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Mycobacterium abscessus(MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-β treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/β receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-β treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-β before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-β did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-β gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-β treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

GraS is a membrane sensor in Staphylococcus aureus that induces mprF and dltABCD expression to alter the surface positive charge upon exposure to cationic human defense peptides (HDPs). The sensing domain of GraS likely resides in the 9-residue extracellular loop (EL). In this study, we assessed a hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strain (COL) for the specific role of two distinct EL mutations: F38G (bulk) and D/35/37/41K (charged inversion). Activation of mprF by polymyxin B (PMB) was reduced in the D35/37/41K mutant versus the D35/37/41G mutant, correlating with reduced surface positive charge; in contrast, these effects were less prominent in the F38G mutant but still lower than those in the parent. These data indicated that both electrostatic charge and steric bulk of the EL of GraS influence induction of genes impacting HDP resistance. Using mprF expression as a readout, we confirmed GraS signaling was pH dependent, increasing as pH was lowered (from pH 7.5 down to pH 5.5). In contrast to PMB activation, reduction of mprF was comparable at pH 5.5 between the P38G and D35/37/41K point mutants, indicating a mechanistic divergence between GraS activation by acidic pH versus cationic peptides. Survival assays in human blood and purified polymorphonuclear leukocytes (PMNs) revealed lower survival of the D35/37/41K mutant versus the F38G mutant, with both being lower than that of the parent. Virulence studies in the rabbit endocarditis model mirrored whole blood and PMN killing assay data described above. Collectively, these data confirmed the importance of specific residues within the EL of GraS in conferring essential bacterial responses for MRSA survival in infections.

Helicobacter pylori is a gram-negative, microaerophilic, and spiral-shaped bacterium and causes gastrointestinal diseases in human. IL-1β is a representative cytokine produced in innate immune cells and is considered to be a key factor in the development of gastrointestinal malignancies. However, the mechanism of IL-1β production by neutrophils during H. pylori infection is still unknown. We designed this study to identify host and bacterial factors involved in regulation of H. pylori-induced IL-1β production in neutrophils. We found that H. pylori-induced IL-1β production is abolished in NLRP3-, ASC-, and caspase-1/11-deficient neutrophils, suggesting essential role for NLRP3 inflammasome in IL-1β response against H. pylori. Host TLR2, but not TLR4 and Nod2, was also required for transcription of NLRP3 and IL-1β as well as secretion of IL-1β. H. pylori lacking cagL, a key component of the type IV secretion system (T4SS), induced less IL-1β production in neutrophils than did its isogenic WT strain, whereas vacA and ureA were dispensable. Moreover, T4SS was involved in caspase-1 activation and IL-1β maturation in H. pylori-infected neutrophils. We also found that FlaA is essential for H. pylori-mediated IL-1β production in neutrophils, but not dendritic cells. TLR5 and NLRC4 were not required for H. pylori-induced IL-1β production in neutrophils. Instead, bacterial motility is essential for the production of IL-1β in response to H. pylori. In conclusion, our study shows that host TLR2 and NLRP3 inflammasome and bacterial T4SS and motility are essential factors for IL-1β production by neutrophils in response to H. pylori.IMPORTANCEIL-1β is a representative pro-inflammatory cytokine and is considered to be a central host factor for the development of gastric cancers. Although neutrophils have been considered to be involved in H. pylori-induced gastric inflammation, the underlying mechanism by which H. pylori triggers IL-1β production in neutrophils remains to be defined. In this study, our data suggested a critical role for the host TLR2 and NLRP3 inflammasome in IL-1β production by neutrophil during H. pylori infection. Moreover, we found the bacterial factors, T4SS and FlaA, to be essential for IL-1β production and NLRP3 activation during the course of H. pylori infection. Our current findings provide detailed molecular genetic mechanisms associated with IL-1β production in neutrophils in response to H. pylori infection, which can serve as innovative anti-inflammatory targets to reduce H. pylori-induced gastric malignancies.

A number of single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been associated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. Such SNPs have been found throughout the mprF ORF, although there are clearly preferred “hot spots” within this gene frequently linked to DAP-R phenotype. These mprF SNPs are often correlated with a gain-in-function phenotype, either in terms of increased production (synthase activity) and/or enhanced translocation (translocase activity) of lysyl-phosphatidylglycerol (L-PG) within its cell membrane. However, it is unclear if multiple hot spot mprF SNPs can accumulate within mprF ORFs and cause additive elevations of DAP minimum inhibitory concentrations (MICs). In this study, we used a previously well-characterized plasmid complementation system in S. aureus Newman ΔmprF mutant to express: (1) single point–mutated forms of mprF ORFs cloned from two DAP-R S. aureus strains (mprF(S295L) or mprF(T345A)) and (2) dual point–mutated forms of mprF ORFs simultaneously harboring SNPs in the central bifunctional domain and synthase domain in MprF, respectively (mprF(S295L+L826F) or mprF(T345A+L826F)). The current study revealed that, although individual hot spot point mutations within mprF ORF can recapitulate signature DAP-R–associated phenotypes (i.e., increased DAP MICs, enhanced surface positive charge, and increased L-PG synthesis), accumulation of such hot spot point mutations paradoxically caused reduction in these latter three metrics.

Mycobacterium tuberculosis is a causative agent leading to pleural effusion, characterized by the accumulation of fluid and immune cells in the pleural cavity. Although this phenomenon has been described before, detailed processes or mechanisms associated with the pleural effusion are still not well understood. Pleural mesothelial cells (PMCs) are specialized epithelial cells that cover the body wall and internal organs in pleural cavity playing a central role in pleural inflammation. Toll-like receptors are expressed in various cell types including mesothelial cells and initiate the recognition and defense against mycobacterial infection. In the present study, we investigated direct immune responses of PMCs against two mycobacterial strains, M. bovis vaccine strain Bacille Calmette-Guérin (BCG) and M. tuberculosis virulent strain H37Rv, and the role of TLR2 in such responses. Infection with BCG and H37Rv increased the production of IL-6, CXCL1, and CCL2 in WT PMCs, which was partially impaired in TLR2-deficient cells. In addition, the activation of NF-κB and MAPKs induced by BCG and H37Rv was suppressed in TLR2-deficient PMCs, as compared with the WT cells. TLR2 deficiency led to the decrease of nitric oxide (NO) production through the delayed gene expression of iNOS in PMCs. TLR2 was also shown to be essential for optimal expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 in PMCs in response to BCG and H37Rv. These findings strongly suggest that TLR2 participates in mycobacteria-induced innate immune responses in PMCs and may play a role in pathogenesis of tuberculosis pleural effusion.

Helicobacter pylori is a gram-negative, microaerophilic, and spiral-shaped bacterium and causes gastrointestinal diseases in human. IL-1β is a representative cytokine produced in innate immune cells and is considered to be a key factor in the development of gastrointestinal malignancies. However, the mechanism of IL-1β production by neutrophils during H. pylori infection is still unknown. We designed this study to identify host and bacterial factors involved in regulation of H. pylori-induced IL-1β production in neutrophils. We found that H. pylori-induced IL-1β production is abolished in NLRP3-, ASC-, and caspase-1/11-deficient neutrophils, suggesting essential role for NLRP3 inflammasome in IL-1β response against H. pylori. Host TLR2, but not TLR4 and Nod2, was also required for transcription of NLRP3 and IL-1β as well as secretion of IL-1β. H. pylori lacking cagL, a key component of the type IV secretion system (T4SS), induced less IL-1β production in neutrophils than did its isogenic WT strain, whereas vacA and ureA were dispensable. Moreover, T4SS was involved in caspase-1 activation and IL-1β maturation in H. pylori-infected neutrophils. We also found that FlaA is essential for H. pylori-mediated IL-1β production in neutrophils, but not dendritic cells. TLR5 and NLRC4 were not required for H. pylori-induced IL-1β production in neutrophils. Instead, bacterial motility is essential for the production of IL-1β in response to H. pylori. In conclusion, our study shows that host TLR2 and NLRP3 inflammasome and bacterial T4SS and motility are essential factors for IL-1β production by neutrophils in response to H. pylori.

Hepatitis B virus infection is a serious global health problem and causes life-threatening liver disease. In particular, genotype C shows high prevalence and severe liver disease compared with other genotypes. However, the underlying mechanisms regarding virological traits still remain unclear. This study investigated the clinical factors and capacity to modulate Type I interferon (IFN-I) between two HBV polymerase polymorphisms rt269L and rt269I in genotype C. This report compared clinical factors between rt269L and rt269I in 220 Korean chronic patients with genotype C infections. The prevalence of preC mutations between rt269L and rt269I was compared using this study's cohort and the GenBank database. For in vitro and in vivo experiments, transient transfection using HBV genome plasmid and HBV virion infection using HepG2-hNTCP-C4 and HepaRG systems and hydrodynamic injection of HBV genome into mice tails were conducted, respectively. This report's clinical data indicated that rt269I vs. rt269L was more significantly related to HBV e antigen (HBeAg) negative serostatus, lower levels of HBV DNA and HBsAg, and disease progression. Our epidemiological study showed HBeAg negative infections of rt269I infections were attributed to a higher frequency of preC mutations at 1896 (G to A). Our in vitro and in vivo studies also found that rt269I could lead to mitochondrial stress mediated STING dependent IFN-I production, resulting in decreasing HBV replication via the induction of heme-oxygenase-1. In addition, we also found that rt269I could lead to enhanced iNOS mediated NO production in an IFN-I dependent manner. These data demonstrated that rt269I can contribute to HBeAg negative infections and liver disease progression in chronic patients with genotype C infections via mitochondrial stress mediated IFN-I production.

Staphylococcus pseudintermedius is considered a primary pathogen of canine skin and soft tissue infections, and the rapid emergence of methicillin-resistant S. pseudintermedius worldwide is a major issue. In the current study, genotypic and phenotypic correlates associated with S. pseudintermedius causing canine otitis externa were evaluated using 41 S. pseudintermedius strains isolated from dogs with otitis externa (n = 26) and healthy dogs (n = 15). The S. pseudintermedius strains were subjected to a comparative analysis of (i) genotypes (multilocus sequence typing, agr, and spa types), (ii) methicillin resistance and SCCmec types, (iii) multidrug resistance (MDR), (iv) biofilm formation, and (v) susceptibility to canine cathelicidin (K9CATH). A high degree of genetic diversity was observed in both groups of S. pseudintermedius strains, regardless of methicillin resistance. Almost all methicillin-resistant strains (>95%) harbored SCCmec V and displayed MDR. Although there was no difference in biofilm formation, S. pseudintermedius strains derived from otitis externa exhibited enhanced resistance to cationic antimicrobial peptide (K9CATH) compared with strains from healthy dogs. The high degree of heterogeneity in MLST, agr, and spa types prevented the identification of correlations between any specific genotype and virulence phenotype in otitis externa caused by S. pseudintermedius, These findings provide an important basis for monitoring and treating canine skin and soft tissue infections in Korea.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in T1D mouse models. Notably, Tregs specific for pancreatic islets were shown more potent than polyclonal Tregs in blocking disease. However, the frequency of antigen-specific Tregs is extremely low and ex vivo expansion has the potential risk to destabilize Tregs leading to an effector phenotype. Here, we developed methods to generate durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells using a combination of lentiviral TCR transduction and FOXP3 homology-directed repair (HDR)-editing. Using TCRs derived from clonally expanded islet-reactive CD4+ T cells in T1D, we generated islet-specific EngTregs that exhibit a Treg-like phenotype. Islet-specific EngTregs effectively suppress proliferation and cytokine production by islet-specific effector T cells (Teff). Notably, EngTregs suppress Teff recognizing the identical peptide as well as bystander Teff recognizing alternative islet antigens. Importantly, islet-specific EngTregs suppress polyclonal endogenous islet-specific T cells derived from PBMC of individuals with T1D. To directly assess the capacity of EngTregs to modulate diabetes in vivo, we established an identical HDR-editing strategy in islet-specific murine cells. Following adoptive transfer, islet-specific mu-EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teff in recipient mice. Collectively, our approach enables the production of EngTregs specific to pancreatic islets with the capacity to efficiently suppress islet specific responses. This approach has the capacity to deliver targeted islet specific therapy to treat or prevent T1D.

Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide–MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I–presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αβ TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRβ-transgenic mice were assessed for QFL-specific TCR α- and β-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3–TRAJ21 α-chain and TRBV5–TRBD1–TRBJ2-7 β-chain gene segments. Furthermore, the CDR3 regions of the α- as well as β-chains were required for QFL ligand recognition. Thus, the αβ TCRs used to recognize the peptide–Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

Antimicrobial-resistant (AR) enterococci have emerged as leading nosocomial pathogens. Transmission of AR Enterococci from animals to humans has been demonstrated. However, there is limited information on the transmission of enterococci from horses to humans. To address this issue, we characterized 260 enterococci isolated from horse-associated samples in Korea in 2013 based on their AR profiles and virulence traits. AR profiling revealed an average ratio of AR enterococci of 23.8%. Seven isolates (2.7%) were multidrug-resistant Enterococcus faecalis. Most tetracycline-resistant enterococci harbored either tetM or tetL or both genes; genes conferring resistance to other antimicrobials were detected at low rates. Biofilm formation and gelatinase activity were observed in 51.1% and 47.7% of isolates, respectively; most were E. faecalis harboring the gelE gene. Evidence of transmission of AR enterococci between horses and their environments was provided by pulsed-field gel electrophoresis, and highlights the risk of AR enterococcus transmission to horse riders and handlers through close contact.

Adoptive transfer of regulatory T cells (Treg) is therapeutic in T1D mouse models. Notably, Treg specific for pancreatic islets are more potent than polyclonal Treg in blocking disease. However, the frequency of antigen-specific Treg is extremely low and ex vivo expansion has the potential to destabilize Treg leading to an effector phenotype. Here, we developed methods to generate durable, antigen-specific engineered (ed)Treg from primary human CD4+ T cells using a combination of lentiviral TCR transduction and FOXP3 homology-directed repair (HDR)-editing. Using TCRs derived from clonally expanded CD4+ T cells in T1D, we generated islet-specific edTreg that exhibit a Treg-like phenotype. Islet-specific edTreg effectively suppress proliferation and cytokine production by islet-specific effector T cells (Teff). Notably, edTreg suppress Teff recognizing the identical peptide as well as bystander Teff recognizing alternative islet antigens. Consistent with this, islet-specific edTreg suppress polyclonal islet-specific T cells derived from PBMC. Further, edTreg expressing TCR with high avidity have superior suppressive capacity to those expressing TCR with low avidity. To directly assess the capacity of edTreg to modulate T1D in vivo, we established an identical HDR-editing strategy in islet-specific murine cells. Adoptively transferred islet-specific mu-edTreg homed to the pancreas and blocked diabetes triggered by islet-specific Teff in recipient mice. Collectively, our approach enables the production of edTreg specific to pancreatic islets with the capacity to efficiently suppress islet specific responses. This approach has the capacity to deliver targeted islet specific therapy to treat or prevent T1D.

The most significant community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Korea is sequence type (ST) 72 with staphylococcal cassette chromosome mec (SCCmec) type IV (ST72-MRSA-IV). Although the impact of CA-MRSA on the clinical outcomes versus healthcare-associated (HA)-MRSA remains unclear, it has recently been revealed that ST5 HA-MRSA-II is associated with higher mortality compared with ST72 CA-MRSA-IV, suggesting higher virulence in ST5 HA-MRSA-II strains. In this investigation, human-/animal-originated ST72-MRSA-IV strains were examined for virulence phenotypes and compared with those of ST5-MRSA-II strains, the established HA-MRSA in Korea. Overall, ST5 HA-MRSA-II strains demonstrated higher levels of resistance to host defense-cationic antimicrobial peptides of human (LL-37), bovine (BMAP-28), and bacterial (polymyxin B) origins versus ST72-MRSA-IV strains via enhanced surface positive charge. Hemolysis profiles, gelatinase activity, and staphylococcal superantigen gene profiles were not different between ST72 CA-MRSA and ST5 HA-MRSA strains. However, ST5 HA-MRSA strains were able to downregulate initial cytokine response in murine macrophages.

In Korea, the major clonal type of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is sequence type 72 (ST72) with staphylococcal cassette chromosome mec (SCCmec) type IV (ST72-MRSA-IV). In this study, we used a previously well-characterized isogenic pair of ST72 vancomycin (VAN) susceptible-and VAN intermediate-MRSA strains (VSSA303 and VISA072) and several VSSA strains complemented with plasmids expressing single-point mutated genes (dprA

This study was conducted to assess emergence of daptomycin-nonsusceptible (DAP-NS) phenotype in DAP-naive patients with invasive Staphylococcus aureus (ISA) infections in Korea. A total of 208 S. aureus clinical isolates were selected from a previous prospective study on ISA infections and evaluated for DAP-NS. Although DAP has never been introduced in Korea, five DAP-NS S. aureus strains (2.4%) were identified among 208 S. aureus strains collected from ISA infections. The DAP-NS phenotype was observed only in methicillin-resistant S. aureus (MRSA) strains, but not in methicillin-susceptible S. aureus strains. One DAP-NS MRSA strain belonged to sequence type 72 (ST72) and four were ST5 MRSA strains, three of which were heteroresistant vancomycin (VAN)-intermediate S. aureus. All these five DAP-NS MRSA strains were from healthcare-associated infections without prior exposure to VAN within 30 days. While the ST72 MRSA strain exhibited DAP-NS phenotype via charge repulsion mechanism, four ST5 DAP-NS S. aureus strains had charge-independent DAP-NS mechanism. None of the five DAP-NS strains displayed significant increase in cell wall thickness, indicating that altered cell wall thickness was not associated with the observed DAP-NS phenotype.

Highlights • TRIF is involved in cytokines and chemokines production by poly I:C and LPS in PMCs. • TRIF mediates iNOS expression and NO production by poly I:C or LPS in PMCs. • TRIF is required for IFN-β gene expression in PMCs stimulated by poly I:C or LPS. • TRIF is essential for optimal production of IL-6, CXCL1, and CCL2 by live G-bacteria., Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF), one of the two major TLRs–adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-β in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

Mycobacterium abscessus is a prominent cause of pulmonary infection in immunosuppressed patients and those with cystic fibrosis. Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic receptor which senses a bacterial peptidoglycan component, muramyl dipeptide (MDP). Although nucleotide-binding oligomerization domain 2 (NOD2) contributes to protect host against various microbial infections, it is still unclear whether NOD2 is essential to regulate host immune responses against M. abscessus infection. In this study, we sought to clarify the role of NOD2 and the underlying mechanism in host defense against M. abscessus infection. Mice were infected intranasally with M. abscessus and sacrificed at indicated time points. Bacterial survival, cytokines production, and pathology in the lungs were determined. Bone marrow-derived macrophages were used to clarify cellular mechanism of NOD2-mediated immune response. Bacterial clearance was impaired, and pathology was more severe in the lungs of NOD2-deficient mice compared with the wild-type mice. In macrophages, NOD2-mediated activation of p38 and JNK were required for production of proinflammatory cytokines and nitric oxide (NO) and expression of iNOS in response to M. abscessus. NO was critical for limiting intracellular growth of the pathogen. Intranasal administration of MDP reduced in vivo bacterial replication and thus improved lung pathology in M. abscessus-infected mice. This study offers important new insights into the potential roles of the NOD2 in initiating and potentiating innate immune response against M. abscessus pulmonary infection.

Mycobacterium abscessus is a prominent cause of pulmonary infection in immunosuppressed patients and those with cystic fibrosis. Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic receptor which senses a bacterial peptidoglycan component, muramyl dipeptide (MDP). Although nucleotide-binding oligomerization domain 2 (NOD2) contributes to protect host against various microbial infections, it is still unclear whether NOD2 is essential to regulate host immune responses against M. abscessus infection. In this study, we sought to clarify the role of NOD2 and the underlying mechanism in host defense against M. abscessus infection. Mice were infected intranasally with M. abscessus and sacrificed at indicated time points. Bacterial survival, cytokines production, and pathology in the lungs were determined. Bone marrow-derived macrophages were used to clarify cellular mechanism of NOD2-mediated immune response. Bacterial clearance was impaired, and pathology was more severe in the lungs of NOD2-deficient mice compared with the wild-type mice. In macrophages, NOD2-mediated activation of p38 and JNK were required for production of proinflammatory cytokines and nitric oxide (NO) and expression of iNOS in response to M. abscessus. NO was critical for limiting intracellular growth of the pathogen. Intranasal administration of MDP reduced in vivo bacterial replication and thus improved lung pathology in M. abscessus-infected mice. This study offers important new insights into the potential roles of the NOD2 in initiating and potentiating innate immune response against M. abscessus pulmonary infection.

We describe 2127 new human leukocyte antigen (HLA) class I alleles found in registered stem cell donors. These alleles represent 28.9% of the currently known class I alleles. Comparing new allele sequences to homologous sequences, we found 68.1% nonsynonymous nucleotide substitutions, 28.9% silent mutations and 3.0% nonsense mutations. Many substitutions occurred at positions that have not been known to be polymorphic before. A large number of HLA alleles and nucleotide variations underline the extreme diversity of the HLA system. Strikingly, 156 new alleles were found not only multiple times, but also in carriers of various parentage, suggesting that some new alleles are not necessarily rare. Moreover, new alleles were found especially often in minority donors. This emphasizes the benefits of specifically recruiting such groups of individuals.

Mechanism of thymic compartmentalization was studied in the transgenic system using the promoter of thymic stromal cotransporter (TSCOT), a cortical thymic epithelium-specific gene. The transgenic 3.1 kb TSCOT (3.1T) and 4.4 kb TSCOT (4.4T) promoters recapitulated the thymic organ and the cortical epithelial cell-specific expression at the newborn stage. However, the 3.1T driving enhanced green fluorescent protein (EGFP; 3.1T-EGFP) or Cre-recombinase (3.1T-CreE) redistributed the expression into the medulla at the adult stages. Two Cre-transgenic lines (3.1T-CreE and 4.4T-CreE), when crossed with the ROSA LacZ or EGFP lines, showed the reporter expression in both the cortex and the medulla. TSCOT promoter activities were also verified in the transient thymic epithelial cell (TEC) population expressing keratin 5 and keratin 8. These indicate that the TSCOT promoter is turned on in the bipotent TEC precursors and regulated in a compartment-specific, developmentally regulated fashion. These transgenic lines provide the useful systems for delineating the specific pathways for TEC lineage development and function.

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (Δ graS ) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of Δ graS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with Δ graS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.

We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/Ly110 expressing subpopulation can be identified among the CDR1 + or 6C3 + cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1 + 6C3 + Ly110 + , CDR1 + 6C3 − / low Ly110 + , CDR1 + Ly110 − . Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1 + or 6C3 + epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process.

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the h exose p hosphate t ransport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli , the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA , hptRS , and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA , hptRS , or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

Aim Classical HLA Class I genes (HLA-A, -B and -C) is thought to be evolved from ancestral gene(s) via duplication events and considered to be homologous. Exon 2 and 3 of those genes encodes Antigen Recognition Sites (ARS). These exons are highly polymorphic and it was believed due to positive selection. We have analyzed over 100,000 human samples that were whole gene sequenced for HLA-A, -B and -C and contained 1188, 1237 and 1185 unique patterns for each gene respectively. Methods We performed whole gene HLA Class I gene sequencing on PacificBio platform as described earlier (Cereb et al. Hum Immunol. 2015 Dec;76(12):963–74). Mean values of nonsynonymous and synonymous divergence (dN and dS, respectively) for all codons for all pairwise comparisons among alleles were determined using SNPGenie, an implementation of the Nei-Gojobori method (Nei and Gojobori, Mol. Biol. Evol. 3:418–426.) for use with next-generation sequencing data (Mol. Biol. Evol. 3:418–426.). Results In summary regarding positive selection, the following exons give signatures of positive selection: (1) HLA-A exons 2, 3, and 5 (weak); (2) HLA-B exons 2 and 3 (weak); and (3) HLA-C exons 1, 6, and 7. Regarding HLA-C, this seems quite revolutionary, and may help to explain previous studies that failed to identify evidence of positive selection specifically favoring amino acid charge differences in the peptide binding residues (PBR) of HLA-C only. HLA-C has the most extreme signatures of positive selection heretofore observed, but in non-PBR exons: dN/dS for exon 1 is 17.94, and dN/dS for exon 7 is 19.02 — both very high values. However, it is noteworthy that these huge ratios are not due to a great increase in dN, but rather to a great decrease in d. By contrast, PBR exons 2 and 3 have overall signatures of weak purifying selection at the HLA-C locus.Overall values for dN at HLA-A, -B, and -C are 0.0305, 0.0306, and 0.0240, respectively. Overall values for dS are 0.0395, 0.0344, and 0.0300, respectively. Therefore, in terms of selectively neutral diversity, diversity is greatest in HLA-A, less in -B, and least in -C. Conclusions Whole gene sequencing of HLA Class I will provide us the foundation to understand the evolutionary forces shaping the regions and the functions of various regions of those genes. N. Cereb: 6. Stock Shareholder; Company/Organization; PacBio and Hitogenetics. C.W. Nelson: 2. Consultant; Company/Organization; Histogenetics. S. Yang: 6. Stock Shareholder; Company/Organization; PacBio and Histogenetics.

