Immunoregulatory cyclophilin a improves low-dose chemotherapy with a modulation of the immune tumor microenvironment in experimental models of melanoma B16 and lymphoma EL4 in vivo.

PURPOSE: Different regimens of low-dose chemotherapy (LDC) are currently being actively developed and introduced into clinical practice. Along with its obvious advantages compared to conventional chemotherapy (low toxicity, prevention of drug resistance), LDC could also stimulate anti-tumor immune responses in a patient by activating effectors of innate and adaptive immunity and diminishing tumor-associated immunosuppression. As non-myeloablative, LDC could be successfully combined with different anti-cancer immunotherapeutic strategies, including immunoregulatory cytokines. Secreted cyclophilin A (CypA) is of particular interest in this respect. Previously, we showed that recombinant human CypA (rhCypA) had pleiotropic immunostimulatory activity and anti-tumor effects. Thus, rhCypA could be potentially proposed as a perspective component of combined therapy with LDC. METHODS: In this work, we evaluated the anti-tumor effects of rhCypA combined with low doses of cyclophosphamide, doxorubicin, dacarbazine, and paclitaxel in the experimental mouse tumor models of melanoma B16 and lymphoma EL4 in vivo. RESULTS: Synergic and potentiating effects of rhCypA combined with LDC were shown in these studies. Furthermore, as a monotherapeutic agent and a component of combined chemoimmunotherapy, rhCypA was shown to modulate the immune tumor microenvironment by enhancing tumor infiltration with macrophages, NK cells, and T cells. It was also found that rhCypA stimulated both systemic and local anti-tumor immune responses. CONCLUSION: RhCypA could be potentially proposed as a perspective component of the combined cancer chemoimmunotherapy.

A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex.

Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.

Cyclosporin A-Based PROTACs Can Deplete Abundant Cellular Cyclophilin A without Suppressing T Cell Activation.

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

Alternate conformations found in protein structures implies biological functions: A case study using cyclophilin A.

Protein dynamics linked to numerous biomolecular functions, such as ligand binding, allosteric regulation, and catalysis, must be better understood at the atomic level. Reactive atoms of key residues drive a repertoire of biomolecular functions by flipping between alternate conformations or conformational substates, seldom found in protein structures. Probing such sparsely sampled alternate conformations would provide mechanistic insight into many biological functions. We are therefore interested in evaluating the instance of amino acids adopted alternate conformations, either in backbone or side-chain atoms or in both. Accordingly, over 70000 protein structures appear to contain alternate conformations only 'A' and 'B' for any atom, particularly the instance of amino acids that adopted alternate conformations are more for Arg, Cys, Met, and Ser than others. The resulting protein structure analysis depicts that amino acids with alternate conformations are mainly found in the helical and ß-regions and are often seen in high-resolution X-ray crystal structures. Furthermore, a case study on human cyclophilin A (CypA) was performed to explain the pre-existing intrinsic dynamics of catalytically critical residues from the CypA and how such intrinsic dynamics perturbed upon Ser99Thr mutation using molecular dynamics simulations on the ns-µs timescale. Simulation results demonstrated that the Ser99Thr mutation had impaired the alternate conformations or the catalytically productive micro-environment of Phe113, mimicking the experimentally observed perturbation captured by X-ray crystallography. In brief, a deeper comprehension of alternate conformations adopted by the amino acids may shed light on the interplay between protein structure, dynamics, and function.

Intracellular and extracellular Cyclophilin a promote cardiac fibrosis through TGF-ß signaling in response to angiotensin â ¡.

Cardiac fibrosis is characterized by abnormal proliferation of cardiac fibroblasts (CFs) and ventricular remodeling, which finally leads to heart failure. Inflammation and oxidative stress play a central role in the development of cardiac fibrosis. CyPA (Cyclophilin A) is a main proinflammatory cytokine secreted under the conditions of oxidative stress. The mechanisms by which intracellular and extracellular CyPA interact with CFs are unclear. Male C57BL/6 J mice received angiotensin Ⅱ (Ang Ⅱ) or vehicle for 4 weeks. Inhibition of CyPA significantly reversed Ang Ⅱ-induced cardiac hypertrophy and fibrosis. Mechanically, TGF-ß (Transforming growth factor-ß) signaling was found to be an indispensable downstream factor of CyPA-mediated myofibroblast differentiation and proliferation. Furthermore, intracellular CyPA and extracellular CyPA activate TGF-ß signaling through NOD-like receptor protein 3 (NLRP3) inflammasome and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, respectively. Pharmacological inhibition of CyPA and its receptor CD147 implemented by Triptolide also attenuated the expression of TGF-ß signaling and cardiac fibrosis in Ang Ⅱ-model. These studies elucidate a novel mechanism by which CyPA promotes TGF-ß and its downstream signaling in CFs and identify CyPA (both intracellular and extracellular) as plausible therapeutic targets for preventing or treating cardiac fibrosis induced by chronic Ang Ⅱ stimulation.

