Effect of tofacitinib on the phenotype and activity of Caco­2 cells in a model of inflammatory bowel disease.

Reports have indicated that autoimmune bowel disorders affect an increasing number of people on every continent; therefore, it is important to better understand inflammatory bowel disease (IBD) and to explore new treatment options for patients suffering from it. Research has indicated the important role of enterocytes in IBD. Understanding the role of the intestinal epithelium in the pathogenesis of IBD may contribute to a better understanding of the inflammatory processes and aid in the identification of potential therapeutic treatments. The present study aimed to evaluate the effects of tofacitinib on Caco-2 cells cultured in an inflammatory environment induced using cytokines naturally found in patients with ulcerative colitis. Tofacitinib is an orally administered inhibitor of Janus kinases (JAKs) which, by modifying the JAK/STAT signaling pathway, reduces the effect of inflammatory cytokines in the gut. Caco-2 cells were used to model the intestinal epithelium and the culture conditions included the proinflammatory cytokine TNFα and tofacitinib. At the end of the culture period, enzymes involved in oxidative stress (superoxide dismutase 1, catalase, nicotinamide adenine dinucleotide phosphate), a marker of apoptosis (Bcl-2) and a key player in intracellular inflammatory signaling (nuclear factor κB) were assessed by quantitative PCR and western blotting. The in vitro phenotype of Caco-2 cells exposed to an inflammatory environment was observed to be similar to that observed in ulcerative colitis. Notably, tofacitinib was able to improve TNFα-induced changes in an in vitro model of ulcerative colitis, and a reduction in the activity of enzymes associated with oxidative stress was observed. In addition, tofacitinib-induced upregulation of Bcl-2 and claudin-1 may contribute to the beneficial effects of tofacitinib on the intestinal epithelium. Tofacitinib appears to have a protective effect on Caco-2 cells. Notably, in the present study, exposure to TNFα stimulated oxidative stress and apoptotic effects, and disrupted intercellular connectivity. The addition of tofacitinib decreased the activity of the examined parameters of oxidative and apoptotic stress, while increasing the activity of the parameter examined to evaluate the degree of intercellular connections. In conclusion, the inhibitory effects of tofacitinib on oxidative stress, as well as its anti-apoptotic and regenerative effects, provide important information regarding the positive effect of tofacitinib on Caco-2 cells, and therefore constitute potential information about the beneficial effect of the evaluated drug in UC.

The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells.

PURPOSE: Human endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs' transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis. MATERIAL AND METHODS: Scratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively. RESULTS: Results revealed reduced ERVW-1 expression in cells exposed to injury (p â€‹< â€‹0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p â€‹< â€‹0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p â€‹< â€‹0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p â€‹< â€‹0.01). CONCLUSION: Results showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

The Current and Promising Oral Delivery Methods for Protein- and Peptide-Based Drugs.

Drugs based on peptides and proteins (PPs) have been widely used in medicine, beginning with insulin therapy in patients with diabetes mellitus over a century ago. Although the oral route of drug administration is the preferred one by the vast majority of patients and improves compliance, medications of this kind due to their specific chemical structure are typically delivered parenterally, which ensures optimal bioavailability. In order to overcome issues connected with oral absorption of PPs such as their instability depending on digestive enzymes and pH changes in the gastrointestinal (GI) system on the one hand, but also their limited permeability across physiological barriers (mucus and epithelium) on the other hand, scientists have been strenuously searching for novel delivery methods enabling peptide and protein drugs (PPDs) to be administered enterally. These include utilization of different nanoparticles, transport channels, substances enhancing permeation, chemical modifications, hydrogels, microneedles, microemulsion, proteolytic enzyme inhibitors, and cell-penetrating peptides, all of which are extensively discussed in this review. Furthermore, this article highlights oral PP therapeutics both previously used in therapy and currently available on the medical market.

Potential Therapeutic Strategies in the Treatment of Metabolic-Associated Fatty Liver Disease.

Metabolic-associated Fatty Liver Disease is one of the outstanding challenges in gastroenterology. The increasing incidence of the disease is undoubtedly connected with the ongoing obesity pandemic. The lack of specific symptoms in the early phases and the grave complications of the disease require an active approach to prompt diagnosis and treatment. Therapeutic lifestyle changes should be introduced in a great majority of patients; but, in many cases, the adherence is not satisfactory. There is a great need for an effective pharmacological therapy for Metabolic-Associated Fatty Liver Disease, especially before the onset of steatohepatitis. Currently, there are no specific recommendations on the selection of drugs to treat liver steatosis and prevent patients from progression toward more advanced stages (steatohepatitis, cirrhosis, and cancer). Therefore, in this Review, we provide data on the clinical efficacy of therapeutic interventions that might improve the course of Metabolic-Associated Fatty Liver Disease. These include the drugs used in the treatment of obesity and hyperlipidemias, as well as affecting the gut microbiota and endocrine system, and other experimental approaches, including functional foods. Finally, we provide advice on the selection of drugs for patients with concomitant Metabolic-Associated Fatty Liver Disease.

Lethargic patient with hepatocirrhosis - more than meets the eye.

Not required for Clinical Vignette.

Inclisiran-A Revolutionary Addition to a Cholesterol-Lowering Therapy.

Hypercholesterolemia plays a crucial role in the development of atherosclerosis, but it remains an undertreated and underdiagnosed disease. Taking into consideration the high prevalence of lipid disorders, long duration of the asymptomatic course of the disease, life-threatening complications resulting from inaccurate therapy, and stringent treatment goals concerning LDL cholesterol level in the prevention of cardiovascular events, novel lipid-lowering therapies have been introduced in the last few years. In this article, a drug belonging to the group of small interfering RNA (siRNA) called inclisiran is described. It is a novel molecule that increases the number of LDL receptors (LDLRs) on the surface of hepatic cells by preventing the formation of proprotein convertase subtilisin/kexin type 9 (PCSK9) responsible for the degradation of LDLRs. With great potential for lowering plasma LDL cholesterol level, high liver specificity, comfortable dosing regimen, and good tolerance without significant adverse effects, it could play an important part in future hypolipemic therapies.

Initial clinical, laboratory and radiological features of SARS-CoV-2-infected patients and their impact on the course of the disease.

BACKGROUND: On March 11, 2020, coronavirus disease (COVID-19) was declared a global threat by the World Health Organization (WHO). It quickly became apparent that reducing inpatient mortality rates and early phase prediction of possible deterioration or severe disease course relied on finding more specific biomarkers. OBJECTIVES: This retrospective study assessed initial clinical, laboratory and radiological features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients and explored their impact on mortality and the course of the disease. Such efforts aimed to facilitate the identification of high-risk patients and to improve the formulation of treatment plans for these individuals. MATERIAL AND METHODS: The cohort comprised 111 consecutive adult inpatients diagnosed with COVID-19 and hospitalized in the Internal Medicine Ward of the University Clinical Center of prof. K. Gibinski of the Medical University of Silesia in Katowice, Poland, a COVID-19 Treatment Unit, between November 16, 2020 and February 15, 2021. All available clinical, laboratory and radiological findings were extracted from electronic records and assessed as possible risk factors for poor prognosis. RESULTS: Clinicasl and radiological features with higher frequency in COVID-19 non-survivors included older age, history of smoking, concomitant cardiovascular diseases, low oxygen saturation (SpO2), and high infection risk assessed on admission as well as high opacity score, percentage of opacity and percentage of high opacity in computed tomography. Non-survivors had decreased serum lymphocytes, monocytes, calcium, magnesium, and hemoglobin oxygen saturation. They also had increased red cell distribution width (RDW), C-reactive protein (CRP), procalcitonin, alkaline phosphatase (ALP), creatinine, blood urea nitrogen (BUN), D-dimer, troponin, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) levels, as well as a base deficit. CONCLUSIONS: This retrospective study identified several markers associated with a fatal course of COVID-19. The early assessment of SARS-CoV-2-infected inpatients should consider these markers.

Modifiable and Non-Modifiable Risk Factors for the Development of Non-Hereditary Pancreatic Cancer.

Pancreatic cancer is becoming an increasing healthcare concern. Though it is a 14th most common cancer worldwide, its incidence is steadily rising. Results of currently available therapies are still not satisfactory. Therefore, great attention should be put on the identification and reduction of risk factors for pancreatic cancer. A thorough up-to-date review of available data on the impact of well-established and novel risk factors of pancreatic cancer development have been performed. Several risk factors associated with lifestyle have significant impact on the risk of pancreatic cancer (i.e., smoking, obesity, alcohol consumption). Physicians should also be aware of the novel findings suggesting increasing role of microbiome, including viral and bacterial infections, in the development of pancreatic cancer. A growing body of evidence suggest also an increased risk during certain occupational exposures. In general, lifestyle seems to be a major contributor in the development of pancreatic cancer. Special attention should be given to individuals with a vicious cluster consisting of metabolic syndrome, tobacco smoking and alcohol consumption. Physicians should urge patients to comply to healthy diet, cessation of smoking and moderation of alcohol consumption, which may halve pancreatic cancer incidence. Further studies are warranted to explore the potential use of therapeutic approach on novel risk factors (e.g., microbiome).

Current status of inherited pancreatic cancer.

BACKGROUND: It is estimated that about 10% of pancreatic cancer cases have a genetic background. People with a familial predisposition to pancreatic cancer can be divided into 2 groups. The first is termed hereditary pancreatic cancer, which occurs in individuals with a known hereditary cancer syndrome caused by germline single gene mutations (e.g., BRCA1/2, CDKN2A). The second is considered as familial pancreatic cancer, which is associated with several genetic factors responsible for the more common development of pancreatic cancer in certain families, but the precise single gene mutation has not been found. AIM: This review summarizes the current state of knowledge regarding the risk of pancreatic cancer development in hereditary pancreatic cancer and familial pancreatic cancer patients. Furthermore, it gathers the latest recommendations from the three major organizations dealing with the prevention of pancreatic cancer in high-risk groups and explores recent guidelines of scientific societies on screening for pancreatic cancers in individuals at risk for hereditary or familial pancreatic cancer. CONCLUSIONS: In order to improve patients' outcomes, authors of current guidelines recommend early and intensive screening in patients with pancreatic cancer resulting from genetic background. The screening should be performed in excellence centers. The scope, extent and cost-effectiveness of such interventions requires further studies.

Cardiovascular Diseases-A Focus on Atherosclerosis, Its Prophylaxis, Complications and Recent Advancements in Therapies.

Long-term consequences of atherosclerosis remain the major culprit of mortality in developed and developing countries [...].

Insight into the Evolving Role of PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the last discovered member of the family of proprotein convertases (PCs), mainly synthetized in hepatic cells. This serine protease plays a pivotal role in the reduction of the number of low-density lipoprotein receptors (LDLRs) on the surface of hepatocytes, which leads to an increase in the level of cholesterol in the blood. This mechanism and the fact that gain of function (GOF) mutations in PCSK9 are responsible for causing familial hypercholesterolemia whereas loss-of-function (LOF) mutations are associated with hypocholesterolemia, prompted the invention of drugs that block PCSK9 action. The high efficiency of PCSK9 inhibitors (e.g., alirocumab, evolocumab) in decreasing cardiovascular risk, pleiotropic effects of other lipid-lowering drugs (e.g., statins) and the multifunctional character of other proprotein convertases, were the cause for proceeding studies on functions of PCSK9 beyond cholesterol metabolism. In this article, we summarize the current knowledge on the roles that PCSK9 plays in different tissues and perspectives for its clinical use.

Exenatide prevents statin-related LDL receptor increase and improves insulin secretion in pancreatic beta cells (1.1E7) in a protein kinase A-dependent manner.

Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.

CD4+ cells in autoimmune thyroid disease.

The phenomenon of autoimmunity develops as a result of the triggering factor released by damaged cells. This leads to an infiltration of CD4+ cells involved in stimulating the effector cells cytotoxicity and stimulating the humoral response. One of the most common autoimmune disorders are autoimmune thyroid diseases, including Hashimoto's thyroiditis and Graves's diseases. Helper T lymphocytes, which are divided into Th1, Th2, Tregs, and the relatively new groups Th17, Th22, and Th9, are involved in the pathogenesis of AITD. CD4+ cell subtypes mature and differentiate by specific transcription factors and in a specific interleukin environment. Not only are Th1 and Th2 cells involved in the development of AITD, but also Th17, Th22, and Th9 lymphocytes and their correlation to Tregs lymphocytes. The plasticity of the CD4+ cells is very important, affecting the balance between these cells, as well the factors modulating their phenotypic variability. Patients with AITD have an increased percentage of Th17, Th22, and Th9 cells as well as defective function of Tregs lymphocytes. The balance between Th17 cells (and also other cytotoxic T cells) and Treg cells is also very important. Understanding the role of CD4 cells in the pathogenesis of AITD may be important not only for the development of the knowledge, but also for determining therapeutic targets.

The treatment of heterozygous familial hypercholesterolemia - a local perspective.

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Pleiotropic Effects of PCSK-9 Inhibitors.

Proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors are a group of drugs whose main mechanism of action is binding to the PCSK-9 molecule, which reduces the degradation of the low-density lipoprotein receptor (LDL-R) and, hence, increases the uptake of low-density lipoprotein cholesterol (LDLc) from the bloodstream as well as reducing its concentration. The effectiveness of three monoclonal antibodies, namely, alirocumab (human IgG1/κ monoclonal antibody, genetically engineered in Chinese hamster ovary cells), evolocumab (the first fully human monoclonal antibody), and bococizumab (humanized mouse antibody), in inhibiting the action of PCSK-9 and reducing LDLc levels has been confirmed. The first two, after clinical trials, were approved by the Food and Drug Administration (FDA) and are used primarily in the treatment of autosomal familial hypercholesterolemia and in cases of statin intolerance. They are currently used both as monotherapy and in combination with statins and ezetimibe to intensify therapy and achieve therapeutic goals following the American Heart Association (AHA) and European Society of Cardiology (ESC) guidelines. However, the lipid-lowering effect is not the only effect of action described by researchers that PCSK-9 inhibitors have. This paper is a review of the literature describing the pleiotropic effects of PCSK-9 inhibitors, which belong to a group of drugs that are being increasingly used, especially when standard lipid-lowering therapy fails. The article focuses on activities other than lipid-lowering, such as the anti-atherosclerotic effect and stabilization of atherosclerotic plaque, the anti-aggregation effect, the anticoagulant effect, the antineoplastic effect, and the ability to influence the course of bacterial infections. In this publication, we try to systematically review the current scientific data, both from our own scientific work and knowledge from international publications.