Mini review: Advances and challenges in CAR-T cell therapy: from early chimeric antigen receptors to future frontiers in oncology

Cell therapy utilizing chimeric antigen receptors (CARs) in conjunction with immune cells, primarily T lymphocytes, is known as CAR-T cell therapy. This innovative approach is revolutionizing the landscape of oncohaematology by precisely targeting specific antigens for elimination. However, despite its promising prospects, CAR-T therapy presents several challenges, including a notable rate of disease relapse, intricate pathologies impeding widespread adoption, prolonged manufacturing timelines, and substantial costs. Looking forward, ongoing research and progress aim to address these challenges to mitigate these constraints, underlining the continuous efforts to enhance the efficacy and accessibility of this transformative therap

Engineering and Assessing Cardiac Tissue Complexity

Cardiac tissue engineering is very much in a current focus of regenerative medicine research as it represents a promising strategy for cardiac disease modelling, cardiotoxicity testing and cardiovascular repair. Advances in this field over the last two decades have enabled the generation of human engineered cardiac tissue constructs with progressively increased functional capabilities. However, reproducing tissue-like properties is still a pending issue, as constructs generated to date remain immature relative to native adult heart. Moreover, there is a high degree of heterogeneity in the methodologies used to assess the functionality and cardiac maturation state of engineered cardiac tissue constructs, which further complicates the comparison of constructs generated in different ways. Here, we present an overview of the general approaches developed to generate functional cardiac tissues, discussing the different cell sources, biomaterials, and types of engineering strategies utilized to date. Moreover, we discuss the main functional assays used to evaluate the cardiac maturation state of the constructs, both at the cellular and the tissue levels. We trust that researchers interested in developing engineered cardiac tissue constructs will find the information reviewed here useful. Furthermore, we believe that providing a unified framework for comparison will further the development of human engineered cardiac tissue constructs displaying the specific properties best suited for each particular application

Amniotic Membrane as a Scaffold for Melanocyte Transplantation in Patients with Stable Vitiligo

Vitiligo is an acquired skin disease that significantly impacts the quality of life of patients. Medical treatment of vitiligo includes the use of melanocyte transplant, but the results are variable. We have treated 4 patients with either focal or generalized stable vitiligo using a graft of autologous melanocytes' culture on a denuded amniotic membrane (AM). A culture biopsy was obtained in every patient and grown in melanocytes' media for 10–14 days after which cells were transferred to a denuded AM and transplanted into the achromic lesions. Patients were followed for up to 6 months using clinical assessment of achromic lesions. Treated areas ranged between 4 cm2 and 210.6 cm2. Response to treatment was excellent in all patients with 90–95% repigmentation success rate. Our results demonstrate that transplantation of autologous melanocytes cultured on AM is a new, simple, and effective treatment for stable vitiligo

Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels

Background: Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A) WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/ CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown. Methods: To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies. Results: Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling. Conclusions: We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML

Histological and ultrastructural comparison of cauterization and thrombosis stroke models in immune-deficient mice

<p>Abstract</p> <p>Background</p> <p>Stroke models are essential tools in experimental stroke. Although several models of stroke have been developed in a variety of animals, with the development of transgenic mice there is the need to develop a reliable and reproducible stroke model in mice, which mimics as close as possible human stroke.</p> <p>Methods</p> <p>BALB/Ca-RAG2^-/-γc^-/-mice were subjected to cauterization or thrombosis stroke model and sacrificed at different time points (48hr, 1wk, 2wk and 4wk) after stroke. Mice received BrdU to estimate activation of cell proliferation in the SVZ. Brains were processed for immunohistochemical and EM.</p> <p>Results</p> <p>In both stroke models, after inflammation the same glial scar formation process and damage evolution takes place. After stroke, necrotic tissue is progressively removed, and healthy tissue is preserved from injury through the glial scar formation. Cauterization stroke model produced unspecific damage, was less efficient and the infarct was less homogeneous compared to thrombosis infarct. Finally, thrombosis stroke model produces activation of SVZ proliferation.</p> <p>Conclusions</p> <p>Our results provide an exhaustive analysis of the histopathological changes (inflammation, necrosis, tissue remodeling, scarring...) that occur after stroke in the ischemic boundary zone, which are of key importance for the final stroke outcome. This analysis would allow evaluating how different therapies would affect wound and regeneration. Moreover, this stroke model in RAG 2^-/-γC ^-/-allows cell transplant from different species, even human, to be analyzed.</p

Myocardial infarction ´through the window´: dual dynamics for cardiac fibroblasts activation

Activated cardiac fibroblasts (CFs) are responsible for the healing of the heart tissue after a myocardial infarction (MI). Based on high throughput technologies, several groups have recently demonstrated their heterogeneity and a unique role of each subpopulation of CFs during the ventricular remodelling process. This is relevant towards the discovery of personalized treatments to control the initial post-MI healing scar that will contribute to preserve ventricular function and prevent the onset of heart failure. However, little is known about the moment that CFs are activated, and which genes are potentially involved in this process. Using a mouse model for MI and single cell RNA-Seq, we demonstrate that the activation of Reparative Cardiac Fibroblasts (RCFs), the CFs responsible for the healing scar, happens within the first week after MI. Interestingly, our data reveals that all CFs show high expression of the top markers genes for RCF in a specific moment, but only few of them finally evolve to an RCF transcriptomic identity. Furthermore, we describe two different molecular dynamics that could give rise to this activation and, in consequence, the appearance of definitive RCFs. Using Spatial Transcriptomics, we localized the genes related to each dynamic in different anatomical regions of the infarcted heart, but, remarkably, only one persists seven days after MI. These results highlight the existence of a specific “window of activation” of RCFs at the beginning of the ventricular remodelling process. This potential ´therapeutical window´ could allow us to regulate the size of the healing scar and, in consequence, the poor prognosis for patients that have suffered an ischemic event.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Spanish Cell Therapy Network (TerCel): 15 years of successful collaborative translational research

In the current article we summarize the 15-year experience of the Spanish Cell Therapy Network (TerCel), a successful collaborative public initiative funded by the Spanish government for the support of nationwide translational research in this important area. Thirty-two research groups organized in three programs devoted to cardiovascular, neurodegenerative and immune-inflammatory diseases, respectively, currently form the network. Each program has three working packages focused on basic science, pre-clinical studies and clinical application. TerCel has contributed during this period to boost the translational research in cell therapy in Spain, setting up a network of Good Manufacturing Practice–certified cell manufacturing facilities– and increasing the number of translational research projects, publications, patents and clinical trials of the participating groups, especially those in collaboration. TerCel pays particular attention to the public-private collaboration, which, for instance, has led to the development of the first allogeneic cell therapy product approved by the European Medicines Agency, Darvadstrocel. The current collaborative work is focused on the development of multicenter phase 2 and 3 trials that could translate these therapies to clinical practice for the benefit of patients

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis

In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1α-expressing cells to obtain osteoblasts lacking Ccr2 (CCR2-Col1αKO). We stimulated PTOA changes in CCR2-Col1αKO and CCR2+/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast-Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2+/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1α-Ccr2 deletion led to less evident improvements. Osteoblast-Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues