Publications by authors named "Karel P Alcedo"

8 Publications

  • Page 1 of 1

CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner.

Cell Mol Gastroenterol Hepatol 2021 Jan 29;12(1):141-157. Epub 2021 Jan 29.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

Background & Aims: Metabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. CD73 is the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. We aimed to determine if CD73 has nonredundant hepatoprotective functions.

Methods: Liver-specific CD73 knockout (CD73-LKO) mice were generated by targeting the Nt5e gene in hepatocytes. The CD73-LKO mice and hepatocytes were characterized using multiple approaches.

Results: Deletion of hepatocyte Nt5e resulted in an approximately 70% reduction in total liver CD73 protein (P < .0001). Male and female CD73-LKO mice developed normally during the first 21 weeks without significant liver phenotypes. Between 21 and 42 weeks, the CD73-LKO mice developed spontaneous-onset liver disease, with significant severity in male mice. Middle-aged male CD73-LKO mice showed hepatocyte swelling and ballooning (P < .05), inflammation (P < .01), and variable steatosis. Female CD73-LKO mice had lower serum albumin levels (P < .05) and increased inflammatory genes (P < .01), but did not show the spectrum of histopathologic changes in male mice, potentially owing to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice showed significant metabolic imbalance, with increased blood urea nitrogen (P < .0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypophosphorylation of AMPK substrates in CD73-LKO livers (P < .0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (P < .001).

Conclusions: Hepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.
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http://dx.doi.org/10.1016/j.jcmgh.2021.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082562PMC
January 2021

Sox9 Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Cell Mol Gastroenterol Hepatol 2021 23;11(5):1437-1462. Epub 2021 Jan 23.

Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Division of Digestive Diseases, Department of Medicine, Emory University, Atlanta, Georgia. Electronic address:

Background & Aims: Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9 transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types.

Methods: Sox9 BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9 mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence.

Results: BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9 BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP BECs. Single Sox9 cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9 remains restricted to BECs and periportal hepatocytes after bile duct ligation.

Conclusions: Our data demonstrate that Sox9 levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.
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http://dx.doi.org/10.1016/j.jcmgh.2021.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024983PMC
January 2021

Alternative Splicing in Hepatocellular Carcinoma.

Cell Mol Gastroenterol Hepatol 2020 8;10(4):699-712. Epub 2020 May 8.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Electronic address:

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases, with more than 850,000 new diagnoses per year globally. Recent trends in the United States have shown that liver cancer mortality has continued to increase in both men and women, while 5-year survival remains below 20%. Understanding key mechanisms that drive chronic liver disease progression to HCC can reveal new therapeutic targets and biomarkers for early detection of HCC. In that regard, many studies have underscored the importance of alternative splicing as a source of novel HCC prognostic markers and disease targets. Alternative splicing of pre-mRNA provides functional diversity to the genome, and endows cells with the ability to rapidly remodel the proteome. Genes that control fundamental processes, such as metabolism, cell proliferation, and apoptosis, are altered globally in HCC by alternative splicing. This review highlights the major splicing factors, RNA binding proteins, transcriptional targets, and signaling pathways that are of key relevance to HCC. We highlight primary research from the past 3-5 years involving functional interrogation of alternative splicing in rodent and human liver, using both large-scale transcriptomic and focused mechanistic approaches. Because this is a rapidly advancing field, we anticipate that it will be transformative for the future of basic liver biology, as well as HCC diagnosis and management.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490524PMC
May 2020

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

Hepatol Commun 2019 Oct 9;3(10):1400-1414. Epub 2019 Aug 9.

Department of Cell Biology and Physiology University of North Carolina at Chapel Hill Chapel Hill NC.

CD73, a cell-surface -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 -linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal -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 ( 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 , 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 -linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. 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.
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http://dx.doi.org/10.1002/hep4.1410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771166PMC
October 2019

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Am J Physiol Cell Physiol 2019 12 28;317(6):C1079-C1092. Epub 2019 Aug 28.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.
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http://dx.doi.org/10.1152/ajpcell.00285.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957383PMC
December 2019

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

Gut Pathog 2018 5;10:51. Epub 2018 Dec 5.

Division of Molecular Microbiology, Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, 4110 Libra Drive, Orlando, FL USA.

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 (-FISH) protocols for simultaneous detection of CD-associated pathogens including subspecies (MAP), , and adherent-invasive 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. -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 -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 -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.
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http://dx.doi.org/10.1186/s13099-018-0278-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280354PMC
December 2018

Frontline Science: RIP2 promotes house dust mite-induced allergic airway inflammation.

J Leukoc Biol 2018 09 27;104(3):447-459. Epub 2018 Jul 27.

Immunity and Pathogenesis Division, Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida, USA.

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 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.
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http://dx.doi.org/10.1002/JLB.4HI0118-017RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113092PMC
September 2018

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

Gut Pathog 2016 14;8:32. Epub 2016 Jun 14.

Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL USA.

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.
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http://dx.doi.org/10.1186/s13099-016-0115-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908774PMC
June 2016