DR30318, a novel tri-specific T cell engager for Claudin 18.2 positive cancers immunotherapy.

BACKGROUND: Claudin 18.2 (CLDN18.2) is a highly anticipated target for solid tumor therapy, especially in advanced gastric carcinoma and pancreatic carcinoma. The T cell engager targeting CLDN18.2 represents a compelling strategy for enhancing anti-cancer efficacy. METHODS: Based on the in-house screened anti-CLDN18.2 VHH, we have developed a novel tri-specific T cell engager targeting CLDN18.2 for gastric and pancreatic cancer immunotherapy. This tri-specific antibody was designed with binding to CLDN18.2, human serum albumin (HSA) and CD3 on T cells. RESULTS: The DR30318 demonstrated binding affinity to CLDN18.2, HSA and CD3, and exhibited T cell-dependent cellular cytotoxicity (TDCC) activity in vitro. Pharmacokinetic analysis revealed a half-life of 22.2-28.6 h in rodents and 41.8 h in cynomolgus monkeys, respectively. The administration of DR30318 resulted in a slight increase in the levels of IL-6 and C-reactive protein (CRP) in cynomolgus monkeys. Furthermore, after incubation with human PBMCs and CLDN18.2 expressing cells, DR30318 induced TDCC activity and the production of interleukin-6 (IL-6) and interferon-gamma (IFN-γ). Notably, DR30318 demonstrated significant tumor suppression effects on gastric cancer xenograft models NUGC4/hCLDN18.2 and pancreatic cancer xenograft model BxPC3/hCLDN18.2 without affecting the body weight of mice.

Correlation Between Systemic Immune-Inflammation Index and Suicide Attempts in Children and Adolescents with First-Episode, Drug-Naïve Major Depressive Disorder During the COVID-19 Pandemic.

Objective: This study aims to investigate the association between the systemic immune-inflammatory index (SII) and suicide attempt (SA) in children and adolescents with first-episode, drug-naïve Major Depressive Disorder (MDD) during the COVID-19 pandemic. Methods: A retrospective study was conducted on 263 MDD patients hospitalized at the Third Hospital of Fuyang City between 2020 and 2022. Patients were categorized into two groups based on the presence of previous SA. The study compared the differences in SII and clinical characteristics between the two groups and used the receiver operating characteristic (ROC) curve to determine the optimal critical value of SII and the area under the curve. Binary logistic regression was used to analyze the independent risk factors for SA. Results: Compared with the patients without SA history, the patients with a personal history of SA had a higher mean HDRS scores (Z=-2.369, p=0.018), higher mean neutrophil count (Z=-2.870, p=0.004), higher mean platelet count (Z=-2.155, p=0.031), and higher mean SII (Z=-3.170, p=0.002). The optimal critical SII determined by the ROC curve was 548.15 (sensitivity = 63.2%, specificity = 83.1%), and the area under the curve was 0.661. After adjusting for gender, age, BMI, illness duration and HDRS score, the risk of total SA in patients with high SII was 8.296 times higher than in those with low SII (OR = 8.296, 95% CI: 3.803-18.095, P < 0.001), The risk of recent SA was 13.922 times higher in patients with high SII than in those with low SII (OR = 13.922, 95% CI: 5.587-34.693, p < 0.001). However, high SII was not a risk factor for past SA (OR = 0.547, 95% CI: 0.062-4.842, P=0.587). Conclusion: SII may be an inexpensive, easily accessible strategy that can assist in determining suicide risk in adolescents with MDD.

Correlation of systemic immune-inflammation index and moderate/major depression in patients with depressive disorders: a large sample cross-sectional study.

Objective: To evaluate the clinical value of systemic immune-inflammation index (SII) based on peripheral blood neutrophil, lymphocyte, and platelet count in evaluating the subtype and severity of depression in patients with depressive disorder. Methods: This retrospective cohort study was conducted in the Third People's Hospital of Fuyang City from January 1, 2020 to December 31, 2022. The data included sociodemographic information at admission, clinical data, discharge diagnosis and inflammatory markers. Patients were divided into low SII group and high SII group according to the optimal threshold of SII determined by receiver operating characteristic curve (ROC curve). Binary logistic regression was used to analyze the correlation between moderate/major depression and SII level. Results: Compared to the low SII group, the high SII group had a higher age level (χ2 = 7.663, p = 0.006), more smokers (χ2 = 9.458, p = 0.002), more moderate/major depression patients (χ2 = 45.645, p < 0.001), and a higher proportion of patients with accompanying somatic symptoms (χ2 = 14.867, p < 0.001). In the final logistic regression model, after controlling for confounding factors, SII at admission was significantly associated with moderate/major depression [ß =1.285, p < 0.001; odds ratio (95% confidence intervals) = 3.614 (2.693-4.850)]. Patients with high SII scores were 3.614 times more likely to have moderate/severe depression than those with low SII scores. We propose a cut-off value of SII =540.78 (sensitivity = 36.4% and specificity = 80.3%) according to the maximum Youden index. Conclusion: Our research indicates that SII may be a useful, repeatable, convenient, and affordable index to identify moderate/major depression in depressive disorder.

Development of a Humanized VHH Based Recombinant Antibody Targeting Claudin 18.2 Positive Cancers.

Claudin 18.2 (CLDN18.2), a tight junction (TJ) family protein controlling molecule exchange between cells, is frequently over-expressed in gastric cancer, pancreatic adenocarcinomas and in a fraction of non-small cell lung cancer cases. The tumor properties indicate that CLDN18.2 could be an attractive drug target for gastric and pancreatic cancers. In this study, we present effective strategies for developing anti-CLDN18.2 therapeutic candidates, based on variable domain of heavy chain of heavy chain antibodies (VHHs). CLDN18.2-specific VHHs were isolated by panning a phage display library from an alpaca immunized with a stable cell line highly expressing CLDN18.2. Humanized VHHs fused with human IgG1 Fc, as potential therapeutic candidates, exhibited desirable binding specificity and affinity to CLDN18.2. In vitro experiments showed that hu7v3-Fc was capable of eliciting both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) on CLDN18.2 positive tumor cells. In the mouse xenograft model, the anti-tumor efficacy of hu7v3-Fc was significantly more potent than Zolbetuximab, the benchmark anti-CLDN18.2 monoclonal antibody. Moreover, in vivo biodistribution using zirconium-89 (89Zr) labeled antibodies demonstrated that hu7v3-Fc (89Zr-hu7v3-Fc) exhibited a better tumor penetration and a faster tumor uptake than Zolbetuximab (89Zr-Zolbetuximab), which might be attributed to its smaller size and higher affinity. Taken together, anti-CDLN18.2 hu7v3-Fc is a promising therapeutic agent for human CLDN18.2 positive cancers. Furthermore, hu7v3 has emerged as a potential module for novel CLDN18.2 related therapeutics.

Regulatory Role of lncRNA NONHSAT089447 in the Dopamine Signaling Pathway in Schizophrenic Patients.

BACKGROUND We previously discovered that 3 long non-coding RNAs (lncRNAs) NONHSAT089447, NONHSAT021545, and NONHSAT041499 were differentially expressed in the peripheral blood of patients with schizophrenia, in comparison to those in normal healthy controls. In this study, we conducted bioinformatic analysis of these 3 lncRNAs and the regulatory role of lncRNA NONHSAT089447 in the dopamine signaling pathway in patients with schizophrenia. MATERIAL AND METHODS There lncRNAs in peripheral blood mononuclear cells (PBMCs) were screened using microarray analysis. Pearson's correlation analysis was performed to assess the levels of co-expressed mRNAs of respective lncRNAs. The Database for Annotation, Visualization and Integrated Discovery (DAVID) software was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes or Genomes (KEGG) enrichment analysis for these lncRNAs. Human neuroblastoma cell lines (SK-N-SH) were cultured and treated with dopamine or olanzapine (OLP), or transfected with siRNA targeting NONHSAT089447 or plasmid expressing NONHSAT089447. Levels of lncRNAs were detected by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Then, mRNA and protein expression of the dopamine receptors DRD1, DRD2, DRD3, DRD4, and DRD5 were measured by RT-PCR and western blot analysis, respectively. RESULTS OLP treatment significantly inhibited the expression of NONHSAT089447. Knockdown of NONHSAT089447 by siRNA decreased DRD3 and DRD5 expression, while overexpression of NONHSAT089447 significantly upregulated expression of DRD3 and DRD5. Western blot analysis confirmed that levels of NONHSAT089447 regulated downstream DRD signaling. CONCLUSIONS Our results revealed that the lncRNA NONHSAT089447 participated in the dopamine signaling pathway via upregulation of DRDs.

hsa_circRNA_104597: a novel potential diagnostic and therapeutic biomarker for schizophrenia.

