A Study of Arginase Expression in Chronic Non-healing Wounds.

Arginase expression has been recently shown to increase in numerous disease states like neurodegeneration, inflammation, and malignancies. Although it has been found to be functionally important in various disease pathologies, little is known about its role in wound healing. Here, we look at the expression of arginase and its isoforms in chronic non-healing wounds and also study the expression of nitric oxide synthase (NOS) and oxidative stress enzymes in them. Wound tissues and blood samples were collected at the time of index presentation and follow-up from 61 chronic non-healing wound cases. The expression patterns of arginase isoenzymes, NOS, superoxide dismutases (SOD), lactic acid dehydrogenase (LDH), and catalase were examined by using enzyme-linked immunosorbent assay, immunohistochemistry, and western blot analysis at the transcript and protein level. We reported a significant decrease of serum arginase levels in chronic nonhealing wounds in the progress of wound healing. Interestingly, tissue arginase levels were found to be increased with improved wound condition at follow-up. Tissue NOS, LDH, and catalase activity were also found to be increased with the progress of healing, whereas SOD levels were downregulated. Our findings reported increased expression at the transcript level of arginase-I and arginase-II in chronic non-healing wounds for the first time. In conclusion, we observed decreased serum arginase levels in completely healed patients as compared to non-healed cases. Our study findings support the hypothesis that inhibition of the activity of arginase delays wound healing. Arginase and iNOS may also find their place in the future as possible biomarkers for wound healing.

Sex Matters: Physiological Abundance of Immuno-Regulatory CD71+ Erythroid Cells Impair Immunity in Females.

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.

Efficacy of expressions of Arg-1, Hep Par-1, and CK19 in the diagnosis of the primary hepatocellular carcinoma subtypes and exclusion of the metastases.

Many conflicts arise using immunohistochemistry of Hepatocellular carcinoma (HCC), some of these conflicts arise from the biliary part within the tumor itself or from liver metastasis. The aim of this study is to investigate the extent of Arg-1, HepPar-1, and CK-19 expressions in the primary HCC subtypes as well as studying of some metastatic cases to find a distinctive immunohistochemical panel utilizing it to differentiate between these entities. MATERIAL AND METHODS: A paraffin-embedded block including 62 cases of primary HCC, and 18 cases diagnosed as metastatic tumors, were subjected for this study using Anti-liver Arginase antibody (ab125134 Cambridge, USA, polyclonal antibody, 3.75 µg/ml), HepPar-1 (polyclonal mouse antibody OCH1E5; 1:600; DAKO, CA, USA), and CK 19 Anti-Cytokeratin 19 antibody (ab15463, rabbit polyclonal antibody; 1:100; Cambridge, USA). The intensity of immunostaining was scored (0 to 3+). Nuclear and cytoplasmic staining with Arg-1 and cytoplasmic for both HepPar-1 and CK 19 are reported. RESULTS: The histopathological patterns were mainly trabecular no= (24, 38.7%), and pseudoglandular (no=14, 22.5%), mixed hepatocellular cholangiocarcinoma was observed in one case (1.6%). Arginase-1 positivity was in 55 cases (88.7%) opposite to 46 (74.19%) and 8 (12.9%) for HepPAr.1% -1 and CK 19, respectively. The intensity of expression was marked in well and moderate differentiation for Arg-1 and HepPar-1and in poorly differentiated for CK 19. Metastatic carcinoma cases revealed two cases positive for Arg-1 (11.1%), 4 cases (22.2%) positive for HepPar-1, and 13 cases (72.2%) positive for CK 19. CONCLUSION: Arg-1 and HepPar-1 are confirmative in the diagnosis of primary HCC in most cases, either separately or collectively but the priority of selection leans more towards Arg-1. Arg-1 and HepPar-1 positive with negative CK 19 expressions give more support to diagnosis of primary HCC while the reverse will support the diagnosis of tumour of biliary origin or liver metastasis.

Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry.

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid-metabolizing enzymes.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Heterotopic Nodules in the Placenta, an Immunohistochemical Re-evaluation of the Diagnosis of Adrenocortical Heterotopia.

BACKGROUND: Rare nodules of heterotopic adrenocortical and hepatic tissue are reported in the placenta. A mechanism for adrenocortical tissue in the placenta has been perplexing, while hepatic tissue is generally considered related to yolk sac primordia. The clear cell morphology of these nodules is similar to the adrenal cortex of the adult; however, the fetal adrenal gland does not usually display clear cells. METHODS: We stained 9 placental nodules, histologically identical to "adrenocortical" heterotopia of the placenta, to determine whether adrenocortical differentiation could be confirmed. These cases include 3 archival cases initially diagnosed as "adrenocortical" heterotopia. RESULTS: Immunohistochemical staining with steroid factor-1 (SF-1), HepPar-1, and Arginase-1 showed that these nodules of clear cells are actually hepatic (SF-1 negative, HepPar-1, and Arginase-1 positive). PAS staining suggests that glycogen accumulation is responsible for the clear cytoplasm. In contrast, a nodule of adrenocortical heterotopia near the testis and the adrenal gland from a 38-week-old neonatal autopsy case confirm SF-1 reactivity as expected. CONCLUSION: We propose that adrenocortical heterotopia in the placenta is a misnomer, and that these subchorionic nodules of clear cells demonstrate hepatic differentiation.

Myeloid-derived suppressor cells exert immunosuppressive function on the T helper 2 in mice infected with Echinococcus granulosus.

Cystic echinococcosis (CE) is a worldwide hazardous zoonotic parasitosis caused by Echinococcus granulosus. CE development involves complex immunological mechanisms, including participation of multiple immune cells and effector molecules. Myeloid-derived suppressor cells (MDSCs) are known to be involved in chronic and acute inflammatory conditions. In this study, we aimed to characterize the immune function of MDSCs in CE to improve the understanding, prevention and treatment of CE. Our results indicated that MDSCs overexpressing Ly6C and Ly6G inhibit the formation and activity of T helper 2 cells in a NO-dependent manner during E. granulosus infection.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages.

Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 µM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 µM (p<0.05) and 250 µM (p<0.01). The POH increased ROS production at both 10 µM and 100 µM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 µM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 µM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Detection and functional evaluation of arginase-1 isolated from human PMNs and murine MDSC.

Immunotherapy has emerged as a potent alternative for cancer treatment, unfortunately, the clinical benefit remains limited to few patients and immunotherapy resistance due to immunosuppressive tumor microenvironment represents the major reason of such a failure. Arginase-1 is one of the enzymes contributing to the establishment of such immunosuppression. Among the human immune cells, polymorphonuclear cells (PMNs) represent the major source of arginase-1, while myeloid-derived suppressor cells (MDSCs) are the main arginase-1 producing cells in mice. Due to arginase-1 potential impact in dampening the immune response, there is a growing interest in assaying arginase-1 levels and functions. Thus, in this chapter we propose how to evaluate the expression and activity of arginase in human peripheral blood-derived PMNs and in MDSCs isolated from tumor-bearing mice.

Preclinical safety and antitumor activity of the arginine-degrading therapeutic enzyme pegzilarginase, a PEGylated, cobalt-substituted recombinant human arginase 1.

Metabolic remodeling contributes to the development and progression of some cancers and exposes them to vulnerabilities, including specific nutrient dependencies that can be targeted therapeutically. Arginine is a semiessential amino acid, and several cancers are unable to endogenously synthesize sufficient levels of arginine for survival and proliferation, most commonly due to reduced expression of argininosuccinate synthase (ASS1). Such cancers are dependent on arginine and they can be targeted via enzyme-mediated depletion of systemic arginine. We report the preclinical safety, antitumor efficacy, and immune-potentiating effects of pegzilarginase, a highly potent human arginine-degrading enzyme. Toxicology studies showed that pegzilarginase-mediated arginine depletion is well tolerated at therapeutic levels that elicit an antitumor growth effect. To determine which tumor types are best suited for clinical development, we profiled clinical tumor samples for ASS1 expression, which correlated with pegzilarginase sensitivity in vivo in patient-derived xenograft (PDx) models. Among the histologies tested, malignant melanoma, small cell lung cancer and Merkel cell carcinoma had the highest prevalence of low ASS1 expression, the highest proportion of PDx models responding to pegzilarginase, and the strongest correlation between low or no ASS1 expression and sensitivity to pegzilarginase. In an immune-competent syngeneic mouse model, pegzilarginase slowed tumor growth and promoted the recruitment of CD8+ tumor infiltrating lymphocytes. This is consistent with the known autophagy-inducing effects of arginine depletion, and the link between autophagy and major histocompatibility complex antigen presentation to T cells. Our work supports the ongoing clinical investigations of pegzilarginase in solid tumors and clinical combination of pegzilarginase with immune checkpoint inhibitors.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Macrophages - a perspective target for antineoplastic immunotherapy.

The review discusses the data on the functional/phenotypic M1/M2 types of macrophages and their chimeric forms, the molecular mechanisms of polarization of these cells, and their role in the development of malignant tumors. Information on the prognostic value of the presence (density and location) of M1/M2 cells in tumor tissue is analyzed. Our own results evidence on the necessity of determination of the functional/phenotypic state of M1/M2 macrophages from different biological niches in the dynamics of tumor growth, in particular in terms of NO level and arginase activity.

Arginase enzymes in the human prostate: A molecular biological and immunohistochemical approach.

