Scavenger receptors mediate increased uptake of irradiated T.gondii extracts by J774 macrophages.

PURPOSE: Protein extracts developed increased immunogenicity without the aid of adjuvants after gamma irradiation. Gamma irradiation of snake venom increased antivenin production by detoxification and enhanced immunity, probably due preferential uptake of irradiated venoms by macrophage scavenger receptors. We studied this uptake of irradiated soluble Toxoplasma gondii extract (STag) by the J774 macrophage cell line similar to antigen presenting cells. MATERIAL AND METHODS: We labeled STag by biosynthesis in living tachyzoites with radioactive amino acids before purification and irradiation or by adding labels as biotin or fluorescein in stored STag, for quantitative studies or subcellular distribution visualization. RESULTS: There was enhanced binding and uptake of irradiated STag into the cells compared to non-irradiated STag. Using fluorescein labeled antigens and morphological assays, we confirmed that cells avidly ingested both native and irradiated proteins but native STag were digested after ingestion while irradiated proteins remained in the cell, suggesting diverse intracytoplasmic pathways. Native or irradiated STag present the same in vitro sensitivity to three types of peptidases. Inhibitors of scavenger receptors (SRs) such as Dextran sulfate (SR-A1 blocker) or Probucol (SR-B blocker) affect the specific uptake of irradiated antigens, suggesting its association with enhanced immunity. CONCLUSIONS: Our data suggests that cell SRs recognize irradiated proteins, mainly SRs for oxidized proteins, leading to antigen uptake by an intracytoplasmic pathway with fewer peptidases that prolongs presentation to nascent major histocompatibility complex I or II and enhances immunity by better antigen presentation.

Toxoplasma gondii in CD36 -/- mice shows lethal infection and poor immunization with probable macrophage immune defects.

Experimental toxoplasmosis is an excellent model for adaptive immune response. Gamma-irradiated tachyzoites or soluble tachyzoite antigen extracts (STag) induce protection against experimental toxoplasmosis in mice. Scavenger receptors recognize irradiated proteins, promote their entry into cells, and lead to antigen presentation. CD36 is a specific scavenger receptor involved in intracellular transport of free fatty acid (FFA), cellular recycling, and intracellular trafficking in lipid rafts outside the lysosomal pathways. CD36 is also associated with an altered immune response, as CD36-/- mice presented some immune defects in the cyst-forming Toxoplasma gondii. We studied T. gondii infection in CD36-/- mice, naïve or immunized, with irradiated T. gondii STags by investigating protection, antibody production, and primed macrophage transplantation. CD36-/- mice presented no resistance against the viable RH tachyzoites, even after immunization with gamma-irradiated STags that protected wild-type mice. The animals presented poor humoral responses to both immunogens despite adequate levels of serum immunoglobulins. CD36-/- mice failed to induce protection against virulent T. gondii infection with inadequate antibody production or an innate response. Irradiated antigens failed to induce antibodies in CD36-/- mice and only produced adequate levels of immunoglobulin G when transplanted with irradiated STag-primed wild-type macrophages. The CD36 pathway is necessary for humoral response against the irradiated antigen; however, several other pathways are also involved in mounting a humoral response against any antigen. CD36 is a multipurpose molecule for FFA and lipid transport, as well as for the immune response, and gamma radiation mimics the innate response by targeting irradiated antigens of this pathway.

Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models.

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH0.25 kGy).Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5 kGy (STag1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag1.5 kGy had higher proportions of CD19+ (cluster of differentiation 19) and CD4+ (cluster of differentiation 14) cells, whereas mice RH0.25 kGy had high proportion of memory CD8+ (cluster of differentiation 8) cells.Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

Behavioral evaluation of BALB/c (Mus musculus) mice infected with genetically distinct strains of Toxoplasma gondii.

In relation to behavioral changes in rodents infected with Toxoplasma gondii (T. gondii), it is believed that the genotype of the infecting strain can have some influence. In this sense, the present work has sought to evaluate the effect of chronic infection by genetically distinct cystogenic strains of T. gondii on the behavior of mice. For this, experimental models of infection with ME-49 (type II) and VEG (type III) strains were developed in isogenic BALB/c mice. ELISA test was performed to evaluate the humoral immune response and real-time PCR test to quantify parasites in the CNS. Behavioral tests such as passive avoidance, open-field and Y-maze tests were also used for, respectively, evaluation of learning and memory, locomotor activity and aversion to feline odor. The results showed that mice infected with VEG strain had higher total IgG level of anti-toxoplasma, higher tissue burden of T. gondii in the CNS, reduction in the long-term memory, lower activity (mobility) and lower aversion to cat urine and l-felinine than mice infected with ME-49 strain. The results suggest that different T. gondii genotypes have a differential impact on behavioral changes in infected mice.

