Detection of Hepatitis B Virus (HBV) Genomes and HBV Drug Resistant Variants by Deep Sequencing Analysis of HBV Genomes in Immune Cell Subsets of HBV Mono-Infected and/or Human Immunodeficiency Virus Type-1 (HIV-1) and HBV Co-Infected Individuals.

UNLABELLED: The hepatitis B virus (HBV) and the human immunodeficiency virus type 1 (HIV-1) can infect cells of the lymphatic system. It is unknown whether HIV-1 co-infection impacts infection of peripheral blood mononuclear cell (PBMC) subsets by the HBV. AIMS: To compare the detection of HBV genomes and HBV sequences in unsorted PBMCs and subsets (i.e., CD4+ T, CD8+ T, CD14+ monocytes, CD19+ B, CD56+ NK cells) in HBV mono-infected vs. HBV/HIV-1 co-infected individuals. METHODS: Total PBMC and subsets isolated from 14 HBV mono-infected (4/14 before and after anti-HBV therapy) and 6 HBV/HIV-1 co-infected individuals (5/6 consistently on dual active anti-HBV/HIV therapy) were tested for HBV genomes, including replication indicative HBV covalently closed circular (ccc)-DNA, by nested PCR/nucleic hybridization and/or quantitative PCR. In CD4+, and/or CD56+ subsets from two HBV monoinfected cases, the HBV polymerase/overlapping surface region was analyzed by next generation sequencing. RESULTS: All analyzed whole PBMC from HBV monoinfected and HBV/HIV coinfected individuals were HBV genome positive. Similarly, HBV DNA was detected in all target PBMC subsets regardless of antiviral therapy, but was absent from the CD4+ T cell subset from all HBV/HIV-1 positive cases (P<0.04). In the CD4+ and CD56+ subset of 2 HBV monoinfected cases on tenofovir therapy, mutations at residues associated with drug resistance and/or immune escape (i.e., G145R) were detected in a minor percentage of the population. SUMMARY: HBV genomes and drug resistant variants were detectable in PBMC subsets from HBV mono-infected individuals. The HBV replicates in PBMC subsets of HBV/HIV-1 patients except the CD4+ T cell subpopulation.

Toll-like receptor 2 ligand, lipoteichoic acid is inhibitory against infectious laryngotracheitis virus infection in vitro and in vivo.

Lipoteichoic acid (LTA) is one of the pathogen associated molecular patterns (PAMPs) that activates toll-like receptor (TLR)2-cluster of differentiation (CD)14 signalling pathway. This recognition elicits antiviral responses that have been recorded against viruses of mammals although such responses have not been characterized adequately against avian viruses. In this investigation, we characterized the LTA induced antiviral responses against infectious laryntotracheitis virus (ILTV) infection in vitro and in vivo. We found that LTA is capable of up regulating mRNA expression of innate proteins in macrophages such as MyD88, iNOS and IL-1ß and reduces the ILTV plaques in vitro. Similarly, we found that LTA treatment of embryonic day 18 (ED18) eggs can lead to the antiviral response against pre-hatch ILTV infection in vivo and is associated with expansion of macrophage populations and expression of IL-1ß and MyD88 in the lung. The data highlight that LTA can be a potential innate immune stimulant that can be used against ILTV infection in chickens.

Bacterial infections in pigs experimentally infected with Nipah virus.

Nipah virus (NiV; Paramyxoviridae) caused fatal encephalitis in humans during an outbreak in Malaysia in 1998/1999 after transmission from infected pigs. Our previous study demonstrated that the respiratory, lymphatic and central nervous systems are targets for virus replication in experimentally infected pigs. To continue the studies on pathogenesis of NiV in swine, six piglets were inoculated oronasally with 2.5 x 10(5) PFU per animal. Four pigs developed mild clinical signs, one exudative epidermitis, and one neurologic signs due to suppurative meningoencephalitis, and was euthanized at 11 days post-inoculation (dpi). Neutralizing antibodies reached in surviving animals titers around 1280 at 16 dpi. Nasal and oro-pharyngeal shedding of the NiV was detected between 2 and 17 dpi. Virus appeared to be cleared from the tissues of the infected animals by 23 dpi, with low amount of RNA detected in submandibular and bronchial lymph nodes of three pigs, and olfactory bulb of one animal. Despite the presence of neutralizing antibodies, virus was isolated from serum at 24 dpi, and the viral RNA was still detected in serum at 29 dpi. Our results indicate slower clearance of NiV from some of the infected pigs. Bacteria were detected in the cerebrospinal fluid of five NiV inoculated animals, with isolation of Streptococcus suis and Enterococcus faecalis. Staphylococcus hyicus was isolated from the skin lesions of the animal with exudative epidermitis. Along with the observed lymphoid depletion in the lymph nodes of all NiV-infected animals, and the demonstrated ability of NiV to infect porcine peripheral blood mononuclear cells in vitro, this finding warrants further investigation into a possible NiV-induced immunosuppression of the swine host.

Modulation of simian immunodeficiency virus neuropathology by dopaminergic drugs.

Drug abuse and human immunodeficiency virus (HIV) infection seem to cause cumulative damage in the central nervous system (CNS). Elevated extracellular dopamine is thought to be a prime mediator of the reinforcing effects of addictive substances. To investigate the possible role of increased dopamine availability in the pathogenesis of HIV dementia, simian immunodeficiency virus (SIV)-infected monkeys were treated with dopaminergic drugs (selegiline or L-DOPA). Both substances increased intracerebral SIV expression, combined with aggravation of infection-related neuropathology and ultrastructural alterations of dendrites in dopaminergic areas (spongiform polioencephalopathy) in asymptomatic animals. Moreover, this treatment resulted in enhanced TNF-alpha expression in the brains of SIV-infected animals. These findings indicate a synergistic interaction between dopamine and SIV infection on microglia activation, leading to increased viral replication and production of neurotoxic substances. Our results suggest that increased dopamine availability through dopaminergic medication or addictive substances may potentiate HIV dementia.

Activation of microglia cells is dispensable for the induction of rat retroviral spongiform encephalopathy.

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9-13 days postinoculation (d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells--as revealed by immunohistochemical staining with monoclonal antibody ED1--were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.

Emergency child mental health services in Poland.

This paper describes emergency child mental health services in Poland. The paper defines common emergencies for children and adolescents, describes who responds and how services are organized. The evaluation process, interventions and treatment follow-up are outlined. The lack of consistency across Poland is highlighted. Recommendations for further training of professionals, as well as improvements in the mental health and legal systems are made.

Enhancement of central nervous system pathology in early simian immunodeficiency virus infection by dopaminergic drugs.

Human immunodeficiency virus infection (HIV) at late stages of the disease is accompanied by neurological complications, including motor, behavioral and cognitive impairment. Using simian immunodeficiency virus (SIV)-infected rhesus monkeys, an animal model of HIV infection, we found that during the asymptomatic SIV infection dopamine (DA) deficits are early components of central nervous system (CNS) dysfunction. To investigate the role of the DA system in SIV infection and to restore the DA deficiency, we administered selegiline, an agent with DAergic and neuroprotective properties, to SIV-infected monkeys. Selegiline increased DA availability but induced CNS vacuolization, SIV encephalitic lesions, and enhanced CNS viral replication during early SIV infection. The pathological changes seem to be mediated by DA, as treatment with L-DOPA, the precursor of DA, had similar effects. We propose that any natural or induced DAergic dysregulation which results in increased DA availability may potentiate HIV-associated neurological disease (ND). Our findings raise new questions regarding the pathogenesis of HIV-ND and generate concerns about the safety of dopaminergic drugs in the clinical management of HIV-infected patients.

Abundant defective viral particles budding from microglia in the course of retroviral spongiform encephalopathy.

A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.

Effects of Selegiline in a retroviral rat model for neurodegenerative disease.

Upon inoculation into neonatal rats, murine leukemia virus (MuLV) NT40 causes a non-inflammatory degeneration of the central nervous system. While microglia cells appear to be the major target cells within the brain parenchyma for neurovirulent MuLV, degenerating neurons do not express retroviral gene products. In order to protect rats from neuronal damage we treated retrovirally infected rats once with monoamine oxidase (MAO) B inhibitor Selegiline which--under different conditions--exerts neuroprotective effects. Unexpectedly, when administered at 17 days post-infection (d.p.i.) a single intraperitoneal dose of Selegilin (1 mg/kg bodyweight) significantly shortened the incubation period for neurological disease. In contrast, Selegiline given in a lower dosage (0.05 mg/kg bodyweight) and/or at a different time point (13 d.p.i.) at the low (0.05 mg/kg bodyweight) and the high dose (1.0 mg/kg bodyweight) had no effect on the outcome of neurological disease. Animals treated with Selegiline (1.0 mg/kg bodyweight at 17 d.p.i.) contained higher amounts of viral loads in the CNS, higher numbers of brain cells expressing major histocompatibility complex class II molecules, and exhibited inhibition of MAO-B in comparison to untreated yet infected (control) animals. Supposedly, Selegiline activated the major target cell population of the CNS for MuLV-NT40, microglia, with the consequence of enhanced susceptibility for retroviral infection and triggered endogenous mechanism(s) involved in the pathogenesis of retroviral neurodegeneration.

Suppression of rat bone marrow cells by Friend murine leukemia virus envelope proteins.

In a retroviral rat model, we have investigated the nontransforming effects of murine leukemia virus FB29 on the bone marrow. Upon intraperitoneal inoculation with murine leukemia virus FB29 of either neonatal or adult rats, bone marrow cells became massively infected within the first 12 days postinoculation. In neonatally inoculated rats, a persistent productive bone marrow infection was established, whereas in rats inoculated as adults, no infected bone marrow cells could be detected beyond 12 days postinoculation. Retroviral infection was most likely cleared by an antiviral immune response (Hein et al., 1995, Virology 211, 408-417). Exposure to virus irreversibly decreased numbers of bone marrow cells staining with monoclonal antibody OX7 by 10-30%. Reduction of OX7+ bone marrow cells by 20% was also observed in vitro, after bone marrow cells from uninfected adult rats had been co-incubated with virus. FB29-envelope proteins were sufficient alone to reduce numbers of OX7+ bone marrow cells, both in vivo and in vitro. According to results on incorporation of propidium iodide, decreased numbers of OX7+ cells were due to cell death. By flow cytometric analyses OX7+ bone marrow cells as well as monocytes/macrophages were identified to be major target cells for infection with FB29 within the bone marrow. Thus, the mechanism(s) responsible for death of OX7+ bone marrow cells might be due to direct toxicity of viral envelope proteins and/or to interactions of viral envelope proteins with cells of the monocytic lineage.

Monoamine metabolite levels in CSF of SIV-infected rhesus monkeys (Macaca mulatta).

Increasing evidence suggests the involvement of the basal ganglia in HIV-1-infected patients. We used SIV-macaques, an animal model of HIV-1 infection, to investigate changes in CSF biogenic amine metabolites over time and compared them with control animals. The dopamine and serotonin metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were analysed by reverse-phase chromatography with electrochemical detection. DOPAC concentrations were significantly increased in SIV-infected animals compared with controls. 5-HIAA and HVA remained unchanged. However, the longitudinal assessment of metabolites up to 3 months post-infection revealed a significant increase in 5-HIAA. Our results reflect the effects of SIV early stage infection on monoamine systems in brain and further validate the simian model for AIDS research.

Nature and sequence of simian immunodeficiency virus-induced central nervous system lesions: a kinetic study.

To determine the onsets and specificities of neuropathological features observed after simian immunodeficiency virus (SIV) infection of macaques, brains of 19 clinically unaffected rhesus monkeys (group A) were examined after intervals ranging from 1 to 48 weeks post-infection and compared to 8 animals with AIDS (group B) as well as to 8 uninfected controls. Based on morphological and virological parameters, seven patterns specific for SIV infection of the central nervous system (CNS) were discerned. In both groups of infected but not control animals, we found mononuclear aggregates in meninges, perivascular space, and choroid plexus stroma (designated pattern 1), isolated infected cells within CNS parenchyma (pattern 2), axonal degeneration (pattern 3), spongy change (pattern 4), microglial proliferation (pattern 5), and small vessel proliferation (pattern 6). SIV encephalitis (pattern 7) was only evident in animals with clinically evident disease. Changes characteristic of patterns 3, 5 and 6 appeared to be chronic and non-progressive, whereas lesions of patterns 1, 2 and 4 appeared to have progressed in animals with AIDS. The main component of mononuclear aggregates in animals of group A were lymphocytes, in contrast to animals of group B, in which macrophages dominated the inflammatory infiltrates. Altogether, our results demonstrate that subtle leukoencephalopathy was a specific feature of clinically silent as well as clinically evident phases of SIV infection. This might explain the neurological impairment of HIV-positive non-AIDS individuals.

Impact of various simian immunodeficiency virus variants on induction and nature of neuropathology in macaques.

To investigate the possible influence of virus tropism on SIV-induced neuropathogenesis, macaques were infected with molecularly cloned SIVmac239 which replicates poorly in cultured macrophages, with SIVmac251/32H which is a macrophage-tropic biologic clone, and with SIVmac251/MPBMC which is an early passage of 32H with enhanced replication competence in macrophages. We found that inflammatory as well as neuropathologic changes were identical in all clinically affected animals, irrespective of the in vitro tropism of the inocula. Moribund animals exhibited SIV encephalitis characterized by overt infection of macrophages/microglia inside the CNS parenchyma. Additionally, neuropathology of moribund animals was characterized by extraparenchymal immunopathology (meninges, perivascular space, choroid plexus stroma) and subtle white matter degeneration with glial changes, often associated with infected macrophages in situ (except in leukoencephalopathy). However, in animals inoculated with the lymphocyte-tropic and enhanced macrophage-tropic inocula, microglia but not blood-derived macrophages were the primary cells infected. Altogether, our results underline the significance of macrophage infection for the development of SIV encephalitis, and suggest that SIVmac239 either undergoes a change in cell tropism in vivo that results in the ability to replicate in macrophages, or else macrophages become more permissive to infection by this virus in the terminal stage of immunosuppressive disease.

Murine leukemia virus-induced neurodegeneration of rats: enhancement of neuropathogenicity correlates with enhanced viral tropism for macrophages, microglia, and brain vascular cells.

A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4-6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry, in situ hybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and microglia, whereas FB29 infected only microglia and those to a lower extent. However, when isolated from adult diseased rats, microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and microglia correlated with enhancement of neuropathogenicity.

Phenotypic and functional characterization of CD8+ T lymphocytes from the central nervous system of rats with coronavirus JHM induced demyelinating encephalomyelitis.

Intracerebral infection of Lewis (LEW) inbred rats with the neurotropic strain of the murine coronavirus JHM (JHMV) frequently results in a monophasic paralytic disease. In contrast, infection of Brown Norway (BN) inbred rats does not lead to clinical disease. Previous findings indicated that in both rat strains brain-infiltrating leukocytes consisted mainly of CD8+ T lymphocytes. Here, we phenotypically as well as functionally characterised this T cell subset after isolation from the central nervous system (CNS). Using JHMV-infected target cells, MHC class I restricted, cytotoxic T lymphocytes were demonstrated to be present in the leukocyte fraction from the CNS of both, susceptible LEW and disease-resistant BN rats. However, compared to infected, but healthy BN rats, diseased LEW rats generated an enhanced cytotoxic immune response which became most prominent at the maximum of neurological disease. Recently published observations from our laboratory demonstrated a strong virus-specific antibody response in the CNS of BN rats. In LEW rats, however, the response was delayed and of low magnitude. This suggests, that consequences of cytotoxic T lymphocyte action in JHMV-infected CNS tissue largely depend on the efficacy of an accompanying virus-specific humoral immune response.

Effects of adoptive immune transfers on murine leukemia virus-infection of rats.

In a rat model, we have investigated the effects of adoptively transferred virus-specific immune cells on an established retroviral infection of various organs. The experimental design required inoculation of neonatal Fisher rats with a molecular clone of Friend murine leukemia virus (F-MuLV; FB29) which resulted in virus-specific immunotolerance, while infection of adult rats lead to a virus-specific humoral and cellular immune response. Adoptive transfer of virus-specific immune cells from immunized to immunotolerant (i.e., neonatally inoculated) rats was performed at around 15 days postpartum, a time when retroviral titers had already reached high levels in serum, spleen, thymus, and central nervous system (CNS). Seven days post-transfer (dpt), virus titers began to decline by 3-5 logs first in sera and at around 11-15 dpt, in spleens and thymi. Approximately 19 days post-transfer viral titers increased again. In the CNS, viral titers appeared not to change after adoptive transfer, although we observed an influx of activated T-cells and natural killer cells (NK-cells), but not of B-cells, into the CNS as well as an upregulation of major histocompatibility complex class I and II molecules between 8 and 21 dpt on both microglia and other brain cells. From these data we conclude that MuLV-infected cells of lymphoid organs can be eliminated by an antiviral immune response. In the CNS, however, most virus-infected cells escaped an immunological attack in spite of the presence of T- and NK-cells and may thus function as a reservoir for MuLVs.

Prevention of retrovirus-induced neurological disease by infection with a nonneuropathogenic retrovirus.

Perinatal infection of susceptible mice with the neurotropic retrovirus CasBrE leads to a noninflammatory spongiform degeneration of the central nervous system with a long incubation period of up to 1 year. Virus replication in infected animals can be suppressed by administration of antiviral antibodies, cytotoxic T cells, or by AZT treatment, which results in partial to complete protection from neurological disease. A highly neuropathogenic chimeric retrovirus, FrCasE, which contains the envelope gene of CasBrE, induces rapid neurodegeneration within only 16 days. Here we report that this fatal disease could be prevented if a nonneuropathogenic Friend murine leukemia virus was administered to mice prior to their infection with FrCasE. This double inoculation led to a substantial reduction of the replication level of FrCasE in spleen and CNS. Only live but not heat-inactivated nonneuropathogenic virus was able to protect from FrCasE-induced neurological disease. The extent of protection was influenced by the viral envelope gene and the kinetics of replication of the nonneuropathogenic virus. These observations in addition to the rapidity of the effect make it likely that competition for replication sites through the mechanism of viral interference is responsible for the protection. Resistance was demonstrable in vivo even when the "protecting" and "challenge" virus belonged to different in vitro interference groups. However, the protection was considerably weaker than that seen between viruses belonging to the same interference group.

Kinetic analysis of spongiform neurodegenerative disease induced by a highly virulent murine retrovirus.

BACKGROUND: A chimeric murine retrovirus, FrCasE, causes a rapid noninflammatory spongiform neurodegenerative disease of the motor system with an incubation period of 15 to 16 days after neonatal inoculation. Neurovirulence is determined by the viral envelope gene, but the neurodegeneration is an indirect consequence of virus infection, because the neurons that degenerate appear not to be infected. EXPERIMENTAL DESIGN: The current study was undertaken to compare the kinetics of lesion development and the expression of viral envelope protein in the central nervous system (CNS). Neonatal mice were inoculated with FrCasE intraperitoneally and were killed at various times for determination of the kinetics of the CNS infection, the distribution of lesion in the CNS, and the distribution of viral envelope protein. In addition, qualitative features of both viral envelope and gag proteins were followed by immunoblot analysis. RESULTS: The lesions induced by FrCasE consisted of vacuolar degeneration but without associated astrocytosis, the lack of an astroglial response being a consequence of the rapidity of the disease process. Vacuoles were observed primarily in the neuropil of the motor centers of spinal cord, brain stem, and cerebral cortex. Lesions appeared in all of these areas during a narrow window of time (< or = 3 days). Cells in which viral envelope protein was detected by immunohistochemistry before the appearance of spongiform degeneration included premigratory cerebellar cortical granule neurons as well as vascular elements in the regions that would ultimately exhibit spongiform degeneration. Two forms of viral envelope protein were detected in the CNS. A 70-kilodalton species appeared first, followed by an approximately 64-kilodalton species, which was detected coincident with the first appearance of spongiform lesions. CONCLUSIONS: Astrocytosis is a secondary reaction to the neuronal cytopathology induced by FrCasE and appears to be dependent on the developmental state of the CNS. The abrupt, diffuse nature of lesion development in this disease suggests a global effect of the virus infection. Cells of the CNS vasculature (either endothelial cells, perivascular microglial cells, or both) as well as cerebellar granule neurons appear to be seminally involved in the pathogenesis of the spongiform degeneration. The two species of viral envelope protein appear to be expressed by different cell types in the CNS.

Retrospective appraisal of intensity of skin postradiation reactions in breast cancer patients treated with telegammatherapy.

Post irradiation skin reaction in patients treated with telegammatherapy is evaluated. Exaggerated reaction was noted in patients older than the mean age of the entire group.

[Survival, complications and quality of life in patients with breast cancer after ovariectomy and hormonal therapy]. / Przezycia, powiklania i jakosc zycia u chorych na raka sutka po kastracji chirurgicznej i terapii hormonalnej.

Seventy one women with breast cancer in clinical stage IIIa were treated by chemotherapy and radical operation on the basis five features, namely: survivals, relapses, metastases, quality of life, and post-therapy complications. The two treatment methods were compared. The dependence between survivals and time elapsed between breast surgery and ovariectomy was evaluated. Women treated by ovariectomy suffer from; menopause symptoms, osteoporosis, blood coagulation distortions more after than women treated by hormonotherapy. Tamoxifen therapy increases the rate of breast cancer relapses and probably it is the cause of breast cancer metastases into liver. Women who underwent hormonal castration are professionally active more after them women treated by ovariectomy. Time elapsed between breast surgery and ovariectomy does not affect survivals in stage IIIa. In stage IIIb however, performing later ovariectomy prolongs survivals.