The nitric oxide (NO) pathway plays a role in maintaining the function of the prostate. An impairment in the activity of the NO system may have an impact in the manifestation of lower urinary tract symptomatology and benign prostatic hyperplasia. Arginase enzymes (Arg) counteract the generation of NO by depleting the intracellular pool of L-arginine, known to be the substrate of the NO synthases. This study investigated the expression of arginase type I and II in the human prostate. Nondiseased prostate tissue was obtained during pelvic surgeries (prostatectomy, cystoprostatectomy). Tissue sections were exposed to antibodies directed against Arg I and II, cGMP, the phosphodiesterase 5 and nNOS. The expression of mRNA transcripts encoding for Arg I and Arg II was investigated using molecular biology. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for Arg I and II, immunofluorescence specific for Arg I was seen in the stromal smooth musculature, and labelling for PDE5 and cyclic GMP was also observed. Nerve fibres containing nNOS were identified running across the smooth musculature. Immunostainings for Arg II did not yield signals. These findings are in support of the notion that, in the prostate, Arg is involved in the modulation of the activity of the NO system.

Overexpression of Arginase-1 is an indicator of poor prognosis in patients with colorectal cancer.

AIM: Arginase-1 (Arg-1) metabolizes l-arginine to l-ornithine and urea. It has been documented to have a role in various malignancies. However, the relationship between Arg-1 expression and clinicopathological characteristics of colorectal cancer (CRC) patients remains to be elucidated. The present study aimed to analyze the expression and prognostic value of Arg-1 in patients with CRC. MATERIAL AND METHODS: The mRNA and protein expressions of Arg-1 in fresh colorectal cancer tissue specimens and the corresponding noncancerous tissue specimens were examined by RT-qPCR (n = 24) and western blot analysis (n = 17). Arg-1 expression levels were determined in paraffin-embedded CRC tissue specimens (n = 236) by immunohistochemistry. The associations of Arg-1 expression and clinicopathological features and clinical prognosis in 236 CRC patients were analyzed. RESULTS: The expression levels of Arg-1 were significantly higher in the CRC tissues compared with the matched noncancerous tissues, and elevated Arg-1 expression was remarkably associated with stage III-IV tumors (P = 0.007), lymph node metastasis (P = 0.019) and a plasma albumin concentration <35 g/l (P = 0.022). Kaplan-Meier analysis indicated that Arg-1 overexpression was associated with adverse prognoses for overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001) in all cases. Further analysis revealed that the patients with high Arg-1 expression had significantly shorter OS and DFS at the advanced stages (III + IV) (P = 0.032 for OS, and P = 0.012 for DFS) but not at the early stages (I + II) (P = 0.194 for OS, and P = 0.065 for DFS). Multivariate analysis revealed that Arg-1 overexpression was an independent prognostic factor for OS (P = 0.002) and DFS (P < 0.001) in patients with CRC. CONCLUSION: The data indicated that Arg-1 overexpression in CRC may be a marker that can discriminate subgroups of patients with a poor prognosis.

Ovarian Sertoli-Leydig Cell Tumor With Heterologous Hepatocytes and a Hepatocellular Carcinomatous Element.

Sertoli-Leydig cell tumors are a group of tumors composed of variable proportions of Sertoli cells, Leydig cells, and sometimes heterologous elements. We describe the case of a 68-yr-old woman who presented with abdominal distention. A computed tomographic scan revealed a large right adnexal mass without evidence of intrahepatic tumors, and a complete cytoreductive surgery was performed. Pathologic examination revealed a moderately differentiated Sertoli-Leydig cell tumor with various heterologous elements, including gastrointestinal-type glands, insular carcinoid, and aggregations of hepatocytes without significant cytologic atypia. Moreover, adjacent to these hepatocytes, extensive overgrowth of highly atypical hepatocyte-like cells, providing a striking morphologic similarity to hepatocellular carcinoma of the liver, was identified. Both the heterologous hepatocytes and hepatocellular carcinomatous tumor cells were immunohistochemically positive for alpha-fetoprotein, hepatocyte paraffin 1, and arginase-1. Some Sertoli cells adjacent to the heterologous hepatocytes were also positive for alpha-fetoprotein and hepatocyte paraffin 1. The present case showed that a tumor morphologically and immunohistochemically analogous to hepatocellular carcinoma of the liver can arise in the ovary, in association with Sertoli-Leydig cell tumors.

Ectopic Fetal Hepatic Tissue in the Placenta.

