Ribosome inactivating proteins from plants inhibiting viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production

Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNβ and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell–cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II+ cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell–cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

The putative role of viruses, bacteria, and chronic fungal biotoxin exposure in the genesis of intractable fatigue accompanied by cognitive and physical disability

Patients who present with severe intractable apparently idiopathic fatigue accompanied by profound physical and or cognitive disability present a significant therapeutic challenge. The effect of psychological counseling is limited, with significant but very slight improvements in psychometric measures of fatigue and disability but no improvement on scientific measures of physical impairment compared to controls. Similarly, exercise regimes either produce significant, but practically unimportant, benefit or provoke symptom exacerbation. Many such patients are afforded the exclusionary, non-specific diagnosis of chronic fatigue syndrome if rudimentary testing fails to discover the cause of their symptoms. More sophisticated investigations often reveal the presence of a range of pathogens capable of establishing life-long infections with sophisticated immune evasion strategies, including Parvoviruses, HHV6, variants of Epstein-Barr, Cytomegalovirus, Mycoplasma, and Borrelia burgdorferi. Other patients have a history of chronic fungal or other biotoxin exposure. Herein, we explain the epigenetic factors that may render such individuals susceptible to the chronic pathology induced by such agents, how such agents induce pathology, and, indeed, how such pathology can persist and even amplify even when infections have cleared or when biotoxin exposure has ceased. The presence of active, reactivated, or even latent Herpes virus could be a potential source of intractable fatigue accompanied by profound physical and or cognitive disability in some patients, and the same may be true of persistent Parvovirus B12 and mycoplasma infection. A history of chronic mold exposure is a feasible explanation for such symptoms, as is the presence of B. burgdorferi. The complex tropism, life cycles, genetic variability, and low titer of many of these pathogens makes their detection in blood a challenge. Examination of lymphoid tissue or CSF in such circumstances may be warranted.

Spatial abundance patterns and recruitment of a virus-affected commercial mollusc fishery

 Infectious pathogens figure prominently among those factors threatening marine wildlife. Mass mortality events caused by pathogens can fundamentally alter the structure of wild fish stocks and depress recruitment rates and yield. In the most severe instances, this can precipitate stock collapses resulting in dramatic economic losses to once valuable commercial fisheries. An outbreak of a herpes-like virus among commercially fished abalone populations in the south-west fishery of Victoria, Australia, during 2006-2007, has been associated with high mortality rates among all cohorts. Long-term records from fishery-independent surveys of blacklip abalone Haliotis rubra (Leach) enabled abundance from pre- and post-viral periods to be analysed to estimate stock density and biomass. The spatial distribution of abundance in relation to physical habitat variables derived from high-resolution bathymetric LiDAR data was investigated. Significant differences were observed in both measures between pre- and post-viral periods. Although there was some limited evidence of gradual stock improvement in recent years, disease-affected reefs have remained below productivity rates prior to the disease outbreak suggesting a reduction in larval availability or settlement success. This was corroborated by trends in sublegal sized blacklip abalone abundance that has yet to show substantial recovery post-disease. Abundance data were modelled as a function of habitat variables using a generalised additive model (GAM) and indicated that high abundance was associated with complex reef structures of coastal waters (<15 m). This study highlights the importance of long-term surveys to understand abalone recovery following mass mortality and the links between stock abundance and seafloor variability.

Investigação química e biológica em hippeastrum breviflorum herb. (amaryllidaceae)

Plantas da família Amaryllidaceae são amplamente utilizadas na medicina popular no tratamento de diversas doenças. Estudos têm mostrado que a atividade biológica destes vegetais está relacionada à presença de alcalóides tetraisoquinolínicos, embora outros compostos não alcaloídicos também tenham sido encontrados. O gênero Hippeastrum encontra-se distribuído predominantemente na América do Sul sendo que no Estado do Rio Grande do Sul foram relatadas seis espécies nativas: H. glaucescens, H. striatum, H. papilium, H. reginae e H. breviflorum. A espécie estudada neste trabalho, H. breviflorum, foi coletada na região de Aparados da Serra (RS) e separada em dois grupos de acordo com a coloração das flores (vermelhas ou brancas). O material vegetal de ambos os grupos foi separado em bulbos, folhas, raízes, frutos e flores e submetido ao método clássico para extração de alcalóides. Empregando-se cromatografia em camada delgada e cromatografia líquida de alta eficiência dos extratos obtidos verificou-se que os dois grupos vegetais são idênticos qualitativamente para um mesmo extrato testado. Foram isolados dez produtos de H. breviflorum sendo que sete compostos tiveram suas estruturas determinadas através do uso de técnicas de ressonância magnética nuclear, de espectrometria de massas e de cromatografia. As outras três substâncias apresentaram-se em quantidade insuficiente para análise. A partir das folhas de H. breviflorum foram isolados dois compostos identificados como etilcafeato e p-hidroxibenzaldeído, nos frutos foram encontrados os flavonóides rutina e quercetina e nos bulbos foram isolados os alcalóides licorina, HB1 e HB5, estes dois últimos inéditos denominados de breviflorina A e beviflorina B. Dentre os compostos e extratos testados quanto à atividade antioxidante frente a DPPH, etilcafeato, quercetina e rutina foram os produtos mais ativos. O composto etilcafeato apresentou inibição do vírus da Herpes equivalente ao controle positivo aciclovir.

