Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.

Lactobacillus delbrueckii UFV-H2b20 increases IFN-γ production and CD39+CD73+ Treg cell numbers in lungs, and protects mice against experimental allergic asthma.

Asthma is a disorder characterized by airflow obstruction, inflammation, declining airway function, bronchial hyperresponsiveness and tissue remodelling. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host". The use of probiotics is becoming increasingly studied and recent evidence has suggested that it may provide therapeutic benefits in asthma and other diseases. Lactobacillus delbrueckii UFV-H2b20 fulfils all the requirements to be classified as probiotic. Previous studies have already shown the ability of L. delbrueckii UFV-H2b20 to stimulate the immune system. Our objective was to evaluate the protective effects of L. delbrueckii UFV-H2b20 in experimental allergic asthma. We used a murine model of ovalbumin-induced allergic airway inflammation to mimic allergic asthma. Oral treatment with L. delbrueckii UFV-H2b20 improves respiratory parameters and inhibits the inflammatory response in the lungs by decreasing the numbers of inflammatory monocytes, eosinophils and alveolar macrophages, as well as IgE levels. Treatment increased the IFN-γ/IL-4 cytokine ratio. Levels of IL-10 in the lungs were also increased in treated animals. Our results also showed that the probiotic administration increases the number of CD39+CD73+ T regulatory lymphocytes in the lung, suggesting a role for purinergic signals in the regulation of inflammation promoted by the treatment. Understanding the mechanisms of modulation of the immune system by probiotics could allow the development of probiotic preparations that are safe and have a direct action. Our results suggest that oral administration of L. delbrueckii UFV-H2b20 could be helpful to treat chronic inflammatory airway diseases, such as asthma.

The adjuvant effect of interleukin-12 in a vaccine against Leishmania major.

Protection induced by vaccination depends on the capacity of the vaccine to elicit an appropriate immune response. In leishmaniasis, protection requires leishmanial-specific CD4+ T helper (TH) cells. Vaccination of BALB/c mice with leishmanial antigens and interleukin-12 (IL-12) promoted the development of leishmanial-specific CD4+ TH1 cells. These mice were resistant to subsequent infection with Leishmania major. Thus, IL-12 is an effective adjuvant for the initiation of protective cell-mediated immunity against leishmaniasis and may be an important component in other vaccines that need to induce cell-mediated immunity.

Early infection with Leishmania major restrains pathogenic response to Leishmania amazonensis and parasite growth.

Experimental models of infection with Leishmania spp. have provided knowledge of several immunological events involved in the resistance mechanism used by the host to restrain parasite growth. It is well accepted that concomitant immunity exists, and there is some evidence that it would play a major role in long-lasting acquired resistance to infection. In this paper, the resistance to Leishmania amazonensis infection in C57BL/6 mice infected with Leishmania major was investigated. C57BL/6 mice, which spontaneously heal lesions caused by infection with L. major, were infected with L. amazonensis at different times before and after L. major. We demonstrated that C57BL/6 mice previously infected with L. major restrain pathogenic responses induced by L. amazonensis infection and decrease parasite burdens by one order of magnitude. Co-infected mice showed production of IFN-gamma in lesions similar to mice infected solely with L. major, but higher TNF-alpha and nitric oxide synthase (iNOS) mRNA expression was observed. Surprisingly, the restrained pathogenic response was not related to IL-10 production, as evidenced by lower levels of both mRNA, protein expression in lesions from co-infected mice and in co-infections in IL-10(-/-) mice. Examination of the inflammatory infiltrate at the site of infection showed a reduced number of monocytes and lymphocytes in L. amazonensis lesions. Additionally, differential production of the CCL3/MIP-1 alpha and CCL5/RANTES was observed. We suggest that the control of lesion progression caused by L. amazonensis in C57BL/6 mice pre-infected with L. major is related to the induction of a down-regulatory environment at the site of infection with L. amazonensis.

Evidence for the occurrence of LDL receptors in extracts of schistosomula of Schistosoma mansoni.

