SARS-CoV-2 B.1.619 and B.1.620 Lineages, South Korea, 2021.

We report the rapid emergence of severe acute respiratory syndrome coronavirus 2 lineages B.1.619 and B.1.620 in South Korea. The surge in frequency in a relatively short time emphasizes the need for ongoing monitoring for new lineages to track potential increases in transmissibility and disease severity and reductions in vaccine efficacy.

IFN-γ-producing NKT cells exacerbate sepsis by enhancing C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils.

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT-cell-deficient mice of C57BL/6 background during cecal ligation and puncture-induced sepsis. The levels of C5a, IFN-γ, and IL-10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d(-/-) mice, while the mortality rate was lower in CD1d(-/-) mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d(-/-) mice. As assessed by intracellular staining, NKT cells expressed IFN-γ, while neutrophils expressed IL-10. Upon coculture, IL-10-deficient NKT cells enhanced IL-10 production by WT, but not IFN-γR-deficient, neutrophils. Meanwhile, CD1d(-/-) mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL-10 administration into CD1d(-/-) mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN-γ-deficient, NKT cells into CD1d(-/-) mice suppressed CD55 expression on neutrophils, but increased IL-10 and C5a levels. Taken together, IFN-γ-producing NKT cells enhance C5a generation via IL-10-mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.

NOD2-mediated suppression of CD55 on neutrophils enhances C5a generation during polymicrobial sepsis.

Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2-/- mice. Nod2-/- mice showed lower levels of C5a, IL-10, and IL-1ß in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2-/- mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1ß-dependent and/or IL-1ß-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.

Invariant NKT cells producing IL-4 or IL-10, but not IFN-gamma, inhibit the Th1 response in experimental autoimmune encephalomyelitis, whereas none of these cells inhibits the Th17 response.

Experimental autoimmune encephalomyelitis (EAE) is mediated by Th1 and Th17 cells. Invariant NKT (iNKT) cells prevent EAE in an IL-4-, IL-10-, and IFN-γ-dependent manner. However, which of the iNKT cell-produced cytokines regulates the Th1 or Th17 response in EAE remains unclear. Wild-type B6 and Jα18(-/-) mice were immunized with MOG(35-55) peptide to address this issue. Clinical scores for EAE, IL-17, and IFN-γ transcript levels, and IL-17- or IFN-γ-expressing CD4(+) T cell percentages in the CNS and draining lymph nodes were higher in Jα18(-/-) than in B6 mice, but all of these parameters in the CNS were reduced by the adoptive transfer of wild-type or IFN-γ-deficient iNKT cells into the Jα18(-/-) mice before immunization. In contrast, adoptive transfer of IL-4- or IL-10-deficient iNKT cells into Jα18(-/-) mice decreased IL-17 transcript levels and the percentage of IL-17-expressing CD4(+) T cells in the CNS but did not affect clinical scores, IFN-γ transcript levels, or the percentage of IFN-γ-expressing CD4(+) T cells in the CNS. Taken together, IL-4- and IL-10-producing iNKT cells inhibit the Th1 cell response, but not the Th17 cell response, although wild-type iNKT cells suppress both the Th1 and Th17 responses in the CNS during EAE. Moreover, IFN-γ-producing iNKT cells have a minimal role in the regulation of the Th1 and Th17 responses in EAE.

A Seroprevalence Study on Residents in a Senior Care Facility with Breakthrough SARS-CoV-2 Omicron Infection.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began spreading rapidly in the community in November 2021, becoming the dominant variant in the Republic of Korea in 2022. Although its pathogenesis in healthy individuals was low, the severity and hospitalization rate was higher in the elderly and immunocompromised patients. We aimed to investigate the immunogenicity in acute and convalescent phases of breakthrough infection by Omicron in elderly individuals. Serological data were assessed by electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay, and plaque-reduction neutralization tests. SARS-CoV-2-specific antibody and immunoglobulin G levels in the acute phase were higher in third dose-vaccinated elderly than in first and second dose-vaccinated patients. The neutralization antibody titer was detected only in third dose-vaccinated patients, and the titer was higher for the Delta than the Omicron variant. In the convalescent phase of Omicron infection, the neutralization antibody titer of vaccinated patients was higher for the Delta than the Omicron variant except in unvaccinated individuals. We demonstrated that the cause of the vulnerability to Omicron variant infection in third dose-vaccinated elderly was due to the low neutralization antibody level against Omicron. A fourth dose of vaccination is required in the elderly to reduce hospitalization and mortality caused by the Omicron variant.

Neutralizing antibody responses in vaccinated and unvaccinated individuals infected with Omicron BA.1 variant.

BACKGROUND: The Omicron variant, with numerous mutations in the spike protein, reduces vaccine-induced immunity, leading to breakthrough infections. However, vaccine protection after infection with the Omicron variant is unclear. AIMS AND METHODS: To compare the neutralizing antibody responses between unvaccinated and vaccinated individuals infected with the Omicron variant, we have collected serial plasma samples from five unvaccinated and four vaccinated individuals with Omicron variant infection, including the first Omicron breakthrough infection case in the Republic of Korea. We evaluated neutralization antibody titers against D614G, Delta, and Omicron using live virus neutralizing assay, and calculated the plaque reduction neutralizing test value. RESULTS: In patients with two-dose vaccinations, neutralizing antibodies against Omicron variant were detected in plasma collected 4-9 days post symptom onset. However, in the plasma from unvaccinated patients and those vaccinated with one dose, neutralizing antibodies against the Omicron variant at the same time point were undetectable. Next, the 1- or 2-dose vaccinated infected groups showed potent cross-neutralizing activity against D614G and Delta variants after 11-14 days. In contrast, the neutralizing antibody titers in the unvaccinated group were low or undetectable. CONCLUSIONS: The major limitation of this study is the small sample size due to the limited samples targeting the first reported cases of Omicron BA.1 variant infection in the Republic of Korea (n = 9). Nevertheless, we found that vaccinated individuals rapidly produced neutralizing antibodies against Omicron, and potent cross-neutralizing antibodies against D614G and Delta upon infection with Omicron.

An outbreak of SARS-CoV-2 reinfection in a long-term care facility in South Korea.

We report a cluster of 12 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection in a long-term care facility in South Korea. There were two outbreaks of SARS-CoV-2 infection in the facility at the beginning and end of October 2021, respectively. All residents in the facility were screened for SARS-CoV-2 infection using RT-PCR as part of the investigation of the second outbreak. Twelve residents, who had infection confirmed during the first outbreak, were found to be re-positive for RT-PCR test at the second outbreak. 8 Of 12 RT-PCR re-positive cases were confirmed as reinfections based on investigation through the whole genome sequencing, viral culture, and serological analysis, despite of the short interval between the first and second outbreaks (29-33 days) and a history of full vaccination for 7 of the 12 re-positive cases. This study suggests that decreased immunity and underlying health condition in older adults makes them susceptible to reinfection, highlighting the importance of prevention and control measures regardless of vaccination status in long-term care settings.

Liver X receptor mediates hepatitis B virus X protein-induced lipogenesis in hepatitis B virus-associated hepatocellular carcinoma.

