Generation of Human Regulatory Dendritic Cells from Cryopreserved Healthy Donor Cells and Hematopoietic Stem Cell Transplant Recipients.

Acute graft versus host disease (GVHD) remains a significant complication following hematopoietic stem cell transplant (HSCT), despite improved human leukocyte antigen (HLA) matching and advances in prophylactic treatment regimens. Previous studies have shown promising results for future regulatory dendritic cell (DCreg) therapies in the amelioration of GVHD. This study evaluates the effects of cryopreservation on the generation of DCreg, the generation of young and older DCreg in serum-free media, and the feasibility of generating DCreg from young and older HSCT patient monocytes. DCregs were generated in X-vivo 15 serum-free media from donor or patient monocytes. This study includes the use of monocytes from young and older healthy, donor, and HSCT patients with varying hematological diseases. Phenotypic differences in cell populations were assessed via flow cytometry while pro-inflammatory and anti-inflammatory cytokine production was evaluated in culture medium. The number of DCreg generated from cryopreserved monocytes of healthy donors was not significantly different from freshly isolated monocytes. DCreg generated from cryopreserved monocytes had comparable levels of co-stimulatory molecule expression, inhibitory molecule expression, and cytokine production as freshly isolated monocytes. Young and older healthy donor monocytes generated similar numbers of DCreg with similar cytokine production and phenotype. Although monocytes from older HSCT patients generated significantly fewer DCreg, DCreg from young and older HSCT patients had comparable phenotypes and cytokine production. Monocytes from young and older myelodysplastic syndrome (MDS) patients generated reduced numbers of DCreg compared to non-MDS-derived DCreg. We demonstrate that the cryopreservation of monocytes from HSCT patients of varying hematological diseases allows for the cost-effective generation of DCreg on an as-needed basis. Although the generation of DCreg from MDS patients requires further assessment, these data support the possibility of in vitro-generated DCreg as a therapy to reduce GVHD-associated morbidity and mortality in young and older HSCT recipients.

XDH genotypes through gene-gene interactions with NUDT15 affect azathioprine-induced leukopenia in Chinese patients.

Aim: To investigate whether genotypes of XDH, GMPS and MOCOS were associated with azathioprine-induced adverse drug reaction (ADR) and had the gene-gene interactions with NUDT15 rs116855232 to induce leukopenia. Methods: Patients who had taken azathioprine were recruited. Genotyping of those gene was performed. Risk factor to ADR was analyzed by logistic regression. The generalized multifactor dimensionality reduction (GMDR) was assessed based on gene-gene interactions with ADR. Results: A total of 111 patients were included in this study, all of whom were Han Chinese. XDH rs2295475 was a risk factor of myelotoxicity (p = 0.022). NUDT15 rs116855232 was a risk factor of myelotoxicity, grade ≥2 leukopenia and drug treatment termination (p-values were <0.05). Rs2295475 and rs116855232 had a gene-gene interaction. The model was associated with grade ≥2 leukopenia (OR: 17.99; 95% CI: 4.11-78.81). Conclusion: Combined testing genotype for rs2295475 and rs116855232 could improve the prediction of azathioprine-induced leukopenia.

Metabolic syndrome related gene signature predicts the prognosis of patients with pancreatic ductal carcinoma. A novel link between metabolic dysregulation and pancreatic ductal carcinoma.

BACKGROUND: Pancreatic cancer is one of the most common malignancies worldwide. In recent years, specific metabolic activities, which involves the development of tumor, caused wide public concern. In this study, we wish to explore the correlation between metabolism and progression of tumor. METHODS: A retrospective analysis including 95 patients with pancreatic ductal adenocarcinoma (PDAC) and PDAC patients from The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), and The Gene Expression Omnibus (GEO) database were involved in our study. Multivariate Cox regression analysis was used to construct the prognosis model. The potential connection between metabolism and immunity of PDAC was investigated through a weighted gene co-expression network analysis (WGCNA). 22 types of Tumor-infiltrating immune cells (TIICs) between high-risk and low-risk groups were estimated through CIBERSORT. Moreover, the potential immune-related signaling pathways between high-risk and low-risk groups were explored through the gene set enrichment analysis (GSEA). The role of key gene GMPS in developing pancreatic tumor was further investigated through CCK-8, colony-information, and Transwell. RESULTS: The prognostic value of the MetS factors was analyzed using the Cox regression model, and a clinical MetS-based nomogram was established. Then, we established a metabolism-related signature to predict the prognosis of PDAC patients based on the TCGA databases and was validated in the ICGC database and the GEO database to find the distinct molecular mechanism of MetS genes in PDAC. The result of WGCNA showed that the blue module was associated with risk score, and genes in the blue module were found to be enriched in the immune-related signaling pathway. Furthermore, the result of CIBERSORT demonstrated that proportions of T cells CD8, T cells Regulatory, Tregs NK cells Activated, Dendritic cells Activated, and Mast cells Resting were different between high-risk and low-risk groups. These differences are potential causes of different prognoses of PDAC patients. GSEA and the protein-protein interaction network (PPI) further revealed that our metabolism-related signature was significantly enriched in immune-related biological processes. Moreover, knockdown of GMPS in PDAC cells suppressed proliferation, migration, and invasion of tumor cells, whereas overexpression of GMPS performed oppositely. CONCLUSION: The results shine light on fundamental mechanisms of metabolic genes on PDAC and establish a reliable and referable signature to evaluate the prognosis of PDAC. GMPS was identified as a potential candidate oncogene with in PDAC, which can be a novel biomarker and therapeutic target for PDAC treatment.

Implementation of Food Safety Management Systems along with Other Management Tools (HAZOP, FMEA, Ishikawa, Pareto). The Case Study of Listeria monocytogenes and Correlation with Microbiological Criteria.

The food industry's failure in planning and designing of and in implementing a Food Safety Management System and its foundation elements leads, in most instances, to compromised food safety and subsequent foodborne illness outbreaks. This phenomenon was noticed, worldwide, for all food processors, but with a much higher incidence in the medium- and small-sized food processing plants. Our study focuses on the importance of Food Safety Management System (FSMS), Critical Control Points Hazard Analysis (HACCP) and the Prerequisite Programs (PRPs) as the foundation of HACCP, in preventing foodborne outbreaks. For emphasis, we make use of the example of organizational food safety culture failures and the lack of managerial engagement which resulted in a multi-state listeriosis outbreak in USA. Moreover, we correlate this with microbiological criteria. Implementation of food safety management systems (ISO 22000:2018) along with incorporation of management tools such as HAZOP, FMEA, Ishikawa and Pareto have proved to be proactive in the maintenance of a positive food safety culture and prevention of cross-contamination and fraud.

The upregulated expression of RFC4 and GMPS mediated by DNA copy number alteration is associated with the early diagnosis and immune escape of ESCC based on a bioinformatic analysis.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that commonly occurs worldwide. Usually, Asia, especially China, has a high incidence of esophageal cancer. ESCC often has a poor outcome because of a late diagnosis and lack of effective treatments. To build foundations for the early diagnosis and treatment of ESCC, we used the gene expression datasets GSE20347 and GSE17351 from the GEO database and a private dataset to uncover differentially expressed genes (DEGs) and key genes in ESCC. Notably, we found that replication factor C subunit 4 (RFC4) and guanine monophosphate synthase (GMPS) were upregulated but have been rarely studied in ESCC. In particular, to the best of our knowledge, our study is the first to explore GMPS and ESCC. Furthermore, we found that high levels of RFC4 and GMPS expression may result from an increase in DNA copy number alterations. Furthermore, RFC4 and GMPS were both upregulated in the early stage and early nodal metastases of esophageal carcinoma. The expression of RFC4 was strongly correlated with GMPS. In addition, we explored the relationship between RFC4 and GMPS expression and tumor-infiltrating immune cells (TILs) in esophageal carcinoma. The results showed that the levels of RFC4 and GMPS increased with a decrease in some tumor-infiltrating cells. Upregulated RFC4 and GMPS with high TILs indicate a worse prognosis. In summary, our study shows that RFC4 and GMPS have potential as biomarkers for the early diagnosis of ESCC and may played a crucial role in the process of tumor immunity in ESCC.

Inhibition of guanosine monophosphate synthetase (GMPS) blocks glutamine metabolism and prostate cancer growth.

Glutamine is a critical nutrient in cancer; however, its contribution to purine metabolism in prostate cancer has not previously been determined. Guanosine monophosphate synthetase (GMPS) acts in the de novo purine biosynthesis pathway, utilizing a glutamine amide to synthesize the guanine nucleotide. This study demonstrates that GMPS mRNA expression correlates with Gleason score in prostate cancer samples, while high GMPS expression was associated with decreased rates of overall and disease/progression-free survival. Pharmacological inhibition or knockdown of GMPS significantly decreased cell growth in both LNCaP and PC-3 prostate cancer cells. We utilized [15 N-(amide)]glutamine and [U-13 C5 ]glutamine metabolomics to dissect the pathways involved and despite similar growth inhibition by GMPS knockdown, we show unique metabolic effects across each cell line. Using a PC-3 xenograft mouse model, tumor growth was also significantly decreased after GMPS knockdown, highlighting the importance of glutamine metabolism and providing support for GMPS as a therapeutic target in prostate cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TRIM21 - A potential novel therapeutic target in cancer.

Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in innate immunity, systemic lupus erythematosus and Sjögren's syndrome. In addition, TRIM21 involvement in cancer proliferation has been observed. However, the clinical significance of TRIM21 and its role in cancer cell proliferation and suppression remains elusive. Here we discuss the effects of TRIM21 on major cancer promoting proteins such as NF-κB, STAT3, BCL2, p53, p27 and Snail, comparing its signaling pathways under normal conditions and in the presence of a variety of carcinogenesis effectors (oncogenic, genotoxic and UV irradiation). Depending on the cancer type and the carcinogenesis effector, TRIM21 may enhance cancer proliferation, or alternatively it may increase the ubiquitination of many cancer-triggering proteins, resulting in their proteasomal degradation. This indicates the importance of TRIM21 in cancer proliferation and/or apoptosis and suggests its potential as a novel cancer therapeutic target.

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

The G115 standardized ginseng extract: an example for safety, efficacy, and quality of an herbal medicine.

Ginseng products on the market show high variability in their composition and overall quality. This becomes a challenge for both consumers and health-care professionals who are in search of high-quality, reliable ginseng products that have a proven safety and efficacy profile. The botanical extract standardization is of crucial importance in this context as it determines the reproducibility of the quality of the product that is essential for the evaluation of effectiveness and safety. This review focuses on the well-characterized and standardized ginseng extract, G115, which represents an excellent example of an herbal drug preparation with constant safety and efficacy within the herbal medicinal products. Over the many decades, extensive preclinical and clinical research has been conducted to evaluate the efficacy and safety of G115. In vitro and in vivo studies of G115 have shown pharmacological effects on physical performance, cognitive function, metabolism, and the immune system. Furthermore, a significant number of G115 clinical studies, most of them double-blind placebo-controlled, have reinforced the findings of preclinical evidence and proved the efficacy of this extract on blood glucose and lipid regulation, chronic obstructive pulmonary disease, energy, physical performance, and immune and cognitive functions. Clinical trials and 50 years of presence on the market are proof of a good safety profile of G115.

TRIM21-SERPINB5 aids GMPS repression to protect nasopharyngeal carcinoma cells from radiation-induced apoptosis.

BACKGROUND: The main strategy against nasopharyngeal carcinoma (NPC) is radiotherapy. However, radioresistance mediated recurrence is a leading clinical bottleneck in NPC. Revealing the mechanism of NPC radioresistance will help improve the therapeutic effect. METHODS: In this study, the role of TRIM21 (tripartite motif-containing 21) in NPC receiving ionizing radiation was firstly examined both in vivo and in vitro. Mass spectrometry analysis was performed to identify the downstream targets of TRIM21. NPC cells with TRIM21 or SERPINB5 (serpin family B member 5) overexpression or knockout were used to determine the epistatic relationship among SERPINB5, GMPS (guanine monophosphate synthase) and TRIM21. Flow cytometry, co-immunoprecipitation, western blot and immunofluorescence were employed to strengthen the results. Finally, immunohistochemistry using 4 radiosensitive and 8 radioresistent NPC patient samples was perform to examine the association between SERPINB5 or GMPS expression and patient radio-sensitivity. RESULTS: As an E3 ligase, TRIM21 was highly expressed in NPC. After ionizing radiation, TRIM21 repressed TP53 expression by mediating GMPS ubiquitination and degradation. Overexpression of TRIM21 protected NPC cells from radiation mediated cell apoptosis in vitro and in vivo. Further analysis revealed that TRIM21 mediated GMPS repression was dependent on SERPINB5, and SERPINB5 served as an adaptor which prevented GMPS from entering into the nucleus and introduced TRIM21 for GMPS ubiquitination. Moreover, the in vitro and in vivo results validated the finding that SERPINB5 promoted NPC cell radioresistance, and the radioresistant patients had higher SERPINB5 expression. CONCLUSIONS: Overall, our data showed that TRIM21-SERPINB5-mediated GMPS degradation facilitated TP53 repression, which promoted the radioresistance of NPC cells. This novel working model related to TP53 suppression provided new insight into NPC radioresistence clinically.