Production and purification of an immunogenic G protein of rabies virus from S2 insect cells

Rabies virus is still a major public health concern in under developed countries of Africa, Asia and Latin America. WHO estimates that almost 55.000 people die from rabies annually. Although Rabies virus strains from dogs still are the main responsible of causing human disease, the increasing case numbers of bat virus strains being transmitted to humans in America raises concerns. The called secondary transmission cycle of rabies involves cats or dogs which are exposed to contaminated bats and then transmit it to humans. Combating this type of transmission requires new strategies for disease control, including new vaccines. Particularly for cats, the current rabies vaccines have demonstrated to cause vaccine-associated sarcoma as a serious side effect. New rabies vaccines using recombinant technology have been proposed using the Rabies Glycoprotein (G) as antigen. The expression of this molecule in S2 insect cells has already proved the ability of this cell line to produce an immunogenic protein. Although this antigen is recognized as the main protein necessary for protection upon immunization, its utilization as a recombinant vaccine has been impaired by difficulties in its production and mainly purification steps. The G protein was expressed as a membrane bound protein in Drosophila melanogaster S2 cells. This membrane G protein was further solubilized using a standardized solution containing detergent and purified in a novel methodology using affinity chromatography resulting in protein recoveries up to 96 %. The membrane protein and the purified G were used for mice immunization, using or not ISCOM adjuvant resulting in good levels of specific antibodies and neutralizing antibodies. When immunized mice were submitted to lethal rabies virus challenge, the preparations showed to confer protection levels similar to that of commercially available veterinary rabies vaccine. A flow path from the production, purification, formulation and immunization against Rabies using a recombinant G protein is presented as a promisor veterinary vaccine candidate

The importance of mitosis as a factor for predicting sentinel lymph node biopsy for thin melanoma

23-year-old female patient, with superficial spreading melanoma (SSM) on the back, Breslow 0.35 mm, Clark II, without ulceration and with 2 mitosis/mm². Patient was submitted to margin enlargement and sentinel biopsy of 2 lymph nodes (left armpit). Histopathology revealed micrometastasis in the subcapsular sinus of both. Following the recommendation of the 2009 American Joint Committee on Cancer Melanoma Staging (AJCC), the patient underwent complete axillary lymphadenectomy. No other lymph nodes were metastatic. The clinical application of dermoscopy has enabled more accurate diagnosis of cutaneous melanoma, probably contributing to a greater proportion of thin melanomas at diagnosis. The mitotic rate was included as an important prognostic factor for thin melanomas by the AJCC, suggesting biopsy for these patientsPaciente, sexo feminino, 23 anos, com melanoma extensivo superficial em dorso, Breslow 0,35 mm, Clark II, sem ulcerações e com 2 mitoses / mm². Foi submetida à ampliação de margem e biópsia de dois linfonodos sentinela (axila esquerda). O exame anatomopatológico mostrou micrometástases, no seio subcapsular de ambos. Seguindo a recomendação do American Joint Commitee on Cancer 2009, a paciente foi submetida à linfadenectomia axilar total, sem outros linfonodos metastáticos. A aplicação da dermatoscopia vem permitindo maior precisão diagnóstica de melanoma cutâneo, contribuindo para maior proporção de melanoma fino ao diagnóstico. A taxa mitótica foi incluída como um importante fator prognóstico para melanomas finos pelo American Joint Commitee on Cancer 2009, sugerindo biópsia para esses pacientesHospital Israelita Albert EinsteinUniversidade Estadual Paulista Julio de Mesquita Filho Faculdade de Medicina de BotucatuUniversidade Anhembi MorumbiSanta Casa de Misericórdia de VotuporangaLaboratório LOCUS Laboratório de Anatomia PatológicaUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL