MicroRNAs and cancer stem cells: therapeutic approaches and future perspectives.

Cancer stem cells (CSCs) are a sub-population of cancer cells that possess characteristics associated with normal stem cells but with the peculiarity that they are tumourigenic. This property allows them to persist in the tumour population, causing relapse and metastasis by giving rise to new tumours. Accordingly, if the CSCs were eliminated, then the tumour would simply regress due to differentiation and cell death. By selectively targeting CSCs, it may be possible to treat patients with aggressive, non-resectable tumours and prevent the tumour from metastasising. MicroRNAs are involved in all biological processes, and several studies have demonstrated their function in human tumourigenesis. Importantly, microRNAs have been implicated in the regulation of stem cells and CSCs. The most important concept to emerge with regard to CSC therapy is still controversial because a number of signalling pathways unique to normal stem cells may also be operating in CSCs, and these offer new targets for therapy. This article reviews how the modulation of microRNAs may revert tumour proliferation in vivo and in vitro and how this approach could be transferred to the clinic. Although the delivery of therapeutic microRNAs to target cells is a challenge that still needs to be overcome, microRNAs offer the advantage that they are small molecules that can be easily transported by body fluids, which makes them good candidates for cancer therapy.

Specific patterns of CD4-associated immunosenescence in vertically HIV-infected subjects.

Vertical transmission of human immunodeficiency virus (HIV) represents an important world-wide health problem although the incidence in developed countries has been drastically reduced by the extensive use of highly active antiretroviral therapy. Vertically HIV-infected subjects have been exposed to the virus during the maturation of their immune systems and have suffered a persistent chronic activation throughout their lifetime; the consequences of this situation for their immune system are not fully understood. The objective of this study was to analyse immunosenescence-related parameters in different CD4 T-cell subsets. Fifty-seven vertically HIV-infected subjects and 32 age-matched healthy subjects were studied. Activation (HLA(-) DR(+) ), senescence (CD28(-) CD57(+) ) and proliferation (Ki67(+) ) were analysed on different CD4 T-cell subsets: naive (CD45RA(+) CD27(+) ), memory (CD45RO(+) CD27(+) ), effector memory (CD45RO(+) CD27(-) ) and effector memory RA (CD45RA(+) CD27(-) ). Compared with healthy subjects, vertically HIV-infected subjects showed increased naive and memory CD4 T-cell frequencies (p 0.035 and p 0.010, respectively) but similar frequencies of both effector subsets. Whereas naive CD4 T cells were not further altered, memory CD4 T cells presented increased levels of senescence and proliferation markers (p <0.001), effector memory CD4 T cells presented increased levels of activation, senescence and proliferation markers (p <0.001) and effector memory RA CD4 T cells presented increased levels of activation and senescence (p <0.001) compared with healthy subjects. Despite long periods of infection, vertically HIV-infected subjects show specific patterns of immunosenescence, revealing a preserved CD4 T-cell homeostasis for subset differentiation and distribution. Nevertheless, excepting the naive subpopulation, all subsets experienced some immunosenescence, pointing to uncertain consequences of the future aging process in these subjects.

Short communication: evaluation of the effect of enfuvirtide in 11 HIV-1 vertically infected pediatric patients outside clinical trials.

The effect of enfuvirtide (ENF) in 11 HIV-1 heavily antiretroviral-experienced children and adolescents enrolled in the HIV-1 Paediatric Spanish cohort was further investigated. Patients who received ENF with novel drugs (etravirine, darunavir, and/or tipranavir) reached and maintained undetectable plasma HIV-1 RNA levels and showed immunological recovery within the first 3 months of therapy that was maintained during the follow-up. Viremia was not fully suppressed in patients who did not combine ENF with novel drugs but interestingly, immunological benefit was observed in half of these patients. Therefore, ENF showed a greater and more stable efficacy when administrated with novel drugs.

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk.

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.

Structural elucidation of a cell wall fungal polysaccharide isolated from Ustilaginoidea virens, a pathogenic fungus of Oriza sativa and Zea mays.

The alkali-extractable water-soluble polysaccharides (F1SS) isolated from the outer cell wall of two strains of Ustilaginoidea virens have been studied by chemical and methylation analyses, and 1D and 2D (1)H and (13)C NMR spectroscopy. The structures of these polysaccharides are very similar, and can be described by the following idealized repeating unit: where n and m are approximately 1 and 2, respectively.