Gamma irradiation of Toxoplasma gondii protein extract improve immune response and protection in mice models.

Gamma radiation induces protein changes that enhance immunogenicity for venoms, used in antivenin production. Coccidian parasites exposed to gamma radiation elicit immune response with protection in mice and man, but without studies on the effect of gamma radiation in soluble acellular extracts or isolated proteins. Toxoplasmosis is a highly prevalent coccidian disease with only one vaccine for veterinary use but with remaining tissue cysts. Total parasite extracts or recombinant proteins used as immunogen induce usually low protection. Here, we study gamma radiation effect on T. gondii extracts proteins (STAG) and its induced immunity in experimental mice models. By SDS-PAGE, protein degradation is seen at high radiation doses, but at ideal dose (1500 Gy), there are preservation of the antigenicity and immunogenicity, detected by specific antibody recognition or production after mice immunization. Immunization with STAG irradiated at 1500 Gy induced significant protection in mice immunized and challenged with distinct T. gondii strains. In their blood, higher levels of specific CD19+, CD3+CD4+ and CD3+CD8+ activated cells were found when compared to mice immunized with STAG. Irradiated T. gondii tachyzoites extracts induce immune response and protection in mice in addition, could be a feasible alternative for Toxoplasma vaccine.

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Dermal dendrocytes FXIIIa+ are essential antigen-presenting cells in indeterminate leprosy.

Indeterminate leprosy (IL) is the early phase of Hansen disease and reword (APCs). Langerhans cells and dermal dendrocytes FXIIIa positive (DDFXIIIa) are the major APCs in the skin and can be identified by the expression of CD1a and FXIIIa, respectively, by immunohistochemical techniques. Plasmacytoid dendritic cells (PDCs) are another type of dermal dendrocytes with a questionable antigen-presenting function and can be highlighted by anti-CD123 expression. To our knowledge, there are no studies evaluating DDFXIIIa and PDC in IL. The purpose was to investigate the involvement of these cells in the pathogenesis of IL. The authors performed a retrospective study on 18 cases of IL (10 confirmed and 8 suspected) to investigate expression of FXIIIa, CD1a, and CD123. The results were compared with normal skin (for CD1a and FXIIIa only). A higher amount of FXIIIa-positive cells (P , 0.05) in confirmed and suspected IL cases was noted when comparing with normal skin. However, CD1a showed no quantitative differences in the epidermis of IL lesions when comparing with normal skin and CD123 expression was negligible. Based on these findings, the authors postulate that Langerhans cells and PDCs do not have a major role in IL and that DDFXIIIa may be the main APCs in IL. Further study is required to establish this.

Thymol and eugenol derivatives as potential antileishmanial agents.

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 µg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples.

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma.

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions.

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC(50) of 11.26 µg/ml against promastigotes and amastigotes of 0.27 µg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests.

Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages.

Leishmaniasis is a parasitic disease caused by the intramacrophage protozoa Leishmania spp. and may be fatal if left untreated. Although pentavalent antimonials are toxic and their mechanism of action is unclear, they remain the first-line drugs for treatment of leishmaniasis. An effective therapy could be achieved by delivering antileishmanial drugs to the site of infection. Compared with free drugs, antileishmanial agent-containing liposomes are more effective, less toxic and have fewer adverse side effects. The aim of this study was to develop novel meglumine antimoniate (MA)-containing liposome formulations and to analyse their antileishmanial activity and uptake by macrophages. Determination of the 50% inhibitory concentration (IC(50)) values showed that MA-containing liposomes were ≥10-fold more effective than the free drug, with a 5-fold increase in selectivity index, higher activity and reduced macrophage toxicity. The concentration required to kill 100% of intracellular amastigotes was ≥40-fold lower when MA was encapsulated in liposomes containing phosphatidylserine compared with the free drug. Fluorescence microscopy analysis revealed increased uptake of fluorescent liposomes in infected macrophages after short incubation times compared with non-infected macrophages. In conclusion, these data suggest that MA encapsulated in liposome formulations is more effective against Leishmania-infected macrophages than the non-liposomal drug. Development of liposome formulations is a valuable approach to the treatment of infectious diseases involving the mononuclear phagocyte system.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii.

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.

Paracoccidioides brasiliensis causing a rib lesion in an adult AIDS patient.

