Nitric oxide and immune response in infection control of Caseous Lymphadenitis / Óxido nítrico e resposta imune no controle de infecção da Linfadenite Caseosa

This study aimed to evaluate aspects of host immune response using an experimental infection model of Corynebacterium pseudotuberculosis (CP) in C57/Black6 wild-type and knockout for nitric oxide (KO-NO) mice. 28 mice were evaluated: 4 wild-type controls; 10 wild-type infected with CP; 4 KO-NO controls; 10 KO-NO infected with CP. Infection procedures were carried out by intraperitoneal inoculation using 107. Infected C57/Black6 KO-NO mice began to die after the 5° day post-inoculation, up until the 14º day. Neutrophils were found in increased numbers in the infiltrate of KO-NO murine peritoneal cavities. Examination of splenic tissue revealed an accumulation of lymphocytes, predominantly CD8 T-cells, in experimental animal groups. KO-NO animals were found to have a predominance of granulomas 7 days post-inoculation, primarily in the lymph nodes. In addition, greater amounts of bacteria were recovered from the mesenteric lymph nodes of KO-NO mice. There was no statistically significant difference in the levels of total IgG and its subclasses 14 days post-inoculation between KO-NO and wild groups. The results suggest the importance of nitric oxide in the process of controlling CP infection, as KO-NO animals were observed to be markedly more affected by infection with this bacterium.(AU)

O objetivo deste estudo foi avaliar os aspectos da resposta imune do hospedeiro, mediante o uso de um modelo experimental de infecção de Corynebacterium pseudotuberculosis (CP) em camundongos C57/Black6 do tipo selvagem e em C57/Black6 knockout para o óxido nítrico (KO-NO). Foram avaliados 28 camundongos: quatro controles de tipo selvagem; 10 do tipo selvagem infectados com CP; quatro controles KO-NO; e 10 KO-NO infectados com CP. A infecção foi realizada via intraperitoneal, usando-se 107. Os animais C57/Black6 KO-NO infectados começaram a vir a óbito no quinto dia pós-inoculação, o que aconteceu até o 14º dia. Um número maior de neutrófilos foi encontrado na sua cavidade peritoneal. O exame do baço revelou um acúmulo de linfócitos, predominantemente células T CD8, nos grupos de animais experimentais. Nos animais KO-NO, foi observada a presença de granulomas, sete dias pós-inoculação, principalmente nos gânglios linfáticos. Além disso, uma maior quantidade de bactérias foi detectada dos linfonodos mesentéricos desses animais. Não houve diferença estatisticamente significante nos níveis séricos IgG total e em suas subclasses aos 14 dias pós-inoculação nos grupos KO-NO e selvagem. Os resultados obtidos sugerem a importância do óxido nítrico no processo de controle da infecção por CP.(AU)

AIM2 inflammasome is associated with disease severity in tegumentary leishmaniasis caused by Leishmania (V.) braziliensis.

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis-associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real-time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2-producing cells was higher in poor responders than in good responders. Although not quite significant, IL-1ß+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions.

Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus.

Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80 °C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP). According to the circular dichroism spectra, the secondary structure of P. furiosus esterase, which is predominantly formed by a ß-sheet structure, is very stable, even after incubation at 120°C. We performed SAXS to determine the low-resolution structure of Pf_Est, which is monomeric in solution at 80 °C and has a molecular weight of 28 kDa. The Km and V max values for this esterase acting on pNPP were 0.53 mmol/L and 6.5 × 10-3 U, respectively. Pf_Est was most active in the immiscible solvents and retained more than 50 % in miscible solvents. Moreover, Pf_Est possesses transesterification capacity, presenting better results when isobutanol was used as an acyl acceptor (2.69 ± 0.14 × 10-2 µmol/min mg) and the highest hydrolytic activity toward olive oil among different types of oils testes in this study. Collectively, these biophysical and catalytic properties are of interest for several biotechnological applications that require harsh conditions, including high temperature and the presence of organic solvents.

Effector memory CD4(+) T cells differentially express activation associated molecules depending on the duration of American cutaneous leishmaniasis lesions.

A high number of Leishmania-responder T cells is found in cutaneous leishmaniasis lesions, suggesting that important immunological events occur at the site of infection. Although activated, cytotoxic and regulatory T cells infiltrating into lesions may influence disease pathogenesis, the role of the T cell differentiation pattern of lymphocytes in lesions is unknown. Our aim was to investigate whether the phase of lesion development (early or late) is influenced by the functional status of cells present in inflammatory infiltrate. Activation, cytotoxity and T cell differentiation molecules were evaluated in lesion mononuclear cells by flow cytometry. The frequency of T cells was correlated with the lesion area (r = 0·68; P = 0·020). CD4(+) CD25(+) T cells predominated over CD4(+) CD69(+) T cells in early lesions (less than 30 days), whereas late lesions (more than 60 days) exhibited more CD4(+) CD69(+) T cells than CD4(+) CD25(+) T cells. The duration of illness was correlated positively with CD4(+) CD69(+) (r = 0·68; P = 0·005) and negatively with CD4(+) CD25(+) T cells (r = -0·45; P = 0·046). Most CD8(+) T cells expressed cytotoxic-associated molecules (CD244(+) ), and the percentages were correlated with the lesion area (r = 0·52; P = 0·04). Both CD4(+) and CD8(+) effector memory T cells (TEM -CD45RO(+) CCR7(-) ) predominated in CL lesions and were significantly higher than central memory (TCM -CD45RO(+) CCR7(+) ) or naive T cells (CD45RO(-) CCR7(+) ). An enrichment of TEM cells and contraction of naive T cells were observed in lesions in comparison to blood (P = 0·006) for both CD4(+) and CD8(+) T cells. Lesion chronicity is associated with a shift in activation phenotype. The enrichment of TEM and activated cytotoxic cells can contribute to immune-mediated tissue damage.

Antigen-triggered interferon-γ and interleukin-10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease.

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = -0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = -0·94) or the IFN-γ : IL-10 ratio (r = -0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.

Decrease in anti-Leishmania IgG3 and IgG1 after cutaneous leishmaniasis lesion healing is correlated with the time of clinical cure.

For better efficiency in the establishment of American tegumentary leishmaniasis clinical cure, the World Health Organization suggests that the clinical criteria are supported by serologic data. The present study aims to investigate the dynamics of IgG subclass production in clinical evolution post-treatment of cutaneous leishmaniasis (CL). Paired sera from 23 subjects with CL resulting from Leishmania braziliensis infection were studied during the active lesion phase (aCL) and after clinical cure post-therapy (hCL), which included an alternative protocol with a low dose of antimony. Anti-Leishmania IgG and its subclasses were measured using ELISA, and the immunoglobulin levels were correlated with patients' clinical data. All of the subjects were clinically healed and did not present relapse during follow-up. Serum levels of anti-Leishmania IgG (r = -0·79; P < 0·0001), IgG1 (r = -0·64, P < 0·001) and IgG3 (r = -0·42, P < 0·045) in hCL were negatively correlated with the duration of clinical cure. After 24 months of clinical cure, 73% of samples were negative for IgG1 and 78% were negative for IgG3. In conclusion, the detection of serum anti-Leishmania IgG1 and IgG3 is an improved laboratory strategy to aid in the decision of interruption of the ambulatory follow-up of CL patients.

Influence of the Notch system in the therapeutic response of American tegumentary leishmaniasis.

