Expression of PD-1 mitigates phagocytic activities TAM in osteosarcoma.

The high expression of programmed death 1 (PD-1) is a hallmark of T cell exhaustion, consequently inhibiting the anti-tumor immunity, tumor-associated macrophages (TAMs) aggravate Osteosarcoma (OS) progression. However, PD-1 expression on TAMs in OS metastasis remains unclear. Here, we used scRNA-Seq of 15500 individual cells from human OS lung metastatic lesion, identified thirteen major cell clusters. Our data revealed that tumor-infiltrating lymphocytes (TILs) OS lung metastatic accompanied by accumulation of exhausted T cells and regulatory T cells (Tregs). CD3+ T cells from human OS lung metastatic exhibited lower proliferation than in primary tissue. Importantly, TAMs mainly comprise immunosuppressive M2 phenotype in OS metastasis. Mechanistically, we found that PD-1 of TAMs inhibits the phagocytic potency, further promoting the progression of OS metastasis. Therefore, the study provides a strong technical support for OS immunotherapy based on PD-1 inhibitors.

AhR may be involved in Th17 cell differentiation in chronic hepatitis B.

Th17 cells which are crucial for host immunity have been demonstrated to increase HBV infection. However, the mechanism of the Th17 cell increase is unknown. Hence, the mechanism of Th17 cell enhancement is important to provide a theoretical foundation for chronic hepatitis B immunotherapy. This study included 15 instances in the healthy control (HC) and 15 cohorts in the chronic hepatitis B (CHB). Their CD4+ T cells were isolated from their peripheral blood and then subjected to RNA transcriptome sequencing. Then, to identify target genes linked to Th17-cell differentiation, DEGs associated with CHB were convergent with the Th17-cell-associated genes from the KEGG database. Hub genes of DEG and target genes linked to Th17 cells were analysed for correlation. The AhR-related genes were located using the GeneMANIA database. To analyse the function of the genes, GO and KEGG pathways were employed. Protein-protein interaction network analysis employed the Metascape, STRING and Cytoscape databases. Finally, Western blotting and RT-qPCR were used to validate AhR. A total of 348 differential genes were identified in CHB patients. CytoHubba was used for screening five hub genes associated with CHB: CXCL10, RACGAP1, TPX2, FN1 and GZMA. This study aimed to determine the mechanism of elevated Th17 cells in CHB. As a result, further investigation using the convergence of DGEs and Th17 cell-related genes identified three target genes: AhR, HLA-DQA1 and HLA-DQB1, all of which were elevated in CHB. The three genes were primarily involved in immune response-related processes, according to the GO enrichment analysis. Correlation analysis of CXCL10, RACGAP1, TPX2, FN1 and GZMA genes with AhR, HLA-DQA1 and HLA-DQB1 revealed that AhR was positively associated with CXCL10 and GZMA genes, which best respond to the severity of CHB disease. Combined with the role of AhR in Th17 cell differentiation, the genes AhR was chosen for confirmation by RT-qPCR and WB in this study. The results showed that the CHB group had higher expression levels of AhR at both RT-qPCR and WB levels. Furthermore, this study's findings revealed that AhR may contribute to the development of CHB by affecting the differentiation of Th17 cells.

m6A Regulator-mediated RNA Methylation Modulates Immune Microenvironment of Hepatitis B Virus-related Acute Liver Failure.

