Societal costs and quality of life associated with arginase 1 deficiency in a European setting - a multinational, cross-sectional survey.

BACKGROUND AND AIMS: Arginase 1 deficiency (ARG1-D) is a ultrarare disease with manifestations that cause mobility and cognitive impairment that progress over time and may lead to early mortality. Diseases such as ARG1-D have a major impact also outside of the health care sector and the aim of this study was to estimate the current burden of disease associated with ARG1-D from a societal perspective. METHODS: The study was performed as a web-based survey of patients with ARG1-D and their caregivers in four European countries (France, Portugal, Spain, United Kingdom). The survey was distributed at participating clinics and included questions on e.g., symptoms (including the Gross Motor Function Classification System, GMFCS, and cognitive impairment), health care use, medication, ability to work, caregiving, and impact on health-related quality-of-life (HRQoL) using the EQ-5D-5L. RESULTS: The estimated total mean societal cost per patient and year was £63,775 (SD: £49,944). The cost varied significantly with both mobility impairment (from £49,809 for GMFCS level 1 to £103,639 for GMFCS levels 3-5) and cognitive impairment (from £43,860 for mild level to £99,162 for severe level). The mean utility score on the EQ-5D-5L for patients was 0.498 (SD: 0.352). The utility score also varied significantly with both mobility impairment (from 0.783 for GMFCS level 1 to 0.153 for GMFCS level 3-5) and cognitive impairment (from 0.738 for mild level to 0.364 for severe level). CONCLUSIONS: Similar to other studies of rare diseases, the study is based on a limited number of observations. However, the sample appear to be reasonably representative when comparing to previous studies of ARG1-D. This study shows that ARG1-D is associated with a high societal cost and significant impact on HRQoL. Earlier diagnosis and better treatment options that can postpone or withhold progression may therefore have a potential for improved HRQoL and savings for the patient, caregiver, and society.

New biomarkers for liver involvement by dengue infection in adult Vietnamese patients: a case-control study.

Liver injury with marked elevation of aspartate aminotransferase enzyme (AST) is commonly observed in dengue infection. To understand the pathogenesis of this liver damage, we compared the plasma levels of hepatic specific, centrilobular predominant enzymes (glutamate dehydrogenase, GLDH; glutathione S transferase-α, αGST), periportal enriched 4-hydroxyphenylpyruvate dioxygenase (HPPD), periportal predominant arginase-1 (ARG-1), and other non-specific biomarkers (paraoxonase-1, PON-1) in patients with different outcomes of dengue infection. This hospital-based study enrolled 87 adult dengue patients, stratified into three groups based on plasma AST levels (< 80, 80-400, > 400 U/L) in a 1:1:1 ratio (n = 40, n = 40, n = 40, respectively. The new liver enzymes in the blood samples from the 4th to 6th days of their illness were measured by commercial enzyme-linked immunosorbent assay (ELISA) or colorimetric kits. Based on the diagnosis at discharge days, our patients were classified as 40 (46%) dengue without warning signs (D), 35 (40.2%) dengue with warning signs (DWS), and 11 (12.6%) severe dengue (SD) with either shock (two patients) or AST level over 1000 U/L (nine patients), using the 2009 WHO classification. The group of high AST (> 400 U/L) also had higher ALT, GLDH, ARG-1, and HPPD than the other groups, while the high (> 400 U/L) and moderate (80-400 U/L) AST groups had higher ALT, αGST, ARG-1, and HPPD than the low AST group (< 80 U/L). There was a good correlation between AST, alanine aminotransferase enzyme (ALT), and the new liver biomarkers such as GLDH, αGST, ARG-1, and HPPD. Our findings suggest that dengue-induced liver damage initiates predominantly in the centrilobular area toward the portal area during the dengue progression. Moreover, these new biomarkers should be investigated further to explain the pathogenesis of dengue and to validate their prognostic utility.

Arginine Deprivation/Citrulline Augmentation with ADI-PEG20 As Novel Therapy for Complications in Type 2 Diabetes.

