GLP overexpression is associated with poor prognosis in Chronic Lymphocytic Leukemia and its inhibition induces leukemic cell death.

Background Heterodimeric methyltransferases GLP (EHMT1/KMT1D) and G9a (EHMT2/KMT1C) are two closely related enzymes that promote the monomethylation and dimethylation of histone H3 lysine 9. Dysregulation of their activity has been implicated in several types of human cancer. Patients and methods Here, in order to investigate whether GLP/G9a exerts any impact on Chronic Lymphocytic Leukemia (CLL), GLP/G9a expression levels were assessed in a cohort of 50 patients and the effects of their inhibition were verified for the viability of CLL cells. Also, qRT-PCR was used to investigate the transcriptional levels of GLP/G9a in CLL patients. In addition, patient samples were classified according to ZAP-70 protein expression by flow cytometry and according to karyotype integrity by cytogenetics analysis. Finally, a selective small molecule inhibitor for GLP/G9a was used to ascertain whether these methyltransferases influenced the viability of MEC-1 CLL cell lineage. Results mRNA analysis revealed that CLL samples had higher levels of GLP, but not G9a, when compared to non-leukemic controls. Interestingly, patients with unfavorable cytogenetics showed higher expression levels of GLP compared to patients with favorable karyotypes. More importantly, GLP/G9a inhibition markedly induced cell death in CLL cells. Conclusion Taken together, these results indicate that GLP is associated with a worse prognosis in CLL, and that the inhibition of GLP/G9a influences CLL cell viability. Altogether, the present data demonstrate that these methyltransferases can be potential markers for disease progression, as well as a promising epigenetic target for CLL treatment and the prevention of disease evolution.

Immunomodulatory effect of IFN-γ licensed adipose-mesenchymal stromal cells in an in vitro model of inflammation generated by SARS-CoV-2 antigens.

In recent years, clinical studies have shown positive results of the application of Mesenchymal Stromal Cells (MSCs) in severe cases of COVID-19. However, the mechanisms of immunomodulation of IFN-γ licensed MSCs in SARS-CoV-2 infection are only partially understood. In this study, we first tested the effect of IFN-γ licensing in the MSC immunomodulatory profile. Then, we established an in vitro model of inflammation by exposing Calu-3 lung cells to SARS-CoV-2 nucleocapsid and spike (NS) antigens, and determined the toxicity of SARS-CoV-2 NS antigen and/or IFN-γ stimulation to Calu-3. The conditioned medium (iCM) generated by Calu-3 cells exposed to IFN-γ and SARS-CoV-2 NS antigens was used to stimulate T-cells, which were then co-cultured with IFN-γ-licensed MSCs. The exposure to IFN-γ and SARS-CoV-2 NS antigens compromised the viability of Calu-3 cells and induced the expression of the inflammatory mediators ICAM-1, CXCL-10, and IFN-ß by these cells. Importantly, despite initially stimulating T-cell activation, IFN-γ-licensed MSCs dramatically reduced IL-6 and IL-10 levels secreted by T-cells exposed to NS antigens and iCM. Moreover, IFN-γ-licensed MSCs were able to significantly inhibit T-cell apoptosis induced by SARS-CoV-2 NS antigens. Taken together, our data show that, in addition to reducing the level of critical cytokines in COVID-19, IFN-γ-licensed MSCs protect T-cells from SARS-CoV-2 antigen-induced apoptosis. Such observations suggest that MSCs may contribute to COVID-19 management by preventing the lymphopenia and immunodeficiency observed in critical cases of the disease.

IFN-γ-Licensed Mesenchymal Stem Cells Are More Susceptible to Death when Exposed to Quorum-Sensing Signal Molecule OdDHL and Less Effective in Inhibiting the Growth of Pseudomonas aeruginosa.

Currently, a series of licensing strategies has been investigated to enhance the functional properties of mesenchymal stem cells (MSCs). Licensing with IFN-γ is one of the most investigated strategies for enhancing the immunosuppressive potential of such cells. However, it is not yet known whether this licensing strategy could interfere with the ability of MSCs to control bacterial growth, which may be relevant considering their clinical potential. In this study, we compared the antimicrobial potential of IFN-γ-licensed and unlicensed MSCs by exposing them to Pseudomonas aeruginosa and its quorum-sensing inducer molecule OdDHL. Our data show that-when challenged with OdDHL-IFN-γ-licensed and unlicensed MSCs present increased levels of the antimicrobial HAMP transcript, but that only IFN-γ-licensed MSCs undergo modulation of CASP1 and BCL2, entering apoptosis. Furthermore, we demonstrate that only IFN-γ-licensed MSCs show modulation in genes involved in apoptosis and tend to undergo cell death when cultured with P. aeruginosa. As a consequence, IFN-γ-licensed MSCs showed lower capacity to control bacterial growth, compared to unlicensed MSCs. Taken together, our observations reveal an increased susceptibility to apoptosis of IFN-γ-licensed MSCs, which compromises their potential to control the bacterial growth in vitro. These findings are relevant to the field of cell therapy, considering the potential applicability of MSCs.

Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas.

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

Induced resistance to ifosfamide in osteosarcoma cells suggests a more aggressive tumor profile.

Aims: Osteosarcoma (OS) is the most common primary malignant bone sarcoma among children and adolescents. Treatment is based on neo-adjuvant and adjuvant chemotherapy, using the standard drugs cisplatin, methotrexate, doxorubicin, and ifosfamide (IFO). Due to the high capacity of tumor resistance, the current work aimed to analyze genes related to cycle control and cell differentiation in OS cells sensitive to and with induced resistance to IFO. This was to assess whether the differentiated expression of these genes may affect resistance to the drug IFO used in OS treatment, and thus establish possible biomarkers of disease progression. Materials and methods: In this work, the treatment-sensitive OS U2OS lineage was used, and the same lineage was submitted to the process of induction of IFO resistance. These cells were evaluated by MTT, migration and proliferation assays and submitted to gene expression analysis. Key findings: The results demonstrate that after induction of resistance to IFO, resistant U2OS cells show a more aggressive tumor behavior, with greater capacity for cell migration, proliferation, and invasion compared to sensitive cells. Gene analysis indicates that resistance-induced cells have differentiated expression of the genes EPB41L3, GADD45A, IER3, OXCT1, UBE2L6, UBE2A ALPL, and EFNB2. Our results suggest new perspectives on possible resistance biomarkers, especially the genes EFNB2 and EPB41L3, given that these genes have rarely been studied their expression linked to osteosarcoma. They show how the resistance induction model can be useful for studies on tumor cell behavior.

Hallmarks of Aging in Macrophages: Consequences to Skin Inflammaging.

The skin is our largest organ and the outermost protective barrier. Its aging reflects both intrinsic and extrinsic processes resulting from the constant insults it is exposed to. Aging in the skin is accompanied by specific epigenetic modifications, accumulation of senescent cells, reduced cellular proliferation/tissue renewal, altered extracellular matrix, and a proinflammatory environment favoring undesirable conditions, including disease onset. Macrophages (Mφ) are the most abundant immune cell type in the skin and comprise a group of heterogeneous and plastic cells that are key for skin homeostasis and host defense. However, they have also been implicated in orchestrating chronic inflammation during aging. Since Mφ are related to innate and adaptive immunity, it is possible that age-modified skin Mφ promote adaptive immunity exacerbation and exhaustion, favoring the emergence of proinflammatory pathologies, such as skin cancer. In this review, we will highlight recent findings pertaining to the effects of aging hallmarks over Mφ, supporting the recognition of such cell types as a driving force in skin inflammaging and age-related diseases. We will also present recent research targeting Mφ as potential therapeutic interventions in inflammatory skin disorders and cancer.

Host DNA repair response to oxidative damage is modulated by Trypanosoma cruzi in a strain-dependent manner.

