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Background: Cutaneous squamous cell carcinoma is a common malignancy, which frequently develops in the areas exposed to the sun. Patients with locally advanced disease in the head and neck region are frequently disqualified from surgical resection and require systemic treatment. Methods: In this report, we present the clinicopathological features and treatment of two patients who received cemiplimab, a monoclonal antibody targeting programmed cell death receptor 1 (PD-1). Results: An 80-year-old female and 82-year-old male patient were admitted to the hospital for the treatment of large tumors diagnosed as squamous cell carcinomas. In both patients, surgical treatment was not recommended due to the large dimensions of the tumors. These patients qualified for systemic treatment with cemiplimab. In the first patient, immunotherapy was interrupted due to adverse events. Nevertheless, a continuous regression of the tumor was observed despite treatment cessation. The second patient experienced a pseudoprogression, which is an increase in the tumor size caused by infiltration of immune cells. The treatment significantly reduced tumor size in both patients, which highly improved their quality of life. Conclusions: Cemiplimab offers clinical benefits in patients with cutaneous squamous cell carcinoma who are ineligible for surgical treatment. Systemic treatment can significantly improve the quality of life and reduce tumor diameters.
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Basal cell carcinoma (BCC) is the most frequent human skin cancer, but metastasizing BCC (MBCC) is extremely rare, developing in approximately 0.0028% to 0.55% of BCC patients. Herein, we report two cases of pulmonary MBCC. The first one developed in a 72-year-old male who underwent surgical resection due to multiple recurrences and adjuvant radiotherapy. Immunohistochemistry showed that neoplastic cells expressed Ber-EP4, CK5/6, p63, EMA (focally), BCL-2, and CD10, but were negative for CK7, CK20, S100, estrogen and progesterone receptors, and TTF-1. The second case is a 64-year-old female treated with vismodegib. Clinicopathological features and differential diagnoses are described.
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4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. METHODS: Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. RESULTS: Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm2 in C to 16.0±2.6 nmol/min/cm2 in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. CONCLUSIONS: This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.
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Nucleótidos de Adenina/metabolismo , Aorta/metabolismo , Aterosclerosis/tratamiento farmacológico , Nucleósidos/farmacología , Piridonas/farmacología , Animales , Aorta/efectos de los fármacos , Aterosclerosis/metabolismo , Evaluación Preclínica de Medicamentos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleósidos/uso terapéutico , Piridonas/uso terapéutico , Proteína Amiloide A Sérica/metabolismoRESUMEN
Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5'NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5'NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.
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Adenosina/metabolismo , Enfermedad de Huntington/metabolismo , Inosina/metabolismo , Miocardio/metabolismo , AMP Desaminasa/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Purina-Nucleósido Fosforilasa/metabolismo , Pirofosfatasas/metabolismoRESUMEN
Huntington's disease (HD) is mainly thought of as a neurological disease, but multiple epidemiological studies have demonstrated a number of cardiovascular events leading to heart failure in HD patients. Our recent studies showed an increased risk of heart contractile dysfunction and dilated cardiomyopathy in HD pre-clinical models. This could potentially involve metabolic remodeling, that is a typical feature of the failing heart, with reduced activities of high energy phosphate generating pathways. In this study, we sought to identify metabolic abnormalities leading to HD-related cardiomyopathy in pre-clinical and clinical settings. We found that HD mouse models developed a profound deterioration in cardiac energy equilibrium, despite AMP-activated protein kinase hyperphosphorylation. This was accompanied by a reduced glucose usage and a significant deregulation of genes involved in de novo purine biosynthesis, in conversion of adenine nucleotides, and in adenosine metabolism. Consequently, we observed increased levels of nucleotide catabolites such as inosine, hypoxanthine, xanthine and uric acid, in murine and human HD serum. These effects may be caused locally by mutant HTT, via gain or loss of function effects, or distally by a lack of trophic signals from central nerve stimulation. Either may lead to energy equilibrium imbalances in cardiac cells, with activation of nucleotide catabolism plus an inhibition of re-synthesis. Our study suggests that future therapies should target cardiac mitochondrial dysfunction to ameliorate energetic dysfunction. Importantly, we describe the first set of biomarkers related to heart and skeletal muscle dysfunction in both pre-clinical and clinical HD settings.
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Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.
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Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina/metabolismo , Válvula Aórtica/enzimología , Bioprótesis , Prótesis Valvulares Cardíacas , Nucleotidasas/metabolismo , Conservación de Tejido/métodos , Animales , Aorta/enzimología , Válvula Aórtica/citología , Válvula Aórtica/trasplante , Cromatografía Líquida de Alta Presión , Desaminación , Desoxirribonucleasa I/metabolismo , Detergentes/química , Xenoinjertos , Hidrólisis , Soluciones Hipotónicas , Cinética , Ribonucleasa Pancreática/metabolismo , Dodecil Sulfato de Sodio/química , PorcinosRESUMEN
AIMS: Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. METHODS AND RESULTS: Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular inflammation and improved endothelial function. CONCLUSIONS: This study highlights the importance of extracellular nucleotides and adenosine metabolism in the atherosclerotic vessel in both experimental and clinical setting. The increased eADA activity marks an early stage of atherosclerosis, contributes to its progression and could represent a novel target for therapy.