OBJECTIVE: Chronic inflammation and oxidative stress mediate the pathological progression of diabetic complications, like diabetic retinopathy (DR), peripheral neuropathy (DPN) and impaired wound healing. Studies have shown that treatment with a stable form of arginase 1 that reduces L-arginine levels and increases ornithine and urea limits retinal injury and improves visual function in DR. We tested the therapeutic efficacy of PEGylated arginine deiminase (ADI-PEG20) that depletes L-arginine and elevates L-citrulline on diabetic complications in the db/db mouse model of type 2 diabetes (T2D). METHODS: Mice received intraperitoneal (IP), intramuscular (IM), or intravitreal (IVT) injections of ADI-PEG20 or PEG20 as control. Effects on body weight, fasting blood glucose levels, blood retinal barrier (BRB) function, visual acuity, contrast sensitivity, thermal sensitivity, and wound healing were determined. Studies using bone marrow-derived macrophages (BMDM) examined the underlying signaling pathway. RESULTS: Systemic injections of ADI-PEG20 reduced body weight and blood glucose and decreased oxidative stress and inflammation in db/db retinas. These changes were associated with improved BRB and visual function along with thermal sensitivity and wound healing. IVT injections of either ADI-PEG20, anti-VEGF antibody or their combination also improved BRB and visual function. ADI-PEG20 treatment also prevented LPS/IFNℽ-induced activation of BMDM in vitro as did depletion of L-arginine and elevation of L-citrulline. CONCLUSIONS: /interpretation: ADI-PEG20 treatment limited signs of DR and DPN and enhanced wound healing in db/db mice. Studies using BMDM suggest that the anti-inflammatory effects of PEG-ADI involve blockade of the JAK2-STAT1 signaling pathway via L-arginine depletion and L-citrulline production.

Toll-Like Receptor 1/2 Postconditioning by the Ligand Pam3cys Tempers Posttraumatic Hyperexcitability, Neuroinflammation, and Microglial Response: A Potential Candidate for Posttraumatic Epilepsy.

Toll-like receptors (TLRs) are activated by endogenous molecules released from damaged cells and contribute to neuroinflammation following traumatic brain injury (TBI) and epilepsy. TLR1/2 agonist tri-palmitoyl-S-glyceryl-cysteine (Pam3cys) is a vaccine adjuvant with confirmed safety in humans. We assessed impact of TLR1/2 postconditioning by Pam3cys on epileptogenesis and neuroinflammation in male rats, 6, 24, and 48 h after mild-to-moderate TBI. Pam3cys was injected into cerebral ventricles 30 min after controlled cortical impact (CCI) injury. After 24 h, rats underwent chemical kindling by once every other day injections of pentylenetetrazole (PTZ) 35 mg/kg until development of generalized seizures. Number of intact neurons, brain expression of proinflammatory cytokine TNF-α, anti-inflammatory cytokine IL-10, and marker of anti-inflammatory microglia arginase1 (Arg1) were determined by immunoblotting. Astrocytes and macrophage/microglia activation/polarization at the contused area was assessed by double immunostaining with Iba1/Arg1, Iba1/iNOS and GFAP/iNOS, specific antibodies. The CCI-injured rats became kindled by less number of PTZ injections than sham-operated rats (9 versus 14 injections, p < 0.0001). Pam3cys treatment returned the accelerated rate of epileptogenesis in TBI state to the sham level. Pam3cys decreased neural death 48 h after TBI. It decreased TNF-α (6 h post-TBI, p < 0.01), and up-regulated IL-10 (p < 0.01) and Arg1 (p < 0.05) 48 h after TBI. The iNOS-positive cells decreased (p < 0.001) whereas Iba1/Arg1-positive cells enhanced (p < 0.01) after Pam3cys treatment. Pam3cys inhibits TBI-accelerated acquisition of seizures. Pam3cys reprograms microglia and up-regulates anti-inflammatory cytokines during the first few days after TBI. This capacity along with the clinical safety, makes Pam3cys a potential candidate for development of effective medications against posttraumatic epilepsy.

CDK8/19 inhibitor enhances arginase-1 expression in macrophages via STAT6 and p38 MAPK activation.

Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPK） even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.

Decreased Memory and Learning Ability Mediated by Bmal1/M1 Macrophages/Angptl2/Inflammatory Cytokine Pathway in Mice Exposed to Long-Term Blue Light Irradiation.

