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Background Alloantibodies directed against antigens of the Kell blood group system are clinically significant. In the Netherlands, the KEL1 antigen is determined in all blood donors. In this study, after phenotyping of KEL:1-positive donors, genotyping analysis was conducted in KEL:1,–2 donors to identify possible KEL*02 variant alleles. Study Design and Methods A total of 407 donors with the KEL:1,–2 phenotype were genotyped for the KEL*01/02 polymorphism, followed by direct sequencing of the KEL gene if the KEL*02 allele was detected. Two K0 patients were also included. Transcript analysis was conducted in two probands with the KEL*02. M05 allele defined by a synonymous mutation (G573G). Flow cytometry analysis to determine the expression of Kell antigen was performed. Results Thirty KEL:1,–2 individuals (30/407, 7.4%) with discrepant KEL*01/02 genotype were identified. Seven novel alleles were identified: KEL*02(R86Q, R281W)mod, KEL*02(L133P)null, KEL*02(436delG)null, KEL*02(F418S)null, KEL*02(R492X)null, KEL*02(L611R)null, and KEL*02(R700X)null. Nine variant alleles described before were detected: KEL*02N.06, KEL*02N.15, KEL*02N.17, KEL*02N.19, KEL*02N.21, KEL*02M.02, KEL*02M.04, KEL*02M.05, and KEL*02(Q362K)mod. A transcript lacking Exon 16 was identified in two probands with the KEL*02M.05 allele as described before. Finally, flow cytometry analysis showed a decreased total Kell expression and a relatively increased KEL1 expression in individuals with the KEL:1,2null or KEL:1,2mod phenotype, compared to KEL:1,2 controls. Conclusion In 7.4% of a group of tested KEL:1,–2 Dutch donors, a KEL*02null or KEL*02mod allele was found. A relatively increased KEL1 antigen expression in KEL:1,2null and KEL:1,2mod individuals suggest that the expression of Kell-XK complexes depends on the availability of the XK protein.

We investigated the migration patterns of hepatitis C virus (HCV) in China. Partial E1 and/or NS5B sequences from 411 volunteer blood donors sampled in 17 provinces and municipalities located in five large regions, the north-northeast, northwest, southwest, central south, and southeast, were characterized. The sequences were classified into eight subtypes (1a,n= 3; 1b,n= 183; 2a,n= 83; 3a,n= 30; 3b,n= 44; 6a,n= 55; 6n,n= 10; 6v,n= 1) and a new subtype candidate. Bayesian evolutionary analysis by sampling trees of the E1 sequences of the five major subtypes revealed distinct migration patterns. Subtype 1b showed four groups: one is prevalent nationwide with possible origins in the north-northeast; two are locally epidemic in the central south and northwest, respectively, and have spread sporadically to other regions; and the fourth one is likely linked to the long-distance dispersion among intravenous drug users from the northwest. Subtype 2a showed two groups: the larger one was mainly restricted to the northwest and seemed to show a trend toward migration via the Silk Road; the smaller one was geographically mixed and may represent descendants of those that spread widely during the contaminated plasma campaign in the 1990s. Subtype 3a exhibited three well-separated geographic groups that may be epidemically unrelated: one showed origins in the northwest, one showed origins in the southwest, and the other showed origins in the central south. In contrast, subtype 3b had a mixture of geographic origins, suggesting migrations from the southwest to the northwest and sporadically to other regions. Structurally resembling the tree for subtype 3a, the tree for subtype 6a showed four groups that may indicate migrations from the central south to southeast, southwest, and northwest. Strikingly, no subtype 6a strain was identified in the north-northeast.IMPORTANCEWith a population of greater than 1.3 billion and a territory of >9.6 million square kilometers, China has a total of 34 provinces and municipalities. In such a vast country, the epidemic history and migration trends of HCV are thought to be unique and complex but variable among regions and are unlikely to be represented by those observed in only one or at best a few provinces and municipalities. However, due to the difficulties in recruiting patients, all previous studies for this purpose have been based only on data from limited regions, and therefore, geographical biases were unavoidable. In this study, such biases were greatly reduced because we utilized samples collected from volunteer blood donors in 17 provinces and municipalities. To our knowledge, this is the first study in which the HCV isolates represented such a large portion of the country, and thus, the results should shed light on the current understanding of HCV molecular epidemiology.

In this study, we characterized the full-length genomes of 16 HCV isolates obtained from patients in a single hospital in China using overlapping PCR followed by DNA sequencing. The obtained genomes are 9,414-9,628 nucleotides in length, and each genome contains a single ORF of 9,021-9,102 nucleotides. Nine genomes represent the common subtypes 1a, 1b, 2a, 2b, 3a, 3b, and 6a, while seven represent the infrequent lineages 1c, 2f, 4d, and 5a, and two novel genotype 6 variants. GZ51969 and GZ52540 are subtype 1b isolates belonging to two unique clusters designated A and B, which account for 29.5% and 59.5% of the 1b infections in China, respectively. ZS542 and GZ98799 represent the first two complete genomes of the provisionally assigned subtype 2f. ZS96 and ZS202 are novel genotype 6 variants that may qualify for two new subtypes. ZS17, ZS537, and ZS631 represent three alien subtypes, namely, 1c, 4d, and 5a, which were detected in China for the first time in this study and may have been recently introduced as a result of globalization. Taken together, these results confirmed a large variety of HCV taxonomic lineages in China through the sequencing of their full-length genomes. These lineages represent six genotypes, 11 subtypes, and two novel variants. They were characterized for achieving a better understanding of the HCV genetic variation patterns and for possible future research applications.