The conservation of genomic integrity and stability is essential for cell survival. DNA Damage Responses (DDRs) are considered of paramount importance for all living beings and involve mechanisms of cell cycle regulation and damage-specific DNA repair pathways. Hydrogen peroxide (H2O2) is a compound that, in supraphysiological concentrations, damages biomolecules including the DNA, causing base modifications and strand breaks. There is evidence that Trypanosoma cruzi, the protozoan that causes Chagas disease, interferes in the host cell's DNA metabolism. In order to investigate the influence of T. cruzi infection over the host cell capacity to withstand and repair DNA damage, we analyzed L6 cells infected with Berenice, and Colombiana T. cruzi strains according to their viability, proliferation, morphology, DNA degradation, expression of DNA repair, and cell cycle genes following H2O2 treatment. It was noted that T. cruzi infection might act as either a stressor or a protective element of host DNA, depending on the strain and H2O2 concentration. Cells infected with Berenice strain and treated with 0.8 mM H2O2 presented a reduced DNA damage response intensity (e.g., BER and HR). Infection with T. cruzi Colombiana prevented the activation of DNA repair pathways in response to 0.8mM and 1.6mM H2O2 (NER and MMR). Nevertheless, since cellular viability was not significantly compromised in Colombiana-infected cells following the oxidative insult, it is possible that the parasite directly influenced the host DNA repair machinery. Our results support the notion that T. cruzi is able to modulate the host cell DNA metabolism in a strain-dependent manner, an event which can be explored in future drug development strategies.

Clinical and biochemical parameters of COVID-19 patients with prior or active dengue fever.

Originated in Wuhan, China, the coronavirus 19 disease (COVID-19) has quickly spread worldwide, reaching countries that already faced other endemics and epidemics. In Brazil, such a concerning situation includes arboviruses, among which the dengue virus stands out. Here, we determined the rate of SARS-CoV-2/dengue virus co-infection in a total of 178 patients with COVID-19 symtoms admitted into a large public hospital of the Federal District of Brazil. Furthermore, we evaluated whether prior or active dengue virus infection influenced hematological, biochemical, and clinical parameters of such patients. One hundred and twelve (63%) individuals tested positive for COVID-19, of which 43 (38.4%) were co-infected with dengue virus, and 50 (44.6%) had antibodies indicative of previous dengue infection. Co-infected patients showed lower numbers of circulating lymphocytes and monocytes, higher glucose rates, and a worse pulmonary condition. Of note, prior infections with dengue virus did not influence clinical parameters, but active dengue fever resulted in higher hospitalization rate. In conclusion, amid the current complex epidemiological scenario in Brazil, our data support the notion that SARS-CoV-2 and dengue co-infection affects an important percentage of COVID-19 patients and leads to worse clinical parameters, requiring greater attention from health authorities.

Epithelial basement membrane of human decellularized cornea as a suitable substrate for differentiation of embryonic stem cells into corneal epithelial-like cells.

The ability to decellularize and recellularize the corneas deemed unsuitable for transplantation may increase the number of available grafts. Decellularized corneas (DCs) may provide a natural microenvironment for cell adhesion and differentiation. Despite this, no study to date has evaluated their efficacy as a substrate for the induction of stem cell differentiation into corneal cells. The present study aimed to compare the efficiency of NaCl and NaCl plus nucleases methods to decellularize whole human corneas, and to investigate the effect of epithelial basement membrane (EBM) of whole DCs on the ability of human embryonic stem cells (hESCs) to differentiate into corneal epithelial-like cells when cultured in animal serum-free differentiation medium. As laminin is the major component of EBM, we also investigated its effect on hESCs differentiation. The decellularization efficiency and integrity of the extracellular matrix (ECM) obtained were investigated by histology, electron microscopy, DNA quantification, immunofluorescence, and nuclear staining. The ability of hESCs to differentiate into corneal epithelial-like cells when seeded on the EBM of DCs or laminin-coated wells was evaluated by immunofluorescence and RT-qPCR analyses. NaCl treatment alone, without nucleases, was insufficient to remove cellular components, while NaCl plus nucleases treatment resulted in efficient decellularization and preservation of the ECM. Unlike cells induced to differentiate on laminin, hESCs differentiated on DCs expressed high levels of corneal epithelial-specific markers, keratin 3 and keratin 12. It was demonstrated for the first time that the decellularized matrices had a positive effect on the differentiation of hESCs towards corneal epithelial-like cells. Such a strategy supports the potential applications of human DCs and hESCs in corneal epithelium tissue engineering.