Acute liver failure (ALF) is a rare and complicated disease with a high mortality rate. Emergency liver transplantation is the only treatment method that can improve the ALF prognosis. However, its clinical application remains limited owing to the aggressive nature of liver transplantation, limited donors, and high postoperative mortality. The study investigated the effect of m6A on the immune microenvironment of hepatitis B virus-related ALF (HBV-ALF). In this study, the gene expression data of 47 normal people and 42 HBV-ALF patients were downloaded from the Gene Expression Omnibu (GEO) database. The known 23 m6A regulators which mediated RNA modification patterns were compared and analyzed in these two groups, and the gene diagnosis model of HBV-ALF patients was established based on the analysis results. In addition, we used unsupervised clustering to identify different m6A RNA methylation modification patterns in HBV-ALF based on m6A regulators, and evaluated the immune infiltration and biological differences in these subtypes. In addition, the relationship between m6A genes and immune cell activation in HBV-ACLF patients was explored by immune infiltration analysis. Nineteen m6A regulators mediated RNA methylation (m6A regulators for short) were differentially expressed in HBV-ALF and control groups. m6A regulators could well distinguish control samples from HBV-ALF samples, and m6A regulators might be used as a basis for diagnosing HBV-ALF patients. Immune cells such as activated CD8 T cells, activated B cells, and activated CD4 T cells might play important roles in HBV-ALF, and m6A regulators were closely associated with immune cell infiltration. ALKBH15, CBLL1, IGF2BP2, IGF2BP3, and ZC3H13 were significantly associated with immune cells. Considering 23 m6A regulators, HBV-ALF patients could be classified into two subtypes (cluster 1 and cluster 2) based on different immune cell infiltration. m6A regulators of the IGFBP and YHDF families have extremely different levels in these two subtypes. Differential immune cell infiltration among these subtypes was observed, a total of 913 differentially expressed genes among different m6A modification patterns was identified, and their biological functions were explored. m6A modification might play a crucial role in the diverse and complex immune microenvironment of HBV-ALF patients.

Risk of hemolysis in Plasmodium vivax malaria patients receiving standard primaquine treatment in a population with high prevalence of G6PD deficiency.

BACKGROUND: Primaquine is essential for the radical cure of Plasmodium vivax malaria, but it poses a potential danger of severe hemolysis in G6PD-deficient (G6PDd) patients. This study aimed to determine whether primaquine is safe in a population with high G6PD prevalence but lacking G6PD diagnosis capacity. METHODS: In Myanmar, 152 vivax patients were gender- and age-matched at 1:3 for G6PDd versus G6PD-normal (G6PDn). Their risk of acute hemolysis was followed for 28 days after treatment with the standard chloroquine and 14-day primaquine (0.25 mg/kg/day) regimen. RESULTS: Patients anemic and non-anemic at enrollment showed a rising and declining trend in the mean hemoglobin level, respectively. In males, the G6PDd group showed substantially larger magnitudes of hemoglobin reduction and lower hemoglobin nadir levels than the G6PDn group, but this trend was not evident in females. Almost 1/3 of the patients experienced clinically concerning declines in hemoglobin, with five requiring blood transfusion. CONCLUSIONS: The standard 14-day primaquine regimen carries a significant risk of acute hemolytic anemia (AHA) in vivax patients without G6PD testing in a population with a high prevalence of G6PD deficiency and anemia. G6PD testing would avoid most of the clinically significant Hb reductions and AHA in male patients.

Extracts of Periplaneta americana alleviate hepatic fibrosis by affecting hepatic TGF-ß and NF-κB expression in rats with pig serum-induced liver fibrosis.

INTRODUCTION: Liver fibrosis is caused by continuous wound healing responses to various harmful stimuli, including viral infection, drugs, alcohol, and autoimmune liver disease. The purpose of this study was to examine the effects of extracts of Periplaneta americana (EPA) in rats with pig serum-induced liver fibrosis to preliminarily assess the antifibrotic effect of EPA. MATERIAL AND METHODS: Seventy rats were randomly divided into 7 groups (10 rats in each group): HC, the healthy control group; FC, the fibrotic control group; TL, low-dose EPA treatment group group; TM, medium-dose EPA group; TH, high-dose EPA treatment group; TC1, Panax notoginseng/Salvia mitiorrhiza treatment control group 1; TC2, colchicine treatment control group 2. TC1 and TC2 were used as the positive control to demonstrate the difference between EPA and the effects of other compounds. The liver fibrosis model was induced by intraperitoneal injection of 0.5 mL pig serum twice a week for 13 weeks in all groups except for the HC group. The hepatic fibrosis model was established at the 7th week, and followingly, the corresponding compounds were administered once a day in all groups for 6 weeks. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity was determined in rat blood serum. We also measured liver fibrosis-related serum markers, including hyaluronic acid (HA), mucin layer (LN), type III pre-collagen (PC-III) and type IV collagen (IV-C). Hematoxylin and eosin (H&E) and Masson stainings were used to assess liver morphology and determine the stage of fibrosis. Immunohistochemistry was used to detect the protein expression of NF-κB, α-smooth muscle actin (α-SMA), transforming growth factor-ß1 (TGF-ß1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in rat liver tissue. RESULTS: Compared with that of the HC group, the liver tissue of the FC group presented obvious liver damage and collagen deposition. The serum levels of ALT, AST, HA, LN, PC-III and IV-C and the expression of NF-κB, α-SMA, TGF-ß1 and TIMP-1 in the FC group were significantly higher than those in the HC group, the EPA treatment groups, the TC1 group and the TC2 group (P < 0.01). The levels of serum ALT, AST, HA, LN, PC-III and IV-C and the expression of α-SMA, NF-κB, TGF-ß1 and TIMP-1 in the TL, TC1 and TC2 groups were significantly higher than those TM and TH groups (P < 0.05). EPA treatment significantly improved liver function, decreased collagen deposition and reversed the pathological changes related to liver fibrosis. CONCLUSIONS: We found that EPA could reduce liver inflammation, suppress liver cell degeneration and necrosis, and reduce the formation of liver fibrous tissue. Its mechanism might be associated with inhibiting the expression of TGF-ß1, TIMP-1, NF-κB and α-SMA to block signal transduction pathways in the hepatic fibrosis process. Therefore, EPA, as a traditional Chinese medicine, might be potentially used to prevent and treat hepatic fibrosis in the future. However, further more experiments are necessary to verify its effectiveness and possible signaling pathways.