Item does not contain fulltext BACKGROUND: Genotyping platforms for common red blood cell (RBC) antigens have been successfully applied in Caucasian and black populations but not in Chinese populations. In this study, a genotyping assay based on multiplex ligation-dependent probe amplification (MLPA) technology was applied in a Chinese population to validate the MLPA probes. Subsequently, the comprehensive distribution of 17 blood group systems also was obtained. STUDY DESIGN AND METHODS: DNA samples from 200 Chinese donors were extracted and genotyped using the blood-MLPA assay. To confirm the MLPA results, a second independent genotyping assay (ID Core+) was conducted in 40 donors, and serological typing of 14 blood-group antigens was performed in 91 donors. In donors who had abnormal copy numbers of an allele (DI and GYPB) determined by MLPA, additional experiments were performed (polymerase chain reaction, sequencing, and flow cytometry analysis). RESULTS: The genotyping results obtained using the blood-MLPA and ID Core+ assays were consistent. Serological data were consistent with the genotyping results except for one donor who had a Lu(a-b-) phenotype. Of the 17 blood group systems, the distribution of the MNS, Duffy, Kidd, Diego, Yt, and Dombrock systems was polymorphic. The Mur and Sta antigens of the MNS system were distributed with a frequency of 9% (18 of 200) and 2% (4 of 200), respectively. One donor with chimerism and one who carried a novel DI*02(A845V) allele, which predicts the depression of Dib antigen expression, were identified. CONCLUSIONS: The blood-MLPA assay could easily identify the common blood-group alleles and correctly predicted phenotype in the Chinese population. The Mur and Sta antigens were distributed with high frequency in a Southern Chinese Han population.

Item does not contain fulltext BACKGROUND AND OBJECTIVES: Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese. MATERIALS AND METHODS: The blood samples of 200 D+, 200 D- and 62 D variant Chinese donors were collected. RhD antigen was routinely typed by serological method. D variant phenotype was determined by an anti-D panel (D-Screen), when RBCs were available. The RHD genotype and its zygosity were analysed with the RH-MLPA technique. When the MLPA was unable to identify a RHD variant, direct sequencing of all exons of the RHD gene was performed. RESULTS: In 200 D+ donors, DD (168/200, 84%), D (12/200, 6%), DDD genotype (1/200) and D variant allele carriers (19/200, 9.5%) were found. In 200 D- donors, six reported RHD alleles, RHD*01EL.01, RHD*01N.03, RHD*01N.05, RHD*01N.16, RHD*DFR2 and RHD*weak partial 15 and one novel RHD*1154T allele were identified in 36.5% (73/200) of them. In 62 D variant donors, three novel RHD alleles, RHD*79_81delCTC, RHD*710T and RHD*689A, and twelve reported alleles, RHD*DVI.3, RHD*weak partial 15, RHD*DVI.4, RHD*01EL.01, RHD*01N.03, RHD*DLO, RHD*DV.5, RHD*D-CE(2-10), RHD*730C, RHD*weak D type 25, 33 and 72, were identified, either alone or in combination. CONCLUSION: The RH-MLPA assay correctly identified the common RHD variant alleles in the Chinese population. However, DNA sequencing was required to identify certain alleles; probes to detect these alleles should be added into the assay.

SummaryCD36 (also known as GPIV) deficiency is known to be responsible for the production of anti-Naka antibodies in different clinical settings such as fetal/neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion refractoriness (PTR) and post-transfusion purpura (PTP). However, no data regarding the relevance of CD36 immunisation is currently available for China. In this study, healthy blood donors were typed for CD36 deficiency using flow cytometry. Nucleotide sequencing was performed to identify the molecular basis underlying the CD36 deficiency. Anti-Naka antibodies in CD36-deficient individuals were analysed by ELISA and flow cytometry. By analysis of 998 healthy blood donors, 18 individuals failed to express CD36 on their platelets. In 5/12 individuals no CD36 expression was detected both on platelets and monocytes. This result suggested that the frequencies of type I CD36 deficiency (platelets and monocytes) and type II CD36 deficiency (platelets only) are approximately 0.5 and 1.3%, respectively. Nucleotide sequencing analysis of type I CD36 deficient individuals revealed eight different mutations; four of them were not described so far. However, 1228–1239de/ ATTGTGCCTATT and 329–330de/AC appear to be the most common mutations related to type I CD36 deficiency in South Chinese population. Further analysis showed that 1/5 type I CD36 deficient individuals developed anti-Naka antibodies. In addition, anti-Naka antibodies could be identified in two cases of thrombocytopenia associated with FNAIT and PTR. In conclusion, more than 0.5% of CD36 type I-deficient individuals are at risk to be immunised through blood transfusion or pregnancy in China. Testing of anti-Naka antibodies should be considered in FNAIT and PTR suspected cases. A registry of CD36-deficient donors should be established to allow treatment of immune-mediated bleeding disorders caused by anti-Naka antibodies.

Spontaneous clearance of hepatitis C virus (HCV) occurs in 10–40% of the infections. Specific human leukocyte antigen (HLA) alleles have been identified in associating with HCV clearance. However, data on the association of HLA with the spontaneous clearance of HCV are scarce in the Chinese population. In the current study we studied the HLA class I and class II genes in 231 Chinese voluntary blood donors who had cleared HCV infection spontaneously compared to 429 subjects with chronic HCV infections. We also studied their IL28B SNP (rs8099917) genotype, since a number of investigators have found a strong association of IL28B with spontaneous or treatment induced HCV clearance. We found that HLA-A*02:01 and DQB1*05:02 distributed differently between the two groups after Bonferroni correction (odds ratio [OR] = 1.839, Pc = 0.024 and OR = 0.547, Pc = 0.016, respectively). Multivariate logistic regression analysis suggested that A*02:01 and DRB1*11:01 (OR = 1.798, P = 0.008 and OR = 1.921, P = 0.005, respectively) were associated with HCV spontaneous clearance, independent of age, gender and IL28B polymorphism. We concluded that in the Chinese population, HLA-A*02:01 and DRB1*11:01 might be associated with the host capacity to clear HCV independent of IL28B, which suggesting that the innate and adaptive immune responses both play an important role in the control of HCV.

