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PGE2, a major end product of cyclooxygenase (COX-2), has an important role in both acute and chronic inflammatory responses . First, we investigated whether DPB could stimulate PGE2 expression in various cell lines. As shown in Fig. 2a, PGE2 levels were significantly increased in DPB-stimulated RAW264.7 macrophages, and IHOK, IGF, and YD38. However, the pH changes occurred slowly in co-culture media with DPB during 24 h of incubation. Therefore, we extracted LPS from DPB, and used 1 μg/ml each of DPB#1-LPS, DPB#2-LPS, and DPB#3-LPS for the next experiments. In RAW264.7, IHOK, IGF, and YD38 cells, PGE2 levels were also increased after DPB-LPS treatment, although the different effects were cell line-specific (Fig. 2b). COX-2 mRNA was also increased by stimulation with DPB#1-LPS, DPB#2-LPS, or DPB#3-LPS (Fig. 2c). Treatment with EETC significantly suppressed the levels of PGE2 and COX-2 that had been increased by DPB-LPS (10 μg/ml) (Fig. 2d and e). YD38 cell viability remained unchanged after treatment with 5 and 30 μg/ml EETC (Fig. 2f). Thus, the EETC inhibited not only the growth of DPB through its anti-bacterial effect but also inflammation by abolishing PGE2 and COX-2 expression, thereby enhancing its preventive effect on gingival and periodontal diseases.Fig. 2Effect of the EETC on DPB-induced PGE2 and COX-2. a The effect of DPB on PGE2 levels in various cell types. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. bacteria-free control medium from each cell line. b The effect of LPS extracted from DPBs (DPB#1-LPS, DPB#2-LPS, and DPB#3-LPS) on PGE2 levels. PBS was used for control. *P < 0.01, # P < 0.001 vs. PBS control medium from each cell line. c The effect of DPBs-LPS on COX-2 expression. mRNA expression were detected by RT-PCR. The inhibitory effects of EETC on PGE2 (d) and COX-2 (e) levels in DPB-LPS-treated cells. DMSO was used for control. Data represent the mean ± SE of three independent experiments performed by triplicate. # P < 0.001 vs. DMSO control (Ctl) medium, *P < 0.01 vs. DPB#1 or DPB#1-LPS medium. f The effect of EETC on the cell viability
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Effect of the EETC on DPB-induced PGE2 and COX-2. a The effect of DPB on PGE2 levels in various cell types. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. bacteria-free control medium from each cell line. b The effect of LPS extracted from DPBs (DPB#1-LPS, DPB#2-LPS, and DPB#3-LPS) on PGE2 levels. PBS was used for control. *P < 0.01, # P < 0.001 vs. PBS control medium from each cell line. c The effect of DPBs-LPS on COX-2 expression. mRNA expression were detected by RT-PCR. The inhibitory effects of EETC on PGE2 (d) and COX-2 (e) levels in DPB-LPS-treated cells. DMSO was used for control. Data represent the mean ± SE of three independent experiments performed by triplicate. # P < 0.001 vs. DMSO control (Ctl) medium, *P < 0.01 vs. DPB#1 or DPB#1-LPS medium. f The effect of EETC on the cell viability
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The Human Inflammation Antibody Array was used to elucidate the molecules related to the DPB-induced inflammation. In the DPB-LPS–stimulated YD38 cells, the inflammatory cytokine profiles were increased for interleukin (IL)-3, IL-16, CXC motif chemokine 10 (CXCL10, IP-10), tumor necrosis factor receptor superfamily member 1A (TNFRSF1A, TNFRI), tumor necrosis factor receptor superfamily member 1B (TNFRSF1B, TNFRII), macrophage colony-stimulating factor 1 (CSF 1, MCSF), and CC motif chemokine 4 (CCL4, MIP-1β) (Fig. 3a). Increased levels of inflammation-related molecules were suppressed by pretreatment with the EETC. In addition, as shown in human protease profiles, proteases that act on inflammation through the regulation of pro-inflammatory cytokines, chemokines, and other proteins were also increased in the DPB-LPS–stimulated YD38 cells (Fig. 3b). The levels of MMP-7 (matrilysin, PUMP-1), KLK 10 (kallikrein 10), CTSC (cathepsin C), MMP-3 (stromelysin-1), and PC9 (proprotein convertase 9) were significantly increased; the induced levels of proteases were also suppressed by pretreatment with the EETC. To validate the tissue damage due to DPB-LPS, YD38 cells were cultured on FITC gelatin-coated coverslips, and matrix degradation was observed by bleaching the fluorescence dye. As shown in Fig. 3c, DPB-LPS stimulated matrix degradation, and the EETC effectively inhibited the DPB-LPS–induced matrix degradation.Fig. 3Effect of EETC on DPB-induced molecules involve in inflammation and tissue damage. a Inflammatory cytokine modulated by DPB-LPS and EETC in gingival epithelial cell. Analysis of culture medium (CM) collected from DMSO-treated cells, which served as the control. Altered factors are indicated with rectangles and circled numbers. The list altered factor is shown as fold-change in the graph. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1#-LPS-treated medium, # P < 0.001 vs. DMSO control medium. b Protease modulated by DPB-LPS and EETC in gingival epithelial cell. Altered factors are indicated with rectangles and circled numbers. The list altered factor is depicted in the graph and is shown as fold-change. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1#-LPS-treated medium, # P < 0.001 vs. DMSO control medium. c Effect of tissue damage by DPB-LPS and/or EETC on FITC-conjugated gelatin matrix-coated coverslips. The data shown are representative of five independent experiments
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Effect of EETC on DPB-induced molecules involve in inflammation and tissue damage. a Inflammatory cytokine modulated by DPB-LPS and EETC in gingival epithelial cell. Analysis of culture medium (CM) collected from DMSO-treated cells, which served as the control. Altered factors are indicated with rectangles and circled numbers. The list altered factor is shown as fold-change in the graph. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1#-LPS-treated medium, # P < 0.001 vs. DMSO control medium. b Protease modulated by DPB-LPS and EETC in gingival epithelial cell. Altered factors are indicated with rectangles and circled numbers. The list altered factor is depicted in the graph and is shown as fold-change. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1#-LPS-treated medium, # P < 0.001 vs. DMSO control medium. c Effect of tissue damage by DPB-LPS and/or EETC on FITC-conjugated gelatin matrix-coated coverslips. The data shown are representative of five independent experiments
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| 100.0 |
RANKL is a critical molecule in osteoclast differentiation and bone resorption. As shown in Fig. 4a, osteoclast formation was clearly detected in medium containing 100 ng/ml of RANKL, whereas TRAP-positive osteoclasts were not detected in the absence of RANKL. In order to observe the effect of DPB-LPS on osteoclast formation, we performed the reaction in media with low levels of RANKL (10 ng/ml), and DPB-LPS was added to cultures of isolated BMMs. The number of TRAP-positive osteoclasts was significantly reduced in response to 10 ng/ml RANKL, as compared with 100 ng/ml RANKL; however, osteoclast formation in 10 ng/ml RANKL condition was significantly stimulated by DPB-LPS treatment. The stimulating effect of DPB-LPS was inhibited by the EETC treatment. The bone resorption activity of mature osteoclasts was measured simultaneously. Consistent with the osteoclast formation studies, the formation of resorption pits was remarkably increased by DPB-LPS treatment and suppressed by the EETC treatment. In addition, the mRNA expression of RANKL was significantly stimulated by DPB-LPS treatment in the hFOB1.19 human osteoblast cells but remained the same for osteopontin (OPG) (Fig. 4b). Treatment with the EETC inhibited RANKL mRNA expression but did not affect OPG mRNA expression in DPB-LPS–treated hFOB1.19 cells (Fig. 4c). Consequently, the increased RANKL/OPG ratio in the DPB-LPS–treated hFOB1.19 cells was remarkably decreased by treatment with EETC. Thus, bone destruction by DPB-LPS was caused by the cooperative stimulation of osteoclast formation in BMMs and of RANKL expression in osteoblasts. EETC could be an important means of preventing bone loss due to oral bacteria.Fig. 4Effect of EETC on DPB-induced osteoclastic bone resorption. a For osteoclast formation, mouse BMMs were treated with M-CSF and RANKL and were stained to detect expression of TRAP. Pit formation was also performed in M-CSF and RANKL on calcium phosphate apatite-coated plates, and light microscopy indicated resorptive pitting. To observe the stimulating effect of DPB-LPS on osteoclast formation and pit formation, both assays were performed at a low concentration of RANKL (10 ng/ml). The graph shows the total number of TRAP-positive multinucleated (≥3 nuclei) osteoclasts (MNC) per well. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. only 10 ng/ml RANKL condition, # P < 0.001 vs. DPB1#-LPS plus 10 ng/ml RANKL condition. b RANKL and OPG mRNA expression was analyzed in hFOB1.19 human osteoblastic cells. Eco-LPS (LPS from Escherichia coli) was the positive control. The data shown are representative of three independent experiments. c Effect of EETC on DPB#1-LPS-induced RANKL and OPG mRNA expression were analyzed by RT-PCR. The ratio of RANKL to OPG was determined after normalization to the intensity of GAPDH. The data shown are representative of three independent experiments. *P < 0.001 vs. control (Ctl), # P < 0.001 vs. DPB#1-LPS condition
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Effect of EETC on DPB-induced osteoclastic bone resorption. a For osteoclast formation, mouse BMMs were treated with M-CSF and RANKL and were stained to detect expression of TRAP. Pit formation was also performed in M-CSF and RANKL on calcium phosphate apatite-coated plates, and light microscopy indicated resorptive pitting. To observe the stimulating effect of DPB-LPS on osteoclast formation and pit formation, both assays were performed at a low concentration of RANKL (10 ng/ml). The graph shows the total number of TRAP-positive multinucleated (≥3 nuclei) osteoclasts (MNC) per well. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. only 10 ng/ml RANKL condition, # P < 0.001 vs. DPB1#-LPS plus 10 ng/ml RANKL condition. b RANKL and OPG mRNA expression was analyzed in hFOB1.19 human osteoblastic cells. Eco-LPS (LPS from Escherichia coli) was the positive control. The data shown are representative of three independent experiments. c Effect of EETC on DPB#1-LPS-induced RANKL and OPG mRNA expression were analyzed by RT-PCR. The ratio of RANKL to OPG was determined after normalization to the intensity of GAPDH. The data shown are representative of three independent experiments. *P < 0.001 vs. control (Ctl), # P < 0.001 vs. DPB#1-LPS condition
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| 100.0 |
Inflammation is the biological response of tissues to infection with pathogens and cell damage . When harmful stimuli cause an inflammatory response, the body’s innate immune system involves the phagocytic activation of leukocytes and macrophages, and the increased release of inflammatory cytokines. Uncontrolled inflammation leads to disease progression because of the excessive production of inflammatory cytokines . DPB are a leading cause of inflammation in periodontal disease . Untreated gingivitis readily progresses to periodontitis and leads to tissue destruction. Bacterial plaque elaborate various compounds that elicit an inflammatory response and lead to destruction of the gingival tissue, which may progress to destruction of the periodontal ligaments. Plaque control is required to prevent further progression of inflammation. In this study, we tried to elucidate the critical factors involved in plaque bacteria-induced inflammation and effective natural anti-inflammatory substances for plaque bacteria control.