Humans are persistently exposed to massive amounts of blue light via sunlight, computers, smartphones, and similar devices. Although the positive and negative effects of blue light on living organisms have been reported, its impact on learning and memory remains unknown. Herein, we examined the effects of widespread blue light exposure on the learning and memory abilities of blue light-exposed mice. Ten-week-old male ICR mice were divided into five groups (five mice/group) and irradiated with blue light from a light-emitting diode daily for 6 months. After 6 months of blue light irradiation, mice exhibited a decline in memory and learning abilities, assessed using the Morris water maze and step-through passive avoidance paradigms. Blue light-irradiated mice exhibited a decreased expression of the clock gene brain and muscle arnt-like 1 (Bmal1). The number of microglia and levels of M1 macrophage CC-chemokine receptor 7 and inducible nitric oxide synthase were increased, accompanied by a decrease in M2 macrophage arginase-1 levels. Levels of angiopoietin-like protein 2 and inflammatory cytokines interleukin-6, tumor necrosis factor-α, and interleukin-1ß were elevated. Our findings suggest that long-term blue light exposure could reduce Bmal1 expression, activate the M1 macrophage/Angptl2/inflammatory cytokine pathway, induce neurodegeneration, and lead to a decline in memory.

Neither injury induced macrophages within the nerve, nor the environment created by Wallerian degeneration is necessary for enhanced in vivo axon regeneration after peripheral nerve injury.

BACKGROUND: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. METHODS: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. RESULTS: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. CONCLUSIONS: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

Tuberculous pleural effusion-induced Arg-1+ macrophage polarization contributes to lung cancer progression via autophagy signaling.

BACKGROUND: The association between tuberculous fibrosis and lung cancer development has been reported by some epidemiological and experimental studies; however, its underlying mechanisms remain unclear, and the role of macrophage (MФ) polarization in cancer progression is unknown. The aim of the present study was to investigate the role of M2 Arg-1+ MФ in tuberculous pleurisy-assisted tumorigenicity in vitro and in vivo. METHODS: The interactions between tuberculous pleural effusion (TPE)-induced M2 Arg-1+ MФ and A549 lung cancer cells were evaluated. A murine model injected with cancer cells 2 weeks after Mycobacterium bovis bacillus Calmette-Guérin pleural infection was used to validate the involvement of tuberculous fibrosis to tumor invasion. RESULTS: Increased CXCL9 and CXCL10 levels of TPE induced M2 Arg-1+ MФ polarization of murine bone marrow-derived MФ. TPE-induced M2 Arg-1+ MФ polarization facilitated lung cancer proliferation via autophagy signaling and E-cadherin signaling in vitro. An inhibitor of arginase-1 targeting M2 Arg-1+ MФ both in vitro and in vivo significantly reduced tuberculous fibrosis-induced metastatic potential of lung cancer and decreased autophagy signaling and E-cadherin expression. CONCLUSION: Tuberculous pleural fibrosis induces M2 Arg-1+ polarization, and M2 Arg-1+ MФ contribute to lung cancer metastasis via autophagy and E-cadherin signaling. Therefore, M2 Arg-1+ tumor associated MФ may be a novel therapeutic target for tuberculous fibrosis-induced lung cancer progression.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Decreased circulating myeloid-derived suppressor cell count at the engraftment is one of the risk factors for multiple myeloma relapse after autologous hematopoietic stem cell transplantation.

Recent studies demonstrated that myeloid-derived suppressor cells (MDSCs) are involved in the pathogenesis and progression of multiple myeloma (MM). Nevertheless, data on the quantitative and functional changes in MDSCs during standard MM treatment remain poorly understood. Here, we determined that monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/-) and granulocytic MDSCs (PMN-MDSC; Lin-HLA-DR-CD33+CD66b+) in MM patients in remission following induction therapy (IT) were significantly increased, while early MDSCs (E-MDSCs; Lin-HLA-DR-CD33+CD66b-) were decreased compared to the donor group. In progression, MM patients had the most pronounced decrease in E-MDSCs and enhanced levels of PMN-MDSCs. IT was accompanied with a decrease in the expression of arginase-1 (Arg-1). In MM patients with relapse or resistance to IT, Arg-1+ cell frequency in M-MDSCs and E-MDSCs, as well as PD-L1+ M-MDSCs, was increased, which may facilitate tumor immunosuppression. G-CSF administration led to a significant increment in the MDSC subsets. At the engraftment, circulating M-MDSC and PMN-MDSCs were temporarily increased, with a gradual decline to the pre-transplant levels in 12 months. The percentage of E-MDSCs was decreased at the leukocyte recovery. Patients with a higher (>Me) M-MDSC count at the engraftment had a shorter post-transplant leukopenia duration (Me 11 vs. 13 days; pU = 0.0086). The advanced MM stage, depth of response, and lower relative count of circulating E-MDSCs at the engraftment were independent risk factors associated with a lower progression-free survival. The obtained data allow us to hypothesize that MDSCs may play a positive role at the stage of leukocyte recovery by ameliorating the long-term anti-tumor response in MM.

Ablative fractional CO2 laser treatment promotes wound healing phenotype in skin macrophages.

OBJECTIVES: Ablative fractional laser (AFL) treatment is a well-established method for reducing signs of skin photoaging. However, the biological mechanisms underlying AFL-induced healing responses and skin rejuvenation remain largely unknown. It is known that macrophages play an important role in orchestrating healing, normalization, and remodeling processes in skin. Macrophage phenotypes are characterized by inflammatory markers, including arginase-1 (Arg1), major histocompatibility class II molecules (MHC II), and CD206. This study aims to explore AFL's effect on macrophage phenotype by evaluating changes in inflammatory markers and the potential concurrent accumulation of Arg1 in the skin. METHODS: Mice (n = 9) received a single AFL treatment on the left side of the back skin (100 mJ/microbeam, 5% density) while the right side of the back remained untreated as control. Treated and untreated skin from each mouse were collected Day 5 posttreatment for flow cytometry and histology analysis. Flow cytometry evaluated the immune infiltration of macrophages and the expression of macrophage inflammatory markers (Arg1, MHC II, and CD206). In addition, Arg1 presence in the skin was evaluated through antibody staining of histology samples and quantification was performed using QuPath image analysis software. RESULTS: Following AFL, the number of macrophages increased 11-fold (p = 0.0053). Phenotype analysis of AFL-treated skin revealed an increase in the percentage of macrophages positive for Arg1 (p < 0.0001) and a decrease in the percentage of macrophages positive for MHC II (p < 0.0001) compared to untreated skin. No significant differences were observed in percentage of CD206-positive macrophages (p = 0.8952). Visualization of AFL-treated skin demonstrated a distinct pattern of Arg1 accumulation that correlated with the microscopic treatment zones (MTZ). Quantification of the percentage of Arg1-positive area in epidermis and dermis showed a significant increase from 3.5% ± 1.2% to 5.2% ± 1.7 (p = 0.0232) and an increase from 2.2% ± 1.2% to 9.6% ± 3.3 (p < 0.0001) in whole skin samples. CONCLUSION: AFL treatment polarizes macrophages toward a wound healing phenotype and induces Arg1 accumulation in the MTZ. We propose that the polarized wound healing macrophages are a major source for the increased Arg1 levels observed in the skin following treatment.

Arginase-1 specific CD8+ T cells react toward malignant and regulatory myeloid cells.

Arginase-1 (Arg1) is expressed by regulatory myeloid cells in the tumor microenvironment (TME), where they play a pro-tumorigenic and T-cell suppressive role. Arg1-specific CD4+ and CD8+ memory T cells have been observed in both healthy individuals and cancer patients. However, while the function of anti-regulatory Arg1-specific CD4+ T cells has been characterized, our knowledge of CD8+ Arg1-specific T cells is only scarce. In the current study, we describe the immune-modulatory capabilities of CD8+ Arg1-specific T cells. We generated CD8+ Arg1-specific T cell clones to target Arg1-expressing myeloid cells. Our results demonstrate that these T cells recognize both malignant and nonmalignant regulatory myeloid cells in an Arg1-expression-dependent manner. Notably, Arg1-specific CD8+ T cells possess cytolytic effector capabilities. Immune modulatory vaccines (IMVs) represent a novel treatment modality for cancer. The activation of Arg1-specific CD8+ T cells through Arg1-based IMVs can contribute to the modulatory effects of this treatment strategy.

Role of myeloid-derived suppressor cells in chronic brucellosis.

Introduction: Human brucellosis, a Brucella infection caused most common zoonosis in the world, remains a serious public health burden in China. Brucella chronic infection always causes immunosuppressive status and results in severe organ or tissue damages. The aim of this work was to study the role of the myeloid-derived suppressor cells (MDSCs) in human chronic brucellosis. Methods: Fifty cases of chronic brucellosis and 40 healthy individual controls were enrolled in this study. We analyzed the frequency and subsets of MDSCs in PBMC between the chronic brucellosis and healthy control groups by flow cytometry. Furthermore, we also measured the inflammatory-related cytokines in serum samples and the MDSCs inhibition ability to the proliferation of T cells in vitro. Results: We found that the frequency of MDSCs in peripheral blood and the level of IL-6 and IL-10 Th2 cytokines and Arginase-1 were significantly increased in chronic brucellosis patients. In addition, we also found that the T cell function was suppressed in vitro by co-culturing with MDSCs from brucellosis patients. Conclusion: Our study described an increase of immunosuppressive MDSCs in peripheral blood of chronic brucellosis patients. These results contribute to the understanding of Brucella persistent infection, which may provide an insight for effective treatment of chronic brucellosis patients in clinical practice.

Transforming growth factor-ß1 and soluble co-inhibitory immune checkpoints as putative drivers of immune suppression in patients with basal cell carcinoma.

The current study compared the levels and possible associations between systemic soluble immune checkpoints (sICPs, n = 17) and a group of humoral modulators of immune suppressor cells (n = 7) in a cohort of patients with basal cell carcinoma (BCC, n = 40) and a group of healthy control subjects (n = 20). The seven humoral modulators of immunosuppressor cells were represented by the enzymes, arginase 1 and fibroblast activation protein (FAP), the chemokine, RANTES (CCL5) and the cytokines, interleukin-10 and transforming growth factor-ß1 (TGF-ß1), as well as the M2-type macrophage markers, soluble CD163 (sCD163) and sCD206. The plasma levels of six co-inhibitory sICPs, sCTLA-4, sLAG-3, sPD-1, sPD-L1, sTIM-3 and sPD-L2 were significantly elevated in the cohort of BCC patients (p<0.001-p<0.00001), while that of sBTLA was significantly decreased (p<0.006). Of the co-stimulatory sICPs, sCD27 and sGITR were significantly increased (p<0.0002 and p<0.0538) in the cohort of BCC patients, while the others were essentially comparable with those of the control participants; of the dual active sICPs, sHVEM was significantly elevated (p<0.00001) and TLR2 comparable with the control group. A correlation heat map revealed selective, strong associations of TGF-ß1 with seven co-stimulatory (z = 0.618468-0.768131) and four co-inhibitory (z = 0.674040-0.808365) sICPs, as well as with sTLR2 (z = 0.696431). Notwithstanding the association of BCC with selective elevations in the levels of a large group of co-inhibitory sICPs, our novel findings also imply the probable involvement of TGF-ß1 in driving immunosuppression in this malignancy, possibly via activation of regulatory T cells. Notably, these abnormalities were present in patients with either newly diagnosed or recurrent disease.

Arginase 1 does not affect RNA m6A methylation in mouse fetal lung.

BACKGROUND: Arginase 1 (Arg1) encodes a key enzyme that catalyzes the metabolism of arginine to ornithine and urea. In our recent study, we found that knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. As the most abundant internal mRNA modification, N6 -methyladenosine (m6 A) has been found to play important roles in lung development and cellular differentiation. However, if the knockdown of Arg1 affects the RNA m6A modification in fetal lungs remains unknown. METHODS: In the current study, the RNA m6A levels and the expression of RNA m6A related enzymes were validated in 13.0 dpc fetal lungs that Arg1 was knocked down by adeno-associated virus carrying Arg1-shRNA, using western blot, immunofluorescence, and RT-qPCR. RESULTS: No statistical differences were found in the expression of methyltransferase, demethylases, and binding proteins in the fetal lungs between AAV-shArg1-injected mice and AAV-2/9-injected mice. Besides, there is no significant change of overall RNA m6A level in fetal lungs from AAV-shArg1-injected mice, compared with that from AAV-2/9-injected mice. CONCLUSIONS: These results indicate that arginase 1 does not affect RNA m6A methylation in mouse fetal lung, and the mechanisms other than RNA m6A modification underlying the effects of Arg1 knockdown on the fetal lung development and their interaction with labor initiation need to be further explored.

Efficacy and safety of pegzilarginase in arginase 1 deficiency (PEACE): a phase 3, randomized, double-blind, placebo-controlled, multi-centre trial.

Background: Arginase 1 Deficiency (ARG1-D) is a rare debilitating, progressive, inherited, metabolic disease characterized by marked increases in plasma arginine (pArg) and its metabolites, with increased morbidity, substantial reductions in quality of life, and premature mortality. Effective treatments that can lower arginine and improve clinical outcomes is currently lacking. Pegzilarginase is a novel human arginase 1 enzyme therapy. The present trial aimed to demonstrate efficacy of pegzilarginase on pArg and key mobility outcomes. Methods: This Phase 3 randomized, double-blind, placebo-controlled, parallel-group clinical trial (clinicaltrials.govNCT03921541, EudraCT 2018-004837-34), randomized patients with ARG1-D 2:1 to intravenously/subcutaneously once-weekly pegzilarginase or placebo in conjunction with their individualized disease management. It was conducted in 7 countries; United States, United Kingdom, Canada, Austria, France, Germany, Italy. Primary endpoint was change from baseline in pArg after 24 weeks; key secondary endpoints were change from baseline at Week 24 in Gross Motor Function Measure part E (GMFM-E) and 2-min walk test (2MWT). Full Analysis Set was used for the analyses. Findings: From 01 May 2019 to 29 March 2021, 32 patients were enrolled and randomized (pegzilarginase, n = 21; placebo, n = 11). Pegzilarginase lowered geometric mean pArg from 354.0 µmol/L to 86.4 µmol/L at Week 24 vs 464.7 to 426.6 µmol/L for placebo (95% CI: -67.1%, -83.5%; p < 0.0001) and normalized levels in 90.5% of patients (vs 0% with placebo). In addition, clinically relevant functional mobility improvements were demonstrated with pegzilarginase treatment. These effects were sustained long-term through additional 24 weeks of subsequent exposure. Pegzilarginase was well-tolerated, with adverse events being mostly transient and mild/moderate in severity. Interpretation: These results support pegzilarginase as the first potential treatment to normalize pArg in ARG1-D and achieve clinically meaningful improvements in functional mobility. Funding: Aeglea BioTherapeutics.

Triptolide inhibits the proinflammatory potential of myeloid-derived suppressor cells via reducing Arginase-1 in rheumatoid arthritis.

Triptolide (TPT) is widely used in the treatment of rheumatoid arthritis (RA). However, its regulatory mechanisms are not fully understood. This study demonstrated that Myeloid-derived suppressor cells (MDSCs) were expanded in both RA patients and arthritic mice. The frequency of MDSCs was correlated with RA disease severity and T helper 17 (Th17) responses. MDSCs from RA patients promoted the polarization of Th17 cells in vitro, which could be substantially attenuated by blocking arginase-1 (Arg-1). TPT inhibited the differentiation of MDSCs, particularly the monocytic MDSCs (M-MDSCs) subsets, as well as the expression of Arg-1 in a dose dependent manner. Alongside, TPT treatment reduced the potential of MDSCs to promote the polarization of IL-17+ T cell in vitro. Consistently, TPT immunotherapy alleviated adjuvant-induced arthritis (AIA) in a mice model, and reduced the frequency of MDSCs, M-MDSCs and IL-17+ T cells simultaneously. The presented data suggest a pathogenic role of MDSCs in RA and may function as a novel and effective therapeutic target for TPT in RA.

The value of echocardiographic indicators combined with serum ARG1 and G6PD in the prognosis evaluation of children with sepsis / 国际检验医学杂志

Objective To investigate the prognostic value of echocardiography indicators combined with se-rum recombinant human arginase 1(ARG1)and glucose-6-phosphate dehydrogenase(G6PD)in children with sepsis.Methods A total of 116 children with sepsis admitted to the hospital from May 2022 to June 2023 were enrolled in the study as the sepsis group.According to the severity of sepsis,the children were further divided into general sepsis group(52 cases),severe sepsis group(38 cases)and septic shock group(26 cases).Ac-cording to the prognosis of the children,the children with sepsis were divided into good prognosis group(84 cases)and poor prognosis group(32 cases).A total of 116 healthy children who underwent physical examina-tion in the hospital during the same period were enrolled as the control group.The left ventricular ejection fraction(LVEF),left ventricular end-diastolic diameter(LVEDD),left ventricular end-diastolic volume(LV-EDV)and early diastolic mitral flow peak velocity(E)were detected by using color Doppler ultrasound.Ser-um ARG1 and G6PD levels were detected by using enzyme-linked immunosorbent assay(ELISA).The echo-cardiographic indexes and serum ARG1 and G6PD levels were compared between the sepsis group and the con-trol group,and among sepsis children with different disease severity and different prognosis.The receiver op-erating characteristic(ROC)curve was used to analyze the predictive value of echocardiographic indexes com-bined with serum ARG1 and G6PD for poor prognosis in children with sepsis.Results Compared with the control group,the sepsis group had significant reductions in LVEF,E,and G6PD(P＜0.05)and significant increases in LVEDD,LVEDV,and ARG1(P＜0.05).With the aggravation of sepsis,the levels of LVEF,E,and G6PD in children with sepsis gradually decreased(P＜0.05),while the levels of LVEDD,LVEDV,and ARG1 gradually increased(P＜0.05).Compared with the good prognosis group,the poor prognosis group had significantly lower levels of LVEF,E,and G6PD(P＜0.05)and significantly higher levels of LVEDD,LV-EDV,and ARG1(P＜0.05).ROC curve analysis showed that the AUC of echocardiographic indexes com-bined with serum ARG1 and G6PD in predicting poor prognosis of children with sepsis was 0.971,and the sensitivity and specificity were 84.4％and 83.2％,respectively.Conclusion The levels of LVEF,E,and G6PD in children with sepsis significantly decreases,and the levels of LVEDD,LVEDV,and ARG1 signifi-cantly increases.Echocardiographic parameters combined with serum ARG1 and G6PD have high predictive value for poor prognosis in children with sepsis.

Anti-inflammatory effect of Celastrol in the ocular tissues of mice with exper-imental autoimmune uveitis and its impact on microglia polarization / 眼科新进展