Desenvolvimento de ferramentas moleculares para indução de tolerância imunológica em transplante

As vacinas de DNA têm sido utilizadas para a indução de imunidade contra antígenos virais e bacterianos. A aplicação de modelos experimentais tem sido explorada visando a indução de tolerância imunológica através da expressão de genes cujos produtos podem modular o sistema imune para um estado de não responsividade. A terapia gênica oferece a possibilidade de manipulação do sistema imune do receptor, através de um sistema de administração de genes específicos sob condições pré-definidas. Sua eficácia depende dos níveis de expressão e da natureza do antígeno, da via de administração assim como de sua distribuição nos tecidos (a qual às vezes depende do promotor utilizado). Porém, sua aplicação clínica é limitada em parte devido aos baixos níveis de expressão obtidos in vivo. A VP22 é uma proteína do tegumento do vírus Herpes simples tipo 1, que tem a propriedade de fazer tráfego intercelular. Estudos recentes têm demonstrado a alta eficiência desta molécula no transporte de proteínas heterólogas como VP22-p53, VP22-β galactosidase e VP22-proteína verde fluorescente. Para a indução de tolerância imunológica, tem sido demonstrado que a persistência do antígeno, pelo menos por algum período, é muito importante. Moléculas do complexo de histocompatibilidade principal (MHC) têm sido utilizadas para induzir tolerância a nível central ou periférico, em diferentes protocolos. Dentre estas, as moléculas da classe I do camundongo, Kb, têm sido utilizadas com sucesso. O objetivo desse trabalho foi de construir duas vacinas recombinantes: pVP22::Kb e pCIneo::Kb. A primeira contém dois genes clonados na mesma pauta de leitura: a cadeia pesada de classe I Kb e VP22. O cDNA que codifica para o Kb foi obtido pela extração de RNA total de baço de um camundongo C57BL/6 (haplótipo H-2b) seguido de transcrição reversa. Este produto foi amplificado pela reação em cadeia da polimerase. Esta molécula também foi obtida pela amplificação direta do gene Kb previamente clonado no sítio EcoR I do plasmídeo pBluescriptIISK (Stratagene®). Ambos os produtos de PCR foram subclonados com extremidades cegas no plasmídeo pCRBluntII (Invitrogen®). Foram obtidos dezenove plasmídeos recombinantes, denominados pCRBluntII::Kb, e um deles foi escolhido e digerido com as enzimas de restrição Spe I e Xba I e defosforilado com a enzima fosfatase alcalina (CIAP). O fragmento digerido foi clonado nos plasmídeos pVP22-myc/His (Invitrogen®) e pCIneo (Promega®) previamente digeridos com a enzima Xba I. Os novos plasmídeos pVP22::Kb e pCIneo::Kb foram utilizados para transfectar a linhagem celular eucariótica CHO. A expressão do mRNA para o Kb foi confirmada pela transcrição reversa e PCR e a expressão da proteína por imunofluorescência e citometria de fluxo.

Análise química e avaliação da atividade antiviral de hypericum connatum lam.

Plantas da família Guttiferae apresentam diversas atividades biológicas sendo Hypericum o gênero mais importante devido às atividades antidepressiva, antibacteriana e antiviral de algumas espécies. Hypericum connatum, utilizado no sul do Brasil para o tratamento de feridas de boca, demonstrou atividade contra o lentivírus, responsável pela imunodeficiência felina. Objetivo: isolar e identificar as substâncias majoritárias de H. connatum e testar a atividade de extratos obtidos das partes aéreas e raízes e das substâncias isoladas frente a duas cepas do herpesvírus simples tipo 1 (HSV-1). Método: foram obtidos frações nhexano, diclorometano e metanol e extratos bruto, aquosos a diferentes temperaturas e hidro-etanólico das partes aéreas e raízes. As frações n-hexano e metanólica das partes aéreas foram submetidas à coluna cromatográfica para o isolamento de substâncias. Os extratos e substâncias isoladas foram testados frente ao HSV-1, cepas KOS e ATCC-VR733. Determinou-se a concentração máxima não tóxica (CMNT) à célula e a concentração que provoca alteração morfológica em 50% das células (CC50) pela técnica da alteração morfológica celular, utilizando-se células VERO, linhagem ATCC CCL-81. A avaliação da atividade antiviral foi realizada em placas de microtitulação e medida pela inibição do efeito citopático (ECP) provocado pelo vírus. Resultados e Conclusões: da fração n-hexano foi isolado hiperbrasilol B, da fração metanólica foram isolados amentoflavona, hiperosídeo e guaijaveriana, além de um flavonol de estrutura ainda não definida (HCN3). A fração n-hexano e o extrato bruto das raízes inibiram o ECP das cepas KOS e ATCC-VR733. Os demais extratos testados não apresentaram atividade antiviral. Dentre as substâncias analisadas, hiperbrasilol B, amentoflavona e HCN3 foram ativos frente às duas cepas. Os flavonóides hiperosídeo e guaijaverina não apresentaram atividade anti-HSV-1.

