Decreased production of interleukin-10 and transforming growth factor-�� in Toll-like receptor-activated intestinal B cells in SAMP1/Yit mice

A unique subset of B cells expressing interleukin‐10 (IL‐10) and transforming growth factor‐β (TGF‐β) plays an essential role in preventing inflammation and autoimmunity. We investigated the presence of this cell subset in intestines and its role in the pathogenesis of ileitis using SAMP1/Yit and age‐matched control AKR/J mice. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and the expressions of B220, CD1d, CD5, Toll‐like receptor 4 (TLR4) and TLR9 in isolated cells were analysed. Purified B cells were stimulated with lipopolysaccharide (LPS) or CpG‐DNA, then IL‐10 and TGF‐β₁ expressions were examined by enzyme immunoassay and flow cytometry. Production of IL‐1β by TLR‐mediated macrophages co‐cultured with or without purified MLN B cells from SAMP1/Yit and AKR/J mice was evaluated. In addition, interferon‐γ (IFN‐γ) production in intestinal T cells co‐cultured with MLN B cells were also assessed in SAMP1/Yit and AKR/J strains. The production levels of IL‐10 and TGF‐β₁ stimulated by LPS and CpG‐DNA were significantly lower in B cells separated from MLNs from the SAMP1/Yit strain. B cells expressing IL‐10 and TGF‐β₁ were mainly located in a population characterized by the cell surface marker CD1d⁺. Interleukin‐1β production by TLR‐activated macrophages co‐cultured with MLN B cells from SAMP1/Yit mice was significantly higher than that of those from AKR/J mice. Interestingly, IFN‐γ production by T cells was noted only when they were co‐cultured with SAMP1/Yit but not the AKR/J B cells. These results are the first to show that disorders of regulatory B‐cell function under innate immune activation may cause disease pathogenesis in a murine model of Crohn’s disease.     

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

OBJECTIVES:

The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2.

INTRODUCTION:

The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.

METHODS:

HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis.

RESULTS:

Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC₅₀ of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2.

CONCLUSIONS:

Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis.     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Fetal calf serum inhibits virus genome expression in Madin-Darby canine kidney cells persistently infected with influenza A virus

A cell line of Madin-Darby canine kidney (MDCK) cells persistently infected with human influenza A virus has been established and designated as MDCK-IVpi cells. Production of progeny virus in MDCK-IVpi cells was suppressed when the cells were incubated in the presence of 10% fetal calf serum (FCS). FCS impaired virus mRNA synthesis in MDCK-IVpi cells, which resulted in a scarcity of virus proteins for virion formation. However, MDCK-IVpi cells well supported the growth of superinfecting heterologous influenza viruses, even in the presence of FCS. A certain fetuin-like substance in FCS might be responsible for the observed inhibition of virus replication.     

Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live-attenuated vaccine candidate in the mouse model

A temperature-sensitive mutant virus unable to replicate at 38 degrees C was recovered from passage 189 (IVpi-189) of Madin-Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi-189 virus-infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non-permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61-24-P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi-189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi-189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi-189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi-189 virus, an avirulent temperature-sensitive mutant, is a promising candidate for use as a live-attenuated vaccine.     

Antiphospholipid antibodies in epilepsy: A systematic review and meta-analysis

Background

Autoimmunity is believed to play an important causative role in the pathogenesis of epilepsy. There are evidences for the presence of autoantibodies in patients with epilepsy. To date, many studies have assessed the presence of antiphospholipid antibodies (aPLs) in epilepsy patients, though the relationship has been inconclusive.

Can T1w/T2w ratio be used as a myelin‐specific measure in subcortical structures? Comparisons between FSE‐based T1w/T2w ratios,GRASE‐based T1w/T2w ratios and multi‐echo GRASE‐based myelin water fractions

