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Tracheal fistula (TF) treatments may involve temporary orthosis and further ablative procedures, which can lead to infection. Thus, TF requires other therapy alternatives development. The hypothesis of this work was to demonstrate the feasibility of a tissue-engineered alternative for small TF in a preclinical model. Also, its association with suture filaments enriched with adipose tissue-derived mesenchymal stromal stem cells (AT-MSCs) was assessed to determine whether it could optimize the regenerative process.Poly (L-Lactic acid) (PLLA) membranes were manufactured by electrospinning and had morphology analyzed by scanning electron microscopy. AT-MSCs were cultured in these scaffolds and in vitro assays were performed (cytotoxicity, cellular adhesion, and viability). Subsequently, these cellular constructs were implanted in an animal small TF model. The association with suture filaments containing attached AT-MSCs was present in one animal group. After 30 d, animals were sacrificed and regenerative potential was evaluated, mainly related to the extracellular matrix remodeling, by performing histopathological (Hematoxylin-Eosin and trichrome Masson) and immunohistochemistry (Collagen I/II/III, matrix metalloproteinases-2, matrix metalloproteinases-9, vascular endothelial growth factor, and interleukin-10) analyses.PLLA membranes presented porous fibers, randomly oriented. In vitro assays results showed that AT-MSCs attached were viable and maintained an active metabolism. Swine implanted with AT-MSCs attached to membranes and suture filaments showed aligned collagen fibers and a better regenerative progress in 30 d.PLLA membranes with AT-MSCs attached were useful to the extracellular matrix restoration and have a high potential for small TF treatment. Also, their association with suture filaments enriched with AT-MSCs was advantageous.

Dendritic cell (DC) vaccines have demonstrated good efficacy in preventing relapse and in increasing survival of patients affected by a variety of both solid and hematological tumors. Most protocols used to generate these cells involve the automated separation of peripheral blood monocytes from patients. This approach requires specialized equipment, which elevates the cost of this type of therapy, potentially limiting the widespread access to patients.In this study, we compare the yield and quality of dendritic cells generated from monocytes and isolated by an automated method or by manual methods using gradient centrifugation.The results demonstrate the equivalence of the 3 methods in relation to the yield and final quality of the product, however with considerable differences between the costs of these procedures. In addition, this study also demonstrates the feasibility of the antigenic pulse with autologous tumor cell lysates, constituting a source of antigens, not only easily obtained and manipulated, but also specific to the patient's tumor.These findings may have important implications for emerging centers interested in using this medical approach and potentially increase the access of a greater number of patients to this therapeutic option.

Even after two years of the pandemic, a completely effective treatment against SARS-CoV-2 has not yet been established. Considering this fact and the emergence of successive new viral variants, the development of therapies based on natural polyclonal antibodies recovered from convalescent plasma remains relevant. This study presents a comparison between different methods of screening antibodies in samples of 41 individuals previously diagnosed with COVID-19. We found a significant correlation between Abbot Architect anti-SARS-CoV-2 IgG and Abbott Allinity SARS-CoV-2 IgG II Quantitative assay intensity of reactivity and neutralizing antibody (nAb) titers. Thus, we propose an initial antibody screening with IgG anti-N Abbott Architect test, with an index of, for example, 3.25 or SARS-CoV-2 IgG II Quantitative Abbott Allinity assay 137.65 AU/mL as good predictors of Nab ≥ 1:80. For the quantitative method, this threshold demonstrated a 100 % sensitivity and 80 % specificity, with 97.3 % accuracy. An interesting observation was the increase in the neutralizing activity of the anti-SARS-CoV-2 antibodies with the longest interval between the end of the symptoms and the collection, demonstrating that the delay in plasma collection does not affect the achievement of adequate nAbs levels. These results demonstrate the possibility of using faster and more widely available commercial serological tests with a good correlation with viral neutralization tests in culture, allowing for optimized large-scale donor selection, which will be of utmost importance for the development of therapies such as hyperimmune immunoglobulin.

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.

