RESUMO
Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.
Assuntos
Fibronectinas/farmacologia , Células de Schwann/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Imunofluorescência , Microscopia Eletrônica de Varredura , Ratos , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestrutura , Nervo Isquiático/fisiologiaRESUMO
A model of latent infection by varicella-zoster virus (VZV) was obtained in the adult rat. Inoculation of VZV-infected cells in the skin led to infection of the peripheral nervous system. Latency was characterized by a long-lasting presence of the viral genome, of selected viral gene transcripts, and of at least one viral protein in the dorsal root ganglia. Reactivation has not been obtained in vivo, but has occurred ex vivo after repeated stresses. Many similarities with VZV latency in humans were found, making this model useful for vaccine and antiviral studies.
Assuntos
Herpes Zoster/genética , Herpesvirus Humano 3/genética , Latência Viral/genética , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , RatosRESUMO
The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 3/genética , Fases de Leitura Aberta , Mapeamento Cromossômico , HumanosRESUMO
The pathogenic mechanisms involved in varicella-zoster virus (VZV) infections remain elusive. The pattern of cutaneous distribution of the IE63 protein and of the gpI (gE) and gpII glycoproteins with their corresponding genome sequences during VZV infections was studied by immunohistochemistry and in situ hybridization. Skin biopsy specimens were obtained from immunocompetent and immunocompromised patients with varicella, herpes zoster, or atypical VZV lesions. The first evidence for VZV infection consisted of the presence of IE63 in keratinocytes. In the vesicles and pustules, the viral transcripts gpI, gpII, and IE63 and the corresponding nucleic acids for gpI and gpII were identified in keratinocytes, sebocytes, Langerhans cells, dermal dendrocytes, monocytes/macrophages, and endothelial cells. The gpI and gpII glycorpoteins were essentially located on the cellular membranes while IE63 expression was generally restricted to the nuclei. In three biopsies of early herpes zoster, viral proteins were disclosed in dermal nerves and in perineurial type I dendrocytes. This was never encountered in varicella. Vasculitic changes and endothelial cell involvement were more prominent in varicella than in herpes zoster. It is concluded that the secondary viremia in varicella that affects the dermal endothelial cells is followed by a cell-to-cell spread to keratinocytes. In herpes zoster, the viral progression through cutaneous nerves primarily extends to the pilosebaceous units with a secondary involvement of epidermal keratinocytes, followed by a further spread to dermal cells.
Assuntos
DNA Viral/análise , Herpes Zoster/genética , Herpesvirus Humano 3/genética , Pele/química , Proteínas Virais/análise , Herpes Zoster/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In SituRESUMO
A 73-year-old man developed an ill-defined fatal vasculitis involving the central nervous system. The case report was published as a clinicopathologic exercise in February 1995 in The New England Journal of Medicine. We restudied the pathologic material and found both varicella zoster virus (VZV) DNA and VZV-specific antigen, but not herpes simplex virus (HSV) or cytomegalovirus (CMV) DNA or HSV- or CMV-specific antigen, in three of the five cerebral arteries examined. The inflammatory response, disruption of the internal elastic lamina, and detection of viral antigen were patchy from one artery to another, as well as within a given artery. A search for VZV should be conducted in cases of vasculitis when both the central and peripheral nervous systems are involved, when focal narrowing is present in large arteries, when brain imaging reveals infarction in gray and white matter, both deep and superficial, and when white matter is disproportionally involved. Zosteriform rash is not required for diagnosis.
Assuntos
Varicela/complicações , Herpesvirus Humano 3/isolamento & purificação , Vasculite/etiologia , Idoso , DNA Viral/análise , Humanos , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Vasculite/patologiaRESUMO
Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields sigmoid curves displaying lower slopes, thus invalidating any quantitative interpretation. We hypothesized that this phenomenon was due to a competition effect between the target (for example an antigen) and related molecules for the binding sites (for example a capture antibody) immobilized onto the solid phase. This has been confirmed experimentally using various target-to-competitor ratios and formulated as a mathematical model. The slope decrease in target detection was related to the proportion of competitor, not in a linear, but in an exponential manner. This mathematical model has been computerized and can be used to correct aberrant sample curves provided the relevant parameters have been previously determined in the same systems.