UNLABELLED: Although hepatitis B virus X protein (HBx) has been implicated in abnormal lipid metabolism in hepatitis B virus (HBV)-associated hepatic steatosis, its underlying molecular mechanism remains unclear. Liver X receptor (LXR) plays an important role in regulating the expression of genes involved in hepatic lipogenesis. Here we demonstrate that LXRalpha and LXRbeta mediate HBV-associated hepatic steatosis. We have found that HBx induces the expression of LXR and its lipogenic target genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and peroxisome proliferator-activated receptor, and this is accompanied by the accumulation of lipid droplets. RNA interference with LXR expression decreases the amount of lipid droplets as well as the expression of the lipogenic genes, and this indicates that HBx-induced lipogenesis is LXR-dependent. LXRalpha and HBx colocalize in the nucleus and are physically associated. HBx induces the transactivation function of LXRalpha by recruiting CREB binding protein to the promoter of the target gene. Furthermore, we have observed that expression of LXR is increased in the livers of HBx-transgenic mice. Finally, there is a significant increase in the expression of LXRbeta (P = 0.036), SREBP-1c (P = 0.008), FAS, and stearoyl-coenyzme A desaturase-1 (P = 0.001) in hepatocellular carcinoma (HCC) in comparison with adjacent nontumorous nodules in human HBV-associated HCC specimens. CONCLUSION: Our results suggest a novel association between HBx and LXR that may represent an important mechanism explaining HBx-induced hepatic lipogenesis during HBV-associated hepatic carcinogenesis.

Role of type II NKT cells in the suppression of graft-versus-host disease.

Natural killer T (NKT) cells, a distinct subset of T cells that recognize glycolipids on CD1d molecules, express both TCR and NK receptors and are critical in regulating various immune responses by modulating the Th1/Th2 balance. Upon activation, NKT cells produce large amounts of IL-4 and IFN-gamma, resulting in the enhancement or inhibition of immune responses. Recent studies have shown that NKT cells are heterogeneous in terms of the expression of a specific Valpha chain of TCR (Valpha14-Jalpha18 in mice and Valpha24-JalphaQ in humans) and reactivity against the glycolipid alpha-galactosylceramide (alpha-GalCer). Accordingly, NKT cells are classified into type I (invariant) and type II (non-invariant) cells in mice and humans. Although the functional roles of type I NKT cells are well characterized in various immune diseases, little is known regarding the function of type II NKT cells. Recent study has demonstrated that type II NKT cells in donor bone marrow play protective roles in graft-versus-host disease (GVHD), in which the complicated immunologic processes are involved. In this review, we discuss the pathogenesis of GVHD and the distinct functions of type II NKT cells in the development of GVHD.

HBx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species.

Hepatitis B virus X protein (HBx) is involved in viral metabolism and progression of liver disease. Iron metabolism plays a significant role in liver disease. In this report, to elucidate the relationship between iron metabolism and HBx, we established the Huh7 cell lines in which HBx was stably expressed (Huh7-HBx). In Huh7-HBx, we observed that transferrin receptor 1 (TfR1) expression decreased and ferritin heavy chain (FtH) expression increased as well as reactive oxygen species (ROS) level increased. We also found that these modulations were caused by the downregulation of iron regulatory protein 1 (IRP1). Furthermore, the levels of total iron and labile iron pool (LIP) were altered in Huh7-HBx. In addition, antioxidant N-acetylcystein (NaC) increased IRP1 expression by depleting HBx-induced ROS. We also confirmed these alterations of TfR1 and FtH in the primary hepatocytes of HBx transgenic mice and in HepG2.2.15 cells that constitutively replicate the intact HBV genome. In conclusion, these results suggest that HBx modulates iron metabolism via ROS leading to pathological status in liver diseases.

GM-CSF and IL-4 produced by NKT cells inversely regulate IL-1ß production by macrophages.

Natural Killer T (NKT) cells are distinct T cell subset that link innate and adaptive immune responses. IL-1ß, produced by various immune cells, plays a key role in the regulation of innate immunity in vivo. However, it is unclear whether NKT cells regulate IL-1ß production by macrophages. To address this, we co-cultured NKT cells and peritoneal macrophages in the presence of TCR stimulation and inflammasome activators. Among cytokines secreted from NKT cells, GM-CSF enhanced IL-1ß production by macrophages via regulating LPS-mediated pro-IL-1ß expression and NLRP3-dependent inflammasome activation, whereas IL-4 enhanced M2-differentiation of macrophages and decreased IL-1ß production. Together, our findings suggest the NKT cells have double-sided effects on IL-1ß-mediated innate immune responses by producing IL-4 and GM-CSF. These findings may be helpful for a comprehensive understanding of NKT cell-mediated regulatory mechanisms of the pro-inflammatory effects of IL-1ß in inflammatory diseases in vivo.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N2(-/-)) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2(-/-) mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1(-)CD44(+) to the NK1.1(+)CD44(+) stage, resulting in accumulation of NK1.1(-)CD44(+) invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2(-/-) mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d(-/-) mice. Unlike N1N2(-/-) mice, RBP-j(lox) (/) (lox) CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus.

CD4+ FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome.

OBJECTIVE: We investigated whether the frequency, phenotype, and suppressive function of CD4+ FOXP3+ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls. DESIGN AND METHODS: Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4-35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (-) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4+ CD25bright were co-cultured with autologous CD4+ CD25- target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function. RESULTS: Despite a lower frequency of CD4+ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3+ Tregs among CD4+ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3+, and CCR4+ CCR6+ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4+ CD25- T cells was significantly impaired in the TS (-) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (-) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group. CONCLUSIONS: The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4+ T cells.

Proteomic analysis of pancreas in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Organ transplantation is limited by the shortage of human organs. Many studies have sought to overcome this hurdle by using animal organs. Porcine organs, especially from miniature pigs, have been used for organ xenotransplantation rather than nonhuman primates. While the molecular profiling for transplantation is well known in humans and rodents, the situation for pigs is almost completely unknown. The present study examined protein regulation of the developing stages of the pancreatic proteome (4 day-old miniature neonate, 19 day-old miniature piglet, and 14 month-old miniature adult pigs) using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen different expressed spots were observed and nine were identified. The data presented within this study provides critical direction relating to the development of pancreas of miniature pigs, which will assist future proteome analysis of the pancreas, and advance our understanding of the hurdles facing xenotransplantation.

Proteomic analysis of domestic pig pancreas during development using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Pig pancreas may be a therapeutic resource for human diabetic patients. However, this potential is hindered by a lack of knowledge of the molecular events of pig pancreas development. In this study, the embryonic day 60, neonate and 6-month protein profiles of pig pancreas were ascertained at using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Twenty four proteins were differentially expressed during pig pancreas development. Among them, 12 spots increased and 7 spots decreased according to development. The expression of 5 protein were highest at birth. Expression of digestive enzymes including trypsin, pancreatic triacylglycerol lipase and pancreatic alpha-amylase was elevated in adults, whereas chymotrypsins were highly expressed in neonates. Proteins that were abundantly expressed during gestation were alpha-1-antitrypsin, alpha-fetoprotein and transferrins. Taken together, we found out that several proteins were significantly up- or down- regulated from pig pancreas based on developmental stage. This study will provide basis for understanding development of pig pancreas.

Proteomic analysis of liver in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Due to the shortage of human organ donors for transplant, various studies of xenotransplantation, or the use of animal organs instead of human organs, have been carried out. The organs of porcine are thought to be safer and of a more suitable size for xenotransplantationthan those of nonhuman primates. Understanding the levels of expression of proteins, and their post-translational regulation, would be very practical between different species and among developing stages, though the molecular profiling for xenotransplantation has been rarely studied for porcine, while that of human and rodent is well known. Here, in this present study, we report protein regulation of the developing stages of liver (4-day old neonate, 19-day old piglet and 14-month old adult miniature pigs) using 2-DE and MALDI-TOF. From images of the three different stages, a total of 8 spotswhich were differently regulated were identified, and 5 spots were identified with MALDI-TOF MS. The data presented within this study provides critical direction relating to the development of livers of miniature pigs, which will assist future proteome analysis of the liver, and advance our understanding of the hurdles facing xenotransplantaion.

