Histopathological and immunohistochemical aspects of American cutaneous leishmaniasis before and after different treatments.

BACKGROUND: The histopathology and immune responses of the healing process of leishmaniasis are still poorly studied. OBJECTIVES: This study aimed to examine the histopathological and immunological aspects of lesions of patients with cutaneous leishmaniasis before and after different therapeutic methods. METHODS: We studied 23 individuals grouped according to the treatments: Glucantime, Glucantime + Leishvacin and Glucantime + Leishvacin associated with Bacillus Calmette-Guerin. For analysis of the histopathological changes present in the dermis and epidermis, histological sections were stained with hematoxylin and eosin. The samples were immunostained before and after treatment to analyze the expression of interferon (IFN)-γ, interleukin (IL) 12, IL-10 and IL-4. RESULTS: Before treatment the presence of intense infiltrates of mononuclear cells was noticed and after treatment, even with a diagnosis of clinical cure, the subjects still showed a moderate inflammatory process. In the immunohistochemical analyses, we noticed a difference between the cytokines, with increased expression of cytokines IFN-γ and IL-12 compared to IL 10 and IL-4, both before and after treatment and, comparatively, the difference in this expression was more intense before treatment. However, the cytokine expression analyzed by treatment group showed no statistically significant difference. CONCLUSION: We conclude that a clinical cure does not always coincide with the histopathological one, and that before treatment there is a predominance of Th1 cytokines. In terms of treatment type, there was no difference in the progression of healing for all the three types of treatment, indicating their clinical equivalence.

Cluster randomised trial to evaluate the effectiveness of a vaccine against cutaneous leishmaniasis in the Caratinga microregion, south-east Brazil.

BACKGROUND: The eco-epidemiological complexity of American cutaneous leishmaniasis (ACL) has made it difficult to devise an efficient strategy for management of the disease, and development of an effective vaccine remains the most promising approach. The objective of the study was to determine the reduction in incidence of ACL following intramuscular administration of two doses of a killed Leishmania (Leishmania) amazonensis vaccine. METHODS: A cluster randomised trial was conducted from 2002 to 2011 in 108 localities in an endemic area of southeast Brazil. Communities were stratified according to population size, and randomly allocated to receive vaccine (n = 50) or placebo (n = 58). The post-vaccination ACL incidence rates in the two groups were compared through covariance analysis. RESULTS: A cyclic fluctuation in the number of cases recorded during the 18-year pre-vaccination period was similar in both groups. Following the vaccination campaign, a significant reduction in the number of cases of ACL was observed in the vaccine group compared with the placebo group. This group also included the individuals who refused to participate in the trial. CONCLUSION: This study demonstrated that the vaccine has been able to confer protection against ACL up to the present time. It is necessary to continue epidemiological surveillance to determine the duration of the vaccine's effectiveness.

Histopathological and immunohistochemical aspects of American cutaneous leishmaniasis before and after different treatments* / Aspectos histopatológicos e imuno-histoquímicos da leishmaniose tegumentar americana antes e depois de diferentes tratamentos

The histopathology and immune responses of the healing process of leishmaniasis are still poorly studied. This study aimed to examine the histopathological and immunological aspects of lesions of patients with cutaneous leishmaniasis before and after different therapeutic methods. We studied 23 individuals grouped according to the treatments: Glucantime, Glucantime + Leishvacin and Glucantime + Leishvacin associated with Bacillus Calmette-Guerin. For analysis of the histopathological changes present in the dermis and epidermis, histological sections were stained with hematoxylin and eosin. The samples were immunostained before and after treatment to analyze the expression of interferon (IFN)-γ, interleukin (IL) 12, IL-10 and IL-4. Before treatment the presence of intense infiltrates of mononuclear cells was noticed and after treatment, even with a diagnosis of clinical cure, the subjects still showed a moderate inflammatory process. In the immunohistochemical analyses, we noticed a difference between the cytokines, with increased expression of cytokines IFN-γ and IL-12 compared to IL 10 and IL-4, both before and after treatment and, comparatively, the difference in this expression was more intense before treatment. However, the cytokine expression analyzed by treatment group showed no statistically significant difference. We conclude that a clinical cure does not always coincide with the histopathological ...

