Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Mechanisms of immune protection in the asexual blood stage infection by Plasmodium falciparum: analysis by in vitro and ex-vivo assays

Mechanisms of immune protection against the asexual blood stage infection by Plasmodium falciparum are reviewed. Recent studies of two independent lines of research developed at the Institute Pasteur, in humans and primate infections clearly indicate an obligatory interaction of antibodies and effector cells to express the anti-parasitic effect.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

A new method for intraoperative localization of epilepsy focus by means of a gamma probe

Objective To evaluate the utility of a new multimodal image-guided intervention technique to detect epileptogenic areas with a gamma probe as compared with intraoperative electrocorticography. Materials and Methods Two symptomatic patients with refractory epilepsy underwent magnetic resonance imaging, videoelectroencephalography, brain SPECT scan, neuropsychological evaluation and were submitted to gamma probe-assisted surgery. Results In patient 1, maximum radioactive count was initially observed on the temporal gyrus at about 3.5 cm posteriorly to the tip of the left temporal lobe. After corticotomy, the gamma probe indicated maximum count at the head of the hippocampus, in agreement with the findings of intraoperative electrocorticography. In patient 2, maximum count was observed in the occipital region at the transition between the temporal and parietal lobes (right hemisphere). During the surgery, the area of epileptogenic activity mapped at electrocorticography was also delimited, demarcated, and compared with the gamma probe findings. After lesionectomy, new radioactive counts were performed both in the patients and on the surgical specimens (ex-vivo). Conclusion The comparison between intraoperative electrocorticography and gamma probe-assisted surgery showed similarity of both methods. The advantages of gamma probe include: noninvasiveness, low cost and capacity to demonstrate decrease in the radioactive activity at the site of excision after lesionectomy.

Efeitos da Luffa operculata sobre o epitélio do palato de rã: aspectos histológicos

Luffa operculata é o nome botânico da buchinha-do-norte ou cabacinha, uma planta medicinal usada popularmente no tratamento das rinites e rinossinusites. Na Europa e nos EUA, está em medicamentos homeopáticos. No Brasil, a infusão (chá) do fruto seco de Luffa operculata é utilizada para inalação ou instilação nasal, resultando em liberação profusa de muco que alivia os sintomas nasossinusais, mas há relatos freqüentes de irritação nasal, epistaxe e anosmia. FORMA DE ESTUDO: Experimental. MATERIAL E MÉTODO: Avaliamos os efeitos da infusão de Luffa operculata em diferentes concentrações, no modelo experimental do palato isolado de rã, examinando 46 palatos após imersão. Quatro grupos (n=10) foram testados com infusão feita em Ringer-rã (solução isotônica): controle; 60mg/l; 600mg/l e 1200mg/l. Um grupo foi testado em água (600mg/l H2O, n=6). Coletamos amostras do epitélio para estudo histológico à microscopia-de-luz e microscopia eletrônica de transmissão. RESULTADOS: Nos palatos tratados, os achados à microscopia-de-luz mostram lesões epiteliais de padrão tóxico, dose-dependentes. Na microscopia eletrônica, aumento dos espaços intercelulares e ruptura de tight junctions apontam para anormalidade no transporte iônico e de fluidos. CONCLUSÕES: A infusão de Luffa operculata, nas concentrações utilizadas popularmente, promove alterações significantes na estrutura e ultraestrutura epitelial deste modelo ex vivo de mucosa respiratória.

Establishment of HTLV-I-infected cell lines from peripheral blood mononuclear cells of Brazilian patients

To investigate epidemiological and pathogenetic features of HTLV-I infection, a cohort of carriers has been followed at the USP Teaching Hospital since 1991. This study describes the establishment of cell lines from peripheral blood mononuclear cells (PBMC) of infected subjects. Ex vivo PBMC were cultured with those from a seronegative donor and morphologic evidence of cell transformation was obtained after 90 days with detection of multinucleated cells exhibiting cerebriform nuclei. Integration of HTLV-I proviral DNA and expression of viral antigens was demonstrated in culture by PCR and immunofluorescence. Cell lines were maintained for 240 days, gradually weaned from exogenous IL-2. Immunophenotyping of cell lines on flow cytometry yielded evidence of cell activation. Establishment of HTLV-I-infected cell lines from ex vivo PBMC is feasible and may be useful for studies on lymphocyte phenotypic changes and on mechanisms of HTLV-induced cell proliferation. Moreover they may be used with diagnostic purposes in immunofluorescence tests.

Abnormal spontaneous interleukin 8 receptor expression: a brief report of two cases

Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.

