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Endometrial cancer (EC) is the most common gynecologic malignancy. While the majority of patients present with early-stage and low-grade EC and have an excellent prognosis, a subset has metastatic disease at presentation or develops distant recurrence after initial treatment of the primary. However, the lack of prognostic biomarkers for metastatic EC is a critical barrier. Arginase 1 (ARG1) regulates the last step of the urea cycle, and an increase in ARG1 has been correlated as a poor prognostic factor in a variety of cancers. In the present study, ARG1 expression was evaluated as a potential prognostic marker for metastatic EC in endometrial hyperplasia and cancer of mice with Pten mutation as well as Pten and Mig-6 double mutations. While Pten mutation in the uterus is not sufficient for distant metastasis, mice with concurrent ablation of Mig-6 and Pten develop distant metastasis. Our immunostaining and RT-qPCR analysis revealed that the expression of ARG1 in early stage of EC as well as endometrial hyperplasia from mice deficient in Mig-6 and Pten mutations significantly increased compared to Pten mutation in the uterus. The results suggest that a high level of ARG1 is associated with poor prognosis in association with EC of mouse. [ABSTRACT FROM AUTHOR]

Subacute spinal cord injury (SCI) displays a complex pathophysiology associated with pro-inflammation and ensuing tissue damage. Microglia, the resident innate immune cells of the CNS, in concert with infiltrating macrophages, are the primary contributors to SCI-induced inflammation. However, subpopulations of activated microglia can also possess immunomodulatory activities that are essential for tissue remodeling and repair, including the production of anti-inflammatory cytokines and growth factors that are vital for SCI recovery. Recently, reports have provided convincing evidence that sex-dependent differences exist in how microglia function during CNS pathologies and the extent to which these cells contribute to neurorepair and endogenous recovery. Herein we employed flow cytometry and immunohistochemical methods to characterize the phenotype and population dynamics of activated innate immune cells within the injured spinal cord of age-matched male and female rats within the first week (7 days) following thoracic SCI contusion. This assessment included the analysis of pro- and anti-inflammatory markers, as well as the expression of critical immunomodulatory kinases, including P38 MAPK, and transcription factors, such as NFκB, which play pivotal roles in injury-induced inflammation. We demonstrate that activated microglia from the injured spinal cord of female rats exhibited a significantly diminutive pro-inflammatory response, but enhanced anti-inflammatory activity compared to males. These changes included lower levels of iNOS and TLR4 expression but increased levels of ARG-1 and CD68 in females after SCI. The altered expression of these markers is indicative of a disparate secretome between the microglia of males and females after SCI and that the female microglia possesses higher phagocytic capabilities (increased CD68). The examination of immunoregulatory kinases and transcription factors revealed that female microglia had higher levels of phosphorylated P38Thr180/Tyr182 MAPK and nuclear NFκB pp50Ser337 but lower amounts of nuclear NFκB pp65Ser536, suggestive of an attenuated pro-inflammatory phenotype in females compared to males after SCI. Collectively, this work provides novel insight into some of the sex disparities that exist in the innate immune response after SCI and indicates that sex is an important variable when designing and testing new therapeutic interventions or interpretating positive or negative responses to an intervention. [ABSTRACT FROM AUTHOR]

Noncoding RNA plays a pivotal role as novel regulators of endothelial cell function. Type 2 diabetes, acknowledged as a primary contributor to cardiovascular diseases, plays a vital role in vascular endothelial cell dysfunction due to induced abnormalities of glucolipid metabolism and oxidative stress. In this study, aberrant expression levels of circHMGCS1 and MIR4521 were observed in diabetes-induced human umbilical vein endothelial cell dysfunction. Persistent inhibition of MIR4521 accelerated development and exacerbated vascular endothelial dysfunction in diabetic mice. Mechanistically, circHMGCS1 upregulated arginase 1 by sponging MIR4521, leading to decrease in vascular nitric oxide secretion and inhibition of endothelial nitric oxide synthase activity, and an increase in the expression of adhesion molecules and generation of cellular reactive oxygen species, reduced vasodilation and accelerated the impairment of vascular endothelial function. Collectively, these findings illuminate the physiological role and interacting mechanisms of circHMGCS1 and MIR4521 in diabetes-induced cardiovascular diseases, suggesting that modulating the expression of circHMGCS1 and MIR4521 could serve as a potential strategy to prevent diabetes-associated cardiovascular diseases. Furthermore, our findings provide a novel technical avenue for unraveling ncRNAs regulatory roles of ncRNAs in diabetes and its associated complications.