Understanding the biology of spontaneous clearance of hepatitis C virus (HCV) infection could lead to improved strategies to prevent the sequelae associated with chronic HCV infection. Chronic infections with hepatitis virus are very common in China, but the factors associated with spontaneous clearance of HCV have not been adequately studied. We evaluated the spontaneous clearance of HCV among 1918 drug users and 1526 HCV-seropositive blood donors in Guangzhou, China. Among participants who were co-infected with hepatitis B virus (HBV), 41.38% of drug users and 39.47% of blood donors had cleared their HCV infection without antiviral therapy compared to 9.41% of drug users and 16.73% of blood donors who were mono-infected with a single virus (P

BACKGROUND: Antibodies against polymorphic structures on human neutrophil antigens (HNAs) play a role in alloimmune-mediated neutropenia and are the leading cause of antibody-mediated transfusion-related acute lung injury (TRALI). This study aimed to determine the frequencies of HNAs in the major Han ethnic group living in Guangdong Province, Southern China. STUDY DESIGN AND METHODS: A total of 493 healthy Chinese Han blood donors from Guangzhou were recruited. DNA samples were isolated and typed for all five HNA-1, -2, -3, -4, and -5 systems using allele-specific polymerase chain reaction approaches. Results were compared with available data from other Chinese cohorts and other Asian and Caucasian populations. RESULTS: In this cohort, the gene frequency for HNA-1a (0.667) was approximately twice that of HNA-1b (0.333). In contrast to Caucasian populations, HNA-1a represents the most frequent allele in the Chinese population. HNA-3 system genotyping revealed comparable frequencies for HNA-3a (0.738) and -3b (0.262) in Chinese and Caucasian populations. Homozygous HNA-3bb individuals were found in 5.64% of our cohort. HNA-4 genotyping revealed no HNA-4bb homozygous individuals. In contrast, HNA-5bb homozygous individuals represented 2.43% of the population. Typing the HNA-2 system for the single-nucleotide polymorphism C42G showed that the C-allele (69%) is overrepresented and is associated with an increased number of HNA-2a–positive neutrophil subpopulations. CONCLUSION: This study describes for the first time the frequencies of all HNA systems, including the newly identified HNA-3, within one cohort of Chinese Han population. Comparison with Caucasian populations may allow assessment of anti-HNA alloimmunization and estimation of alloimmune neutropenia and TRALI incidence in Chinese populations.

SUMMARY. Recently, we studied hepatitis C virus (HCV) sera-prevalence among 559 890 first-time volunteer blood donors in China. From randomly selected 450 anti-HCV positive donors, we detected HCV RNA in 270 donors. In this study, we amplified HCV E1 and/or NS5B sequences from 236 of these donors followed by DNA sequencing and phylogenetic analysis. The results indicate new trends of HCV infection in China. The HCV genotype distribution differed according to the donors region of origin. Among donors from Guangdong province, we detected subtypes 6a, 1b, 3a, 3b, 2a, and 1a at frequencies of 49.7%, 31.0%, 7.6%, 5.5%, 4.1%, and 2.1%, respectively. Among donors from outside Guangdong, we detected 1b, 2a, 6a, 3b, 3a, 6e, and 6n at frequencies 57.1%, 13.2%, 11.0%, 9.9%, 4.4%, 2.2%, and 2.2%, respectively. Although we found no significant differences among regions in age or gender, subtype 6a was more common (P < 0.001) in donors from Guangdong than those from elsewhere, whilst subtypes 1b (P < 0.02) and 2a (P < 0.001) were more frequent outside Guangdong. Disregarding origins, the male/female ratio was higher for subtype 6a-infected donors (P < 0.05) than for subtype 1b donors, whilst the mean age of subtype 2a donors was 8‐10 years older (P < 0.05) than that for all other subtypes. Detailed phylogenetic analysis of our sequence data provides further insight into the transmission of HCV within China, and between China and other countries. The predominance of HCV 6a among blood donors in Guangdong is striking and mandates studies into risk factors for its acquisition.

Chronic infection with hepatitis C virus (HCV) is a major and growing public health problem. Globally, approximately 170 million people are infected with HCV; however, the prevalence varies greatly among countries, from 0.2% to 26%.1-6 The rapid global spread of HCV is believed to have occurred primarily because of efficient transmission through blood transfusion and parenteral exposures with contaminated equipment.7 In countries where HCV antibody screening and nucleic acid amplification testing (NAT) are mandatory for all blood donors, new HCV-infected donors often have a history of injecting illicit drugs, contaminated medical procedures, or other parenteral exposures. Risk factors also include tattooing, ear and body piercing, household or sexual contact, and unknown sources of infection.8 One of the most consistent characteristics of HCV is its ability to cause chronic infection in a high percentage of individuals. Chronic HCV infection often leads to liver cirrhosis and hepatocellular carcinoma.9,10 As blood-borne pathogens, both HCV and the human immunodeficiency virus (HIV) were frequently detected among paid blood donors in China during the early 1990s.11-14 To improve the safety of blood supply and reduce the risk of transfusion-transmitted diseases, the Chinese government has, since 1998, outlawed the use of paid blood donors. As a result, Chinese blood banks now rely on various other methods to recruit blood donors; these include employer-organized donor recruitment and the use of student donors, replacement donors, and in the recent past, only volunteer donors. This transition in the blood donor recruitment methods in China has been associated with a gradual decrease in the seroprevalence of anti-HCV among donors.13 The HCV seroprevalence among the general population of China has been estimated to be about 3.2%, while among paid blood donors the prevalence has been 5.7% or higher.15-17 However, among employer-organized donors and volunteer donors, the prevalence has ranged between 1.1, 2.3, and 0.46%, respectively13,18,19 Although a progressive decrease in the overall prevalence of anti-HCV has been observed among donors in some regions, the HCV prevalence among first-time volunteer blood donors in China has not been reported. From January 2004 to December 2007, a total of 559,890 first-time volunteer blood donors were evaluated at the Guangzhou Blood Center, in Guangdong Province. In this study, we report the prevalence of anti-HCV in this cohort of donor population.