BACKGROUND: The evolution and therapeutic outcome of American tegumentary leishmaniasis (ATL) depend upon many factors, including the balance between Th1 and Th2 cytokines to control parasite multiplication and lesion extension. Other cytokines known for their role in inflammatory processes such as interleukin IL-17 or IL-18 as well as factors controlling keratinocyte differentiation and the inflammatory process in the skin, like the Notch system, could also be involved in the disease outcome. Notch receptors are a group of transmembrane proteins that regulate cell fate decisions during development and adulthood in many tissues, including keratinocyte differentiation and T-cell lineage commitment, depending on their activation by specific groups of ligands (Delta-like or Jagged). OBJECTIVES: To compare the in situ expression of Notch system proteins (receptors, ligands and transcriptional factors) and cytokines possibly involved in the disease outcome (IL-17, IL-18, IL-23 and transforming growth factor-ß) in ATL cutaneous and mucosal lesions, according to the response to therapy with N-methyl glucamine. METHODS: Cutaneous and mucosal biopsies obtained from patients prior to therapy with N-methyl glucamine were analysed by immunohistochemistry and real-time polymerase chain reaction. RESULTS: Notch receptors and Delta-like ligands were found increased in patients with ATL, particularly those with poor response to therapy or with mucosal lesions. CONCLUSIONS: The increase of Notch receptors and Delta-like ligands in patients with a poor response to treatment suggests that these patients would require a more aggressive therapeutic approach or at least a more thorough and rigorous follow-up.

Therapeutic failure in American cutaneous leishmaniasis is associated with gelatinase activity and cytokine expression.

Cutaneous lesions caused by Leishmania braziliensis infection occasionally heal spontaneously, but with antimonials therapy heal rapidly in approximately 3 weeks. However, about 15% of the cases require several courses of therapy. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are gelatinases that have been implicated in other chronic cutaneous diseases and skin re-epithelialization. These enzymes are controlled by their natural inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] and by some cytokines. Uncontrolled gelatinase activity may result in intense tissue degradation and, consequently, poorly healing wounds. The present study correlates gelatinase activity to therapeutic failure of cutaneous leishmaniasis (CL) lesions. Our results demonstrate an association between gelatinase activity and increased numbers of cells making interferon (IFN)-γ, interleukin (IL)-10 and transforming growth factor (TGF)-ß in lesions from poor responders. Conversely, high levels of MMP-2 mRNA and enhanced MMP-2 : TIMP-2 ratios were associated with a satisfactory response to antimonials treatment. Additionally, high gelatinolytic activity was found in the wound beds, necrotic areas in the dermis and within some granulomatous infiltrates. These results indicate the importance of gelatinase activity in the skin lesions caused by CL. Thus, we hypothesize that the immune response profile may be responsible for the gelatinase activity pattern and may ultimately influence the persistence or cure of CL lesions.

The skin homing receptor cutaneous leucocyte-associated antigen (CLA) is up-regulated by Leishmania antigens in T lymphocytes during active cutaneous leishmaniasis.

The cutaneous leucocyte-associated antigen receptor (CLA) can direct Leishmania-specific T lymphocytes towards inflamed skin lesions. Homing receptors [CLA, lymphocyte-associated antigen 1 (LFA-1) or CD62L] were analysed in lymphocytes from blood and cutaneous leishmaniasis (CL) lesions. CL patients with active lesions (A-CL) presented lower levels of T lymphocytes expressing the CLA(+) phenotype (T CD4(+) = 10.4% +/- 7.5% and T CD8(+) = 5.8% +/- 3.4%) than did healthy subjects (HS) (T CD4(+) = 19.3% +/- 13.1% and T CD8(+) = 21.6% +/- 8.8%), notably in T CD8(+) (P < 0.001). In clinically cured patients these percentages returned to levels observed in HS. Leishmanial antigens up-regulated CLA in T cells (CLA(+) in T CD4(+) = 33.3% +/- 14.1%; CLA(+) in T CD8(+) = 22.4% +/- 9.4%) from A-CL but not from HS. An enrichment of CLA(+) cells was observed in lesions (CLA(+) in T CD4(+) = 45.9% +/- 22.5%; CLA(+) in T CD8(+) = 46.4% +/- 16.1%) in comparison with blood (CLA(+) in T CD4(+) = 10.4% +/- 7.5%; CLA(+) in T CD8(+) = 5.8% +/- 3.4%). Conversely, LFA-1 was highly expressed in CD8(+) T cells and augmented in CD4(+) T from peripheral blood of A-CL patients. In contrast, CD62L was not affected. These results suggest that Leishmania antigens can modulate molecules responsible for migration to skin lesions, potentially influencing the cell composition of inflammatory infiltrate of leishmaniasis or even the severity of the disease.

