Application of explanatory sequential design of mixed methods research in conciliating qualitative and quantitative findings on social stressors and to examine the social problem solving orientation in substance abuse population

Substance abuse has been a problem with every society and across every generation. The increasing number of substance abusers in Hong Kong causes a huge impact to our healthcare system. With the consideration in the complexity of psychosocial nature of this population, the utility of both qualitative and quantitative research methods have been becoming increasingly accepted in health sciences. This is a pioneer project in Hong Kong that employed mixed methods research for substance abuse populations which incorporates evidence of pre-treatment individual characteristics and other specific-tailored treatment factors in promoting changes over time. This study aimed at developing a comprehensive understanding of how individual factors, different social stressors and social problem solving orientation in shaping their behavior. This study covered two phases. The first one was to collect quantitative data in subjects’ ability in problem recognition, treatment readiness, social values and their orientation in social problem solving. A regression model of relapse prediction was constructed, in which, social problem solving was the most prominent factor, followed by treatment readiness, problem recognition and emotional problem. In the second phase of the study, individual semi-structure interview, and qualitative focus group activities of free-listing and pile-sorting were employed to collect qualitative data on the impact of various social stressors like stress from peers, from family, and from work or study. In the final step, we interpreted findings from these two subsets of quantitative and qualitative data. Results indicated stress from peers was the most prominent single stressor of substance abusers, which yielded similar impact as the combination of cross-product of stress from family and from work or study. Moreover, most subjects adopted negative orientation in their social problem solving, in which, stress from work or study noted with the highest response rate in negative orientation in their social problem solving

Sulfonylurea is associated with higher risks of ventricular arrhythmia or sudden cardiac death compared with metformin: A population-based cohort study

Background Commonly prescribed diabetic medications such as metformin and sulfonylurea may be associated with different arrhythmogenic risks. This study compared the risk of ventricular arrhythmia or sudden cardiac death between metformin and sulfonylurea users in patients with type 2 diabetes. Methods and Results Patients aged ≥40 years who were diagnosed with type 2 diabetes or prescribed antidiabetic agents in Hong Kong between January 1, 2009, and December 31, 2009, were included and followed up until December 31, 2019. Patients prescribed with both metformin and sulfonylurea or had prior myocardial infarction were excluded. The study outcome was a composite of ventricular arrhythmia or sudden cardiac death. Metformin users and sulfonylurea users were matched at a 1:1 ratio by propensity score matching. The matched cohort consisted of 16 596 metformin users (47.70% men; age, 68±11 years; mean follow‐up, 4.92±2.55 years) and 16 596 sulfonylurea users (49.80% men; age, 70±11 years; mean follow‐up, 4.93±2.55 years). Sulfonylurea was associated with higher risk of ventricular arrhythmia or sudden cardiac death than metformin hazard ratio (HR, 1.90 [95% CI, 1.73–2.08]). Such difference was consistently observed in subgroup analyses stratifying for insulin usage or known coronary heart disease. Conclusions Sulfonylurea use is associated with higher risk of ventricular arrhythmia or sudden cardiac death than metformin in patients with type 2 diabetes

Induction of Protective CD4+ T Cell-Mediated Immunity by a Leishmania Peptide Delivered in Recombinant Influenza Viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4+ Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK158–173 CD4+ peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK158–173-specific CD4+ T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK158–173 triggers LACK158–173-specific Th1-biased CD4+ T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2–4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4+ T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK158–173 led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The role of SOCS3 and related proteins in inflammatory processes

© 2011 Dr. Hiu KiuThis thesis investigates two functional aspects of SOCS3 in inflammation: (1) the physiological role of SOCS3 in the non-haemopoietic compartment, with particular interest in endothelial cells; and (2) whether there is functional overlap between SOCS3 and SOCS1, SOCS2 and CIS in regulation of cytokine signaling. In vivo mouse models were integral to both studies. The former study employed endothelial-specific Cre transgenic mice to generate and study mice with SOCS3-deficient endothelium. To investigate whether there are shared and overlapping functions within the SOCS family, the latter study focused on characterization of double knockout mice with pair-wise deletion of SOCS3 and SOCS1, SOCS2 or CIS in hematopoietic cells. Although SOCS2 had previously been reported to regulate SOCS3 expression, this thesis demonstrates that there is no physiological role for SOCS2 in the regulation of SOCS3 protein expression and activity in primary hematopoietic cells. With this clarification, examination of mice with additional SOCS2 deletion demonstrated accelerated inflammatory disease onset in mice with haemopoietic-specific SOCS3 deficiency. Analyses of mice lacking both SOCS3 and SOCS1 and SOCS3 and CIS also revealed accelerated mortality in comparison to the single knockout mice. While the pathology observed in each of the double knockout mice, particularly in SOCS2/SOCS3 and CIS/SOCS3 double deficient mice, resembled that of the single mutants, neither G-CSF nor IL-6 signalling dysregulation in SOCS3-deficient cells was augmented in double-deficient cells. As a miscellany of haemopoietic alterations were observed in each of the double deficient mice, other mechanisms must be also involved and underpin the bases of acceleration of the disease in these mice. Further studies will be required to define which signaling pathways are involved. Collectively, this is the first in vivo study that demonstrates functional interaction among SOCS family proteins in modulation of inflammation

Suppressive Oligodeoxynucleotides Promote the Development of Th17 Cells

<div><p>Synthetic oligonucleotides containing repetitive TTAGGG motifs mimic the immunosuppressive activity of telomeric DNA. These suppressive oligonucleotides (Sup ODN) are effective in the treatment/prevention of various inflammatory and autoimmune diseases in mice. The therapeutic activity of Sup ODN was originally attributed to the inhibition of Th1 cell activation. Current results indicate that Sup ODN also promote the maturation of naive CD4⁺ T cells into Th17 effectors. The generation of Th17 cells is linked to the prolonged activation of signal transducer and activator of transcription (STAT)3 mediated by suppressor of cytokine signaling 3 (SOCS3) inhibition. <i>In vivo</i> studies show that treatment with Sup ODN promotes Th17 responsiveness under physiological conditions, increasing host resistance to <i>Candida albicans</i> infection. These findings support the development of Sup ODN to suppress pathological inflammatory conditions and improve host resistance to fungal pathogens.</p></div

Gene expression by Th17 cells.

<p>Naive T cells from C57BL/6 mice were cultured under Th17 polarizing conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067991#pone-0067991-g001" target="_blank">Fig 1</a>. mRNA was isolated on day 3 and the expression of genes encoding T-bet, IL-23R, IL-33 and IL-10 determined by RT-PCR. Relative mRNA levels were calculated by comparison to Th17 cells generated in the absence of ODN after normalization to GAPDH mRNA levels. Each bar represents the mean+SD of 3 independent experiments. **, p<0.01.</p

Effect of Suppressive ODN on STAT3, SOCS3 and IL-17.

<p>Naive T cells from C57BL/6 mice were cultured under Th17 polarizing conditions for 20 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067991#pone-0067991-g001" target="_blank">Fig 1</a>. A) The level of STAT3 phosphorylation was determined by flow cytometry using a phospho-specific anti-STAT3 Ab. Results reflect the increase over baseline in phosphorylated STAT3 vs peak levels (see methods section for details). B,C) mRNA was isolated and analyzed by RT-PCR. The level of SOCS3 and IL-17 expression was normalized to GAPDH levels in the same sample. Changes in SOCS3 expression determined by comparison to cells cultured under Th0 biased conditions. Data represent the mean+SD of 3 independent experiments. *, p<0.05; ***, p<0.001.</p

Suppressive ODN promote the maturation of Th17 cells.

<p>Highly purified naive CD4⁺ T cells from female C57BL/6 mice were incubated for 2 h with 1 µM of suppressive or control ODN and then cultured for 3 days under Th0 or Th17 polarizing conditions (see methods section for details). (A) Frequency of cells producing IL-17A and IFN_γ in samples as determined by intracellular staining and flow cytometry. (B) Combined results (from 4 independent experiments) showing the mean+SD of the effects of treatment on the frequency of IL-17A⁺ cells. (C) Level of IL17A and IL-17F protein in culture supernatants of cells stimulated under Th17 polarizing conditions for 3 days (mean+SD of <u>4</u> experiments). (D) Level of mRNA encoding RORC determined by RT-PCR. mRNA levels in cells treated under Th17 polarizing conditions are compared to those generated under Th0 conditions after normalization to GAPDH mRNA for each sample. Each bar represent the mean+SD of 3 independent experiments. *, p<0.05; ***, p<0.001.</p