Serological Evidence of Bovine Herpesvirus 5 (BoHV-5) in Cattle at the Colombia's Eastern Plains

Several diseases involve the nervous system of cattle, among which infections with Rabies Virus and Bovine Herpes Virus 5 (BoHV-5) are noteworthy. In order to detect seropositive animals to BoHV-5, 156 Brahman-Zebu bovines blood samples from Colombia's eastern plains were analyzed through seroneutralization assay; the area has a history of animals dying with nervous symptoms and which rule out the disease of rabies. All animals were over one year old and randomly selected from two different herds reporting no vaccination for infectious bovine rhinotracheitis in a period exceeding one year. Results indicated seropositivity for BoHV-5 in 91 cases (58.4%), of which 88 were also seropositive for bovine herpes virus 1 (BoHV-1), while 41 were seronegative for both agents. 22/64 seronegative cases for BoHV-5 were seropositive for BoHV-1 and 2/43 seronegative cases for BoHV-1 were seropositive for BoHV-5, and consequently, these animals could be only infected by encephalitis herpes virus. With these initial findings, emphasis its placed on the need establish the true impact of the disease in Colombia and proposes the epidemiological surveillance of cattle in the region studied in order to establish mechanisms for control of viral infection.

Mutagenic Activity of Glycoallkaloids from Solanum palinacanthum Dunal (Solanaceae) in the Brazilian cerrado

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise quantitativa das células CD4 e sua importância no diagnóstico de lesões orais em pacientes HIV positivos

The oral manifestations due to HIV infection are, a lot of times, the first clinical signs of the disease. These injuries may also function as beepers and sentries of the curse and progression of the HIV infection and AIDS. The objective of this work was to evaluate the prevalence of the oral injuries in HIV positive patients, relating them with the CD4+ cells counting and the viral load in patients from the Hospital of Infected contagious Gizelda Trigueiro in Natal-RN. One hundred and one patients were evaluated, where after the clinical exam of the oral cavity, these ones were conducted to the peripheral blood collection for the counting of CD4+ lymphocytes. We observed a prevalence of 25,6%, that is, 31 cases. The Oral Candidiasis was the most commum injure, followed by Oral Hairy Leukoplakia, linear gingival erytema, lips herpes, gingivitis and periodontitis - HIV. The average counting of cells CD4+ of the injury carrying patients was of 250 cells/mm3. We did not observe relation between the presence of injuries and the viral load of the individuals

Unilateral Facial Paralysis Caused by Ramsay Hunt Syndrome

The Ramsay Hunt syndrome is a rare disease caused by an infection of the geniculate ganglion by the varicella-zoster virus. The main clinical features of the syndrome are as follows: Bell palsy unilateral or bilateral, vesicular eruptions on the ears, ear pain, dizziness, preauricular swelling, tingling, tearing, loss of taste sensation, and nystagmus. We describe a 23-year-old white woman, who presented with facial paralysis on the left side of the face, pain, fever, ear pain, and swelling in the neck and auricular region on the left side. She received appropriate treatment with acyclovir, vitamin B complex, and CMP nucleus. After 30 days after presentation, the patient did not show any signs or symptoms of the syndrome. At follow-up at 1 year, she showed no relapse of the syndrome.

Crystal structure of human purine nucleoside phosphorylase complexed with acyclovir

In human, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This work reports the first crystallographic Study of human PNP complexed with acyclovir (HsPNP:Acy). Acyclovir is a potent clinically useful inhibitor of replicant herpes simplex virus that also inhibits human PNP but with a relatively lower inhibitory activity (K-i=90muM). Analysis of the structural differences among the HsPNP:Acy complex, PNP apoenzyme, and HsPNP:Immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design. (C) 2003 Published by Elsevier B.V.

Antiviral activities of plants occurring in the state of Minas Gerais, Brazil: Part 2. Screening Bignoniaceae species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)