ABSTRACT
BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.
Subject(s)
Human T-lymphotropic virus 1 , Vaccines, DNA , Mice , Humans , Animals , CD8-Positive T-Lymphocytes , Granzymes/genetics , Tumor Necrosis Factor-alpha , Vaccines, DNA/genetics , Viral Proteins/metabolism , Vaccinia virus/genetics , DNA , Basic-Leucine Zipper Transcription Factors , Retroviridae Proteins/geneticsABSTRACT
Regional lymph node status impacts survival in dogs with malignant mammary tumors. However, few studies have evaluated extracapsular extension and tumor implants in regional lymph node metastases in dogs with mammary carcinoma. Therefore, 84 cases of mammary carcinomas with metastases in inguinal and/or axillary lymph nodes from female dogs of different breeds and a total of 139 metastatic lymph nodes were evaluated by routine histological staining. Clinical and pathological characteristics of primary tumors as well as the presence of extracapsular extension and tumor implants in the lymph nodes were analyzed, in addition to survival. One to 5 lymph nodes were evaluated in each case. Extracapsular extension and tumor implants were present in 17.9% and 39.3% of cases, respectively. The simultaneous presence of extracapsular extension and tumor implants were associated with an increased risk of death (hazard ratio 10.46). In addition, "special type carcinomas", high histological grade (grade III), and presence of extracapsular extension associated with tumor implants were related to a worse prognosis and shorter survival times (p < 0.05). Based on these results, we highlight the importance of identifying extracapsular extension and tumor implants in dogs with metastatic mammary carcinomas, as they are associated with a higher risk of death and shorter survival.
Subject(s)
Carcinoma , Dog Diseases , Lymphatic Metastasis , Animals , Carcinoma/veterinary , Dogs , Extranodal Extension , Female , Lymph Nodes , Prognosis , Survival AnalysisABSTRACT
Recombinant virus vectors represent a promising strategy for vaccine research. Among available viral vectors, members of the Poxviridae family-especially the modified Vaccinia virus Ankara (MVA)-stand out as immunogenic and safe vaccine platforms. Because MVA usually does not produce plaques in cell culture, visible selection markers such as the green fluorescent protein (GFP) are frequently incorporated into the constructions in order to facilitate the recognition of recombinants. However, these genetic markers have to be removed before any clinical trial. Here, we evaluated the acute responses generated in mice immunized with a MVA vector in which the GFP marker was not removed. We observed no differences in neutrophil, monocyte, or total leucocyte recruitment among animals inoculated with MVA or MVA-GFP. Likewise, there were no differences in neutrophil activation between mice groups. Hepatic functions were not altered in either MVA or MVA-GFP-inoculated mice, and we observed no histopathological alterations in different tissues from virus-inoculated animals. In conclusion, the presence of GFP is innocuous to immunized animals and do not alter acute physiopathological responses to the MVA vector. We suggest that keeping the GFP marker may be a good strategy for vaccine development, production, and evaluation.
Subject(s)
Green Fluorescent Proteins/adverse effects , Vaccines, Attenuated/immunology , Vaccinia virus/genetics , Vaccinia virus/immunology , Viral Vaccines/immunology , Animals , Female , Green Fluorescent Proteins/genetics , Mice , Mice, Inbred BALB C , Monocytes/immunology , Neutrophils/immunology , Smallpox/prevention & control , Vaccination , Vaccines, DNAABSTRACT
Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484) foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB) (T1), albumina sérica bovina (BSA) (T2) ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+) (T3), no cultivo embrionário in vitro, a uma atmosfera de 5 por cento CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF) e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63) foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P<0,05) do que em T1 e T3. A taxa de blastocistos foi similar (P>0,05) entre T2 e T3, porém menor (P<0,01) do que em T1. A proporção do sexo dos embriões não diferiu (P>0,05) entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.
Subject(s)
Animals , Female , Cattle , Culture Media , Embryonic Structures , Fertilization in VitroABSTRACT
Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484) foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB) (T1), albumina sérica bovina (BSA) (T2) ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+) (T3), no cultivo embrionário in vitro, a uma atmosfera de 5% CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF) e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63) foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P<0,05) do que em T1 e T3. A taxa de blastocistos foi similar (P>0,05) entre T2 e T3, porém menor (P<0,01) do que em T1. A proporção do sexo dos embriões não diferiu (P>0,05) entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.(AU)
The effect of culture media on the development and on the sex ratio of bovine embryos fertilized in vitro was studied. Cumulus oocyte-complexes from slaughterhouse ovaries were matured and fertilized in vitro. Zygotes (n= 484) were randomly allotted to different culture media and cultured with their cumulus cells in CR2aa medium and an atmosphere of 5% CO2 in air at 38.8ºC. The fetal calf serum (FCS), bovine seric albumin (BSA) or BSA plus insulin:transferrin:selenium and vitamins (BSA+) supplementation effect on embryo culture was evaluated. Cleavage rate was assessed at 72-76h post-fertilization (PF) and blastocyst rate on days 7 and 8 PF. The blastocysts (n= 63) were also sexed using polymerase chain reaction. Cleavage rate for BSA medium supplemented was higher (P<0.05) than FCS and BSA+. Blastocyst rates for BSA and BSA+ media were similar (P>0.05), but lower (P<0.01) than FCS. Culture medium FCS supplemented affected blastocyst development but not the sex ratio.(AU)
Subject(s)
Animals , Female , Cattle , Embryonic Structures , Culture Media , Fertilization in VitroABSTRACT
PURPOSE: The aim of this preliminary study was to present an alternative method to assess the anal sphincters by ultrasonography using a conventional ultrasound surface probe by transperineal approach. METHODS: Transperineal ultrasonography was performed in 20 asymptomatic volunteers to assess the anal sphincters. Ultrasonographic findings were compared with conventional anal endosonography pictures available in the literature. RESULTS: Images of the anal sphincters obtained by transperineal ultrasound were found to be similar to those produced by conventional anal endosonography. Internal and external anal sphincters were easily demonstrated in addition to mucosal and submucosal layers. CONCLUSIONS: Transperineal ultrasonography is a new technique that enables imaging of anal sphincters and anal canal structures with potential application in functional and inflammatory anorectal disorders.