Infection Of Peritoneal Macrophages Research Articles

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection.

BackgroundReactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date.MethodsIn this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/−) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production.ResultsIn vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils.ConclusionROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Interleukin-6: a potential biomarker of the success of tuberculosis treatment

Background: The present measure of the success of TB treatment is fraught with problems. A cytokine biomarker(s), a qualitative and quantitative reflection of the treatment success, is thus warranted. Because TB treatment is expected to affect macrophage and mycobacteria interactions and, consequently cytokine(s) elaboration, a biomarker(s) seems crucial to assess its success. Methods & Materials: We studied the effect of anti-TB drugs isoniazid (INH), rifampicin (RIF), streptomycin (STP) and ethambutol (EMB) on IL-1β, IL-6, IL-10, IL-12 p40 and IL-12 p70 elaboration by mouse peritoneal macrophages (PMs) infected with M. tuberculosis H37Rv for 6 h, 24 h, 4 days and 7 days, in vitro, by using a multiplex suspension cytokine array system. Results: INH, at 6 h, at 0.4 μg/ml (MIC: 0.2 μg/ml) and not at 0.1 and 0.2 μg/ml, and at 24 h and on D 4 and D 7, at all the tested concentrations, significantly (p < 0.001) suppressed IL-6 elaboration. RIF, at 6 h, lacked any effect on IL-6 elaboration at 0.4 μg/ml (MIC: 0.8 μg/ml), whereas at 0.8 and 1.6 μg/ml, and at all the tested concentrations at 24 h and on D 4 and D 7, significantly (p < 0.001) inhibited IL-6 elaboration. STP, at all the time-points, at 2.5 μg/ml (MIC: 5 μg/ml), lacked any effect on IL-6 elaboration; however, at 5 and 10 μg/ml it caused significant (p < 0.001) inhibition. Surprisingly, EMB at 4, 8 and 16 μg/ml (MIC: 8 μg/ml) suppressed IL-6 elaboration at all the time-points studied. M. tuberculosis-infected untreated controls, in contrast to the uninfected ones, irrespective of the time-points, elaborated significantly (p < 0.001) high concentrations of IL-6 (infected controls, 365 ± 30.41–777.5 ± 74.45 pg/ml; uninfected controls, 15.25 ± 1.77–21 ± 1.41 pg/ml) only. Curiously, all the other cytokines (IL-1β, IL-10, IL-12 p40 and IL-12 p70) were unaffected by the drug treatments. Conclusion: M. tuberculosis -infected PMs, in vitro, elaborated IL-6 as the only major cytokine, which was significantly inhibited by anti-TB drugs. Therefore, IL-6 can be developed as a potential biomarker or biosignature to assess the success of the treatment of TB.

Kinetoplastid Membrane Protein-11 as a Vaccine Candidate and a Virulence Factor in Leishmania.

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. In Leishmania amazonensis, KMP-11 is expressed in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures at the cell surface, flagellar pocket, and intracellular vesicles. More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. In this connection, we have shown that addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide production. The doses of KMP-11, the IL-10 levels, and the intracellular amastigote loads were strongly, positively, and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10-neutralizing antibodies, but not by isotype controls. The neutralizing antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. All these data indicate that KMP-11 acts as a virulence factor in L. amazonensis infection.

Nitric oxide stimulates early egress of Toxoplasma gondii tachyzoites from Human foreskin fibroblast cells.

BackgroundEgress is a vital step in the life cycle of Toxoplasma gondii which attracts attentions of many groups. Previous studies have shown that exogenous nitric oxide (NO) stimulates the early egress of T. gondii from infected peritoneal macrophages, a kind of immune cells. However, because Toxoplasma forms cysts in brain and muscle tissues, the development of autonomous immunity in non-immune cells is vital for limiting parasite burden and cyst formation. Therefore, we attempted to investigate whether exogenous NO could induce the early egress of T. gondii from infected non-immune cells.MethodsT. gondii tachyzoites were cultured in human foreskin fibroblast (HFF) cells and were then treated with NO released by sodium nitroferricyanide (III) dihydrate (SNP). The egressed parasites were analysed by flow cytometry.ResultsThe results showed that NO induced the early egress of parasites from HFF cells before completing their intracellular life cycles. We also found that the occurrence of egress was dependent on intracellular calcium (Ca2+) levels and the mobility of the parasite. Compared with freshly isolated tachyzoites, the developmental ability and virulence of egressed tachyzoites presented no difference.ConclusionsTaken together, our findings demonstrate a novel assay for the analysis of egress signalling mechanisms and an avenue of parasite clearance by hosts of T. gondii.

Kaurenoic Acid Possesses Leishmanicidal Activity by Triggering a NLRP12/IL-1β/cNOS/NO Pathway.