Myeloid-derived suppressor cells (MDSCs) have been known to be a key factor in the regulation of the immune system under numerous conditions such as tumors, infections, autoimmune diseases, and transplantations. In contrast to the proposed deleterious role of MDSCs in tumors and infections, MDSCs with their suppressive function are now proved to have the beneficial potential of suppressing the autoimmune response and promoting tolerance to transplantation. Therefore, the expansion of MDSCs could be a promising therapeutic strategy for many diseases. In this study, we aimed to identify FDA-approved drugs that could aid in the expansion of functional MDSCs. We performed a high-throughput screening (HTS) of FDA-approved drugs based on the in vitro human MDSC-differentiation system and identified finasteride (FIN) to have the best potency to aid the generation of human MDSCs. The FIN-induced MDSCs were quite similar to monocytic MDSCs with regard to their surface phenotype, morphology, immunosuppressive function, and related gene expression. Next, we aimed to determine the mechanism of action of FIN and found that FIN induced the expansion of MDSCs through up-regulation of the COX2/PGE2 pathway by enhancing the activity of COX2 promoter. In addition, the administration of indomethacin (IND), a COX2 inhibitor, abrogated the effect of FIN. Based on these results, we suggested that FIN could find applications in the future in the expansion of MDSCs. Further development of FIN-like compounds could be a novel strategy for generating functional MDSCs for immunosuppressive therapies in various immune disorder conditions.

Five hepatitis C virus (HCV) complete genome sequences (Th846, 537796, QC227, QC216 and QC99) from a blood donor in Thailand and three Asian immigrants and one Caucasian in North America were determined. Phylogenetically, they represent the first complete genomes for subtypes 6c, 6l, 6o, 6p and 6q, respectively. Similarity analysis showed no evidence of inter- or intrasubtype recombination. Further analysis in conjunction with partial sequences from the Los Alamos HCV database led to the identification of other closely related isolates from south-eastern Asia or immigrants from that region. However, Th846 did not cluster with any reference sequence and is the sole isolate of subtype 6c reported so far. This study completes the full genome sequencing of all 17 assigned HCV genotype 6 subtypes (6a–6q). The utility of this panel of complete sequences for accurate detection and classification of infection, and for estimating the origin of this genotype of HCV, is discussed.

An HCV outbreak occurred in 2012 in China, affecting hundreds of patients. We characterized HCV subtype 2a and 6a sequences from 60 and 102 patients, respectively, and co-analyzed them with 82 local controls and 103 calibrating references. The close grouping of the patients׳ sequences contrasted sharply with the diversity of local controls. Scaled by the calibrating references, the emergence of patients׳ isolates was estimated at 2–5 years before sampling. In contrast, the controls intermingled with the calibrating references that were much older. For both subtypes, the major and minor clusters could be defined, with the closeness to indicate linked transmission. Conclusion : HCV sequences from the study patients grouped into three subtype 2a and two subtype 6a clusters, in addition to three 6a solitary branches, representing descendants of eight earlier strains that were distinct and otherwise sporadic. Due to iatrogenic transmission through reusing needles, five strains were highly selected and preferentially spread.

Here, the complete genome sequences for three hepatitis C virus (HCV) variants identified from China and belonging to genotype 6 are reported: km41, km42 and gz52557. Their entire genome lengths were 9430, 9441 and 9448 nt, respectively; the 5′ untranslated regions (UTRs) contained 341, 342 and 339 nt, followed by single open reading frames of 9045, 9045 and 9057 nt, respectively; the 3′ UTRs, up to the poly(U) tracts, were 41, 51 and 52 nt, respectively. Phylogenetic analyses showed that km41 is classified into subtype 6k and km42 into subtype 6n. Although gz52557 clustered distantly with subtype 6g, it appeared to belong to a distinct subtype. Analysis with 53 and 105 partial core and NS5B region sequences, respectively, representing 17 subtypes from 6a to 6q and three unassigned isolates of genotype 6 in co-analyses demonstrated that gz52557 was equidistant from all of these isolates, indicating that it belongs to a novel subtype. However, based on a recent consensus that three or more examples are required for a new HCV subtype designation, it is suggested that gz52557 remains unassigned to any subtype.

In one of our recent studies, two HCV genotype 6 variants were identified in patients from Hong Kong and Guangxi in southern China, with injection drug use and HIV-1 co-infection. We report the complete genomic sequences for these two variants: GX004 and HK6554. Their entire genome lengths were 9,468 and 9,462 nt; the 5' UTRs were 338 nt followed by single ORFs of 9,069 nt; the 3' UTRs were 61 and 55 nt including 29 and 23 nt poly(U) tracks. Phylogenetic analysis using a maximum likelihood method showed that HK6554 was classified into subtype 6g and GX004 represented the first complete genome sequence for subtype 6e. Further analysis with reference sequences in three different genomic regions revealed that GX004 closely clustered with a group of subtype 6e variants, which were previously exclusively found in Vietnam and recently increasingly identified in injection drug users from the Guangxi province in southern China that borders Vietnam. This suggests that subtype 6e could become epidemic in southern China by network transmission among injection drug users.

