A longitudinal and prospective study of Epstein-Barr virus load in AIDS-related non-Hodgkin lymphoma.

BACKGROUND: Epstein-Barr virus (EBV) may be causally associated with non-Hodgkin Lymphoma (NHL) in HIV-infected patients. OBJECTIVES: To compare EBV load in whole blood in AIDS-NHL patients, HIV non-AIDS patients and non-HIV-infected persons, and to prospectively measure EBV load in whole blood in AIDS-NHL patients. STUDY DESIGN: Longitudinal and prospective study. RESULTS: We observed no statistical difference in EBV load between AIDS-NHL (3.69log(10) copies/mL [interquartile range (IQR): 2.89-4.27]) and HIV non-AIDS patients (3.08log(10) copies/mL [IQR: 1.29-3.57]) but AIDS-NHL patients had significantly higher EBV loads than HIV-negative controls (1.19log(10) copies/mL [IQR: 0.00-3.29]). We noticed an inverse correlation between CD4+ lymphocytes count and EBV load in patients with AIDS-NHL (r(2)=0.41, P=0.01). In the longitudinal study, the mean EBV load three months after NHL diagnosis decreased significantly (mean difference=-1.69log(10) copies/mL [95% confidence interval: -0.32; -3.04]; P=0.03) under chemotherapy but was still elevated in patients with relapses or no response to chemotherapy. CONCLUSION: Although EBV load seems a suboptimal marker for the diagnosis of AIDS-NHL, we observed a significant decrease of EBV load in patients treated with chemotherapy and a strong association between NHL outcome and EBV load in whole blood.     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.     

Partial purification and characterization of B cell growth factor constitutively secreted by human T-cell hybridoma

Human T cell hybridomas were established by fusion of PHA-activated PBL with the 8-azaguanine resistant human T-leukemic cell line CEM-CM3. High levels of B cell growth factor (BCGF) activity were detected in the supernatants of hybridoma C8-2B2 and its subclones. Hybridoma C8-2B2, in addition to the Leu 3a, also expressed the OKT11 surface marker which was not detectable on the parent CEM-CM3 cells. BCGF from the culture supernatant was purified by combined use of salt fractionation and gel filtration to 36.6 fold with 23.9% recovery of activity. The BCGF produced by hybridoma C8-2B2 has a molecular weight range of 16,000–20,000 in two major electrophoretically different forms with pI values of 6.4 and 7.4.     

Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation

Recent evidence indicates that bone-marrow-derived stromal cells (MSCs) have a histology coherent with endothelial cells that may enable them to contribute to tumor angiogenesis through yet undefined mechanisms. In this work, we investigated the angiogenic properties of murine MSCs involved in extracellular matrix degradation and in neovascularization that could take place in a hypoxic environment such as that encountered in tumor masses. MSCs were cultured in normoxia (95% air and 5% CO(2)) or in hypoxia (1% oxygen, 5% CO(2), and 94% nitrogen). We found that hypoxic culture conditions rapidly induced MSC migration and three-dimensional capillary-like structure formation on Matrigel. In vitro, MSC migration was induced by growth-factor- and cytokine-enriched conditioned media isolated from U-87 glioma cells as well as from MSCs cultured in hypoxic conditions, suggesting both paracrine and autocrine regulatory mechanisms. Although greater vascular endothelial growth factor levels were secreted by MSCs in hypoxic conditions, this growth factor alone could not explain their greater migration. Interestingly, matrix metalloproteinase (MMP)-2 mRNA expression and protein secretion were downregulated, while those of membrane-type (MT)1-MMP were strongly induced by hypoxia. Functional inhibition of MT1-MMP by a blocking antibody strongly suppressed MSC ability to migrate and generate capillary-like structures. Collectively, these data suggest that MSCs may have the capacity to participate in tumor angiogenesis through regulation of their angiogenic properties under an atmosphere of low oxygen that closely approximates the tumor microenvironment.     

Comparison of α-Tocopherol, α-Tocopherol Acetate,and α-Tocopheryl Polyethylene Glycol Succinate 1000 Absorption by Caco-2 TC7 Intestinal Cells

(1) Background: vitamin E is often supplemented in the form of tocopherol acetate, but it has poor bioavailability and can fail to correct blood tocopherol concentrations in some patients with severe cholestasis. In this context, α-tocopheryl polyethylene glycol succinate 1000 (TPGS) has been of value, but very little is known about the mechanisms of its absorption. The aim of our work was to evaluate the mechanisms of absorption/secretion of TPGS compared to tocopherol acetate (TAC) and α-tocopherol by human enterocyte-like Caco-2 TC7 cells. (2) Methods: two weeks post-confluence Caco-2 cells were incubated with tocopherol- or TAC- or TPGS-rich mixed micelles up to 24 h and, following lipid extraction, TAC and tocopherol amounts were measured by high performance liquid chromatography (HPLC) in apical, cellular, and basolateral compartments. (3) Results: at equivalent concentrations of tocopherol in the apical side, the amounts of tocopherol secreted at the basolateral pole of Caco-2 cells are (i) significantly greater when the tocopherol is in the free form in the micelles; (ii) intermediate when it is in the TAC form in the micelles (p < 0.001); and (iii) significantly lower with the TPGS form (p < 0.0001). Interestingly, our results show, for the first time, that Caco-2 cells secrete one or more esterified forms of the vitamin contained in TPGS at the basolateral side.     