Aim We have been sequencing whole gene HLA Class I at large scale at Histogenetics on PacBio RSII® platform since the beginning of July of 2016. To date we have sequenced around 185,000 samples for whole gene Class I. We have found 1645, 1697 and 1664 unique sequences for A, B and C genes, respectively. Most of the intronic sequence information comes from the introns flanking Antigen Recognition Site (ARS) encoding introns. In this study we wanted to analyze all intronic sequences to determine their evolutionary process. Methods We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al. Hum Immunol. 2015 Dec;76(12):963–74). Exons were translated, aligned at the codon level using the ClustalW algorithm in MEGA7 (default settings), and the alignment was back-imposed on the nucleotide sequence. Intronic sequence diversity (d), and exonic nonsynonymous (dN) and synonymous (dS) sequence diversity, were calculated based on all pairwise comparisons of unique alleles using SNPGenie (adaptation of Nei-Gojobori method). Results For HLA-A, dS-exons = 0.0404 while d-introns = 0.0236; for HLA-B, dS-exons = 0.0347 while d-introns = 0.0094; and for HLA-C, dS-exons = 0.0305 while d-introns = 0.0186. Conclusions Our results are consistent with the hypothesis that diversity at synonymous coding sites has been elevated because of linkage to nonsynonymous sites, which experience overdominant (positive balancing) selection. In contrast, intronic sites are less closely linked to nonsynonymous coding sites than are synonymous coding sites, making them more likely to experience recombinational separation from coding sites and subsequent drift, which has acted to homogenize the sequences over evolutionary time thus resulting in locus-specific sequences un the introns.

Aim Recent advances in sequencing, sample and library preparation technologies made it possible to sequence entire genes, haplotypes and genome in human. In this study, we used four approaches; amplicon based whole gene sequencing, capture probe based MHC haplotype sequencing, PacBio Sequel system based whole genome sequencing and whole genome mapping based on Bionanogenomics’ Technology to study one individual’s MHC. We wanted to compare the information that we have obtained from each technology and confirm the haplotype predictions. Methods We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al., Hum Immunol. 2015 Dec;76(12):963–74). We performed MHC region target sequence capture using Roche-NimbleGen SeqCap EZ Library. Whole genome sequencing We prepared 40 to 50 kb libraries with 15 to 20 kb cut off size selection and followed manufacturer’s protocol thereafter. Sequencing performed on PacBio Sequel® platform using v2 chemistry and v4 software. Movie time for these large fragments were set to 600 min and secondary analysis is done with SMRTLink’s (v4.0.0) SMRT Analysis Application: de novo Assembly (HGAP 4). To get 85× coverage, we have sequenced the same library in 62 different SMRT® Cell 1 M v2. Whole genome de novo assembly of PacBio sequencing is being performed by DNANexus. BioNano genome mapping was performed by Bio Nano genomics. Results Haplotype determination of the individual tested with whole gene amplicon sequencing performed based on the family studies and is being compared to haplotypes determined by other methods. Conclusions Now thanks to novel technologies we can decipher the complex genetic structure of MHC and other immune response genes with higher precision. This in turn will increase our ability to understand the genetic basis of the complex diseases.

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (Δ graS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The Δ graS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo , the Δ graS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.

We have characterized 372 novel human leukocyte antigen (HLA) class II alleles identified in newly registered stem cell donors, this includes 281 HLA-DRB1 alleles, 89 HLA-DQB1 alleles and 2 HLA-DPB1 alleles. Most novel alleles were single nucleotide variants when compared to their respective most homologous alleles. In 66.4% of all novel alleles non-synonymous nucleotide variations were identified, in 30.4% synonymous substitutions and in 3.2% nonsense mutations. Ninty-three (25.0%) novel alleles were found in several individuals; most often these were novel HLA-DRB1 alleles. Lastly, we underline the importance of recruiting ethnic minority donors in countries such as Germany and the United States, as novel alleles were frequently found among these groups.

How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-γ (IFN-γ) alleles and by inducing IL-12 receptor β2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-γ synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce THfate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.

A total of 42,160 individuals were typed for HLA-A and HLA-B by both serology and PCR-based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)-based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or PCR using sequence-specific primers (PCR-SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA-A assignments, 13% for HLA-B, and 3% for both HLA-A and -B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA-based testing correctly typed nearly 99% of the donors at HLA-A, more than 98% at HLA-B, and more than 97% at both HLA-A and -B validating this methodology for registry typing.

DNA-based typing of HLA class I alleles of the HLA-A and HLA-B loci using sequence-specific oligonucleotide primers and/or probes has been used for the large-scale typing of individuals for the National Marrow Donor Program unrelated donor registry. Typing was performed by 16 laboratories at a low level of resolution (e.g. A*01, B*07). The results of blinded quality control analysis for the first 12 months of the project show the typing to be highly accurate, specific and reliable. The total error rate based on 11,545 HLA-A and 11,428 HLA-B assignments was 1.1% for HLA-A and 1.9% for HLA-B. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 64,180 donor samples tested at the same time by the laboratories.

BACKGROUND Transforming growth factor beta-1 (TGF beta 1) has been suggested to play a role in the development, growth or progression of hepatocellular carcinoma (HCC). Genotype and serum titer of HCV also affect the occurrence of HCC in chronic hepatitis C. In this study, we were to evaluate the effects of genotype or serum titer of HCV on the expression of TGF beta 1. We also intended to examine the correlation between the up-regulation of TGF beta 1 and the association with HCC in patients with chronic hepatitis C. METHODS We studied 19 patients with chronic hepatitis C and 18 with HCC associated with HCV infection. HCV genotype was determined by line probe reverse hybridization assay and the amount of HCV-RNA was quantitated by branched DNA signal amplification assay. Serum TGF beta 1 level was measured by enzyme linked immunosorbent assay. RESULTS HCV genotypes of patients with HCC were similar to those without it. Serum HCV-RNA titer was higher in genotype 1b than in non-1b (p < 0.05). Serum TGF beta 1 levels were higher in HCC than in chronic hepatitis (p < 0.05). However, there was no significant difference in the serum TGF beta 1 level between genotype 1b and non-1b. Also, it was not correlated with the serum HCV-RNA titer or alanine aminotransferase levels. CONCLUSION TGF beta 1 seems to be overexpressed in HCC compared to that of chronic hepatitis C: it was not affected by serum ALT levels, genotype or serum HCV titer. It is suggested that TGF beta 1 may be associated with the malignant transformation of hepatocyte or the progression of HCV-associated HCC.