Dialysate cyclophilin A as a predictive marker for historical peritonitis in patients undergoing peritoneal dialysis.

Introduction: No markers have been used to diagnose historical peritoneal dialysis (PD)-related peritonitis. Cyclophilin A (CypA) is associated with glucose toxicity and inflammation. We hypothesize that dialysate CypA can be a marker for historical peritonitis (at least 3 months free from peritonitis). Method: An enzyme-linked immunosorbent assay kit was used to measure the concentration of dialysate CypA. Clinical and laboratory data were collected to correlate with historical peritonitis. Mann-Whitney U test and Chi-square test were used for analysis. Receiver operating characteristic (ROC) analysis was used to evaluate predictive power. Results: Out of a total of 31 patients who had undergone PD for at least 2 years, 18 had no history of PD-related peritonitis, while 13 had experienced PD-related peritonitis at least once. Overall, the patients in this population were in good health (normal white blood cell count, no anemia, normal electrolyte and serum albumin levels). There were no significant differences between patients with and without a history of peritonitis, except for blood white blood cell count (5650.6 ± 1848.4 vs. 7154.6 ± 2056.8, p = 0.032) and dialysate CypA value (24.27 ± 22.715 vs. 54.41 ± 45.63, p = 0.020). In the univariate analysis, only the dialysate CypA level showed a statistically significant association with historical peritonitis (HR = 1.030, 95 % CI = 1.010-1.062, p = 0.046). The AUC for dialysate CypA (>34.83 ng/mL) was 0.748, with a sensitivity of 0.615 and specificity of 0.833. Conclusion: PD peritonitis poses a significant threat to the long-term use of peritoneal dialysis. Based on our study, even in the absence of concurrent infection, dialysate CypA can serve as a predictive marker for historical peritonitis, demonstrating high predictive power along with fair sensitivity and good specificity.

Circ_005077 accelerates myocardial lipotoxicity induced by high-fat diet via CyPA/p47PHOX mediated ferroptosis.

The long-term high-fat diet (HFD) can cause myocardial lipotoxicity, which is characterized pathologically by myocardial hypertrophy, fibrosis, and remodeling and clinically by cardiac dysfunction and heart failure in patients with obesity and diabetes. Circular RNAs (circRNAs), a novel class of noncoding RNA characterized by a ring formation through covalent bonds, play a critical role in various cardiovascular diseases. However, few studies have been conducted to investigate the role and mechanism of circRNA in myocardial lipotoxicity. Here, we found that circ_005077, formed by exon 2-4 of Crmp1, was significantly upregulated in the myocardium of an HFD-fed rat. Furthermore, we identified circ_005077 as a novel ferroptosis-related regulator that plays a role in palmitic acid (PA) and HFD-induced myocardial lipotoxicity in vitro and in vivo. Mechanically, circ_005077 interacted with Cyclophilin A (CyPA) and inhibited its degradation via the ubiquitination proteasome system (UBS), thus promoting the interaction between CyPA and p47phox to enhance the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for ROS generation, subsequently inducing ferroptosis. Therefore, our results provide new insights into the mechanisms of myocardial lipotoxicity, potentially leading to the identification of a novel therapeutic target for the treatment of myocardial lipotoxicity in the future.

Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, informing antiretroviral therapies that contain lenacapavir.

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

Role of cyclophilin A in aggravation of myocardial ischemia reperfusion injury via regulation of apoptosis mediated by thioredoxin-interacting protein.