Efficacy of directly-observed chloroquine-primaquine treatment for uncomplicated acute Plasmodium vivax malaria in northeast Myanmar: A prospective open-label efficacy trial.

BACKGROUND: Chloroquine (CQ) and primaquine (PQ) remain the frontline drugs for radical cure of uncomplicated P. vivax malaria in the Greater Mekong Sub-region (GMS). Recent reports of decreased susceptibility of P. vivax to CQ in many parts of the GMS raise concerns. METHODS: From April 2014 to September 2016, 281 patients with uncomplicated P. vivax infection attending clinics in border settlements for internally displaced people in northeast Myanmar were recruited into this study. Patients were treated with standard regimen of 3-day CQ and concurrent 14-day PQ (3.5 mg/kg total dose) as directly observed therapy, and followed for recurrent parasitemia within 28 days post-patency. RESULTS: Within the 28-day follow-up period, seven patients developed recurrent parasitemia, resulting in a cumulative rate of parasite recurrence of 2.6%. Five of the seven parasitemias recurred within two weeks, and two of those failed to clear within seven days, indicating high-grade resistance. CONCLUSION: Although failure of CQ/PQ treatment of P. vivax was relatively infrequent in northeast Myanmar, this study nonetheless confirms that CQ/PQ-resistant strains do circulate in this area, some of them of a highly resistant phenotype. It is thus recommended that patients who acquire vivax malaria in Myanmar be treated an artemisinin-combination therapy along with hypnozoitocidal primaquine therapy to achieve radical cure.

Morphological and molecular characteristics of Sarcocystis bertrami from horses and donkeys in China.

While Sarcocystis parasites from the muscles of donkey and horse have been characterized as different species, similarities between the parasites in these host raises questions about this assignment (Levine and Tadros, 1980; Matuschka, 1983; Odening et al., 1995b). To resolve this, we examined the tissue cysts of Sarcocystis collected from donkeys and horses were studied by morphological and molecular methods. Morphological studies performed by light microscopy (LM) revealed that each of two types of cysts were present in samples from each host type. Under LM, villar protrusions (VP) were sometimes observed on the larger (Type I) and smaller (Type II) of these cyst types; when present, these were sometimes short and sometimes long. By electron microscopy (EM), VPs from both horse and donkey cysts were found to share similar structures, appearing to be typical of 'type 11a' VPs found on the Sarcocystis wall of Sarcocystis fayeri as described by Dubey et al., 1977. The VP of cysts in both horses and donkeys contained microtubules extending from the villar tips to the ground substance (GS). Ovoid, osmiophilic bodies (OB) were found along the length of the microtubules within the villi, but this feature was not found in all VP. To understand the phylogeny of the parasites, a portion of the coxI gene was sequenced from 22 isolated cysts (9 from donkeys and 13 from horses). Phylogenetic relationships were reconstructed from these sequences and the closest homologues available in GenBank, revealing that all of the samples, regardless of host origin or morphological appearance under LM, grouped in one clade. Ours is the first attempt to combine morphological measurements with coxI sequences in assessing such equine parasites; the results confirm a close relationship of the parasites from horse and donkey with S. fayeri. Further, the data suggest that the cysts in each host likely belong to the same species. As the first named species was Sarcocystis bertrami, we propose S. bertrami (syn. Sarcocystis fayeri) as the descriptor for this parasite of both horses and donkeys. Ultimately, this finding will only be validated by cross-transmission infection experiments that score the ability of parasite isolates from one Equus to infect the other.