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The in vitro anti-bacterial activity of EETC against DPB was evaluated. Disc diffusion assay results showed that EETC effectively suppressed the DPB growth and the anti-bacterial activity of EETC were increased with increasing concentration of extract. This concentration of EETC was not cytotoxic to gingival epithelial cells, gingival fibroblasts and macrophage. The induction of inflammatory factors and proteases that contributed to the inflammatory reaction were also abolished by EETC. These results indicate that EETC could effectively suppress the inflammatory responses induced by plaque bacteria. The collective data indicate that EETC is useful in reducing bacterial plaque accumulation and gingival inflammation, thereby preventing periodontal disease. Since the efficacy and safety of EETC has been established, it can be developed into a mouthwash without added alcohol. Many commercially available mouthwashes contain alcohol, which increases the risk of oral cancers and dry mouth.
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| 99.94 |
LPS is also known as lipoglycans and endotoxins. The molecules is a component of the outer membrane of the cell wall of gram-negative bacteria, and is one of the most powerful bacterial virulence factors in the pro-inflammatory response . Initiation of the inflammatory response by LPS stimulates a strong immune response. LPS acts as the prototypical endotoxin through binding to the CD14/TLR4/MD2 receptor complex in many cell types, but especially in monocytes, dendritic cells, macrophages, and B cells, which promotes the secretion of pro-inflammatory cytokines, nitric oxide, and eicosanoids . Therefore, LPS is a central molecule in the pathogenesis of certain bacterial infections . In this study, in vitro co-culture with gingival epithelial cells and plaque bacteria caused pH changes of the culture medium during 24 h of incubation. Therefore, LPS was extracted from DPBs and its capacity to induce inflammation was verified. DPB-LPS significantly increased the levels of inflammatory factors in gingival epithelial cells, gingival fibroblasts and macrophages. EETC also effectively suppressed the levels of DPB-LPS-induced inflammatory factors.
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EETC significantly suppressed growth of DPB and DPB-mediated periodontal disease. Antibiotics are used to both treat and prevent bacterial infections . Antibiotics like penicillin and erythromycin used to be highly effective against many bacterial species and strains. They are now less effective because these organisms have developed increased resistance to the drugs . Dental plaque, which adheres to the gingival sulcus, consists mainly of bacterial cells, salivary polymers, and bacterial extracellular products. Bacterial LPS stimulates the host cells, including macrophages, fibroblasts, and epithelial cells, which abundantly produce cytokines, thereby triggering a direct or an indirect immune response through inflammatory processes . The growth of bacterial biofilms increases antibiotic resistance and has been implicated in the etiopathogenesis of gingivitis and periodontal disease by inducing inflammation.
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| 99.9 |
An effective substance is needed that will prevent and treat bacterial plaque-mediated disease by controlling bacterial growth and the inflammatory reaction. The fruits of the T. chebula, which has been amply verified as being medicinally valuable, has been used to treat cancer, cardiovascular diseases, paralysis, leprosy, ulcers, gout, arthritis, epilepsy, cough, fever, diarrhea, gastroenteritis, skin disorders, urinary tract infection, and wound infections [10, 24]. T. chebula fruit is also effective in the treatment of bacterial infections [25, 26]. Clinical trials of T. chebula fruit extract as a mouthwash preparation have reported reduced plaque accumulation and gingival inflammation [27, 28]. The present findings provide mechanistic details for these beneficial effects.
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| 96.75 |
Most dental care products exert their antibacterial effects by eliminating oral bacteria. Instead, LPS from other types of bacterial cells, such as E. coli and Pseudomonas aeruginosa, have been used to induce the inflammation. Inflammatory events related to DPB-derived endotoxins have not been evaluated as intensively [29, 30], and the value of anti-microbial dental care products to treat periodontal disease has not been sufficiently verified. Thus, there was a need to simultaneously investigate inflammatory events due to DPB-derived endotoxin and elucidate an effective anti-microbial component for periodontal disease. Here we demonstrate that DPB and DPB- significantly increase inflammation-associated elements such as COX-2 and PGE2. Many inflammatory molecules and proteases are also upregulated by DPB-LPS and lead to matrix damage. In addition, DPB-LPS stimulation promotes osteoclast formation, causing bone resorption. The risk of bone loss tends to increase in patients with inflammatory conditions . Inflammatory cytokines modulate osteoblast and osteoclast activity . Interleukins (IL), tumor necrosis factor (TNF), and tumor necrosis factor receptor superfamily members (TNFRSF) perturb bone homeostasis, leading to increased cartilage degradation and bone resorption by osteoclasts and inhibiting bone formation by osteoblasts .
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| 99.94 |
In the present study, IL-3, IL-16, CXCL10, TNFRSF1A, TNFRSF1B, M-CSF 1, and CCL4 were increased in DPB-LPS-stimulated gingival epithelial cells. Proteases including MMP-3, MMP-7, KLK 10, CTSC, and PC9, which were also increased by DPB-LPS stimulation, might also contribute to the inflammatory reaction, thereby leading to matrix degradation and bone destruction. However, the levels of inflammation-related molecules and osteoclastic bone resorption were significantly reduced by treatment with EETC. EETC also exhibited anti-bacterial properties by arresting the growth of DPB. These findings suggest that the EETC could be used to treat DPB-mediated periodontal disease.
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Osteoclast differentiation and bone resorption activity require stimulation by the receptor activator of nuclear factor-kappaB (RANKL) expressed on osteoblasts . In the present study, increased RANKL mRNA expression was observed in DPB-LPS-stimulated hFOB1.19 human osteoblast cells. In addition, DPB-LPS stimulated osteoclast formation and bone resorption under RANKL-limited conditions. These results are consistent with the results of previous studies showing that LPS stimulated osteoclastogenesis [35–37]. These results indicate that DPB-LPS promotes functional osteoclast formation through osteoblast-dependent and osteoblast-independent pathways. DPB-LPS-induced osteoclast formation and bone resorption were also significantly abolished by the EETC treatment.
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In conclusion, treatment with the EETC inhibits the growth of DPB as well as DPB-induced inflammation, and effectively abolishes DPB-LPS-induced osteoclastic bone resorption in vitro. EETC is an effective botanical chemopreventive agent that can modulate DPB-induced inflammatory factors involved in gingivitis and periodontal disease. EETC can be considered a promising anti-bacterial and anti-oral inflammatory agent capable of preventing the development of gingivitis and periodontitis. Further research is needed to isolate and identify the beneficial chemical constituents in the extract that could be exploited for pharmaceutical use.
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| 99.94 |
Human norovirus (HuNoV) is a major causative agent of gastroenteritis in humans (Green, 2013). The HuNoV genogroup II (GII), in particular, is frequently detected in outbreaks. The HuNoV GII strains can be classified into 22 genotypes (Kroneman et al., 2013). Moreover, the most worldwide prevalent HuNoV GII genotypes belong to GII genotype 2 (GII.2), GII.3, GII.4, GII.6, and GII.17 (Centers for Disease Control and Prevention. CaliciNet Data [cited 2016])1. Since national surveillance began, nearly 3 million cases of NoV gastroenteritis have been recorded, and Japan was experiencing its second most serious norovirus outbreak during November 2016 to February 2017 (National Institute of Infectious Diseases. Japan. Infectious Gastroenteritis. [cited 4th April 2017, in Japanese])2 Importantly, HuNoV GII.2 emerged as a major cause of this outbreak in Japan, although the GII.4 strains were the most prevalent genotype during the past 10 years (National Institute of Infectious Diseases. Japan. Flash report of norovirus in Japan [cited 4th April 2017, in Japanese])3.
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| 99.8 |
Very recent studies suggested that the evolutionary patterns of human and animal NoV genotypes are distinct (Kobayashi et al., 2015, 2016). Although all viral proteins may act as antigens, the HuNoV VP1 protein is also involved in viral infection. Furthermore, HuNoV frequently experiences recombination at the ORF1/ORF2 junction, resulting in new chimera viruses with different types of the RNA-dependent RNA polymerase (RdRp) genes and capsid (VP1) genes. Most studies have focused on the molecular evolution of HuNoV GII.4. Only a few examined that gene in other HuNoV genotypes, including GII.2. To gain insight into this process, we examined the molecular evolution of the GII.2 RdRp and VP1 genes, including chimera viruses, based on the full genome analyses of those detected in Japan over a period of 10 years (2004–2015 seasons).