Leishmania amazonensis (L. amazonensis) infection can cause severe local and diffuse injuries in humans, a condition clinically known as American cutaneous leishmaniasis (ACL). Currently, the therapeutic approach for ACL is based on Glucantime, which shows high toxicity and poor effectiveness. Therefore, ACL remains a neglected disease with limited options for treatment. Herein, the in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against L. amazonensis were investigated. KA exhibited a direct antileishmanial effect on L. amazonensis promastigotes. Importantly, KA also reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALB/c mice. Mechanistically, KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase- (cNOS-) dependent manner, subverting the NO-depleting escape mechanism of L. amazonensis. Furthermore, KA induced increased production of IL-1β and expression of the inflammasome-activating component NLRP12. These findings demonstrate the leishmanicidal capability of KA against L. amazonensis in macrophage culture by triggering a NLRP12/IL-1β/cNOS/NO mechanism.

Neutrophils Increase or Reduce Parasite Burden in Trypanosoma cruzi-Infected Macrophages, Depending on Host Strain: Role of Neutrophil Elastase

Neutrophils are involved in the initial steps of most responses to pathogens and are essential components of the innate immune response. Due to the ability to produce and release various soluble mediators, neutrophils may participate in the regulation of the inflammatory response. Little is known about the role of neutrophils during protozoan infections including infection by Trypanosoma cruzi. In the present study we investigated the importance of inflammatory neutrophils on macrophage activation and T. cruzi replication in vitro, in cells obtained from BALB/c mice and C57Bl/6 mice. Co-cultures of BALB/c apoptotic or live neutrophils with infected peritoneal macrophages resulted in increased replication of the parasites and in the production of TGF-β and PGE2. The treatment with anti-TGF-β neutralizing antibody and COX inhibitor blocked the parasite replication in vitro. On the other hand, co-cultures of T. cruzi infected macrophages with live neutrophils isolated from C57BL/6 mice resulted in decreased number of trypomastigotes in culture and increased production of TNF-α and NO. The addition of anti-TNF-α neutralizing antibody or elastase inhibitor resulted in the abolishment of macrophage microbicidal effect and increased parasite replication. Addition of elastase to infected macrophages reduced the replication of the parasites, and on the other hand, addition of a selective inhibitor of iNOS increased parasite growth, suggesting the role of NO in this system. Our findings reveal that neutrophils may regulate T. cruzi experimental infection and determine susceptibility and resistance to infection.

Characterization of Immune Responses to Yersinia pestis (Indian Isolate) Infection in Mouse Model

Yersinia pestis, causative agent of plague, is one of the deadliest pathogens around globe. Innate immune response is first line of host defense against pathogens. Here, we have investigated innate and adaptive immune response in plague infected mice, gene expression levels of TLR1-9 and CD14, MyD88, NF-A„¸B, TNF-α, MAPKp38, IL-1β were studied in peritoneal macrophages of plague infected mice in a time dependent manner (0 h, 24 h, 48 h, 72 h, 96 h and 120 h of post infection) by qRT-PCR. We also evaluated the immune response to Yersinia outer proteins (Yops) in Y. pestis infected mice. Selected genes (11 numbers) of Y. pestis encoding virulent factors viz, YpkA, YopH, YopM, V antigen, Pla, YopN, YopJ, YopE, YopK, F1 and pH6 antigen were amplified by PCR, cloned and expressed in Escherichia coli . To study the IgG and its isotypes level, ELISA and immunoblotting were performed using purified recombinant antigens. The major antigens recognized by murine plague infected sera were V antigen, YopH, YopM, YopE, F1 but very weak immunoreaction was observed in case of Pla. We observed a significant difference in IgG isotypes (IgG1, IgG2a, IgG2b and IgG3) to V antigen and F1, whereas in case of YopH and YopE (IgG1 and IgG2b) only. We also observed significant increase in the expression of CD14 at 48 h post infection, TLR4 and MyD88 at 72 h post infection in Y. pestis infected mouse peritoneal macrophages. Our findings suggest that both innate and humoral immune responses are crucial for protection.

Anti-herpes virus activities of Achyranthes aspera: An Indian ethnomedicine, and its triterpene acid

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC50 64.4μg/ml for HSV-1 and 72.8μg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC50 6.8μg/ml) and HSV-2 (EC50 7.8μg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2–6h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48–72h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2–6h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.