A histopatologia e as respostas imunológicas do processo de cura da leishmaniose são ainda pouco estudadas. Este estudo teve como objetivo avaliar os aspectos histopatológicos e imunológicos das lesões de pacientes com leishmaniose tegumentar, antes e após diferentes métodos terapêuticos. Foram estudados 23 indivíduos agrupados de acordo com os tratamentos: Glucantime, Glucantime + Leishvacin e Glucantime + Leishvacin associado com Bacilo Calmette-Guerin. Para a análise das alterações histopatológicas presentes na derme e epiderme, cortes histológicos foram corados com hematoxilina e eosina. Para avaliar a expressão de interferon (IFN)-γ, interleucina (IL) 12, IL-10 e IL-4 foi utilizada a técnica de imuno-histoquímica antes e após o tratamento. Antes do tratamento houve um intenso infiltrado de células mononucleares, após o tratamento, mesmo com um diagnóstico de cura clínica, apresentou-se ainda um moderado processo inflamatório. Na análise imuno-histoquímica, notamos uma diferença entre as citocinas, com expressão aumentada de citocinas IFN-γ e IL-12 em comparação com IL-10 e IL-4 tanto antes quanto depois do tratamento, e comparativamente, a diferença nesta expressão mostrou-se mais intensa antes do tratamento. No entanto, a expressão das citocinas analisadas por grupo de tratamento não mostraram diferenças ...

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with American cutaneous leishmaniasis.

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.

In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune(®) induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+ and B-lymphocytes, whereas Leishmune(®) promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-γ/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-γ/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL).

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis.

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06 percent and 85.9 percent, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Increase of NK cells and proinflammatory monocytes are associated with the clinical improvement of diffuse cutaneous leishmaniasis after immunochemotherapy with BCG/Leishmania antigens.

Diffuse cutaneous leishmaniasis (DCL) is characterized by disseminated lesions and the absence of a specific cellular immune response. Here, the immunochemotherapy outcome of a patient with DCL from Amazonian Brazil infected with Leishmania (Leishmania) amazonensis is presented. After several unsuccessful chemotherapy treatment regimens and many relapses, a monthly immunotherapy scheme of L. amazonensis PH8 plus L. (Viannia) braziliensis M2903 monovalent vaccines associated with Bacillus Calmette-Guerin (BCG) was established, one round of which also included an M2903 vaccine associated with intermittent antimonial treatment. Temporary healing of all lesions was achieved, although Leishmania skin tests were negative and interferon gamma was not detected in mononuclear cell cultures stimulated with Leishmania antigens. The frequencies of CD16 (+)CD56(+) NK cells (approximately 2x) and CD14 (+)CD16(+) proinflammatory monocytes (approximately 8x) increased in peripheral blood, and CD56 (+) lymphocytes were found infiltrating the lesions. An association between the increase of the frequency of innate immune system cells and the healing of lesions is shown, suggesting that this protocol of immunotherapy reduced the parasite load and activated NK cells and monocytes.

Alterations in phenotypic profiles of peripheral blood cells from patients with human American cutaneous leishmaniasis following treatment with an antimonial drug and a vaccine.

