Search Results (516)

The C-kinase-activated protein phosphatase-1 (PP1) inhibitor of 17 kDa (CPI-17) is a specific inhibitor of the PP1 catalytic subunit (PP1c) and the myosin phosphatase (MP) holoenzyme. CPI-17 requires the phosphorylation of Thr38 in the peptide segment ³⁵ARV(P)TVKYDRREL⁴⁶ for inhibitory activity. CPI-17 regulates myosin phosphorylation in smooth muscle contraction and the tumorigenic transformation of several cell lines via the inhibition of MP. A phosphospecific antibody (anti-CPI-17^pThr38) against the phosphorylation peptide was used to determine the phosphorylation levels in cells. We found that phospho-CPI-17 and its closely related proteins are not present in HeLa and MCF7 cells after inducing phosphorylation by inhibiting phosphatases with calyculin A. In contrast, cross-reactions of proteins in the 40–220 kDa range with anti-CPI-17^pThr38 were apparent. Searching the protein database for similarities to the CPI-17 phosphorylation sequence revealed several proteins with 42–75% sequence identities. The LIMK2-1 isoform emerged as a possible PP1 inhibitor. Experiments with Flag-LIMK2-1 overexpressed in tsA201 cells proved that LIMK2-1 interacts with PP1c isoforms and is phosphorylated predominantly by protein kinase C. Phosphorylated LIMK2-1 inhibits PP1c and the MP holoenzyme with similar potencies (IC50 ~28–47 nM). In conclusion, our results suggest that LIMK2-1 is a novel phosphorylation-dependent inhibitor of PP1c and MP and may function as a CPI-17-like phosphatase inhibitor in cells where CPI-17 is present but not phosphorylated upon phosphatase inhibition. Full article

Phospholipase A1 (Ves a 1), a major toxin from Vespa affinis venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting Naja naja PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope–epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of V. affinis venom allergy. Full article

Background: Canine parvovirus (CPV) is a highly pathogenic virus that predominantly affects puppies, with mortality rates exceeding 70%. Although commercial multivalent live attenuated vaccines (MLV) are widely employed, their efficacy is often compromised by maternal antibody interference. Consequently, the development of novel vaccines remains imperative for effective CPV control. Methods: Recombinant CPV VP2 protein (rVP2) and canine interlukine 12 protein (rcIL-12) were expressed using the Bac-to-Bac baculovirus expression system and the biological activity of these proteins was assessed through hemagglutination, Cell Counting Kit-8 (CCK8) and IFN-γ induction assays. The combined immunoenhancement effect of rVP2 and rcIL-12 protein was evaluated in puppies. Results: Both rVP2 and rcIL-12 were successfully expressed and purified, exhibiting confirmed antigenicity, immunogenicity, and bioactivity. Co-administration of rVP2 with rcIL-12 elicited higher neutralizing antibody titer (6–7 times higher), complete challenge protection efficiency (no clinical symptoms and tissue and organ lesions), fewer viral shedding (decreasing significantly 8-day post challenge) and superior viral blockade (lower viral load in the organism) compared to rVP2 alone. Conclusions: Our findings demonstrate that rVP2 co-administered with rcIL-12 induces robust protective immunity in puppies and significantly mitigated the inhibitory effects of maternal antibodies. This represents a promising strategy for enabling earlier vaccination in puppies and rational design of CPV subunit vaccines. Full article

