CD73 is a Critical Regulator of Hepatocytes in Homeostasis and Disease

The enzymatic dephosphorylation of 5´-nucleotides like adenosine 5´-monophosphate (AMP) is a key step in purine salvage and purinergic signaling. This reaction occurs inside the cell or in the extracellular space. Ecto-5´-nucleotidase (CD73) is the major enzyme catalyzing the formation of extracellular adenosine from AMP. CD73-generated adenosine controls tissue homeostasis and responses related to inflammation, ischemia, fibrosis, and cancer. However, the specific functions of CD73 in liver homeostasis and diseases have not been fully elucidated. The objectives of the study were to 1) define the cell-specific role of CD73 in the liver, 2) determine how hepatocyte CD73 regulates alcohol-induced liver injury, and 3) examine CD73 regulation in human hepatocellular carcinoma. We generated and characterized a hepatocyte-specific CD73 knockout mouse (CD73-LKO) model using serological, biochemical, and histological assays. We found that CD73-LKO mice developed spontaneous liver injury in a sex- and age-dependent manner, which was associated with hypoactivation of AMPK, the key regulator of cellular energy metabolism. Furthermore, CD73-generated extracellular adenosine mediated AMPK activity upon cellular uptake through nucleoside transporters. Given that hepatocyte CD73 and adenosine exerts basal hepatoprotection, we then determined its role in alcohol-induced liver injury, a leading risk factor for chronic liver diseases. The loss of hepatocyte CD73 predisposed mice to more severe liver injury. In contrast, alcohol exposure in WT mice resulted in mild injury, concomitant with CD73 induction and enhanced enzymatic activity. In vitro assays revealed a non-enzymatic CD73 binding protein function for bioactive lipids, which are generated upon alcohol exposure. These data suggest CD73-mediated dual protection in alcoholic liver. In humans, chronic alcohol-induced liver injury leads to hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide. Because hepatocyte CD73 is critical in normal and injured liver, we examined its regulation in HCC. CD73 was expressed in tumor and non-tumor liver tissues. However, tumor-specific CD73 altered N-linked glycosylation led to its cytoplasmic redistribution and a reduction in the enzymatic activity, suggesting a potential impairment in CD73-mediated liver protection. In conclusion, these studies provide further understanding of CD73 biology in the liver and has implications for the potential therapeutic use of CD73 in chronic liver diseases.Doctor of Philosoph

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Abstract Background Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn’s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease

Tumorâ Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151913/1/hep41410_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151913/2/hep41410.pd

Rhb-104 Triple Antibiotics Combination In Culture Is Bactericidal And Should Be Effective For Treatment Of Crohn\u27S Disease Associated With Mycobacterium Paratuberculosis

Background: Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as an etiological agent of Crohn\u27s disease (CD), a debilitating chronic inflammatory bowel disease. Clarithromycin (CLA), clofazimine (CLO), rifabutin (RIF) and other antibiotics have been used individually or in combinations with other drugs to treat mycobacterial diseases including CD. The treatment has varied by regimen, dosage, and duration, resulting in conflicting outcomes and additional suffering to the patients. RHB-104, a drug formula with active ingredients composed of (63.3 %) CLA, (6.7 %) CLO, and (30 %) RIF, has been recently subjected to investigation in an FDA approved Phase III clinical trial to treat patients with moderate to severe CD. In this study, we determined the efficacy of RHB-104 active ingredients against MAP strains isolated from the blood, tissue, and milk of CD patients. Based on fluorescence quenching technology using the Bactec MGIT Para-TB medium, we determined the minimum inhibitory concentration (MIC) of CLA, CLO, RIF individually and in dual and triple combinations against 16 MAP clinical strains and 19 other mycobacteria. Results: The MIC of all drugs against 35 different mycobacteria ranged between 0.25-20 μg/mL. However, the MIC of RHB-104 active ingredients regimen was the lowest at 0.25-10 μg/mL compared to the MIC of the other drugs at 0.5-20 μg/mL. The components of RHB-104 active ingredients at their individual concentrations or in dual combinations were not effective against all microorganisms compared to the triple combinations at MIC level. The MIC of CLA-CLO, CLA-RIF, and CLO-RIF regimens ranged between 0.5-1.25 μg/mL compared to 0.25 μg/mL of bactericidal effect of the triple combination. Conclusion: The data clearly demonstrated that lower concentrations of the triple combination of RHB-104 active ingredients provided synergistic anti-MAP growth activity compared to individual or dual combinations of the drugs. Consequently, this is favorable and should lead to tolerable dosage that is desirable for long-term treatment of CD and Mycobacterium avium complex disease

