RESUMO
Subtractive suppressive hybridization was used to identify differentially expressed genes in subcuticular tissues from white spot syndrome virus(WSSV)-infected shrimp kept at different temperatures. Subtractive libraries I and II contained genes expressed at 26 and 33 degrees C, respectively. Three hundred and seventy-nine insert positive clones were selected to confirm differential expression by dot-blot hybridization. Twenty-two clones from library I and eight from library II were sequenced. All sequences from Library I corresponded to white spot syndrome virus genes. From library II, five clones were homologous with previously reported expressed sequence tags of Litopenaeus vannamei, two had similarity with beta-actin and one transcript represented an unknown gene. Over-expression of VP15 in shrimp at 26 degrees C was further confirmed by real-time polymerase chain reaction (PCR), whereas beta-actin expression was similar in animals kept at both temperatures. Together, our results show that hyperthermia reduces the expression of WSSV genes on shrimp subcuticular epithelial cells.
Assuntos
Células Epiteliais/virologia , Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/fisiologia , Temperatura Alta , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/genética , Animais , Células Epiteliais/imunologia , Biblioteca Gênica , Penaeidae/genética , Reação em Cadeia da Polimerase , Vírus da Síndrome da Mancha Branca 1/imunologiaRESUMO
We have previously reported that white spot syndrome virus-infected Penaeus vannamei (also called Litopenaeus vannamei) maintained at 32 degrees C show higher survival rates and a significant increase in number of apoptotic cells when compared to infected shrimp kept at 26 degrees C. As apoptosis plays an important part in the antiviral response of invertebrates, we hypothesized that this process would reduce WSSV replication, allowing the shrimp to control the disease and survive. To test this hypothesis, shrimp were orally infected and maintained at either 26 degrees C (Group 1) or 32 degrees C (Group 2), DNA was extracted from haemolymph collected at various times from 6 to 216 h post-infection, and the number of viral units was quantified by real time PCR using SYBR Green. In parallel, histological examination was carried out to confirm the WSSV infection and to rule out concomitant diseases. Linear regression of real time PCR units (rtPCRU) of WSSV from Group 1 showed a significant increase with time post-infection (r2 = 0.7383; p < 0.001). Conversely, there was no increase in rtPCRU with time post-infection in Group 2 (r2 = 0.142), indicating that hyperthermia inhibited, either directly or indirectly, viral replication. In addition, comparison between the groups showed no difference in WSSV rtPCRU up to 48 h post-infection. After 72 h, shrimp from Group 1 had a significantly higher viral rtPCRU (ANOVA, p < 0.001). We conclude that hyperthermia-associated WSSV rtPCRU reduction could reflect either an increase in the shrimp antiviral response, or a direct negative effect on viral replication, or both.
Assuntos
Temperatura Alta , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/crescimento & desenvolvimento , Animais , Primers do DNA/química , Modelos Lineares , Reação em Cadeia da Polimerase/métodos , Carga Viral/veterinária , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/patogenicidadeRESUMO
Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.
Assuntos
Criopreservação/métodos , Penaeidae , Preservação do Sêmen/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Citometria de Fluxo , Glicerol/farmacologia , Masculino , Metanol/farmacologia , Fatores de TempoRESUMO
Heat shock proteins (Hsp) are moving from the category of basically intracellular chaperone molecules to important proteins in both innate and acquired immune responses, with great potential for clinical application as immunomodulators. Both proinflammatory and regulatory Hsp-reactive T cells have been described in animal models of autoimmune diseases. To investigate the role of autoreactivity to Hsp60 and Hsp70 in human transplantation, we analyzed, sequentially, peripheral blood mononuclear cell proliferation and cytokine production before and at different time points after renal transplantation, as well as the modulation of proliferation to Hsp in the presence of exogenous cytokines. Proliferation to Hsp60 and Hsp70 in the pretransplantation (pre-Tx) period was significantly associated with rejection episodes in the first months post-Tx. In contrast, IL-4 production was significantly associated with absence of rejection. Addition of exogenous IL-4 distinctly modulated the proliferative response to Hsp60; inhibiting proliferation in 83% of patients in the early post-Tx period (0-6 months), in which rejection episodes occurred, and inducing proliferation in 62.5% of patients in the later period (>12-24 months), when no rejection was observed. Characterization of autoreactive anti-Hsp60 regulatory T cells may permit new approaches to control the proinflammatory response to the graft, as well as aggressive autoimmunity.