Schistosomula of Schistosoma mansoni were shown to contain proteins on their surface membranes which bind iodinated human low density lipoproteins (125I-LDL). Treatment of the parasites with trypsin decreased the binding in comparison with untreated controls. Membrane-bound, acetone-insoluble proteins were extracted from the schistosomula with Triton X-100 and the extract in liposome form was incubated with 125I-LDL at room temperature. After incubation a complex was formed between the proteins present in the extract and 125I-LDL, as shown by a filter binding assay. 125I-LDL binding to filters was proportional to the amount of protein in the extract; it was inhibited by unlabelled LDL and VLDL and by EDTA. Binding of 125I-LDL to proteins present in the liposome suspension containing the Triton X-100 extract followed saturation kinetics, indicating the occurrence of receptors for lipoproteins in the extract.

Infection with Leishmania major induces interleukin-12 production in vivo.

Experimental infections of mice with the protozoan parasite Leishmania major provide an excellent model for defining the conditions required for generation of CD4+ Th1 and Th2 cells in vivo. Since interleukin-12 (IL-12) has been implicated in the development of Th1 cells, we investigated whether L. major stimulates IL-12 production in vitro or in vivo. Surprisingly, macrophages cultured in vitro failed to produce IL-12 following L. major infection. In contrast, lymph node cells from C3H mice infected for 2 days with L. major produced elevated levels of IL-12. In order to determine if the inability to stimulate IL-12 production was limited to in vitro infections, we infected macrophages in vivo by inoculating L. major into the peritoneal cavity. Peritoneal cells isolated 24 h later exhibited a significant increase in the number of cells producing IL-12. In addition, supernatants harvested from these cells following culture contained elevated levels of IL-12. These data indicate that L. major infection induces increased IL-12 production in mice.

Effects of antihypertensive therapy on platelet cytosolic calcium responses to low density lipoprotein cholesterol.

This study examines the effects of antihypertensive therapy on platelet cytosolic calcium [Ca2+]i responses to low-density lipoprotein cholesterol (LDL) and vasopressin (AVP) in 15 patients (50-80 years) participating in the Hypertension Optimal Treatment International Study. All patients (diastolic blood pressure (DBP) > or = 100 mm Hg and < or = 115 mm Hg) were treated with the calcium antagonist felodipine (10 mg p.o.) with or without addition of enalapril (up to 20 mg daily as needed) to lower diastolic pressures to < 85 mm Hg. This antihypertensive therapy lowered DBP (104 +/- 0.8 to 78 +/- 1.6 mm Hg, P < 0.0001), but had no effect on basal [Ca2+]i or AVP-stimulated [Ca2+]i responses. Basal platelet [Ca2+]i following antihypertensive therapy (49 +/- 3.4 ng/ml) were not different from those prior to therapy (52 +/- 4.7 ng/ml). Additionally, [Ca2+]i responses to AVP following therapy (554 +/- 74 units) were not different from those prior to treatment (595 +/- 49 units). Following antihypertensive therapy, [Ca2+]i responses to 200 micrograms/ml of LDL were decreased fourfold (P < 0.05). These results suggest that antihypertensive therapy with a calcium channel blocker may potentially impact the atherogenic process by reducing the platelet [Ca2+]i rise, and potentially the aggregatory response, to LDL.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house flies (LD50 80.0 ng/house fly). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house fly). The amino-acid sequences of these toxins were established and found to be similar (60-95%) to previously described beta-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure-function relationships that may be responsible for the differences in bioactivity displayed by these toxins.

Screening of expression libraries using ELISA: identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom.

The present report describes the use of ELISA with cDNA expression libraries in the identification of immunogenic proteins. The methodology described was applied using libraries constructed with mRNA isolated from Tityus serrulatus and Tityus bahiensis venom glands. In addition we describe for the first time the sequence of a neurotoxin from Tityus bahiensis venom gland named TbTx5 whose amino acid sequencing showed 93% similarity with the Tityus bahiensis TbTx IV-5 neurotoxin. The methodology described can be used for the generation of an immunogenic bank in order to contribute to genome and proteome projects.

Immune responses of germfree mice to experimental infection with Trypanosoma cruzi.