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Adenosina Desaminasa/metabolismo , Aterosclerosis/metabolismo , Adenosina/metabolismo , Inhibidores de la Adenosina Desaminasa/uso terapéutico , Animales , Aorta/enzimología , Aorta/metabolismo , Apolipoproteínas E/fisiología , Aterosclerosis/tratamiento farmacológico , Células Cultivadas , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Pentostatina/farmacología , Receptores de LDL/fisiologíaRESUMEN
Ecto-5'-nucleotidase (e5NT, CD73) is an enzyme that is highly expressed in endothelium and is involved in the extracellular nucleotide catabolism. CD73 converts AMP to adenosine that via specific subtypes of P1 receptor mediates cytoprotection involving diverse mechanisms such as vasodilatation, suppression of inflammation, inhibition of thrombosis and anti-adrenergic effect. Physiological intravascular concentration of adenosine is in nanomolar range, but could become micromolar in response to various forms of stress. Endothelium is a major site for both CD73 mediated production of adenosine and its cytoprotective effect. Nucleotides (predominantly ATP or ADP) that could be released from different cells via controlled specific of unspecific mechanisms constitute a major source of substrate for adenosine production via CD73. Direct effects of extracellular nucleotides (mediated by P2 receptors) are typically opposite to adenosine P1 mediated activities. Retention of nucleotides and decreased adenosine production due to loss of CD73 function may have negative implications and could be important cause of various pathologies. Protective role of CD73 was indicated in ectopic calcification, atherosclerosis, rejection after xenotransplantation and thrombosis. Reduced activity of CD73 due to lymphocyte contact with endothelium increases its permeability that leads to enhanced leukocyte transmigration. Upregulation of endothelial CD73 may therefore be protective in a number of cardiovascular pathologies. Such effect has been confirmed for some common drugs such as statins and it could be part of its pleiotropic portfolio. Activation of CD73 could be a new target for specific treatment strategy that in particular will enhance endothelial protection.
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5'-Nucleotidasa/fisiología , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Enfermedades Vasculares/enzimología , Animales , Proteínas Ligadas a GPI/fisiología , Humanos , Enfermedades Vasculares/diagnóstico , Vasodilatación/fisiologíaRESUMEN
Atherosclerosis is a consequence of diverse pathologies that could be affected by signaling mediated by nucleotides and their metabolites. Concentration of specific nucleotide derivatives in the proximity of purinergic receptors is controlled by extracellular enzymes such as ecto-nucleoside triphopsphate diphosphohydrolase (eNTPD), ecto-5'-nucleotidase (e5NT), and ecto-adenosine deaminase (eADA). To estimate changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall, aortoiliac bifurcation of ApoE/LDLr (-/-) mice was perfused with solution containing adenosine-5'-triphosphate (ATP), adenosine-5'-monophosphate (AMP) or adenosine. Formation of the product of eNTPD, e5NT or eADA was measured by high performance liquid chromatography (HPLC). The most significant difference between ApoE/LDLr (-/-) and wild-type mice was several times higher rate of conversion of adenosine to inosine catalyzed by eADA activity. This highlights potential decrease in intravascular adenosine concentration in atherosclerosis.
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Aorta/patología , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Espacio Extracelular/metabolismo , Arteria Ilíaca/patología , Nucleótidos/metabolismo , Receptores de LDL/deficiencia , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Espacio Extracelular/enzimología , Ratones , Ratones Noqueados , Receptores de LDL/genéticaRESUMEN
AMP-regulated protein kinase (AMPK) is involved in numerous regulatory processes and its role in control of cardiac energy metabolism is particularly important. This activity could be affected by AMP-deaminase (AMPD) since substrate of AMPD is AMPK activator. Hearts of male mouse, fed for six weeks with normal or high-fat diet, were fractionated to enrich AMPK activity. Purified fraction was incubated with AMARA peptide for up to 5 minutes and then conversion of AMARA to pAMARA was determined by liquid chromatography-mass spectrometry (LC/MS) using mass detector. Activity of AMPK in heart was 0.038±0.012 pmol/min/mg protein for mice fed high-fat diet and that was not different to control (0.032±0.01 pmol/min/mg protein). We observed change in AMPD activity. It was 5.39±1.5 nmol/mg tissue/min in heart of mice fed high-fat diet while in heart of mice fed low-fat diet it was 2.29±0.32 nmol/mg tissue/min. Data we present indicate that while total AMPK activity is not changed decrease in AMPD activity may affect AMPK signaling in diabetic heart.