RESUMO
We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating: a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis; an early and late p38 MAPK activation; an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Citotoxinas/farmacologia , Inflamação/induzido quimicamente , Astrócitos/metabolismo , Linhagem Celular , Citotoxinas/metabolismo , Citometria de Fluxo , HumanosRESUMO
Syncytial giant-cell hepatitis is a rare but severe form of hepatitis that is associated with autoimmune diseases, drug reactions, and viral infections. We used serologic, molecular, and immunohistochemical methods to search for an infectious cause in a case of syncytial giant-cell hepatitis that developed in a liver-transplant recipient who had latent infection with variant B of human herpesvirus 6 (HHV-6B) and who had received the organ from a donor with variant A latent infection (HHV-6A). At the onset of the disease, the detection of HHV-6A (but not HHV-6B) DNA in plasma, in affected liver tissue, and in single micromanipulated syncytial giant cells with the use of two different polymerase-chain-reaction (PCR) assays indicated the presence of active HHV-6A infection in the patient. Expression of the HHV-6A-specific early protein, p41/38, but not of the HHV-6B-specific late protein, p101, was demonstrated only in liver syncytial giant cells in the absence of other infectious pathogens. The same markers of HHV-6A active infection were documented in serial follow-up samples from the patient and disappeared only at the resolution of syncytial giant-cell hepatitis. Neither HHV-6B DNA nor late protein was identified in the same follow-up samples from the patient. Thus, HHV-6A may be a cause of syncytial giant-cell hepatitis.
Assuntos
Doença de Caroli/cirurgia , Hepatite/virologia , Herpesvirus Humano 6/isolamento & purificação , Transplante de Fígado/efeitos adversos , Infecções por Roseolovirus/complicações , Adulto , Anticorpos Antivirais/sangue , DNA Viral/sangue , Transmissão de Doença Infecciosa , Células Gigantes , Glucocorticoides/uso terapêutico , Rejeição de Enxerto , Hepatite/tratamento farmacológico , Hepatite/patologia , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/imunologia , Humanos , Fígado/patologia , Masculino , Infecções Oportunistas/complicações , Infecções por Roseolovirus/transmissão , Carga Viral , Latência ViralRESUMO
A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in several cerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusual infectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such an activity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showed a blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection, two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). When neutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein( s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samples disclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reported in previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymes Mn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathway among the causes of the DNA damage induced by the cytotoxic transmissible agent under study.
Assuntos
Isquemia Encefálica/líquido cefalorraquidiano , Isquemia Encefálica/imunologia , Proteínas do Líquido Cefalorraquidiano/imunologia , Testes de Neutralização/métodos , Deficiências na Proteostase/líquido cefalorraquidiano , Deficiências na Proteostase/imunologia , Animais , Isquemia Encefálica/sangue , Células Cultivadas , Proteínas do Líquido Cefalorraquidiano/sangue , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Citotoxinas/sangue , Citotoxinas/líquido cefalorraquidiano , Citotoxinas/imunologia , Fibroblastos/citologia , Fibroblastos/imunologia , Heme Oxigenase-1/sangue , Heme Oxigenase-1/líquido cefalorraquidiano , Humanos , Imunoglobulina G/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/líquido cefalorraquidiano , Lúpus Eritematoso Sistêmico/imunologia , Neuroglia/citologia , Neuroglia/imunologia , Estresse Oxidativo/fisiologia , Deficiências na Proteostase/sangue , Coelhos , Superóxido Dismutase/sangue , Superóxido Dismutase/líquido cefalorraquidianoRESUMO
Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.
Assuntos
Proteínas Amiloidogênicas/análise , Príons/análise , Agregados Proteicos , Proteínas Amiloidogênicas/isolamento & purificação , Proteínas Amiloidogênicas/toxicidade , Linhagem Celular Tumoral , Cromatografia por Troca Iônica , Fracionamento por Campo e Fluxo , Humanos , Luz , Microscopia de Força Atômica , Tamanho da Partícula , Príons/isolamento & purificação , Príons/toxicidade , Espalhamento de RadiaçãoRESUMO
Positive results were attained when human peripheral blood lymphocytes (PBLs) were investigated for their ability to propagate a transmissible cytotoxic activity (TCA) isolated on VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. In consideration of this finding it can be assumed that "in vivo" blood lymphocytes contributed to give rise to the TCA detected "in vitro" in the CSF inoculum.
Assuntos
Citotoxinas/metabolismo , Linfócitos/metabolismo , Animais , Apoptose , Técnicas de Cultura de Células , Líquido Cefalorraquidiano/citologia , Chlorocebus aethiops , Testes Imunológicos de Citotoxicidade , Citotoxinas/líquido cefalorraquidiano , Humanos , Linfócitos/sangue , Linfócitos/citologia , Células VeroRESUMO
We report an unusual case of documented Bartonella henselae genotype I from hepatic tissue in an Italian immunocompetent girl presenting with erythema nodosum and hepatic granulomata. Polymerase chain reaction (PCR) was performed on biopsied liver sample to confirm the etiologic role of B. henselae and to identify the genetic variant of this organism. A PCR on the same liver biopsy for parvovirus B19 was also positive, but the clinical meaning of this was not clear.