1. The immune responses to Trypanosoma cruzi infection of germfree mice were compared to the responses of infected conventional mice. Two groups (40 animals in each group) of 2-month old female CFW germfree and conventional mice were used. The IgM and IgG which bound to the surface of T. cruzi epimastigotes determined by ELISA were significantly lower in germfree than in conventional mice (1/3 and 1/5 for IgM and IgG, respectively). 2. After infection there was a three-fold increase in the circulating levels of these immunoglobulins in germfree but not in conventional mice. Twenty-one days after T. cruzi inoculation, both IgG and IgM levels were similar in germfree and conventional animals. 3. Footpad swelling after T. cruzi-antigen inoculation was initially four-fold more intense in germfree than in conventional mice. 4. These results suggest that the reduced humoral immune response of germfree mice during the initiation of experimental Chagas' disease may be responsible for the more severe parasitism when compared to conventional mice.

Effect of an essential fatty acid deficient diet on experimental infection with Trypanosoma cruzi in germfree and conventional mice.

1. To investigate the possible role of essential fatty acid deficiency in host cell/parasite interaction, weanling germfree (GF) and conventional (CV) CFW mice maintained on an essential fatty acid-deficient (-) or a control (+) diet for 110 days were infected with Trypanosoma cruzi. 2. Blood parasitemia indicated that the disease was milder in the animals maintained on the essential fatty acid-deficient diet than in the GF and CV controls (maximum parasitemia: GF+ 33,300, GF-26,200, CV+ 17,100 and CV- 6,400 trypomastigotes/ml blood). 3. Survival 30 days after infection was 12% for GF+, 28% for GF-, 37% for CV+ and 65% for CV- mice. 4. Linoleic and arachidonic acid levels were significantly lower in animals kept on the essential fatty acid-deficient diet (GF-: 28.0 +/- 9.3, 23.4 +/- 8.6; CV-: 37.6 +/- 5.8, 19.9 +/- 3.6) than in controls (GF+: 164.4 +/- 48.8, 162.6 +/- 45.8; CV+: 147.1 +/- 26.5, 107.5 +/- 23.6) confirming the deficiency. 5. Before the infection, succinic dehydrogenase levels were higher in liver of all CV mice (4.52 micrograms phosphate/mg fresh tissue) than in GF mice (0.84 micrograms phosphate/mg fresh tissue), whereas the opposite was true for 5'-nucleotidase levels in brain and liver, respectively (GF: 2.84 and 3.18 micrograms phosphate/mg fresh tissue; CV: 1.25 and 1.54 micrograms phosphate/mg fresh tissue). 6. The disease was milder in deficient than in control animals in both the GF and CV groups on the basis of parasitemia and survival, indicating that fatty acid-deficient mice are partially protected against Chagas' disease. The mechanism underlying this phenomenon requires further investigation.

Immune responses and protection induced in mice by an industrialized vaccine against American cutaneous leishmaniasis.

An industrialized vaccine against American cutaneous leishmaniasis was compared to a laboratory made vaccine in its ability to induce cellular and humoral immune responses in mice. No differences were observed between seric IgG levels or lymphoblastic proliferation response of mice immunized with either vaccine. Antigenic composition, evaluated by SDS-PAGE, was identical in both preparations. Protection induced in mice against a challenge with infective parasites was also compared. The level of protection obtained with the industrialized vaccine was comparable to that induced by the laboratory made preparation. The results showed that the industrialization process did not alter the efficacy of the vaccine.

Filamentous fungi and media for cellulase production in solid state cultures.

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.

Characteristics of a tandem system of ionization chambers in X-ray beams, mammography level.

Two parallel plate ionization chambers (inserted in slab phantoms) recently assembled at IPEN were studied in relation to their operational characteristics for use in quality control of X-ray beams, mammography level. The chambers present only one difference: one has an inner collecting electrode made of graphite and the other, of aluminum. These chambers make up a tandem system, which may be employed to verify X-ray beams energy constancy, by the confirmation of half-value layers and effective energies, and to determinate air kerma rates. The chambers presented good results for the operational tests, as recommended internationally.

Filamentous fungi and media for cellulase production in solid state cultures

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.

Lactobacillus delbrueckii UFV-H2b20 induces type 1 cytokine production by mouse cells in vitro and in vivo.

Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-alpha by peritoneal cells and IFN-gamma by spleen cells from germ-free or monoassociated Swiss/NIH mice and LPS-hyporesponsive mice (around 40 ng/mL for IL-12 p40, 200 pg/mL for TNF-alpha and 10 ng/mL for IFN-gamma). Heat treatment of L. delbrueckii did not affect the production of these cytokines. Lysozyme treatment decreased IL-12 p40 production by peritoneal cells from C3H/HeJ mice, but did not affect TNF-alpha production by these cells or IFN-gamma production by spleen cells from the same mouse strain. TNF-alpha production by peritoneal cells from Swiss/NIH L. delbrueckii-monoassociated mice was inhibited by lysozyme treatment. When testing IL-12 p40 and IFN-gamma levels in sera from germ-free or monoassociated Swiss/NIH mice systemically challenged with Escherichia coli we observed that IL-12 p40 was produced at marginally higher levels by monoassociated mice than by germ-free mice (40 vs 60 ng/mL), but IFN-gamma was produced earlier and at higher levels by monoassociated mice (monoassociated 4 and 14 ng/mL 4 and 8 h after infection, germfree 0 and 7.5 ng/mL at the same times). These results show that L. delbrueckii UFV-H2b20 stimulates the production of type 1 cytokines in vitro and in vivo, therefore suggesting that L. delbrueckii might have adjuvant properties in infection in which these cytokines play a major role.

Immune response of bovines stimulated by synthetic vaccine SBm7462 against Rhipicephalus (Boophilus) microplus.

Ten-month-old calves Bos taurus taurus were immunized with three doses of SBm7462 with saponin as an adjuvant at 30-day intervals and were evaluated for IgG isotypes, phenotype circulating lymphocytes and changes in the lymph nodes (LN). SBm7462 stimulated the production of predominantly IgG1-isotype IgG antibodies. The lymph nodes exhibited activation at the seventh day after the first immunization, with areas of paracortical and interfollicular hyperplasia and the early formation of germinal centers (GC). Fifteen days after the first immunization, the GC exhibited compartmentalization of cellular populations, a light zone (LZ), a dark zone (DZ) and a mantle. At the same time, hyperplasia of the medullary cords was observed with cells associating with DC cells. Seven days after the first immunization, apoptosis in the DZ and in the paracortical region became evident. By day 15, there was an increase in the medullary cords, which became more numerous at days 35 and 42. PAP-positive cells were found in the paracortical region, medullary cords and GC 7 days after the first immunization. At day 35, there were further strongly PAP-positive cells in the medullary cords. By comparison, none of these changes were observed in the lymph nodes of control groups at any of the days analyzed. The number of CD21(+) lymphocytes increased in the immunized groups after the first inoculation, with a maximum number observed at 15 and 10 days after the first and third immunizations, respectively. Compared to pre-immunization counts, the percentage of WC1(+) gammadelta T-lymphocytes displayed more variation, increasing 5 days after the second immunization but decreasing over the following days. According to the results, the synthetic anti Rhipicephalus microplus vaccine elicits a complete immune response being T-dependant.

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice.

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.

Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions.

In order to investigate the importance of the host microbiota on differentiation of T cell subsets in response to infection, Swiss/NIH germ-free mice and conventional (microbiota-bearing) mice were infected with Leishmania major, and lesion development, parasite loads, and cytokine production were assessed. Germ-free mice failed to heal lesions and presented a higher number of parasites at the site of infection than their conventional counterparts. In addition, histopathological analysis indicated a higher density of parasitized macrophages in lesions from germ-free mice than in conventional mice. The initial production of interleukin (IL)-12 and interferon-gamma (IFN-gamma) in germ-free mice was comparable to the conventional controls. Also, germ-free mice produced elevated levels of IFN-gamma and lower levels of IL-4 throughout the course of infection, suggesting the development of a Th1 response. Macrophages from germ-free mice exposed to IFN-gamma and infected with amastigotes in vitro were not as efficient at killing parasites as macrophages from conventional animals. These observations indicate that the microbiota is not essential for the development of Th1 immune responses, but seems to be important for macrophage activation.