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To investigate the molecular evolution of the HuNoV VP1 and RdRp genes, 950 stool specimens were collected from various areas (13 prefectures) of Japan during the 2004–2015 seasons. These samples were obtained from patients with acute gastroenteritis due to HuNoV infections, in compliance with the Food Sanitation Law and the Law Concerning the Prevention of Infections and Medical Care for Patients of Infections of Japan. The personal data related to these samples were anonymized. RNA was extracted from 10% PBS suspensions of the specimens, and the HuNoV genomes were comprehensively analyzed by next-generation sequencing as described (Matsushima et al., 2015). Of 950 samples, the complete genome sequences of 538 strains were obtained (a success rate of 57%). Next, HuNoV genotypes were confirmed with the Norovirus Typing Tool (Version1.0), based on the nucleotide sequences of RdRp and VP1 genes as described by Kroneman et al. (2011). GII.2 strains were selected from these all genotyped strains, and then a few of strains having the undetermined base sequences (e.g., N, Y, R, and V) were omitted. Finally, 51 GII.2 strains were used for evolutionary analyses for the present study (Supplementary Table 1). The obtained nucleotide sequences for the GII.2 strains were deposited in GenBank under the accession numbers LC209431 to LC209481.
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Time-scale evolutionary analyses were performed using the Bayesian Markov Chain Monte Carlo method (MCMC) with the BEAST package v1.8.3 (Drummond and Rambaut, 2007) and Tracer4 as a demographic model. Substitution models were calculated with Kakusan4 (Tanabe, 2011). The substitution model for the VP1 or the RdRp gene was the GTR-Γ or GTR-Γ invariant model, respectively. Based on Akaike's Information Criterion for MCMC values, we used the random local clock as a clock model, and used the logistic growth model (VP1 gene) or the constant size model (RdRp gene) as a tree model. Convergence was evaluated with an effective sample size (acceptable more than 200). The MCMC chain length was 3 × 108 steps with sampling every 1,000 steps for the MCMC tree of the VP1 gene. To exactly estimate the evolutionary rates and topologies of the MCMC tree of the RdRp gene, we bound two independent data of the MCMC chains5. The MCMC chain length was 2 × 108 steps and 4 × 108 with sampling every 5,000 steps for the MCMC tree of the RdRp gene. Statistical analyses were performed with the Welch's t-test in Excel 2013.
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Four genotypes of the GII.2 strains, including GII.P2-GII.2 (13 strains), GII.Pe-GII.2 (one strain), GII.P12-GII.2 (one strain), and GII.P16-GII.2 (36 strains), were determined by the Norovirus Typing Tool (Figure 1). Of them, GII.P16-GII.2 strains were the most prevalent genotype after 2009. The single GII.P12-GII.2 and GII.Pe-GII.2 strains were detected in 2004 and 2014 respectively. The GII.P2-GII.2 strains were detected throughout the investigation periods.
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Based on the VP1 gene sequences, we constructed a time-scale evolutionary tree (Figure 2A). The phylogeny of the VP1 gene showed that GII.2 strains could be divided according to the type of RdRp gene. GII.P16-GII.2 could be subdivided into three clusters of strains in 2009–2010, 2010–2012, and 2012–2014. In addition, the phylogenetic divergence of the GII.P16-GII.2 strains might be wider than that of the GII.P2-GII.2 strains. The tree shows that the most recent common ancestor (MRCA) of the present GII.2 strains appeared in 1956 (mean ± 95% highest posterior densities [HPD]: 1945–1966). Subsequently, GII.P2-GII.2 virus strain emerged in 2000 (mean ± 95% HPD: 1998–2001). Moreover, the GII.P16-GII.2 strains detected in 2010–2012 diverged from a common ancestor of the GII.P2-GII.2 strains at 2002 (mean ± 95% HPD: 2001–2004). The GII.P16-GII.2 strains detected in 2009–2010 and 2012–2014 diverged at 2005 (mean ± 95% HPD: 2004–2007). The evolutionary rate of these VP1 genes was 2.987 × 10−3 substitutions/site/year (mean ± 95% HPD: 2.496–3.486 × 10−3 substitutions/site/year).
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Phylogenetic trees of VP1 (A) and RdRp (B) genes of the genotype GII.2 constructed by the Bayesian MCMC method. We analyzed VP1 gene of 50 strains, and RdRp gene of 49 strains, excluding 100%—matched homologous strains. Reference strains in these trees were indicated in bold letters. Gray bar shows 95% HPD. The scale bar represents actual time (year).
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We also constructed a time-scale evolutionary tree of the RdRp gene (Figure 2B). The tree shows that the MRCA of RdRp of the present GII.2 strains was in the year 1696 (mean ± 95% HPD: 1542–1837). The common ancestor of the GII.P16-GII.2 strains diverged in 1858 (mean ± 95% HPD: 1747–1950) and formed two clusters. Moreover, the GII.P16-GII.2 strains detected in 2010–2012 diverged at 1989 (mean ± 95% HPD: 1972–2003), whereas the GII.P16-GII.2 strains detected in 2009–2010 and 2012–2014 diverged at 1986 (mean ± 95% HPD: 1968–2002). The common ancestor of the GII.P2-GII.2, GII.P12-GII.2, and GII.Pe-GII.2 diverged in 1828 (mean ± 95% HPD: 1741–1913). The RdRp gene of GII.P2-GII.2 diverged in 1992 (mean ± 95% HPD: 1984–2000). The evolutionary rate of these RdRp genes was 1.314 × 10−3 substitutions/site/year (mean ± 95% HPD: 0.698–1.95 × 10−3 substitutions/site/year).
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| 100.0 |
Next, we compared the evolutionary rates of the GII.P16-GII.2 and GII.P2-GII.2 strains. To gain statistical significance, we collected the nucleotide sequences of the GII.P2-GII.2 strains (25 strains) from GenBank, but we could not collect a sufficient number of the GII.P12-GII.2 and GII.Pe-GII.2 sequences from the GenBank to reach statistical significance. The evolutionary rate of GII.P16-GII.2 (1.838 × 10−3 substitutions/site/year; mean ± 95% HPD: 1.237–2.456 × 10−3 substitutions/site/year) was greater than that of GII.P2-GII.2 (1.712 × 10−3 substitutions/site/year; mean ± 95% HPD: 0.957–2.41 × 10−3 substitutions/site/year) (p = 7.891 × 10−135).
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| 100.0 |
A previous report suggested that the evolution of VP1 may be influenced by the activities of RdRp (Bull et al., 2010). Collectively, our bioinformatics data also showed that the evolution of the GII.2 VP1 gene was accelerated by a recombination of ORF1, including the RdRp gene. However, additional in vitro studies regarding the mutation rates of RdRp of the GII.P2 and GII.P16 may be needed to clarify the hypothesis of the relationships between and VP1 and RdRp genes in this study. Furthermore, GII.2 variant strains were detected in the present season (2016/17 season), and thus, further genetic studies may be needed to prove this hypothesis.
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| 100.0 |
Here we report the molecular evolution of the VP1 and RdRp genes in HuNoV GII.2. Our main findings and hypothesis are as follows. (1) Four genotypes of GII.2 (GII.P2-GII.2, GII.P16-GII.2, GII.P12-GII.2, and GII.Pe-GII.2) were detected in Japan in 2004–2015. (2) A common ancestor of the current GII.2 virus strains circulated around 1956. (3) VP1 gene evolution seems to depend on the RdRp gene. The VP1 gene in a prevalent HuNoV genotype GII.2 might evolve uniquely by transfer of the RdRp gene.
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| 100.0 |
FM designed and performed the research, analyzed the data and wrote the manuscript. KN, YD, and KH performed the research and analyzed the data. FM, SY, YU, MS, MI, NS (Sakon), NS (Shigemoto), RO, and AO contributed samples and analyzed the data; and HK and KK designed and supervised the research, analyzed the data, and wrote the manuscript. All authors contributed, read, and approved the manuscript.
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other
| 99.94 |
Although infants with perinatal HIV infection can now expect to survive childhood, they face life-long treatment with antiretrovirals (ARVs) in order to maintain a healthy immune system. Starting antiretroviral therapy (ART) before 3 months of age yields good clinical, immunological, and developmental outcomes (Violari et al., 2008; Laughton et al., 2012; Cotton et al., 2013; Crowell et al., 2015). However, it is known that ART cannot completely reverse the neurological effects of HIV (Laughton et al., 2013; van Arnhem et al., 2013; Whitehead et al., 2014) and neurodevelopmental delay and neurocognitive deficits remain (Govender et al., 2011; Donald et al., 2014; Wilmshurst et al., 2014; Musielak and Fine, 2015). In addition, neurotoxic effects of ART itself (Robertson et al., 2012) may be detrimental to brain development.
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review
| 99.9 |
Abnormalities of the basal ganglia are frequently seen in HIV-infected (HIV+) children and adults (George et al., 2009; Hoare et al., 2014), and subcortical structures may contain the highest levels of the virus (Tornatore et al., 1994). Neuroimaging shows that HIV+ children may have shape and volume alterations in subcortical gray matter (Lewis-de los Angeles et al., 2016; Yadav et al., 2017) and calcification of the basal ganglia (Govender et al., 2011), despite ART initiation during childhood.
