Eriocitrin Inhibits Angiogenesis by Targeting VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways.

Eriocitrin, a flavanone found in peppermint and citrus fruits, is known to possess many physiological activities. However, the anti-angiogenic effects of eriocitrin are yet to be fully elucidated. Therefore, the objective of this research was to explore the anti-angiogenic effects of eriocitrin both in vitro and in vivo as well as its underlying mechanism. Anti-angiogenic effects of eriocitrin were evaluated utilizing in vitro models of angiogenesis, including inhibition of tube formation, and induction of apoptosis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay in chick embryos was also performed to evaluate the in vivo effects of eriocitrin on angiogenesis. Results showed significant eriocitrin effects on proliferation, tube formation, migration, and apoptosis in HUVECs. Furthermore, in vivo analysis revealed that eriocitrin significantly suppressed the formation of new blood vessels. In particular, it regulated MAPK/ERK signaling pathway and VEGFR2, inhibited the downstream PI3K/AKT/mTOR signaling pathway, and activated apoptosis signals such as caspase cascades. In HUVECs, the expression of matrix metalloproteinases (MMP-2 and MMP-9) exhibited an inhibitory effect on angiogenesis through the suppression of the signaling pathway. Therefore, eriocitrin presents potential for development into an antiangiogenic therapeutic agent.

Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma.

BACKGROUND: Major histocompatibility complex (MHC) class I chain-related protein (MIC) is a stress-induced ligand released from multiple myeloma (MM) cells during progression, and soluble MIC impairs natural killer group 2D (NKG2D) activating receptor-mediated recognition and function of natural killer (NK) cells. However, whether clearing soluble MIC with a monoclonal antibody (mAb) can restore NK cell activity of MM patients remains undetermined. METHODS: We analyzed The Cancer Genome Atlas (TCGA) Multiple Myeloma Research Foundation (MMRF) CoMMpass data set to examine the prognostic significance of MIC expression in MM. We examined the level of soluble MIC in paired peripheral blood (PB) and bone marrow (BM) plasma of patients with MM at diagnosis by ELISA. We evaluated the correlation between the level of soluble MIC and immunophenotype of NK cells from MM patients by multicolor flow cytometry. We also generated MIC-overexpressing MM cell line and characterized the cytotoxic function of patient NK cells in the presence of soluble MIC, and examined the impact of clearing soluble MIC with a humanized mAb (huB10G5). RESULTS: We characterize the importance of MICA in MM by revealing the significantly better overall survival of patients with high MICA expression from TCGA MMRF CoMMpass data set. The level of soluble MICA is more highly elevated in MM than in precursor stages, and the concentration of soluble MICA is higher in BM plasma than in PB. The concentration of soluble MICA in BM was correlated with myeloma burden, while it was negatively correlated with the frequency of NKG2D+ NK cells in diagnostic BM aspirates of MM patients. Soluble MICA downregulated NKG2D expression and decreased cytotoxicity of MM patient NK cells ex vivo, which were reversed by a humanized soluble MIC-clearing mAb (huB10G5) with enhanced degranulation of NK cells. CONCLUSIONS: Our findings indicate targeting soluble MIC with huB10G5 might be a viable therapeutic approach to promote NKG2D-dependent cellular immunotherapy outcome in MM.

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater.

Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 108 to 105 colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 105 CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety.

Phase I Study: Safety and Efficacy of an Ex Vivo-Expanded Allogeneic Natural Killer Cell (MG4101) with Rituximab for Relapsed/Refractory B Cell Non-Hodgkin Lymphoma.

The prognosis of non-Hodgkin lymphoma (NHL) remains poor, with an unmet need for novel therapies. MG4101, an ex vivo-expanded allogeneic natural killer (NK) cell, can enhance rituximab antibody-dependent cytotoxicity in relapsed/refractory (r/r) B cell non-Hodgkin lymphoma. This study assessed the safety and efficacy of MG4101 plus rituximab for patients with r/r NHL. Patients received escalating doses of i.v. MG4101 plus rituximab every 2 weeks. IL-2 was administered s.c. after MG4101 treatment. Fludarabine plus cyclophosphamide was administered i.v. before rituximab treatment in cycles 1, 3, and 5. A 3+3 design was used to determine the maximum tolerated dose (MTD) and maximum feasible dose. Assessments were performed over a 6-cycle period, with an extended maintenance period of up to 8 cycles. Nine patients received 3 different doses of MG4101 and rituximab. MTD could not be determined because of the absence of dose-limiting toxicity. Treatment-related adverse events, mostly grade 1 or 2, occurred in 89% of patients. Only 1 patient experienced grade 1 cytokine release syndrome. MG4101 persisted for at least 7 days in 7 patients. Four patients achieved a partial response and 1 patient attained a complete response, for an overall response rate of 55.6%. Two patients showed prolonged responses and low exhaustion marker levels in T cells. For allogeneic NK cell therapy, strategies including the use of the high-affinity hFcγRIIIaV158 variant of the KIR B/x haplotype with lymphodepleting chemotherapy may be promising options for improving clinical efficacy in the antibody combination therapeutic setting as an off-the-shelf product. MG4101 plus rituximab presented a favorable safety profile and overall response rate in patients with r/r NHL.

Development and Evaluation of a Next-Generation Sequencing Panel for the Multiple Detection and Identification of Pathogens in Fermented Foods.

These days, bacterial detection methods have some limitations in sensitivity, specificity, and multiple detection. To overcome these, novel detection and identification method is necessary to be developed. Recently, NGS panel method has been suggested to screen, detect, and even identify specific foodborne pathogens in one reaction. In this study, new NGS panel primer sets were developed to target 13 specific virulence factor genes from five types of pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium, respectively. Evaluation of the primer sets using singleplex PCR, crosscheck PCR and multiplex PCR revealed high specificity and selectivity without interference of primers or genomic DNAs. Subsequent NGS panel analysis with six artificially contaminated food samples using those primer sets showed that all target genes were multi-detected in one reaction at 108-105 CFU of target strains. However, a few false-positive results were shown at 106-105 CFU. To validate this NGS panel analysis, three sets of qPCR analyses were independently performed with the same contaminated food samples, showing the similar specificity and selectivity for detection and identification. While this NGS panel still has some issues for detection and identification of specific foodborne pathogens, it has much more advantages, especially multiple detection and identification in one reaction, and it could be improved by further optimized NGS panel primer sets and even by application of a new real-time NGS sequencing technology. Therefore, this study suggests the efficiency and usability of NGS panel for rapid determination of origin strain in various foodborne outbreaks in one reaction.

Mouse feeding study and microbiome analysis of sourdough bread for evaluation of its health effects.

Sourdough bread fermented with yeast and lactic acid bacteria (LAB) is thought to have various beneficial health effects. However, its beneficial effects were not fully evaluated with in vivo mouse model. To evaluate these effects in vivo, a mouse feeding study and microbiome analysis of white bread containing 40% sourdough (WBS) and yeast-leavened white bread (WB) were performed. Although feed consumption and body weight increased with WBS, the glycemic index was reduced, suggesting a diabetes-lowering effect, probably due to the presence of dietary fiber and short-chain fatty acids (SCFA). In addition, a mineral absorption test showed that WBS increased magnesium absorption owing to phytate degradation during fermentation. Interestingly, WBS decreased total cholesterol and triglycerides, probably due to the dietary fiber and SCFA in LAB. In addition, the ratio of low- and high-density lipoprotein was decreased in WBS, implying potential risk reduction for cardiovascular disease. An immunomodulatory assay of WBS revealed that pro-inflammatory cytokines TNF-α and IL-6 were decreased, suggesting anti-inflammatory activity. Gluten degradation by fermentation and antioxidation activity of menaquinol/ubiquinol by gut microbiota also supported the anti-inflammatory activity of sourdough bread. Furthermore, some beneficial gut bacteria, including Akkermansia, Bifidobacterium, and Lactobacillus, were increased in WBS. In particular, Akkermansia has been associated with anti-inflammatory properties. Consequently, WBS has beneficial effects on health, including decreased glycemic index and cholesterol, increased mineral availability and absorption, anti-inflammatory properties, and establishment of healthy gut microbiota.

Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open-label, non-randomised, phase 1 trials in healthy adults.

BACKGROUND: We assessed the safety and immunogenicity of two recombinant DNA vaccines for COVID-19: GX-19 containing plasmid DNA encoding the SARS-CoV-2 spike protein, and GX-19N containing plasmid DNA encoding the SARS-CoV-2 receptor-binding domain (RBD) foldon, nucleocapsid protein, and plasmid DNA encoding the spike protein. METHODS: Two open-label non-randomised phase 1 trials, one of GX-19 and the other of GX-19N were done at two hospitals in South Korea. We enrolled healthy adults aged 19-49 years for the GX-19 trial and healthy adults aged 19-54 years for the GX-19N trial. Participants who tested positive by serological testing for SARS-CoV-2 were excluded. At 4-week intervals, the GX-19 trial participants received two vaccine doses (either 1·5 mg or 3·0 mg), and the GX-19N trial participants received two 3·0 mg doses. The vaccines were delivered intramuscularly using an electroporator. The participants were followed up for 52 weeks after first vaccination. Data collected up to day 57 after first vaccination were analysed in this interim analysis. The primary outcome was safety within 28 days after each vaccination measured in the intention-to-treat population. The secondary outcome was vaccine immunogenicity using blood samples collected on day 43 or 57 after first vaccination measured in the intention-to-treat population. The GX-19 (NCT044445389) and GX-19N (NCT04715997) trials are registered with ClinicalTrials.gov. FINDINGS: Between June 17 and July 30, 2020, we screened 97 individuals, of whom 40 (41%) participants were enrolled in the GX-19 trial (20 [50%] in the 1·5 mg group and 20 [50%] in the 3·0 mg group). Between Dec 28 and 31, 2020, we screened 23 participants, of whom 21 (91%) participants were enrolled on the GX-19N trial. 32 (52%) of 61 participants reported 80 treatment-emergent adverse events after vaccination. All solicited adverse events were mild except one (2%) case of moderate fatigue in the 1·5 mg GX-19 group; no serious vaccine-related adverse events were detected. Binding antibody responses increased after second dose of vaccination in all groups (p=0·0002 in the 1·5 mg GX-19 group; p<0·0001 in the 3·0 mg GX-19; and p=0·0004 for the spike protein and p=0·0001 for the RBD in the 3·0 mg GX-19N group). INTERPRETATION: GX-19 and GX-19N are safe and well tolerated. GX-19N induces humoral and broad SARS-CoV-2-specific T-cell responses. GX-19N shows lower neutralising antibody responses and needs improvement to enhance immunogenicity. FUNDING: The Korea Drug Development Fund, funded by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare.

Longitudinal Assessment of Antisevere Acute Respiratory Syndrome Coronavirus 2 Immune Responses for Six Months Based on the Clinical Severity of Coronavirus Disease 2019.

BACKGROUND: There is insufficient data on the longevity of immunity acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19). The study population comprised asymptomatic (n = 14), symptomatic/nonpneumonic (n = 42), and pneumonic (n = 41) patients. RESULTS: The anti-SARS-CoV-2 immunoglobulin class G and neutralizing antibody (NAb) titers lasted until 6 months after diagnosis, with positivity rates of 66.7% and 86.9%, respectively. Older age, prolonged viral shedding, and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. Severe acute respiratory syndrome coronavirus 2-specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. CONCLUSIONS: In conclusion, most (>85%) patients carry NAb until 6 months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.

