Advances In Therapeutic Approaches Research Articles

Importance of CD8 Tex cell-associated gene signatures in the prognosis and immunology of osteosarcoma

As a highly aggressive bone malignancy, osteosarcoma poses a significant therapeutic challenge, especially in the setting of metastasis or recurrence. This study aimed to investigate the potential of CD8-Tex cell-associated genes as prognostic biomarkers to reveal the immunogenomic profile of osteosarcoma and guide therapeutic decisions. mRNA expression data and clinical details of osteosarcoma patients were obtained from the TCGA database (TARGET-OS dataset). The GSE21257 dataset (from the GEO database) was used as an external validation set to provide additional information on osteosarcoma specimens. 84 samples from the TARGET-OS dataset were used as the training set, and 53 samples from the GSE21257 dataset served as the external validation cohort. Univariate Cox regression analysis was utilized to identify CD8 Tex cell genes associated with prognosis. The LASSO algorithm was performed for 1000 iterations to select the best subset to form the CD8 Tex cell gene signature (TRS). Final genes were identified using the multivariate Cox regression model of the LASSO algorithm. Risk scores were calculated to categorize patients into high- and low-risk groups, and clinical differences were explored by Kaplan–Meier survival analysis to assess model performance. Prediction maps were constructed to estimate 1-, 3-, and 5 year survival rates for osteosarcoma patients, including risk scores for CD8 Texcell gene markers and clinicopathologic factors. The ssGSEA algorithm was used to assess the differences in immune function between TRS-defined high- and low-risk groups. TME and immune cell infiltration were further assessed using the ESTIMATE and CIBERSORT algorithms. To explore the relationship between immune checkpoint gene expression levels and the two risk-defined groups. A CD8 Tex cell-associated gene signature was extracted from the TISCH database and prognostic markers including two genes were developed. The high-risk group showed lower survival, and model performance was validated by ROC curves and C-index. Predictive plots were constructed to demonstrate survival estimates, combining CD8 Tex cell gene markers and clinical factors. This study provides valuable insights into the molecular and immune characteristics of osteosarcoma and offers potential avenues for advances in therapeutic approaches.

Highlight of 2023: From fundamental studies to clinical trials, the importance of NK cells against cancer.

In this article for the Highlights of 2023 Series, we discuss how various factors affect the ability of natural killer (NK) cells to fight tumors. For instance, tumor cells can hinder NK cell function by reducing surface protrusions or increasing HLA-E expression via platelets. Lower UTX protein levels in male NK cells also decrease their cytotoxicity compared with females. Fortunately, recent advancements in therapeutic approaches have emerged, including the development of a comprehensive atlas of NK cell heterogeneity within the tumor microenvironment, as well as a trispecific engager molecule that has shown promise in enhancing the anti-tumor functions of NK cells.

Mechanical Support in High-Risk Pulmonary Embolism: Review Article

Acute pulmonary embolism (PE) may manifest with mild nonspecific symptoms or progress to a more severe hemodynamic collapse and sudden cardiac arrest. A substantial thrombotic burden can precipitate sudden right ventricular strain and failure. Traditionally, systemic thrombolytics have been employed in such scenarios; however, patients often present with contraindications, or these interventions may prove ineffective. Outcomes for this medically complex patient population are unfavorable, necessitating a compelling argument for advanced therapeutic modalities or alternative approaches. Moreover, patients frequently experience complications beyond hemodynamic instability, such as profound hypoxia and multiorgan failure, necessitating assertive early interventions to avert catastrophic consequences. The existing data on the utilization of mechanical circulatory support (MCS) devices are not exhaustive. Various options for percutaneous MCS devices exist, each possessing distinct advantages and disadvantages. There is an imminent imperative to develop a tailored approach for this high-risk patient cohort to enhance their overall outcomes.

Molecular Regulation of NLRP3 Inflammasome Activation During Parasitic Infection.

Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis,and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium,Leishmania, Toxoplasma gondii, Entamoeba histolytica,Trypanosoma cruzi and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites' growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome's action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.

Abstract 7097: Integrated proteomics and metabolomics reveals new insight into the synergistic interaction of valproic acid plus simvastatin in prostate cancer xenograft model associated with down-modulation of YAP and TAZ signaling

Abstract Background: Despite advancements in therapeutic approaches, including taxane-based chemotherapy and androgen receptor targeting agents, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease, underlying the need of novel strategies that can target the complexities of this disease and bypass the development of drug-resistance mechanisms. We previously demonstrated the synergistic antitumor interaction of the antiepileptic with histone deacetylase inhibitory activity valproic acid (VPA), and the lipid-lowering drug simvastatin (SIM). This combination sensitizes mCRPC cells to docetaxel treatment and revert docetaxel-resistance, both in vitro and in vivo models, by targeting cancer stem cells compartment via mevalonate pathway/YAP axis modulation. Methods: Proteomic and metabolomic/lipidomic analysis on tumor samples were performed by LC-MS/MS-based shotgun proteomics and by 1H-NMR approach, respectively. Progenesis QI for proteomics v. 4.2 was used as label-free quantification platform. Functional annotation analysis were performed by using the DAVID v6.8 (Database for Annotation, Visualization and Integrated Discovery); g:Profiler WikiPathways databases; Reactome version 66; Ingenuity Pathway Analysis software. Partial least squares-discriminant analysis (PLS-DA) and Loading plot were performed by Metabo Analyst v5.0 tool. Results: Tumor samples derived from 22Rv1 mCRPC cells xenografted mice, treated or not with VPA/SIM combination were characterized by a combined proteomic and metabolomic/lipidomic approach. In deep bioinformatics analysis confirmed a specific impact of VPA/SIM on Hippo pathway, with a clear down regulation of YAP and its targets (CTGF, CycD1 ANKH1) in treated vs untreated tumors, functionally related with modulation of cancer-related extracellular matrix remodelling and metabolic reprogramming. Treatment also downregulated TEAD transcription factor mediating YAP-induced expression, along with the upregulation of 14-3-3, responsible for YAP cytoplasmic retention and degradation, paralleled by the up-regulation of YAP up-stream negative regulators such as LATS1 and AMPK. VPA/SIM also modulated intermediates of TCA, urea cycles and glycolysis and decreased cholesterol and fatty acids. Conclusion: In summary, this study provides further insights into the molecular mechanism of the VPA/SIM antitumor effect suggesting the potential of repurposing these two generic and safe drugs in combination with standard antitumor therapies also in other tumors besides mCRCP. Citation Format: Federica Iannelli, Rita Lombardi, Susan Costantini, Maria Serena Roca, Laura Addi, Francesca Bruzzese, Elena Di Gennaro, Alfredo Budillon, Biagio Pucci. Integrated proteomics and metabolomics reveals new insight into the synergistic interaction of valproic acid plus simvastatin in prostate cancer xenograft model associated with down-modulation of YAP and TAZ signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7097.

In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Flavonoids and Flavonoid-Based Nanoparticles for Osteoarthritis and Rheumatoid Arthritis Management