Objective To investigate the anti-inflammatory action of Celastrol in the ocular tissues of mice with ex-perimental autoimmune uveitis(EAU)and its effect on microglia polarization.Methods A total of 36 healthy B10.RⅢmice at 6-8 weeks of age were selected and randomly divided into the normal control group,EAU solvent control group and Celastrol intervention group,with 12 mice in each group.The interphotoreceptor retinoid-binding protein(IRBP)161-180 and Freund's complete adjuvant were mixed by thorough emulsification and injected subcutaneously into the bilateral thighs and tails of mice in the EAU solvent control group and the Celastrol intervention group with a total volume of 200 μL and 50 μg IRBP 161-180 in each mouse.On 7-14 days after immunization,mice in the Celastrol intervention group received a daily intraperitoneal injection of 0.5 mg·kg-1 Celastrol,and mice in the EAU solvent control group were injected with an equivalent dose of sterile Phosphate Buffered Saline solution.On the 14th day after immunization,the anterior segment of mice in each group was observed by slit-lamp microscope and Hematoxylin and Eosin(HE)staining of tissue sections was performed;the clinical and histopathological scores of mice in each group were obtained by reference to the Caspi grading standards;immunofluorescence staining was used to observe the activation of microglia in the eyes of mice;Western blot was used to detect the protein expression levels of inducible nitric oxide synthase(iNOS)and arginase-1(Argl)in the reti-na;quantitative real-time PCR was used to detect the relative mRNA expression of inflammatory factors in the retina,such as tumor necrosis factor(TNF)-α,interleukin(IL)-1β and IL-6.GraphPad Prism 9.0 was used for data analysis.Results On the 14th day after immunization,it was observed by the slit-lamp microscope that the anterior segment of mice in the EAU solvent control group was markedly congested with dilated iris blood vessels,corneal edema,and anterior chamber exudation;the inflammation in the anterior segment of mice in the Celastrol intervention group was markedly at-tenuated,and the iris blood vessels were seen to be mildly congested.Compared with the normal control group,the clini-cal scores of mice in the EAU solvent control group and the Celastrol intervention group were significantly elevated(both P＜0.05);the clinical scores of mice in the Celastrol intervention group were lower than those in the EAU solvent control group(P＜0.05).HE staining results showed that on the 14th day after immunization,mice in the EAU solvent control group showed severe retinal folds and detachment with diffuse infiltration of inflammatory cells,while mice in the Celastrol intervention group showed slight structural damage to the retina and a small amount of inflammatory cell infiltration.Com-pared with the normal control group,the histopathological scores of mice in the EAU solvent control group and the Celas-trol intervention group were significantly elevated(both P＜0.05);the histopathological scores of mice in the Celastrol in-tervention group were lower than those in the EAU solvent control group(P＜0.05).The intraocular Iba1+cell densities of mice in the normal control,EAU solvent control and Celastrol intervention groups were(1.00±0.12)％,(36.07± 4.57)％,and(1.83±0.36)％,respectively.Compared with the normal control group,the number of Iba1+cells in the eyes of mice in the EAU solvent control group and the Celastrol intervention group significantly increased(both P＜0.05);compared with the EAU solvent control group,the number of Iba1+cells in the eyes of mice in the Celastrol intervention group was significantly reduced(P＜0.05).Compared with the normal control group,the expression levels of iNOS and Arg1 proteins in the retinas of mice in the EAU solvent control group were significantly elevated(both P＜0.01);compared with the EAU solvent control group,the expression of iNOS protein in the retinas of mice in the Celastrol intervention group was significantly reduced(P＜0.01).Compared with the normal control group,the relative mRNA expressions of TNF-α.IL-1β,and IL-6 in the retinas of mice in the EAU solvent control group was significantly elevated(all P＜0.05);compared with the EAU solvent control group,the relative mRNA expressions of TNF-α,IL-1 β,and IL-6 in the retinas of mice in the Celastrol intervention group significantly decreased(all P＜0.05).Conclusion Celastrol inhibits Ml microglia activation and reduces the production of retinal inflammatory factors TNF-α,IL-1 β and IL-6 in EAU mice,thereby attenuating the in-flammatory reaction.

Discovery of a new marker to identify myeloid cells associated with metastatic breast tumours.

BACKGROUND: Myeloid cells play an essential role in cancer metastasis. The phenotypic diversity of these cells during cancer development has attracted great interest; however, their functional heterogeneity and plasticity have limited their role as prognostic markers and therapeutic targets. METHODS: To identify markers associated with myeloid cells in metastatic tumours, we compared transcriptomic data from immune cells sorted from metastatic and non-metastatic mammary tumours grown in BALB/cJ mice. To assess the translational relevance of our in vivo findings, we assessed human breast cancer biopsies and evaluated the association between arginase 1 protein expression in breast cancer tissues with tumour characteristics and patient outcomes. RESULTS: Among the differentially expressed genes, arginase 1 (ARG1) showed a unique expression pattern in tumour-infiltrating myeloid cells that correlated with the metastatic capacity of the tumour. Even though ARG1-positive cells were found almost exclusively inside the metastatic tumour, ARG1 protein was also present in the plasma. In human breast cancer biopsies, the presence of ARG1-positive cells was strongly correlated with high-grade proliferating tumours, poor prognosis, and low survival. CONCLUSION: Our findings highlight the potential use of ARG1-positive myeloid cells as an independent prognostic marker to evaluate the risk of metastasis in breast cancer patients.