Assuntos
Proteínas de Choque Térmico/imunologia , Interleucina-4/metabolismo , Transplante de Rim/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Autoimunidade/imunologia , Proliferação de Células/efeitos dos fármacos , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Criança , Ensaio de Imunoadsorção Enzimática , Rejeição de Enxerto/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/farmacologia , Teste de Histocompatibilidade , Humanos , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/farmacologia , Interleucina-4/imunologia , Interleucina-4/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Estudos Longitudinais , Pessoa de Meia-Idade , Fito-Hemaglutininas/farmacologia , Valor Preditivo dos TestesRESUMO
Apoptosis plays a critical role in development and maintenance of multicellular organisms. It has also been described as an anti-viral mechanism in both insects and vertebrates. In fact, to escape the immune system and to increase their spread, some viruses such as baculovirus produce anti-apoptotic molecules. Conversely, a recent report showing a positive correlation between the number of apoptotic cells and the severity of white spot syndrome virus (WSSV) infection in Penaeus monodon suggested that apoptosis might be the cause of death in viral-infected shrimp. Searching for the mechanisms involved in the beneficial effect of hyperthermia for WSSV-infected Litopenaeus vannamei (also called Penaeus vannamei) and considering that hyperthermia increases apoptosis in other experimental models, we investigated the presence of apoptosis by Tdt-mediated dUTP nick-end label (TUNEL), from 4 of 168 h in 3 groups of 50 L. vannamei juveniles. Group 1 consisted of experimentally infected shrimp (intramuscular injection of 3 x 10(7) viral copies) kept at 25 degrees C, Group 2 of similarly infected shrimp kept at 32 degrees C and Group 3 of uninjected shrimp kept at 32 degrees C. Apoptosis was found only in WSSV-infected individuals. Shrimp at 25 degrees C were positive for apoptotic cells in 48 (16%) of their examined tissues or organs, compared to 62 (21%) for those at 32 degrees C. Moreover, shrimp at 32 degrees C also had a significantly higher overall mean apoptotic index (AI) than shrimp at 25 degrees C (p < 0.05). Comparison of mean AI at 72, 96 and 120 h post-infection showed that individuals at 32 degrees C presented a significantly higher values than those at 25 degrees C. These results suggested that hyperthermia might facilitate apoptosis in WSSV-infected L. vannamei and might be one of the mechanisms responsible for increased survival of infected shrimp maintained at 32 degrees C.
Assuntos
Apoptose , Vírus de DNA/patogenicidade , Hipertermia Induzida/veterinária , Penaeidae/virologia , Animais , Aquicultura , Vírus de DNA/genética , Temperatura Alta , Marcação In Situ das Extremidades Cortadas/veterinária , Distribuição Aleatória , Fatores de Tempo , Carga Viral/veterinária , VirulênciaRESUMO
A INH é uma das drogas capazes de induzir a formaçäo de auto-anticorpos e, em alguns casos, uma síndrome semelhante ao LE. Os autores avaliaram longitudinalmente, no soro de 24 pacientes com tuberculose tratados com INH, a presença de A-AH e fraçöes às quais säo dirigidos. O fator antinuclear foi positivo em dois pacientes tratados e anticorpos IgM anti-histona foram detectados em 4/24 (6%); estes também foram observados em um paciente pré-tratamento. Soros com IgM-AH que apresentavam reatividade forte por Elisa reconheciam todas as fraçös de histona no western blotting e os com reatividade menor ligavam-se somente à fraçäo H1. Näo se verificou correlaçä com manifestaçöes clínicas de LE