High incidences of graft failure, graft-vs.-host disease (GVHD), and serious infections following unrelated donor (URD) marrow transplantation, despite apparent human leukocyte antigen (HLA) identity, may reflect the presence of molecular disparities, including those for HLA-C alleles between the patient and the URD. The level of these disparities could be significant, because as many as 42 alleles are currently known for HLA-C locus. We studied 84 patients and 251 potential URDs to evaluate 1) the extent of HLA-C disparity between the patient and the URD identified by serology for HLA-A and -B and by DNA typing for -DRB1 and 2) the level of HLA-C disparity between patients and URDs matched by high-resolution DNA typing for HLA-A, -B, and -DRB1. The DNA typing was performed at the Memorial Sloan Kettering Cancer Center, and the serotyping was provided by the registries. Of 251 URDs matched by HLA-A and -B serology and -DRB1 (sA_sB_dnaDRB1 ); 94, 75, and 82 were 6/6, 5/6, and 4/6 matches, respectively. Of 94 sA_sB_dnaDRB1 6/6 URDs, 51 (54.3%) were matched for both HLA-C alleles. In contrast, 31 (41.3%) 5/6 (p=0.12) and 15 (18.3%) 4/6 (p < 0.01) sA_sB_dnaDRB1 URDs were matched for both HLA-C alleles. Following DNA typing for HLA-A and -B, 52 (55.3%) of 94 6/6, 30 (40%) of 75 5/6, and 25 (30.5%) of 82 4/6 sA_sB_dnaDRB1 URDs remained 6/6, 5/6, and 4/6 matches at the DNA level (dnaA_B_DRB1). HLA-C disparities continued to exist in the dnaA_B_DRB1 URD group. Of 54 dnaA_B_DRB1 6/6 URDs, 41 (75.9%) were matched for both HLA-C alleles. Only 45.3% of the 5/6 (p=0.01) and 22.2% of the 4/6 (p < 0.01) dnaA_B_DRB1 URDs were matched for both HLA-C alleles. In the 6/6 category, the frequency of HLA-C matching improved (75.9 vs. 54.3%; p=0.01) following DNA matching for HLA-A and -B. In comparison to mismatching for HLA-B locus, mismatching for either HLA-DRB1 or -A resulted in a lower odds ratio for HLA-C disparity. The presence of a common haplotype in the sA_sB_dnaDRBl (p=0.06) URD category improved the level of HLA-C matching. We identified alleles that are associated with high (B*1501, B*4402, B*5101, DRB1*0101, A*0201, A*1101, A*2301, and A*3201) or low (B*0702, B*0801, B*1302, B*3502, DRB1*0301, DRB1*1104, A*0101, A*3001, and A*6801) probability of HLA-C disparity. Overall, sA_sB_dnaDRB1 as well as dnaA_B_DRB1 matched URDs for non-Caucasian patients were more likely to have HLA-C disparity in comparison to the matched URDs of Caucasian patients. However, a high incidence of HLA-C disparities was identified even in the URDs for Caucasian patients. Whether the disparities demonstrated by this study contribute to the higher immunological complications noted following URD bone marrow transplantation is unclear. Outcome analysis and studies aimed at understanding the functional role of HLA-C may provide an answer.Biol Blood Marrow Transplant 1999;5(2):77-85.

High incidences of graft failure and graft-versus-host disease in the recipients of bone marrow transplantations (BMT) from unrelated donors (URD) may reflect the existence of allelic disparities between the patient and the URD despite apparent HLA identity at HLA-A, HLA-B, and HLA-DRB1 loci. To identify the extent and pattern of allelic disparities at HLA-A and HLA-B loci, 128 patients and 484 potential URD were evaluated by DNA typing. DNA typing for HLA-A, HLA-B, and HLA-DRB1 was performed at Memorial Sloan Kettering Cancer Center. HLA-A and HLA-B serotyping on URD was provided by the registries. By original typing (serology for HLA-A and HLA-B; DNA typing for DRB1) 187, 164, and 133 URD were 6/6, 5/6, and 4/6 matches, respectively. Following DNA typing, however, only 52.9% of the originally 6/6 matched URD remained 6/6, while 38.5%, 7.5%, and 1.1% were found to be 5/6, 4/6, and 3/6 matches. The level of disparity was higher in the originally 5/6 (P< .01) and 4/6 (P < .01) matched URD. A higher level of disparity was seen for HLA-B as compared to HLA-A. In addition, a serotype related variation was also noticed. For example, 24.1% of HLA-A2 and 60.1% of HLA-B35 seromatched URD were genotypically disparate, but no disparities were seen for HLA-A1 and HLA-B8. A higher percentage of HLA-A (67.4%) compared with HLA-B (35.4%) serologic homozygous URD remained genotypically homozygous (P = .01). The level of allelic disparity was lower (P < .01 for 6/6; P = .02 for 5/6) if the patient had one of the 15 most common haplotypes (A1B8DR3, A2B7DR15, A3B7DR15, etc) in comparison to the rest of the group. Outcome studies will answer the question whether these disparities are associated with a higher rate of immunological complications seen with URD-BMT.

The protein-coding sequences of the major histocompatibility complex (MHC) genes are characterized by extraordinarily high polymorphism, apparently maintained by balancing selection, which favors diversity in the peptide-binding domains of the MHC glycoproteins. Here we report that the introns flanking the polymorphic exons of the human MHC class I loci HLA-A, -B, and -C genes have been relatively conserved and have become locus-specific apparently as a result of recombination and subsequent genetic drift, leading to homogenization within loci over evolutionary time. Thus, HLA class I genes have been shaped by contrasting evolutionary forces maintaining polymorphism in the exons and leading to conservation in the introns. This study provides the first extensive analysis of the introns of a highly polymorphic gene family.

Purpose The aim of the present study was to evaluate the clinical characteristics of the primary Epstein-Barr virus (EBV) hepatitis with elevation of both serum alkaline phosphatase (ALP) and γ-glutamyltransferase (γ-GT) levels in children. Materials and Methods A retrospective study was performed by reviewing of the medical records of 36 patients who were diagnosed with primary EBV hepatitis. The patients were divided into 2 groups: patients with elevated serum ALP and γ-GT levels (group 1) and patients without (group 2). Results The classic features of infectious mononucleosis (fever, pharyngitis and/or tonsillitis, and cervical lymphadenitis) were seen in 20 (57.1%) of group 1 patients and 18 (50.0%) of group 2 patients. Hepatitis with elevated serum ALP and γ-GT levels were present in 14 (38.9%) of the all patients. Of these patients, Jaundice occurred in only 2 (5.6%). The mean levels of aspartate aminotransferase and alanine aminotransferase (ALT) as well as the number of patients with ALT greater than 400 IU/L were significantly different between the groups (177 IU/L vs. 94 IU/L, 418 IU/L vs. 115 IU/L, and 50.0% vs. 13.6%; p=0.001, p=0.001, p=0.026, respectively). The mean duration of elevated serum ALT levels was 17.5 days in group 1 and 9.0 days in group 2 (p=0.013). All patients recovered fully without any chronic or serious complications. Conclusion Primary EBV hepatitis with predominant biochemical abnormalities of the elevation of ALP and γ-GT is frequent and mostly anicteric. This may represent a benign disease, but a delay in recovery of liver function as well.

