Tissue Parasite Load Research Articles

Toll Receptors Type-2 and CR3 Expression of Canine Monocytes and Its Correlation with Immunohistochemistry and Xenodiagnosis in Visceral Leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ−/XENO−). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ+/XENO+) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b+TLR2+ and CD11b+MHCII+, higher values were obtained from dogs with IHQ−/XENO− than dogs with IHQ+/XENO+ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b+TLR2+ and NO with higher values for dogs with IHQ−/XENO− than dogs with IHQ+/XENO+), led to the conclusion that IHQ−/XENO− dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.

Myeloid-Derived Suppressor Cells Infiltrate the Heart in Acute Trypanosoma cruzi Infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed in the acute and chronic phases of the disease, is characterized by a mononuclear cell inflammatory infiltrate. We previously identified a myeloid cell population in the inflammatory heart infiltrate of infected mice that expressed arginase I. In this study, we purified CD11b(+) myeloid cells from the heart and analyzed their phenotype and function. Those CD11b(+) cells were ∼70% Ly6G(-)Ly6C(+) and 25% Ly6G(+)Ly6C(+). Moreover, purified CD11b(+)Ly6G(-) cells, but not Ly6G(+) cells, showed a predominant monocytic phenotype, expressed arginase I and inducible NO synthase, and suppressed anti-CD3/anti-CD28 Ab-induced T cell proliferation in vitro by an NO-dependent mechanism, activity that best defines myeloid-derived suppressor cells (MDSCs). Contrarily, CD11b(+)Ly6G(+) cells, but not CD11b(+)Ly6G(-) cells, expressed S100A8 and S100A9, proteins known to promote recruitment and differentiation of MDSCs. Together, our results suggest that inducible NO synthase/arginase I-expressing CD11b(+)Ly6G(-) myeloid cells in the hearts of T. cruzi-infected mice are MDSCs. Finally, we found plasma l-arginine depletion in the acute phase of infection that was coincident in time with the appearance of MDSCs, suggesting that in vivo arginase I could be contributing to l-arginine depletion and systemic immunosuppression. Notably, l-arginine supplementation decreased heart tissue parasite load, suggesting that sustained arginase expression through the acute infection is detrimental for the host. This is, to our knowledge, the first time that MDSCs have been found in the heart in the context of myocarditis and also in infection by T. cruzi.

Taenia crassiceps upper limb fasciitis in a patient with untreated acquired immunodeficiency syndrome and chronic hepatitis C infection – the role of surgical debridement

We report a rare case of human Taenia crassiceps infection in a 47-year-old female patient with untreated acquired immunodeficiency syndrome and chronic hepatitis C infection. Little experience exists regarding the appropriate treatment of this infection. Usually, a combination of anthelmintic drugs is applied. Whether surgical measures are indicated have not been clarified. In our patient, initial surgery showed an abscess and fluid collection with numerous transparent cysts localised in the subcutaneous tissue of the cubital fossa. Parasitological and pathological examinations identified these structures as larvae of the cestode T.crassiceps. After treatment with anthelmintic medications, the patient was discharged in good condition. However, the patient presented with the clinical symptoms of an acute fasciitis of the right upper extremity 7 days later. The deteriorating general condition entailing a pre-septical state demanded emergency debridement and fasciectomy of the right arm. After the surgery, the patient recovered fully. Surgical treatment appears to be an important measure to reduce the tissue parasite load in patients with severe immunodeficiency. It also has to be questioned whether the bioavailability and the penetration of the drugs commonly administered is sufficiently high to treat such a fulminant infection alone.

<em>In vivo</em> Imaging of Transgenic <em>Leishmania</em> Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

<em>In vivo</em> Imaging of Transgenic <em>Leishmania</em> Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

Reduction of parasite levels in blood improves pregnancy outcome during experimentalTrypanosoma cruziinfection

