Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

Comparative evaluation of the DPP(®) CVL rapid test for canine serodiagnosis in area of visceral leishmaniasis.

We investigated the performance of the DPP(®) canine visceral leishmaniasis (CVL) rapid test, a novel immunochromatographic assay launched by BioManguinhos (Brazil), which was recently included in the new Brazilian protocol for screening CVL in serological surveys. The present study compared the DPP(®) with the ELISA and IFA produced by BioManguinhos (Brazil) both with L. major-like antigens and with in-house tests using Leishmania infantum chagasi (in-house ELISA and in-house IFA). We analyzed the sera from clinically symptomatic (n=47) and asymptomatic (n=38) infected dogs from an endemic area of CVL, as well as from healthy (n=18) dogs, in addition to the sera of dogs (n=81) infected with other pathogens. The DPP(®) and the in-house ELISA showed a sensitivity of 90.6% and 94.1%, respectively, and specificity of 95.1% and 97.5%, respectively, and both presented cross-reactivity only with the sera of dogs with babesiosis, 44% for the DPP(®) and 22% for the in-house ELISA. The clinical groups were detected equally by the two assays. The ELISA BioManguinhos, IFA BioManguinhos, and in house-IFA showed a good sensitivity, 90.6%, 96.5% and 89.4%, respectively, but very low specificity, 77.8%, 69.1% and 65.8%, respectively, due to the high cross-reactivity with the sera from the animals harboring other pathogens. The in-house ELISA provided the highest accuracy (95.8%), followed by the DPP(®) (92.7%), ELISA BioManguinhos (84.3%), IFA BioManguinhos (83.1%), and in-house IFA (78.0%). The simultaneous use of the DPP(®) and ELISA BioManguinhos reached a sensitivity of 99.1% and 82.1% when used sequentially. In conclusion, the DPP(®) performed well as serological test for CVL, and detected both asymptomatic and symptomatic dogs in equal proportions. Although its sensitivity is not ideal yet, discarding the IFA and including the DPP(®) improved the accuracy of the new Brazilian CVL diagnostic protocol, particularly of detecting truly infected dogs. Moreover, considering the higher specificity of DPP(®) (95.1% vs 77.8%), positive predictive value (95.1% vs 81.1%) and positive likelihood value (18.3% vs 4.1%) in comparison with the ELISA BioManguinhos, the use of DPP(®) as a confirmatory test instead of a screening test is suggested.

Effects of salivary gland homogenate from wild-caught and laboratory-reared Lutzomyia longipalpis on the evolution and immunomodulation of Leishmania (Leishmania) amazonensis infection.

We investigated the effects of Lutzomyia longipalpis salivary glands homogenate of wild-caught and laboratory-reared vectors on the lesion evolution and immunomodulation of the infection caused by Leishmania (Leishmania) amazonensis. To compare the effect of both salivary glands homogenate (SGH), C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 10(6) promastigotes in the presence or not of SGH from wild-caught and laboratory-colonized sand flies. Comparing SGH groups, the lesion size was lower in mice co-inoculated with wild-caught SGH, as the parasitism and the infiltration of macrophages at the inoculation site. Wild-caught SGH also determined lower production of IL-4 and IL-10 but higher IL-12 levels compared with laboratory-reared SGH. Our findings address a probable bias by using SGH from laboratory-colonized sand flies instead of wild-caught vector SGH in studies concerning saliva effects. A possible mild influence of sand fly saliva in natural infections caused by Leishmania is also speculated, as infection is transmitted by wild and not by laboratory-reared vectors.

Insulin-like growth factor (IGF)-I affects parasite growth and host cell migration in experimental cutaneous leishmaniasis.

While the control or progression of leishmaniasis depends on host immune responses, the initial inflammatory process represents a key event. This process involves the participation of several cytokines and growth factors induced during inflammation as well as factors already present at the site of infection such as insulin-like growth factor (IGF)-I. We have previously demonstrated a potential role for IGF-I in experimental cutaneous leishmaniasis based on the significant increase in lesion size seen in mice injected with Leishmania promastigotes preactivated with IGF-I. In the present study we show that preactivation of Leishmania (Leishmania) amazonensis promastigotes with IGF-I induces an increase in the actual number of parasites at the lesion site from seven days postinfection, in addition to a more intense inflammatory infiltrate. There was a higher numerical density of polymorphonuclear neutrophils from 3 to 24 h, and of mononuclear cells from 48 h of infection onward. A higher density of polymorphonuclear neutrophils and mononuclear cells harboring parasites was also observed. The most important observation, however, was that more parasites per cell were present, revealing that IGF-I appears to favour parasite growth within the macrophages. These results strongly suggest an important role for IGF-I in the development of cutaneous leishmaniasis, where it influences both the inflammatory process and parasite growth.

Detection of specific antibody isotypes and subtypes before and after treatment of American visceral leishmaniasis.

