Sebastião Alves Pinto

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNgamma KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Graces insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNgamma KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNgamma KO mice to improve the possibility to isolate New World Leishmania species.

Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10

BackgroundThe interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis.MethodsIL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP.ResultsWe showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals.ConclusionsThese data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

Achados de fibrobroncoscopia em pacientes com diagnóstico de neoplasia pulmonar

OBJECTIVE: To compile fiberoptic bronchoscopy findings in patients diagnosed with lung cancer and to correlate those with histopathological findings. METHODS: This was a retrospective study involving 212 patients with a confirmed diagnosis of lung cancer by cytological evaluation of BAL specimens or by histopathological evaluation of endobronchial or transbronchial biopsy specimens. The data were collected at the Respiratory Endoscopy Sector of Hospital Sao Salvador, located in the city of Goiânia, Brazil, between 2005 and 2010. The endoscopic findings were classified as endoscopically visible tumor, endoscopically invisible tumor, mucosal injury, as well as being classified by the presence/type of secretion. The visible tumors were also classified according to their location in the tracheobronchial tree. RESULTS: Endobronchial mass (64%) and mucosal infiltration (35%) were the main endoscopic findings. The histological type was determined in 199 cases, the most prevalent types being squamous carcinoma, in 78 (39%), adenocarcinoma, in 42 (21%) small cell carcinoma, in 24 (12%), and large cell carcinoma, in 2 (1%). More than 45% of the visible tumors were at the upper bronchi. Squamous carcinoma (n = 78) was most commonly visualized as an endobronchial mass (in 74%), mucosal infiltration (in 36%), luminal narrowing (in 10%), or external compression (in 6%). CONCLUSIONS: Our results show that the endobronchial mass is the most common bronchoscopic finding that is suggestive of malignancy. Proportionally, mucosal infiltration is the most common finding in small cell carcinoma. In adenocarcinoma, luminal narrowing, external compression, mucosal injury, and endobronchial secretion prevail.

Improvements in obtaining New World Leishmania sp from mucosal lesions: Notes on isolating and stocking parasites

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Pneumonia lipoide secundária ao uso prolongado de óleo de prímula

Lipoid pneumonia is an underdiagnosed disease that is caused by the aspiration of lipid particles into the lungs. Although most of the reported cases have been associated with the use of mineral oil as a laxative, other lipid substances can also cause the disease. We report the case of a 50-year-old female patient with a complaint of productive cough who was initially diagnosed with bronchial hyperresponsiveness and gastroesophageal reflux disease (GERD). The patient was treated for GERD. Because the productive cough persisted, the patient underwent chest CT, fiberoptic bronchoscopy, and open lung biopsy. She was diagnosed with lipoid pneumonia. The patient was questioned regarding the use of lipid substances, and she reported the chronic use of evening primrose oil. After the discontinuation of the substance and the maintenance of GERD treatment, her condition improved.

Human Interleukin-32 gamma Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice

ABSTRACT Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32β transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum. IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.

Identification and Biological Characterization of Leishmania (Viannia) guyanensis Isolated from a Patient with Tegumentary Leishmaniasis in Goias, a Nonendemic Area for This Species in Brazil

The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil.

Case Report: Atypical Cutaneous Leishmaniasis in a Patient with Mixed Leishmania guyanensis and Leishmania amazonensis Infection

The disseminated form of leishmaniasis is a serious and rare disease, being diagnosed in 2% of the cutaneous cases registered per year in Brazil. The main characteristic is the appearance of multiple pleomorphic lesions on the cutaneous surface. A 68-year-old male from the rural area of Tocantins, Brazil, presented atypical disseminated cutaneous leishmaniasis (ACL). The clinical course and histopathological and immunological findings presented a mixed pattern that hindered diagnosis and therapeutic management. Molecular typing revealed a mixed infection with Leishmania (V.) guyanensis and Leishmania (L.) amazonensis. Molecular identification of the agents responsible for ACL is important for adequate therapeutic planning, minimizing the possibility of sequellae that impact the quality of life of the patient.

Necrose de canto medial associado a hanseníase virchowiana: relato de caso

In the present case we deal with a medial eyelid necrosis and injury of the canalicular system in a patient in treatment for lepromatous leprosy. Histology of the necrotic lesion showed granulomatous inflammatory reaction with accumulation of histiocytes and presence of alcohol-acid resistant bacilli. After medical treatment, the patient had a spontaneous recovery of the medial canthus architecture but with complete destruction of the canalicular system.