Assuntos
Calibragem/normas , Técnicas Imunoenzimáticas/normas , Imunoglobulina G/normas , Espectrofotometria , Pesos e Medidas/normas , Algoritmos , Animais , Ligação Competitiva , Humanos , Isotipos de Imunoglobulinas/normas , Camundongos , Coelhos , Espectrofotometria/métodos , Espectrofotometria/normasRESUMO
Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation procedures. The conjugates were tested by enzyme immunoassay and their efficiency was characterized by the antibody and enzyme concentrations needed to obtain an arbitrary OD value. The suitability of antibody for conjugation through NH2 groups was tested by fluorodinitrobenzene (FDNB). HRP conjugates were produced by two variants of the sodium periodate procedure and two variants of the glutaraldehyde method, as well as by the heterobifunctional linker N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Two of the four antibodies were coupled by a third variant of the periodate method, through their carbohydrate moieties. The periodate-mediated conjugations, using sugar moieties on the enzyme, provided the most efficient HRP conjugates, regardless of the antibody subclass or isoelectric point. The glutaraldehyde procedures consistently gave the worst results. AP conjugates were prepared using the same methods. The most efficient and reproducible AP conjugates with all four monoclonal antibodies were obtained using the SPDP procedure. The efficiency of the other methods differed from one antibody to another.
Assuntos
Anticorpos Monoclonais , Técnicas Imunoenzimáticas , Fosfatase Alcalina , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Concanavalina A , Dinitrofluorbenzeno , Hidrazinas , Imunoquímica , Ponto Isoelétrico , CamundongosRESUMO
Am important aspect of human immunodeficiency virus (HIV-1) infection is the regulation of its expression by nuclear factor kappa B (NF-kappa B) by redox-controlled signal transduction pathways. In this study, we demonstrate that selenium supplementation can effectively increase glutathione peroxidase (GPx) activity in latently infected T lymphocytes. The Se-supplemented cells exhibited an important protection against the cytotoxic and reactivating effects of hydrogen peroxide (H2O2). Concomitantly, NF-kappa B activation by H2O2 was also decreased in Se-supplemented cells. Selenium stimulation of GPx activity also induces a protective effect against cell activation by tumor necrosis factor alpha (TNF-alpha) but less significantly by phorbol esters such as PMA. These Se-mediated effects were specific because they were not found when AP-1 DNA-binding activity was studied after H2O2-induced stress. Hyperthermia was also studied because it could promote intracellular electron leakage in electron transport chains. Elevating the temperature to 42 degrees C did not induce NF-kappa B directly. Rather, it sensitized infected cells to subsequent oxidative stress by H2O2, demonstrating the importance of hyperthermia, often associated with opportunistic infections in the development of immunodeficiency. In this case, Se induced partial protection against the sensitizing effect of hyperthermia.
Assuntos
Glutationa Peroxidase/metabolismo , HIV-1/fisiologia , Antioxidantes/metabolismo , Linhagem Celular , DNA Viral/metabolismo , Infecções por HIV/etiologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Temperatura Alta , Humanos , Peróxido de Hidrogênio/farmacologia , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Selênio/farmacologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/virologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Pain typically accompanies acute herpes zoster and, in a proportion of patients, it persists well beyond rash healing. Pain must therefore be analyzed in trials of antiviral agents in herpes zoster, but different methods have been used to analyze pain in recent published trials. These reports are reviewed and their methodological strengths and weaknesses examined. Based on this review, recommendations for the design and analysis of future trials of antiviral agents in herpes zoster are proposed. The principal recommendation is that antiviral efficacy should be evaluated both by distinguishing post-herpetic neuralgia from acute pain and by considering pain as a continuum. The primary endpoint should address both the prevalence and duration of post-herpetic neuralgia and should be examined in those patients who have post-herpetic neuralgia. Adopting the proposed recommendations in design and analysis of future trials should facilitate comparison across trials of the efficacy of antiviral agents in the treatment of herpes zoster.
Assuntos
Antivirais/uso terapêutico , Herpes Zoster/tratamento farmacológico , Herpes Zoster/fisiopatologia , Medição da Dor , Ensaios Clínicos como Assunto , Previsões , Humanos , Projetos de PesquisaRESUMO
AIMS: To study the cutaneous and visceral distribution of herpes simplex virus (HSV) and varicella zoster virus (VZV) in fatal infections. METHODS: Standard histology, immunohistochemistry (monoclonal antibodies VL8 and VL2 and polyclonal antibody IE63 directed against VZV; monoclonal antibodies IBD4 and HH2 and polyclonal antibodies directed against HSVI and HSVII) and in situ hybridisation (anti-HSV and anti-VZV probes) were applied to formalin fixed, paraffin wax sections. RESULTS: On histological examination, Herpesviridae infection was evident in various organs including the lungs, liver and skin. In addition, immunohistochemistry and in situ hybridisation revealed the presence of HSV and VZV antigens and nucleic acids in several cell types and tissues showing no cytopathological alterations suggestive of Herpesviridae infection. The organs with histological evidence of infection also contained VZV or HSV antigens and their genes. CONCLUSIONS: These findings suggest that organ failure in disseminated VZV and HSV infections is primarily caused by HSV or VZV induced cell damage and lysis. They also indicate that immunohistochemistry and in situ hybridisation can provide an accurate, type-specific diagnosis on formalin fixed, paraffin wax embedded tissue even when classic histological and cytological characteristics are lacking.