Exon identity influences splicing induced by exonic variants and in silico prediction efficacy

BackgroundMinigenes and in silico prediction tools are commonly used to assess the impact on splicing of CFTR variants. Exon skipping is often neglected though it could impact the efficacy of targeted therapies. The aim of the study was to identify exon skipping associated with CFTR variants and to evaluate in silico predictions of seven freely available software.MethodsCFTR basal exon skipping was evaluated on endogenous mRNA extracted from non-CF nasal cells and on two CFTR minigene banks. In silico tools and minigene systems were used to evaluate the impact of CFTR exonic variants on exon skipping.ResultsData showed that out of 65 CFTR variants tested, 26 enhanced exon skipping and that in silico prediction efficacy was of 50%-66%. Some in silico tools presented predictions with a bias towards the occurrence of splicing events while others presented a bias towards the absence of splicing events (non-detection including true negatives and false negatives). Classification of exons depending on their basal exon skipping level increased prediction rates up to 80%.ConclusionThis study indicates that taking basal exon skipping into account could orientate the choice of the in silico tools to improve prediction rates. It also highlights the need to validate effects using in vitro assays or mRNA studies in patients. Eventually, it shows that variant-guided therapy should also target exon skipping associated with variants.     

Amoeboid Microglial Cells and not Astrocytes Synthesize TNF-alpha in Swiss Mouse Brain Cell Cultures

The role of tumour necrosis factor (TNF-alpha) in brain physiology and pathology has been the focus of several studies. However, the source of this lymphokine in the central nervous system and the regulation of its synthesis is still poorly understood. We have therefore used purified astrocytes and brain macrophages in culture to compare the abilities of these two cell types to synthesize TNF-alpha and its mRNA. We find that, in the Swiss mouse, no significant TNF activity or TNF-alpha mRNA are produced by astrocytes, even following activation with lipopolysaccharides (LPS). On the other hand, purified microglial cells express a cytotoxic activity able to kill TNF-sensitive LM cells. Part of this activity is released into the culture medium and part remains bound to the membrane after mild paraformaldehyde treatment, demonstrating the existence in the culture of the soluble and membrane-bound forms of TNF activity. The fact that amoeboid microglial cells, and not astrocytes, are the actual source of TNF in brain cultures was further demonstrated by Northern blot analysis and in situ hybridization using a TNF-alpha specific oligonucleotide probe. The definition of the cell type which, in the CNS, is responsible for TNF synthesis will allow the regulation of this lymphokine to be analysed and opens the way for a better understanding of the interactions between amoeboid microglial cells and the other cell types which make up the nervous system.     

Extreme temperament and diagnosis. A study in a psychiatric sample of consecutive children

We report on an epidemiological-clinical study of the New York (NY) Longitudinal Study temperament model in a consecutive sample of children (N = 814) referred to a child psychiatric center. Temperament comparisons in this clinical population were made by using temperament normative values obtained in previous random samples of the general population in the greater Quebec City (Canada) area. Different clinical diagnostic groups (externalized disorders, developmental delays, and mixed disorders) were derived from a review of the entire hospital charts in which the interrater reliability was tested and performed "blind" to temperament scores. The diagnostic groups were confirmed through discriminant function analyses. The results (1) replicated, in this child psychiatric population, two factors of temperament similar to those previously found in random samples of our general population; (2) showed, in the psychiatric population of children, an overproportion of difficult temperaments on both factors; (3) confirmed conversely that a large proportion of children referred for a disorder did not present with an extreme temperament, and, therefore, an extreme temperament and a clinical disorder were not equivalent; and (4) suggested a specificity in the relationship between particular temperament factors and the type of clinical problem. Temperament factor 1 (withdrawal from new stimuli, low adaptability, high intensity, and negative mood) was found to be more associated with externalized disorders (opposition, conduct, or attention-deficit disorders), whereas temperament factor 2 (low persistence, high sensory threshold, and high mobility) was found to be more associated with specific developmental delays. The findings provided leads for future clinical research on temperament, family functioning, and child psychiatric diagnoses.     

Robustness of case-control studies of genetic factors to population stratification: magnitude of bias and type I error.

Case-control studies of genetic factors are prone to a special form of confounding called population stratification, whenever the existence of one or more subpopulations may lead to a false association, be it positive or negative. We quantify both the bias (in terms of confounding risk ratio) and the probability of false association (type I error) in the most unfavorable situation in which only one high-risk subpopulation is hidden within the studied population, considering different scenarios of population structuring and varying sample sizes. In accord with previous work, we find that the bias is likely to be small in most cases. In addition, we show that the same applies to the associated type I error whenever the subpopulation is small in proportion. For instance, when the hidden subpopulation makes up 5% of the entire population, with an allelic frequency of 0.25 (versus 0.10) and a disease rate that is double, then the estimated bias is 1.07 and the type I error associated with a sample of 500 cases and 500 controls is 8% (instead of 5%). We also show that the type I error is substantially greater for a rare allele (frequency of 0.1) than for a common allele (frequency of 0.5) and analyze the pattern of increase of vulnerability to stratification bias with sample size. Based on our findings, we may therefore conclude that with moderate sample sizes the type I error associated with population stratification remains very limited in most realistic scenarios.