Ma Ángeles Muñoz-Fernández

The role of tumour necrosis factor, interleukin 6, interferon-γ and inducible nitric oxide synthase in the development and pathology of the nervous system

Proinflammatory cytokines, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma and interleukin (IL)-6, have multiple effects in the central nervous system (CNS) not strictly cytotoxic being involved in controlling neuronal and glial activation, proliferation, differentiation and survival, thus influencing neuronal and glial plasticity, degeneration as well as development and regeneration of the nervous system. Moreover, they can contribute to CNS disorders, including multiple sclerosis. Alzheimers disease and human immunodeficiency virus-associated dementia complex. Recent results with deficient mice in the expression of those cytokines indicate that they are in general more sensible to insults resulting in neural damage. Some of the actions induced by TNF-alpha, and IFN-gamma, including both beneficial and detrimental, are mediated by inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) production. NO produced by iNOS may be beneficial by promoting the differentiation and survival of neurons. IL-6 does not induce iNOS, explaining why this cytokine is less often involved in this dual role protection pathology. Some of the proinflammatory as well as the neurotrophic effects of those cytokines also involve upregulation of cell adhesion molecules (CAM). Those apparently conflicting results may be reconciled considering that proinflammatory cytokines are involved in promoting the disease, mostly by inducing expression of CAM leading to alteration of the blood-brain barrier integrity, whereas they have a protective role once disease is established due to its immunosuppressive or neurotrophic role. Understanding the dichotomy pathogenesis/neuroprotection of those cytokines may provide a rationale for better therapeutic strategies.

The Spanish HIV BioBank: a model of cooperative HIV research

BackgroundThe collection of samples from HIV-infected patients is the beginning of the chain of translational research. To carry out quality research that could eventually end in a personalized treatment for HIV, it is essential to guarantee the availability, quality and traceability of samples, under a strict system of quality management.MethodsThe Spanish HIV BioBank was created with the objectives of processing, storing and providing distinct samples from HIV/AIDS patients, categorized according to strictly defined characteristics, free of charge to research projects. Strict compliance to ethical norms is always guaranteed.ResultsAt the moment, the HIV BioBank possesses nearly 50,000 vials containing different prospective longitudinal study sample types. More than 1,700 of these samples are now used in 19 national and international research projects.ConclusionThe HIV BioBank represents a novel approach to HIV research that might be of general interest not only for basic and clinical research teams working on HIV, but also for those groups trying to establish large networks focused on research on specific clinical problems. It also represents a model to stimulate cooperative research among large numbers of research groups working as a network on specific clinical problems. The main objective of this article is to show the structure and function of the HIV BioBank that allow it to very efficiently release samples to different research project not only in Spain but also in other countries.

Clinical Outcomes Improve with Highly Active Antiretroviral Therapy in Vertically HIV Type-1–Infected Children

Background. Use of antiretroviral therapy has resulted in a decrease in morbidity and mortality rates in human immunodeficiency virus type 1 (HIV-1)-infected children.Methods. We performed a retrospective study involving 427 children to determine the effectiveness of different antiretroviral therapy protocols on clinical outcome. The follow-up period was divided into 5 calendar periods (CPs): CP1 (1980-1989), before antiretroviral therapy was administered; CP2 (1990-1993), when monotherapy was administered; CP3 (1994-1996), when combined therapy was administered; CP4 (1997-1998), when </=50% of children were receiving highly active antiretroviral therapy (HAART); and CP5 (1999-2003), when >/=60% of children were receiving HAART.Results. Children experienced a progressive increase in the CD4(+) cell count and decrease in the viral load from 1997 onwards. A lower number of AIDS cases and deaths occurred during CP5 than during the other CPs (P<.01), with a relative risk of an absence of AIDS of >20 and a relative risk of survival of >30. The AIDS rate was >50% in CP1; we observed a very strong decrease to 14% in CP2, to 16% in CP3, to 7% in CP4, and to 2% in CP5. The mortality rates in CP2 and CP3 were >6% and thereafter decreased to 0.5% in CP5. The relative risks for no hospital admission in CP4 and CP5 were >3.5. The total rates of hospital admission in CP1, CP2, and CP3 were >30%; we observed a decrease in CP4 and CP5. The duration of hospitalization decreased during the follow-up period, and it was higher in CP1 (~30 days) than in the other periods.Conclusions. We observed that HAART produces a decrease in adverse clinical outcomes (i.e., hospital admission, AIDS, and death) in children with vertical HIV-1 infection in Madrid, Spain.

In vivo delivery of siRNA to the brain by carbosilane dendrimer.

Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents in the same nanocompose, along with the added provisions of site-directed delivery, delivery across the blood-brain-barrier (BBB), and controlled release of therapeutics. We studied the viability of dendrimers and dendriplexes in human primary astrocytes, as well as their uptake by these astrocytes. Functional validation was performed by using specific siRNA against HIV-1 Nef to interfere to HIV-1 infectivity. A high efficiency in Nef silencing, reducing HIV-1 infectivity was observed in astrocytes treated with dendriplexes compared with control or siRandom treated astrocytes. More interestingly, we studied the biodistribution of the second generation of carbosilane dendrimer loaded with FITC (2G-(SNMe3I)11-FITC) in vivo, in BALB/c mice. Dendriplexes were inoculated into BALB/c mice by the retro-orbital venous plexus, and their localization was determined after 1 and 24h post-injection. Dendriplexes were detected inside the brain by a sensitive imaging system of fluorescent imaging in vivo (IVIS Lumina), and by confocal microscopy analysis of sections of OCT-embedded tissues. The 2G-(SNMe3I)11-FITC dendrimer transported efficiently siRNA into the brain, crossing the BBB. Moreover, this dendrimer successfully delivered and transfected siRNA to HIV-infected human primary astrocytes and achieved gene silencing without causing cytotoxicity. These results highlight the potential of this nanoformulation in the treatment of neurological disorders.