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review
| 98.6 |
Magnetic resonance spectroscopy (MRS) measures brain metabolites including creatine (Cr), involved in energy metabolism, N-acetyl-aspartate (NAA), reflecting neuronal integrity, and glutamate (Glu), the primary excitatory neurotransmitter, as well as choline (Cho) and myo-Inositol (Ins). Ins is considered a glial marker and Cho reflects cell membrane turnover – both present in larger concentrations in glial cells than in neurons. Assessment of metabolites associated with neuronal integrity — NAA and Glu, and those associated with glial proliferation — Ins and Cho, can present a sensitive description of metabolic events underlying, and even preceding, the structural changes in basal ganglia seen in HIV+ children.
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| 100.0 |
Early single voxel spectroscopy (SVS) studies in children with HIV encephalopathy (HIVE) found lower NAA/Cr ratios in the basal ganglia compared to HIV+ children without encephalopathy and controls (Pavlakis et al., 1995; Lu et al., 1996). In earlier studies where most HIV+ children did not have encephalopathy, no alterations in basal ganglia NAA were found (Lu et al., 1996; Keller et al., 2004; Prado et al., 2011). More recently, Mbugua et al. (2016) reported higher absolute NAA levels in HIV+ children initiating ART before 12 weeks compared to a control group comprising mostly (80%) HIV-exposed uninfected (HEU) children.
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review
| 99.56 |
Findings on glial metabolites in the basal ganglia are less consistent. Keller colleagues and Lu colleagues found lower Cho (Keller et al., 2004) and Cho/Cr (Lu et al., 1996) in HIV+ children than controls. However, other studies found higher basal ganglia Cho/Cr (Ashby et al., 2015) and Ins/Cr (Ashby et al., 2015) in HIV+ children, as well as higher absolute Cho (Mbugua et al., 2016) in HIV+ children who started ART before 12 weeks compared to children who initiated treatment later and a predominantly HEU control group.
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review
| 99.5 |
Although earlier studies do not report ART use (Pavlakis et al., 1995; Lu et al., 1996), basal ganglia neurometabolite alterations reported subsequently were observed even in HIV+ children on ART (Keller et al., 2004; Gabis et al., 2006; Ashby et al., 2015; Mbugua et al., 2016). However, these studies used relatively small sample sizes (between 8 and 45 HIV+ subjects) spanning a wide age range and starting ART at varying stages of childhood. One study examined basal ganglia neurometabolites in young children who all initiated ART before 18 months of age (Mbugua et al., 2016). In that study, lower CD4/CD8 ratio at enrollment, aged 6–8 weeks, was associated with lower basal ganglia NAA and Cho at age 5 years, despite early ART initiation and viral load suppression.
|
review
| 99.3 |
The aim of this study was to investigate whether immune system impairment in infancy remains associated with lower NAA and Cho in the basal ganglia at age 7 and at age 9 in an expanded group from the same cohort studied by Mbugua et al. (2016). Because increased basal ganglia Cho and NAA in HIV+ children who initiated ART before 12 weeks was attributed to the use of primarily HEU controls (Mbugua et al., 2016), we aimed to compare HIV+ children to HIV-unexposed controls. In addition, although some studies report neurocognitive deficits in HEU children (Van Rie et al., 2008; Kerr et al., 2014), none have investigated the effects of perinatal HIV and ART exposure on neurometabolite levels in childhood. Our second aim therefore, was to investigate differences in basal ganglia neurometabolite levels between HEU and HIV-unexposed children (controls) at ages 7 and 9.
|
study
| 100.0 |
Participants included 78 HIV+ children from the Children with HIV Early Antiretroviral therapy (CHER) trial (Violari et al., 2008; Cotton et al., 2013) and 53 HIV-uninfected (HIV-) children from the same community enrolled in a longitudinal neuroimaging study (Holmes et al., 2017) in Cape Town, South Africa.
|
study
| 99.94 |
On the CHER trial, infants with CD4+ percentage (CD4%) of at least 25% were randomized to begin ART early (before 12 weeks of age) and have treatment interrupted after either 40 or 96 weeks, or to have ART deferred until CD4% < 20% (25% in the first year) or on presentation of clinical symptoms of disease. A small group with CD4% < 25% were randomized to the early treatment arms only.
|
other
| 99.9 |
The first line ART regimen consisted of Zidovudine (ZDV) + Lamivudine (3TC) + Lopinavir-Ritonavir (LPV/r, Kaletra®) (Violari et al., 2008; Cotton et al., 2013). All HIV+ children started ART before 76 weeks of age and received regular clinical and immunological follow-up. All but nine children had plasma HIV RNA below detectable limits (<400 RNA copies/mL) by 2 years of age.
|
other
| 97.3 |
The HIV- children, comprising both unexposed (control) children born to HIV-seronegative mothers (N = 32) and HEU children (N = 21) born to HIV+ mothers, were recruited from a linked vaccine trial (Madhi et al., 2010). HEU children were exposed to treatment for prevention of mother-to-child transmission (PMTCT), mostly zidovudine antenatally from 28 to 34 weeks and a single dose of nevirapine (NVP) to the mother and zidovudine for a week and a single dose of NVP to the infant.
|
study
| 99.94 |
Neuroimaging was performed without sedation according to protocols approved by the Human Research Ethics Committees of the Universities of Cape Town and Stellenbosch. Parents/guardians provided written informed consent and children provided oral assent. A senior radiologist reviewed all structural scans, and children with abnormalities were excluded from analysis.
|
other
| 99.9 |
On both scanners, the protocol included a high-resolution T1-weighted multiecho magnetisation prepared rapid gradient echo acquisition (MEMPRAGE; Van der Kouwe et al., 2008) FOV 224 mm × 224 mm, TR 2530 ms, TI 1160 ms, TE’s = 1.53/3.19/4.86/6.53 ms, bandwidth 650 Hz/px, 144 slices, 1.3 mm × 1.0 mm × 1.0 mm) and single voxel 1H-MRS (PRESS: 1.5 cm × 1.5 cm × 1.5 cm voxel; TR 2000 ms, TE 30 ms, 64 averages) in the basal ganglia with Chemical Shift Selective (CHESS) water suppression. A water reference was acquired in the same voxel without water suppression. On the Allegra, an EPI volumetric navigated (vNav) PRESS (Hess et al., 2011) sequence was used that applies prospective motion- and shim correction throughout the acquisition and has been shown to provide high quality repeatable spectra in young children (Hess et al., 2013). The basal ganglia voxel comprised approximately 60% gray matter and anatomically represents the frontal limb of the internal capsule and part of the caudate nucleus, putamen, and globus pallidus (representative voxel placement shown in Figure 1). Shimming was performed over the voxel volume, first automatically using the scanner’s “Advanced” adjustment, then manually if necessary to reduce the spectral linewidths reported by the scanner.
|
study
| 100.0 |
LCModel (Provencher, 2001) was used to perform eddy current correction and to calculate metabolite ratios to creatine, as well as absolute concentrations using the water scaling method (Ernst et al., 1993; Kreis et al., 1993). SPM12 software was used to segment the MEMPRAGE regions corresponding to the SVS voxel into gray matter, white matter, and cerebrospinal fluid (CSF) for partial volume correction and water concentration calculation. Spectra were eliminated if the quality was poor (signal-to-noise ratio < 7, line width at half the peak maximum > 0.07 parts per million as reported by LCModel).
|
study
| 100.0 |
Because metabolite levels are scanner-dependent and measured in institutional units, data from scans at different ages were treated independently with separate cross-sectional analyses at each age, to examine differences in Cr, Cho, NAA, Glu, and Ins between HIV+/HEU children and HIV-unexposed controls. Linear regression models in the R programming language (R Core Team, 2013) were used, with age at scan, gender, ethnicity, and voxel gray matter content as confounders. To ensure that results were not driven by influential outliers, all analyses were repeated excluding concentration estimates removed more than 1.5 times the interquartile range from the median value for that metabolite.
|
study
| 100.0 |
At age 7, spectra from 17 HIV+ (38%), 6 HEU (43%), and 7 control children (33%) did not meet the quality control criteria and were excluded. At age 9, data from 1 HIV+ child were excluded. Scans from 37 HIV+ children, 10 HEU children and 13 control children provided data at both 7 and 9 years. Figure 1 shows the voxel placement and example basal ganglia spectra from the Allegra and Skyra scanners.
|
study
| 100.0 |
Demographic data for subjects included at age 7 and age 9 are presented in Table 1. At age 7 there was no difference in birth weight or age between groups. At the 9-year scan there was no difference in birth weight between groups, but the HIV+ children were younger than the control (p = 0.0003) and HEU (p = 0.00004) children.
|
study
| 100.0 |
Clinical data for the HIV+ children are presented in Table 2. Plasma HIV RNA was undetectable (<400 copies/mL) in 91% of the children (all except 4) at the 7-year and 97% (all except 2) at the 9-year scan. At the 7-year scan 3 children had not yet restarted ART after interruption, but by the 9-year scan all children were on ART.
|
study
| 99.94 |
No differences in metabolites relative to the control group were found at age 7 (Table 3). However, at 9 years, both HIV+ and HEU had lower NAA and Glu than control children (Figure 2 and Table 3). HEU children also had lower Cr and Cho than control children.
|
study
| 100.0 |
There was no relationship between CD4% at enrollment and neurometabolites at age 7 or 9 years (Table 4 and Supplementary Table 1). However, at age 7, lower CD4/CD8 ratio at enrollment was associated with lower Cho levels. Also, there was a weak association between lower CD4% at scan and NAA. The same relationships were not evident at age 9, but at this age lower CD4/CD8 at enrollment was associated with lower Ins, and a CDC stage C diagnosis showed a trend-level association with lower NAA and higher Ins. Relationships of CD4/CD8 ratio at enrollment to Cho at ages 7 and 9 are illustrated in Figure 3.