IgG antibodies, signaling via the inhibitory receptor, FcγRIIb, are potent inhibitors of IgE-mediated mast cell activation. We have previously reported that in addition to blocking mast cell degranulation, inhibitory IgG signals shut down a proinflammatory transcriptional program in which mast cells produce cytokines and chemokines known to drive type 2 tissue inflammation. To determine whether such effects of allergen-specific IgG can modulate allergic inflammation in vivo, we examined the airways of mice sensitized to ovalbumin (OVA) by intraperitoneal injection and then challenged with intranasal OVA. Pretreatment with allergen-specific IgG significantly reduced the recruitment of inflammatory cells, including macrophages and eosinophils, into the lungs of OVA-sensitized mice. The bronchoalveolar lavage fluid of OVA-challenged mice contained elevated levels of chemokine ligands (CCL2 and CCL24) and interleukin-5, a response that was markedly blunted in animals receiving allergen-specific IgG. IgG-treated animals exhibited attenuated allergen-induced production of IgE, IL-4, and IL-13, along with impaired OVA-induced goblet cell hyperplasia and Muc5ac expression and suppressed airway hyperresponsiveness, consistent with a shift away from a Th2 response. Using mice with a lineage-specific deletion of FcγRIIb, we demonstrated that each of these protective effects of IgG was dependent upon the expression of this receptor on mast cells. Overall, our findings establish that allergen-specific IgG can reduce allergen-driven airway inflammation and airway hyperresponsiveness and point to a mechanistic basis for the therapeutic benefit of aeroallergen-specific IgG therapy. Full article

Human Interleukin-6 (hIL-6) is a pro inflammatory cytokine that binds to its receptor, IL-6Rα followed by binding to gp130 and subsequent dimerization to form a hexamer signaling complex. As a critical inflammation mediator, hIL-6 is associated with a diverse range of diseases and monoclonal antibodies in clinical use that either target IL-6Rα or hIL-6 to inhibit signaling. Here, we perform high-throughput structure-based computational screening using ensemble docking for small-molecule antagonists for which the target conformations were taken from 600 ns long molecular dynamics simulations of the apo protein. Prior knowledge of the contact sites from binary complex studies and experimental work was incorporated into the docking studies. The top 20 scoring ligands from the in silico studies after post analysis were subjected to in vitro functional assays. Among these compounds, the ligand with the second-highest calculated binding affinity experimentally showed an ~84% inhibitory effect on IL6-induced STAT3 reporter activity at 10 μM concentration. This finding may pave the way for designing small-molecule inhibitors of hIL-6 of therapeutic significance. Full article

Hemophilia, an X-linked bleeding disorder, is characterized by a deficiency in coagulation factors. It manifests as spontaneous bleeding, leading to severe complications if not properly managed. In contrast, acquired hemophilia is an autoimmune condition marked by the development of inhibitory antibodies against coagulation factors. Both forms present significant diagnostic and therapeutic challenges, highlighting the need for advanced genetic, molecular, laboratory, and clinical assessments. Recent advances in artificial intelligence have opened new avenues for the management of hemophilia. Machine learning and deep learning technologies enhance the ability to predict bleeding risks, optimize treatment regimens, and monitor disease progression with greater precision. Artificial intelligence-driven applications in medical imaging have also improved the detection of joint damage and hemarthrosis, ensuring timely interventions and better clinical outcomes. Moreover, the integration of artificial intelligence into clinical practice holds the potential to transform hemophilia care through predictive analytics and personalized medicine, promising not only faster and more accurate diagnoses but also a reduction in long-term complications. However, ethical considerations and the need for data standardization remain critical for its widespread adoption. The application of artificial intelligence in hemophilia represents a paradigm shift towards precision medicine, with the promise of significantly improving patient outcomes and quality of life. Full article

Tumor diseases are characterized by high interindividual and intratumoral heterogeneity (ITH). The development and progression of neoplasms outline complex networks of extracellular and cellular signals that have yet to be fully elucidated. This narrative review provides a comprehensive overview of the literature related to the cellular and molecular mechanisms underlying the heterogeneity of the tumor mass. Furthermore, it examines the possible role of the tumor microenvironment in the development and support of the neoplasm, in order to highlight its potential in the construction of a diagnostic–therapeutic approach to precision medicine. Many authors underline the importance of the tumor microenvironment (TME) as it actively takes part in the growth of the neoplastic mass and in the formation of metastases and in the acquisition of resistance to anticancer drugs. In specific body districts, the ideal conditions occur for the TME establishment, particularly the inflammatory state, the recruitment of cell types, the release of specific cytokines and growth factors, hypoxic conditions. These components actively intervene by enabling tumor progression and construction of physical barriers shaped by the extracellular matrix that contribute to forming peripheral tolerance by intervention of myeloid precursors and the polarization of M2 macrophages. In recent years, ITH and the TME have assumed an important position in cancer research and pharmacology as they enable understanding the dense network of communication existing between the neoplasm and the surrounding environment, and to monitor and deepen the effects of drugs with a view to develop increasingly precise and effective therapies. In the last decade, knowledge of TME has been exploited to produce targeted molecular agents (inhibitory small molecules, monoclonal antibodies, gene therapy). Nonetheless, the bibliography shows the need to study ITH through new prognostic and predictive biomarkers (e.g., ctDNA and CTCs) and to increase its basic biology knowledge. Precision medicine is a new opportunity in the treatment of oncological diseases that is transforming the development of new drug approaches and their clinical use. Biology and biotechnologies are providing the bases for this revolution. Full article

It is becoming evident that the therapeutic effect of reawakening the immune response is to limit tumor cell growth and expansion. The use of immune checkpoint inhibitors, like blocking antibodies against programmed cell death receptor (PD) 1 and/or cytotoxic T lymphocyte antigen (CTLA) 4 alone or in combination with other drugs, has led to unexpected positive results in some tumors but not all. Several other molecules inhibiting lymphocyte antitumor effector subsets have been discovered in the last 30 years. Herein, we focus on the leukocyte-associated immunoglobulin (Ig)-like receptor 1 (LAIR1/CD305). LAIR1 represents a typical immunoregulatory molecule expressed on almost all leukocytes, unlike other regulatory receptors expressed on discrete leukocyte subsets. It bears two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the intracytoplasmic protein domain involved in the downregulation of signals mediated by activating receptors. LAIR1 binds to several ligands, such as collagen I and III, complement component 1Q, surfactant protein D, adiponectin, and repetitive interspersed families of polypeptides expressed by erythrocytes infected with Plasmodium malariae. This would suggest LAIR1 involvement in several cell-to-cell interactions and possibly in metabolic regulation. The presence of both cellular and soluble forms of LAIR would indicate a fine regulation of the immunoregulatory activity, as happens for the soluble/exosome-associated forms of PD1 and CTLA4 molecules. As a consequence, LAIR1 appears to play a role in some autoimmune diseases and the immune response against tumor cells. The finding of LAIR1 expression on hematological malignancies, but also on some solid tumors, could open a rationale for the targeting of this molecule to treat neoplasia, either alone or in combination with other therapeutic options. Full article

Murine double minute 2 (MDM2) is involved in various cancers and is an attractive target. The RING domain of MDM2 has been discussed as an alternative target to stabilize p53. Designing drugs to target the RING domain of MDM2 is an alternative approach to preventing MDM2-mediated deactivation of p53. In this study, we obtained a human VH single-domain antibody and revealed its regulatory effects and mechanisms. The RING domain of MDM2 was synthesized using a chemical synthesis method, and antibodies against the MDM2 RING domain were screened from a human VH single-domain antibody library and expressed intracellularly. A nuclear localization sequence was designed to ensure intrabody efficiency. The binding activity of the individually cloned antibodies was detected using ELISA. MTT and flow cytometry assays were used to detect the reactions related to intrabody in vitro. The combination and its influence on MDM2 were detected using immunoprecipitation assays, confocal microscopy, and Western blotting. The effects on apoptosis-related mitochondrial pathways downstream of p53 were examined using Western blotting. The influence on cell cycle distribution and cyclin-related proteins was detected using flow cytometry and Western blotting. A549 cell xenografts were constructed to assess the effect of intrabodies on growth in vivo. The molecular mechanisms of MDM2 and p53 were studied using Western blotting. Eight individual cloned antibodies were positive compared to the signals on the BSA-coated plates, especially intrabodies VH-HT3. In A549 and MCF-7 cell lines, VH-HT3 exhibited significant inhibitory effects on cell proliferation and apoptosis. VH-HT3 co-localized with MDM2 in the nucleus and cytoplasm. The specific combination of VH-HT3 triggered no significant effect on MDM2 activity for p53 degradation but upregulated the levels of factors downstream of p53, especially those in the mitochondrial apoptosis pathway. Moreover, VH-HT3 induced cell cycle arrest, and the expression of cyclin-related proteins was consistent with this observation. VH-HT3 also retarded the growth of A549 xenografts in vivo. Further tests suggested that VH-HT3 inhibited MDM2 function by increasing HIPK2 levels and activating p53 at the Ser46 site. VH-HT3, prepared from a human VH single-domain antibody library, inhibited p53 activity and produced a tumor-suppressive effect. The intrabody VH-HT3 is a candidate for the development of novel MDM2 inhibitors. Full article

Glioblastoma, classified as a grade IV astrocytoma, is an aggressive and malignant primary brain tumor with no known cure. Despite the implementation of standard medical and surgical treatment protocols, the disease often progresses with unsatisfactory outcomes. This study aimed to evaluate the cytotoxic, proapoptotic, and antimetastatic effects of anti-angiogenic monoclonal antibody bevacizumab combined with the sphingosine-1-phosphate receptor modulator fingolimod on rat glioma C6 cells. The cytotoxicity of bevacizumab and fingolimod was evaluated using the MTT assay. Proapoptotic activity was assessed through flow cytometric analyses, including Annexin V–FITC staining, caspase 3/7 activation, and mitochondrial membrane potential measurements. Morphological changes were examined using confocal microscopy. Antimetastatic effects were evaluated via anti-migration and colony formation assays. The combination of bevacizumab and fingolimod exhibited antiproliferative, cytotoxic, proapoptotic, and antimetastatic effects on C6 glioma cells at low IC₅₀ concentrations. Based on growth inhibitory, proapoptotic, and antimetastatic activities on C6 glioma cells, the combination of bevacizumab and fingolimod demonstrates significant growth-inhibitory, proapoptotic, and antimetastatic activities against C6 glioma cells, suggesting its potential as a promising pharmacotherapeutic approach for the treatment of glioblastoma. Full article

Immunotherapies targeting the programmed cell death-1 ligand 1 (PD-L1) and programmed cell death 1 (PD-1) pathway sparked a revolution in cancer treatment. These breakthrough therapies work by disrupting the interaction between PD-1—expressed on T cells—and its ligand PD-L1, commonly found on the surface of cancer cells. By using monoclonal antibodies to block this binding, the immune system is unleashed to fight cancer more effectively. However, PD-L1’s role extends far beyond immune evasion. When situated on cancer cells, PD-L1 transmits inhibitory signals through PD-1, silencing the effector functions of T cells. However, PD-L1 also engages in reverse signaling, also called intrinsic signaling, delivering intracellular instructions that contribute to cancer cell survival, even in the absence of PD-1 binding. This signaling cascade shields cancer cells from apoptosis, drives proliferation, regulates DNA damage responses, and even functions as a co-transcriptional transactivator, amplifying cancer’s ability to thrive. The intricate mechanisms behind PD-L1’s intrinsic signaling are under intense investigation. In this review, we provide a historical perspective on the discoveries leading to PD-L1’s structure, signaling motifs, and interacting partners, shedding light on its multifaceted roles and the promising therapeutic possibilities ahead. Full article