Enzyme replacement therapy with agalsidase alfa in children with Fabry disease.

AIM: To assess the effects of enzyme replacement therapy (ERT) in children with Fabry disease. METHODS: Safety and efficacy of ERT with agalsidase alfa, 0.2 mg/kg infused over 40 minutes every 2 weeks for 23 weeks, were studied in a multicentre open-label trial in nine boys and four girls. Median age at the start of the study was 11.0 years (range 3.5-18 years). RESULTS: Fifty-four adverse events were reported in 11 patients. No serious adverse events related to ERT were reported. Twelve of the 54 adverse events were considered possibly or probably related to ERT. Infusion reactions (8 mild, 3 moderate) occurred in four boys, in seven infusions. One boy developed IgG antibodies, although he continued to make good clinical progress. At the end of the study, two of the four boys and the one girl on regular pain medication at baseline had stopped taking analgesics. Brief Pain Inventory (BPI) scores decreased in most patients by week 12 and were sustained until the end of the study. This change was greater in the boys, who had higher (worse) BPI scores at baseline. Pain-related quality of life (QoL) scores also decreased during the study. Plasma globotriaosylceramide concentrations and urinary globotriaosylceramide:sphingomyelin ratios decreased after 12 and 23 weeks of therapy, particularly in the boys. Increases in sweat volume were recorded in three out of five of the boys and in one of two girls tested after 23 weeks of treatment. CONCLUSION: ERT with agalsidase alfa in children with Fabry disease is well tolerated and, in the short term, appears to decrease pain and to improve pain-related QoL.

alpha-galf I -->6-alpha-mannopyranoside side chains in Paracoccidioides brasiliensis cell wall are shared by members of the Onygenales, but not by galactomannans of other fungal genera.

The water-soluble polysaccharide fraction of the cell wall alkali extract (F1SS) from the mycelial phase of the dimorphic fungus Paracoccidioides brasiliensis is compared with F1SS polysaccharides obtained from the Onygenalean mycelial fungi Ascocalvatia alveolata, Onygena equina and Aphanoascus terreus. These polymers were exclusively composed of mannose and galactose. Data from methylation and NMR analyses reveal that F1SS polysaccharides from the four fungi contain the same residues although in different proportions: [-->2,6)-alpha-D-Manp-(1 -->]; [2)-alpha-D-Manp-(1 -->]; [ -->6)-alpha-D-Manp-(1 -->]; and [alpha-D-Galf-(1 -->]. In P. brasiliensis, the repeating unit of the polysaccharide consists of a backbone of [(1 -->6)-alpha-D-Manp] substituted at the 0-2 position by the disaccharide [alpha-D-Galf-(1 -->6)-alpha-D-Manp-(1 -->], while the remaining 0-2 positions are substituted by single residues of mannose or short chains of (1 -->2)-mannose. The other species had a lower proportion of galactofuranose-containing side chains and higher proportion of mannose-containing side chains. The similarities found among the F1SS polysaccharides from P. brasiliensis and the Onygenalean A. alveolata, A. terreus and O. equina, reveal the close relatedness of all these fungi, show differences with polysaccharides from other fungal genera and agree with the molecular evidence provided in the scientific literature for the placement of P. brasiliensis within the Onygenales.

Linfohistiocitosis hemofagocítica (I) / Hemophagocytic lymphohistiocytosis

La linfohistiocitosis hemofagocítica (LHH) es una enfermedad rara con una alta morbimortalidad. Se caracteriza por una disfunción de las células efectoras del sistema inmunológico, que puede ser debida a un defecto congénito o adquirido. Clínicamente, presenta un cuadro de fiebre, hepatosplenomegalia y síntomas neurológicos. A nivel de parámetros analíticos, observamos citopenias, alteración en la coagulación, cambios en el perfil lipídico, elevación de citocinas inflamatorias, e infiltración de órganos por histiocitos y fenómenos de hemofagocitosis. Recientemente, se ha detectado un defecto a nivel de células inmunes efectoras, caracterizado por la ausencia de la actividad natural killer (NK). El tratamiento inicial debe ir encaminado a la supresión del ambiente de hiperinflamación presente en estos niños; el trasplante de medula ósea es el tratamiento de elección en los casos de LHH congénita. En la actualidad, los enfermos de LHH pueden beneficiarse de nuevas técnicas diagnósticas específicas y mejores medidas terapéuticas (AU)

[T-cell subsets variation during clinical and immunological progression in vertically HIV-infected children]. / Variacion de las subpoblaciones de células T según la progresión clínica e inmunológica en niños infectados verticalmente por HIV-1.