|
study
| 100.0 |
This study presents SVS basal ganglia neurometabolite data in a larger sample of older HIV+ children than previously studied, 91% of whom had undetectable viral loads at 7 and 97% at 9 years, and all of whom had started ART before 76 weeks of age. Although we find no neurometabolite differences at age 7, lower NAA and Glu is apparent at age 9.
|
study
| 100.0 |
Contrary to findings in a subset of the same children at age 5 (Mbugua et al., 2016), we do not find an effect of immune health in infancy on basal ganglia NAA at either 7 or 9 years, suggesting that subsequent health, or other events during childhood, have a stronger influence on neuronal integrity at these older ages. However, as at age 5, higher CD4/CD8 ratio at enrollment remains associated with higher basal ganglia Cho at age 7, suggesting the effect of early immune health on glial cells may persist for some time.
|
study
| 100.0 |
The p-values presented here should, however, be interpreted with caution, as due to the number of statistical tests the family-wise error rate is greater than 5%. Findings significant at p < 0.05 would not have survived correction for multiple comparisons. Although the observed associations may therefore represent type I errors, they nevertheless present a starting point for investigation in future studies.
|
study
| 99.94 |
Similar to previous findings in children without HIVE, at age 7 we find no differences in NAA or other metabolites between HIV+ children and controls. Although NAA/Cr levels have been shown to be decreased in basal ganglia in children with HIVE (Pavlakis et al., 1995; Lu et al., 1996), in HIV+ children without encephalopathy NAA levels have been reported to remain unchanged (Keller et al., 2004). In our own previous study of the same cohort at age 5 years, children who initiated ART before 12 weeks had higher NAA and choline compared to the uninfected group of which 80% were HIV exposed (Mbugua et al., 2016). Notably, at age 9 we find lower NAA and Glu (but not NAA/Cr or Glu/Cr, Supplementary Table 2) in HIV+ children, even though only six children in our 9-year old HIV+ group had a prior HIVE diagnosis.
|
study
| 99.94 |
The reason these differences in NAA and Glu are not observed at age 7 may be that the normal age-related increase in NAA (Holmes et al., 2017) is altered in HIV (Keller et al., 2004), such that NAA is no different from controls at age 7 but has dropped below control levels by age 9. At age 5 (Mbugua et al., 2016), only two of the uninfected children were HIV-unexposed, and the rest HEU, so that we cannot say with any certainty how NAA levels in HIV+ children compared to those of HIV-unexposed children at age 5. This makes it difficult to interpret these findings.
|
study
| 99.94 |
No previous study has reported reduced basal ganglia Glu in HIV+ children; together with reduced NAA this suggests neuronal cell loss. Neurons in the basal ganglia may be more vulnerable than other regions to damage via excitotoxicity because of their greater density of NDMA receptors (Lipton et al., 1991) or because of increased blood–brain barrier damage in these structures associated with high plasma viral load (Avison et al., 2004).
|
study
| 99.94 |
Although in basal ganglia both lower (Lu et al., 1996; Keller et al., 2004) and higher (Ashby et al., 2015) Cho/Cr and Cho have previously been found in HIV+ children, we found no difference in basal ganglia Cho between HIV+ children and controls at either age. In adults, elevated basal ganglia Cho/Cr normalizes after ART (Sailasuta et al., 2012). In these children, ART may have caused normalization of basal ganglia Cho levels, without preventing cell loss. However, the strongest neurometabolite effect observed in regressions with clinical data (B/SE, Table 4), is that of Cho at 7 years against CD4/CD8 at enrollment. This replicates the finding in the same cohort at age 5 (Mbugua et al., 2016), suggesting that better immune health in infancy may be associated with a greater cell density in childhood. Although the regression coefficient for Cho is 20 times smaller by 9 years of age, at 9 years the regression coefficient for Ins against baseline CD4/CD8 is similarly large.
|
study
| 100.0 |
An alternative reason for the lack of group differences at age 7 may be the lower SNR obtained from the single channel head coil on the Allegra scanner, resulting in higher standard deviations for metabolite concentrations estimated with LCModel at age 7, even though spectra still met strict quality control criteria. The standard errors for the regressions at each age were comparable, showing similar between-subject variability on each scanner. However, effect sizes were much larger in the 9 year old data (Table 3), suggesting greater sensitivity to detect differences on the Skyra. It is notable, however, that no group differences were found at age 7 even when regressions were weighted by the inverse of the metabolite standard deviations, to provide heavier weighting to metabolite concentrations estimated with greater precision by LCModel. Moreover, in our study on the same cohort at age 5 years, we were able to detect in data from the same scanner, with similar spectra of similar quality, and in a smaller sample, metabolite differences between HIV+ children initiating ART before and after 12 weeks (Mbugua et al., 2016).
|
study
| 100.0 |
Alterations in NAA and Glu observed at age 9, but not 5 and 7, suggest that basal ganglia neurons may be particularly affected by perinatal HIV/ART and that neuronal damage may be ongoing in this region despite early ART and viral suppression. The results should be considered exploratory, and suggest that longitudinal investigation should be performed to clarify the timing and persistence of these effects during childhood.
|
study
| 99.94 |
Surprisingly, at age 9 we detect reductions in several neurometabolites in HEU children relative to controls. Notably, regression coefficients for NAA, Glu, and Cr suggest larger reductions in HEU than HIV+ children, and a reduction in Cho is seen relative to controls in HEU but not HIV+ children. Although few neuroimaging studies have been done in HEU children, one diffusion tensor imaging (DTI) study in infants identified a region with high fractional anisotropy in the cerebellum (Tran et al., 2016) and another did not detect DTI or brain volume differences between HEU and control children (Jahanshad et al., 2015). Ours is the first to demonstrate neurometabolite alterations related to HIV/ART exposure. One previous study found elevated white matter Cho/Cr and NAA/Cr ratios in neonates exposed to HIV and ART in utero, however, the HIV status of these infants was not determined (Cortey et al., 1994).
|
study
| 99.94 |
Although at age 9 HIV+ children on ART show no difference in Cho level to control children, in HEU children Cho levels are lowered, suggesting that cell density may be reduced. In addition, we found reduced Cr, reflecting lowered energy metabolism, as well as reduced NAA and Glu, possibly reflecting loss of neurons or reduction in normal neurotransmission. Together, these neurometabolite reductions support the suggestion of loss of both neuronal and glial cells.
|
study
| 100.0 |
It is not clear why HIV exposure should be associated with greater basal ganglia neurometabolite reductions than HIV infection. It is likely that in the context of this study HEU children experience some of the same environmental and social stressors as HIV+ children, as well as nutritional and health challenges, but do not have access to the same level of clinical care as the HIV+ children under follow-up on the CHER trial. This may have resulted in poorer neurodevelopmental outcomes for HEU children.
|
study
| 99.94 |
Concern has also been raised about mitochondrial dysfunction in HEU children due to in utero and postpartum exposure to ARVs, particularly Zidovudine (Poirier et al., 2003), which may affect neurodevelopment. Neuroimaging in ART-exposed children without symptoms may show abnormalities similar to those in children with congenital mitochondrial disease (Tardieu et al., 2005). Reassuringly, however, the Surveillance Monitoring for ART Toxicities (SMARTT) cohort of the Pediatric HIV/AIDS Cohort Study, including more than 3500 children, recently found no association between in utero exposure to ART drugs and cognitive or academic scores in school-age children (Van Dyke et al., 2016).
|
review
| 99.5 |
Although most studies in HEU children under 3 years of age found no neurodevelopmental differences to controls when confounding factors were controlled (Alimenti et al., 2006; Gómez et al., 2009; Williams et al., 2010; Springer et al., 2012; Ngoma et al., 2014), studies from resource-limited settings suggest that HEU infants in Africa demonstrate subtle cognitive and motor impairment, and expressive language delay (Boivin et al., 1995; Le Doare et al., 2012).
|
review
| 98.2 |
Similarly, studies of school-aged HEU children demonstrate subtle deficits compared to control children, particularly in language-related cognitive performance (Van Rie et al., 2008; Kerr et al., 2014; Milligan and Cockcroft, 2017). A recent longitudinal study reported that neurodevelopment of HEU children is initially similar to their HIV-unexposed peers, but neurocognitive performance starts to fall behind that of HIV-unexposed children during childhood (Smith et al., 2017). This might correspond to a lack of normal age-related increase in Cho, Glu, and NAA in the basal ganglia during childhood (Holmes et al., 2017), which could explain our observations of reduced levels relative to control children at age 9.
|
study
| 99.94 |
It is, however, notable that a recent longitudinal study in the same cohort showed no differences in linear metabolite change between HEU and controls between 5 and 10 years, using a smaller sample of 9-year-old children scanned on a different scanner a few months earlier (Holmes et al., 2017). A limitation of the current study is that data were acquired on different scanners at each age, which does not allow straightforward examination of change in metabolites with age. The cross-sectional analyses presented here suggest that the neurometabolite increase with age may not be linear in one or both of these groups, with a difference manifesting only at the later end of this age range in the basal ganglia.
|
study
| 100.0 |
Future work should investigate the association of basal ganglia metabolites with cognitive performance. Although the basal ganglia are critically involved in motor control, they are also implicated in language processing (Booth et al., 2007). It would be interesting to determine whether basal ganglia neurometabolite alterations are related to subtle cognitive and language deficits in HEU children.
|
study
| 99.94 |
This study conforms to the ethical guidelines and principles of the international Declaration of Helsinki, and was approved by the Faculty of Health Sciences Human Research Ethics Committees of both the Universities of Cape Town and Stellenbosch. Parents/guardians provided written informed consent and children oral assent.