Arthritis, a global health burden comprising osteoarthritis and rheumatoid arthritis, demands advanced therapeutic approaches. In this context, flavonoids, a diverse group of naturally occurring compounds abundant in fruits, vegetables, and medicinal plants, have emerged as promising candidates for mitigating the inflammatory processes associated with arthritic conditions. This review aims, first, to provide a comprehensive exploration of the potential of flavonoids, focusing on specific compounds such as quercetin, epigallocatechin-3-gallate (EGCG), apigenin, luteolin, fisetin, silibinin, kaempferol, naringenin, and myricetin. The second section of this review delves into the anti-arthritic activities of these flavonoids, drawing insights from clinical trials and scientific studies. Each flavonoid is scrutinized individually to elucidate its mechanisms of action and therapeutic efficacy in the context of both osteoarthritis and rheumatoid arthritis. The third section of this review highlights the challenges associated with harnessing flavonoids for anti-inflammatory purposes. Bioavailability limitations pose a significant hurdle, prompting the exploration of innovative strategies such as the use of nanoparticles as delivery vehicles. In response to these challenges, the fourth section focuses on the emerging field of flavonoid-based nanoparticles. This includes detailed discussions on quercetin, EGCG, fisetin, and naringenin-based nanoparticles, highlighting formulation strategies and preclinical evidence supporting their potential in arthritis management. The targeted delivery to inflammatory sites and the exploration of synergistic combinations with other compounds are also discussed as promising avenues to enhance the therapeutic impact of flavonoids. This review consolidates current knowledge on flavonoids and their nanoformulations as potential therapeutic interventions for osteoarthritis and rheumatoid arthritis. By addressing challenges and presenting future research directions, this review aims to contribute to the advancement of innovative and effective strategies for alleviating the global burden of arthritis.

The protective effects of statins in traumatic brain injury.

Traumatic brain injury (TBI), often referred to as the "silent epidemic", is the most common cause of mortality and morbidity worldwide among all trauma-related injuries. It is associated with considerable personal, medical, and economic consequences. Although remarkable advances in therapeutic approaches have been made, current treatments and clinical management for TBI recovery still remain to be improved. One of the factors that may contribute to this gap is that existing therapies target only a single event or pathology. However, brain injury after TBI involves various pathological mechanisms, including inflammation, oxidative stress, blood-brain barrier (BBB) disruption, ionic disturbance, excitotoxicity, mitochondrial dysfunction, neuronal necrosis, and apoptosis. Statins have several beneficial pleiotropic effects (anti-excitotoxicity, anti-inflammatory, anti-oxidant, anti-thrombotic, immunomodulatory activity, endothelial and vasoactive properties) in addition to promoting angiogenesis, neurogenesis, and synaptogenesis in TBI. Supposedly, using agents such as statins that target numerous and diverse pathological mechanisms, may be more effective than a single-target approach in TBI management. The current review was undertaken to investigate and summarize the protective mechanisms of statins against TBI. The limitations of conducted studies and directions for future research on this potential therapeutic application of statins are also discussed.

Impaired Brain-Heart Axis in Focal Epilepsy: Alterations in Information Flow and Implications for Seizure Dynamics

Abstract This study delves into functional Brain-Heart Interplay (BHI) dynamics during interictal periods before and after seizure events in focal epilepsy. Our analysis focuses on elucidating the causal interaction between cortical and Autonomic Nervous System (ANS) oscillations, employing Electroencephalography and Heart Rate Variability series. The dataset for this investigation comprises 47 seizure events from 14 independent subjects, obtained from the publicly available Siena Dataset.Our findings reveal an impaired brain-heart axis especially in the Heart-to-Brain functional direction. This is particularly evident in bottom-up oscillations originating from sympathovagal activity during the transition between pre-ictal and post-ictal periods. These results indicates a pivotal role of the ANS in epilepsy dynamics. Notably, the Brain-to-Heart information flow targeting cardiac oscillations in the low-frequency band does not display significant changes. However, there are noteworthy changes in cortical oscillations, primarily originating in central regions, influencing heartbeat oscillations in the high-frequency band.Our study conceptualize seizures as a state of hyperexcitability and a network disease affecting both cortical and peripheral neural dynamics.Our results pave the way for a deeper understanding of BHI in epilepsy, which holds promise for the development of advanced diagnostic and therapeutic approaches also based on bodily neural activity for individuals living with epilepsy.

Current advancements in therapeutic approaches in orthopedic surgery: a review of recent trends.

Recent advancements in orthopedic surgery have greatly improved the management of musculoskeletal disorders and injuries. This review discusses the latest therapeutic approaches that have emerged in orthopedics. We examine the use of regenerative medicine, including stem cell therapy and platelet-rich plasma (PRP) injections, to accelerate healing and promote tissue regeneration. Additionally, we explore the application of robotic-assisted surgery, which provides greater precision and accuracy during surgical procedures. We also delve into the emergence of personalized medicine, which tailors treatments to individual patients based on their unique genetic and environmental factors. Furthermore, we discuss telemedicine and remote patient monitoring as methods for improving patient outcomes and reducing healthcare costs. Finally, we examine the growing interest in using artificial intelligence and machine learning in orthopedics, particularly in diagnosis and treatment planning. Overall, these advancements in therapeutic approaches have significantly improved patient outcomes, reduced recovery times, and enhanced the overall quality of care in orthopedic surgery.

Diabetic Cardiomyopathy and Cell Death: Focus on Metal-Mediated Cell Death.

Cardiac myocyte death is an essential initiator of the pathogenesis and progression of various etiological cardiomyopathies, including diabetic cardiomyopathy (DCM), a disease that has been reported since 1972. Cardiac cell death has been detected in the hearts of patients with diabetes and in animal models, and the role of cell death in the pathogenesis of DCM has been extensively investigated. The first review by the authors, specifically focusing on "Cell death and diabetic cardiomyopathy," was published in the journal, Cardiovascular Toxicology in 2003. Over the past two decades, studies investigating the role of cardiac cell death in the pathogenesis of DCM have gained significant attention, resulting in the discovery of several new kinds of cell death involving different mechanisms, including apoptosis, necroptosis, pyroptosis, autophagy, ferroptosis, and cuproptosis. After the 20th anniversary of the review published in 2003, we now provide an update with a focus on the potential role of metal-mediated cell death, ferroptosis, and cuproptosis in the development of DCM in compliance with this special issue. The intent of our review is to further stimulate work in the field to advance the body of knowledge and continue to drive efforts to develop more advanced therapeutic approaches to prevent cell death, particularly metal-dependent cell death, and, ultimately, to reduce or prevent the development of DCM.

Diallyl Trisulfide Induces ROS-Mediated Mitotic Arrest and Apoptosis and Inhibits HNSCC Tumor Growth and Cancer Stemness.

Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3-this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.

[Metabolic steatotic liver disease: the move towards an inclusive definition].

Steatotic liver disease is the most common liver pathology worldwide, comprises a wide range of liver diseases linked to metabolic dysfunction, excessive alcohol consumption, drug reactions and infectious and genetic origins. Following several years of deliberation, the major liver disease societies have recently adopted a new nomenclature and updated diagnostic criteria for steatotic liver diseases, aimed at better reflecting our evolving understanding of their pathophysiology. This article summarizes these newly adopted designations, explores the basis for these nomenclatures, presents recent epidemiological data and discusses new diagnostic criteria and recent advances in therapeutic approaches for steatotic liver disease.

NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment.

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

Unveiling the Discrepancy in Multiple Myeloma Risk Classification between Clinical Practice and SKY92 Test and the Impact on Survival

Introduction:Despite the advancements in therapeutic approaches for multiple myeloma (MM), which have led to improve the survival rates, a subset of high-risk patients still faces limited benefits from current treatments. Therefore, it is crucial to accurately identify specific patient groups who would benefit from tailored therapies through risk stratification. In this study, we aim to assess the risk classification of patients based on conventional risk markers currently used in clinical practices and with the gene expression profiling (GEP)-based SKY92 risk classifier (SkylineDx, San Diego). This approach could provide further insights for optimizing risk-adapted treatment compared to the standard guidelines used in clinical practice. Methods: The PRospective Observational Multiple Myeloma Impact Study (PROMMIS; NCT02911571) trial, a prospective US multicenter study, enrolled a total of 251 newly diagnosed MM patients (NDMM). The patients underwent risk stratification using two distinct approaches. Firstly, physicians assessed their risk of progression at the time of diagnosis according to their routine practice guidelines. Then, the patients were classified into risk groups using the SKY92 molecular test. The analysis involved the processing of RNA extracted from CD138-positive plasma cells (≥80% purity), which were isolated through immunomagnetic separation. SKY92 test was carried out either at the enrolling institute or by sending samples to a central laboratory. For 221 patients, progression-free survival (PFS) and overall survival (OS) data were available, with measurements taken from date of diagnosis; the median follow-up period was 38 months. Survival analysis was performed utilizing Cox proportional hazards model. Results: Based on conventional risk classification, 123 (49%) NDMM were classified as standard-risk and 128 (51%) patients as high-risk. However, SKY92 yielded a noticeable difference in classification, with 179 (71%) patients being categorized as standard-risk and 72 (29%) patients as high-risk. In specific 23 standard-risk and 79 high-risk (n=102, 41%) patients accordingly with clinical practice assessment were classified differently by the SKY92 test. Standard-risk and high-risk groups identified by current clinical practice classification showed a significant difference in PFS (HR: 1.6, 95%CI 1.0-2.4, p=0.031), but not in OS (HR: 1.9, 95%CI 0.9-4.0, p=0.10). With SKY92 risk stratification, instead, a significant difference was observed between the two risk groups in both PFS (HR: 2.1, 95%CI 1.4-3.1, p<0.001) and OS (HR: 3.7, 95%CI 1.8-7.6, p<0.001); furthermore, the median PFS for standard-risk and high-risk patients was, respectively, 50 and 26 months. The median OS was not reached within the 60-month timeframe for both groups. For the n=221 MM patients with survival data, we observed that most patients classified as standard-risk based on clinical practice classification were also categorized as standard-risk according to SKY92 analysis (83 out of 105, 79%). However, among those initially identified as high-risk by conventional risk classification, 71 out of 116 (61%) patients exhibit no significant difference in PFS (HR: 1.1, 95%CI 0.6-1.8, p=0.8) and OS (HR: 0.6, 95%CI 0.2-1.9, p=0.4) when compared to standard-risk patients, as determined by both risk classification methods ( Figure 1). Conversely, only 22 out of 72 (30%) patients initially identified as high-risk by SKY92 and as standard-risk by clinical practice show no significance difference for PFS (HR: 1.1, 95%CI 0.5-2.4, p=0.8) and OS (HR: 0.9, 95%CI 0.2-4.1, p=0.9) compared with standard-risk patients determined by both tests. Forty-five patients identified as HR by both risk classification methods show highly significant differences for PSF (HR: 2.9, 95%CI 1.7-4.9, p<0.001) and OS (HR: 4.1, 95%CI 1.8-9.6, p<0.001) compare with standard-risk group. Conclusions: These findings based on prospective real-world clinical data, collected in a prospective US multicenter trial confirm the effectiveness of the GEP SKY92 on risk stratification both for PFS and OS. These results support the added value of integrating SKY92 into clinical practice for precise risk assessment of patients. Figure1: PFS and OS based on the combination of the clinical risk assessment (CRA) and the SKY92 risk.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p<0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p<0.01).

TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, Is a Promising Anti-Myeloma Agent Regardless of Types of Cytogenetic/Genetic Abnormality