GVHD-derived plasma as a priming strategy of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. METHODS: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1ß, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. RESULTS: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. CONCLUSIONS: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases.

Unraveling KDM4 histone demethylase expression and its association with adverse cytogenetic findings in chronic lymphocytic leukemia.

The acquisition of complex karyotypes is related to the progression of chronic lymphocytic leukemia (CLL) and patients with this condition have a poor prognosis. Despite recent advances in the classification of prognosis in CLL patients, understanding of the molecular mechanisms that lead to genomic instability and progression of this disease remains inadequate. Interestingly, dysregulated expression of KDM4 members is involved in the progression of several cancer types and plays a role in the DNA damage response; however, the gene expression profile and the importance of KDM4 members in CLL are still unknown. Here, we assessed the gene expression profile of KDM4A, KDM4B, and KDM4C in 59 CLL samples and investigated whether these histone demethylases have any influence on the prognostic markers of this leukemia. KDM4A gene expression was higher in CLL patients as compared with control samples. In contrast, CLL samples showed decreased levels of the KDM4B transcript in relation to control cases, and no difference was detected in KDM4C expression. Furthermore, patients with positive expression of ZAP-70 had lower expression of KDM4B and KDM4C as compared with ZAP-70-negative patients. More importantly, patients with low expression of these histone demethylases had higher leukemic cell numbers and displayed adverse cytogenetic findings and the acquisition of a complex karyotype. The present data clearly show that the expression of KDM4 members is dysregulated in CLL and impact the prognosis of this leukemia. These findings are useful for a better understanding of the impact of epigenetics on CLL progression.

Stem cells in cardiovascular diseases: turning bad days into good ones.

During the past decade, several types of stem cells have been investigated as promising therapeutic agents for cardiovascular diseases (CVDs). Among them, mesenchymal stem cells (MSCs) were the most investigated stem cell population. Hundreds of clinical trials later, results remain disappointing and far from the revolutionary improvements expected for heart function. In the present review, we address strategies under investigation to boost MSC therapy for CVDs. Pluripotent stem cells (PSCs) are also intended to reach clinical applications for CVDs, but here we suggest that, in the short term, the major impact of PSCs in the cardiovascular field might be at the bench and not the bedside.

Production of Human Endothelial Cells Free from Soluble Xenogeneic Antigens for Bioartificial Small Diameter Vascular Graft Endothelization.

Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease, but most lack adequate saphenous vein or other conduits for bypass procedures and would benefit from a bioartificial conduit. This study aimed to produce human endothelial cells (hECs) in large scale, free from xenogeneic antigens, to develop a small diameter, compatible vessel for potential use as a vascular graft. Human adipose-derived stromal cells (hASCs) were isolated, cultured, and differentiated in the presence of human serum and used for the reendothelization of a decellularized rat aorta. hASC derived ECs (hASC-ECs) expressed VEGFR2, vWf and CD31 endothelial cell markers, the latter in higher levels than hASCs and HUVECs, and were shown to be functional. Decellularization protocol yielded aortas devoid of cell nuclei, with preserved structure, including a preserved basement membrane. When seeded with hASC-ECs, the decellularized aorta was completely reendothelized, and the hASC-ECs maintained their phenotype in this new condition. hASCs can be differentiated into functional hECs without the use of animal supplements and are capable of reendothelizing a decellularized rat aorta while maintaining their phenotype. The preservation of the basement membrane following decellularization supported the complete reendothelization of the scaffold with no cell migration towards other layers. This approach is potentially useful for rapid obtention of compatible, xenogeneic-free conduit.