Aim: To assess whether expression of circular RNAs (circRNAs) in peripheral blood mononuclear cells can serve as a biomarker for diagnosis and/or therapeutic response in people living with schizophrenia (SZ). Materials & methods: Differentially expressed circRNAs were screened via microarray in nine individuals living with SZ and nine healthy controls, then quantified using real-time quantitative reverse transcription PCR in SZ (n = 102) and healthy control (n = 103) groups. CircRNAs were re-assessed twice in 30 randomly selected individuals living with SZ after 4- and 8-week antipsychotic treatments. Results: Five circRNAs were differentially expressed between groups. Only hsa_circRNA_104597, which was downregulated in the SZ group, was significantly upregulated after 8-week treatment. Conclusion: Dysregulation of hsa_circRNA_104597 may serve as a novel potential diagnostic and therapeutic biomarker for SZ.

Deletion of Macrophage Mineralocorticoid Receptor Protects Hepatic Steatosis and Insulin Resistance Through ERα/HGF/Met Pathway.

Although the importance of macrophages in nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) has been recognized, how macrophages affect hepatocytes remains elusive. Mineralocorticoid receptor (MR) has been implicated to play important roles in NAFLD and T2DM. However, cellular and molecular mechanisms are largely unknown. We report that myeloid MR knockout (MRKO) improves glucose intolerance, insulin resistance, and hepatic steatosis in obese mice. Estrogen signaling is sufficient and necessary for such improvements. Hepatic gene and protein expression suggests that MRKO reduces hepatic lipogenesis and lipid storage. In the presence of estrogen, MRKO in macrophages decreases lipid accumulation and increases insulin sensitivity of hepatocytes through hepatocyte growth factor (HGF)/Met signaling. MR directly regulates estrogen receptor 1 (Esr1 [encoding ERα]) in macrophages. Knockdown of hepatic Met eliminates the beneficial effects of MRKO in female obese mice. These findings identify a novel MR/ERα/HGF/Met pathway that conveys metabolic signaling from macrophages to hepatocytes in hepatic steatosis and insulin resistance and provide potential new therapeutic strategies for NAFLD and T2DM.

Mineralocorticoid Receptor Deficiency in Macrophages Inhibits Neointimal Hyperplasia and Suppresses Macrophage Inflammation Through SGK1-AP1/NF-κB Pathways.

OBJECTIVE: Restenosis after percutaneous coronary intervention remains to be a serious medical problem. Although mineralocorticoid receptor (MR) has been implicated as a potential target for treating restenosis, the cellular and molecular mechanisms are largely unknown. This study aims to explore the functions of macrophage MR in neointimal hyperplasia and to delineate the molecular mechanisms. APPROACH AND RESULTS: Myeloid MR knockout (MMRKO) mice and controls were subjected to femoral artery injury. MMRKO reduced intima area and intima/media ratio, Ki67- and BrdU-positive vascular smooth muscle cells, expression of proinflammatory molecules, and macrophage accumulation in injured arteries. MMRKO macrophages migrated less in culture. MMRKO decreased Ki67- and BrdU-positive macrophages in injured arteries. MMRKO macrophages were less Ki67-positive in culture. Conditioned media from MMRKO macrophages induced less migration, Ki67 positivity, and proinflammatory gene expression of vascular smooth muscle cells. After lipopolysaccharide treatment, MMRKO macrophages had decreased p-cFos and p-cJun compared with control macrophages, suggesting suppressed activation of activator protein-1 (AP1). Nuclear factor-κB (NF-κB) pathway was also inhibited by MMRKO, manifested by decreased p-IκB kinase-ß and p-IκBα, increased IκBα expression, decreased nuclear translocation of p65 and p50, as welll as decreased phosphorylation and expression of p65. Finally, overexpression of serum-and-glucocorticoid-inducible-kinase-1 (SGK1) attenuated the effects of MR deficiency in macrophages. CONCLUSIONS: Selective deletion of MR in myeloid cells limits macrophage accumulation and vascular inflammation and, therefore, inhibits neointimal hyperplasia and vascular remodeling. Mechanistically, MR deficiency suppresses migration and proliferation of macrophages and leads to less vascular smooth muscle cell activation. At the molecular level, MR deficiency suppresses macrophage inflammatory response via SGK1-AP1/NF-κB pathways.