Direct engagement of TLR4 in invariant NKT cells regulates immune diseases by differential IL-4 and IFN-γ production in mice.

During interaction with APCs, invariant (i) NKT cells are thought to be indirectly activated by TLR4-dependently activated APCs. However, whether TLR4 directly activates iNKT cells is unknown. Therefore, the expression and function of TLR4 in iNKT cells were investigated. Flow cytometric and confocal microscopic analysis revealed TLR4 expression on the surface and in the endosome of iNKT cells. Upon LPS stimulation, iNKT cells enhanced IFN-γ production, but reduced IL-4 production, in the presence of TCR signals, depending on TLR4, MyD88, TRIF, and the endosome. However, enhanced TLR4-mediated IFN-γ production by iNKT cells did not affect IL-12 production or CD1d expression by DCs. Adoptive transfer of WT, but not TLR4-deficient, iNKT cells promoted antibody-induced arthritis in CD1d(-/-) mice, suggesting that endogenous TLR4 ligands modulate iNKT cell function in arthritis. Furthermore, LPS-pretreated WT, but not TLR4-deficient, iNKT cells suppressed pulmonary fibrosis, but worsened hypersensitivity pneumonitis more than untreated WT iNKT cells, indicating that exogenous TLR4 ligands regulate iNKT cell functions in pulmonary diseases. Taken together, we propose a novel direct activation pathway of iNKT cells in the presence of TCR signals via endogenous or exogenous ligand-mediated engagement of TLR4 in iNKT cells, which regulates immune diseases by altering IFN-γ and IL-4 production.

Comparison of Invariant NKT Cells with Conventional T Cells by Using Gene Set Enrichment Analysis (GSEA).

BACKGROUND: Invariant Natural killer T (iNKT) cells, a distinct subset of CD1d-restricted T cells with invariant Vαß TCR, functionally bridge innate and adaptive immunity. While iNKT cells share features with conventional T cells in some functional aspects, they simultaneously produce large amount of Th1 and Th2 cytokines upon T-cell receptor (TCR) ligation. However, gene expression pattern in two types of cells has not been well characterized. METHODS: we performed comparative microarray analyses of gene expression in murine iNKT cells and conventional CD4(+)CD25(-)γδTCR(-) T cells by using Gene Set Enrichment Analysis (GSEA) method. RESULTS: Here, we describe profound differences in gene expression pattern between iNKT cells and conventional CD4(+)CD25(-)γδTCR(-) T cells. CONCLUSION: Our results provide new insights into the functional competence of iNKT cells and a better understanding of their various roles during immune responses.

CD4(+)CD25(+) regulatory T cells attenuate Hypersensitivity Pneumonitis by suppressing IFN-gamma production by CD4(+) and CD8(+) T cells.

HP results from the repeated inhalation of environmental antigens; however, the roles of CD4(+)CD25(+) T(reg) cells in HP are unknown. Therefore, we investigated the functions of CD4(+)CD25(+) T(reg) cells in SR-induced murine HP. More severe HP was observed in CD4(+)CD25(+) T(reg) cell-depleted mice than in control mice in terms of histological alterations, inflammatory cell numbers in BALF, and the serum level of SR-specific IgG, which were restored by the adoptive transfer of CD4(+)CD25(+) T(reg) cells. The CD4(+)CD25(+) T(reg) cell-depleted mice also showed elevated levels of IFN-gamma, TGF-beta, and reduced IL-4 production in the lungs. Moreover, IL-10 production of CD4(+)CD25(+) T(reg) cells and direct contact between CD4(+)CD25(+) T(reg) cells and CD4(+) or CD8(+) T cells in BALF resulted in reduced IFN-gamma production. Taken together, CD4(+)CD25(+) T(reg) cells play a protective role in SR-induced HP by suppressing IFN-gamma production by T cells.