The susceptibility or resistance of a vertebrate host to leishmaniasis is related to the species of Leishmania and to the host immune response of the host. In the present study, the phenotypic profiles of the peripheral blood cells of patients with American cutaneous leishmaniasis (ACL) were evaluated before and after receiving three different therapeutic regimens. The study population comprised 24 patients, living in an ACL-endemic area of Caratinga (MG, Brazil), who had been diagnosed as ACL-positive on the basis of characteristic lesions, the Montenegro skin reactivity test, and/or positive parasitology. Subjects were divided into three groups and received treatment regimens based on (i) the pentavalent antimonial (SbV) N-methyl meglumine antimoniate (Glucantime), (ii) the vaccine Leishvacin, or (iii) SbV in association with the vaccine. Comparative analyses of peripheral mononuclear cells prior to and after treatment revealed that the therapeutic regimens induced no significant differences in the percentages of CD3+ and CD4+ T lymphocytes, CD19+ B lymphocytes, or CD16+ and CD56+ natural killer cells. Additionally, the CD4/CD8 and CD3/CD19 ratios remained unaltered by any of the treatments applied. Most previous studies in the field have focused on the analysis of peripheral blood from ACL patients following in vitro stimulation with either Leishmania antigens or mitogens. The ex vivo cellular immune phenotypic profiles determined in the present study, however, revealed that different ACL treatments did not significantly alter either the immune response exhibited by a patient prior to therapy or the expected cure rate.

T-cell-derived cytokines, nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune.

It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n=6) and Leishmune (n=6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune, with the Leishvaccine triggering a mixed, IFN-gamma and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune induced an immunological pattern characterized by enhanced levels of IFN-gamma, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune, the ability of both immunobiologicals to activate T-cell-derived IFN-gamma synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.

Advances in flow cytometric serology for canine visceral leishmaniasis: diagnostic applications when distinct clinical forms, vaccination and other canine pathogens become a challenge.

We have previously reported the applicability of flow cytometry anti-fixed Leishmania infantum chagasi promastigotes IgG (FC-AFPA-IgG) as a novel serological device for laboratorial diagnosis of CVL. Herein, we validate throughout a blind study applied into a broader range of coded sera samples that FC-AFPA-IgG at serum dilution 1:8192 have an outstanding performance to discriminate the serological reactivity of canine visceral leishmaniasis (CVL, n=64) and Leishmune vaccines (VAC, n=62) and non-infected controls (NI, n=25). Moreover, we have evaluated the performance of indirect immunofluorescence antibody test (IFAT) and the crude-antigen enzyme-linked immunosorbent assay (ELISA) in parallel with FC-AFPA-IgG, to discriminate the seroreactivity of NI, CVL and VAC. Our data demonstrated that both ELISA and FC-AFPA-IgG showed similar performance to detect the seronegativity in 100% of NI, whereas FC-AFPA-IgG displayed better performance to exclude seropositivity in 100% of VAC. The high kappa agreement indexes observed suggested similar performance between these two serological testes when distinct clinical forms of CVL become a challenge. Furthermore, the FC-AFPA-IgG applied at sera dilution 1:8192 showed a remarkable performance to discriminate CVL from other co-endemic canine infections with high co-negativity in dogs infected with Trypanosoma cruzi and Leishmania braziliensis (86% and 84%, respectively). In conclusion, the data presented here re-emphasize the applicability of FC-AFPA-IgG as an innovative methodology able to discriminate post-infection imunomediated seroreactivity from that triggered by prophylactic immunization with minor cross-reactivity with other relevant canine pathogens, which may contribute as a supplementary assay for the CVL immunodiagnosis.

Systemic and compartmentalized immune response in canine visceral leishmaniasis.

Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.

Despite Leishvaccine and Leishmune trigger distinct immune profiles, their ability to activate phagocytes and CD8+ T-cells support their high-quality immunogenic potential against canine visceral leishmaniasis.

Phenotypic features of peripheral blood leukocytes have been investigated as a pre-requisite to characterize the protective immunity attributed to both Leishvaccine and Leishmune. Our results showed that either those vaccine were accompanied by distinct profiles on innate immune compartment. While Leishvaccine promoted early changes in phenotypic features of neutrophils and eosinophils with late involvement of monocytes, Leishmune induced early and persistent activation of neutrophils and monocytes, without changes on eosinophil activation status. Regarding the adaptive immunity, Leishvaccine sponsored a mixed profile, associated with phenotypic changes of T and B-lymphocytes. Major phenotypic changes in CD4+ T-cells with transient activation of CD8+ T-cell, besides decreased frequency of B-cell expressing CD32 were the hallmark of Leishvaccine. In contrast, Leishmune was associated with phenotypic changes in T-lymphocytes, particularly in CD8+ T-cells, and selective up-regulation of CD3+CD5+LowCD8+ cells. We hypothesized that this dissimilar alteration in immunological events would represent phenomenon directly related with the molecular nature of these vaccines besides the distinct adjuvants employed. However, it is important to emphasize that both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered priority vaccines with a high-quality immunogenic potential against CVL.