Paracoccidioidomycosis is a systemic mycosis with a geographic distribution that is limited to Central and South America; Brazil has the highest number of cases. Severe disseminated disease caused by paracoccidioidomycosis was observed in acquired immunodeficiency syndrome patients who live or have resided in endemic paracoccidioidomycosis areas. Here we describe a male patient admitted to a large public hospital with diffuse nodular infiltrates observed in chest radiographs and with erosion at the second rib near the sternum. Blood tests showed anti-human immunodeficiency virus antibodies, a human immunodeficiency virus viral load of 59,700 (4.8 log), and CD4 144/mm(3), with negative serology result for fungal infections. Aspirate of the rib lesion showed cells with a typical morphology of Paracoccidioides brasiliensis, aside from benign inflammatory cells. The histology of the rib biopsy showed typical granulomas and immunostained fungal cells. Although there was no growth in the Sabouraud cultures, Paracoccidioides brasiliensis gp43 and rDNA genes were detected in the aspirate by polymerase chain reaction. Therapy with amphotericin resulted in complete recovery. This type of bone lesion is rare and has been described primarily in the juvenile form of paracoccidioidomycosis; it must be included in the differential diagnosis of bone lesions in adult acquired immunodeficiency syndrome patients of endemic areas.

TGF-beta and mesenchymal hepatic involvement after visceral leishmaniasis.

The liver involvement in the human visceral leishmaniasis (VL) has been related to parasitism and activated Kupffer cells with further occasional fibrotic alterations, especially after long-term disease without treatment. However, fibrotic alterations have been reported after therapy, whose clinical finding is the persistence of hepatomegaly. Fibrotic involvement of the liver after therapy was never well understood, and the aim of this study was to evaluate this finding through ultrastructural and morphometric analysis. A case-control study was performed with 20 patients (15 cases and five controls). Cases included patients with persistent hepatomegaly (residual) after treatment of VL submitted to liver biopsy to exclude other causes of liver enlargement, including serum tests of viral hepatitis. The material was evaluated by electron microscopy allowing ultrastructural with morphometric analysis of medium portion of hepatic lobule. Narrow sinusoidal lumen and prominent Kupffer cells were found with insignificant alterations of hepatocytes, pit, and endothelial cells. On ultrastructural analysis, the enlargement of the space of Disse was due to fibrous collagen, increase of number of Ito cells, and nonfibrous extracellular matrix that were associated with Kupffer cells enlargement. Immunohistochemistry showed an intense expression of TGF-beta in patients with VL. These findings suggest a production of TGF-beta by Kupffer cells that resulted in the characteristic fibrotic involvement of the liver. Residual hepatomegaly in visceral leishmaniasis could result from sustained Kupffer cell activation with perihepatocytic fibrosis.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion.

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

Lung involvement in childhood measles: severe immune dysfunction revealed by quantitative immunohistochemistry.

Measles, accounting for nearly 1 million deaths each year, presents intense involvement of lymphoid organs and the lungs. The immune response in situ in the lungs was determined in blocks recovered from 42 necropsies of children who died from measles determined by immune cell phenotype (CD4, CD8, CD20, CD45RO, CD68, natural killer [NK], and antigen S-100 B [S100]) and cytokine production (interferon, tumor necrosis factor, interleukin [IL]-1, IL-2, IL-4, IL-10, and IL-12). Compared with the lungs of age-paired controls, patients with measles presented severe depletion of CD4+, CD20+, CD68+, NK+, and S100+ cells in alveolus- and bronchus-associated lymphoid tissue without depletion of CD8+ cells. Most of these features were similar in both forms of measles lung involvement, Hecht giant cell, or interstitial pneumonia, but S100+ cells were depleted in bronchus-associated lymphoid tissue from patients with Hecht pneumonia, which also occurs more frequently in malnourished children. IL-10- and IL-12-producing cells were depleted in patients with measles, whereas IL-1-, interferon-, and IL-4-producing cells were more frequently seen in the alveolus of patients with measles compared with controls. Quantitative in situ immune cell phenotype and function in the lung in measles demonstrated severe immune dysfunction, with loss of key cells, such as dendritic, CD4+, and NK+ cells, and deficient cytokine production, which allows for a better comprehension of local reactions in this process.

Supplementation of CXCL12 (CXCL12) induces homing of CD11c+ dendritic cells to the spleen and enhances control of Plasmodium berghei malaria in BALB/c mice.