Molecular mechanisms and biological importance of Plasmodium falciparum erythrocyte rosetting

Rosetting, i.e. the spontaneous binding of uninfected to malaria infected erythrocytes and endothelial cytoadherence may hinder the blood flow and lead to serve Plasmodium falciparum malaria. Falciparum isolates obtained from unconscious patients all form rosettes and/or express a significantly higher man rosetting rate than isolates from patients with uncomplicated malaria. Furthermore, sera of patients with cerebral malaria are devoid of anti-rosetting activity while sera from patients with mild disease carry high levels of anti-rosetting antibodies. The presence of anti-rosetting antibodies also seems important for the efficient interaction of rosetting infected rbc and leucocytes. Two parasite derived rosetting ligands of Mr 22k and Mr28K named "rosettins, have been found on the surface of rosetting infected erythrocytes. CD36 has in at least some strains of parasites been found to function as a rosetting receptor on the uninfectederythrocyte. Heparin disrupts rosettes of P. falciparum in vitro and inhibits the sequestration of rosetting cells ex vivo. In conclusion, rosetting seems a crucial factor in the development of cerebral malaria and treatment of patients with anti-rosetting substances might become an effectivew adjunct in the treatment of severe malaria.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 μM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 μM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 μM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 μM) is optimal for bioimaging, whereas a high concentration (200 μM CdTe) could be toxic to cells. Taken together, our data indicate that 2 μM QD can be used for the successful long-term study of the parasite-vector interaction in real time.

A química medicinal de N-acilidrazonas: novos compostos-protótipos de fármacos analgésicos, antiinflamatórios e anti-trombóticos

In this article are described new bioactive N-acylhydrazone (NAH) derivatives, structurally designed as optimization of aryl hydrazones precursors planned by molecular hybridization of two 5-lipoxigenase inhibitors, e.g. CBS-1108 and BW-755c. The analgesic, antiedematogenic and anti-platelet aggregating profile of several isosteric compounds was investigated by using classic pharmacological assays in vivo and ex-vivo, allowing to identify new potent peripheric analgesic lead, a new anti-inflammatory and an antithrombotic agent. During this study was discovered dozen of active NAH compounds clarifying the structure-activity relationship for this series of NAH derivatives, indicating the pharmacophore character of the N-acylhydrazone functionality.

Equipamento brasileiro de detecção gama intra-operatória para biópsia de linfonodo sentinela

OBJETIVO: Testar a eficácia de equipamento de detecção gama intra-operatória (DGI) desenvolvido pelo IPEN (Brasil), em procedimentos de biópsia de linfonodo sentinela (BLS) no melanoma e no câncer de mama. MÉTODOS: Foram estudados 40 pacientes portadores de melanoma ou câncer de mama com indicação para realização de BLS.Todos pacientes foram submetidos à linfocintilografia e a BLS ocorreu entre 2 a 24 horas após a mesma. Concomitantemente à DGI, realizou-se o mapeamento linfático com corante vital. Foram feitas leituras com o equipamento convencional Neoprobe® 1500 e com o equipamento em teste (IPEN) dos valores de captação do sítio de injeção do radiofármaco, do LS in vivo e ex vivo e da captação de fundo. Foi registrado se o LS estava corado e se o cirurgião teve facilidade para encontrá-lo. Nos primeiros 20 pacientes utilizou-se o equipamento convencional e depois o de teste; nos outros 20, utilizou-se primeiro o equipamento em teste, com objetivo de verificar se o mesmo identificava primariamente o LS. RESULTADOS: Dos quarenta pacientes, 33 eram portadores de tumor de mama e sete de melanoma cutâneo; variação da idade: 21 a 68 anos (mediana= 46 anos); 35 mulheres e 5 homens. Em apenas um paciente o LS não foi encontrado, nem pela DGI nem pelo corante vital. Não houve diferença estatística entre as razões ex vivo/fundo obtidas com os dois equipamentos (p=0, 2583-ns). CONCLUSÃO: É possível realizar o procedimento de BLS com o equipamento brasileiro desenvolvido pelo IPEN, com facilidade e sem prejuízo para o paciente.

Injeção intraoperatória de dextran-500-99m_tecnécio para identificação do linfonodo sentinela em câncer de mama

OBJETIVO: avaliar a eficácia da injeção intraoperatória para identificação do LS em câncer de mama com o uso do Dextran 500-99m-tecnécio (Tc) e analisar o tempo para marcação do linfonodo sentinela (LS) axilar. MÉTODOS: estudo prospectivo realizado entre abril de 2008 e junho de 2009 que incluiu 74 biópsias de LS em pacientes com câncer de mama em estádios T1N0 e T2N0. Após a indução anestésica, injetou-se de 0,5 a 1,5 mCi (18 a 55 MBq) de Dextran 500-99m-Tc filtrado 0,22 µm num volume de 5 mL de acordo com a técnica de injeção subareolar para a biópsia do LS. Após a marcação com o radiofármaco injetou-se 2 mL de azul patente. O tempo entre a injeção e a marcação na região axilar, a contagem com o probe do LS in vivo, ex vivo, background e o número de LS identificados foram documentados. Os dados foram analisados por meio da estatística descritiva pelo programa SPSS, versão 18. RESULTADOS: identificamos o LS em 100% dos casos. A taxa de identificação com o probe foi de 98% (73/74 casos). Um caso (1,35%) estava marcado apenas com o azul. A dose média do radiofármaco aplicada foi 0,97 mCi±0,22. O tempo para marcação na região axilar, após a injeção subareolar, foi de 10,7 minutos (±5,7 min. ). Foram identificados, em média, 1,66 linfonodos marcados com o radioisotopo. CONCLUSÃO: o procedimento para identificação do LS com injeção intraoperatória do radiofármaco é oncologicamente seguro, apresentando conforto ao paciente e agilidade à equipe cirúrgica.

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.