When an unusual intraplacental lesion is identified during pathologic examination, it becomes of substantial import to determine whether it represents a normal structure, metastasis from the mother, or a primary benign tumor, including those secondary to abnormal embryologic development versus a primary malignant placental tumor. In this case report, we identified an incidental nest of intraplacental cells with nondiagnostic morphology and negative initial Glypican-3 stain in a healthy 35-wk gestation. This negative result prompted a broadening of the differential before ultimately determining this lesion was indeed ectopic liver with positive Arginase-1 and HepPar-1 staining. This may represent the mature hepatocyte phenotype within the lesional cells of this near-term birth, a dichotomy not previously discussed in the literature, which focuses on the fetal hepatocyte phenotype, also rarely seen. In this report, we summarize the previous literature regarding intraplacental ectopic liver, and we propose a sensitive approach to suspected ectopic liver of the placenta that may be sufficient to capture both the fetal and mature hepatocyte immunophenotypes. This approach may extend to other related pathologies including assessment of suspected intraumbilical hepatocytes.

Clinicopathological features and differential diagnosis of hepatocellular carcinoma in extrahepatic metastases.

Extrahepatic metastasis of hepatocellular carcinoma (HCC) may cause a diagnostic problem. All 195 cases of histologic and immunostained sections were reviewed retrospectively in one center. The expression of arginase-1 (Arg-1), hepatocyte paraffin-1 (HepPar-1), glypican-3 (GPC-3), and α-Fetoprotein (AFP) was evaluated. Eighty cases of metastatic tumors of the liver were also collected to verify their effectiveness. Totally 151 cases had previous history of HCC, in whom 49 had history of liver transplantation. Forty-four cases were diagnosed as metastatic HCC at initial presentation. The most common extrahepatic metastatic sites were bone (57%), followed by lung, lymph node, etc. Around 19 cases were positive for 1 marker, 22 were positive for 2 markers, 95 were positive for 3 markers, and 59 were positive for 4 markers. With the number of antibody increased in the panel, the negative cases decreased. The sensitivity of ARG, GPC-3, HepPar-1, and AFP was 82.6%, 89.2%, 83.6% and 53.8%, and the specificity was 98.3%, 94.8%, 96.2% and 100%, respectively. These data suggest that the panel of ARG-1, GPC-3, HepPar-1 and AFP has a high sensitivity and specificity to differentiate HCC from non-HCC. This study indicated that HCC should be considered when diagnosing metastasis of unclear origin. It is recommended to use the panel of ARG-1, GPC-3, HepPar-1 and AFP to differentiate HCC from non-HCC in extrahepatic metastasis, because of their sensitivity and specificity, especially in poorly differentiated lesions.

Tobacco and Antiretrovirals Modulate Transporter, Metabolic Enzyme, and Antioxidant Enzyme Expression and Function in Polarized Macrophages.

BACKGROUND: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically activated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage. OBJECTIVE: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy. METHODS: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa. RESULTS: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication. CONCLUSION: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir.

Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have attracted attention as they can modulate key cancer-related activities, yet TAM represent a heterogenous group of cells that remain incompletely characterized. In growing tumors, TAM are often referred to as M2-like macrophages, which are cells that display immunosuppressive and tumorigenic functions and express the enzyme arginase 1 (Arg1). Methods: Here we combined high resolution intravital imaging with single cell RNA seq to uncover the topography and molecular profiles of immunosuppressive macrophages in mice. We further assessed how immunotherapeutic interventions impact these cells directly in vivo. Results: We show that: i) Arg1+ macrophages are more abundant in tumors compared to other organs; ii) there exist two morphologically distinct subsets of Arg1 TAM defined by previously unknown markers (Gbp2b, Bst1, Sgk1, Pmepa1, Ms4a7); iii) anti-Programmed Cell Death-1 (aPD-1) therapy decreases the number of Arg1+ TAM while increasing Arg1- TAM; iv) accordingly, pharmacological inhibition of arginase 1 does not synergize with aPD-1 therapy. Conclusion: Overall, this research shows how powerful complementary single cell analytical approaches can be used to improve our understanding of drug action in vivo.

A subset of well-differentiated hepatocellular carcinomas are Arginase-1 negative.

Hepatocellular carcinoma (HCC) is a common malignancy both in the United States of America and worldwide. Despite the refinement of imaging techniques in at-risk populations, a needle biopsy diagnosis remains an important diagnostic tool for HCC in many cases. Various immunohistochemical markers have been developed to facilitate this diagnosis, such as HepPar-1, glypican-3 and, most recently, arginase-1. Amongst them, arginase-1 has been shown to have superior sensitivity and specificity than the others. Performance of arginase-1 has been reported to be excellent for diagnosis of well-differentiated HCCs, with some tail-off in sensitivity for poorly differentiated tumors. Our experience has suggested that a subset of well-differentiated HCCs can be negative for arginase-1. We examined 68 consecutive confirmed cases of well-differentiated HCC diagnosed on needle biopsy, and found 7 (10%) to be completely negative for arginase-1. This finding is of fundamental clinical importance in view of previous studies that have shown arginase-1 to be always positive in well-differentiated HCC.