Psychological maladjustment of Asian and African peacekeepers in Liberia and its related factors.

This study aimed to investigate the maladjustment of Asian (Bangladeshi, Pakistani) and African (Nigerian, Namibian, Ghanaian) peacekeepers and its major influence factors. By random cluster sampling, 300 Asian peacekeepers and 271 African peacekeepers were administered the military psychological maladjustment scale (MPMS) and risk factors questionnaire. Investigation at Day 7 and Day 120 into the peacekeeping deployment period indicated that MPMS total score and factor scores of the Asian peacekeepers were significantly lower than those of the African peacekeepers (p < .01). The total score and each factor score of MPMS of the Asian peacekeepers significantly decreased (p < .01); for the African peacekeepers, only the factor score of emotional disorder of MPMS significantly decreased (p < .05). Stepwise regression analysis showed that the education duration was the influence factor for the emotional disorder factor score in the Asian peacekeepers, and the two factors were positively correlated. Age, military service duration, education duration and marital status were the major influence factors for the MPMS factors of the African peacekeepers, among which age was negatively correlated with the total score and each factor score, and military service duration, education duration and marital status were positively related. We conclude that the Asian peacekeepers are more adaptable and resilient than the African peacekeepers. Education duration was the major influence factor for Emotional Disorder in the Asian peacekeepers. The major influence factors for maladjustment in the African peacekeepers were age, military service duration, education duration and marital status.

Relationship between acute stress and sleep disorder in grass-root military personnel: mediating effect of social support.

BACKGROUND: Sleep disorder induced by acute stress has always been an important topic for study among the general population. However, the mediating effect of social support between acute stress and sleep disorder has rarely been reported before. METHODS: A total of 2,411 grass-root military personnel were randomly selected by cluster sampling, and administered the Chinese Military Personnel Sleep Disorder Scale, Military Acute Stress Scale and Social Support Rating Scale. RESULTS: The total score of acute stress scale was positively correlated with the total score and factor scores of sleep disorder scale (r = 0.209 ~ 0.465, P < 0.01); The total score of social support scale was positively correlated with the total score of acute stress scale and the total score and factor scores of sleep disorder scale (r = 0.356 ~ 0.537, P < 0.01). The analysis of mediating effects showed that lack of social support partially mediated between acute stress and the factors of sleep disorder. The analysis of structural equation model showed that acute stress not only had a direct effect on sleep disorder (the path coefficient was 0.29, P = 0.000), but also on lack of social support (the path coefficient was 0.39, P = 0.000); lack of social support had a direct effect on sleep disorder (the path coefficient was 0.48, P = 0.000). CONCLUSIONS: Acute stress and lack of social support are two significant factors of sleep disorder in grass-root military personnel. Well-established social support could alleviate sleep disorder induced by acute stress. Lack of social support was a partial mediator between acute stress and sleep disorder.

Scene-trait coping style of military rescuers in Wenchuan earthquake.

OBJECTIVE: This study aimed to explore the scene-trait coping style of military rescuers in Wenchuan earthquake in an effort to provide scientific evidences for mental intervention program for Chinese military personnel. METHODS: By cluster sampling, a total of 151 military rescuers and 331 control servicemen were administered the military personnel scene-trait coping style scale (MPSTCSS). RESULTS: All active coping factor scores, and passive coping factor scores of affection, health and economy in the rescuer group were significantly higher than those in control group (P < 0.05). The 21-above age subgroup, the 3-year plus service subgroup, and the officer subgroup had significantly higher active coping factor scores on military tasks, military experience and personal development than those of the 21-below age subgroup, 3-year minus service subgroup and the soldier subgroup, respectively (P < 0.05 or P < 0.01). CONCLUSION: The earthquake relief servicemen can cope with stressful situations better than control group by taking active coping style. The officers, servicemen older than 21 years, and servicemen with more service duration than 3 years could usually take active coping style.

Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using L-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93%, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 µmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg(2+) and was slightly stimulated by the monovalent cation K(+). It showed an improved thermostability, about 50% of the enzyme activity was retained even after preincubation at 50 °C for 2 h.

GAP promoter library for fine-tuning of gene expression in Pichia pastoris.

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (ß-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.