Assuntos
Infecções por Bartonella/diagnóstico , Bartonella henselae/genética , Eritema Nodoso/etiologia , Parvovirus B19 Humano/genética , Antibacterianos/uso terapêutico , Infecções por Bartonella/complicações , Infecções por Bartonella/tratamento farmacológico , Bartonella henselae/classificação , Pré-Escolar , Claritromicina/uso terapêutico , Eritema Nodoso/tratamento farmacológico , Feminino , Granuloma/tratamento farmacológico , Granuloma/microbiologia , Humanos , Imunocompetência , Hepatopatias/tratamento farmacológico , Hepatopatias/microbiologia , Infecções por Parvoviridae/complicações , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/genética , Reação em Cadeia da PolimeraseRESUMO
When inoculated into cell cultures to search for cytopathic viruses, six out of 384 cerebrospinal fluid (CSF) samples from patients with different neurological disorders proved to have a transmissible cytotoxic activity (TCA) not correlated to a conventional infectious agents. Properties shown by a TCA previously detected in the CSF sample of a patient with brain ischemia (Portolani et al., 2005) were shared by each of the newly isolated TCAs. We conclude that independently of the neurological clinical picture shown by the patient, the TCA detected in the CSF samples under study could have the same origin.
Assuntos
Doenças do Sistema Nervoso/líquido cefalorraquidiano , Doenças do Sistema Nervoso/virologia , Adolescente , Adulto , Idoso , Animais , Isquemia Encefálica/líquido cefalorraquidiano , Chlorocebus aethiops , Efeito Citopatogênico Viral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dobramento de Proteína , Células VeroRESUMO
We investigated a transmissible cytotoxicity isolated in VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. Amorphous aggregates formed by subunities of approximately 11 nm of diameter were detected in ultracentrifugates from partially purified cytotoxic cell preparations in the absence of virion-like particles which might justify the trasmissibility of this cytotoxic activity. Results of chemico-physical studies provided indications on the presence in the CSF of two protease-resistant acidic glycoproteins of about 39 and 27 kDa, respectively. The conformational change of a proteinic molecule may associate with particular properties such as tendency to aggregation, resistance to proteolysis, cytotoxicity. Considering that these same properties are shared by proteins present in the CSF sample under study, a hypothesis to pursue is that the CSF inoculum we isolated contained misfolded proteins formed in vivo following the ischemic injury of brain tissue. As far as the in vitro transmissibility of the cytotoxic activity, this could take place following the reproduction of the alterations of those proteins, independently of the original cause(s) which have fostered their formation in vivo.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Líquido Cefalorraquidiano/química , Citotoxinas/isolamento & purificação , Isquemia/líquido cefalorraquidiano , Animais , Apoptose/efeitos dos fármacos , Fracionamento Celular/métodos , Células Cultivadas , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/toxicidade , Chlorocebus aethiops , Cromatografia por Troca Iônica/métodos , Citotoxinas/química , Citotoxinas/toxicidade , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Humanos , Microscopia Eletrônica de Varredura/métodos , Fatores de TempoRESUMO
A 7-year-old male presented sudden-onset left hemiparesis, left-sided paresthesia, central paralysis of the left VII cranial nerve, and subsequent headache. Magnetic resonance scans were obtained 24 hours after admission. T(2)-weighted images disclosed hyperintensities located mainly in the posterior portion of the lenticular nucleus and in the head and body of the right caudate nucleus. A diagnosis of ischaemic stroke was made on the basis of neuroradiologic findings. Laboratory tests undertaken to establish the cause of stroke revealed parvovirus B19 infection preceding the neurologic abnormalities. In the absence of other known risk factors for stroke the possibility of parvovirus B19's being correlated with stroke onset is discussed.
Assuntos
Encefalite Viral/complicações , Infecções por Parvoviridae/complicações , Parvovirus B19 Humano/isolamento & purificação , Acidente Vascular Cerebral/virologia , Criança , DNA Viral/análise , Encefalite Viral/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Infecções por Parvoviridae/patologia , Parvovirus B19 Humano/genética , Acidente Vascular Cerebral/patologiaRESUMO
Keshan disease is a cardiomyopathy of unknown origin reported in some areas of China. Because of epidemiologic features, this disease was ascribed to an infectious agent, likely a Coxsackie virus, but it has also been thought to depend on selenium deficiency, mainly because selenite is effective in its prophylaxis. We examined the hypothesis that pharmacological activity of selenite on Coxsackie virus growth was associated with prevention of Keshan disease. We studied the antiviral effects of three selenium compounds on Coxsackie virus B5 replication: five microM selenite reduced viral replication, whilst 10 microM selenate and selenomethionine did not exhibit any antiviral activity. The inhibitory activity of selenite on viral replication was due to its toxicity following its interaction with thiols, as that activity could be blocked by dithiothreitol, a sulfhydryl-protecting agent known to reverse several toxic effect of selenite. Zinc, another inhibitor of selenite toxicity, also counteracted the antiviral effect of selenite. The selenium compounds showed only limited activity against herpes simplex 1 virus and IHD strain of vaccinia virus. A direct inhibitory effect of selenite on Coxsackie virus replication might explain the efficacy demonstrated by this compound in the prophylaxis of Keshan disease.