In malaria, parasitaemia is controlled in the spleen, a multicomponent organ that undergoes changes in its cellular constituents to control the parasite. During this process, dendritic cells (DCs) orchestrate the positioning of effector cells in a timely manner for optimal parasite clearance. We have recently demonstrated that CXCL12 [stromal cell-derived factor-1 (CXCL12)] supplementation partially restores the ability to control parasitaemia in Plasmodium berghei-infected mice. In the present study, we investigated the nature of the DCs involved by flow cytometry and immunohistochemistry of CD11c(+) cells. Flow cytometry of bone marrow cells showed that infection with P. berghei did not alter the proportion of CD11c(+) cells present in this haematopoietic compartment, while CXCL12 supplementation of naïve uninfected mice induced only minor increases in the population of CD11c(+) cells. In the spleen, P. berghei infection alone resulted in an increase in CD11c(+) cells as compared with naïve animals. Exogenously administered CXCL12 in the absence of infection resulted in a significant expansion of the splenic CD11c(+) population, and this effect was even more pronounced in infected and supplemented mice. Immunohistochemistry revealed that CD11c(+) cells infiltrated the perivascular areas and marginal zone of the spleen in infected animals treated with CXCL12, suggesting that this chemokine induces homing of CD11c(+) dendritic cells to the splenic compartment. Our results show that small amounts of CXCL12 supplementation are effective in recruiting DCs to the spleens of both uninfected and infected mice, suggesting the participation of CXCL12 and CD11c(+) cells in the establishment of an adequate environment in the spleen for malaria control.

Targeting Leishmania (L.) chagasi amastigotes through macrophage scavenger receptors: the use of drugs entrapped in liposomes containing phosphatidylserine.

OBJECTIVES: We devised liposome-entrapped antimony with the negatively charged lipid phosphatidylserine-liposome-entrapped antimony (Sb-LP)-in order to improve their targeting to infected macrophages through the interaction with scavenger receptors (SRs). METHODS: SR production was indirectly evaluated by its mRNA synthesis in infected and uninfected peritoneal macrophages using RT-PCR. The interaction and cytotoxicity of Sb-LP with SRs and their metabolism were determined by incubation with macrophages in the presence of cytochalasin B, chloroquine or different competitive ligands, with determination of the 50% inhibitory concentration (IC50) in vitro in infected macrophages. The intracellular trafficking of Sb-LP was evaluated by confocal microscopy using trapped fluorescent dyes. RESULTS: Our results showed an up-regulation of macrophage SR mRNA during the initial steps of Leishmania (L.) chagasi infection. By competitive ligand assays, we demonstrated the preferential uptake of Sb-LP by macrophage SRs. Sb-LP was 16-fold more effective (IC50=14.11 microM) than the free drug (IC50=225.9 microM) against L. (L.) chagasi-infected macrophages. The binding and uptake of Sb-LP in macrophages were shown to be energy-dependent and were reduced in the presence of cytochalasin B, showing the dependency of the cell microfilament system. Confocal analysis using trapped fluorescent dyes showed fluorescence of parasites or in their close proximity, compatible with the localized delivery of the liposomes. CONCLUSIONS: The uptake of Sb-LP was reduced in infected macrophages, despite their effectiveness and targeting ability, suggesting a low metabolic rate in infected macrophages that could be overcome by the higher efficiency of the liposomal formulation. These in vitro results suggest that liposomes could improve the therapeutic index of old drugs, such as pentavalent antimony, via targeted delivery to Leishmania-infected cells.

Mucosal leishmaniasis: in situ characterization of the host inflammatory response, before and after treatment.

Mucosal leishmaniasis (ML) generally shows progressive tissue destruction, not yet fully elucidated, associated with an intense inflammatory response. To contribute to the understanding of this process and of how treatment interferes with it, we studied several anatomopathological parameters, including those analyzed by immunohistochemistry, such as Leishmania antigens, cells participating in the immune response and cytokine expression. Biopsies were taken from 20 patients with ML before and after treatment. A mixed Th1 and Th2 pattern response occurred inside ML before treatment, persist after treatment. Nevertheless, this mixed response was smaller than in active lesions, with reduced but present numbers of cells expressing TNF-alpha, IFN-gamma and IL-4 and sustained numbers of cells expressing IL-10. We may conclude that specific treatment causes a reduction of inflammatory lesions and disappearance of amastigote forms of Leishmania although the factors related to the pathogenesis of the lesion, such as T CD4+ and T CD8+ lymphocytes and Leishmania antigens, persist in treated lesions. The maintenance of these inflammatory patterns may be due to a specific host-parasite relationship response, strongly indicating the need for continuous surveillance of LM patients at risk of reactivation, despite effective cicatrization after therapy.