Infection with a myotropic Trypanosoma cruzi clone induces maternal fertility failure. In the current work, we evaluated whether reduction of maternal parasitaemia before mating has beneficial effects on pregnancy outcome. Female mice were subjected to benznidazole (Bz) treatment after infection. On day 30 of therapy, mating was assessed and pregnancy outcome was determined on day 14 of gestation. Fetal resorptions diminished in T. cruzi-infected Bz-treated mice compared with T. cruzi-infected untreated mice. This was in agreement with the reduction in the blood/solid tissue parasite load and with the percentage of necrotic foci in placental samples from T. cruzi-infected Bz-treated females. To study eventual changes in the immune homeostasis of T. cruzi-infected Bz-treated mice, activation of the immune system was evaluated at the end of Bz therapy and before mating. We found specific IgG1 reduction resulting in a predominance of specific IgG2a, reduced numbers of CD69+ CD4+ cells and diminished frequency and numbers of CD44+ T cells. Concanavalin A-stimulated splenocytes from T. cruzi-infected Bz-treated mice produced higher amounts of IFN-gamma than T. cruzi-infected untreated mice. These results indicate that reduction of maternal parasite load improves pregnancy outcome. These findings correlate with a favourable modulation of the immune response.

The roles of type I IFN receptor and neutrophils in regulating innate immunity to Leishmania parasites (133.26)

Abstract Type I IFNs exert diverse effector/regulatory functions in innate and adaptive immune responses to viruses and bacteria; however, their roles in parasitic infections are less clear. It has been reported that parasite-induced type I IFNs are critical for NO-dependent disease control, and that administration of IFN-β has a dose-dependent protective effect in L. major-infected mice. Surprisingly, we found that following infection with L. amazonensis parasites, IFNAR-/- mice developed significantly attenuated disease with low levels of cytokines and sera antigen-specific IgG. The marked reduction in tissue parasite loads and inflammatory responses even at 3 days of infection in IFNAR-/- mice suggests the possibility of neutrophil-mediated parasite clearance. First, although WT and IFNAR-/- mice were efficient in recruiting CD11b+F4/80+Gr1+ peritoneal neutrophils at 6 h post-injection of parasites, knockout mice displayed unique phenotypic changes in Gr1lowCD11bhigh macrophages and Gr1highCD11blow neutrophils at 24 h. Second, in addition to release more neutrophil elastase and myeloperoxidase, IFNAR-/- not STAT1-/- neutrophils had significantly higher rates of spontaneous and infection-induced apoptosis than did their WT controls. Third, while macrophages alone responded comparably to parasites, the interactions between macrophages and IFNAR-/- neutrophils (but not WT and STAT1-/- neutrophils), greatly enhanced parasite killing. Finally, adoptive transfer of WT bone marrow neutrophils into IFNAR-/- mice did enhance tissue parasite loads. This study suggests an important role for type I IFNs in regulating neutrophil turnover and Leishmania infection and provides new information on innate immunity to protozoan parasites.

Role of Natural Killer Cells in Modulating Dendritic Cell Responses toLeishmania amazonensisInfection

The importance of the interaction between natural killer (NK) cells and dendritic cells (DCs) in the expansion of antiviral and antitumor immune responses is well-documented; however, limited information on DC-NK cell interaction during parasitic infections is available. Given that some Leishmania parasites are known to prevent or suppress DC activation, we developed a DC-NK cell coculture system to examine the role of NK cells in modulating the functions of Leishmania-infected DCs. We found that the addition of freshly isolated, resting NK cells significantly promoted the activation of DCs that were preinfected with Leishmania amazonensis promastigotes and that these activated DCs, in turn, stimulated NK cell activation mostly via cell contact-dependent mechanisms. Notably, L. amazonensis amastigote infection failed to activate DCs, and this lack of DC activation could be partially reversed by the addition of preactivated NK (ANK) cells but not resting NK cells. Moreover, the adoptive transfer of ANK cells into L. amazonensis-infected mice markedly increased DC and T-cell activation and reduced tissue parasite loads at 1 and 3 weeks postinfection. These results suggest differential roles of DC-NK cell cross talk at different stages of Leishmania infection and provide new insight into the interplay of components of the innate immune system during parasitic infection.

Diffuse Intralobular Liver Fibrosis in Dogs Naturally Infected with Leishmania (Leishmania) chagasi

The aim of this study was to evaluate the diffuse intralobular fibrosis in dogs naturally infected with Leishmania (Leishmania) chagasi. One hundred five infected animals with positive serologic tests for Leishmania were divided into two clinical groups: 69 symptomatic animals and 36 asymptomatic. Special staining with Gomori, Heidenhain, Silver, and Picrosirius Red was applied to characterize fibrilopoesis. The tissue parasite load was measured by immunohistochemistry and associated histomorphometric analyses. Intralobular fibrosis was observed in all dogs, and more collagen deposition was confirmed in the infected animals than in the controls by these histomorphometric studies. There were significant differences among the distinct clinical groups. In fact, symptomatic dogs showed an increased collagen deposition in the liver compared with asymptomatic ones. A peculiar diffuse intralobular fibrosis, where the collagen fibers encircled small groups of hepatocyte(s), was observed in two cases (1.9%).