|
other
| 99.94 |
EM, BL, and AvdK were involved in the study design and acquisition of data. FR, MH, and FL were involved in data and statistical analyses. FR drafted the work and all other authors provided critical revision of the manuscript. FR, MH, EM, BL, ED, and MC provided interpretation of data.
|
other
| 99.94 |
Bullous pemphigoid (BP) is an autoimmune skin disease characterized by the binding of autoantibodies directed against two hemidesmosal proteins of the dermal–epidermal junction, namely BP180 and BP230 (1–6). The disease typically presents in the elderly with a generalized pruritic blistering eruption (7–9), which results from an inflammatory associated disruption of the dermal–epidermal junction induced by the binding of autoantibodies onto their targets. Clinical criteria for typical skin eruption of BP include the absence of associated external mucous membrane (almost exclusively oral) involvement, the absence of skin atrophy, and the absence of head and neck predominant involvement (10). However, clinical presentation of BP can be polymorphic, notably during the early, pre-bullous stage of the disease or in atypical variants, in which full-blown bullous lesions may be absent (11, 12). Beside, blisters and erosions arising on mucosal membranes, mainly within the oral cavity, may be observed in up to 20% of BP patients (6, 11–13), without identified pathophysiologic mechanism(s) associated with their development.
|
review
| 99.9 |
Despite the interest of the research community in always better understanding BP pathophysiology, no study demonstrated whether mucosal involvement occurred as a consequence of BP extent and severity or whether skin and mucosal lesions occurred concomitantly. Recently a clinical activity score named BP Disease Area Index (BPDAI) was proposed as an international consensus to evaluate both the disease extent and the location and type of skin lesions (9). The BPDAI also has the advantage to measure separate scores for skin and mucous membrane activity. Besides, the skin BPDAI score also evaluates separately the ultimate skin lesions, i.e., blisters/erosions, and the early, pre-bullous inflammatory skin lesions, i.e., urticaria/erythema, and their extent as well as the residual damages. Thus, such specific clinical score may be useful to better characterize those BP patients with associated mucosal involvement and improve their monitoring.
|
review
| 99.7 |
Immunological and biological investigations in BP brought strong evidence that autoantibodies to BP180 are pathogenic and play a key role in subepidermal blister formation (14–20). Biopsy specimens from BP lesional skin exhibit dense inflammatory infiltration of eosinophils, basophils, neutrophils, lymphocytes, and mast cells in the dermis (1, 11, 13, 21, 22). Inflammatory cells infiltration and activation release cytokines and proteases that may create an auto-amplification loop reported to induce dermal–epidermal separation (23–27). However, variations in this pathophysiological process are still missing to explain why some BP patients will exhibit mucosal involvement and other not. In this matter, a more precise clinical characterization of BP patients with mucosal involvement may help to point out variations in the autoimmune and inflammatory responses in this particular BP subset.
|
review
| 99.7 |
We here performed a prospective study on series of consecutive BP patients with and without mucosal involvement. To further understand why some BP patients display mucosal blisters or erosions and other not, we compared the total BPDAI and its different components with other clinical and immunological parameters of disease activity at baseline, including the number of daily new blisters and the anti-BP180 and anti-BP230 autoantibody titers.
|
study
| 100.0 |
A prospective, single-center study was conducted in the Dermatology Department of the Reims University Hospital (French Referral Center for Autoimmune Bullous Diseases) between September 2013 and July 2017. The investigation was conducted under the approval of the Ethic Committee of the University Hospital of Reims (CNIL authorization DR-2013-320), and all of the subjects gave their informed and written consent before participating in the study in accordance with the Helsinki Declaration. Consecutive patients with newly diagnosed BP were included in this prospective study. Patients were diagnosed as having BP using the following criteria: clinical features typical of BP with the presence of at least three of four well-established criteria according to Vaillant et al. (10); subepidermal blister on skin biopsy, and deposits of IgG and/or C3 in a linear pattern along the epidermal basement membrane zone by direct immunofluorescence. Non-inclusion criteria were administration of a specific BP treatment for more than 2 days, pregnancy and expected survival after BP diagnosis shorter than 3 months.
|
study
| 99.94 |
Clinical data recorded at baseline were gender, age, number of daily new blisters (determined over a 3-day period), and BPDAI. BPDAI was calculated according to the International Pemphigoid Committee recommendations (9). At baseline, BPDAI was recorded. The total BPDAI computes two scores: total BPDAI activity and total BPDAI damage. The total BPDAI activity score is the arithmetic sum of the three subcomponents—cutaneous blisters/erosions, cutaneous urticaria/erythema, and mucosal blisters/erosions. The total BPDAI damage score is the arithmetic sum of the items rated regionally for damage caused by more permanent features such as post-inflammatory hyperpigmentation, scarring, and other. BPDAI also takes into account lesion number and size thresholds and skin lesions are rated based on the regions affected. Scores can range from 0 to 360 for BPDAI total activity (maximum 240 for total skin activity and 120 for mucosal activity) and 0 to 12 for BPDAI damage, with higher scores indicating greater disease activity or damage. On the basis of previous literature (28), severe BP at baseline was defined as a total BPDAI score ≥56. BP patients who did not fulfill these criteria were classified as having moderate BP.
|
study
| 100.0 |
Anti-BP180 and anti-BP230 autoantibodies were detected in serum and in blister fluids (BFs) using specific commercially available enzyme-linked immunosorbent assays (ELISAs) following the manufacturer’s instructions (MBL, Nagoya, Japan). Anti-BP180 and anti-BP230 antibodies’ detection was routinely performed on classically used dilution with no further dilution when titers were high. ELISA values were expressed as units per milliliters of serum (U/mL), and the 9 U/mL cut-off value recommended by the manufacturer was used in both anti-BP180 and anti-BP230 ELISAs (29–32).
|
study
| 100.0 |
Quantitative variables were described as mean ± SD and qualitative data as number and percentage. Kolmogorov–Smirnov test was performed to compare BP population distribution. The Kolmogorov–Smirnov test works by comparing the cumulative distribution of the two data sets and computes a P value that depends on the largest discrepancy between distributions. In contrast to Mann–Whitney test that is mostly sensitive to changes in the median, the Kolmogorov–Smirnov test is sensitive to changes in the shape, spread, or median of the two distributions. Chi2 tests were used for comparison between qualitative variables and nonparametric, Mann–Whitney tests for comparison between quantitative values. Owing to absence of normal distribution, comparisons between groups were performed using Spearman’s correlation coefficient to explore the relationship between continuous variables (BPDAI with immunological parameters, immunological parameters in serum with those in BF). A P value <0.05 was considered statistically significant. Multivariate analyses were then performed using stepwise logistic regressions, with enter and removal limits set at 0.20 and factors significant at P = 0.20 included.
|
study
| 100.0 |
A total of 97 patients with newly diagnosed BP were included in this study, 79 had typical clinical manifestations of BP and 18 presented with mucosal lesions in additional to skin blisters or erosions. In those BP patients with mucosal involvement, indirect immunofluorescence on salt-split skin was either positive with labeling of the epidermal side of the cleavage or negative. Using the same technique, two patients with positive immunolabeling on the dermal side only were subsequently excluded from the group with typical clinical BP presentation.
|
study
| 100.0 |
The clinical characteristics of those 77 patients with typical BP features (81%, 51 women and 26 men) and of the 18 BP patients with mucosal involvement (19%, 11 women and 7 men) are shown in Table 1. In the 18 BP patients with unconventional mucosal involvement, oral mucosal lesions were observed in 17 cases and anogenital mucosae lesions in 1 case. Among the 17 patients with oral lesions, 1 patient also displayed anogenital mucosae involvement, and another 1 had concomitant pharyngeal mucosa lesions. In those 18 BP patients, neither conjunctival nor nasal mucosa involvement was observed. Occurrence of mucosal lesions was correlated neither with the gender (P = 0.78) nor with the age (P = 0.24) of patients.
|
study
| 99.94 |
At the time of diagnosis, the number of patients with at least 10 daily new blisters was significantly higher in BP patients with mucosal involvement (P = 0.02) compared with patients with typical BP (Table 1), whereas the number of daily new blisters only tended to be higher in BP patients with mucosal involvement (P = 0.07).
|
study
| 99.94 |
Total BPDAI mean score was 39.6 ± 27 at baseline in the whole BP population (Table 1). Based on a cut-off BPDAI value of 56 (see Materials and Methods), the percentage of BP patients with a severe disease was significantly lower in BP patients without mucosal involvement (20.8%) as compared to those with mucosal involvement (44.4%) (Table 1; P = 0.04). Deeper analysis showed that compared with BP patients with a typical disease, BP patients with mucosal involvement displayed a higher total BPDAI score (Table 1; P = 0.008), but also higher skin BPDAI and blister/erosion BPDAI scores (Table 1; P = 0.021 and P = 0.001, respectively). By contrast, the erythema/urticaria BPDAI score was not different between those two groups of patients (Table 1; P = 0.91).
|
study
| 100.0 |
Analysis of the whole BP population distribution according to BPDAI score revealed that most patients had a mild to moderate disease and that the higher the BPDAI was, the lower the number of BP patients was (Figure 1A). Such a distribution was also observed for BP patients without mucosal involvement (Figure 1B), but was significantly different in BP patients with mucosal involvement (D = 0.6154; P = 0.008). Indeed, BP patients with mucosal involvement were evenly distributed from moderate to severe disease (Figure 1C). To further understand these discrepancies, the same analysis was then performed using the separate skin activity. The relative distribution according to the erythema/urticaria BPDAI score of BP patients without mucosal involvement superposed with the one of BP patients with mucosal involvement (D = 0.1667; P = 0.991) (Figure 2A). Analysis of the distribution according to the blisters/erosions BPDAI revealed a high percentage of patients with mucosal involvement when the BPDAI values increased and a low percentage of those patients when the BPDAI values were low (D = 0.33; P = 0.433) (Figure 2B).
