2, 3) and monoether (6, 7) compounds in a dose‐dependent manner.
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