Toxoplasma gondii: Effects of exogenous nitric oxide on egress of tachyzoites from infected macrophages

Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated cells of warm-blood vertebrates. Invasion and egress by this protozoan parasite, both of which are crucial for its life cycle, are rapid events that are dependent upon parasite motility. A variety of chemicals and molecules have been utilized to induce Toxoplasma early egress from host cells. Here, we aimed to determine whether nitric oxide (NO) could induce egress of T. gondii tachyzoites from infected cells. Infected macrophages were collected from C57BL/6 mice and treated with different doses of sodium nitroferricyanide (III) dihydrate (SNP) which releases nitric oxide into cell culture medium. The pattern of parasite egress was analyzed by flow cytometry. The results showed that exogenous NO released by SNP could trigger egress of T. gondii tachyzoites from infected peritoneal macrophages which then underwent necrosis after parasite egress. Our findings provided a novel approach to study the interactions between host immune responses and T. gondii.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo

Encephalitozoon cuniculi (Phylum Microsporidia) infects a wide range of mammals, and replicates within resting macrophages. Activated macrophages, conversely, inhibit replication and destroy intracellular organisms. These studies were performed to assess mechanisms of innate immune responses expressed by macrophages to control E. cuniculi infection. Addition of reactive oxygen and nitrogen species inhibitors to activated murine peritoneal macrophages statistically significantly, rescued E. cuniculi infection ex vivo. Mice deficient in reactive oxygen species, reactive nitrogen species, or both survived ip inoculation of E. cuniculi, but carried significantly higher peritoneal parasite burdens than wild-type mice at 1 and 2 weeks post inoculation. Infected peritoneal macrophages could still be identified 4 weeks post inoculation in mice deficient in reactive nitrogen species. L-tryptophan supplementation of activated murine peritoneal macrophage cultures ex vivo failed to rescue microsporidia infection. Addition of ferric citrate to supplement iron, however, did significantly rescue E. cuniculi infection in activated macrophages and further increased parasite replication in non-activated macrophages over non-treated resting control macrophages. These results demonstrate the contribution of reactive oxygen and nitrogen species, as well as iron sequestration, to innate immune responses expressed by macrophages to control E. cuniculi infection.

The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice

Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-γ and have smaller parasite load, after 8 weeks of infection. Furthermore, after 1 week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.

The role of MHC Ib in selecting functional CD8 T cell during acute LCMV infection (36.38)

Abstract Conventional MHC Ia &amp; II molecules (H-2K/D/L &amp; I-A/E in mouse) select α/β CD8 &amp; CD4 T cell development, respectively. Although unconventional MHC class Ib, mainly encoded in H-2 Q, T &amp; M regions in mouse, contains more than 20 MHC Ib genes, their functions remain largely unknown. We developed a mouse model, in which 3 genes, Kb, Db &amp; CIITA were deleted, thus called triple knock-out (TKO) mouse. In TKO, CD8 T cells are selected only by MHC Ib. Previously we have shown that α/β CD8 T cells develop well in TKO. Functionally, we observe that TKO mice mount a protective immune response against in vivo acute infections of LCMV. The protection is CD8 T cell mediated &amp; β2m dependent. Rapid production of IFNγ and Granzyme B is detected in expended CD8 cells. Intriguingly, TKO mice do not appear to mount a memory response to a second infection. We report that splenocytes, from in vivo LCMV primed TKO, can be restimulated in vitro, by LCMV infected peritoneal macrophages or bone marrow derived dendritic cells, to produce IFNγ, but little IL-2. This MHC Ib-dependent response appears to be partially mediated by Qa-2. Therefore we have established an in vitro system to analyze MHC Ib-restricted T cell responses to LCMV &amp; we are attempting to map the MHC Ib restriction element(s) and target epitopes. Collectively, our results suggest that MHC Ib can select functional CD8 T cells &amp; these T cells participate in, at least under some circumstances, protective immune responses to pathogens.

Human Peritoneal Macrophages From Ascitic Fluid Can be Infected by a Broad Range of HIV-1 Isolates

Macrophages are major HIV target cells. They support both productive and latent HIV-1 infection. Susceptibility of primary macrophages to HIV depends on the anatomical location and activation state of the cells. We demonstrate that peritoneal macrophages (PMs) are abundant in ascitic fluid of patients with liver cirrhosis and are susceptible to HIV-1 infection. PMs expressed CD68, a differentiation marker, exhibited phagocytic activity, and survived in culture for 2 months without additional growth factors. Freshly isolated PMs were susceptible to HIV-1 R5 strains but not to X4-T-cell line-adapted strains. Interestingly, after 7 days in culture, PMs acquired susceptibility to X4-T-cell line-adapted strains. HIV entry inhibitors, TAK779 and AMD3100, blocked HIV infection of PMs, indicating that infection by R5 and X4 strains was mediated by CCR5 and CXCR4, respectively. Although PMs did not express detectable cell surface levels of CXCR4 and CCR5, they did express mRNAs of these HIV coreceptors and responded to stimulation by their natural ligands, SDF-1alpha and RANTES. PMs were susceptible to HIV-1 X4, R5, and X4R5 primary isolates. PMs after 7 days in culture produced greater amounts of X4 and X4R5 HIV than freshly isolated PMs. The day-7 PMs were more susceptible to R5 infection in a single-cycle infection assay, but there was no increase in viral production in a multiple-round infection assay. The level of CXCR4 mRNA and production of CC-chemokines (MIP-1alpha, MIP-1beta, and RANTES) increased significantly during 7 days in culture. Our results indicate that PMs are susceptible to receptor-mediated infection by a broad range of HIV strains. These primary macrophages could provide a valuable system for investigating the role of primary macrophages in HIV pathogenesis.