|
study
| 100.0 |
Distribution of the whole bullous pemphigoid (BP) patients (n = 95) (A) and of BP patients without (n = 77) (B) and with (n = 18) (C) mucosal involvement according to total BP Disease Area Index (BPDAI) values at the time of diagnosis. In the whole BP group and in the subgroup of BP without mucosal involvement, the number of BP included decrease when the values of the BPDAI increased, whereas BP patients with mucosal involvement were evenly distributed. Comparison of the cumulative distribution by the Kolmogorov–Smirnov test showed significant difference between BP patients with (C) and without (B) mucosal involvement (D = 0.6154; P = 0.008).
|
study
| 100.0 |
Distribution of both bullous pemphigoid (BP) patients with (white column) and without (gray column) mucosal involvement according to erythema/urticaria BP Disease Area Index (BPDAI) values (A) and blisters/erosions BPDAI values (B) at the time of diagnosis. No statistical differences were observed between the distributions of these two BP groups according to the Kolmogorov–Smirnov test, although a clear different distribution could be visualized within the low (0–19) and within the high (50–79) values of the blisters/erosions BPDAI values (B).
|
study
| 100.0 |
In the aim to analyze the clinical features through the BPDAI scores with regards to the autoimmune response, we first measured anti-BP180 and anti-BP230 autoantibody concentrations in the BF of patients with BP. Anti-BP180 and anti-BP230 antibodies were detected in the BF of 32 (86.5%) and 16 (43.2%) patients with a mean titer of 79.4 ± 51.2 and 31.2 ± 40.3 U/mL, respectively (Table 2A). Subgroup analysis of BP patients with and without mucosal involvement showed no significant difference both within the number of BP patients who produced these antibodies and within the mean BF titer related to each antibody. We then investigated whether serum autoantibody concentrations reflected those measured in the BF of the same patients to seek a relationship between BPDAI scores and autoantibody production in the whole BP patients included. Anti-BP180 and anti-BP230 antibodies titers within the BF were highly and significantly correlated with those measured in the serum (r = 0.78 and r = 0.73; P < 0.0001, respectively) (Table 2B). Such a correlation was also found for the anti-BP180 antibody titers within the two subsets of patients with or without mucosal involvement (r = 0.74 and r = 0.89; P < 0.0001 and P = 0.01, respectively) (Table 2B). Anti-BP230 antibody titers in BF from typical BP patients were highly and significantly correlated with those measured in the serum of these patients (r = 0.74; P < 0.0001), whereas only a high tendency was found for BP patients with mucosal involvement (r = 0.75; P = 0.07) (Table 2B).
|
study
| 100.0 |
Subsets of BP patients with or without mucosal involvement were compared and P-value was calculated using Chi2 tests for comparison between qualitative variables and nonparametric, Mann–Whitney tests for comparison between quantitative values. Spearman’s correlation coefficient was used to explore the relationship between immunological parameters in serum with those in BF.
|
study
| 100.0 |
Anti-BP180 antibodies were found in the serum of 77 patients (81.9%) with a mean titer of 63.9 ± 50.8 U/mL (Table 3). Subgroup analysis of BP patients with and without mucosal involvement showed no significant difference both within the number of BP patients who produced anti-BP180 antibodies and within the mean serum titer for this autoantibody (Table 3). In BP patients without mucosal involvement at baseline, serum anti-BP180 ELISA values were significantly correlated with all BPDAI scores except mucosal BPDAI, i.e., total, skin, blister/erosion, and erythema/urticaria BPDAI scores (P < 0.0001, Figures 3A–D, Table 4A).
|
study
| 100.0 |
Correlation of serum anti-BP180 NC16A titers with total BP Disease Area Index (BPDAI) (A,E), total skin BPDAI (B,F), blister/erosion BPDAI (C,G), and erythema/urticaria BPDAI (D,H) scores in bullous pemphigoid patients without (A–D) or with (E–H) mucosal involvement. The correlation coefficients and statistical significances were calculated according to Spearman’s correlation test and are summarized in Table 4.
|
study
| 100.0 |
In BP patients with mucosal involvement at baseline, anti-BP180 ELISA values were only correlated with the erythema/urticaria BPDAI score (P = 0.03). Within this subgroup of BP patients, only a correlation tendency was observed with the total BPDAI score (P = 0.09) and the skin BPDAI score (P = 0.06), whereas no correlation could be drawn with the blister/erosion BPDAI score (P = 0.30) (Figures 3E–H, Table 4A). Finally, no correlation was found between the mucous membrane part of BPDAI score and the anti-BP180-NC16A ELISA values.
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study
| 100.0 |
Anti-BP230 antibodies were found in the serum of 44 BP patients (47.3%) with a mean titer of 29.1 ± 37 U/mL (Table 3). Interestingly, 39 BP patients without mucosal involvement (52%) but only 5 among BP patients with mucosal involvement (27.8%) had a positive anti-BP230 ELISA value (P = 0.06). Such a difference tendency was also observed when analyzing the anti-BP230 mean titer (32.2 ± 37.8 and 16.2 ± 30.9 U/mL; P = 0.07, respectively) (Table 3). At baseline, anti-BP230 antibody serum concentrations were correlated with none of the BPDAI scores within these two groups (Table 4B).
|
study
| 100.0 |
In univariate analysis, clinical features associated with mucosal involvement at baseline were a number of daily new blisters >10 (P = 0.02), a higher total, skin and blister/erosion BPDAI (P = 0.004, P = 0.02, P = 0.001, respectively), and a severe disease according BPDAI (P = 0.04). The absence of anti-BP230 autoantibody also showed a tendency of association with a mucosal involvement (P = 0.08). By contrast, univariate analysis revealed no relationship between anti-BP180 autoantibody serum concentration and mucosal involvement. In multivariate analysis, the absence of serum anti-BP230 autoantibody was the only factor independently associated with mucosal involvement (OR 7.8; 95% CI, 3.1–19.6) (P < 0.0001).
|
study
| 100.0 |
This study is the first one to compare at baseline the clinical characteristics by means of the BPDAI score along with the concentrations of anti-BP180 and anti-BP230 antibodies in BP patients with and without mucosal involvement. Analysis of separate and independent BPDAI sub-scores highlighted that skin lesions, more specifically blisters, and erosions were more substantial in patients with mucosal involvement than in typical BP. Such clinical characteristics were associated with the absence of anti-BP230 autoantibody in the serum of BP patients with mucosal involvement. Furthermore, we showed that mucosal lesions are clinically mainly related to disease severity, but not only. Altogether, our results suggest that compared to the classical pathophysiological processes previously defined in BP (12, 16, 17, 24, 33–36), specific immunological mechanisms should be triggered to end with mucosal lesions.
|
study
| 99.94 |
In BP patients with mucosal involvement, skin and mucosal lesions are likely to concomitantly occur during the disease process. Although our results showed that mucosal involvement in BP was associated with disease severity, we also observed mucosal involvement in BP patient with mild disease. Actually, there was a broad distribution of total BPDAI score among BP patients with mucosal involvement, but the percentage of patients with mucosal involvement increased along with the BPDAI values (Figure 1C). This suggests that mucous membrane involvement in patients with mild BP is not just a consequence of the extent or severity of the disease, and therefore that some BP patients are more prone to develop mucosal lesions than others. Thus, we can speculate that specific biological/immunological mechanisms responsible for these lesions are activated in those BP patients. This further raised the question of the event on which a break of tolerance outside of the skin tissue could originate. Occurrence of mucosal lesions in some patients with BP recalls the variation in the number of sites involved in patients with mucous membrane pemphigoid (37). Previous study suggested that regulatory T (Treg) cells play an indispensable role in maintaining self-tolerance in BP leading to an increase in autoreactive Th2, Th1, and B cells that can recognize different domains of BP180 (38–40). The role of Treg cells in pemphigoid diseases was further demonstrated by mean of mice models (41). Although Treg cells account for less than 10% of peripheral CD4+ T cells, recent studies have reported that tissue resident Treg cells can even control non-immunological processes. Both Treg types demonstrated plasticity and can convert to Th17 cells according to the cytokine environment (42). In this line, we and other groups demonstrated the presence of cytokines such as IL-1, IL-6, IL-23, TGFβ, etc, in BP skin lesions. We also demonstrated that the cytokines panel more likely originated from BP epiphenomenon that still remained to be elucidated (26). Such variations could account for variations in a deficit in Treg cells and favor the development of tissue damages. In turn, skin remodeling could impact on both the number and the Treg homing at different levels of the skin compartments. Although aging has been associated with a loss of central immune tolerance in elderly patients as in BP (43), further investigations are now required to determine the events specifically responsible for the mucosal involvement in patients with BP, especially of the oral cavity.