To determine hepatitis C virus (HCV) genotype distribution in China, a total of 148 HCV RNA positive serum samples were collected from nine geographic areas and subjected to RT-PCR followed by direct DNA sequencing and phylogenetic analysis of the core, E1, and NS5B regions. HCV was genotyped in 139 (93.9%) samples. Among them subtype 1b was the most predominant [66% (92/139)] followed by 2a [14% (19/139)]. Of 92 subtype 1b isolates, 35 (38%) and 30 (33%) formed two clusters, designated groups A and B. Group A was prevalent throughout China, while group B was predominant in the central and southern regions. In three cities in the Pearl River Delta, subtype 6a replaced 2a as the second most predominant subtype, and in Kunming (southwest) multiple HCV genotypes/subtypes were present. New variants of HCV genotype 6 were discovered in three samples from Kunming and one in Guangzhou in the Pearl River Delta.

The novel MICA*002:04 allele differs from MICA*002:01 by a single mutation at position 486 (C>A) in codon 20 (GCC>GCA) in exon 3.

We characterized the full-length genomes of 22 hepatitis C virus genotype 6 (HCV-6) isolates: 10 from Vietnam (classified into subtypes 6e, 6h, 6p, 6r, 6s, and 6u), one from China (confirmed as a new subtype 6xd), and 11 from the Lao PDR (representing a new subtype 6xe plus eight novel variants). With these 22 new genomes, HCV-6 now has a diverse and extended taxonomic structure, comprised of 28 assigned subtypes (denoted 6a-6xe) and 27 unassigned lineages, all of which have been represented by full-length genomes. Our phylogenetic analyses also included many partially-sequenced novel variants of HCV-6 from Lao PDR. This revealed that Lao HCV isolates are genetically very diverse and are phylogenetically distributed in multiple lineages within genotype 6. Our results suggest that HCV-6 has been maintained in Laos, a landlocked country, since the common ancestor of genotype 6 and indicates historical dispersal of HCV-6 across Southeast Asia., Highlights • We characterized 22 full-length genomes of HCV-6. • They confirmed two new subtypes 6xd and 6xe plus eight novel variants. • We also reanalyzed many partially-sequenced novel HCV-6 variants from Lao PDR. • They are phylogenetically distributed across the whole of genotype 6. • They indicate historical dispersal of HCV-6 across Southeast Asia.

We previously reported that sphingosine kinase 1 (SphK1), an enzyme that catalyzes the production of sphingosine- 1-phosphate (SIP), is upregulated in human gastric cancer and predicts poor clinical outcome. In the present study, we used known differential effects of UV irradiation on human MGC-803 gastric cancer cells to determine their effect on SphK1 activity. Ectopic expression of SphK1 in MGC-803 gastric cancer cells markedly enhanced their resistance to UV irradiation, whereas silencing endogenous SphK1 with shRNAs weakened this ability. Furthermore, these antiapoptotic effects were significantly associated with decrease of Bim, an apoptosis-related protein. We further demonstrated that SphK1 could downregulate the transcriptional activity of forkhead box O3a (FoxO3a) by inducing its phosphorylation, which was found to be associated with the PI3K/Akt signaling. Taken together, our study supports the theory that SphK1 confers resistance to apoptosis in gastric cancer cells via the Akt/FoxO3a/Bim pathway.

Different hepatitis C virus (HCV) genotypes exhibit differences in disease pathogenesis and progression, as well as disease outcomes and response to therapy. Tracking the change of HCV genotypes in various epidemiological settings is critical for both disease surveillance and the development of improved antiviral treatment. Here, we tracked the changes in the prevalence of the HCV genotypes in China between 2004-2007 and 2008-2011. HCV-RNA-positive sera were collected from volunteer blood donors during the period 2008-2011. The genotypes were determined by phylogenic analysis using the NS5B and E1 sequences. Geographical and demographic distribution patterns related to the HCV genotypes obtained in 2008-2011 were compared with our previous study, which recorded data in the period 2004-2007. Pearson chi-square test and t-test were used to statistically analyze the results. In 2008-2011, HCV subtypes 1b and 6a were detected in 43.8 % (184/420) and 34.3 % (144/420), respectively. The male/female ratio was found to be higher for HCV genotype 6 than for genotypes 1 and 2. When compared with the period of 2004-2007, although no significant difference was found in gender or age for genotypes 1, 2, 3 and 6, the subtype 6a frequency was significantly increased from 11 % to 26.5 % in the blood donors from outside of Guangdong Province in 2008-2011. A pattern of increase in HCV subtype 6a was found in blood donors outside of Guangdong Province, indicating that HCV subtype 6a has rapidly spread from Guangdong to other regions of China over the past 10 years. Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2185-1) contains supplementary material, which is available to authorized users.

Alloantibodies directed against antigens of the Kell blood group system are clinically significant. In the Netherlands, the KEL1 antigen is determined in all blood donors. In this study, after phenotyping of KEL:1-positive donors, genotyping analysis was conducted in KEL:1,-2 donors to identify possible KEL*02 variant alleles.A total of 407 donors with the KEL:1,-2 phenotype were genotyped for the KEL*01/02 polymorphism, followed by direct sequencing of the KEL gene if the KEL*02 allele was detected. Two K0 patients were also included. Transcript analysis was conducted in two probands with the KEL*02. M05 allele defined by a synonymous mutation (G573G). Flow cytometry analysis to determine the expression of Kell antigen was performed.Thirty KEL:1,-2 individuals (30/407, 7.4%) with discrepant KEL*01/02 genotype were identified. Seven novel alleles were identified: KEL*02(R86Q, R281W)mod, KEL*02(L133P)null, KEL*02(436delG)null, KEL*02(F418S)null, KEL*02(R492X)null, KEL*02(L611R)null, and KEL*02(R700X)null. Nine variant alleles described before were detected: KEL*02N.06, KEL*02N.15, KEL*02N.17, KEL*02N.19, KEL*02N.21, KEL*02M.02, KEL*02M.04, KEL*02M.05, and KEL*02(Q362K)mod. A transcript lacking Exon 16 was identified in two probands with the KEL*02M.05 allele as described before. Finally, flow cytometry analysis showed a decreased total Kell expression and a relatively increased KEL1 expression in individuals with the KEL:1,2null or KEL:1,2mod phenotype, compared to KEL:1,2 controls.In 7.4% of a group of tested KEL:1,-2 Dutch donors, a KEL*02null or KEL*02mod allele was found. A relatively increased KEL1 antigen expression in KEL:1,2null and KEL:1,2mod individuals suggest that the expression of Kell-XK complexes depends on the availability of the XK protein.

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192-1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope (1231)PTGSGKSTK(1239) (EP05) or core motif (1373)IPFYGKAI(1380) (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59-79% chronic and weakly with 30-58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.

In China, the prevalence of chronic hepatitis B virus (HBV) carriers declined from 9.5% to 7.2% in general population aged 1–59 years between 1992 when universal infant vaccination program was implemented nationwide and 2006. Approximately 75% to 90% reduction in hepatitis B surface antigen (HBsAg)-positivity was achieved in children, the HBsAg prevalence being reduced to 3% or less than 1% in children aged 5–14 years or 100 IU/L of sero-protective anti-HBs level in college students. The implementation of a systematic hepatitis B boosting vaccination program for non- and low-responder children and adolescents at age 15–17 is suggested in China. Such strategy might be cost-saving and a significant health benefit for the Chinese population.

Myeloid-derived suppressor cells (MDSCs) are known to accumulate under some pathologic conditions and suppress immune system in a variety of ways. This study aims to evaluate the significance of MDSCs in chronic Hepatitis C (CHC) patients.14 CHC patients and healthy donors were enrolled and subject to antiviral therapy including Peg-INF-alpha and Ribavirin for 48 weeks. The peripheral blood mononuclear cells (PBMCs) were collected at different weeks post-therapy and MDSC frequency was analyzed by flow cytometry. The correlation between MDSCs level with CHC disease parameters was analyzed by Spearman's rank test. The suppressive function of MDSCs from CHC patients and the underlying mechanism was further evaluated.A significant elevation of MDSCs was observed in the peripheral blood of treatment-naive CHC patients compared with healthy donors. The level of MDSCs in CHC patients correlated with plasma HCV-RNA (r = 0.7164, p = 0.0039), blood aminotransaminase (r = 0.6116, p = 0.021), and activated CD38(+) T cells (CD4(+): r = 0.6649, p = 0.0095; CD8(+): r = 0.6189, p = 0.0189). Initiation of clinical therapy reduced MDSC levels as early as 4 weeks, while it rebounded at week 12 post-therapy in patients. CHC-derived MDSCs could suppress T cell function in an arginase-1-dependent manner, that was distinct from the HCV core protein-generated MDSCs as previously reported.Our study reveals a significant correlation between MDSC levels with HCV disease progression, and their response to antiviral therapy. The arginase-1-dependent mechanism of MDSCs from CHC patients indicates that arginase-1 may be promising target for HCV immunotherapy.

Background Both HCV genotypes and viral loads are predictors of therapeutic outcomes among patients treated with α-interferon plus ribavirin; however, such correlation has only been studied for genotypes 1, 2, and 3 but not for genotype 6. Methodology/Findings 299 voluntary blood donors were recruited who were HCV viremic. Their mean age was 31.8; the male/female ratio was 3.82 (225/59). The viral loads of HCV were measured using the COBAS AmpliPrep/COBAS TaqMan test (CAP/CTM) while HCV genotypes were determined by direct sequencing the partial NS5B region. HCV genotypes 1, 2, 3, and 6 were determined in 48.9%, 8.7%, 12.3%, and 30.1% of the donors, respectively, and the levels of mean viral loads in genotype 1 and 6 significantly higher than that of 2 and 3 (P

Background Recently in China, HCV 6a infection has shown a fast increase among patients and blood donors, possibly due to IDU linked transmission. Methodology/Findings We recruited 210 drug users in Shanwei city, Guangdong province. Among them, HCV RNA was detected in 150 (71.4%), both E1 and NS5B genes were sequenced in 136, and 6a genotyped in 70. Of the 6a sequences, most were grouped into three clusters while 23% represent emerging strains. For coalescent analysis, additional 6a sequences were determined among 21 blood donors from Vietnam, 22 donors from 12 provinces of China, and 36 IDUs from Liuzhou City in Guangxi Province. Phylogeographic analyses indicated that Vietnam could be the origin of 6a in China. The Guangxi Province, which borders Vietnam, could be the first region to accept 6a for circulation. Migration from Yunnan, which also borders Vietnam, might be equally important, but it was only detected among IDUs in limited regions. From Guangxi, 6a could have further spread to Guangdong, Yunnan, Hainan, and Hubei provinces. However, evidence showed that only in Guangdong has 6a become a local epidemic, making Guangdong the second source region to disseminate 6a to the other 12 provinces. With a rate of 2.737×10−3 (95% CI: 1.792×10−3 to 3.745×10−3), a Bayesian Skyline Plot was portrayed. It revealed an exponential 6a growth during 1994–1998, while before and after 1994–1998 slow 6a growths were maintained. Concurrently, 1994–1998 corresponded to a period when contaminated blood transfusion was common, which caused many people being infected with HIV and HCV, until the Chinese government outlawed the use of paid blood donations in 1998. Conclusions/Significance With an origin from Vietnam, 6a has become a local epidemic in Guangdong Province, where an increasing prevalence has subsequently led to 6a spread to many other regions of China.