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation by sequencing at a level that should allow the genome-wide detection of most variants with frequencies as low as 1%. However, in the major histocompatibility complex (MHC), only the top 10 most frequent haplotypes are in the 1% frequency range whereas thousands of haplotypes are present at lower frequencies. Given the limitation of both the coverage and the read length of the sequences generated by the 1000 Genomes Project, the highly variable positions that define HLA alleles may be difficult to identify. We used classical Sanger sequencing techniques to type the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes in the available 1000 Genomes samples and combined the results with the 103,310 variants in the MHC region genotyped by the 1000 Genomes Project. Using pairwise identity-by-descent distances between individuals and principal component analysis, we established the relationship between ancestry and genetic diversity in the MHC region. As expected, both the MHC variants and the HLA phenotype can identify the major ancestry lineage, informed mainly by the most frequent HLA haplotypes. To some extent, regions of the genome with similar genetic or similar recombination rate have similar properties. An MHC-centric analysis underlines departures between the ancestral background of the MHC and the genome-wide picture. Our analysis of linkage disequilibrium (LD) decay in these samples suggests that overestimation of pairwise LD occurs due to a limited sampling of the MHC diversity. This collection of HLA-specific MHC variants, available on the dbMHC portal, is a valuable resource for future analyses of the role of MHC in population and disease studies. © 2014 Gourraud et al.

Chronic myelogenous leukemia (CML) cells are characterized by a t(9;22) translocation, which can encode one of two chimeric P210 bcr-abl fusion proteins, comprising products of either the b2a2 or the b3a2 exon junction. The junctional sequences represent potentially immunogenic tumor-specific antigens. Despite their intracellular location, the fusion proteins might be recognized immunologically by T lymphocytes if peptides, derived from these unique sequences, are capable of presentation by the major histocompatibility complex molecules. We previously found that four peptides, 9 to 11 amino acids long, spanning the b3a2 CML breakpoint bind with high or intermediate affinity to purified HLA class I molecules A3, A11, B8, or both A3 and A11. We tested the ability of these peptides to elicit specific class I restricted cytotoxic T lymphocytes (CTLs) in vitro in HLA-matched healthy donors. In addition, a longer b3a2 CML-breakpoint-derived peptide, 25 aminoacids in length (b3a2–25), was studied for its ability to induce peptide-specific, class II-mediated, T-cell proliferation. In four of four HLA-A3 donors tested, CML-A3/A11-peptide specific CTLs were induced that killed an allogeneic HLA-A3-matched peptide pulsed leukemia cell line. In two of three HLA-A3 donors, the CML-A3/A11 peptide was able to induce killing of autologous and allogeneic HLA- matched peptide-pulsed peripheral blood mononuclear cells (PBMC). CML- A3 peptide induced peptide specific CTLs in one of the four HLA A3 donors tested. No killing was observed in two HLA-B8 and two HLA-A11 donors. PBMC from seven donors were also tested for anti b3a2–25 peptide proliferation in a thymidine incorporation assay. Specific proliferation was detected in three donors, all of the HLA-DR11 haplotype. These data represent the first evidence of a cytolytic human immune response against CML bcr-abl oncogene-derived peptides and provide a rationale for developing peptide-based vaccines for this disease.

Current typing technology for class I HLA antigens uses serological and/or isoelectric focusing gel electrophoresis. DNA typing for the HLA class I antigens can accurately identify the class I genotype of individuals and cell lines. Here, we report correlation of DNA typing results with serological and IEF results for the B17 group. The B17 antigens are relatively common, being carried by almost 9% of Caucasians and 28% of blacks. In this study, five 10th International Histocompatibility Workshop cell lines carrying B17 and 106 individuals in 61 families carrying B17 were DNA typed for B17 using B17-allele-specific amplification and sequence specific oligonucleotide probe hybridization pattern analysis. 38 (55.07%) out of 69 unrelated haplotypes had B*5701, 23 (33.33%) had B*5801, 6 (8.70%) had B*5702, and 2 (2.90%) had B*5802. DNA typing results correlated well with serological and isoelectric focusing results. In general, there was high degree of agreement between all three methods, although heterozygosity for B17 poses a particular problem for serological and IEF methodology. Both B*5701 and B*5801 have the same electrophoretic mobility on IEF gel, corresponding to B17.2, B*5702 corresponds to B17.1, while B*5802 corresponds to B17.3.

Aim One of the factors that needs to be considered for a successful hematopoietic stem cell transplantation is the ABO Rh compatibility between patient and donor. Until recently, most donor registries did not have ABO Rh typing data on donors due to logistics and expenses that are involved. Furthermore, most donor recruitments are currently done using buccal swabs or saliva samples that precludes serotyping for ABO Rh groups. However, these specimen types are suitable for DNA typing of ABO Rh genes. We have developed a method based on Illumina MiSeq platform to genotype ABO Rh genes and build an algorithm to predict the phonotypes. This additional information on donors could speed up the search process for the patients who need hematopoietic stem cell transplant. Methods: We designed 9 Exon-based primer pairs for the 7 exons for the glycosyltransferase gene. Exon 7 required 3 overlapping amplicons for full coverage. For Rh genes, we designed 10 primer sets to amplify all 10 exons. Previously, 1000 serotyped DNA samples of Caucasian ethnicity were genotyped to predict genotype-phenotype correlation. We applied a phenotyping algorithm based on all the exon sequences, incorporating known serological motif sequences in exons 6 and 7. This algorithm predicted ABO type with 99.9% accuracy and Rh with 100%. Results We blindly tested 1376 samples of diverse ethnic origin. We found that 12 (0.87%) samples for ABO and 31 (2.23%) samples for Rh had discordant results with serotyping. We typed close to 1 million individuals as of today, and we have discovered many novel sequences for each exon as shown in the table below: We have found 144 cases with new motifs in exon 7 that changed B blood group genotype to O. Thirteen had a point substitution, and 131 had a 1 bp deletion. Conclusion Large scale molecular typing for ABO and Rh is now feasible. Molecular basis for some ambiguous serotyped could be identified with this approach. ABO and Rh information in donor files will help accelerate the donor search process .