Given the growing popularity of T₁‐weighted/T₂‐weighted (T₁w/T₂w) ratio measurements, the objective of the current study was to evaluate the concordance between T₁w/T₂w ratios obtained using conventional fast spin echo (FSE) versus combined gradient and spin echo (GRASE) sequences for T₂w image acquisition, and to compare the resulting T₁w/T₂w ratios with histologically validated myelin water fraction (MWF) measurements in several subcortical brain structures. In order to compare these measurements across a relatively wide range of myelin concentrations, whole‐brain T₁w magnetization prepared rapid acquisition gradient echo (MPRAGE), T₂w FSE and three‐dimensional multi‐echo GRASE data were acquired from 10 participants with multiple sclerosis at 3 T. Then, after high‐dimensional, non‐linear warping, region of interest (ROI) analyses were performed to compare T₁w/T₂w ratios and MWF estimates (across participants and brain regions) in 11 bilateral white matter (WM) and four bilateral subcortical grey matter (SGM) structures extracted from the JHU_MNI_SS ‘Eve’ atlas. Although the GRASE sequence systematically underestimated T₁w/T₂w values compared to the FSE sequence (revealed by Bland–Altman and mountain plots), linear regressions across participants and ROIs revealed consistently high correlations between the two methods (r² = 0.62 for all ROIs, r² = 0.62 for WM structures and r² = 0.73 for SGM structures). However, correlations between either FSE‐based or GRASE‐based T₁w/T₂w ratios and MWFs were extremely low in WM structures (FSE‐based, r² = 0.000020; GRASE‐based, r² = 0.0014), low across all ROIs (FSE‐based, r² = 0.053; GRASE‐based, r² = 0.029) and moderate in SGM structures (FSE‐based, r² = 0.20; GRASE‐based, r² = 0.17). Overall, our findings indicated a high degree of correlation (but not equivalence) between FSE‐based and GRASE‐based T₁w/T₂w ratios, and low correlations between T₁w/T₂w ratios and MWFs. This suggests that the two T₁w/T₂w ratio approaches measure similar facets of subcortical tissue microstructure, whereas T₁w/T₂w ratios and MWFs appear to be sensitized to different microstructural properties. On this basis, we conclude that multi‐echo GRASE sequences can be used in future studies to efficiently elucidate both general (T₁w/T₂w ratio) and myelin‐specific (MWF) tissue characteristics.     

A comparison of multiple imputation methods for missing data in longitudinal studies

Background

Multiple imputation (MI) is now widely used to handle missing data in longitudinal studies. Several MI techniques have been proposed to impute incomplete longitudinal covariates, including standard fully conditional specification (FCS-Standard) and joint multivariate normal imputation (JM-MVN), which treat repeated measurements as distinct variables, and various extensions based on generalized linear mixed models. Although these MI approaches have been implemented in various software packages, there has not been a comprehensive evaluation of the relative performance of these methods in the context of longitudinal data.

Method

Using both empirical data and a simulation study based on data from the six waves of the Longitudinal Study of Australian Children (N?=?4661), we investigated the performance of a wide range of MI methods available in standard software packages for investigating the association between child body mass index (BMI) and quality of life using both a linear regression and a linear mixed-effects model.

Results

In this paper, we have identified and compared 12 different MI methods for imputing missing data in longitudinal studies. Analysis of simulated data under missing at random (MAR) mechanisms showed that the generally available MI methods provided less biased estimates with better coverage for the linear regression model and around half of these methods performed well for the estimation of regression parameters for a linear mixed model with random intercept. With the observed data, we observed an inverse association between child BMI and quality of life, with available data as well as multiple imputation.

Conclusion

Both FCS-Standard and JM-MVN performed well for the estimation of regression parameters in both analysis models. More complex methods that explicitly reflect the longitudinal structure for these analysis models may only be needed in specific circumstances such as irregularly spaced data.

     

Influence of ionic crosslinkers (Ca2+/Ba2+/Zn2+) on the mechanical and biological properties of 3D Bioplotted Hydrogel Scaffolds

Three dimensional (3D) bioplotting requires appropriate crosslinkers to crosslink the hydrogel precursor while simultaneously maintaining the viability of embedded cells. However, the evaluation and comparison of various types of crosslinkers in bioplotting remains underexplored to date. This paper presents our study of the influence of three ionic crosslinkers—calcium chloride (CaCl₂), barium chloride (BaCl₂), and zinc chloride (ZnCl₂)—on the mechanical and biological properties of 3D bioplotted alginate scaffolds. The scaffold mechanical properties characterized included the elastic modulus, swelling, and degradation while the biological properties considered included Schwann cell viability and surface morphology. The mechanical and biological properties of the bioplotted scaffolds were both dependent on the crosslinkers used for fabrication; specifically, crosslinking ions resulted in the elastic modulus of the hydrogels decreasing in the order BaCl₂>CaCl₂>ZnCl₂ over 42 days while Schwann cell viability decreased in the order CaCl₂>BaCl₂>ZnCl₂ over 7 days. Taken together, these results offer insights that are effective in terms of manipulating the 3D bioplotting process so as to tune and optimize the mechanical and biological performance of the plotted scaffolds for tissue engineering applications.