Project description:Three experiments, corresponding to the three figures in the article, are represented in this set. Each experiment was an ex vivo treatment time course as described in the paper. For each of the experiments, mRNA was isolated at the indicated time points, cDNA was directly prepared by reverse transcription in the presence of Cy5-labeled dUTP, and the expression profiled using Cy3-labeled cDNA prepared by reverse transcription of Stratagene Universal Human Reference RNA as a control. One experiment, corresponding to Figure 1 in the paper, is a set of infection, or mock-infection time courses, in which each of three cell types: primary human dermal fibroblasts, primary human macrophages, or HELA cells were respectively infected with Monkeypox virus, Vaccinia virus, or Ebola virus, or mock-infected. Infected cells or mock-infected cells were harvested and lysed at the indicated times after infection and mRNA isolated for analysis following the protocol described above. The second experiment, corresponding to Figure 2 in the paper, is a set of treatments of human dermal fibroblasts, each in 24-well plates with either: PBS only (mock), interferon alpha (IFN-alpha) at 0.6 pM final concentration (Sigma, St. Louis, MO), tumor necrosis factor alpha (TNF-alpha) at 0.6 pM final concentration (Sigma), PMA at 25ng/mL final concentration plus ionomycin at 1micromolar final concentration, polyinosinic-polycytidylic acid as potassium salt (poly(I-C)) at 100 microg/mL final concentration (Sigma), Escherichia coli 055:B5 lipopolysaccharide (LPS) at 1microg/mL final concentration (Sigma), or dexamethasone at 1 micromolar final concentration (Sigma). Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. The third experiment, corresponding to Figure 3 in the paper, is a set of infections or mock-infections of human dermal fibroblasts, or human primary macrophages, with Monkeypox virus or Vaccinia virus, respectively, followed by treatment with either ionomycin + phorbol myristic acid (PMA) or with poly(I-C). The intention of the experiment was to investigate whether prior infection altered the response of the cells to the chemical agents. Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. Description of sample characteristics: Time: Time after infection or Mock infection Infection: Ebola-Zaire/killed Monkeypox Virus/Mock infection/Monkaypox Virus/None/Pre infection/Vaccinia NY/Vaccinia WR Compound Based Treatment: Dexamethasone/Interferon-alpha/Ionomycin + PMA/LPS/PBS/polyinosinic-polycytidylic acid/TNF-alpha Cell Type: Primary Human Dermal Fibroblast/Primary Human Macrophage/HeLa Figure in article: Numbers group slides that are represented in the same figure of the article. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment
Project description:Three experiments, corresponding to the three figures in the article, are represented in this set. Each experiment was an ex vivo treatment time course as described in the paper. For each of the experiments, mRNA was isolated at the indicated time points, cDNA was directly prepared by reverse transcription in the presence of Cy5-labeled dUTP, and the expression profiled using Cy3-labeled cDNA prepared by reverse transcription of Stratagene Universal Human Reference RNA as a control. One experiment, corresponding to Figure 1 in the paper, is a set of infection, or mock-infection time courses, in which each of three cell types: primary human dermal fibroblasts, primary human macrophages, or HELA cells were respectively infected with Monkeypox virus, Vaccinia virus, or Ebola virus, or mock-infected. Infected cells or mock-infected cells were harvested and lysed at the indicated times after infection and mRNA isolated for analysis following the protocol described above. The second experiment, corresponding to Figure 2 in the paper, is a set of treatments of human dermal fibroblasts, each in 24-well plates with either: PBS only (mock), interferon alpha (IFN-alpha) at 0.6 pM final concentration (Sigma, St. Louis, MO), tumor necrosis factor alpha (TNF-alpha) at 0.6 pM final concentration (Sigma), PMA at 25ng/mL final concentration plus ionomycin at 1micromolar final concentration, polyinosinic-polycytidylic acid as potassium salt (poly(I-C)) at 100 microg/mL final concentration (Sigma), Escherichia coli 055:B5 lipopolysaccharide (LPS) at 1microg/mL final concentration (Sigma), or dexamethasone at 1 micromolar final concentration (Sigma). Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. The third experiment, corresponding to Figure 3 in the paper, is a set of infections or mock-infections of human dermal fibroblasts, or human primary macrophages, with Monkeypox virus or Vaccinia virus, respectively, followed by treatment with either ionomycin + phorbol myristic acid (PMA) or with poly(I-C). The intention of the experiment was to investigate whether prior infection altered the response of the cells to the chemical agents. Cells were harvested and lysed at the indicated times and mRNA isolated for analysis following the protocol described above. Description of sample characteristics: Time: Time after infection or Mock infection Infection: Ebola-Zaire/killed Monkeypox Virus/Mock infection/Monkaypox Virus/None/Pre infection/Vaccinia NY/Vaccinia WR Compound Based Treatment: Dexamethasone/Interferon-alpha/Ionomycin + PMA/LPS/PBS/polyinosinic-polycytidylic acid/TNF-alpha Cell Type: Primary Human Dermal Fibroblast/Primary Human Macrophage/HeLa Figure in article: Numbers group slides that are represented in the same figure of the article.