The MICA gene encodes nonclassical major histocompatibility complex class I molecules, centromeric to HLA-B and telomeric to HLA-DRB1. The MICA genes are polymorphic. The immune response against MICA may correlate with a decrease in graft survival after transplantation. However, data on the frequency of MICA polymorphisms in different populations are limited. In this study, we determined MICA allelic frequencies in a Han population living in Guangdong Province in south China. A total of 15 MICA alleles were identified using sequence-based typing. The most frequent allele was MICA*010 (22.22%), followed by MICA*002:01(18.56%), MICA*008:01(16.32%), and MICA*019(14.93%). The MICA null gene (MICA*Del) exhibited a frequency of 1.743% in this population. MICA and HLA, MICA-HLA-B, and MICA-HLA-A/HLA-B/HLA-DRB1 haplotype frequencies were estimated. The most common 2-, 3- and 4-locus haplotypes were HLA-B*40:01-MICA*008:01 (13.70%), HLA-A*11:01-B*40:01-MICA*008:01(8.25%), and HLA-A*33:03-B*58:01-DRB1*03:01-MICA*002:01(5.22%). A new MICA allele, MICA*061, was identified and appears to be evolutionarily related to MICA*012:01. This study provides high-resolution information on the distribution of haplotypes with MICA, HLA-A, HLA-B, and HLA-DRB1 in China. This information should help determine the mechanisms underlying diseases and allotransplant rejection associated with MICA polymorphisms in the southern Chinese Han population.

Antibodies against polymorphic structures on human neutrophil antigens (HNAs) play a role in alloimmune-mediated neutropenia and are the leading cause of antibody-mediated transfusion-related acute lung injury (TRALI). This study aimed to determine the frequencies of HNAs in the major Han ethnic group living in Guangdong Province, Southern China.A total of 493 healthy Chinese Han blood donors from Guangzhou were recruited. DNA samples were isolated and typed for all five HNA-1, -2, -3, -4, and -5 systems using allele-specific polymerase chain reaction approaches. Results were compared with available data from other Chinese cohorts and other Asian and Caucasian populations.In this cohort, the gene frequency for HNA-1a (0.667) was approximately twice that of HNA-1b (0.333). In contrast to Caucasian populations, HNA-1a represents the most frequent allele in the Chinese population. HNA-3 system genotyping revealed comparable frequencies for HNA-3a (0.738) and -3b (0.262) in Chinese and Caucasian populations. Homozygous HNA-3 bb individuals were found in 5.64% of our cohort. HNA-4 genotyping revealed no HNA-4 bb homozygous individuals. In contrast, HNA-5 bb homozygous individuals represented 2.43% of the population. Typing the HNA-2 system for the single-nucleotide polymorphism C42G showed that the C-allele (69%) is overrepresented and is associated with an increased number of HNA-2a-positive neutrophil subpopulations.This study describes for the first time the frequencies of all HNA systems, including the newly identified HNA-3, within one cohort of Chinese Han population. Comparison with Caucasian populations may allow assessment of anti-HNA alloimmunization and estimation of alloimmune neutropenia and TRALI incidence in Chinese populations.

Background: Typing of human platelet antigens (HPA) has proven to be useful in some clinical situations related to platelet alloimmunization. Objective: The objective of this study was to investigate HPA 1–16 and to determine genotype and allele frequencies by polymerase chain reaction-sequence specific primer (PCR-SSP) in apheresis platelet donors in Guangzhou Blood Center. Methods: A total of 200 random samples from donors were involved in the study. Genotype and allele frequencies of HPA 1 to 16 were detected by PCR-SSP method. Results: The frequencies obtained from these donors were 99·50 and 0·50% for HPA-1a and -1b; 96·25 and 3·75% for HPA-2a and -2b; 54·25 and 45·75% for HPA-3a and -3b; 99·50 and 0·50% for HPA-4a and -4b; 99·00 and 1·00% for HPA-5a and -5b; 97·00 and 3·00% for HPA-6a and -6b and 42·25 and 57·75% for HPA-15a and -15b. There is only a/a homozygosis detected in HPA-7, -8, -9, -10, -11, -12, -13, -14 and -16. In this study, none of HPA-1b/ 1b, -2b/2b, -5b/5b homozygosis were detected which were found in other racial groups. One homozygosis of HPA-6b/6b in 200 individuals was detected which was not found in a study involving 1000 Chinese (Feng et al., 2006). Conclusion: The HPA-3 and -15 appear to the highest priority and HPA-2, -6, -5 -1 and -4 to be the second priority in Chinese Cantonese when it comes to the diagnosis of neonatal alloimmune thrombocytopenia and to provide the HPA-matched platelet for patients with platelet transfusion refractoriness. The PCR-SSP method makes it possible to detect genotype of HPA-1 to -16 in less than 4 h and to establish a donor database for HPA genotype in a blood bank.