Assuntos
Herpes Simples/virologia , Herpes Zoster/virologia , Herpesvirus Humano 3/isolamento & purificação , Intestinos/virologia , Simplexvirus/isolamento & purificação , Pele/virologia , Adulto , Idoso , Estado Terminal , Evolução Fatal , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Fígado/virologia , Pulmão/virologia , MasculinoRESUMO
Varicella zoster virus immediate-early (IE) proteins are intracellular regulators of viral gene expression. Some of them (IE62 and IE63) are found in large amounts in infected cells but are also components of the virion tegument. Several IE and early genes are transcribed during latency, while late genes are not. Recently, we demonstrated the presence of protein IE 63 in dorsal root ganglia of persistently infected rats as well as in normal human ganglia; other IE proteins have been found since in human ganglia. Cell-mediated immunity (CMI) to IE 62 has been evidenced. We found both humoral immunity and CMI to IE 63 in immune adults. In elderly zoster-free individuals, CMI to IE 63 remained high. The differences in the CMI to IE 63 among young adults, elderly people and immunocompromized patients have to be analyzed according to their status relative to zoster, to determine whether the decrease in CMI, particularly to IE proteins, could be responsible for viral reactivation and for the onset of shingles. Hopefully, the waning of the CMI to VZV IE 63 and perhaps to other IE proteins could become a predictive marker for herpes zoster and reimmunization, not only with the vaccine strain, but also with purified IE proteins could help prevent zoster at old age.
Assuntos
Herpesvirus Humano 3/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Proteínas Virais/fisiologia , Latência Viral , Animais , Vacina contra Varicela/genética , Vacina contra Varicela/imunologia , Herpesvirus Humano 3/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Transativadores/genética , Transativadores/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Varicella zoster virus (VZV) is responsible for a primary infection (varicella) and, upon reactivation, zoster, which in immunocompromised patients, may both lead to life-threatening disseminated disease. There is a great need for antiviral compounds that are effective inhibitors of VZV replication and for rapid and accurate methods for evaluating viral sensitivity to candidate anti-VZV drugs. With the monoclonal antibody (mAb) (VL8), which is directed against the gpI of VZV, and using the fluorescence-activated cell sorter (FACS) we could readily demonstrate expression of the VZV gpI antigen at 3-4 days after VZV infection. (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMC) were shown to be potent inhibitors of VZV replication by this assay. HPMPA and HPMPC were also active against thymidine kinase-deficient (TK-) VZV whereas BVDU was not. The flow cytometric method based on the use of mAb VL8 may be of considerable help for the early diagnosis of VZV infection and evaluation of viral sensitivity to antiviral drugs.
Assuntos
Citometria de Fluxo , Herpesvirus Humano 3/isolamento & purificação , Organofosfonatos , Proteínas do Envelope Viral/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Monoclonais/imunologia , Antivirais/farmacologia , Células Cultivadas , Cidofovir , Citosina/análogos & derivados , Citosina/farmacologia , Nucleotídeos de Desoxiuracil/farmacologia , Avaliação de Medicamentos , Herpesvirus Humano 3/crescimento & desenvolvimento , Herpesvirus Humano 3/imunologia , Humanos , Compostos Organofosforados/farmacologia , Timidina Quinase/deficiência , Proteínas do Envelope Viral/isolamento & purificação , Replicação Viral/efeitos dos fármacosRESUMO
Hemoblot, a new immunological faecal occult blood test, produced by Gamma, Angleur, Belgium, was characterized and compared with another immunological test (HemeSelect, SmithKline Diagnostics, USA) and with a guaiac test (Hemoccult II, SmithKline Diagnostics). The analytical sensitivity of Hemoblot is 0.15 mg haemoglobin/g faeces and the test is specific for human haemoglobin. In addition, 135 symptomatic patients who had to undergo a colonoscopy were tested using the three tests. Two criteria were considered for the analysis: (1) the blood criterion: any pathology likely to cause colorectal or other bleeding; and (2) the precancerous-cancerous criterion: the pathology being either a colorectal polyp > 0.5 cm or a colorectal cancer. Considering both criteria, the sensitivity of Hemoblot was significantly higher than the sensitivity of Hemoccult: 38% and 23%, respectively, for the blood criterion; and 54% and 29% for the precancerous-cancerous criterion. Sensitivity and specificity did not differ statistically between Hemoblot and HemeSelect but Hemoblot was faster and simpler to perform. It could be widely used in mass screening.
Assuntos
Neoplasias do Colo/diagnóstico , Sangue Oculto , Neoplasias Retais/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Pólipos do Colo/diagnóstico , Colonoscopia , Ensaio de Imunoadsorção Enzimática , Hemorragia Gastrointestinal/diagnóstico , Guaiaco , Hemoglobinas/análise , Humanos , Técnicas Imunoenzimáticas , Pólipos Intestinais/diagnóstico , Programas de Rastreamento , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/diagnóstico , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells.
Assuntos
Herpesvirus Humano 3/fisiologia , Herpesvirus Humano 3/patogenicidade , Animais , Varicela/fisiopatologia , Varicela/virologia , Modelos Animais de Doenças , Gânglios Espinais/virologia , Genes Virais , Cobaias , Herpes Zoster/fisiopatologia , Herpes Zoster/virologia , Herpesvirus Humano 3/isolamento & purificação , Humanos , Neurônios/virologia , Fases de Leitura Aberta , Dor , Ratos , Proteínas Virais/biossíntese , Latência ViralRESUMO
Various biological processes, such as photosensitization or inflammatory reactions, can generate singlet oxygen (1O2) as one of the major oxidative species. Because this oxidant can be generated either extracellularly or intracellularly, it can cause severe damage to various biological macromolecules, even to those deeply embedded inside the cells such as DNA. Sublethal biological modifications induced by different DNA-damaging agents can promote various cellular responses initiated by the activation of various cellular genes and certain heterologous viruses. Since 1O2 fulfils essential prerequisites for a genotoxic substance, we have examined the effects of an oxidative stress, mediated by this species, on cells harbouring a heterologous promoter-leader sequence derived from the human immunodeficiency virus type 1 (HIV-1). Our results demonstrate that HIV-1 long terminal repeat (LTR), integrated into the cellular DNA of epithelial cells, can be transactivated following an oxidative stress mediated by 1O2. In addition, using HIV-1 latently infected promonocytes or lymphocytes, it can be shown that virus reactivation can be induced through a sublethal dose of 1O2 generated intracellularly. An extracellular generation of 1O2 can promote a substantial lethal effect without HIV-1 reactivation. These data may be relevant to the understanding of the events converting a latent infection into a productive one and to the appearance of the acquired immune deficiency syndrome.
Assuntos
HIV-1/genética , Oxigênio/farmacologia , Regiões Promotoras Genéticas , Sobrevivência Celular/efeitos da radiação , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Dano ao DNA , Relação Dose-Resposta à Radiação , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Cinética , Luz , Fotoquímica , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos da radiação , Oxigênio Singlete , Ativação Transcricional , Transfecção , Integração ViralRESUMO
Varicella-zoster virus is a Herpesvirus responsible for three distinct clinical features: chicken pox (varicella), shingles (herpes zoster) and post-zosterian pain (post-herpetic neuralgia). Neurological features of these diseases such as complications of chicken pox, viral latency in sensory ganglia and reactivation as shingles with concurrent and possibly subsequent prolonged pain, are the sequels of the invasion of the peripheral nervous system during primary infection. Prevention is achieved by vaccination with a live attenuated virus strain and therapy calls for specific antiviral agents. In many respects, vzv behaves differently from close relatives. In particular, viral latency in the nervous system is basically different from that of other Herpesviridae. The recent discovery of the expression of some viral regulatory proteins during latency, although it had always been considered that vzv latency was silent, and demonstration that these proteins are immunogenic open new avenues concerning the immune control of vzv reactivation.
Assuntos
Herpesvirus Humano 3/fisiologia , Ativação Viral , Latência Viral , Animais , HumanosRESUMO
We report a case of lethal varicella developed in an immunocompromised man in his seventies. A study by immunohistochemistry and in situ hybridization was conducted revealing the presence of the glycoprotein gpI specific for VZV and its corresponding nucleic acids found in multiple foci of most tissues and organs, except in the brain and paravertebral sympathetic ganglia.