We have investigated the relationship among peripheral blood T-cell subsets with immunological and clinical categories, and viral load (VL) in 65 HIV-1-infected children on stable antiretroviral therapy (ART): 26 (40%) children on combination therapy with 2 nucleoside inhibitors, and 39 (60%) children on highly active antiretroviral therapy (HAART). T-cell subsets were determined by flow cytometry. VL was quantified using a standardized molecular method. Naïve CD4+ T-cells (CD45RA+CD62L+) were lower in children with low %CD4+ T-cells, but neither in children with advanced stage of illness nor with high VL. By contrast, naïve CD8+ T-cells were lower in children with low %CD4+ T-cells, advanced stage of illness and high VL. Memory (CD45RO+) and activated (CD38+, HLA-DR+ and CD38+HLA-DR+) CD4+ and CD8+ T-cells were higher in children with low %CD4+ T-cells, advanced stage of illness and high VL. However, CD4+CD38+ T-cells were higher in HIV-children with CD4+ > 25% than in the control group (p < 0.001) and were diminished in children with low %CD4+ T-cells. Naïve and memory CD4+ and CD8+ T-cells are more tightly dependent on the immunological category than on clinical category or plasma VL. Furthermore, our data indicate an association between low %CD4+ T-cells, high VL and high expression of cellular activation markers, although not with advanced clinical stage, possibly due to ART.

Structure of a cell wall polysaccharide isolated from Hypocrea gelatinosa.

The structure of a polysaccharide isolated from the cell wall of Hypocrea gelatinosa has been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysacharide has an irregular structure, idealized as follows: [carbohydrate structure in text].

Variacion de las subpoblaciones de células T según la progresión clínica e inmunológica en niños infectados verticalmente por HIV-1. / [T-cell subsets variation during clinical and immunological progression in vertically HIV-infected children]

We have investigated the relationship among peripheral blood T-cell subsets with immunological and clinical categories, and viral load (VL) in 65 HIV-1-infected children on stable antiretroviral therapy (ART): 26 (40

) children on combination therapy with 2 nucleoside inhibitors, and 39 (60

) children on highly active antiretroviral therapy (HAART). T-cell subsets were determined by flow cytometry. VL was quantified using a standardized molecular method. Na´ve CD4+ T-cells (CD45RA+CD62L+) were lower in children with low

CD4+ T-cells, but neither in children with advanced stage of illness nor with high VL. By contrast, na´ve CD8+ T-cells were lower in children with low

CD4+ T-cells, advanced stage of illness and high VL. Memory (CD45RO+) and activated (CD38+, HLA-DR+ and CD38+HLA-DR+) CD4+ and CD8+ T-cells were higher in children with low

CD4+ T-cells, advanced stage of illness and high VL. However, CD4+CD38+ T-cells were higher in HIV-children with CD4+ > 25

than in the control group (p < 0.001) and were diminished in children with low

CD4+ T-cells. Na´ve and memory CD4+ and CD8+ T-cells are more tightly dependent on the immunological category than on clinical category or plasma VL. Furthermore, our data indicate an association between low

CD4+ T-cells, high VL and high expression of cellular activation markers, although not with advanced clinical stage, possibly due to ART.

Infecciones oportunistas en pacientes pediátricos con infección por VIH / Opportunistic infections in HIV-infected children

Desde que comenzaron a describirse los primeros casos de SIDA en 1981, las infecciones oportunistas ocuparon un papel importante en la morbilidad y en la mortalidad de los pacientes con infección por el VIH. Estudios epidemiológicos realizados en adultos demuestran que desde la introducción de los regímenes antirretrovirales potentes, la incidencia de episodios oportunistas ha disminuido considerablemente y, por tanto, se cuestiona el papel de la profilaxis en pacientes con infección por VIH en quienes se ha conseguido una respuesta antiviral adecuada. Sin embargo, el porcentaje de descenso no es el mismo para cada infección oportunista. En este trabajo se presenta una extensa revisión sobre cada uno de los microrganismos implicados en la incidencia de infecciones oportunistas (AU)

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin.

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.

Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides.

The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of beta-(1-->6)-galactofuranose fully substituted at O-2 by terminal residues of alpha-glucopyranose and alpha-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted beta-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.