Embryonic stem (ES) cells have the ability to undergo indefinite self-renewal in vitro and give rise during development to derivatives of all three primary germ layers (ectoderm, endoderm, and mesoderm), which make them a highly prized reagent in cell and gene therapy. Efficient introduction of various genes of interest into primate ES cells has proven to be difficult. Here, we demonstrated that the self-inactivating HIV-1-based lentiviral vectors constructed by MultiSite gateway technology are efficient tools for the transduction of cynomolgus monkey ( Macaca fasicularis) ES (cmES) cells. After antibiotic selection, all of the transduced cells can stably express the reporter gene (humanized Renilla GFP or dTomato) while maintaining their stem cell properties, including continuous expression of stem cell markers, alkaline phosphatase (AKP), OCT-4, SSEA-4, and TRA-1-60, formation of embryoid bodies in vitro and teratomas in vivo containing derivatives of three embryonic germ layers. This approach will provide a useful tool for both gene function studies and in vivo cell tracking of stem cells.

Platelet transfusion refractoriness (PTR) is the major complication of long-term platelet supportive care. To improve the effectiveness of platelet transfusion therapy in PTR patients, we aimed to establish a platelet donor registry in our region (Guangzhou, China) by typing the human leukocyte antigen (HLA) and human platelet antigen (HPA). Blood donors (n = 864) from our population were genotyped for HLA-A, HLA-B and HPA systems by polymerase chain reaction amplification with sequence-specific primer(PCR-SSP) techniques. Using this cohort, we compared the results of platelet transfusions (matched vs. random) in 23 patients with PTR. Matched platelets were selected either by HLA antigen matching or by HLA antibody matching, as predicted by antibody specificity prediction (ASP) analysis. Significantly higher platelet recovery (PPR) values were obtained with HLA-matched platelets in comparison with random platelets. No significant difference in PPR was observed between HLA matching and ASP methods. In two patients, platelet-specific alloantibodies (alloabs) (anti-HPA-3b and anti-HPA-5b) were detected besides HLA class I alloabs. Transfusion with HLA- and HPA-compatible platelets in both the patients resulted in significantly higher PPR when compared with HLA-compatible platelet transfusion alone. In this study, we demonstrated that the establishment of an HLA- and HPA-typed platelet aphaeresis donor registry is useful to improve the treatment outcome of PTR patients and to maintain a long-term platelet transfusion strategy.

Background Although hepatitis C virus (HCV) genotype 6 is classified into 21 subtypes, 6a–6u, new variants continue to be identified. Objective To characterize the full-length genomes of three novel HCV genotype 6 variants: KMN02, KM046 and KM181. Study design From sera of patients with HCV infection, the entire HCV genome was amplified by RT-PCR followed by direct DNA sequencing and phylogenetic analysis. Results The sera contained HCV genomes of 9461, 9429, and 9461 nt in length, and each harboured a single ORF of 9051 nt. The genomes showed 95.3–98.1% nucleotide similarity to each other and 72.2–75.4% similarity to 23 genotype 6 reference sequences, which represent subtypes 6a–6u and unassigned variants km41 and gz52557. Phylogenetic analyses demonstrated that they were genotype 6, but were subtypically distinct. Based on the current criteria of HCV classification, they were designed to represent a new subtype, 6v. Analysis of E1 and NS5B region partial sequences revealed two additional related variants, CMBD-14 and CMBD-86 that had been previously reported in northern Thailand and sequences dropped into Genbank. Conclusion Three novel HCV genotype 6 variants were entirely sequenced and designated subtype 6v.

Subtype 1b is the most common strain ofHepatitis C virus(HCV) in China. Here, the molecular epidemiology and epidemic history of this strain were investigated by conducting phylogenetic and population genetic analyses of E1 and NS5B gene sequences sampled from nine Chinese cities. The phylogenetic analysis indicated the presence of two clusters of Chinese strains that did not include reference strains from other countries, suggesting that these clusters represent two independent chains of HCV transmission within China. The remaining Chinese isolates were more closely related to reference strains from other countries. The date of origin and past population dynamics of the two groups were investigated using a new population genetic method, the Bayesian skyline plot. The estimated dates of origin of both groups coincide with the period of the Chinese ‘Cultural Revolution’ during the years 1966–1976. Both groups grew at a rapid exponential rate between ∼1970 and ∼1990, after which transmission slowed considerably. Possible explanations for the groups' fast spread and subsequent slowdown are discussed, including parenteral transmission by unsafe injection, iatrogenic transmission by infected blood or blood products and improvements in blood safety since 1990. These results shed light on HCV transmission in China and may help to predict the future burden of HCV-related disease in the country.

Background In China, high prevalence of HBV and HCV parallels with the growing epidemic of syphilis and HIV in the general population poses a great threat to blood safety. This study investigated the prevalence of serologic markers for transfusion transmissible infections (TTIs) among four Chinese blood centers. Methods We examined whole blood donations collected from January 2000 through December 2010 at four Chinese blood centers. Post-donation testing of TTIs (HIV, HBV, HCV and syphilis) were conducted using two different enzyme-linked immunosorbent assay kits for each seromarker. The prevalence of serologic markers for TTIs (%) was calculated and additional analysis was conducted to examine donor characteristics associated with positive TTIs serology. Results Of the 4,366,283 donations, 60% were from first-time donors and 40% were from repeated donors. The overall prevalence of HIV, HBsAg, HCV and syphilis was 0.08%, 0.86%, 0.51% and 0.47%, respectively. The prevalence profile of TTIs varied among different blood centers and appeared at relatively high levels. Overall, the prevalence of HBsAg and HCV demonstrated a decline trend among four blood centers, while the prevalence of HIV and syphilis displayed three different trends: constantly steady, continually increasing and declining among different centers. Conclusions This study reflects the risk of TTIs has been greatly reduced in China, but blood transfusion remains an ongoing risk factor for the spread of blood-borne infections, and further work and improvements are needed to strengthen both safety and availability of blood in China.