Assuntos
Cardiomiopatias/virologia , Enterovirus Humano B/crescimento & desenvolvimento , Selenito de Sódio/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Cardiomiopatias/prevenção & controle , Chlorocebus aethiops , Ditiotreitol/farmacologia , Interações Medicamentosas , Enterovirus Humano B/efeitos dos fármacos , Cinética , Compostos de Selênio/farmacologia , Selenito de Sódio/antagonistas & inibidores , Selenito de Sódio/uso terapêutico , Compostos de Sulfidrila/farmacologia , Células Vero , Sulfato de Zinco/farmacologiaRESUMO
A case of primary infection by human herpesvirus 6 (HHV-6) variant A in a 54-year-old woman, which occurred at the same time as the onset of encephalomyelitis, is reported. The correlation between the two events is discussed. It is speculated that, during the early phase of the infection, the HHV-6 spread to the central nervous system and triggered a pathogenic process that initially developed without symptoms. When the neurological disorders appeared, HHV-6 had already established a latent state: only the virus carried by infected blood cells was detected in the cerebrospinal fluid.
Assuntos
Encefalomielite/virologia , Herpesvirus Humano 6/classificação , Herpesvirus Humano 6/isolamento & purificação , Infecções por Roseolovirus/complicações , Vértebras Cervicais/diagnóstico por imagem , DNA Viral/sangue , DNA Viral/líquido cefalorraquidiano , Encefalomielite/diagnóstico por imagem , Feminino , Variação Genética , Herpesvirus Humano 6/genética , Humanos , Pessoa de Meia-Idade , Radiografia , Infecções por Roseolovirus/diagnóstico por imagem , Infecções por Roseolovirus/virologiaRESUMO
An HHV-6 variant A infection is described in a 75 year-old man in association with meningoencephalitis identified at autopsy. The patient presented with fever and anorexia, then he developed altered consciousness, motor weakness, progressive lethargy, and coma, and died 21 days after hospital admission. Histopathological examination showed perivascular lymphocytic infiltrates in the central nervous system (CNS). Serum and cerebral spinal fluid (CSF) samples drawn from the patient were tested for viruses by a nested polymerase chain reaction (nPCR). HHV-6 primers A and C [Aubin et al., 1991: J Clin Microb 29: 367-372] and HS6AE and HS6AF from [Dewhurst et al. (1993): J Clin Microb 31: 416-418] disclosed a 750 bp genomic product of HHV-6 in both types of biological samples. Restricted site analysis showed that the HHV-6 DNA amplified belonged to the variant A of the virus. Short sequences of HHV-6 DNA could also be detected in the DNA extracted from formalin-fixed, paraffin-embedded sections of CNS tissues by use of one (GM5 and GM6) of three pairs of HHV-6 primers that were selected. Immunohistochemical examination of brain sections, employing a specific monoclonal antibody directed against the HHV-6 gp 102 protein, detected the viral antigen in neurons and glial cells.
Assuntos
Herpesvirus Humano 6/isolamento & purificação , Meningoencefalite/virologia , Infecções por Roseolovirus/diagnóstico , Idoso , DNA Viral/análise , Herpesvirus Humano 6/genética , Humanos , Masculino , Meningoencefalite/diagnósticoRESUMO
Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.
Assuntos
Ameloblastoma/patologia , Neoplasias Maxilomandibulares/patologia , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ameloblastoma/etiologia , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Maxilomandibulares/etiologia , Masculino , Microdissecção/instrumentação , Microdissecção/métodos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/análise , Proteínas Oncogênicas Virais/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Reação em Cadeia da PolimeraseRESUMO
Previous infection, both of bacterial and viral origin, is reported to represent an independent risk factor for ischemic stroke in children and young adults. The authors describe the case of an immunocompetent young woman who developed a middle cerebral artery thrombosis and stroke in course of a recurrence of human parvovirus B19 (PVB19) infection. A previously healthy 25-year-old woman developed right ataxic hemiparesis, 5 days after the onset of a flulike syndrome. Magnetic resonance imaging of the brain revealed acute multiple left frontal-parietal ischemic lesions. Conventional and magnetic resonance angiograms revealed a stenosis in the left middle cerebral artery. Nested polymerase chain reaction detected PVB19-specific DNA sequences in the cerebrospinal fluid and blood, and serology showed high titers of high avidity immunoglobulin G against PVB19. After 10 days, the patient's recovery was nearly complete. One month later, PVB19 disappeared from the serum, whereas it persisted in the peripheral blood mononuclear cells. This case report suggests that PVB19 infection may play a trigger role in the development of ischemic stroke, and that it should be considered in the screening of infectious risk factors for cerebrovascular diseases in young adults.