CD4+ and CD8+ T lymphocyte regeneration after anti-retroviral therapy in HIV-1-infected children and adult patients.

Previous studies have shown a slow recovery of naive CD4+ T cell counts after anti-retroviral therapy in HIV-1-infected adults, which is in accordance with thymus atrophy after puberty. Here we investigate whether or not different patterns of naive CD4+ and CD8+ T cell repopulation are present in adult and child patients undergoing anti-retroviral treatment. Thus, 25 adults under highly active anti-retroviral therapy and 10 children under combined anti-retroviral therapy were retrospectively analysed for T cell subpopulations at baseline (T0) and around week 12 (T1) and week 24 (T2) of anti-retroviral treatment. Mean serum HIV-1 RNA levels dropped in both groups. Recovery of T cells in adults was characterized by a heterogeneous response between patients, with only 44% of them increasing their naive CD4+ and CD8+ T cell counts at T1, and changes in mean total CD4+ T cells were mainly shaped by memory cells. Otherwise, children were characterized by an early increase in naive T cells. Thus, at T1, all children analysed had a strong rise in CD4+ (from 389 +/- 116 to 569 +/- 121 cells/microl; P < 0.01), and nine out of 10 also in naive CD8+ T cells (from 244 +/- 58 to 473 +/- 85 cells/microl; P < 0.05). However, no significant correlation between age and naive repopulation was observed (P = 0. 22) in children. Thus, children had the earlier and greater increases in naive T cell subsets than adults, probably due to a more active thymus, with the potential for immune reconstitution when HIV-1 replication is controlled.

[Clinico-radiological correlation of pulmonary complications of pediatric HIV infection]. / Correlación clínico-radiológica de las complicaciones pulmonares de la infección VIH en Pediatría.

Fifty-five percent of children with HIV infection, aged two months to ten years, were admitted at our hospitals because of respiratory conditions. Pulmonary complications found at admission in these children were lymphoid interstitial pneumonitis, Pneumocystis carinii pneumonia, fungal over-infection, tuberculosis, and bacterial complications. Also, non-specific infectious bronchial conditions, probably of viral origin. The most representative chest-X rays of these pulmonary conditions were analyzed; together with data from clinical records a clinico-radiological diagnosis was obtained.

Galactomannans from the cell walls of species of Paecilomyces sect. Paecilomyces and their teleomorphs as immunotaxonomic markers.

An alkali-extractable and water-soluble fraction (F1S) was obtained from cell walls of Paecilomyces variotii and species of the related genera Talaromyces, Byssochlamys and Thermoascus. The structure of the main polysaccharide of these fractions was studied and found to consist of a core of (1 --> 6)-alpha-mannopyranose partially substituted at 0.2 by chains of galactofuranose and shorter chains of mannopyranose. The differences in the regularity of the branching points and the length of the galactofuranose side chains are useful to distinguish between species. These differences were detected by immunological methods, since highly specific polyclonal antibodies were raised against these polysaccharides. Mycelium of P. variotii CBS 990.73A was stained by indirect immunofluorescence. The polysaccharides studied in this work differ from the one described for species from section Isarioidea, and this is another indication of the heterogeneity of the genus Paecilomyces.

TWEAK induces angiogenesis and proliferation of endothelial cells.

TWEAK is a recently described member of the Tumor Necrosis Factor (TNF) ligand family whose transcripts are present in a wide variety of human tissues (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997) J. Biol. Chem. 272, 32401-32410). TWEAK is a weak inducer of apoptosis in transformed cells when administered with interferon-gamma or cycloheximide (Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu Y. M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997) J. Biol. Chem. 272, 32401-32410; Masters, S. A., Sheridan, J. P., Pitti, R. M., Brush, A. G., and Ashkenazi, A. (1998) Curr. Biol. 8, 525-528) and also promotes IL-8 secretion in cultured cells. We report here that picomolar concentrations of recombinant soluble TWEAK induce proliferation in a variety of normal human endothelial cells and in aortic smooth muscle cells and reduce culture requirements for serum and growth factors. Blocking antibodies to Vascular Endothelial Growth Factor (VEGF) do not significantly inhibit TWEAK-induced proliferation, indicating that TWEAK does not function indirectly through up-regulation of VEGF. Pellets containing TWEAK induce a strong angiogenic response when implanted in rat corneas, suggesting a role for TWEAK in vasculature formation in vivo.