Impairment of Infectivity and Immunoprotective Effect of a LYT1 Null Mutant of Trypanosoma cruzi

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 10(3) trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-γ, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania ( Leishmania) chagasi

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania ( Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.

Viscerotropic growth pattern of Leishmania tropica in BALB/c mice is suggestive of a murine model for human viscerotropic leishmaniasis

Leishmania (L.) tropica is a causative agent of cutaneous leishmaniasis, and occasionally of visceral or viscerotropic leishmaniasis in humans. Murine models of Leishmania infection have been proven to be useful for elucidation of mechanisms for pathogenesis and immunity in leishmaniasis. The aim of this study was to establish a murine model for human viscerotropic leishmaniasis, and the growth pattern of L. tropica was studied in different tissues of BALB/c mice in order to find out whether the parasite visceralizes in this murine model. L. major was used as a control as this species is known to cause a progressive infection in BALB/c mice. L. tropica or L. major was injected into the footpad of mice, and thickness of footpad, parasite loads in different tissues, and the weight of the spleen and lymph node were determined at different intervals. Results showed that L. tropica visceralizes to the spleen and grows there while its growth is controlled in footpad tissues. Dissemination of L. tropica to visceral organs in BALB/c mice was similar to the growth patterns of this parasite in human viscerotropic leishmaniasis. The BALB/c model of L. tropica infection may be considered as a good experimental model for human diseases.

Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (Leishmania) major infection

Heat shock proteins (HSPs) are highly conserved among different organisms. A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic and immunotherapeutic effects against Mycobacterium tuberculosis infection in mice. Here, BALB/c mice were immunized with mycobacterial DNA-hsp65 or with DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable microspheres (MHSP/TDM), and challenged with Leishmania (Leishmania) major. MHSP/TDM conferred protection against L. major infection, as indicated by a significant reduction of edema and parasite loads in infected tissues. Although high levels of interferon-gamma and low levels of interleukin (IL)-4 and IL-10 were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals immunized with MHSP/TDM displayed a consistent Th1 immune response, i.e., significantly higher levels of anti-soluble Leishmania antigen (SLA) immunoglobulin G (IgG)2a and low anti-SLA IgG1 antibodies. These findings indicate that encapsulated MHSP/TDM is more immunogenic than naked hsp65 DNA, and has great potential to improve vaccine effectiveness against leishmaniasis and tuberculosis.

Effect of rSAG-1(P30) immunisation on the circulating and tissue parasites in guinea pigs as determined by quantitative PCR

The efficacy of immunisation with Toxoplasma gondii recombinant protein (rSAG-1) was evaluated in the guinea pig model. For the infectious challenge, two strains, namely, strain C56 (10,000 tachyzoites) and strain 76K (100 cysts), were used to infect a group of 32 guinea pigs each. The circulating, cerebral and pulmonary parasite loads were determined with the real-time polymerase chain reaction (PCR) after immunisation. With the C56 strain, immunisation showed high activity with a reduction of greater than 1 log of the circulating and tissue parasite loads. Thus, there was a significantly lower circulating parasite load in the rSAG-1 + adjuvant group (0.5+/-1.5 Eq parasites/ml) as compared to that in the control group (67+/-110 Eq parasites/ml; p<0.05). In the same manner, the cerebral parasite load was much lower in the rSAG-1 + adjuvant group (10+/-20 Eq parasites/g) than that in the control group (339+/-291 Eq parasites/g; p<0.01). On the other hand, with the 76K strain, the effect of immunisation was much less and that only on the pulmonary parasite load [p(lung)<0.05]. This could be due to the use of different strains and stages of the parasite and/or the difference in the route of administration for challenge. The quantitative PCR technique used has shown a good correlation with animal inoculation, and when associated with the guinea pig model, it seems to be a useful and reproducible technique for future vaccine studies.