Doping in poly(o-ethoxyaniline) nanostructured films studied with atomic force spectroscopy (AFS).

The study of intermolecular interactions at interfaces is essential for a number of applications, in addition to the understanding of mechanisms involved in sensing and biosensing with liquid samples. There are, however, only a few methods to probe such interfacial phenomena, one of which is the atomic force spectroscopy (AFS) where the force between an atomic force microscope tip and the sample surface is measured. In this study, we used AFS to estimate adhesion forces for a nanostructured film of poly(o-ethoxyaniline) (POEA) doped with various acids, in measurements performed in air. The adhesion force was lower for POEA doped with inorganic acids, such as HCl and H(2)SO(4), than with organic acids, because the counterions were screened by the ethoxy groups. Significantly, the morphology of POEA both in the film and in solution depends on the doping acid. Using small-angle X-ray scattering (SAXS) we observed that POEA dissolved in a mixture of dimethyl acetamide exhibits a more extended coil-like conformation, with smaller radius of gyration, than for POEA in water, as in the latter POEA solubility is lower. In AFS measurements in a liquid cell, the force curves for a POEA layer displayed an attractive region for pH>or=5 due to van der Waals interactions, with no contribution from a double-layer since POEA was dedoped. In contrast, for pH

Can interferon-gamma and interleukin-10 balance be associated with severity of human Leishmania (Viannia) braziliensis infection?

Suitable levels of interferon (IFN)-gamma and interleukin (IL)-10 seem to favour the outcome of cutaneous leishmaniasis (CL), while high IFN-gamma and low IL-10 production are associated with severity of mucosal leishmaniasis (ML). Considering that cytokine balance is important for the maintenance of protective responses in leishmaniasis, our aim was to investigate leishmanial antigens-induced IFN-gamma and IL-10 levels maintained in healed individuals who had different clinical outcomes of Leishmania infection. Thirty-three individuals who recovered from L. braziliensis infection were studied: cured CL (CCL), cured ML (CML), spontaneous healing of CL (SH) or asymptomatic individuals (ASY). Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) in culture supernatants of L. braziliensis-stimulated peripheral blood mononuclear cells (PBMC). IFN-gamma levels were higher in CML (7593 +/- 5994 pg/ml) in comparison to SH (3163 +/- 1526 pg/ml), ASY (1313 +/- 1048 pg/ml) or CCL (1897 +/- 2087 pg/ml). Moreover, cured ML cases maintained significantly lower production of IL-10 (127 +/- 57.8 pg/ml) in comparison to SH (1373 +/- 244 pg/ml), ASY (734 +/- 233 pg/ml) or CCL (542 +/- 375 pg/ml). Thus, a high IFN-gamma/IL-10 ratio observed in CML can indicate unfavourable cytokine balance. On the other hand, no significant difference in the IFN-gamma/IL-10 ratio was observed when CCL individuals were compared to SH or ASY subjects. In conclusion, even after clinical healing, ML patients maintained a high IFN-gamma/IL-10 secretion profile in response to leishmanial antigens. This finding can explain a delayed down-modulation of exacerbated inflammatory responses, which can be related in turn to the necessity of prolonged therapy in ML management. Conversely, lower IFN-gamma/IL-10 balance observed in CCL, SH and ASY individuals can represent a better-modulated immune response associated with a favourable prognosis.

Development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis.

