Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is t... more Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-chall...
Background: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phleboto... more Background: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. Methodology/Principal Findings: Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-b. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-c expression. Conclusions/Significance: Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.
T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activ... more T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activation requires signals provided by costimulatory molecules. Herein we evaluated the role of costimulatory molecules on cytokine production and T cell surface molecule expression by peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients. PBMC from CL patients were stimulated with soluble Leishmania antigen (SLA, 10 µg/ml), in the presence or absence of soluble CTLA4-Ig to block CD28-B7 interaction or in the presence or absence of anti-human CD40L to block CD40-CD40L interaction. Supernatants were harvested to evaluate tumor necrosis factor alpha (TNF-a), interleukin 10 (IL-10), transforming growth factor beta (TGF-b) and interferon gamma (IFN-c) production by ELISA. Cells were harvested after 48 h of culture, stained for specific activation markers and analyzed by flow cytometry. Results show that the blockade of CD28-B7 interaction by CTLA4-Ig downmodulated IFN-c, IL-10, and TNF-a secretion by PBMC from CL patients. No alteration was detected on either TGF-b production or the expression of CTLA44 or CD25 on CD4 + and CD8 + T cells. When the CD40-CD40L interaction was blockade using anti-CD40L, we did not observe changes in cytokine production or in surface molecule expression. The blockade of the CD28-B7 interactions by CTLA4-Ig also did not alter cytokine production in volunteers immunized against tetanus toxoid (TT). Taken together, these data suggest that the interaction of CTLA4 and CD28-B7 is a TGF-b-independent mechanism that specifically downmodulates the immune response in cutaneous leishmaniasis patients.
Toward obtaining a more comprehensive understanding of factors governing activation and/or functi... more Toward obtaining a more comprehensive understanding of factors governing activation and/or function during visceral leishmaniasis (VL), we have compared active disease (pre-treatment) versus post-chemotherapy immune response in VL patients by means of ex vivo staining with different cell markers. Our results show that during active disease, the frequency of T cells positive for CD25, CTLA-4 and CD45RO was significantly lower in VL patients compared with healthy controls, whereas cells staining positive for Annexin V and CD95 were significantly higher. In all cases, chemotherapy was able to restore these frequencies to normal levels. Interestingly, significant differences in the frequency of CD18 and in the frequency of CD45RO-positive cells were observed in the CD81 T cell subset. These two frequencies were also significantly higher in bone marrow when compared with peripheral blood, suggesting a possible compartmentalization of certain CD81 T cell populations during active disease. Given that CD81 T cells have been shown to play an essential role in immunity to infection with Leishmania, our data indicate that the lower frequency of CD181 and CD45RO1 lymphocytes in the bone marrow CD81 T cell subset may be considered a biomarker of acute VL.
In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly sal... more In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Mφs), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Mφs, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Mφs, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SG...
Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to m... more Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. Results Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations...
Periodontal diseases are well recognized as a result of the interaction between host immune respo... more Periodontal diseases are well recognized as a result of the interaction between host immune response and pathogenic bacteria species of the dental biofilm. 1 During the last few decades, evidence has linked periodontal infections with an increased risk of systemic complications, including rheumatoid arthritis, respiratory and cardiovascular diseases, diabetes mellitus and preterm low birth weight. 2-4 Although the precise mechanisms that explain the relationship between a r c h i v e s o f o r a l b i o l o g y 5 5
It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly sa... more It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-␥ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-␥/IL-10 and IFN-␥/TGF- in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-␥/IL-10 and IFN-␥/TGF- ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.
A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promast... more A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells
Background: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infant... more Background: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches. Methodology/Principal Findings: In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1610 7 parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35610 5 parasites in their lymph nodes. An increase of IFN-c and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection. Conclusion: The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.
Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is t... more Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-chall...
Background: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phleboto... more Background: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. Methodology/Principal Findings: Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-b. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-c expression. Conclusions/Significance: Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.
T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activ... more T cell-mediated immunity is critical in resistance against Leishmania parasites, and T cell activation requires signals provided by costimulatory molecules. Herein we evaluated the role of costimulatory molecules on cytokine production and T cell surface molecule expression by peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis (CL) patients. PBMC from CL patients were stimulated with soluble Leishmania antigen (SLA, 10 µg/ml), in the presence or absence of soluble CTLA4-Ig to block CD28-B7 interaction or in the presence or absence of anti-human CD40L to block CD40-CD40L interaction. Supernatants were harvested to evaluate tumor necrosis factor alpha (TNF-a), interleukin 10 (IL-10), transforming growth factor beta (TGF-b) and interferon gamma (IFN-c) production by ELISA. Cells were harvested after 48 h of culture, stained for specific activation markers and analyzed by flow cytometry. Results show that the blockade of CD28-B7 interaction by CTLA4-Ig downmodulated IFN-c, IL-10, and TNF-a secretion by PBMC from CL patients. No alteration was detected on either TGF-b production or the expression of CTLA44 or CD25 on CD4 + and CD8 + T cells. When the CD40-CD40L interaction was blockade using anti-CD40L, we did not observe changes in cytokine production or in surface molecule expression. The blockade of the CD28-B7 interactions by CTLA4-Ig also did not alter cytokine production in volunteers immunized against tetanus toxoid (TT). Taken together, these data suggest that the interaction of CTLA4 and CD28-B7 is a TGF-b-independent mechanism that specifically downmodulates the immune response in cutaneous leishmaniasis patients.
Toward obtaining a more comprehensive understanding of factors governing activation and/or functi... more Toward obtaining a more comprehensive understanding of factors governing activation and/or function during visceral leishmaniasis (VL), we have compared active disease (pre-treatment) versus post-chemotherapy immune response in VL patients by means of ex vivo staining with different cell markers. Our results show that during active disease, the frequency of T cells positive for CD25, CTLA-4 and CD45RO was significantly lower in VL patients compared with healthy controls, whereas cells staining positive for Annexin V and CD95 were significantly higher. In all cases, chemotherapy was able to restore these frequencies to normal levels. Interestingly, significant differences in the frequency of CD18 and in the frequency of CD45RO-positive cells were observed in the CD81 T cell subset. These two frequencies were also significantly higher in bone marrow when compared with peripheral blood, suggesting a possible compartmentalization of certain CD81 T cell populations during active disease. Given that CD81 T cells have been shown to play an essential role in immunity to infection with Leishmania, our data indicate that the lower frequency of CD181 and CD45RO1 lymphocytes in the bone marrow CD81 T cell subset may be considered a biomarker of acute VL.
In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly sal... more In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Mφs), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Mφs, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Mφs, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SG...
Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to m... more Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. Results Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations...
Periodontal diseases are well recognized as a result of the interaction between host immune respo... more Periodontal diseases are well recognized as a result of the interaction between host immune response and pathogenic bacteria species of the dental biofilm. 1 During the last few decades, evidence has linked periodontal infections with an increased risk of systemic complications, including rheumatoid arthritis, respiratory and cardiovascular diseases, diabetes mellitus and preterm low birth weight. 2-4 Although the precise mechanisms that explain the relationship between a r c h i v e s o f o r a l b i o l o g y 5 5
It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly sa... more It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-␥ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-␥/IL-10 and IFN-␥/TGF- in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-␥/IL-10 and IFN-␥/TGF- ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids.
A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promast... more A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells
Background: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infant... more Background: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches. Methodology/Principal Findings: In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1610 7 parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35610 5 parasites in their lymph nodes. An increase of IFN-c and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection. Conclusion: The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.
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