Evaluation of an immunochemotherapeutic protocol constituted of N-methyl meglumine antimoniate (Glucantime) and the recombinant Leish-110f + MPL-SE vaccine to treat canine visceral leishmaniasis.

The evaluation of the efficacy of an immunochemotherapy protocol to treat symptomatic dogs naturally infected with Leishmania chagasi was studied. This clinical trial had the purpose to test the combination of N-methyl meglumine antimoniate (Glucantime and the second generation recombinant vaccine Leish-110f plus the adjuvant MPL-SE to treat the canine leishmaniasis (CanL). Thirty symptomatic naturally infected mongrel dogs were divided into five groups. Animals received standard treatment with Glucantime or treatment with Glucantime Leish-110f + MPL-SEas immunochemotherapy protocol. Additional groups received Leish-110f + MPL-SE only, MPL-SE only, or placebo. Evaluation of haematological, biochemical (renal and hepatic function) and plasmatic proteins, immunological (humoral and cellular immune response) and the parasitological test revealed improvement of the clinical parameters and parasitological cure in dogs in both chemotherapy alone and immunochemotherapy cohorts. However, the immunotherapy and immunochemotherapy cohorts had reduced number of deaths, higher survival probability, and specific cellular reactivity to leishmanial antigens, in comparison with chemotherapy cohort only and control groups (adjuvant alone and placebo). These results support the notion of using well-characterized recombinant vaccine as an adjunct to improve the current chemotherapy of CanL.

Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis.

While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania (Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti-Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8+ T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21+ B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8+ lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.

Histopathological and immunohistochemical investigations of the hepatic compartment associated with parasitism and serum biochemical changes in canine visceral leishmaniasis.

The immunopathological evaluation of the hepatic compartment associated with parasitism and biochemical findings are essential for understanding the genesis of hepatomegaly in canine visceral leishmaniasis (CVL). Three clinical groups of dogs naturally infected with Leishmania chagasi [i.e., asymptomatic (AD, n=12), oligosymptomatic (OD, n=12) and symptomatic (SD, n=17)] were assessed and compared with a group of non-infected dogs (NID, n=11). Intense reaction of the Kupffer cells, capsule and portal inflammation, and the presence of intralobular granulomas, were observed in the different clinical groups. Dogs in the SD group presented a higher frequency of parasitism compared with the AD group. Inflammatory alterations were more intense in the SD group and were associated with parasitism. Our results indicated an association between histological liver changes and the progression of biochemical alterations according to progression of clinical forms of CVL, and the direct relationship between clinical symptoms and frequency of hepatic parasitism.

Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoa Leishmania chagasi (syn. L. infantum) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vaccination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a promising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8(+) that would be related to the control of tissue parasitism. In addition, a higher production of anti-Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated.

The influence of copper, selenium and zinc on the response to the Montenegro skin test in subjects vaccinated against American cutaneous leishmaniasis.

We evaluated the relationship between the trace elements copper, zinc and selenium and the response to the Montenegro skin test (MST) in 172 volunteers vaccinated against American cutaneous leishmaniasis. The MST diameter was categorized as negative and in quartiles of positive response, constituting five groups. Trace element serum levels were analyzed by coupled plasma atomic emission spectrometry and hydride generation atomic absorption spectrometry, with study subjects classified into two groups depending on low or high levels of trace elements observed. MST-positive subjects had an MST diameter (mean+/-SD) of 10.35+/-4.64mm, with copper, selenium and zinc serum levels of 1433.7+/-665.7microg/l, 88.6+/-39microg/l and 999.2+/-366microg/l, respectively. The MST diameter was significantly different in the selenium groups only. The selenium levels also differed with the quartiles of the MST diameters of individuals testing positive (P<0.05). Our findings suggest that trace elements, particularly selenium, should be measured in future vaccine trials so that vaccine immunogenicity and response can be assessed and compared between different studies.