Potentiation by cigarette smoke of macrophage function against Leishmania donovani infection

Cigarette smoke is able to induce the generation of reactive oxygen species and pro-inflammatory cytokines, which are mediators of macrophage function and therefore, we have investigated the ability of cigarette smoke to activate Leishmania donovani infected peritoneal macrophage. Cultured peritoneal macrophages were either left untreated or treated with aqueous cigarette smoke extract prior to L. donovani infection. Parasite burden was assessed by giemsa staining. The level of intracellular reactive oxygen species was determined by FACS analysis. PCR was performed to analyze mRNA levels of cytokines. NF- kappaB activity was assessed by EMSA and reporter assay. A pre-treatment with cigarette smoke extract (CSE) causes a decrease in parasite burden, an increase in intracellular ROS level, up-regulation of pro-inflammatory cytokines, reduced expression of immuno-suppressive cytokine and boosting of NF-kappaB activity in L. donovani-infected macrophage. Low concentration of cigarette smoke extract (CSE) counteracts L. donovani infection-mediated suppression of macrophage function without affecting host cell viability. This study reveals a new role of cigarette smoke extract (CSE) as an activator of macrophage function.

Morphine-induced Neuroimmunomodulation in Murine Visceral Leishmaniasis: The Role(s) of Cytokines and Nitric Oxide

Opioid modulation of host resistance to infectious diseases is well documented; however, not much is known during visceral leishmaniasis (VL). Low doses of morphine, administered subcutaneously in Leishmania donovani-infected BALB/c mice, on days 0 and +15, significantly (p < 0.05) suppressed (1 mg/kg/day) or even sterile-cleared (2 mg/kg/day) the infection; paradoxically, high doses (10 and 30 mg/kg/day) exacerbated the infection. In vitro, low concentration (1 x 10(-9) and 1 x 10(-11) M) morphine treatment of L. donovani-infected mouse peritoneal macrophages (PM), endowed them with significant (p < 0.05) leishmanicidal activity, whereas a high-concentration (1 x 10(-5) M) treatment augmented intramacrophage parasite growth. Naloxone pre-treatment of infected-mice (4 mg/kg x 2) and of infected-PM (1 x 10(-5) M), blocked only the morphine low dose/concentration-induced protective effect. The splenocytes from protected mice and morphine low concentration-treated infected-PM, elaborated significantly (p < 0.05) enhanced levels of interleukin-12, interferon-gamma, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor and nitrite in the culture medium; a high dose/concentration suppressed their elaboration. Curiously, only morphine high dose/concentration-treated infected mice splenocytes and infected PM, produced significantly (p < 0.05) increased quantity of transforming growth factor-beta1. Aminoguanidine, significantly (p < 0.05) blocked the morphine low dose/concentration-induced protective effect, in vivo and in vitro. This first study demonstrates dose-dependent biphasic modulatory effects of morphine in L. donovani-infected mice and PM, in vitro, apparently via nitric oxide-dependent mechanisms. These results thus demonstrate the implications of opiate abuse on the efficacy assessment of antileishmanial drugs and vaccines, and on the reactivation of latent VL in areas where both drug abuse and VL are rampant.

Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection.

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated "O" chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-gamma(-/-)), BALB/c, Toll-like receptor 2 knockout (TLR2(-/-)), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-gamma(-/-) and TLR2(-/-) mice, which showed elevated rectal temperatures that were sustained for approximately 15 days with transient increases in splenic weights. Reinfection of IFN-gamma(-/-) and TLR2(-/-) mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2(-/-) mouse spleens was observed, greater colonization was evident in the IFN-gamma(-/-) mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-gamma(-/-) macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2(-/-) macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.

Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H(2)O(2), NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H(2)O(2) production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.

&lt;![CDATA[&lt;B&gt;Effects of Brazilian propolis on &lt;I&gt;Leishmania amazonensis&lt;/i&gt;&lt;/B&gt;]]&gt;

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 µg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

Effects of Brazilian propolis on Leishmania amazonensis

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 microg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.