First list of parasitoids in flies of medical, veterinary, and forensic importance in Rio Grande do Sul – Brazil / Primeira lista de parasitoides em moscas de importância médica, veterinária e forense no Rio Grande do Sul - Brasil

Necrophagous flies are of great importance for human and animal health. In places where their development occurs, parasitoids can be used as a tool to control these dipterans. In Brazil, the fauna of these parasitoids has been investigated in some regions. However, in Rio Grande do Sul, it is known the occurrence of only one species. Thus, this study aimed to create the first list of parasitoids in flies of medical and veterinary importance in Southern Brazil. Collections took place in the municipality of Pelotas (31º 42' S; 52º 18' W). Three replicates consisting of a 500 g bovine liver chunk placed in a tray were exposed to open air for 20 days. Then pupae were individualized and observed until their emergence. We identified 4,882 adult flies of Calliphoridae, Fanniidae, Muscidae, and Sarcophagidae. 4,040 parasitoids emerged, belonging to eight species, of which Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis, and Tachinephagus zealandicus are new records in Rio Grande do Sul state. Also, three undescribed species of the genus Aphaereta were collected. In spite of being the first attempt to inventory the fauna of hymenopteran parasitoids, this study may help in the development of management programs of these dipterans in the region.

Moscas necrófagas possuem grande importância para a sanidade humana e animal. Onde estas se desenvolvem ocorrem parasitoides que podem ser utilizados como ferramentas para o controle desses dípteros. No Brasil, a fauna desses parasitoides vem sendo investigada em algumas regiões e no Rio Grande do Sul é conhecida a ocorrência de apenas uma espécie. Portanto, o objetivo deste estudo foi criar a primeira lista de espécies de parasitoides de moscas de importância médica e veterinária ocorrentes no extremo sul do Brasil. As coletas foram realizadas no município de Pelotas (31º 42' S; 52º 18' O). Foram montadas três réplicas de bandejas contendo, cada uma, 500 g de fígado bovino, expostas ao ar livre durante 20 dias. Na sequência, as pupas foram individualizadas e observadas até a emergência. Foram identificados 4.882 adultos pertencentes às famílias Calliphoridae, Fanniidae, Muscidae e Sarcophagidae. Emergiram 4.040 parasitoides, pertencentes a oito espécies, das quais Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis e Tachinephagus zealandicus são novas ocorrências para o Rio Grande do Sul. Foram coletadas três espécies ainda não descritas do gênero Aphaereta. Apesar de ainda ser um primeiro esforço de inventariar a fauna de himenópteros parasitoides, este estudo pode auxiliar no desenvolvimento de programas de manejos desses dípteros na região.

First list of parasitoids in flies of medical, veterinary, and forensic importance in Rio Grande do Sul Brazil

Abstract Necrophagous flies are of great importance for human and animal health. In places where their development occurs, parasitoids can be used as a tool to control these dipterans. In Brazil, the fauna of these parasitoids has been investigated in some regions. However, in Rio Grande do Sul, it is known the occurrence of only one species. Thus, this study aimed to create the first list of parasitoids in flies of medical and veterinary importance in Southern Brazil. Collections took place in the municipality of Pelotas (31º 42 S; 52º 18 W). Three replicates consisting of a 500 g bovine liver chunk placed in a tray were exposed to open air for 20 days. Then pupae were individualized and observed until their emergence. We identified 4,882 adult flies of Calliphoridae, Fanniidae, Muscidae, and Sarcophagidae. 4,040 parasitoids emerged, belonging to eight species, of which Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis, and Tachinephagus zealandicus are new records in Rio Grande do Sul state. Also, three undescribed species of the genus Aphaereta were collected. In spite of being the first attempt to inventory the fauna of hymenopteran parasitoids, this study may help in the development of management programs of these dipterans in the region.