Amine and ammonium functionalization of chloromethylsilane-ended dendrimers. Antimicrobial activity studies

Novel amine- and ammonium-terminated carbosilane dendrimers of type G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(2)}](x) or G(n)-[Si{CH(2)O-(C(6)H(4))-3-NMe(3)(+)I(-)}](x) have been synthesized and characterized up to second generation by phenolysis of (chloromethyl)silyl-terminated dendrimers with 3-dimethylamine phenol and subsequent quaternization with methyl iodide. Quaternized carbosilane dendrimers are stable in protic solvents and can be solubilised in water after the addition of less than 1% of dimethyl sulfoxide. A study of the antimicrobial activity of these cationic dendrimers of first and second generation against both gram-positive and gram-negative bacteria is also described. The results obtained demonstrate that the new ammonium-terminated carbosilane dendrimers can be considered as multivalent biocides.

Water-soluble carbosilane dendrimers protect phosphorothioate oligonucleotides from binding to serum proteins

Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioate oligodeoxynucleotides (ODN) with the anionic detergent sodium dodecyl sulfate disrupted the complexes indicating that the nature of the union in such dendriplexes is merely electrostatic. However, dendriplexes were not dissociated by serum proteins like bovine or human serum albumins, as assessed by gel electrophoresis and fluorescence experiments. This would imply a dendrimer-mediated protective effect able to prevent ODN interactions with serum proteins and additionally could translate into a reduction of the ODN doses needed to achieve the biological effects. The employment of carbosilane dendrimers as carriers may solve the problem of ODN kidnapping by plasmatic proteins as a key drawback for therapeutics involving ODNs. As examples, transfection processes on normal primary peripheral blood cells and diagnosis of HIV infection in the presence of serum have been assayed.

Viral phenotype affects the thymic production of new T cells in HIV-1-infected children.

ObjectiveTo determine whether viral phenotype has any effect on thymic production of new T cells in HIV-1-infected children. DesignDifferences in CD4+ T-cell counts and a marker of thymic output [T-cell antigen receptor (TCR) rearrangement excision circles (TRECs)], between HIV-1-infected children with non-syncytium-inducing (NSI) and syncytium-inducing (SI) viral strains were determined. Patients and methodsA cross-sectional study in 90 samples from vertically HIV-1-infected-children (median age 4.9 years) treated with combination therapy, and a longitudinal study in three children that underwent a change from NSI to SI phenotype were carried out. Viral load, viral phenotype, CD4+ T-cell counts, and quantification of TRECs values were determined. ResultsChildren with SI virus showed significant lower levels of CD4+ T cells and a lower thymic production of new T cells than children with NSI. These reductions were independent of the treatment and the age of the children. However, there were no differences in viral load with the phenotype between those groups. In children with both NSI and SI viral phenotype, there was a significant correlation between CD4+ T-cell counts and TRECs values. ConclusionThe decrease of CD4+ T cells in presence of T-tropic viruses would be mainly due to a lower production of new CD4+ T cells as consequence of the inhibitory effect of these T-tropic strains on thymic function. This effect is not due either to the amount of circulating virus or to the replication kinetics of those strains, but rather depends on the ability of T-tropic viruses to infect T-cell precursors using CXCR4 receptors, which are highly expressed in immature thymocytes.

TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimers disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

Establishment and replenishment of the viral reservoir in perinatally HIV-1-infected children initiating very early antiretroviral therapy

Initiation of combined antiretroviral therapy within the first 12 weeks of life in vertically human immunodeficiency virus type 1-infected children favors the establishment of low-level proviral reservoirs. Nevertheless, treatment discontinuation in these patients may lead to rapid and irreversible expansion of reservoir size.

HIV-1 infection and neurocognitive impairment in the current era.

Brain HIV‐1‐infection may result in a syndrome of profound cognitive, behavioral and motor impairment known as AIDS dementia complex (ADC) in adults and HIV‐related encephalopathy in children. Although the introduction of highly active antiretroviral therapy (HAART) has prolonged and improved the lives of infected individuals, it is clear that HAART does not provide complete protection against neurological damage in HIV/AIDS. HIV‐1 associated dementia is a complex phenomenon, which could be the result of several mechanisms caused by those players using different intracellular signaling pathways. Understanding the causes of neurodegeneration during HIV‐1 infection and the factors which certain individuals develop disease can provide researches on new therapeutic targets to positively affect disease outcomes. Controlling CNS viral replication with HAART is an essential primary approach, but it should be complemented with adjunctive CNS‐directed therapeutics. Understanding the nature of HIV‐1 infection within the CNS as well as inflammatory responses will ultimately lead to the elimination of HIV‐associated neurocognitive disorders. Copyright