Introduction: Despite advancements in therapeutic approaches, treatment outcome for multiple myeloma (MM) remains unsatisfactory. One critical factor of the poor outcomes is the complex inter-patient and intra-clonal heterogeneity of cytogenetic, genetic, and molecular abnormalities. To overcome this, the exploration of therapeutic target molecules that play a universal and essential role in MM regardless of the types of molecular profile is crucial even in the post-genomic era. We have previously demonstrated that RSK2 Ser227 at the N-terminal kinase domain (NTKD), as the downstream signaling hub of the RAS/ERK pathway, plays essential roles in the proliferation and survival of myeloma cells (Shimura Y, Mol Cancer Ther 2012). We also identified that RSK2-NTKD is constitutively activated by constitutive active PDPK1, the master serine-threonine kinase of >20 AGC kinases, in myeloma cells of approximately 90% of MM patients regardless of cytogenetic abnormalities (Chinen Y, Cancer Res 2014, Tatekawa S, Br J Haematol 2017). Moreover, the combinatory blockade of RSK2 and AKT, one of the other important molecules regulated by PDPK1, exhibited synergistic anti-myeloma efficacy through the modulation of various critical gene sets involved in myeloma pathophysiology, such as MYC (Isa R, Int J Mol Sci 2022). Based upon these, this study investigates the anti-myeloma potency of TAS0612 (Taiho Oncology Inc., Princeton, NJ) that targets RSK, including RSK2, AKT, and S6K, and inhibits the bindings to their respective substrate molecules (Shibata T, Mol Cancer Ther 2020). Methods and Results: Firstly, we examined the activation status of RSK, AKT, and S6K in eight human myeloma-derived cell lines (HMCLs) with diverse genetic and molecular profiles at the protein level. RSK2 Ser227 was found to be commonly activated through phosphorylation in all HMCLs examined. In contrast, the activation patterns of p-AKT Ser473 and p-S6K Thr389 were inconsistent among eight HMCLs, and depending on cell types. Despite these cell type-dependent effects, the exposure to TAS0612 resulted in the accumulation of p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in the majority of HMCLs examined, and this led to the subsequent inactivation of their respective target substrates, such as p-YB1 Ser102, p-PRAS Thr246, and p-S6 Ser235/236, through dephosphorylation. In accompany with these molecular effects, the modified MTT assay showed the dose-dependent growth inhibitory effects of TAS0612 in all cell lines with various types of cytogenetic/genetic abnormalities with IC50 values mostly below 0.5 μM (48 hours). In addition, flow cytometry analyses for the cell cycle analysis and the apoptosis assay by the counterstaining of propidium iodide and Annexin-V showed that TAS0612 induced G1/S cell cycle arrest and apoptotic cell death. Furthermore, patient-derived primary myeloma cells were labeled with anti-CD138 MicroBeads, and were positively isolated from bone marrow mononuclear cells from patients with newly diagnosed MM (n=7) and relapsed/refractory MM (n=11), and, then, were treated by TAS0612. As the results, the ex vivo treatment of 2.5-5.0 μM TAS0612 for 72 hours showed robust cell reductive effects on primary-myeloma cells from all 18 patients examined irrespective of cytogenetic profile. To clarify the precise molecular effects of TAS0612 treatment in myeloma cells, we conducted a comprehensive molecular analysis with TAS0612 treatment by gene expression profiling. The results showed that TAS0612 treatment significantly modulated the gene sets involved in Myc signaling, mTOR signaling, and the core serum response (CSR). Additionally, TAS0612 caused the concomitant upregulation of several bona-fide tumor suppressor genes, including TP53INP, HBP1, FBOX32, and YPEL3, while the downregulation of oncogenes LYAR and HSPA1. Intriguingly, according to MMRF CoMMpass Study, 8 of 18 upregulated genes by TAS0612 in HMCLs were reported to be associated with favorable survival outcomes when highly expressed in myeloma cells. Conclusion: Our findings strongly support the potential of TAS0612 as an effective anti-myeloma agent. Further preclinical investigations are necessary to substantiate these results and facilitate its future clinical development.

Periodontal vaccines: Where we are now and where we can go

Edward Jenner devised and established the notion of vaccination in the late 18th century, using the cross-protection offered by the cowpox virus, which is not dangerous to humans. In adults, periodontal diseases with their multi-microbial etiology are a leading cause of tooth loss. Current treatment approaches have only been able to stop the disease’s progression; neither do they totally cure the condition nor stop it from returning. As a result, there is a need for more advanced therapeutic approaches, which may include vaccines that target potential periodontal bacteria. A boon to periodontics is the periodontal vaccination. No periodontal vaccine study has been able to fully meet all of the criteria for the ideal periodontal vaccine. The goal of the periodontal vaccination is to pinpoint the antigens implicated in the periodontitis damaging process that antibodies would be induced to guard against. In the future, periodontal vaccinations might become a supplement to mechanical therapy. To reduce the morbidity associated with periodontal disease in humans, eliminating the worldwide burden of periodontal disease would be the demanding primary function of any periodontal vaccination. This paper focuses on bringing to light the current approaches to periodontal vaccinations and what the future might have in store as far as this issue is concerned.

Changes in neonatal morbidity, neonatal care practices, and length of hospital stay of surviving infants born very preterm in the Netherlands in the 1980s and in the 2000s: a comparison analysis with identical characteristics definitions

BackgroundThis study evaluates changes in the neonatal morbidity, the neonatal care practices, and the length of hospital stay of surviving very preterm (VP) infants born in the Netherlands in the 1980s and in the 2000s; a period over which historical improvements were introduced into neonatal care. We, herein, also study whether these changes in neonatal morbidity, neonatal care practices and length of hospital stay are associated with sociodemographic, prenatal, and infant characteristics.MethodsTwo community-based cohorts from 1983 (POPS) and 2002−03 (LOLLIPOP) have provided the perinatal data for our study. The analysis enrolled 1,228 participants born VP (before the 32nd week of gestation) and surviving to 2 years of age without any severe congenital malformation. A rigorous harmonisation protocol ensured a precise comparison of the cohorts by using identical definitions of the perinatal characteristics.ResultsIn 2003, mothers were older when giving birth, had higher multiple birth rates, and significantly more parents had received higher education. In 2003, less VP infants had severe intraventricular haemorrhage and sepsis and relatively more received continuous positive airway pressure, mechanical ventilation and caffeine therapy than in 1983. Antenatal corticosteroids and surfactant therapy were provided only in 2003. The length of the stay in the neonatal intensive care unit and in hospital had decreased in 2003 by 22 and 11 days, respectively. Differences persisted after adjustment for sociodemographic, prenatal, and infant characteristics.ConclusionsNeonatal morbidities of the surviving VP infants in this study have not increased, and exhibit improvements for various characteristics in two cohorts born 20 years apart with comparable gestational age and birth weight. Our data suggest that the improvements found are associated with more advanced therapeutic approaches and new national protocols in place, and less so with sociodemographic changes. This analysis provides a basis for further comparative analyses of the health and the development of VP children, particularly with regard to long-term outcomes.

Hantavirus: an overview and advancements in therapeutic approaches for infection.

Hantaviruses are a significant and emerging global public health threat, impacting more than 200,000 individuals worldwide each year. The single-stranded RNA viruses belong to the Hantaviridae family and are responsible for causing two acute febrile diseases in humans: Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Currently, there are no licensed treatments or vaccines available globally for HTNV infection. Various candidate drugs have shown efficacy in increasing survival rates during the early stages of HTNV infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir and vandetanib. Immunotherapy utilizing neutralizing antibodies (NAbs) generated from Hantavirus convalescent patients show efficacy against HTNV. Monoclonal antibodies such as MIB22 and JL16 have demonstrated effectiveness in protecting against HTNV infection. The development of vaccines and antivirals, used independently and/or in combination, is critical for elucidating hantaviral infections and the impact on public health. RNA interference (RNAi) arised as an emerging antiviral therapy, is a highly specific degrades RNA, with post-transcriptional mechanism using eukaryotic cells platform. That has demonstrated efficacy against a wide range of viruses, both in vitro and in vivo. Recent antiviral methods involve using small interfering RNA (siRNA) and other, immune-based therapies to target specific gene segments (S, M, or L) of the Hantavirus. This therapeutic approach enhances viral RNA clearance through the RNA interference process in Vero E6 cells or human lung microvascular endothelial cells. However, the use of siRNAs faces challenges due to their low biological stability and limited in vivo targeting ability. Despite their successful inhibition of Hantavirus replication in host cells, their antiviral efficacy may be hindered. In the current review, we focus on advances in therapeutic strategies, as antiviral medications, immune-based therapies and vaccine candidates aimed at enhancing the body's ability to control the progression of Hantavirus infections, with the potential to reduce the risk of severe disease.