Frontline Science: Rip2 Promotes House Dust Mite–Induced Allergic Airway Inflammation

House dust mites (HDMs) are one of the most significant environmental allergens in the establishment of the so-called “Atopic March.” It is known that the immune response to HDM is Th2 dominant, but the innate mechanisms leading to HDM-induced type 2 responses are still not completely understood. A number of innate immune receptors have been implicated in the response to HDM including toll-like receptors, C-type lectin receptors, and protease activated receptors. NOD2 is a member of the NOD-like receptor family, which has been reported to be involved in the establishment of type 2 immunity and in blocking respiratory tolerance. NOD2 mediates its effects through its downstream effector kinase, receptor interacting protein (RIP2). It has not been shown if RIP2 is involved in the innate response to HDM and in the resulting generation of type 2 immunity. Furthermore, the role of RIP2 in modulating allergic airway inflammation has been controversial. In this study, we show that RIP2 is activated in airway epithelial cells in response to HDM and is important for the production of CCL2. Using a murine HDM asthma model, we demonstrate that lung pathology, local airway inflammation, inflammatory cytokines, HDM-specific IgG 1 antibody production, and HDM-specific Th2 responses are all reduced in RIP2 knockout mice compared to WT animals. These data illustrate that RIP2 can be activated by a relevant allergic stimulus and that such activation can contribute to allergic airway inflammation. These findings also suggest that RIP2 inhibitors might have some efficacy in down-regulating the inflammatory response in type 2 dominated diseases

Development Of Multiplex Pcr And Multi-Color Fluorescent In Situ Hybridization (M-Fish) Coupled Protocol For Detection And Imaging Of Multi-Pathogens Involved In Inflammatory Bowel Disease

Background: Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn\u27s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods: The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results: Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion: We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Abstract Background Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn’s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC

RHB-104 triple antibiotics combination in culture is bactericidal and should be effective for treatment of Crohn’s disease associated with Mycobacterium paratuberculosis

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as an etiological agent of Crohn’s disease (CD), a debilitating chronic inflammatory bowel disease. Clarithromycin (CLA), clofazimine (CLO), rifabutin (RIF) and other antibiotics have been used individually or in combinations with other drugs to treat mycobacterial diseases including CD. The treatment has varied by regimen, dosage, and duration, resulting in conflicting outcomes and additional suffering to the patients. RHB-104, a drug formula with active ingredients composed of (63.3 %) CLA, (6.7 %) CLO, and (30 %) RIF, has been recently subjected to investigation in an FDA approved Phase III clinical trial to treat patients with moderate to severe CD. In this study, we determined the efficacy of RHB-104 active ingredients against MAP strains isolated from the blood, tissue, and milk of CD patients. Based on fluorescence quenching technology using the Bactec MGIT Para-TB medium, we determined the minimum inhibitory concentration (MIC) of CLA, CLO, RIF individually and in dual and triple combinations against 16 MAP clinical strains and 19 other mycobacteria. RESULTS: The MIC of all drugs against 35 different mycobacteria ranged between 0.25–20 μg/mL. However, the MIC of RHB-104 active ingredients regimen was the lowest at 0.25–10 μg/mL compared to the MIC of the other drugs at 0.5–20 μg/mL. The components of RHB-104 active ingredients at their individual concentrations or in dual combinations were not effective against all microorganisms compared to the triple combinations at MIC level. The MIC of CLA–CLO, CLA–RIF, and CLO–RIF regimens ranged between 0.5–1.25 μg/mL compared to 0.25 μg/mL of bactericidal effect of the triple combination. CONCLUSION: The data clearly demonstrated that lower concentrations of the triple combination of RHB-104 active ingredients provided synergistic anti-MAP growth activity compared to individual or dual combinations of the drugs. Consequently, this is favorable and should lead to tolerable dosage that is desirable for long-term treatment of CD and Mycobacterium avium complex disease