Project description:Despite being rarely reported, improved diagnostic and prognostic indicators are necessary for treating malignant melanoma in rabbits. In this study, two cases of primary skin lesions, on the scrotum and on eyelid, with systemic metastases, were examined. The tumors formed intra-dermally by sheet-like proliferation of polymorphic cells, with anisocytosis and varying amount of melanin granules. Tumors had displaced almost 50% of the lung and liver tissue, and tumor metastasis was the cause of early death in both rabbits. Ki-67-positive population was high in both, and it was found to be useful in assessing the outcome and malignancy. In addition, Melan-A, HMB-45, PNL2 and S100 established a useful immunohistochemical panel for the diagnosis of melanocytic tumor in rabbits.
Project description:Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Even though the eradication of variola virus - the causative agent of human smallpox M-bM-^@M-^S succeeded, with the end of vaccinations several other orthopoxviruses emerged as potential threat to human health. For instance, animal-borne monkeypox virus, cowpox virus and closely related vaccinia virus are all capable of establishing zoonotic infections in humans. The disease caused by each virus differs in terms of expression and severity, but we still know little about the reasons for these different phenotypes. They may be explained by the unique repertoire of host cell modulating factors encoded by each virus. In this study, we aimed at characterizing the specific modulation of the host cells gene expression profile by orthopoxvirus infection. In our study we analyzed changes in host cell gene expression of HeLa cells after infection with cowpox virus, monkeypox virus or vaccinia virus and compared these to each other and to the gene expression profile of non-infected cells using Agilent Whole Genome Microarray technology. We could identify major differences in viral modulation of host cell immune response genes, especially an induction of genes involved in leukocyte migration and Toll-like receptor signalling in cowpox and monkeypox virus infected cells. This was not observed following vaccinia virus infection. If these differences contribute to the different clinical manifestation of cowpox, monkeypox and vaccinia virus infections in certain host species remains to be elucidated. We analyzed the gene expression profile of HeLa cells wich were either mock-infected or infected with Vaccinia virus strain IHD-W, Cowpox virus strain Brighton Red or Monkeypox virus strain MSF#6 at a multiplicity of infection of 5. Experiments were performed in duplicate. At 6 h post infection total RNA was isolated from infected cells and used for microarray analysis.
Project description:Cryptosporidium cuniculus is a zoonotic parasite responsible for cryptosporidiosis cases and outbreaks in both humans and rabbits. Since there are no molecular Cryptosporidium spp. infection data in rabbits (Oryctolagus cuniculus) from Spain, our aim was to gather information about this parasite in wild European rabbits from Tenerife, Canary Islands (Spain). A total of 100 faecal samples were collected from rabbits from eight municipalities of Tenerife. Microscopic analysis showed that 4.0% of the samples presented structures compatible with Cryptosporidium oocyst. A nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA (rRNA) gene fragments was carried out, and sequencing confirmed the identity of C. cuniculus in one sample (1.0%). The sample was successfully subtyped using nested PCR analysis of the 60-kDa glycoprotein (gp60) gene as the subtype VbA26R3. This study confirms the presence of C. cuniculus in wild rabbits from Tenerife, providing new information on the occurrence of this zoonotic parasite. Further studies are required to better understand the epidemiology of Cryptosporidium spp. in wild rabbits in Spain and their possible public health repercussions.