|
study
| 99.94 |
Although the analysis of skin BPDAI highlighted an increase in the blisters/erosions activity in BP patients with mucosal involvement, serum titers of autoantibodies directed against BP180-NC16A, the dominant autoantigen in BP, were not different between patients with and without mucosal involvement. Of note, Hofmann et al. showed that the presence of autoantibodies against both the BP180 N- and C-terminal of the ectodomain was associated with the presence of mucosal lesions (44). Then, Di Zenzo et al. (21) and Mariotti et al. (45) found that autoantibody reactivity against three extracellular epitopes of BP180 (NC16A, AA 1,080–1,107 and AA 1,331–1,404) appeared to be related to the presence of both skin and mucosal involvement in BP patients. By contrast, in our study, the uni- and multivariate analysis highlighted that the absence of anti-BP230 antibody was the only factor independently associated with mucosal involvement in BP. This unconventional result could imply that the immune response may be shifted toward other autoantibodies in those BP patients with mucosal involvement which could be related to a lower threshold of tolerance breakdown in line with a reduced level of Treg cells or of Treg homing as mentioned above. Based on a recent publication (20), we can hypothesize that the loss of immune tolerance curbs both the innate and the adaptive immune responses, which leads to the release of a specific panel of proteases associated with blister formation and tissue remodeling, and subsequently to a shift of the epitope targeted as compared with the classical pathophysiological mechanisms associated with BP. Altogether, this could be reflected by a reduced activity of the adaptive immune response toward the hemidesmosomal proteins of the basal keratinocyte layer and an increased activity toward the antigenic cryptic domains of molecules involved in the anchoring of those basal keratinocyte to the basal membrane structure. To address those mechanisms, it would be relevant to compare in future prospective studies the number and the activity of Treg in biopsy specimens from either skin or mucosa BP lesions with others samples collected from patients with inflammatory non-autoimmune diseases.
|
study
| 99.94 |
The presence of other autoantibodies may interfere with the classical pathophysiological process of BP (12, 16, 17, 24, 33–36, 46). In BP patients without mucosal involvement, the serum concentration of anti-BP180 NC16A autoantibodies was well correlated with both the skin erythema/urticaria and the blisters/erosions BPDAI scores, which is in line with the fact that anti-BP180 autoantibody binding onto their target induced an inflammatory response leading to blister formation (14–18). By contrast, the correlation between anti-BP180 autoantibody titer and the skin blisters/erosions BPDAI score was not evidenced for BP patients with mucosal involvement. Of note, the BPDAI investigated the presence of both blisters and erosions. Thus, considering that the number of daily new blisters only displayed a higher tendency in BP patients with mucosal involvement, the highly significant difference in the BPDAI scores associated to blisters and erosions lesions may be related to the presence of erosions rather than to new blisters. This implies that the mechanisms leading to skin blisters/erosions and maybe to mucosal involvement too, rely on either different or additional trigger than anti-BP180-NC16A autoantibodies in BP patients with mucosal lesion. It was proposed that differential epitope recognition of BP180 could be associated with distinct clinical severity (5). However, additional studies are necessary to define the panel of autoantibodies associated with BP with mucosal lesions, to decipher the pathogenic role of these autoantibodies as well the immunologic mechanisms associated to their production.
|
study
| 99.94 |
Several limitations of the study are to be acknowledged, mainly its relatively limited number of BP patients with mucosal involvement. However, the prospective collection of clinical data including BPDAI assessment allowed a good evaluation of the percentage of BP patients with mucosal involvement, which was very close to results from previous large series of BP patients (5, 6, 21, 32, 44, 45) and from a recent retrospective study performed in our dermatology department (manuscript under consideration). Another weakness is the lack of investigations regarding biopsy specimens or BFs from oral mucous membrane for pathophysiological purpose. Unfortunately, recent intact oral blisters were rare and too transient for an effective, systematic sampling. To avoid biases, our present results were based on clinical or biological data which could systematically be collected in all consecutive patients. Although we observed that mucosal involvement also occurred in patients with mild or moderate disease, we here showed that mucosal involvement was more frequent in patients with severe BP according to the BPDAI score. Actually, in those severe BP patients, mucosal involvement could represent a severity criterion but not a clinical atypia. Considering all elements discussed above, further investigations should help defining the pathophysiological particularities of severe BP patients with mucosal involvement. Finally, in patients with moderate or minimal BP disease, other diagnoses could be considered, including anti-P200 pemphigoid and epidermolysis bullosa acquisita, which was very unlikely in our present series since indirect immunofluorescence on salt-split skin was either positive with labeling of the epidermal side of the cleavage or negative. The latter BP patients may represent an interesting subset of patients to identify in future studies the specific mechanisms responsible for mucous membrane lesions with the ultimate goal to propose innovative targeted therapy to preserve the quality of life for those elderly fragile patients.
|
study
| 99.94 |
This study was carried out in accordance with the recommendations of the “Commission Nationale de l’Informatique et des libertés (CNIL, authorization DR-2013-320)” and approved by the Ethic Committee of the University Hospital of Reims. All subjects gave written informed consent in accordance with the Declaration of Helsinki.
|
other
| 99.94 |
Since 2003, multiple highly pathogenic avian influenza A (HPAI) H5 subtypes, including H5N1, H5N2, H5N6, and H5N8, have generated severe epidemics and thus not only tremendous economic losses in the domestic poultry industry, but also serious threats to human health worldwide (Jhung and Nelson, 2015). As of October 3, 2016, at least 856 cases of human infection with avian influenza A (H5N1) virus in 16 countries had been reported to the World Health Organization, among which 452 had ended in death, for an apparent case fatality rate of 52.8% (WHO, 2016). As the natural reservoir for avian influenza viruses (AIVs), wild bird populations can be infected by many such viruses, including the H3, H5, and H7 subtypes AIVs, and thus play a critical role in AIV epidemiology and ecology (Claes et al., 2016; Dhingra et al., 2016; Kang et al., 2017). Thus far, results of the phylogenetic analysis of the hemagglutinin (HA) gene have revealed multiple clades and subclades of H5 subtype AIVs. Among them, H5N6 has replaced H5N1 as the dominant subtype in southern China (Bi et al., 2016a), while clade 2.3.4.4 of AIVs is now considered to be the dominant in China (Saito et al., 2015; Claes et al., 2016). Given recent suggestions that clade 2.3.4.4 of AIVs has become increasingly pathogenic to domestic poultry and wild birds (Claes et al., 2016; Sun et al., 2016), AIV virulence is likely affects multiple factors and depends upon both antigenic drift and the AIV-infected strain in the host immunity (Tscherne and Garcia-Sastre, 2011).
|
review
| 99.7 |
An AIV replicates primarily in the respiratory system (Sturm-Ramirez et al., 2004), from where it spreads to the brain and lymphoid tissues by way of infection. Such infection induces batteries of receptors and triggers a signaling cascade that ultimately activates the host’s immune response. As part of that process, for example, the endosomal Toll-like receptor (TLR) 3 and sphingosine-1-phosphate-1 receptor (S1PR1) recognize double-stranded viral RNA released during the uncoating of an internalized virus (Barton, 2007; Teijaro et al., 2011). During AIV infection in mammals, the endosomal TLR 7/8, which recognizes single-stranded viral RNA, can prompt the production of interferon (IFN)-α and IFN-β (Diebold et al., 2004). As MacDonald et al. (2008) have shown, when TLR7/8 are activated by AIV infection in host cells, the recognition of viral RNA results in the secretion of proinflammatory cytokines (e.g., IL-1β, IL-6) and antiviral cytokines (e.g., IFNs). By extension, the expression of proinflammatory cytokines and IFNs influences both viral clearance and the manifestation of clinical symptoms. At the same time, since major histocompatibility complex (MHC) classes I and II antigen presentation molecules used for AIV uptake activated cellular immunity and humoral immunity of B cells (i.e., IFN-γ) and T cells (i.e., CD3+, CD4+, CD8+), MHC molecules likely play a role in activating host innate immune response to AIV infection (Gromme and Neefjes, 2002; Williams et al., 2002).
|
study
| 99.8 |
In China’s Sichuan Province on May 7, 2014, the first-ever fatal case of human infection by a reassortant H5N6 AIV involved a 49-year-old man with a history of exposure to live poultry. To date, 14 additional cases of human infection with the H5N6 virus in China’s Sichuan, Guangdong, Jiangxi, and Yunnan Provinces—10 of which ended in death—documented by the World Health Organization and World Organisation for Animal Health were characterized as posing a potential risk to public health1.
|
other
| 99.5 |
In studies conducted during 2014–2015, we performed epidemic surveillance of AIVs among wild birds at nature reserves in southern China, isolated two novel reassortant HPAI H5N6 viruses, and conducted genetic and phylogenetic analyses to elucidate their molecular features (Kang et al., 2017). By extension, in the present study, we investigated the pathogenicity and transmissibility of the viruses in chickens. In addition, to assess the role of the host innate immune response of H5N6-infected chickens, we examined a complex expression profile of pattern recognition receptors (PRRs), proinflammatory cytokines, chemokines, and MHC molecules in the brain, lung, spleen, and bursa of Fabricius.
|
study
| 100.0 |
All animal experiments were conducted in ABSL-3 facilities and in accordance with the guidelines of South China Agricultural University’s Institutional Animal Care and Use Committee. All animal protocols were approved by the Committee on the Ethics of Animal Experiments of the ABSL-3 Committee of South China Agricultural University (approval no. L102012017001K).
|
other
| 99.94 |
Two H5N6 viruses—namely, A/oriental magpie-robin/Guangdong/SW8/2014 (SW8) and A/Pallas’s sandgrouse/Guangdong/ZH283/2015 (ZH283)—used in this study were grown and purified three times in Madin–Darby canine kidney cells by standard plaque assay. The stocks of H5N6 viruses were propagated in 9-day-old specific pathogen-free (SPF) chicken eggs at 37°C for 72 h per the procedure (Yuan et al., 2014). Allantoic fluid pooled from multiple eggs was taken for centrifugation for 2 min at 8,000 rpm, from which the supernatant was harvested and subsequently frozen in aliquots at -80°C for further characterization. The 50% egg infectious dose (EID50) titer for egg-grown virus was determined by 10-fold serial dilutions and the titration of each virus in 9-day-old SPF eggs using Reed and Muench’s (1938) method. Six-week-old SPF white leghorn chickens (Guangdong Wens Dahuanong Biotechnology Co., Ltd, Yunfu, China) were held in isolator cages with a feeding space of 117 m3 throughout the duration of each experiment.
|
study
| 100.0 |
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