Dynamic changes in circulating PD-1+CD8+ T lymphocytes for predicting treatment response to PD-1 blockade in patients with non-small-cell lung cancer.

BACKGROUND: The predictive value of immune monitoring with circulating CD8+ T lymphocytes for treatment response to programmed cell death protein 1 (PD-1) inhibitors has not been explored in non-small-cell lung cancer (NSCLC), prompting us to investigate whether dynamic changes in PD-1+CD8+ T lymphocytes have predictive value for durable clinical benefit (DCB) and survival after PD-1 blockade. METHODS: Patients with recurrent and/or metastatic NSCLC treated with PD-1 inhibitors were enrolled (discovery cohort; n = 94). Peripheral blood was obtained immediately before and after one cycle of treatment with PD-1 blockade. Phenotyping of circulating CD8+ T lymphocytes was conducted using multi-colour flow cytometry. Predictive values of dynamic changes in circulating PD-1+CD8+ T lymphocytes during the first cycle were validated in an independent cohort (validation cohort; n = 54) of a prospective trial with a PD-1 inhibitor (NCT03486119). RESULTS: Circulating PD-1+CD8+ T lymphocytes were enriched with effector/memory populations with elevated expression of activation- and exhaustion-related markers. Reduction in the frequency of PD-1+ cells among CD8+ T lymphocytes after one cycle of treatment was associated with a higher probability of DCB and superior survival outcomes in the discovery cohort. Similar results were obtained in the analysis of tumour antigen NY-ESO-1-specific CD8+ T lymphocytes and the validation cohort. Mechanistically, PD-1 molecule expression on CD8+ T lymphocytes suppresses the effector functions of tumour antigen-specific CD8+ T lymphocytes. CONCLUSIONS: Dynamic changes in circulating PD-1+CD8+ T lymphocytes predict clinical, and survival benefit from PD-1 blockade treatment in NSCLC, providing a useful tool to identify patient subgroups who will optimally benefit from PD-1 inhibitors.

Increased frequency of CD4+CD57+ senescent T cells in patients with newly diagnosed acute heart failure: exploring new pathogenic mechanisms with clinical relevance.

Recent animal studies showed T cells have a direct pathogenic role in the development of heart failure (HF). However, which subsets of T cells contribute to human HF pathogenesis and progression remains unclear. We characterized immunologic properties of various subsets of T cells and their clinical implications in human HF. Thirty-eight consecutive patients with newly diagnosed acute HF (21 males, mean age 66 ± 16 years) and 38 healthy control subjects (21 males, mean age 62 ± 12 years) were enrolled. We found that pro-inflammatory mediators, including CRP, IL-6 and IP-10 and the frequencies of CD57+ T cells in the CD4+ T cell population were significantly elevated in patients with acute HF compared to control subjects. A functional analysis of T cells from patients with acute HF revealed that the CD4+CD57+ T cell population exhibited a higher frequency of IFN-γ- and TNF-α- producing cells compared to the CD4+CD57- T cell population. Furthermore, the frequency of CD4+CD57+ T cells at baseline and its elevation at the six-month follow-up were significantly related with the development of cardiovascular (CV) events, which were defined as CV mortality, cardiac transplantation, or rehospitalization due to HF exacerbation. In conclusion, CD4+CD57+ senescent T cells showed more inflammatory features and polyfunctionality and were associated with clinical outcome in patients with acute HF. More detailed study for senescent T cells might offer new opportunities for the prevention and treatment of human HF.

Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets.

Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10-20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection.

Tumor Necrosis Factor and Regulatory T Cells.

CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells play major roles in the maintenance of immune homeostasis. In this review, we comprehensively describe the relationship between tumor necrosis factor (TNF) and Treg cells, focusing on the effects of TNF on Treg cells and on TNF-producing Treg cells. Contradictory results have been reported for the effect of TNF on the suppressive activity of Treg cells. In patients with rheumatoid arthritis, TNF has been shown to reduce the suppressive activity of Treg cells. Meanwhile, however, TNF has also been reported to maintain the suppressive activity of Treg cells via a TNFR2-mediated mechanism. In addition, Treg cells have been found to acquire the ability to produce TNF under inflammatory conditions, such as acute viral hepatitis. These TNF-producing Treg cells exhibit T helper 17-like features and hold significance in various human diseases.

Herpes Zoster DNA Vaccines with IL-7 and IL-33 Molecular Adjuvants Elicit Protective T Cell Immunity.

Herpes zoster (HZ), or shingles, is caused by the reactivation of latent varicella-zoster virus (VZV) from the sensory ganglia when VZV-specific T-cell immunity is decreased because of aging or immunosuppression. In the present study, we developed HZ DNA vaccine candidates encoding VZV proteins and cytokine adjuvants, such as IL-7 and IL-33. We immunized C57BL/6 mice with DNA plasmids encoding VZV glycoprotein E (gE), immediate early (IE) 63, or IE62 proteins and found that robust VZV protein-specific T-cell responses were elicited by HZ DNA vaccination. Co-administration of DNA plasmids encoding IL-7 or IL-33 in HZ DNA vaccination significantly enhanced the magnitude of VZV protein-specific T-cell responses. Protective immunity elicited by HZ DNA vaccination was proven by challenge experiments with a surrogate virus, vaccinia virus expressing gE (VV-gE). A single dose of HZ DNA vaccine strongly boosted gE-specific T-cell responses in mice with a history of previous infection by VV-gE. Thus, HZ DNA vaccines with IL-7 and IL-33 adjuvants strongly elicit protective immunity.

IL-17A-Producing Foxp3+ Regulatory T Cells and Human Diseases.

CD4+Foxp3+ regulatory T (Treg) cells play major roles in immune homeostasis. While CD4+Foxp3+ Treg cells act to suppress other immune effector cells, there is growing evidence that they also produce pro-inflammatory cytokines, such as IL-17A, in inflammatory conditions. The pro-inflammatory cytokine milieu, toll-like receptor (TLR) signaling, and specific transcription factors are important for the production of IL-17A by CD4+Foxp3+ Treg cells. In particular, IL-17A-producing CD4+Foxp3+ Treg cells express RORγt, the T helper (Th) 17-specific transcription factor, in addition to Foxp3. IL-17A-producing CD4+Foxp3+ Treg cells are also involved in the pathogenesis of various diseases. Here we review the mechanisms underlying the induction of IL-17A-producing CD4+Foxp3+ Treg cells and the roles of these cells in human disease.

Nano-patterning of a stainless steel microneedle surface to improve the dip-coating efficiency of a DNA vaccine and its immune response.

DNA vaccination with microneedles (MNs) into the skin represents a potential therapeutic approach for the clinical treatment of viral diseases as well as for intradermal genetic immunization. In this study, we investigated a DNA vaccination against the severe fever with thrombocytopenia syndrome virus (SFTSV) delivered by nano-patterned microneedles (nMNs) to improve the efficiency compared to a conventional MN vaccination. Because DNA vaccinations delivered by coated MNs have major disadvantages such as a poor coating efficiency and immunogenicity, additional excipients are necessary. Therefore, we developed nMNs to improve the affinity of stainless steel for plasmid DNA vaccinations. The results show that the nMNs have an improved DNA vaccine loading capacity because their surfaces have an increased hydrophilicity from the high surface/volume ratio. The cytocompatibility analysis also showed a higher cell proliferation when using the nMNs. Finally, the in vivo experiments with balb/c mice vaccinated with the SFTSV DNA vaccine-coated nMNs generated a higher level of cellular immune responses than that of the unmodified MNs.