Overview of the improvement of the ring-stage survival assay-a novel phenotypic assay for the detection of artemisinin-resistant Plasmodium falciparum.

Artemisinin resistance in Plasmodium falciparum threatens the remarkable efficacy of artemisinin-based combination therapies worldwide. Thus, greater insight into the resistance mechanism using monitoring tools is essential. The ring-stage survival assay is used for phenotyping artemisinin-resistance or decreased artemisinin sensitivity. Here, we review the progress of this measurement assay and explore its limitations and potential applications.

Th17/Treg imbalance is an indicator of liver cirrhosis process and a risk factor for HCC occurrence in HBV patients.

OBJECTIVE: To determine the impact of T helper 17 cell (Th17)/regulatory T cell (Treg) ratio imbalance on the process and prognosis of hepatitis B virus (HBV)-related liver cirrhosis. METHODS: Patients with HBV who refused to accept any therapy from were recruited from 2009 to 2014 and followed-up to August 2016. Based on a liver stiffness measurement (LSM), the patients were divided into a low LSM group and a high LSM group. After propensity score matching, 150 patients were included. The peripheral blood of all subjects was tested using a flow cytometer to measure the Th17 and Treg cell frequencies and the Th17/Treg ratio. Spearman rank correlation, log-rank test, Cox regression model, and receiver operating characteristic (ROC) curves were applied to analyze the correlation between the Th17/Treg ratio and LSM level and the risk factors of HCC occurrence and to predict the Th17/Treg ratio for the LSM level. RESULTS: Th17 and Treg cell frequencies and the Th17/Treg ratio were significantly different between the low and high LSM groups. The Th17/Treg ratio showed a strong positive association with LSM level (r=0.631, P<0.01). Furthermore, the Th17/Treg ratio was an independent factor influencing the HCC occurrence, which was tested using a Cox regression model. The area under the ROC curve of the Th17/Treg ratio for predicting LSM level was 0.783 (95% CI 0.705-0.861). CONCLUSION: Th17/Treg ratio imbalance is an indicator of the liver cirrhosis process and a risk factor for HCC occurrence in patients infected with HBV.

Seasonal dynamics and microgeographical spatial heterogeneity of malaria along the China-Myanmar border.

Malaria transmission is heterogeneous in the Greater Mekong Subregion with most of the cases occurring along international borders. Knowledge of transmission hotspots is essential for targeted malaria control and elimination in this region. This study aimed to determine the dynamics of malaria transmission and possible existence of transmission hotspots on a microgeographical scale along the China-Myanmar border. Microscopically confirmed clinical malaria cases were recorded in five border villages through a recently established surveillance system between January 2011 and December 2014. A total of 424 clinical cases with confirmed spatial and temporal information were analyzed, of which 330 (77.8%) were Plasmodium vivax and 88 (20.8%) were Plasmodium falciparum, respectively. The P. vivax and P. falciparum case ratio increased dramatically from 2.2 in 2011 to 4.7 in 2014, demonstrating that P. vivax malaria has become the predominant parasite species. Clinical infections showed a strong bimodal seasonality. There were significant differences in monthly average incidence rates among the study villages with rates in a village in China being 3-8 folds lower than those in nearby villages in Myanmar. Spatial analysis revealed the presence of clinical malaria hotspots in four villages. This information on malaria seasonal dynamics and transmission hotspots should be harnessed for planning targeted control.

A more appropriate white blood cell count for estimating malaria parasite density in Plasmodium vivax patients in northeastern Myanmar.