Aim Recent advances in Next Generation sequencing technologies have enabled us to provide higher resolution typing for HLA in Antigen Recognition Sites (ARS) faster and more cost effectively compared to Sanger Sequencing. Using these technologies, we can also sequence whole gene and long range HLA Class I and II alleles that were amplified in one long amplicon (>3 kb). PacBio RSII platform’s Single molecule, real-time (SMRT®) sequencing, generating average long reads of 10–15 kb, allows us to obtain in-phase long sequence reads. We have introduced and applied the PacBio RSII platform in our lab for NMDP recruitment samples using whole gene (Class I) and long range (Class II) amplicons as sequencing templates. We present our findings of extensive variation beyond ARS especially in introns of Class I genes. Methods We performed whole gene Class I amplification on the genomic DNA samples using locus-specific primers at 5 ′ and 3 ′ UTRs. Whole gene products were sequenced on the PacBio RSII platform. In parallel, we performed Class I and Class II exon-based amplicon sequencing on the gDNA samples using the Illumina MiSeq platform. We analyzed the data based on 3.20.0 allele DB using our in house program. Results A total of 891 out of 3589 samples showed novel variations with 359 unique patterns. Most new variations were observed in introns of HLA-B, in particular intron 2 and 6 being the most active. Exon 6 showed the most novel variation among the exons having HLA-A the most. HLA-C did not have any new variation in exon 6 in this group. Only 4 novel alleles were observed in the ARS region, one in HLA-B and three in HLA-C. Most alleles that were named based on the ARS sequences had no or incomplete published sequences for the other regions of the gene at the completion of this study. Conclusion The whole gene sequencing of class I alleles is essential in determining the structural integrity and expressivity of the genes that could not be determined solely based on ARS. Intronic variation may be involved in modulation of the expression. Variation in intra cytoplasmic domain coding exons (Exons 6, 7, 8) may play a role in modulating signal transduction in Class I genes. Establishing a database with complete gene sequences will help further studies in clarifying the biological significance of those variations. S. Yang: Stock Shareholder; Company/Organization; PacBio .

The HLA-B35 serotype represents a group of antigens detectable by IEF, cytotoxic T cells, and by sequencing analysis. Four isotypes and eight alleles have been thus far reported. We have determined the relative frequencies of these B35 subtypes in a group of 203 unrelated people. Dot blot hybridization of PCR amplified products was performed using 23 sequence-specific oligo probes designed based on the EMBL HLA class I sequence database. The amplification was achieved by a pair of group-specific primers, producing approximately 600 bp fragments. By hybridization pattern analysis, we found that four alleles represent over 95% of the B35+ population, with relative frequency of 48.2% for B*3501, 23.7% for B*3502, 15.2% for B*3503, and 8.0% for B*3508. We also identified 3 individuals with B*3504 and one with B*3505, and seven samples with new patterns. B*3501 and B*3503 exactly correlated with the most common isotype B35.3, B*3502 and B*3504 with B35.2, B*3508 may be the B35.1 IEF isotype. The B*3505 was identified from an individual with B35 IEF variant form. Our study shows that the B35 antigen has a wide distribution of alleles, and that many more B35-related alleles may yet to be uncovered.

We have identified locus-specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA-A, -B, and -C loci in the amplification of genomic DNA. PCR products generated using each of the locus-specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non-classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin-ddUTP-labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus-specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR-based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.

Activated CD8 + T cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector functions of CD8 + T cells. We now show that Eomesodermin ( Eomes ), a paralogue of T-bet , is induced in effector CD8 + T cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8 + T cells, including interferon-γ (IFN-γ), perforin, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8 + T cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.

HLA-C gene products are the most poorly understood of the HLA class I molecules because they express at low level on the cell surface compared to HLA-A and -B. However, recent evidence shows that HLA-C molecules are functionally competent in eliciting T-cell responses and in controlling NK-cell recognition. Approximately 20 to 50% of HLA-C alleles type "blank" in most populations. To provide a better definition of the HLA-C alleles, we analyzed 98 extensively characterized B-cell lines from the 10th International Histocompatibility Workshop. Selective HLA-C-specific DNA amplification of exons 2 and 3 from DNA prepared from the cell panel was achieved with the use of two sets of locus-specific primers. We used 64 sequence-specific oligonucleotide probes (SSOPs) complementary to variable sites in exons 2 and 3 to generate hybridization patterns. Twenty-five alleles were found among these patterns, including seven new alleles in the homozygous cell lines and seven potential new alleles in heterozygous cell lines. Differences between the new alleles and known alleles were generally small. Five major groups were identified in the Cw "blank" cells by the SSOP patterns. In addition, linkage between HLA-B specificities and HLA-C alleles was similar to previous observations. The present study demonstrated that SSOP typing was effective in identifying new alleles in homozygous typing cells but not in the heterozygous cells. Also, DNA typing can facilitate the identification of all HLA-C alleles, including those that serologically type as blanks. The HLA-C locus may be more polymorphic than was previously recognized.

Background/Aims: We intended to evaluate the association between specific human leukocyte antigen (HLA)-DRB1 gene polymorphism and antiviral response to lamivudine (LAM) therapy in chronic hepatitis B (CHB) patients. Methods: Six-digit HLA-DRB1 genotypes were determined using sequence-based typing in 334 CHB patients initially treated with LAM for at least 12 months. Antiviral response was evaluated every 3–6 months during LAM therapy. Results: Median age of the subjects was 43 years (range, 16–72). Median duration of LAM therapy was 69 months (range, 13–140). Median baseline serum hepatitis B virus (HBV DNA) level was 7.0 log10 copies/ml (range, 5.5–9.1). At 12 months of LAM therapy, serum HBV DNA was undetectable by solution hybridization method in 308 (88%) patients. Among 25 HLA-DRB1 alleles identified, HLA-DRB1*090102, *080302, and *070101 were the most frequent alleles (>10%). HLA-DRB1*010101 was identified in 5.4% (18/334). The frequency of the HLA-DRB1*010101 allele was significantly lower in patients with virological response at 12 months of LAM therapy than in patients without it (4.2 vs. 19.2%, p = 0.025). The other HLA-DRB1 alleles were not associated with virological response. HBeAg loss/seroconversion and alanine aminotransferase normalization were not associated with HLA-DRB1 alleles. Conclusion: The HLA-DRB1*010101 allele is closely associated with poor virological response to initial LAM therapy in CHB patients.

The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS ), and show that (i) the expression of two key determinants of net positive surface charge ( mprF and dlt ) is dependent on the cotranscription of both graR and vraG , (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF -dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG , the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.