APAF-1 is a central component of the intrinsic pathway of apoptosis, where APAF-1 dysregulation results in the development of diverse human neoplasms. The aim of this study was to characterize the mRNA expression levels of APAF-1 transcripts in low-risk and high-risk MDS and to elucidate whether the expression levels of APAF-1 transcripts are modulated with increased apoptosis in CD34(+) MDS cells undergoing erythroid differentiation.APAF-1 (NM_181861) expression was verified, by quantitative RT-PCR, in bone marrow aspirates from 33 patients with myelodysplastic syndromes (MDS), at the time of diagnosis, and in erythroid differentiation cultures from CD34(+) from normal donors and patients with MDS.APAF-1 expression was significantly higher in low-risk, compared to high-risk MDS, according to IPSS (P0.0001), FAB (P = 0.0265), and cytogenetic risk (P = 0.0134). Low-risk MDS-derived differentiated erythroid cells demonstrated an increased expression of APAF-1, compared with normal cells, accompanied by an augmented rate of apoptosis.Increased expression of APAF-1 in low-risk disease and its positive correlation with the apoptotic rate observed during the erythroblast differentiation of low-risk MDS cells may indicate that the modulation of APAF-1, at the transcriptional level, participates in the pathophysiology of MDS.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis. In addition to HSC defects, an important role is also played by the hematopoietic microenvironment niche that has as key component the mesenchymal stromal cells (MSC). The MSC of MDS patients have morphological and functional abnormalities. Recently, our group identified new possible target genes involved in MDS pathophysiology through microarray analysis of MSC from MDS patients. An interesting underexpressed gene found was SPINT2, a gene that encodes a transmembrane protein which inhibits the hepatocyte growth factor activator (HGFA), the enzyme responsible for the conversion of hepatocyte growth factor (HGF) into its active form. SPINT2 is downregulated in some types of solid cancer and correlated with their prognostic and progression; however, the functional role of SPINT2 in MDS remains unknown. We herein investigated the role of SPINT2 in MSC, studying HGF and SDF1 secretion and cell adhesion with normal CD34+, P39 and U937 cells by silencing SPINT2 gene in HS5 and HS-27a stromal cell lines. We also investigated, using qPCR, SPINT2 and HGF mRNA expression in MSC and total bone marrow (BM) cells from 56 untreated MDS patients (WHO 35 low-risk, 21 high-risk) and 28 healthy donors. To inhibit SPINT2, specific shRNA expressing lentiviral vectors targeting SPINT2 gene or no specific sequence were used. The HGF and SDF1 secretion in cell supernatant from the cells silenced or not for SPINT2 was measured by BioPlex after 6, 12, 24 and 48h. The cell-cell adhesion of CD34+, P39 or U937 cells onto transduced stromal cells and the adhesion molecule profile were analyzed by flow cytometry. We observed a significant decrease in SPINT2 mRNA expression of MDS MSC (P=0.006) and MDS BM cells (P=0.03) compared to normal cells. Further, HGF mRNA expression of MDS MSC was significantly increased (P=0.01) compared to normal cells. Spearman analysis showed a negative significant correlation between SPINT2 and HGF expressions (P=0.01;r2=0.60). In both stromal cell lines, SPINT2 inhibition resulted in a significant increase in HGF secretion after 24 and 48h and a significant increased secretion of SDF1 after 48h. Moreover, SPINT2 silencing induced a significant increased adherence of CD34+, P39 and U937 cells onto stromal cells probably due to the alteration in integrin expression, since an increase in CD49b and CD49d and decrease in CD49e expressions were also observed in cells silenced for SPINT2. Considering that SPINT2 limits signaling via HGF pathway by inhibiting HGF activation through HGFA, the SPINT2 underexpression in MDS would allow the conversion of the inactive HGF monomer to an active heterodimer. Interestingly, a significant decrease of SPINT2 and increase of HGF expressions were observed in MSC of MDS patients, compared to normal cells. Moreover, recent studies have demonstrated that HGF serum levels are significantly increased in MDS patients and dependent on MDS severity. In addition, SPINT2 inhibition in stromal cells significantly increased HGF secretion by these cells. The increased HGF secretion can result in an autocrine regulation which induces the production and secretion of SDF1 by stromal cells themselves. Cytokines secretions provided by MSC are required for adhesion, survival and proliferation of HSC cells. In this way, in MDS MSC, SPINT2 underexpression and, consequently, increased HGF and SDF1 secretion, may lead to an increased adhesion between MSC and normal or malignant HSC cells. Corroborating our hypotheses, we found overexpression of integrins CD49b and CD49d, which mediate cell-MSC interaction, and a decrease in CD49e expression, an integrin that promotes interaction with extracellular matrix. The interaction onto MSC contributes to the maintenance of the stem and malignant cell properties, such as self-renewal, survival and proliferation. Cytokine secretion and cell adhesion onto MSC is important for MDS physiology. Hence, we demonstrate for the first time that, in MDS, SPINT2 plays a role in the HGF and SDF1 secretion by MSC, resulting in an alteration in cell-cell adhesion and molecule adhesion profile. In view of these data, the SPINT2 expression alteration in MDS MSC may constitute a particular mechanism of MDS pathophysiology and maintenance of self-renewal, homing and proliferation of HSC and malignant clones in MDS.Support: FAPESP, CNPq Disclosures: No relevant conflicts of interest to declare.