Mixed cytokine profile during active cutaneous leishmaniasis and in natural resistance.

Most studies on immune response in human cutaneous leishmaniasis evaluate patients with active disease in comparison with healthy uninfected controls or patients that have had the lesions healed, however, little is known about the immune response associated with natural resistance. In this paper we evaluate the cytokine expression patterns of T-cells and the plasmatic levels of nitrite and nitrate in patients with localized cutaneous leishmaniasis (LCL) as well as endemic non-infected individuals with positive (MST) and negative (NI) Montenegro skin test, without previous history of leishmanial lesions. Our results demonstrated an increased number of IFN-gamma+ and TNF-alpha+ T-cells and high level of plasma nitrite and nitrate in LCL patients. Moreover, we have observed that early in infection (LCL equal/less than 60 days of lesion evolution), Leishmania patients present predominance in IL-4+ and IL-10+ T-lymphocytes. However, this is a transitory phenomenon, since patients with older lesions (LCL more than 60 days of lesion evolution) show a predominant Type-1 immune profile, suggesting that disease development may depend on a transient deregulation of T-cell response, during the initial phase of infection. Interestingly MST displayed a basal mixed Type-0 cytokines profile. However, the low frequency of IL-4+ T-cells, high IFN-gamma+/IL-10+ cell ratio as well as elevated nitrite and nitrate plasma levels observed in MST, suggested that despite basal levels of cytokines, a high proportion of Type-1 over Type-2 cytokines would count to prevent parasite growth and lesion development.

Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi.

The role of anti-leishmanial immune response underlying the susceptibility/resistance during canine visceral leishmaniasis (CVL) has been recognized throughout ex vivo and in vitro investigations. Recently, we demonstrated that immunoglobulin levels (Igs), as well as the parasite load are relevant hallmarks of distinct clinical status of CVL. To further characterize and upgrade the background on this issue, herein, we have evaluated, in Leishmania (Leishmania) chagasi naturally infected dogs, the relationship between tissue parasitism (skin, bone marrow, spleen, liver and lymph node), the CVL clinical status (asymptomatic (AD), with no suggestive signs of the disease; oligosymptomatic (OD), with maximum three clinical signs-opaque bristles; localized alopecia and moderate loss of weight; symptomatic (SD), serologically positive with severe clinical signs of visceral leishmaniasis), and the humoral immunological profile of anti-Leishmania immunoglobulins (IgG, IgG1, IgG2, IgM, IgA and IgE). Our major statistically significant findings revealed distinct patterns of tissue parasite density within L. chagasi-infected dogs despite their clinical status, pointing out the spleen and skin as the most relevant sites of high parasitism during ongoing CVL. Parasite density of bone marrow and spleen were the most reliable parasitological markers to decode the clinical status of CVL. Moreover, the parasite density of bone marrow better correlates with most anti-Leishmania Igs reactivity. Additionally, a prognostic hallmark for canine visceral leishmaniasis was found, highlighting strong correlation between IgG1 and asymptomatic disease, but with IgA, IgE and IgG2 displaying better association with symptomatic disease. The new aspects of this study highlighted pioneer findings that correlated the degree of tissue parasite density (low (LP), medium (MP) and high (HP) parasitism) with distinct patterns of anti-Leishmania Igs reactivity. In this scope, our data re-enforce the anti-Leishmania IgG but with IgA reactivity as the better marker for overall tissue parasitism. The association between clinical status, Ig profile and the tissue parasitism support a novel investigation on the impact of humoral immune response and susceptibility/resistance mechanism during ongoing CVL.