The conventional method of estimating parasite densities employ an assumption of 8000 white blood cells (WBCs)/µl. However, due to leucopenia in malaria patients, this number appears to overestimate parasite densities. In this study, we assessed the accuracy of parasite density estimated using this assumed WBC count in eastern Myanmar, where Plasmodium vivax has become increasingly prevalent. From 256 patients with uncomplicated P. vivax malaria, we estimated parasite density and counted WBCs by using an automated blood cell counter. It was found that WBC counts were not significantly different between patients of different gender, axillary temperature, and body mass index levels, whereas they were significantly different between age groups of patients and the time points of measurement. The median parasite densities calculated with the actual WBC counts (1903/µl) and the assumed WBC count of 8000/µl (2570/µl) were significantly different. We demonstrated that using the assumed WBC count of 8000 cells/µl to estimate parasite densities of P. vivax malaria patients in this area would lead to an overestimation. For P. vivax patients aged five years and older, an assumed WBC count of 5500/µl best estimated parasite densities. This study provides more realistic assumed WBC counts for estimating parasite densities in P. vivax patients from low-endemicity areas of Southeast Asia.

Correction: Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border.

[This corrects the article DOI: 10.1371/journal.pone.0134593.].

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary disease that predisposes red blood cells to oxidative damage. G6PD deficiency is particularly prevalent in historically malaria-endemic areas. Use of primaquine for malaria treatment may result in severe hemolysis in G6PD deficient patients. In this study, we systematically evaluated the prevalence of G6PD deficiency in the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD fluorescent spot test. The overall prevalence of G6PD deficiency in the study population was 29.6% (523/1770), among which 27.9% and 30.6% were males and females, respectively. From these G6PD deficient samples, 198 unrelated individuals (147 females and 51 males) were selected for genotyping at 11 known G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and one in intron 11) using a multiplex SNaPshot assay. Mutations with known association to a deficient phenotype were detected in 43.9% (87/198) of cases, intronic and synonymous mutations were detected alone in 34.8% (69/198) cases and no mutation were found in 21.2% (42/198) cases. Five non-synonymous mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T, and Viangchan 871G>A were detected. Of the 87 cases with known deficient mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations, respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested females and males, respectively (P<0.05). It is noteworthy that 24 subjects carrying the Mahidol mutation and two carrying the Kaiping mutation also carried the 1311C>T/IVS11nt93T>C SNPs. Further studies are needed to determine the enzyme levels of the G6PD deficient people and presence of additional G6PD mutations in the study population.

Therapeutic responses of Plasmodium vivax malaria to chloroquine and primaquine treatment in northeastern Myanmar.

Chloroquine-primaquine (CQ-PQ) continues to be the frontline therapy for radical cure of Plasmodium vivax malaria. Emergence of CQ-resistant (CQR) P. vivax parasites requires a shift to artemisinin combination therapies (ACTs), which imposes a significant financial, logistical, and safety burden. Monitoring the therapeutic efficacy of CQ is thus important. Here, we evaluated the therapeutic efficacy of CQ-PQ for P. vivax malaria in northeast Myanmar. We recruited 587 patients with P. vivax monoinfection attending local malaria clinics during 2012 to 2013. These patients received three daily doses of CQ at a total dose of 24 mg of base/kg of body weight and an 8-day PQ treatment (0.375 mg/kg/day) commencing at the same time as the first CQ dose. Of the 401 patients who finished the 28-day follow-up, the cumulative incidence of recurrent parasitemia was 5.20% (95% confidence interval [CI], 3.04% to 7.36%). Among 361 (61%) patients finishing a 42-day follow-up, the cumulative incidence of recurrent blood-stage infection reached 7.98% (95% CI, 5.20% to 10.76%). The cumulative risk of gametocyte carriage at days 28 and 42 was 2.21% (95% CI, 0.78% to 3.64%) and 3.93% (95% CI, 1.94% to 5.92%), respectively. Interestingly, for all 15 patients with recurrent gametocytemia, this was associated with concurrent asexual stages. Genotyping of recurrent parasites at the merozoite surface protein 3α gene locus from 12 patients with recurrent parasitemia within 28 days revealed that 10 of these were the same genotype as at day 0, suggesting recrudescence or relapse. Similar studies in 70 patients in the same area in 2007 showed no recurrent parasitemias within 28 days. The sensitivity to chloroquine of P. vivax in northeastern Myanmar may be deteriorating.