Objective(s): Macrophages exhibit versatile phenotypes, with M1 macrophages releasing inflammatory cytokines and possessing microbicidal activities, while M2 macrophages release anti-inflammatory cytokines and contribute to tissue repair. The M1/M2 imbalance plays a significant role in various pathological processes. Crocin, known for its antioxidant properties and ability to eliminate free radicals, has been investigated for its potential anti-inflammatory effects. We examined the effect of the primary activation state of macrophages on their phenotype switching when exposed to crocin.Materials and Methods: The crocin impact on macrophage viability was evaluated by MTT. TNF-α, IL-6, and IL-10 secretion, as well as Nos2/Arg1 ratio, were measured in cells treated with crocin or LPS+IFN-γ (M1 inducers), in cells concurrently treated with crocin and LPS+IFN-γ or in cells pretreated with crocin before M1 induction.Results: Crocin did not show any toxicity at the concentration of 500 µM or lower. When uncommitted macrophages were exposed to crocin (25-100 µM), it elevated certain M1 activity indicators, including Nos2/Arg1 ratio and TNF-α secretion, but not IL-6. Crocin in concurrent treatment with LPS+IFN-γ prevented the increase in M1 indicators, Nos2/Arg1 ratio, and TNF-α secretion. However, pretreatment of cells with crocin before the addition of LPS+IFN-γ did not reverse M1 induction in macrophages; instead, it further increased the Nos2/Arg1 ratio and TNF-α secretion. IL-10 was not detectable in any of the experimental groups.Conclusion: It appears that the modulatory effects of crocin on macrophage M1/M2 phenotype switching partly depend on the presence or absence of inflammatory mediators and, accordingly, the initial state of macrophage commitment.

Abstract Background Arginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein. Methods C57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT–qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit. Results ARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased. Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown. The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes. Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression. Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients. Conclusions ARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients. Graphical abstract

Objective(s): Macrophages exhibit versatile phenotypes, with M1 macrophages releasing inflammatory cytokines and possessing microbicidal activities, while M2 macrophages release antiinflammatory cytokines and contribute to tissue repair. The M1/M2 imbalance plays a significant role in various pathological processes. Crocin, known for its antioxidant properties and ability to eliminate free radicals, has been investigated for its potential anti-inflammatory effects. We examined the effect of the primary activation state of macrophages on their phenotype switching when exposed to crocin. Materials and Methods: The crocin impact on macrophage viability was evaluated by MTT. TNF-α, IL-6, and IL-10 secretion, as well as Nos2/Arg1 ratio, were measured in cells treated with crocin or LPS+IFN-γ (M1 inducers), in cells concurrently treated with crocin and LPS+IFN-γ or in cells pretreated with crocin before M1 induction. Results: Crocin did not show any toxicity at the concentration of 500 μM or lower. When uncommitted macrophages were exposed to crocin (25-100 μM), it elevated certain M1 activity indicators, including Nos2/Arg1 ratio and TNF-α secretion, but not IL-6. Crocin in concurrent treatment with LPS+IFN-γ prevented the increase in M1 indicators, Nos2/Arg1 ratio, and TNF-α secretion. However, pretreatment of cells with crocin before the addition of LPS+IFN-γ did not reverse M1 induction in macrophages; instead, it further increased the Nos2/Arg1 ratio and TNF-α secretion. IL-10 was not detectable in any of the experimental groups. Conclusion: It appears that the modulatory effects of crocin on macrophage M1/M2 phenotype switching partly depend on the presence or absence of inflammatory mediators and, accordingly, the initial state of macrophage commitment. [ABSTRACT FROM AUTHOR]

The effect of recombinant human chorionic gonadotropin (hCG) at pregnancy-appropriate concentrations (10 and 100 IU/mL) on differentiation and functional activity of myeloid-derived suppressor cells (MDSCs) was studied. The object of the study was isolated CD11b+ cells that were converted to the MDSC phenotype by two-step activation with GM-CSF cytokines, IL1β and lipopolysaccharide (LPS). After a week of cultivation, the total MDSC level was determined considering the subpopulations M-MDSC and PMN-MDSC, the expression of arginase-1 (Arg1) and indoleamn-2,3-dioxydiogenase (IDO) in these cells, as well as the cytokine profile in cell culture supernatant. It was shown that hCG increased the total number of MDSCs, and its lower concentration (10 IU/mL) contributed to the differentiation of the M-MDSC subpopulation. hCG did not affect the expression of IDO expression in MDSCs, but there was a tendency to increase IDO expression under the influence of hCG at a concentration of 10 IU/mL. CD11b+ cells converted to the MDSC phenotype had a low Arg 1 content, making it impossible to evaluate the effect of the hormone on the expression of this enzyme. Evaluation of the cytokine profile by multiplex analysis showed that hCG did not modulate cytokine production in the culture of CD11b+ cells converted the MDSC phenotype. This is the first time that hCG has been shown to induce differentiation of MDSCs. [ABSTRACT FROM AUTHOR]

Background: Arginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein. Methods: C57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT–qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit. Results: ARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased. Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown. The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes. Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression. Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients. Conclusions: ARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients. Plain English summary: Fibrotic cataracts can be classified as anterior subcapsular cataract or posterior capsular opacification depending on where fibrosis occurs. The mechanism of fibrotic cataracts is not fully understood. Fibrotic opacities induced by trauma, inflammation, or radiation can accumulate underneath the anterior lens capsule, causing anterior subcapsular cataract. Posterior capsular opacification is one of the most common complications of phacoemulsification with intraocular lens implantation, with a high incidence in young patients. We show for the first time that ARG1 can regulate EMT in fibrotic cataracts. TGF-β2 is the main cause of fibrosis in LECs. The expression of ARG1 and fibronectin in LECs increased after TGF-β2 treatment or mouse lens capsular injury. We investigated the specific molecular mechanisms by which ARG1 regulates EMT in fibrotic cataracts. The mRNA expression of enzymes of the arginase-related pathway was decreased due to knockdown of ARG1 expression in HLECs. These effects were reversed by ARG1 overexpression. Additionally, knockdown of ARG1 decreased collagen 1A1, fibronectin, and vimentin expression; superoxide levels; and cell migration and increased NO levels. These effects were reversed by ARG1 overexpression. Pharmacological blockade of the ARG1 pathway with CB-1158 reduced the proliferation of LECs and decreased fibronectin, α-SMA, collagen 1A1, and vimentin expression in mouse lenses. We believe that ARG1 promotes the production of collagen 1A1 by directly activating the arginase pathway and leads to lens fibrosis by reducing NO production and increasing superoxide levels, providing a new mechanism for the prevention and treatment of fibrotic cataracts. 2rJpd8qCKY4LBSvBN5cEnm Video Abstract [ABSTRACT FROM AUTHOR]

Background: Multiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by activated neuroglial cells, CNS infiltrating leukocytes, and their reciprocal interactions through inflammatory signals. An inflammatory stimulus triggers inducible nitric oxide synthase (NOS2), a pro-inflammatory marker of microglia/macrophages (MG/Mφ) to catalyze sustained nitric oxide production. NOS2 during neuroinflammation, has been associated with MS disease pathology; however, studies dissecting its role in demyelination are limited. We studied the role of NOS2 in a recombinant β-coronavirus-MHV-RSA59 induced neuroinflammation, an experimental animal model mimicking the pathological hallmarks of MS: neuroinflammatory demyelination and axonal degeneration. Objective: Understanding the role of NOS2 in murine-β-coronavirus-MHV-RSA59 demyelination. Methods: Brain and spinal cords from mock and RSA59 infected 4–5-week-old MHV-free C57BL/6 mice (WT) and NOS2-/- mice were harvested at different disease phases post infection (p.i.) (day 5/6-acute, day 9/10-acute-adaptive and day 30-chronic phase) and compared for pathological outcomes. Results: NOS2 was upregulated at the acute phase of RSA59-induced disease in WT mice and its deficiency resulted in severe disease and reduced survival at the acute-adaptive transition phase. Low survival in NOS2-/- mice was attributed to (i) high neuroinflammation resulting from increased accumulation of macrophages and neutrophils and (ii) Iba1 + phagocytic MG/Mφ mediated-early demyelination as observed at this phase. The phagocytic phenotype of CNS MG/Mφ was confirmed by significantly higher mRNA transcripts of phagocyte markers-CD206, TREM2, and Arg1 and double immunolabelling of Iba1 with MBP and PLP. Further, NOS2 deficiency led to exacerbated demyelination at the chronic phase as well. Conclusion: Taken together the results imply that the immune system failed to control the disease progression in the absence of NOS2. Thus, our observations highlight a protective role of NOS2 in murine-β-coronavirus induced demyelination. [ABSTRACT FROM AUTHOR]

Abstract Background Sepsis is a life-threatening multi-organ dysfunction caused by the dysregulated host response to infection. Sepsis remains a major global concern with high mortality and morbidity, while management of sepsis patients relies heavily on early recognition and rapid stratification. This study aims to identify the crucial genes and biomarkers for sepsis which could guide clinicians to make rapid diagnosis and prognostication. Methods Preliminary analysis of multiple global datasets, including 170 samples from patients with sepsis and 110 healthy control samples, revealed common differentially expressed genes (DEGs) in peripheral blood of patients with sepsis. After Gene Oncology (GO) and pathway analysis, the Weighted Gene Correlation Network Analysis (WGCNA) was used to screen for genes most related with clinical diagnosis. Also, the Protein-Protein Interaction Network (PPI Network) was constructed based on the DEGs and the hub genes were found. The results of WGCNA and PPI network were compared and one shared gene was discovered. Then more datasets of 728 experimental samples and 355 control samples were used to prove the diagnostic and prognostic value of this gene. Last, we used real-time PCR to confirm the bioinformatic results. Results Four hundred forty-four common differentially expressed genes in the blood of sepsis patients from different ethnicities were identified. Fifteen genes most related with clinical diagnosis were found by WGCNA, and 24 hub genes with most node degrees were identified by PPI network. ARG1 turned out to be the unique overlapped gene. Further analysis using more datasets showed that ARG1 was not only sharply up-regulated in sepsis than in healthy controls, but also significantly high-expressed in septic shock than in non-septic shock, significantly high-expressed in severe or lethal sepsis than in uncomplicated sepsis, and significantly high-expressed in non-responders than in responders upon early treatment. These all demonstrate the performance of ARG1 as a key biomarker. Last, the up-regulation of ARG1 in the blood was confirmed experimentally. Conclusions We identified crucial genes that may play significant roles in sepsis by WGCNA and PPI network. ARG1 was the only overlapped gene in both results and could be used to make an accurate diagnosis, discriminate the severity and predict the treatment response of sepsis.

Stroke is a serious worldwide disease that causes death and disability, more than 80% of which is ischemic stroke. The expression of arginase 1 (Arg1), a key player in regulating nitrogen homeostasis, is altered in the peripheral circulation after stroke. Growing evidence indicates that ischemic stroke also induces upregulated Arg1 expression in the central nervous system, especially in activated microglia and macrophages. This implies that Arg1 may affect stroke progression by modulating the cerebral immune response. To investigate the effect of Arg1+ microglia/macrophages on ischemic stroke, we selectively eliminated cerebral Arg1+ microglia/macrophages by mannosylated clodronate liposomes (MCLs) and investigated their effects on behavior, neurological deficits, and inflammatory responses in mice after ischemic stroke. More than half of Arg1+ cells, mainly Arg1+ microglia/macrophages, were depleted after MCLs administration, resulting in a significant deterioration of motility in mice. After the elimination of Arg1+ microglia/macrophages, the infarct volume expanded and neuronal degenerative lesions intensified. Meanwhile, the absence of Arg1+ microglia/macrophages significantly increased the production of pro-inflammatory cytokines and suppressed the expression of anti-inflammatory factors, thus profoundly altering the immune microenvironment at the lesion site. Taken together, our data demonstrate that depletion of Arg1+ microglia/macrophages exacerbates neuronal damage by facilitating the inflammatory response, leading to more severe ischemic injury. These results suggest that Arg1+ microglia/macrophages, as a subpopulation regulating inflammation, is beneficial in controlling the development of ischemia and promoting recovery from injury. Regulation of Arg1 expression on microglia/macrophages at the right time may be a potential target for the treatment of ischemic brain injury. [ABSTRACT FROM AUTHOR]

Background: Psoriasis is a common chronic skin inflammatory disease. Understanding the pathogenesis of psoriasis and identifying novel therapeutic targets are under investigation. Methods: Gene expression profiles were obtained from GSE13355, GSE30999 and GSE54456 datasets to identify differentially expressed genes (DEGs) between psoriasis and normal controls. Enrichment analysis was used to identify the biological functions and pathways of common genes from three groups of DEGs. Protein-protein interaction (PPI) network was then constructed to identify key genes according to degree of connectivity. Expression of genes was detected by the method of qRT-PCR and immunohistochemistry. The infiltration of immune cells of psoriasis were quantified and detected by flow cytometry. Results: A total of 146 common genes were identified between psoriasis and normal controls. They were significantly enriched in IL-17, chemokine, and NOD-like receptor (NLR) signaling pathway. Ten key genes were selected with bigger degree of connectivity through PPI network, and ARG1 and CXCL2 had better predictive ability based on ROC curves. Increased expression of ARG1 and CXCL2 in psoriasis patients were verified by qRT-PCR and immunohistochemistry method. In addition, a lot of immune cells were upregulated in psoriasis compared to healthy controls through ssGSEA and flow cytometry. Conclusion: ARG1 and CXCL2 may serve as biomarkers and potential therapy for psoriasis. This may be related to the immune response and NLR pathway. [ABSTRACT FROM AUTHOR]

Objective: To explore the relationship between plasma arginase-1 (ARG1) and early transarterial chemoembolization (TACE) refractoriness in patients with hepatocellular carcinoma (HCC) and develop nomograms for predicting early TACE refractoriness. Methods: A total of 200 patients with HCC, treated with TACE, were included in the study, including 120 in the training set and 80 in the validation set. Pretreatment enzyme-linked immunosorbent assay was used to detected the plasma ARG1 levels of the patient, and independent predictors of early TACE refractoriness were determined using a multivariate logistic regression model, based on which a predictive model was developed using a nomogram. Results: Risk of early TACE refractoriness was negatively correlated with plasma ARG1 levels, and multivariate logistic analysis showed tumour size (OR = 1.138, 95% CI = 1.006-1.288, P = 0.041), multiple tumors (OR=4.374, 95% CI = 1.189-16.089, P = 0.026), platelet count (OR = 0.990, 95% CI = 0.980-0.999, P = 0.036), and plasma ARG1 levels (OR = 0.209, 95% CI = 0.079-0.551, P = 0.002) to be independent prognostic factors for early TACE refractoriness. The AUC value for the nomogram of the training cohort was 0.786 (95% CI = 0.702-0.870), and the validation set AUC value was 0.833 (95% CI = 0.791-0.875). The decision curve analysis suggested that the nomogram had good clinical utility. Conclusion: High plasma ARG1 expression was associated with a lower incidence of early TACE refractoriness. The nomogram constructed based on four independent prognostic factors could facilitate an individualised prediction of the incidence of early TACE refractoriness. [ABSTRACT FROM AUTHOR]

Arginases are often overexpressed in human diseases, and they are an important target for developing anti-aging and antineoplastic drugs. Arginase type 1 (ARG1) is a cytosolic enzyme, and arginase type 2 (ARG2) is a mitochondrial one. In this study, a dataset containing 2115-FDA-approved drug molecules is virtually screened for potential arginase binding using molecular docking against several ARG1 and ARG2 structures. The potential arginase ligands are classified into three categories: (1) Non-selective, (2) ARG1 selective, and (3) ARG2 selective. The evaluated potential arginase ligands are then compared with their clinical use. Remarkably, half of the top 30 potential drugs are used clinically to lower blood pressure and treat cancer, infection, kidney disease, and Parkinson's disease thus partially validating our virtual screen. Most notable are the antihypertensive drugs candesartan, irbesartan, indapamide, and amiloride, the antiemetic rolapitant, the anti-angina ivabradine, and the antidiabetic metformin which have minimal side effects. The partial validation also favors the idea that the other half of the top 30 potential drugs could be used in therapeutic settings. The three categories greatly expand the selectivity of arginase inhibition. [ABSTRACT FROM AUTHOR]

Macrophages play essential roles throughout the wound repair process. Nevertheless, mechanisms regulating the process are poorly understood. MAFB is specifically expressed in the macrophages in hematopoietic tissue and is vital to homeostatic function. Comparison of the skin wound repair rates in macrophage-specific, MAFB-deficient mice (Mafbf/f::LysM-Cre) and control mice (Mafbf/f) showed that wound healing was significantly delayed in the former. For wounded GFP knock-in mice with GFP inserts in the Mafb locus, flow cytometry revealed that their GFP-positive cells expressed macrophage markers. Thus, macrophages express Mafb at wound sites. Immunohistochemical (IHC) staining, proteome analysis, and RT-qPCR of the wound tissue showed relative downregulation of Arg1, Ccl12, and Ccl2 in Mafbf/f::LysM-Cre mice. The aforementioned genes were also downregulated in the bone marrow-derived, M2-type macrophages of Mafbf/f::LysM-Cre mice. Published single-cell RNA-Seq analyses showed that Arg1, Ccl2, Ccl12, and Il-10 were expressed in distinct populations of MAFB-expressing cells. Hence, the MAFB-expressing macrophage population is heterogeneous. MAFB plays the vital role of regulating multiple genes implicated in wound healing, which suggests that MAFB is a potential therapeutic target in wound healing. [ABSTRACT FROM AUTHOR]

Background: Sepsis is a life-threatening multi-organ dysfunction caused by the dysregulated host response to infection. Sepsis remains a major global concern with high mortality and morbidity, while management of sepsis patients relies heavily on early recognition and rapid stratification. This study aims to identify the crucial genes and biomarkers for sepsis which could guide clinicians to make rapid diagnosis and prognostication. Methods: Preliminary analysis of multiple global datasets, including 170 samples from patients with sepsis and 110 healthy control samples, revealed common differentially expressed genes (DEGs) in peripheral blood of patients with sepsis. After Gene Oncology (GO) and pathway analysis, the Weighted Gene Correlation Network Analysis (WGCNA) was used to screen for genes most related with clinical diagnosis. Also, the Protein-Protein Interaction Network (PPI Network) was constructed based on the DEGs and the hub genes were found. The results of WGCNA and PPI network were compared and one shared gene was discovered. Then more datasets of 728 experimental samples and 355 control samples were used to prove the diagnostic and prognostic value of this gene. Last, we used real-time PCR to confirm the bioinformatic results. Results: Four hundred forty-four common differentially expressed genes in the blood of sepsis patients from different ethnicities were identified. Fifteen genes most related with clinical diagnosis were found by WGCNA, and 24 hub genes with most node degrees were identified by PPI network. ARG1 turned out to be the unique overlapped gene. Further analysis using more datasets showed that ARG1 was not only sharply up-regulated in sepsis than in healthy controls, but also significantly high-expressed in septic shock than in non-septic shock, significantly high-expressed in severe or lethal sepsis than in uncomplicated sepsis, and significantly high-expressed in non-responders than in responders upon early treatment. These all demonstrate the performance of ARG1 as a key biomarker. Last, the up-regulation of ARG1 in the blood was confirmed experimentally. Conclusions: We identified crucial genes that may play significant roles in sepsis by WGCNA and PPI network. ARG1 was the only overlapped gene in both results and could be used to make an accurate diagnosis, discriminate the severity and predict the treatment response of sepsis. [ABSTRACT FROM AUTHOR]

Multi-drug-resistant bacterial infections, which have become a global threat, lack effective treatments. The discoveries of non-antibiotics with different modes of antibacterial action, such as methylsulfonylmethane (MSM), are a promising new treatment for multi-drug-resistant pathogens. We constructed a mouse peritonitis infection model to evaluate the effects of MSM against methicillin-resistant Staphylococcus aureus (MRSA) infection. The time-kill kinetics of MSM against MRSA and the effect of MSM on the integrity of bacterial cell membrane were measured. Viability effects of MSM on THP1 cells were performed by CCK-8 cytotoxicity assay. Systematic inflammatory factor levels of mice were detected using ELISA. The immune response of peritoneal macrophages during MRSA-infection was evaluated using RNA sequencing. Gene Ontology function, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, and correlation analyses were applied to analysis RNA sequencing data. RT-qPCR, western blotting and flow cytometry were performed to analysis the gene and protein expression levels of macrophages. In in vitro experiments, MSM did not show significant killing effects on the growth of MRSA directly and did not destroy bacterial membrane integrity. MSM also displayed no significant effects on the proliferative capacity of THP1 cells. However, MSM treatment protected mice against a lethal dose MRSA-infection and decreased systemic inflammation. MSM upregulated metabolic pathway in peritoneal macrophages, especial glycolysis, during MRSA infection. MSM increased the expression of M2 markers (such as Arg1), promoted phosphorylation of STAT3 (which regulates M2 polarization), and decreased the expression of M1 markers in peritoneal macrophages. Additionally, MSM treatment increased the expression of H3K18 lactylation specific target genes, including Arg1. GNE-140, the LDHA-specific inhibitor of glycolysis, blocked the MSM-induced Arg1 expression in this disease model. MSM protects against MRSA infection through immunomodulation. MSM promotes the expression of Arg1 by lactate-H3K18la pathway to control macrophage to M2 polarization; it firstly provides therapeutic potential for drug-resistant infections and sepsis. • MSM relieves inflammation and improves mice survival rate after MRSA infection. • MSM promotes M2 polarization of peritoneal macrophages via H3K18la-Arg1 pathway. • MSM could be a potential therapeutic agent against lethal MRSA-induced sepsis. [ABSTRACT FROM AUTHOR]

Objective: To study the effect of polydatin on the injury of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). Methods: SD rats were induced to develop PAH injury by a single subcutaneous injection of MCT (60 mg/kg). From the second day, rats in the administration group were orally given sildenafil (20 mg/kg) and polydatin (30 or 60 mg/kg) for 3 weeks. At the end of the experiment, right ventricular hypertrophy (RVH) index of SD rats was calculated, pathological damage was assessed by HE staining, transcription levels of target genes were detected by RT-PCR and Elisa, and expression levels of Endothelial-to-mesenchymal transition (EndMT) related proteins were detected by immunohistochemistry (IHC) and immunofluorescence (IF). Finally, molecular docking analysis was used to verify the interaction of polydatin on the main targets. Results: Polydatin could significantly restore the body function, reduce MCT-induced PAH injury, reduce serum biochemical indices; polydatin could effectively inhibit EndMT process by decreasing the expression of N-cadherin, β-catenin and vimentin; polydatin could down-regulate TAGLN expression and increase PECAM1 expression to reduce pulmonary vascular remodeling. The interaction between polydatin and EndMT target was confirmed by molecular docking operation. Conclusion: Pharmacological experiments combined with Combining molecular docking was first used to clarify that polydatin can reduce the pulmonary endothelial dysfunction and pulmonary vascular remodeling induced by MCT by inhibiting EndMT. The results of the study provide new ideas for the further treatment of PAH injury. [ABSTRACT FROM AUTHOR]

Factors promoting chronification of migraine remain almost unknown. In the current research, we studied genetic characteristics of chronic migraine (CM) in comparison with healthy population control aiming to reveal new genetic biomarkers of this disabling disorder. The study included 16 female patients diagnosed with CM. Genetic analysis of blood samples was performed using our specifically designed PGRNseq-NDD panel for targeted sequencing of genes implicated in inflammation, immunoreactivity, neurodegeneration, metabolism and detoxification of drugs, xenobiotics, and endogenous substances. Genotype and allele frequencies were compared using either the standard Pearson's chi-squared test or the two-sided Fisher's exact test and Bonferroni correction for multiple tests. Out of 81 tested genes, our analyses revealed significant difference in genotype distribution between CM patients versus healthy control in four genes MOCS2 (rs62359375), ARG1 (rs34504481), CES2 (rs8192925), and SLCO2B1 (rs548541129) which variants were not previously described. Since these genes are implicated in metabolism of xenobiotics and endogenous substances, these data suggest the role of impaired detoxication processes predisposing to migraine chronification. [ABSTRACT FROM AUTHOR]

Objective: To study the effect of polydatin on the injury of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT).Methods: SD rats were induced to develop PAH injury by a single subcutaneous injection of MCT (60 mg/kg). From the second day, rats in the administration group were orally given sildenafil (20 mg/kg) and polydatin (30 or 60 mg/kg) for 3 weeks. At the end of the experiment, right ventricular hypertrophy (RVH) index of SD rats was calculated, pathological damage was assessed by HE staining, transcription levels of target genes were detected by RT-PCR and Elisa, and expression levels of Endothelial-to-mesenchymal transition (EndMT) related proteins were detected by immunohistochemistry (IHC) and immunofluorescence (IF). Finally, molecular docking analysis was used to verify the interaction of polydatin on the main targets.Results: Polydatin could significantly restore the body function, reduce MCT-induced PAH injury, reduce serum biochemical indices; polydatin could effectively inhibit EndMT process by decreasing the expression of N-cadherin, β-catenin and vimentin; polydatin could down-regulate TAGLN expression and increase PECAM1 expression to reduce pulmonary vascular remodeling. The interaction between polydatin and EndMT target was confirmed by molecular docking operation.Conclusion: Pharmacological experiments combined with Combining molecular docking was first used to clarify that polydatin can reduce the pulmonary endothelial dysfunction and pulmonary vascular remodeling induced by MCT by inhibiting EndMT. The results of the study provide new ideas for the further treatment of PAH injury.

Arginases are often overexpressed in human diseases, and they are an important target for developing anti-aging and antineoplastic drugs. Arginase type 1 (ARG1) is a cytosolic enzyme, and arginase type 2 (ARG2) is a mitochondrial one. In this study, a dataset containing 2115-FDA-approved drug molecules is virtually screened for potential arginase binding using molecular docking against several ARG1 and ARG2 structures. The potential arginase ligands are classified into three categories: (1) Non-selective, (2) ARG1 selective, and (3) ARG2 selective. The evaluated potential arginase ligands are then compared with their clinical use. Remarkably, half of the top 30 potential drugs are used clinically to lower blood pressure and treat cancer, infection, kidney disease, and Parkinson’s disease thus partially validating our virtual screen. Most notable are the antihypertensive drugs candesartan, irbesartan, indapamide, and amiloride, the antiemetic rolapitant, the anti-angina ivabradine, and the antidiabetic metformin which have minimal side effects. The partial validation also favors the idea that the other half of the top 30 potential drugs could be used in therapeutic settings. The three categories greatly expand the selectivity of arginase inhibition.

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC–MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.

Cyclooxygenase 2 (COX-2) is a key enzyme in the synthesis of prostaglandins. Recent studies have shown that overexpression of COX-2 can reduce the antitumor effect of the immune system by inhibiting the proliferation of B and T lymphocytes. Programmed cell death ligand 1 (PD-L1) was the first functionally characterized ligand of programmed cell death protein 1. It plays an important role in maintaining peripheral and central immune tolerance by combining with programmed cell death protein 1. Arginase 1 (ARG1) can process L -arginine in the local microenvironment and affect the function of T cells, resulting in immune escape. In this study, COX-2, PD-L1, and ARG1 expression in human pituitary adenoma (PA) and their relationship were investigated, which provided an initial theoretic basis for further study of the immune escape mechanism in PA in cellular and animal experiments. The protein expression of COX-2, PD-L1, and ARG1 in 55 PA samples was detected by immunohistochemistry, with 10 normal brain tissues as the control group. The location of COX-2, PD-L1, and ARG1 in PA cells was studied by double immunofluorescence colocalization. The results of immunohistochemistry were further verified by Western blot. The expression of COX-2, PD-L1, and ARG1 in PA was significantly higher than that in normal brain tissue. In functional PA (FPA) and nonfunctional PA (NFPA), there was no significant difference in the expression of COX-2 and PD-L1, whereas ARG1 was higher in NFPA. Moreover, the protein expression level of COX-2 was positively correlated with that of PD-L1 and ARG1, and the expression of PD-L1 was positively correlated with that of ARG1. Immunofluorescence confocal imaging showed that COX-2, PD-L1, and ARG1 were all expressed in the cytoplasm of PA cells, and the physical positions of COX-2, PD-L1, and ARG1 were partially coincident. These findings indicate that overexpression of COX-2, PD-L1, and ARG1 may be involved in the pathogenesis of PA. ARG1 plays a more important role in the development of NFPA. By upregulating the expression of PD-L1, COX-2 may promote the expression of ARG1, forming the COX-2/PD-L1/ARG1 signal pathway in promoting the occurrence and development of PA. Perhaps further study of the pathogenesis of PA can start with the mechanism of immune escape. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophagespecific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine. [ABSTRACT FROM AUTHOR]

Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer's disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrPSc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states. [ABSTRACT FROM AUTHOR]

Abstract Background Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity. Methods CB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively. Results CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers. Conclusions These results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

In 2018, African swine fever broke out in China, and the death rate after infection was close to 100%. There is no effective and safe vaccine in the world. In order to better characterize and understand the virus–host-cell interaction, quantitative proteomics was performed on porcine alveolar macrophages (PAM) infected with ASFV through tandem mass spectrometry (TMT) technology, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The proteome difference between the simulated group and the ASFV-infected group was found at 24 h. A total of 4218 proteins were identified, including 306 up-regulated differentially expressed proteins and 238 down-regulated differentially expressed proteins. Western blot analysis confirmed changes in the expression level of the selected protein. Pathway analysis is used to reveal the regulation of protein and interaction pathways after ASFV infection. Functional network and pathway analysis can provide an insight into the complexity and dynamics of virus–host cell interactions. Further study combined with proteomics data found that ARG1 has a very important effect on ASFV replication. It should be noted that the host metabolic pathway of ARG1-polyamine is important for virus replication, revealing that the virus may facilitate its own replication by regulating the level of small molecules in the host cell.

Cerebral ischemia induces neuroinflammation and microglial activation, in which activated microglia upregulate their proliferative activity and change their metabolic states. In activated microglia, l-arginine is metabolized competitively by inducible nitric oxide synthase (iNOS) and arginase (Arg), which then synthesize NO or polyamines, respectively. Our previous study demonstrated that Sema4D deficiency inhibits iNOS expression and promotes proliferation of ionized calcium-binding adaptor molecule 1 (Iba1)-positive (Iba1 +) microglia in the ischemic cortex, although the underlying mechanisms were unclear. Using middle cerebral artery occlusion, we tested the hypothesis that Sema4D deficiency alters the balance of l-arginine metabolism between iNOS and Arg, leading to an increase in the production of polyamines, which are an essential factor for cell proliferation. In the peri-ischemic cortex, almost all iNOS + and/or Arg1 + cells were Iba1 + microglia. In the peri-ischemic cortex of Sema4D-deficient (Sema4D−/−) mice, the number of iNOS + Arg1 − Iba1 + microglia was smaller and that of iNOS − Arg1 + Iba1 + microglia was greater than those of wild-type (WT) mice. In addition, urea and polyamine levels in the ischemic cortex of Sema4D−/− mice were higher than those of WT mice; furthermore, the presence of Sema4D inhibited polyamine production in primary microglia obtained from Sema4D−/− mice. Finally, microglia cultured under polyamine putrescine-supplemented conditions demonstrated increased proliferation rates over non-supplemented controls. These findings indicate that Sema4D regulates microglial proliferation at least in part by regulating the competitive balance of l-arginine metabolism. • Sema4D deficiency promotes microglial proliferation after cerebral ischemia. • Sema4D deficiency shifts the l-arginine metabolism of microglia to polyamine production in the ischemic cortex. • The polyamine putrescine promotes microglial proliferation in vitro. [ABSTRACT FROM AUTHOR]

• MDSCs are found in the burn wound and uninjured skin, but not the skin of naïve mice. • TLR expression is suppressed on burn wound MDSCs. • Burn wound MDSCs constitutively produced both pro- and anti-inflammatory cytokines. • Burn wound M-MDSCs expressed both iNOS and ARG1. Myeloid-derived suppressor cells (MDSCs) have been identified in the burn wound, however their characterization is incomplete. To study this, mice were subjected to a major burn and skin cells were isolated 3 days thereafter for analysis. Significant infiltration of the burn wound with MDSCs was observed as compared with uninjured skin. The skin of naïve mice did not contain MDSCs. Characterization of the cells showed that 33% of MDSCs in the wound were monocytic (M)-MDSCs, which was significantly less than that found in uninjured skin (52%). In contrast, polymorphonuclear (PMN)-MDSCs were greater in the burn wound as compared with uninjured skin. Burn wound TLR expression by both MDSCs subsets was decreased as compared with uninjured skin. Wound MDSCs produced pro- and anti-inflammatory cytokines and iNOS was present in both MDSC subsets, whereas ARG1 was only present in M-MDSCs. In conclusion, both M- and PMN-MDSCs infiltrate burn wound with after injury, however, they displayed decreased TLR expression, suggesting receptor down-regulation. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in murine and patient hepatocellular carcinoma (HCC), despite reduced expression of arginine synthesis genes. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth. [Display omitted] • Arginine is increased in liver cancer despite suppression of the urea cycle • Increased uptake and decreased conversion to polyamines maintain high arginine • Arginine binds the transcription regulator RBM39 to promote oncogenic metabolism • RBM39 dependency in liver cancer could be exploited by molecular glue degraders Arginine is a second messenger-like molecule that promotes liver tumorigenesis by binding to RBM39 and controlling metabolic gene expression. RBM39 depletion by the molecular glue degrader indisulam blocks pro-oncogenic metabolic reprogramming in hepatocellular carcinoma, suggesting that RBM39 is a therapeutic target in liver cancer. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner. [ABSTRACT FROM AUTHOR]

mRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5′UTR and 3′UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5′ UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5′UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs. [ABSTRACT FROM AUTHOR]