We performed a serological study with sera from 92 patients with confirmed sporotrichosis registered between 1999 and 2004 in two hospitals in Rio de Janeiro State, Brazil. The clinical presentation of sporotrichosis was distributed as follows: lymphocutaneous, 67%; fixed cutaneous, 23%; disseminated cutaneous, 8%; and extracutaneous, 2%. Sera were assayed by ELISA against a cell wall antigen of Sporothrix schenckii, SsCBF, that we have previously described. The cross-reactivity was determined with 77 heterologous sera. The serological test showed a sensitivity of 90% and a global efficiency of 86%. A group of 55 patients with several clinical presentations of sporotrichosis was clinically and serologically followed-up for at least 6 months. We observed by ELISA data a decrease in the antibody serum titers which correlated with the progress in healing. An HIV-positive patient with meningeal sporotrichosis was serologically followed-up for over 2 years. Serum and cerebrospinal fluid specimens were examined and significant antibodies levels against the antigen SsCBF were detected. Our results strongly suggest that this serological test is valuable for the differential diagnosis and follow-up of all clinical forms of sporotrichosis.

Flow cytometric analysis of cellular infiltrate from American tegumentary leishmaniasis lesions.

BACKGROUND: CD4+ and CD8+ T lymphocytes play different roles in the outcome of leishmaniasis. However, T-cell distribution in lesions shows significant variability in in situ immunocytochemical studies. OBJECTIVES: In this report flow cytometry was used to determine the predominant T-cell subsets in leishmaniasis lesions, and their relationship with Leishmania-responsive circulating T cells. PATIENTS AND METHODS: Mononuclear cells from lesions or peripheral blood (PBMC) of 34 cutaneous (CL), four mucosal (ML) and four disseminated leishmaniasis were phenotypically characterized by flow cytometry. Leishmania-responsive T cells were obtained after in vitro stimulation of PBMC with leishmanial antigens. RESULTS/CONCLUSIONS: Variable amounts of gammadelta lymphocytes were present in all lesions, with no association with duration of illness. The highest percentages of interleukin-2R- and interferon-gammaR-positive cells were observed in ML lesions and could render these T cells more susceptible to the effects of these cytokines. The distribution of intralesional T-lymphocyte subsets was quite variable (CD4+ > CD8+ = 18 cases, CD8+ > CD4+ = 12 cases and CD4+ congruent with CD8+ = 4 cases) without any association with clinical parameters, and could explain the controversy regarding proportions of these T-cell subsets in leishmaniasis lesions. Low percentages of Leishmania-reactive CD8+ T cells were observed in blood while an enrichment of CD8+ cells was shown in the inflammatory infiltrate, suggesting that local immunoregulatory factors could favour the recruitment and/or proliferation of local CD8+ lymphocytes. Increased percentages of CD8+ cells observed in older lesions are consistent with the hypothesis that they can mediate healing, although their involvement in tissue damage cannot be ruled out. It is possible that these mechanisms can influence the clinical outcome or even the response to therapy.

Responses of Leishmania (Viannia) braziliensis cutaneous infection to N-methylglucamine antimoniate in the rhesus monkey (Macaca mulatta) model.

The antileishmanial efficacy of the reference drug N-methylglucamine antimoniate (Glucantime) was evaluated in groups of rhesus monkeys with acute and chronic Leishmania (Viannia) braziliensis cutaneous infection. The therapeutic responses in experimental animals to either a low dose (5 mg/kg body wt/day for 28 days) or a routine dose (20 mg/kg/day for 28 days) of pentavalent antimony were similar to those reported in the human disease. Primates were cured of their lesions after treatment, but with cryptic parasitism and/or relapse. The rhesus model of L. (V.) braziliensis cutaneous leishmaniasis therefore provides an additional resource for preclinical trials with newer drugs.

Leishmania (Viannia) braziliensis-induced chronic granulomatous cutaneous lesions affecting the nasal mucosa in the rhesus monkey (Macaca mulatta) model.

The present studies on infections with Leishmania (Viannia) braziliensis in rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmania antibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions in L. (V.) braziliensis-infected M. mulatta was similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease.

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79 percent in attenuated parasite-vaccinated monkeys, versus 75 percent in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.