Sera from patients with American visceral leishmaniasis (AVL) were studied before and after treatment based on their antibody isotypes and subtypes. The study was comprised of 33 Brazilian patients with well-defined diagnosis of AVL and 39 clinically healthy individuals. Antileishmanial antibody isotypes and subtypes were observed in almost all patients, except IgA that was detected in about 63% of them. The sensitivity and specificity of the immunofluorescence assay in the detection of antibody isotypes (IgG and IgM) and subtypes (IgG1, IgG2, IgG3, and IgG4) were high with no statistical difference, ranging from 0.937 to 1.000 and from 0.954 to 1.000, respectively. All IgG antibodies and its subtypes had their levels reduced after treatment. However, the IgG4 had an early decay and its conversion to negative was significantly high in children. Moreover, the profile of IgG4 before treatment corresponded to a unimodal curve that shifted to a patent bimodal curve after treatment, indicative of therapeutic success. Thus, the IgG4 shows to be a suitable immunological marker for the assessment of chemotherapy in AVL patients or communities. Our findings suggest that IgG4 correlates with IL-4 that also decreases after therapy.

An evaluation of clinical, serologic, anatomopathologic and immunohistochemical findings for fifteen patients with mucosal leishmaniasis before and after treatment.

Treatment of mucosal leishmaniasis (ML) can be controlled by clinical examination and by serologic titers by the indirect immunofluorescence serologic reaction (IISR). We studied the correlation between the presence of antigen in tissue determined by immunohistochemistry, the IISR titers and the anatomopathologic findings in fifteen patients with ML before and after healing of the lesions as determined by otorhinolaryngologic evaluation, and evaluated these parameters to determine which of them could be useful during follow-up. Tissue antigens became negative in four patients (group A) after treatment, with a statistically significant reduction or negativity of IISR titers (p < 0.05). This did not occur in patients in whom the antigen persisted after treatment (group B), suggesting that serologic follow-up should be performed together with the search for tissue antigen, a combination which, to our knowledge, has not been used in previous studies. The negativity of tissue antigens and the behavior of IIRS titers in group A patients probably indicate a lower possibility of recurrence. Upon anatomopathologic examination the inflammatory process was found to persist after treatment even in group A, suggesting that the permanence of inflammatory activity even in clinically healed lesions is possibly correlated with the presence of the antigen or of some unknown factor.

Experimental visceral leishmaniasis: sequential events of granuloma formation at subcutaneous inoculation site.

Hamsters were inoculated with IO7 Leishmania (Leishmania) chagasi amastigotes in the hind footpads and killed at 7, 15, 30, 45, 60, 75 and 90 days after infection. We observed mononuclear inflammatory infiltrates with many parasites on the 7th and 15th days of infection. On the 30th day there was early granuloma formation. After 45 days the lesion was characterized by well defined epithelioid granuloma with multinuclear giant cells whose cytoplasm showed Schaumann bodies. Non-particulate antigenic material was present in the macrophage cytoplasm and between the lamellae of the Schaumann bodies. Granuloma formation has an important role for the control of infection at the inoculation site. The results indicate that dissemination of the infection must occur in the first 45 days, before granuloma formation has taken place.

Cutaneous leishmaniasis of the New World: diagnostic immunopathology and antigen pathways in skin and mucosa.

Non-specific chronic inflammation and/or granulomatous reaction are the main histopathological manifestations of cutaneous and mucocutaneous leishmaniasis of the New World. Plasma cell infiltration associated with collagen and vascular changes are data suggestive but not diagnostic of the disease. Specific diagnosis is only possible through demonstration of the parasite in the tissue examined. It is noteworthy that the parasites are usually scanty and difficult to demonstrate in the lesions. Biopsies from 40 patients with cutaneous or mucocutaneous leishmaniasis were examined using the immunofluorescence and immunoperoxidase techniques in order to demonstrate the parasite and/or antigen in the tissues. Nineteen biopsies showed non-specific chronic inflammation and 21 a granulomatous reaction. Parasites were found in 20% of the routine biopsies. The positivity through indirect immunofluorescence was 88.46% in frozen sections of fresh material and 89.28% in paraffin embedded tissue. The antigen positivity with the immunoperoxidase technique was 64.51%. Antigen was detected as amastigotes and also as diffuse material in the macrophage cytoplasm and adsorbed in the epithelial basement membrane and vessel walls. There was no difference in the positivity of antigen according to the type of inflammatory reaction.

Interstitial pneumonitis in human visceral leishmaniasis.

The involvement of the lung in 13 cases of human visceral leishmaniasis was studied. Interstitial pneumonitis with mononuclear cells was found in 76.8% of the cases; 53.8% also had foci of septal fibrosis. Leishmania were seen within macrophages in 3 cases only. However, all 10 interstitial pneumonitis cases showed PAP-positive material using specific L. donovani (MHOM/BR/72/LD 46) antiserum. 3 cases with no interstitial pneumonitis were PAP-negative. A short discussion about clinical aspects and the course of the disease is presented.