Perfil neuropsicológico de personas adultas con VIH/SIDA

Los déficit neurológicos en el virus de inmunodeficiencia humana VIH, específicamente las alteraciones en el funcionamiento cognitivo, han estado presentes desde el inicio de su propagación y han sido una de las principales manifestaciones a lo largo de todas las etapas del virus. No obstante, gracias a los avances de la terapia antirretroviral se ha dado un aumento de la expectativa de vida de los pacientes, dándose de la misma manera un incremento en los déficits anteriormente mencionados. El objetivo del presente estudio es describir el perfil neuropsicológico de los pacientes con VIH y establecer si existen relaciones entre las funciones que se encuentren deterioradas, el tiempo de diagnóstico y la terapia antiretroviral. Para esto se llevo a cabo un estudio descriptivo de tipo exploratorio con el fin de medir las características de las funciones neuropsicológicas en un grupo de 24 pacientes pertenecientes al programa especial B24 del Hospital Universitario Mayor y Hospital de Barrios Unidos MÉDERI en Bogotá, Colombia. Para esto, se utilizó un protocolo de pruebas neuropsicológicas: Mini Mental StateExamination (MMSE), WAIS-III (sub-pruebas dígitos, letras y números, aritmética y semejanzas), Curva de aprendizaje auditivo verbal de Rey (RAVLT), WMS-III (sub-pruebas de recobro de historias y Localización), TMT A y B, Set Test de Isaacs, Figura Compleja de Rey y Test de Stroop. Dentro de los resultados se encontró que la medida de edad fue de 50 con un total de 19 hombres y 5 mujeres. Las funciones con mayor predominio de deterioro fueron la atención sostenida y alternante, la memoria declarativa, las funciones ejecutivas (específicamente en el control inhibitorio) y la velocidad de procesamiento, los pacientes presentan un rango de deterioro cognitivo leve (GDS 3). Se concluyó que el perfil de deterioro es mixto y que es necesario ampliar la muestra para obtener resultados más precisos en cuanto a las diferencias de acuerdo al tiempo de diagnóstico y la terapia antiretroviral.

The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64's biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

From Rational Design of Drug Crystals to Understanding of Nucleic Acid Structures: Lamivudine Duplex

A DNA-like duplex of nucleosides is probable to exist even without the 5`-phosphate groups needed to assemble the chain backbone. However, double-stranded helical structures of nucleosides are unknown. Here, we report a duplex of nucleoside analogs that is spontaneously assembled due to stacking of the neutral and protonated molecules of lamivudine, a nucleoside reverse transcriptase inhibitor (NTRI) widely used in anti-HIV drug combinatory medication. The left-handed lamivudine duplex has features similar to those of i-motif DNA, as the face-to-face base stacking and the helix rise per base pair. Furthermore, the protonation pattern on alternate bases expected for it DNA-like duplex stabilized by pairing of neutral and protonated cytosine fragments was observed for the first time in the lamivudine double-stranded helix. This structure demonstrates that hydrogen bonds can substitute for covalent phosphodiester linkage in the stabilization of the duplex backbone. This interesting example of spontaneous molecular self-organization indicates that the 5`-phosphate group could not be a requirement for duplex assembly.

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.

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)

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal ptant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 celts, and the 50% inhibitory concentration (IC50) were 22.01 mu g/ml (selectivity index (SI) = 2.83) and 21.10 mu g/mi (SI = 2.95), respectivety. At the highest concentration of the drug (40 mu g/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains. (C) 2006 Elsevier GmbH. All rights reserved.

Antiviral activity of plants occurring in the state of minas gerais (Brazil): Part III

A total of 24 extracts from 14 plant species collected at the state of Minas Gerais, Brazil, and belonging to five botanical families (Annonaceae, Apocynaceae, Ochnaceae, Polygonaceae and Vitaceae) was screened for cytotoxicity in cultured Vero cells and for antiviral activity against human herpes virus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV). The highest cytotoxicity (CC 50 < 10 μg/mL) was observed for the ethanol extracts from Annona coriacea fruits and seeds. Extracts from Hancornia speciosa, Ouratea castaneafolia and O. semisrrata were the only ones that have shown activity against all the three viruses assayed. Extracts from Polygonum spectabile, Hancornia speciosa, Himatanthus phagedaenica, Ouratea spectabilis and O. semiserrata were the most active against HSV-1 (EC 50 < 50 mg/mL), with favorable SI values (8.0 to 10.0). Hancornia speciosa and Anaxagorea dolichocarpa were the most active against EMCV (EC 50 50 - 100 μg/mL), with reasonable SI values (5.2 to 6.1), while moderate to low activity (EC 50 > 100 μg/mL) was observed for Ouratea spectabilis and O. semiserrata. A total of 7 plant species, Ouratea semiserrata, O. spectabilis, O. castanaeafolia, Rollinia laurifolia, Cissus erosa, Polygonum spectabile, and Hancornia speciosa, were active against VACV, disclosing EC50 < 50 μg/mL and SI values ranging from 6.6 to 67.3. In total, 10 out of the 14 species were selected from a literature survey on plants used to treat viral diseases in Brazil; these species were responsible for 70% of the positive results.

Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology

We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (beta-L-2',3'-dideoxy-3'-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC=3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N-H center dot center dot center dot O or N-H center dot center dot center dot N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1'-endo and C1'-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.

Evaluation of Viral Load Thresholds for Predicting New World Health Organization Stage 3 and 4 Events in HIV-Infected Children Receiving Highly Active Antiretroviral Therapy

Background: This study evaluated a wide range of viral load (VL) thresholds to identify a cut-point that best predicts new clinical events in children on stable highly active antiretroviral therapy (HAART). Methods: Cox proportional hazards modeling was used to assess the adjusted risk for World Health Organization stage 3 or 4 clinical events (WHO events) as a function of time-varying CD4, VL, and hemoglobin values in a cohort study of Latin American children on HAART >= 6 months. Models were fit using different VL cut-points between 400 and 50,000 copies per milliliter, with model fit evaluated on the basis of the minimum Akaike information criterion value, a standard model fit statistic. Results: Models were based on 67 subjects with WHO events out of 550 subjects on study. The VL cut-points of >2600 and >32,000 copies per milliliter corresponded to the lowest Akaike information criterion values and were associated with the highest hazard ratios (2.0, P = 0.015; and 2.1, P = 0.0058, respectively) for WHO events. Conclusions: In HIV-infected Latin American children on stable HAART, 2 distinct VL thresholds (>2600 and >32,000 copies/mL) were identified for predicting children at significantly increased risk for HIV-related clinical illness, after accounting for CD4 level, hemoglobin level, and other significant factors.

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Hypermetabolism and altered substrate oxidation in HIV-infected patients with lipodystrophy

Objective: Human immunodeficiency virus type 1 (HIV)-associated lipodystrophy syndrome compromises body composition and produces metabolic alterations, such as dyslipidemia and insulin resistance. This study aims to determine whether energy expenditure and substrate oxidation are altered due to human HIV-associated lipodystrophy syndrome. Methods: We compared energy expenditure and substrate oxidation in 10 HIV-infected men with lipodystrophy syndrome (HIV+LIPO+), 22 HIV-infected men without lipodystrophy syndrome (HIV+LIPO-), and 12 healthy controls. Energy expenditure and substrate oxidation were assessed by indirect calorimetry, and body composition was assessed by dual-energy X-ray absorptiometry. The substrate oxidation assessments were performed during fasting and 30 min after eucaloric breakfast consumption (300 kcal). Results: The resting energy expenditure adjusted for lean body mass was significantly higher in the HIV+LIPO+ group than in the healthy controls (P = 0.02). HIV-infected patients had increased carbohydrate oxidation and lower lipid oxidation when compared to the control group (P < 0.05) during fasting conditions. After the consumption of a eucaloric breakfast, there was a significant increase in carbohydrate oxidation only in the HIV+LIPO- and control groups (P < 0.05), but there was no increase in the HIV+LIPO+ group. Conclusion: Hypermetabolism and alteration in substrate oxidation were observed in the HIV+LIPO+ group. (C)2012 Elsevier Inc. All rights reserved.

Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods

Abstract Background An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Results Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Conclusions Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

Mechanismus der Apoptose, Gentoxizität und DNA-Reparatur in Herpesvirus-Thymidinkinase-exprimierenden Säugerzellen nach Behandlung mit Antiherpes-Virustatika vom Typ der Nukleosidanaloga

Um Cytotoxizität und Gentoxizität nukleosidischer Antiherpes-Virustatika zu untersuchen, wurden stabile CHO-Klone etabliert, die Thymidinkinase (TK) des Herpes simplex-Virus Typ 1 (HSV-TK) oder des Varicella zoster-Virus (VZV-TK) exprimieren. In HSV-TK-exprimierenden Zellen wurde das Purinanalogon Ganciclovir (GCV) effizient in die genomische DNA eingebaut, worauf in den nächsten Replikationsrunden DNA-Strangbrüche und Aberrationen entstehen und Apoptose ausgelöst wird. GCV-induzierte Apoptose wird hauptsächlich über den mitochondrialen Weg vermittelt, wobei das anti-apoptotische Protein Bcl-2 im Mittelpunkt steht. Nach GCV-Behandlung konnte eine Caspase-9-vermittelte post-translationale Spaltung von Bcl-2 nachgewiesen werden. Das 23 kDa-großes Bcl-2-Fragment wirkt im Gegensatz zum intakten Bcl-2-Protein pro-apoptotisch und verstärkt die Cytochrom C-Freisetzung und damit die Aktivierung der Caspase-9, die Bcl-2 spaltet, was zu einem positiven 'Amplifikationsloop' des mitochondrialen apoptotischen Weges führt. In weiteren Experimenten wurde gezeigt, daß in die DNA inkorporiertes GCV durch Basenexzisionsreparatur repariert wird, wobei die DNA-Polymerase ß eine entscheidende Rolle spielt. Diese Reparatur führte zu einer signifikanten Reduktion der Apoptose und Klastogenität und damit zur Resistenzsteigerung gegenüber GCV. In VZV-TK-exprimierenden Zellen wurde gezeigt, daß Brivudin (BVDU), gleichermaßen Apoptose und Nekrose induzierte. Für die BVDU-induzierte Cytotoxizität konnte die Hemmung der Thymidylatsynthetase als Ursache identifiziert werden. Im Gegensatz zur GCV-induzierten Apoptose war für die BVDU-induzierte Apoptose der Rezeptor (Fas/CD95/APO-1)-vermittelte Weg von vorrangiger Bedeutung.

Metodi biomolecolari per il follow up dei pazienti HIV positivi

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon attivation, its analysis, in addition to RNA viral load, could be considered a useful marker during the follow-up of infected individuals, to evaluate reservoir status, especially in HAART-treated patients when RNA viral load is undetectable by current techniques and the antiretroviral efficacy of new, more potent therapeutic regimens. Standardized methods for the measurement of the two most significant forms of proviral DNA, total and non-integrated, are currently lacking, despite the widespread of molecular biology techniques. In this study, total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load, CD4 cell count and serological parameters, were determined by quantitative analysis in peripheral blood mononuclear cells (PBMC) in naïve or subsequently HAART-treated patients with acute HIV-1 infection in order to establish the role of these two DNA proviral forms in the course of HIV infection. The study demonstrated that HAART-treated individuals show a significant decrease in both total and 2-LTR circular HIV-1 DNA proviral load compared with naïve patients: these findings confirm that HIV-1 reservoir decay correlates with therapeutic effectiveness. The persistence of small amounts of 2-LTR HIV-1 DNA form, which is considered to be a molecular determinant of infectivity, in PBMC from some patients demonstrates that a small rate of replication is retained even when HAART is substantially effective: HAART could not eradicate completely the infection because HIV is able to replicate at low levels. Plasma-based viral RNA assays may fail to demonstrate the full extent of viral activity. In conclusion, the availability of a new standardized assay to determine DNA proviral load will be important in assessing the true extent of virological suppression suggesting that its quantification may be an important parameter in monitoring HIV infection.

How HIV takes advantage of the cytoskeleton in entry and replication

The host cell cytoskeleton plays a key role in the life cycle of viral pathogens whose propagation depends on mandatory intracellular steps. Accordingly, also the human immunodeficiency virus type 1 (HIV-1) has evolved strategies to exploit and modulate in particular the actin cytoskeleton for its purposes. This review will recapitulate recent findings on how HIV-1 hijacks the cytoskeleton to facilitate entry into, transport within and egress from host cells as well as to commandeer communication of infected with uninfected bystander cells.