Sclerostin, encoded by the SOST gene, is a novel bone anabolic target for bone diseases. Humanized anti-sclerostin antibody, romosozumab, was approved for treatment of postmenopausal osteoporosis by the US Food and Drug Administration (FDA), but with a black-box warning on cardiovascular risk. The clinical data regarding cardiovascular events from various pre-marketing and post-marketing studies of romosozumab were inconsistent. Overall, the cardiovascular risk of sclerostin inhibition could not be excluded. The restriction of romosozumab in patients with cardiovascular disease history would be necessary. Moreover, genome-wide association study (GWAS) analyses of SOST variants revealed inconsistent results of the association between SOST variations and cardiovascular diseases. Further research incorporating larger sample sizes and functional analyses are necessary. In analyses of serum/tissue sclerostin levels in patients with cardiovascular diseases, the results were controversial but indicated an association between sclerostin and the presence/severity/outcomes of cardiovascular diseases. Nonclinical studies in rodents indicated the inhibitory effect of sclerostin on inflammation, aortic aneurysm, atherosclerosis, and vascular calcification. Sclerostin loop3 participated in the inhibitory effect of sclerostin on bone formation, while the cardiovascular protective effect of sclerostin was independent of sclerostin loop3. Macrophagic sclerostin loop2–apolipoprotein E receptor 2 (ApoER2) interaction participated in the inhibitory effect of sclerostin on inflammation in vitro. Sclerostin in human aortic smooth muscle cells participated in the reduction in calcium deposition. The role of sclerostin in cardiovascular system deserves further investigation. Full article

Background/Objectives: Protein aggregation, particularly the aggregation of antibody-based drugs, has long been a significant challenge in downstream processes and formulation. While the inhibitory effects of excipients on aggregation have been extensively studied using early experimental characterization methods, complete formulation research requires significant amounts of antibodies and time, resulting in high research costs. Methods: This study proposed a quick and small-scale position-restrained simulation method which elucidated the mechanism of the reversible self-association (RSA) of antibodies and the influence of excipients on RSA under different conditions. We also validated the rationality of rapid and small-scale simulations through long-term (>1 μs) and large-scale (>1,000,000 atoms) simulations. Results: Through combing with simple stability characterization, the effects of different excipients on monomer residual content and the trend shown with concentration changes after thermal incubation were found to be similar to those observed in the simulations. Additionally, the formulation proposed by the simulations was validated using experimental characterization. Conclusions: Simulations and experiments revealed the mechanism and showed consistent trends, providing better understanding for aggregation research. Full article

Background/Objectives: According to recent guidelines, including the guideline on bioanalytical method validation issued by the European Medicine Agency, the stability of clinical analytes should be known. We summarize human α₁-proteinase inhibitor (A1PI) and urea stability data in relevant matrices, as these analytes are usually measured in clinical A1PI studies. Methods: Stability samples with appropriate A1PI concentrations were prepared in a citrated human reference plasma pool and a matrix mimicking bronchoalveolar lavage (BAL) solution. These samples were kept at −20 °C and −60 °C for up to 24 months. A1PI protein was measured with a nephelometric method and an enzyme-linked immunosorbent assay using paired commercially available polyclonal antibodies. A1PI elastase inhibitory activity was determined with an elastase complex formation immunosorbent assay that combines A1PI complex formation with a solid phase-immobilized elastase and immunological detection of the formed A1PI-elastase complex and urea in samples kept at −20 °C only with an enzymatic assay. Results: Overall, the stability criterion (100 ± 20%) was met for the analytes A1PI protein and A1PI activity at both temperatures during storage of BAL-mimicking and plasma samples for 15 and 24 months, respectively; urea was stable in both matrices at −20 °C for 18 months. Plasma samples showed smaller drops in concentration over storage time than BAL-mimicking samples. As expected, the reduction of A1PI elastase inhibitory activity was more pronounced than that of A1PI protein. Interestingly, the analyte concentration did not significantly influence the results in either of the sample matrices. Conclusions: The data confirmed the appropriate stability of the three analytes in the matrices of citrated plasma and BAL-mimicking samples for at least up to 15 months. Full article

Monoclonal antibodies (mAbs) targeting various pathways in cancer therapy play crucial roles in enhancing the immune system’s ability to recognise and eliminate tumour cells. These therapies are designed to either block inhibitory immune checkpoint pathways or to target specific tumour cell markers for direct destruction. Additionally, mAbs can modulate the tumour microenvironment, enhance antibody-dependent cellular cytotoxicity, and inhibit angiogenesis, further amplifying their therapeutic impact. Below is a summary of monoclonal antibodies targeting key pathways, along with their indications and mechanisms of action, which are reviewed based on therapeutic mechanisms. Full article