Resumo Moscas necrófagas possuem grande importância para a sanidade humana e animal. Onde estas se desenvolvem ocorrem parasitoides que podem ser utilizados como ferramentas para o controle desses dípteros. No Brasil, a fauna desses parasitoides vem sendo investigada em algumas regiões e no Rio Grande do Sul é conhecida a ocorrência de apenas uma espécie. Portanto, o objetivo deste estudo foi criar a primeira lista de espécies de parasitoides de moscas de importância médica e veterinária ocorrentes no extremo sul do Brasil. As coletas foram realizadas no município de Pelotas (31º 42 S; 52º 18 O). Foram montadas três réplicas de bandejas contendo, cada uma, 500 g de fígado bovino, expostas ao ar livre durante 20 dias. Na sequência, as pupas foram individualizadas e observadas até a emergência. Foram identificados 4.882 adultos pertencentes às famílias Calliphoridae, Fanniidae, Muscidae e Sarcophagidae. Emergiram 4.040 parasitoides, pertencentes a oito espécies, das quais Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis e Tachinephagus zealandicus são novas ocorrências para o Rio Grande do Sul. Foram coletadas três espécies ainda não descritas do gênero Aphaereta. Apesar de ainda ser um primeiro esforço de inventariar a fauna de himenópteros parasitoides, este estudo pode auxiliar no desenvolvimento de programas de manejos desses dípteros na região.

First list of parasitoids in flies of medical, veterinary, and forensic importance in Rio Grande do Sul - Brazil / Primeira lista de parasitoides em moscas de importância médica, veterinária e forense no Rio Grande do Sul - Brasil

Necrophagous flies are of great importance for human and animal health. In places where their development occurs, parasitoids can be used as a tool to control these dipterans. In Brazil, the fauna of these parasitoids has been investigated in some regions. However, in Rio Grande do Sul, it is known the occurrence of only one species. Thus, this study aimed to create the first list of parasitoids in flies of medical and veterinary importance in Southern Brazil. Collections took place in the municipality of Pelotas (31º 42' S; 52º 18' W). Three replicates consisting of a 500 g bovine liver chunk placed in a tray were exposed to open air for 20 days. Then pupae were individualized and observed until their emergence. We identified 4,882 adult flies of Calliphoridae, Fanniidae, Muscidae, and Sarcophagidae. 4,040 parasitoids emerged, belonging to eight species, of which Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis, and Tachinephagus zealandicus are new records in Rio Grande do Sul state. Also, three undescribed species of the genus Aphaereta were collected. In spite of being the first attempt to inventory the fauna of hymenopteran parasitoids, this study may help in the development of management programs of these dipterans in the region.

Moscas necrófagas possuem grande importância para a sanidade humana e animal. Onde estas se desenvolvem ocorrem parasitoides que podem ser utilizados como ferramentas para o controle desses dípteros. No Brasil, a fauna desses parasitoides vem sendo investigada em algumas regiões e no Rio Grande do Sul é conhecida a ocorrência de apenas uma espécie. Portanto, o objetivo deste estudo foi criar a primeira lista de espécies de parasitoides de moscas de importância médica e veterinária ocorrentes no extremo sul do Brasil. As coletas foram realizadas no município de Pelotas (31º 42' S; 52º 18' O). Foram montadas três réplicas de bandejas contendo, cada uma, 500 g de fígado bovino, expostas ao ar livre durante 20 dias. Na sequência, as pupas foram individualizadas e observadas até a emergência. Foram identificados 4.882 adultos pertencentes às famílias Calliphoridae, Fanniidae, Muscidae e Sarcophagidae. Emergiram 4.040 parasitoides, pertencentes a oito espécies, das quais Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis e Tachinephagus zealandicus são novas ocorrências para o Rio Grande do Sul. Foram coletadas três espécies ainda não descritas do gênero Aphaereta. Apesar de ainda ser um primeiro esforço de inventariar a fauna de himenópteros parasitoides, este estudo pode auxiliar no desenvolvimento de programas de manejos desses dípteros na região.

First list of parasitoids in flies of medical, veterinary, and forensic importance in Rio Grande do Sul - Brazil.

Necrophagous flies are of great importance for human and animal health. In places where their development occurs, parasitoids can be used as a tool to control these dipterans. In Brazil, the fauna of these parasitoids has been investigated in some regions. However, in Rio Grande do Sul, it is known the occurrence of only one species. Thus, this study aimed to create the first list of parasitoids in flies of medical and veterinary importance in Southern Brazil. Collections took place in the municipality of Pelotas (31º 42' S; 52º 18' W). Three replicates consisting of a 500 g bovine liver chunk placed in a tray were exposed to open air for 20 days. Then pupae were individualized and observed until their emergence. We identified 4,882 adult flies of Calliphoridae, Fanniidae, Muscidae, and Sarcophagidae. 4,040 parasitoids emerged, belonging to eight species, of which Nasonia vitripennis, Spalangia cameroni, Spalangia chontalensis, and Tachinephagus zealandicus are new records in Rio Grande do Sul state. Also, three undescribed species of the genus Aphaereta were collected. In spite of being the first attempt to inventory the fauna of hymenopteran parasitoids, this study may help in the development of management programs of these dipterans in the region.

Eukaryotic translation initiation factor 6 is a novel regulator of reactive oxygen species-dependent megakaryocyte maturation.

BACKGROUND: Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. OBJECTIVES: To determine whether eIF6 activity is necessary for BM development. METHODS: We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. RESULTS: We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. CONCLUSIONS: Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.

The waiting room as a relational space: young patients and their families' experience in a day hospital.

BACKGROUND: Waiting in hospital is a condition of high stress for patients and their families, especially in childhood. The literature has investigated the emotional experiences of patients and their families, recognizing the need for a comfortable environment, attention from the staff, information and sharing emotions with others. Waiting time in day hospitals is a topic that has not yet been investigated in the literature, but is nevertheless interesting for researchers. This exploratory study investigates the experience of waiting young patients and their families go through during treatments in day hospitals. METHOD: Fifty children and adolescents from ages 7 to 15 years, admitted to the day hospital of a paediatric haematology and oncology ward of an Italian hospital, completed the Emotional Reaction Instrument (ERI) and the Child Drawing: Hospital. Their parents or relatives completed a semi-structured interview on waiting. RESULTS: The data showed that the young patients displayed a low level of anxiety and negative emotion. In contrast, the adults' experience of waiting in the day hospital entailed boredom, anxiety and concern for the emotional state of their children. These conditions can be alleviated by relationships and sharing emotions with other adults. CONCLUSIONS: This study has shown that day hospital waiting rooms should be organized and should be experienced by adults and children as relational spaces. This could provide useful suggestions in order to improve the organization of day hospital waiting rooms.

Semantic memory activation in individuals at risk for developing Alzheimer disease.

OBJECTIVE: To determine whether whole-brain, event-related fMRI can distinguish healthy older adults with known Alzheimer disease (AD) risk factors (family history, APOE epsilon4) from controls using a semantic memory task involving discrimination of famous from unfamiliar names. METHODS: Sixty-nine cognitively asymptomatic adults were divided into 3 groups (n = 23 each) based on AD risk: 1) no family history, no epsilon4 allele (control [CON]); 2) family history, no epsilon4 allele (FH); and 3) family history and epsilon4 allele (FH+epsilon4). Separate hemodynamic response functions were extracted for famous and unfamiliar names using deconvolution analysis (correct trials only). RESULTS: Cognitively intact older adults with AD risk factors (FH and FH+epsilon4) exhibited greater activation in recognizing famous relative to unfamiliar names than a group without risk factors (CON), especially in the bilateral posterior cingulate/precuneus, bilateral temporoparietal junction, and bilateral prefrontal cortex. The increased activation was more apparent in the FH+epsilon4 than in the FH group. Unlike the 2 at-risk groups, the control group demonstrated greater activation for unfamiliar than familiar names, predominately in the supplementary motor area, bilateral precentral, left inferior frontal, right insula, precuneus, and angular gyrus. These results could not be attributed to differences in demographic variables, cerebral atrophy, episodic memory performance, global cognitive functioning, activities of daily living, or depression. CONCLUSIONS: Results demonstrate that a low-effort, high-accuracy semantic memory activation task is sensitive to Alzheimer disease risk factors in a dose-related manner. This increased activation in at-risk individuals may reflect a compensatory brain response to support task performance in otherwise asymptomatic older adults.

Semantic memory activation in amnestic mild cognitive impairment.

Cognitively intact older individuals at risk for developing Alzheimer's disease frequently show increased functional magnetic resonance imaging (fMRI) brain activation presumably associated with compensatory recruitment, whereas mild cognitive impairment (MCI) patients tend not to show increased activation presumably due to reduced neural reserve. Previous studies, however, have typically used episodic memory activation tasks, placing MCI participants at a performance disadvantage relative to healthy elders. In this event-related fMRI study, we employed a low effort, high accuracy semantic memory task to determine if increased activation of memory circuits is preserved in amnestic MCI when task performance is controlled. Fifty-seven participants, aged 65-85 years, comprised three groups (n = 19 each): amnestic MCI patients; cognitively intact older participants at risk for developing Alzheimer's disease based on having at least one ApoE epsilon4 allele and a positive family history of Alzheimer's disease (At Risk); and cognitively intact participants without Alzheimer's disease risk factors (Control). fMRI was conducted on a 3T MR scanner while participants performed a famous name discrimination task. Participants also underwent neuropsychological testing outside the scanner; whole brain and hippocampal atrophy were assessed from anatomical MRI scans. The three groups did not differ on demographic variables or on fame discrimination performance (>87% correct for all groups). As expected, the amnestic MCI participants demonstrated reduced episodic memory performance. Spatial extent of activation (Fame--Unfamiliar subtraction) differentiated the three groups (Control = 0 ml, At Risk = 9.7 ml, MCI = 34.7 ml). The MCI and At Risk groups showed significantly greater per cent signal change than Control participants in 8 of 14 functionally defined regions, including the medial temporal lobe, temporoparietal junction, and posterior cingulate/precuneus. MCI participants also showed greater activation than Controls in two frontal regions. At Risk, but not MCI, participants showed increased activity in the left hippocampal complex; MCI participants, however, evidenced increased activity in this region when hippocampal atrophy was controlled. When performance is equated, MCI patients demonstrate functional compensation in brain regions subserving semantic memory systems that generally equals or exceeds that observed in cognitively intact individuals at risk for Alzheimer's disease. This hyperactivation profile in MCI is even observed in the left hippocampal complex, but only when the extent of hippocampal atrophy is taken into consideration.

The MTHFD1 gene is not involved in cleft lip with or without palate onset among the Italian population.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is the most common orofacial malformation, having a non-Mendelian and multifactorial aetiology. It has been shown that polymorphic variants of genes encoding key proteins of folate and methionine metabolism might be important maternal risk factors for having a child with these craniofacial anomalies. The aim of this study was to evaluate the role of two polymorphisms of the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene, the A1958G and the G401A variants, on the risk of CL/P in the Italian population. A1958G and G401A polymorphism genotyping of MTHFD1 was performed on 216 CL/P triads, (patient and parents), for this study by restriction endonuclease digestion of PCR products. Linkage disequilibrium between markers and disease was tested using both pairwise and haplotype analyses. In our case-parents triad design no significant association between MTHFD1 and the disease is evident. Our data do not support MTHFD1 involvement in CL/P onset among the Italian population.

The nuclear envelope, human genetic diseases and ageing.

Here we present an overview of the experimental evidence and of the conceptual basis for the involvement of lamins and nuclear envelope proteins in a group of genetic diseases collectively referred to as laminopathies. Some of these diseases affect a specific tissue (skeletal and/or cardiac muscles, subcutaneous fat, peripheral nerves), while others affect a variety of tissues; this suggests that the pathogenic mechanism of laminopathies could reside in the alteration of basic mechanisms affecting gene expression. On the other hand, a common feature of cells from laminopathic patients is represented by nuclear shape alterations and heterochromatin rearrangements. The definition of the role of lamins in the fine regulation of heterochromatin organization may help understanding not only the pathogenic mechanism of laminopathies but also the molecular basis of cell differentiation and ageng.

In vitro apoptotic cell death during erythroid differentiation.

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.

Pathogenesis of viral hepatitis.

The aim of our research is to use animal models to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The results herein discussed provide insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV and HCV infections.

Inositol-specific phospholipase C in low and fast proliferating hepatoma cell lines.

Inositol lipid cycle, among the pletora of signalling events, is directly involved in cell growth. It is located both in the cytoplasm and in the nucleus. Disturbances may cause uncontrolled proliferation of the cell and ultimately cancer. The phosphatidyl inositol phospolipase C (PLC) is a key enzyme in the hydrolysis of polyphosphoinositides (PIs) and could be differently involved in the normal and pathological cell growth. We report immunochemical and immunocytochemical demonstrations that the PLC isoforms are present in both cytoplasmic and nuclear compartments of low and fast proliferating hepatoma cells. The PLC activity is increased in fast proliferating cells, in which PLC delta1 and to a greater extent PLC delta4 are more expressed at cytosolic level, suggesting an involvement of PI specific PLCs in the progression of cell cycle and in the control of cell proliferation and possibly of neoplastic cell growth.

Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver.

We have previously reported that intrahepatic NK T cells activated by alpha-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-gamma produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.

Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes.

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the human myeloid K562 cell line. TRAIL stimulated caspase 3 and nitric oxide synthase (NOS) activities, and both pathways cooperate in mediating inhibition of K562 survival/growth. This was demonstrated by the ability of z-VAD-fmk, a broad inhibitor of effector caspases, and N-nitro-L-arginine methyl ester (L-NAME), an NOS pharmacologic inhibitor, to completely (z-VAD-fmk) or partially (L-NAME) suppress the TRAIL-mediated inhibitory activity. Moreover, z-VAD-fmk was able to block TRAIL-mediated apoptosis and cell cycle abnormalities and increase of NOS activity. The addition of the NO donor sodium nitroprusside (SNP) to K562 cells reproduced the cytostatic effect of TRAIL without inducing apoptosis. When TRAIL was associated to SNP, a synergistic increase of apoptosis and inhibition of clonogenic activity was observed in K562 cells as well as in other myeloblastic (HEL, HL-60), lymphoblastic (Jurkat, SupT1), and multiple myeloma (RPMI 8226) cell lines. Although SNP greatly augmented TRAIL-mediated antileukemic activity also on primary leukemic blasts, normal erythroid and granulocytic cells were less sensitive to the cytotoxicity mediated by TRAIL with or without SNP. These data indicate that TRAIL promotes cytotoxicity in leukemic cells by activating effector caspases, which directly lead to apoptosis and stimulate NO production, which mediates cell cycle abnormalities. Both mechanisms seem to be essential for TRAIL-mediated cytotoxicity.

Characterization of nuclear RNases that cleave hepatitis B virus RNA near the La protein binding site.

Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5' end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5' of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Noncytolytic control of viral infections by the innate and adaptive immune response.

This review describes the contribution of noncytolytic mechanisms to the control of viral infections with a particular emphasis on the role of cytokines in these processes. It has long been known that most cell types in the body respond to an incoming viral infection by rapidly secreting antiviral cytokines such as interferon alpha/beta (IFN-alpha/beta). After binding to specific receptors on the surface of infected cells, IFN-alpha/beta has the potential to trigger the activation of multiple noncytolytic intracellular antiviral pathways that can target many steps in the viral life cycle, thereby limiting the amplification and spread of the virus and attenuating the infection. Clearance of established viral infections, however, requires additional functions of the immune response. The accepted dogma is that complete clearance of intracellular viruses by the immune response depends on the destruction of infected cells by the effector cells of the innate and adaptive immune system [natural killer (NK) cells and cytotoxic T cells (CTLs)]. This notion, however, has been recently challenged by experimental evidence showing that much of the antiviral potential of these cells reflects their ability to produce antiviral cytokines such as IFN-gamma and tumor necrosis factor (TNF)-alpha at the site of the infection. Indeed, these cytokines can purge viruses from infected cells noncytopathically as long as the cell is able to activate antiviral mechanisms and the virus is sensitive to them. Importantly, the same cytokines also control viral infections indirectly, by modulating the induction, amplification, recruitment, and effector functions of the immune response and by upregulating antigen processing and display of viral epitopes at the surface of infected cells. In keeping with these concepts, it is not surprising that a number of viruses encode proteins that have the potential to inhibit the antiviral activity of cytokines.

Nuclear covalently closed circular viral genomic DNA in the liver of hepatocyte nuclear factor 1 alpha-null hepatitis B virus transgenic mice.

The role of hepatocyte nuclear factor 1alpha (HNF1 alpha) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1 alpha-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1 alpha transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1 alpha protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1 alpha-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1 alpha-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.