A diagram of test results and their representative alleles.

<p>A diagram demonstrated the number of samples grouped by their results and the alleles which could give each result. In the AS-PCR step, all samples were tested: 82 gave negative results and were reported as negative for screening, following which 73 positive AS-PCR samples were then tested in the DDB step, giving 47 negative and 26 positive results. The only falsely screened result was from <i>HLA-B*15</i>:<i>31</i>.</p

Results of AS-PCR and DDB.

<p><b>(a)</b> Electrophoresis was applied by using 2.5% agarose gel with 100V for 30 minute. A control product was 301 bp long while the specific PCR product was 137 bp long. The alleles with PCR product included <i>HLA-B*15</i>:<i>02</i> (No. 1 and 8), <i>B*15</i>:<i>21</i> (No. 2), <i>B*15</i>:<i>25</i> (No. 3), <i>B*15</i>:<i>11</i> (No. 4), <i>B*15</i>:<i>32</i> (No. 5), <i>B*15</i>:<i>01</i> (No. 6), <i>B*15</i>:<i>31</i> (No. 7), and <i>B*46</i>:<i>01</i> (No. 4, 8). <i>HLA-B*15</i>:<i>13</i> (No. 9) and other common alleles (No. 10–16) gave negative results in the PCR step. <b>(b)</b> The samples analyzed in the PCR step (No. 1–9) were subsequently examined by DDB. (NTC: “no template control”).</p

Sequences of primers and probe used for <i>HLA-B*15</i>:<i>02</i> screening by AS-PCR and DDB.

<p>Sequences of primers and probe used for <i>HLA-B*15</i>:<i>02</i> screening by AS-PCR and DDB.</p

Flowchart of Subjects Participating in the Different Phases of Randomized Crossover Trial.

<p>Flowchart of Subjects Participating in the Different Phases of Randomized Crossover Trial.</p

Baseline Characteristics, Patients Who Received Placebo First Compared to Those That Received Pitavastatin First.

<p>Baseline Characteristics, Patients Who Received Placebo First Compared to Those That Received Pitavastatin First.</p

Cost-effectiveness analysis of HLA-B*13:01 screening for the prevention of co-trimoxazole-induced severe cutaneous adverse reactions among HIV-infected patients in Thailand

Studies found a strong association between HLA-B*13:01 allele and co-trimoxazole-induced severe cutaneous adverse reactions (SCARs). Genetic screening before initiation of co-trimoxazole may decrease the incidence of co-trimoxazole-induced SCARs. This study aims to evaluate the cost-effectiveness of HLA-B*13:01 screening before co-trimoxazole initiation in HIV-infected patients in Thailand. A combination of a decision tree model and a Markov model was used to estimate lifetime costs and outcomes of two strategies including 1) HLA-B*13:01 screening before co-trimoxazole initiation and 2) usual practice from a societal perspective. Alternative drugs are not considered because dapsone (the second-line drug) also presents a genetic risk. Input parameters were obtained from literature, government documents, and part of the TREAT Asia HIV Observational Database (TAHOD). One-way sensitivity analyses and probabilistic analyses were performed to determine robustness of the findings. HLA-B*13:01 screening resulted in 0.0061 quality-adjusted life years (QALYs) loss with an additional cost of 370 THB ($11.84). At the cost-effectiveness threshold of 160,000 THB ($5,112.85), the probability of the genetic screening strategy being cost-effective is 9.54%. This analysis demonstrated that HLA-B*13:01 allele screening before initiation of co-trimoxazole among HIV-infected patients is unlikely to be cost-effective in Thailand. Our findings will help policymakers make an evidence-informed decision making.</p

Lipid Measurements, Patients Who Received Placebo First Compared to Those That Received Pitavastatin First.

<p>Lipid Measurements, Patients Who Received Placebo First Compared to Those That Received Pitavastatin First.</p

P2Y₆ receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium

<div><p>One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-β, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (β-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y₆R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca²⁺ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y₆ purinergic receptors.</p></div

Inactivated-H5N1 increases phosphorylation of ERK1/2 and p38 MAPK.

<p>Immunoblot analysis of ERK1/2 <i>(A)</i> and p38 MAPK <i>(B)</i> protein expression in 16HBE14o- cells treated with inactivated-H5N1. Cells were untreated or treated with PD98059 (50 μM) or SB203580 (10 μM) as appropriate. One hour later, cells were treated for 30 min with vehicle, allantoic fluid or inactivated-H5N1 before being harvested for immunoblot analysis for the presence of ERK1/2, phospho-ERK1/2 (P-ERK1/2), p38 MAPK or phospho-p38 MAPK (P-p38). Upper panel, representative gels for immunoblot analysis of ERK1/2 and phosphorylated ERK1/2 (P-ERK1/2) <i>(A)</i> or p38 MAPK and phosphorylated p38 MAPK (P-p38) <i>(B)</i>. Lower panel, quantitative analysis of the amount of P-ERK1/2 (<i>A</i>) or P-p38 MAPK <i>(B)</i>, expressed as fold change, using ImageJ software (NIH). Values are means ± SEM from at least 3 sets of experiments. * and *** indicates <i>p</i> < 0.05 and <i>p</i> < 0.001, respectively (one-way ANOVA with Student-Newman-Keuls post-hoc test).</p

The cellular signaling mechanism by which inactivated-H5N1 increases mRNA for IL-6 and CXCL8 involves intracellular Ca²⁺, PLC, PKC, ERK1/2 and p38 MAPK.

<p>16HBE14o- cells were incubated with BAPTA-AM (50 μM), U73122 (10 μM), BIM (1 μM), Gö6976 (10 μM) <b><i>(</i></b><i>A and B)</i>, or with SN-50 (10 μM), PD98059 (50 μM), SB203580 (10 μM) or SP600125 (10 μM) <i>(C and D)</i>, for 1 hr before being exposed to inactivated-H5N1. Three hours later, mRNA expression of IL-6 <i>(A and C)</i> or that of CXCL8 <i>(B and D)</i> was analyzed. All data were normalized with corresponding control groups treated with inactivated-H5N1 (open bars) and reported as a percentage. <i>(E)</i> Cells were treated with SB203580 (10 μM) for 1 hr before incubation with inactivated-H5N1 (20 μg/ml hemagglutinin). Thirty-six hours later, the cell-free culture medium was harvested and analyzed for the presence of IL-6 and CXCL8 proteins as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176974#pone.0176974.g001" target="_blank">Fig 1</a>. Data are presented as a percentage of control (treated with inactivated-H5N1, open bar). Values are means μ SEM from at least 3 sets of experiments. *, ** and *** indicates <i>p</i> < 0.05, <i>p</i> < 0.01 and <i>p</i> < 0.001 compared with control, respectively (unpaired Student’s <i>t</i>-test).</p