An ex vivo multiparametric flow cytometry assay using human whole blood to simultaneously measure cytotoxicity and leishmanicidal activities.

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.

Morphofunctional changes of female germinal epithelium to support spermatozoa along the annual reproductive cycle in an inseminating catfish (Trachelyopterus galeatus, Auchenipteridae).

The reproductive system of some fish species presents elaborate mechanisms by which the females store spermatozoa inside their ovaries, keeping them viable for fertilization for an extended period of time. However, as intriguing as this sperm storage is, it is not yet understood how the sperm can remain viable in the ovary. Aiming to understand this phenomenon, the epithelium covering the ovarian lamellae, that is, the germinal epithelium, of the Cangati (Trachelyopterus galeatus), an inseminating catfish, was evaluated taking into account the different stages of the annual reproductive cycle. The germinal epithelium morphology changed during the annual reproductive cycle, presumably in preparation to receive the spermatozoa and keep them viable until fertilization. There was a progressive increase of the epithelium height. Also the number of intercellular junctions, desmosomes, and extended tight junctions, apparently increased forming chains that could be regarded as a barrier to isolate the sperm from the female immune system. Synthetic organelles were active releasing cytoplasmic granules and secretion in the epithelial enfolds in which the spermatozoa were deeply embedded. Concomitantly, oogonium nests were formed in the germinal epithelium during early folliculogenesis.

Spermatozoon and its relationship with the ovarian lamellae in the internally inseminating catfish Trachelyopterus galeatus.

We examined the spermatozoa and their relationship with the ovarian lamellae in the catfish Trachelyopterus galeatus by classical light microscopy, high-resolution light microscopy, and transmission electron microscopy. Trachelyopterus galeatus is an internally inseminating species the spermatozoon of which presented an elongated cylindrical head (12.3 +/- 1.5 microm), elongated midpiece (5.0 +/- 0.7 microm), and flagellum (23.9 +/- 2.8 microm). Fertilized eggs or embryos were not found in its ovaries. Spermatozeugmata were demonstrated for the first time in this species. At the ultrastructural level, the anterior region of the head was devoid of chromatin with its shape being rounded with a hyaline tip in frontal sections and flattened in sagittal sections. The proximal centriole and most of the distal centriole were contained within a nuclear fossa. Mitochondria with lamellar cristae, as well as glycogen granules, were located just caudal to the nuclear fossa and distally in the midpiece. A single row of accessory microtubules ran peripherally in the midpiece. The flagellar axoneme had the typical 9 + 2 arrangement, having electron-dense and electron-lucent A-tubules at different points along the flagellum; flagellar fins were lacking. The ovarian lamellae were covered by a simple cuboidal epithelium. In maturing/mature females, spermatozoa were free in the ovarian lumen or inserted in pits of the lamellar epithelial cells. Tight junctions and desmosomes were seen between the epithelial cells. In addition to nourishment of the spermatozoon, the lamellar epithelial cells may play a role in protecting the spermatozoa against the female immune system.

Plasmablastic lymphoma in a previously undiagnosed AIDS patient: a case report.

BACKGROUND: Plasmablastic lymphoma (PBL) is an unusual non-Hodgkin lymphoma (NHL) most commonly found in the head and neck region. The majority of cases are seen in adult HIV-positive patients, although PBL has been reported in HIV-negative patients. The diagnosis of PBL serves as an AIDS-defining illness. METHODS: We report a case of PBL localized to the oral cavity in a previously undiagnosed AIDS patient. The lesion manifested as solitary, ulcerated, and markedly tender. PBL was confirmed by immunohistochemical profile and subsequent tests confirmed AIDS diagnosis. The patient was prescribed highly active antiretroviral therapy (HAART) and concomitant local low dose radiation therapy prior to initiation of chemotherapy. RESULTS: Complete local clinical response was observed after 4 weeks of treatment with HAART and radiation therapy. The response sustained in this patient in the subsequent 11 months following diagnosis. CONCLUSIONS: The diagnosis of PBL has a unique immunophenotypic profile and should raise suspicion for AIDS in these patients. HAART added to treatment has shown improved survival.