BACKGROUND: The progression and persistence of myocardial ischemia/reperfusion injury (MI/RI) are strongly linked to local inflammatory responses and oxidative stress. Cyclophilin A (CypA), a pro-inflammatory factor, is involved in various cardiovascular diseases. However, the role and mechanism of action of CypA in MI/RI are still not fully understood. METHODS: We used the Gene Expression Omnibus (GEO) database for bioinformatic analysis. We collected blood samples from patients and controls for detecting the levels of serum CypA using enzyme-linked immunosorbent assay (ELISA) kits. We then developed a myocardial ischemia/reperfusion (I/R) injury model in wild-type (WT) mice and Ppia-/- mice. We utilized echocardiography, hemodynamic measurements, hematoxylin and eosin (H&E) staining, immunohistochemistry, enzyme-linked immunosorbent assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the role of CypA in myocardial I/R injury. Finally, we conducted an in vitrostudy, cell transfection, flow cytometry, RNA interference, and a co-immunoprecipitation assay to clarify the mechanism of CypA in aggravating cardiomyocyte apoptosis. RESULTS: We found that CypA inhibited TXNIP degradation to enhance oxidative stress-induced cardiomyocyte apoptosis during MI/RI. By comparing and analyzing CypA expression in patients with coronary atherosclerotic heart disease and in healthy controls, we found that CypA was upregulated in patients with Coronary Atmospheric Heart Disease, and its expression was positively correlated with Gensini scores. In addition, CypA deficiency decreased cytokine expression, oxidative stress, and cardiomyocyte apoptosis in I/R-treated mice, eventually alleviating cardiac dysfunction. CypA knockdown also reduced H2O2-induced apoptosis in H9c2 cells. Mechanistically, we found that CypA inhibited K48-linked ubiquitination mediated by atrophin-interacting protein 4 (AIP4) and proteasomal degradation of TXNIP, a thioredoxin-binding protein that mediates oxidative stress and induces apoptosis. CONCLUSION: These findings highlight the critical role CypA plays in myocardial injury caused by oxidative stress-induced apoptosis, indicating that CypA can be a viable biomarker and a therapeutic target candidate for MI/RI.

Thai traditional medicines reduce CD147 levels in lung cells: Potential therapeutic candidates for cancers, inflammations, and COVID-19.

ETHNOPHARMACOLOGICAL RELEVANCE: The cluster of differentiation 147 (CD147) is identified as the signaling protein relevant importantly in various cancers, inflammations, and coronavirus disease 2019 (COVID-19) via interacting with extracellular cyclophilin A (CypA). The reduction of CD147 levels inhibits the progression of CD147-associated diseases. Thai traditional medicines (TTMs): Keaw-hom (KH), Um-ma-ruek-ka-wa-tee (UM), Chan-ta-lee-la (CT), and Ha-rak (HR) have been used as anti-pyretic and anti-respiratory syndromes caused from various conditions including cancers, inflammations, and infections. Thus, these medicines would play a crucial role in the reduction of CD147 levels. AIM OF THE STUDY: This article aimed to investigate the effects of KH, UM, CT, and HR for reducing the CD147 levels through in vitro study. Additionally, in silico study was employed to screen the active compounds reflexing the reduction of CD147 levels. MATERIALS AND METHODS: The immunofluorescent technique was used to evaluate the reduction of CD147 levels in human lung epithelial cells (BEAS-2B) stimulated with CypA for eight extracts of KH, UM, CT, and HR obtained from water decoction (D) and 70% ethanol maceration (M) including, KHD, UMD, CTD, HRD, KHM, UMM, CTM, and HRM. RESULTS: UM extracts showed the most efficiency for reduction of CD147 levels in the cytoplasm and perinuclear of BEAS-2B cells stimulated with CypA. Phenolic compounds composing polyphenols, polyphenol sugars, and flavonoids were identified as the major chemical components of UMD and UMM. Further, molecular docking calculations identified polyphenol sugars as CypA inhibitors. CONCLUSIONS: UMD and UMM are potential for reduction of CD147 levels which provide a useful information for further development of UM as potential therapeutic candidates for CD147-associated diseases such as cancers, inflammations, and COVID-19.

Inhibitors of Cyclophilin A: Current and Anticipated Pharmaceutical Agents for Inflammatory Diseases and Cancers.

Cyclophilin A, a widely prevalent cellular protein, exhibits peptidyl-prolyl cis-trans isomerase activity. This protein is predominantly located in the cytosol; additionally, it can be secreted by the cells in response to inflammatory stimuli. Cyclophilin A has been identified to be a key player in many of the biological events and is therefore involved in several diseases, including vascular and inflammatory diseases, immune disorders, aging, and cancers. It represents an attractive target for therapeutic intervention with small molecule inhibitors such as cyclosporin A. Recently, a number of novel inhibitors of cyclophilin A have emerged. However, it remains elusive whether and how many cyclophilin A inhibitors function in the inflammatory diseases and cancers. In this review, we discuss current available data about cyclophilin A inhibitors, including cyclosporin A and its derivatives, quinoxaline derivatives, and peptide analogues, and outline the most recent advances in clinical trials of these agents. Inhibitors of cyclophilin A are poised to enhance our comprehension of the molecular mechanisms that underpin inflammatory diseases and cancers associated with cyclophilin A. This advancement will aid in the development of innovative pharmaceutical treatments in the future.

In vitro and in vivo validation of the antiviral effect of hCypA against SARS-CoV-2 via binding to the RBD of spike protein.

The novel coronavirus disease 2019 has stimulated the rapid development of new biological therapeutics to inhibit SARS-CoV-2 infection; however, this remains a challenging task. In a previous study using structural analysis, we revealed that human cyclophilin A inhibits the entry of SARS-CoV-2 into host cells by interfering with the interaction of the receptor-binding domain of the spike protein with angiotensin-converting enzyme 2 on the host cell surface, highlighting its potential for antiviral therapy. For a comprehensive experimental validation, in this study, we verified the antiviral effects of human cyclophilin A against SARS-CoV-2, including its variants, using in vitro assays and experiments on an in vivo mouse model. Human cyclophilin A demonstrated a highly effective antiviral effect, with an 85% survival rate upon SARS-CoV-2 infection. It also reduced viral titers, inflammation in the lungs and brain, and cytokine release in the serum, suggesting a controlled immune response and potentially faster recovery. Overall, our study provides insights into the potential of human cyclophilin A as a therapeutic agent against SARS-CoV-2, which should guide future clinical trials that might provide an additional therapeutic option for patients.

The extracellular cyclophilin A-integrin ß2 complex as a therapeutic target of viral pneumonia.

The acute respiratory virus infection can induce uncontrolled inflammatory responses, such as cytokine storm and viral pneumonia, which are the major causes of death in clinical cases. Cyclophilin A (CypA) is mainly distributed in the cytoplasm of resting cells and released into the extracellular space in response to inflammatory stimuli. Extracellular CypA (eCypA) is upregulated and promotes inflammatory response in severe COVID-19 patients. However, how eCypA promotes virus-induced inflammatory response remains elusive. Here, we observe that eCypA is induced by influenza A and B viruses and SARS-CoV-2 in cells, mice, or patients. Anti-CypA mAb reduces pro-inflammatory cytokines production, leukocytes infiltration, and lung injury in virus-infected mice. Mechanistically, eCypA binding to integrin ß2 triggers integrin activation, thereby facilitating leukocyte trafficking and cytokines production via the focal adhesion kinase (FAK)/GTPase and FAK/ERK/P65 pathways, respectively. These functions are suppressed by the anti-CypA mAb that specifically blocks eCypA-integrin ß2 interaction. Overall, our findings reveal that eCypA-integrin ß2 signaling mediates virus-induced inflammatory response, indicating that eCypA is a potential target for antibody therapy against viral pneumonia.

Autophagy inhibitors 3-MA and BAF may attenuate hippocampal neuronal necroptosis after global cerebral ischemia-reperfusion injury in male rats by inhibiting the interaction of the RIP3/AIF/CypA complex.

Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.

Prognostic Significance of Tumor-Associated Inflammation in Renal Cell Carcinoma.

This study analyzed tumor-associated inflammation by assessing the expression of cyclophilin A (CypA) and TNF in samples of kidney tumors of various histological types. It was shown that different histological types of renal cell carcinoma differed by the expression of these proteins. Thus, the highest expression of CypA and TNF was observed in papillary and chromophobe kidney cancer, although no correlation with overall bacterial load was found for these tumors. In the case of clear cell renal cell carcinoma, the expression of proinflammatory factors was observed in only half of the cases and directly correlated with the presence of resident bacteria, serving as a favorable prognostic factor for the disease.

The Mechanism and Potential Therapeutic Effects of Cyclosporin, Cyclophilin, Probiotics and Syndecan-1 in an Animal Model of Inflammatory Bowel Disease.

Background: Inflammatory bowel diseases (IBDs) have several treatment modalities including immunoregulators, like cyclosporine A, an immunosuppressant that interacts with cytoplasmic cyclophilin A, and probiotics. Aims: This study explored and compared the possible role of syndecan-1 in the IBD pathogenic process as well as the effectiveness of cyclophilin A, cyclosporine A, and their combination in the management of IBDs in the presence of probiotics. Methodology: IBD was induced in a total of 112 mice equally divided between syndecan-1 knock-out (KO) and Balb/c wild-type mice, using 2% dextran sulfate sodium (DSS) followed by intraperitoneal treatment with cyclosporine A, cyclophilin A, or a combination of both. In addition, a daily dose of probiotics was given in their drinking water. The animals were monitored for clinical signs and symptoms and checked for gross pathologies in the abdomen after 3 weeks. Descending and sigmoid colon biopsies were collected and fixed for routine microscopy or frozen for protein extraction and molecular testing for IL-6, CD3, CD147, and beta 1 integrins as well as pAkt expression. Results: The data showed that the induction of IBD in the syndecan-1 KO mice was more severe at the clinical, histological, and molecular levels than in the wild type. The combined CypA-CyA treatment showed no added inhibitory effect compared to single-drug treatment in both strains. Probiotics added to the combination was more effective in the wild type and, when used alone, its inhibition of IL-6 was the highest. As for the CD147 marker, there were more suppressions across the various groups in the KO mice except for the probiotics-alone group. Concerning CD3, it was significantly increased by the CypA-CyA complex, which led to more inflammation in the KO mice. Probiotics had little effect with the combination. In relation to beta 1 integrins, the CypA-CyA combination made no significant difference from CyA alone, and adding probiotics to the combination resulted in higher beta 1 integrin expression in the KO mice. As for pAkt, it was very well expressed and upregulated in both strains treated with DSS, but the effect was much larger in the KO mice. In brief, the CypA-CyA complex showed a decrease in the expression of pAkt, but there was no added effect of both drugs. Probiotics along with the complex had a similar reduction effects in both strains, with a greater effect in the wild-type mice, while probiotics alone led to a similar reduction in pAkt expressions in both strains. Conclusions: The differential effects of CyA, CypA, probiotics, and their combinations on the various inflammatory markers, as well as the histological alterations and clinical signs and symptoms, speak in favor of a clear role of syndecan-1 in reducing inflammation. However, probiotics need to be considered after more explorations into the mechanisms involved in the presence of CypA and CyA especially since pAkt is less active in their presence.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

Cyclophilin A: An Independent Prognostic Factor for Survival in Patients with Metastatic Colorectal Cancer Treated with Bevacizumab and Chemotherapy.

Colorectal cancer (CRC) ranks as second most common cause of cancer-related deaths. The CRC management considerably improved in recent years, especially due to biological therapies such as bevacizumab. The lack of predictive or prognostic biomarkers remains one of the major disadvantages of using bevacizumab in the CRC management. We performed a prospective study to analyze the prognostic and predictive roles of three potential serum biomarkers (Cyclophilin A (CypA), copeptin and Tie2) investigated by ELISA in 56 patients with metastatic CRC undergoing bevacizumab and chemotherapy between May 2019 and September 2021 at baseline and after one and six months of therapy. We showed that low levels of CypA at baseline and after one month of treatment were associated with better overall survival (OS) (42 versus 24 months, p = 0.029 at baseline; 42 versus 25 months, p = 0.039 after one month). For copeptin and Tie2, Kaplan-Meier curves showed no correlation between these biomarkers and OS or progression-free survival. When adjusting for baseline and post-treatment factors, a multivariate Cox analysis showed that low values of CypA at baseline and after one month of treatment were independent prognostic factors for OS and correlated with a better prognosis in metastatic CRC patients.

Exploring azomethine ylides reactivity with acrolein through cycloaddition reaction and computational antiviral activity assessment against hepatitis C virus.

CONTEXT: The regioselectivity and diastereoselectivity of the 1,3-dipolar cycloaddition reaction between azomethine ylides and acrolein were investigated. The DFT studies revealed that the favored pathway leads to the formation of cis-cycloadduct pyrrolidine and these computational findings align with experimental observations. The cis-cycloadduct pyrrolidine product serves as an advanced intermediate in the synthesis of a hepatitis C virus inhibitor. For this, the antiviral activity of cis-cycloadduct pyrrolidine against cyclophilin A, the co-factor responsible for hepatitis C virus, was also evaluated through molecular docking simulations which revealed intriguing interactions and a high C-score, which were further confirmed by molecular dynamics simulations, demonstrating stability over a 100-ns simulation period. Furthermore, the cis-cycloadduct pyrrolidine exhibits favorable drug-like properties and a better ADMET profile compared to hepatitis C virus inhibitor. METHODS: Chemical reactivity studies were performed using DFT method by the functional B3LYP at 6-31G (d, p) computational level by GAUSSIAN 16 program. Frontal molecular orbitals theory used to investigate HOMO/LUMO interactions between azomethine ylides and acrolein. Findings of this approach were confirmed by global reactivity indices and electron displacement was investigated based on Fukui functions. Furthermore, the activation energies were determined after frequency calculations using TS Berny algorithm and transition states were confirmed by the presence of a single imaginary frequency. Moreover, antiviral activity of cis-cycloadduct was explored through molecular docking using Surflex-Dock suite SYBYL X 2.0, and molecular dynamics simulation using GROMACS program. Finally, drug-like properties were investigated with SwissADME and ADMETlab 2.0.