TNF but not Fas ligand provides protective anti- L. major immunity in C57BL/6 mice

The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNF-deficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF –/–, B6. gld and C57BL/6. gld x TNF –/– (B6. gld.TNF –/–) double-negative mice. Visceral, fatal disease was only observed in B6.TNF –/– mice, but not in B6 gld mice. The course of infection and the immune response of B6. gld.TNF –/– mice were similar to those of B6.TNF –/– mice. B6. gld.TNF –/– mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF –/– mice, whereas the tissue parasite load and the iNOS expression of B6. gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen.

QUANTIFICATION OF LEISHMANIA INFANTUM PARASITES IN TISSUE BIOPSIES BY REAL-TIME POLYMERASE CHAIN REACTION AND POLYMERASE CHAIN REACTION–ENZYME-LINKED IMMUNOSORBENT ASSAY

Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.

Inducible nitric oxide synthase is not essential for control of Trypanosoma cruzi infection in mice.

Immune control of many intracellular pathogens, including Trypanosoma cruzi, is reported to be dependent on the production of nitric oxide. In this study, we show that mice deficient in inducible nitric oxide synthase (iNOS or NOS2) exhibit resistance to T. cruzi infection that is comparable to that of wild-type mice. This is the case for two iNOS-deficient mouse strains, Nos2(tm1Lau) and Nos2 N5, infected with the Brazil or Tulahuen strain of T. cruzi. In all cases, blood parasitemia, tissue parasite load, and survival rates are similar between wild-type and iNOS-deficient mice. In contrast, both wild-type and Nos2(tm1Lau) mice died within 32 days postinfection when treated with the nitric oxide synthase inhibitor aminoguanidine. Increased transcription of NOS1 or NOS3 is not found in iNOS-knockout (KO) mice, indicating that the absence of nitric oxide production through iNOS is not compensated for by increased production of other NOS isoforms. However, Nos2(tm1Lau) mice exhibit enhanced expression of tumor necrosis factor alpha, interleukin-1, and macrophage inflammatory protein 1alpha compared to that of wild-type mice, and these alterations may in part compensate for the lack of iNOS. These results clearly show that iNOS is not required for control of T. cruzi infection in mice.

Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rVV) vectors expressing relevant antigens has been shown to enhance specific cellular immune responses and to elicit protection against a variety of pathogens in animal models. In this paper, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a plasmid carrying the gene for the LACK antigen from Leishmania infantum (DNA-LACK) followed by a booster with a rVV containing the same gene (rVV-LACK). Thereafter, animals were challenged with L. infantum to induce visceral leishmaniasis (VL). In the vaccinated dogs as compared with the controls, the outcome of the infection after challenge with a high inoculum (10 8) of L. infantum stationary promastigotes was assessed by tissue parasite load, specific anti- Leishmania antibody production, cytokine level and development of clinical signs of leishmaniasis. We observed a 60% protection against infection in dogs immunized by DNA-LACK prime/rVV/-LACK boost while two doses of DNA-LACK did not elicit protection against the disease. The interleukin 4 (IL-4), interferon gamma (IFNγ) and IL-12 (p40 subunit) cytokine mRNA expression profiles in PBMC as well as lymphocyte proliferative response and the IgG2/IgG1 ratios specific for LACK suggest that in vaccinated animals there is triggering of cellular immune responses. This type of DNA/rVV prime/boost immunization approach may have utility against visceral leishmaniasis in dogs.

Mice lacking the chemokine receptor CCR1 show increased susceptibility to Toxoplasma gondii infection.

Chemokines are critical for the recruitment of effector immune cells to sites of infection. Mice lacking the chemokine receptor CCR1 have defects in neutrophil trafficking and proliferation. In the present study, we tested the susceptibility of CCR1 knockout mice to infection with the obligate intracellular protozoan parasite Toxoplasma gondii. In comparison with parental wild-type mice, CCR1(-/-) mice exhibited dramatically increased mortality to T. gondii in association with an increased tissue parasite load. No differences were observed in Ag-specific T cell proliferation or in cytokine responses between mutant and wild-type mice. However, the influx of PMNs to the peripheral blood and to the liver were reduced in CCR1(-/-) mice during early infection. Our results suggest that CCR1-dependent migration of neutrophils to the blood and tissues may have a significant impact in controlling parasite replication.