Swallowing disorders and associated factors in older adults living in nursing homes.

PURPOSE: The aim of this study was to identify the frequency of swallowing disorders and the associated factors in older adults living in nursing homes. METHODS: This is a cross-sectional study with a sample of 73 older adults (≥ 60 years; mean age = 80 ± 7.49 years; female = 82.2%) living in five non-profit Brazilian nursing homes. Demographic and clinical data were collected. The frequency of swallowing disorders was determined by the Volume-Viscosity Swallow Test (VVS-T). Covariables with a p-value less than 0.20 according to Pearson's chi-square test or Fisher's exact test were included in the multiple logistic regression analysis model. The level of significance was 5%. RESULTS: The frequency of swallowing disorders was 63%. The multiple logistic regression model revealed that the chance of an older adult living in a nursing home presenting with swallowing disorders increased by 8% with each 1-year increase in age. Decreases in oral intake level improved the chance of a negative result in the VVS-T by approximately four times, and an individual with a FOIS level below seven was almost 11 times more likely to have a swallowing disorder. CONCLUSION: The frequency of swallowing disorders in older adults living in nursing homes is high and is associated with age and oral intake. The management care team should be aware of the early detection of these conditions to prevent complications of oropharyngeal dysphagia.

Characterizing temporal and spatial recruitment of systemically administered RPE65-programmed bone marrow-derived cells to the retina in a mouse model of age-related macular degeneration.

PURPOSE: Previously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer. METHODS: C57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP+ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP+ RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP+ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections. RESULTS: GFP+ RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP+ BMDC into the RPE layer and that these GFP+ human RPE65 expressing cells co-localized with murine RPE65. No GFP+ cells were observed in the neural retina or RPE layer of normal uninjured control eyes. CONCLUSIONS: Our study shows that systemically administered GFP+ RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

Combined dermatan sulfate and endothelial progenitor cell treatment: action on the initial inflammatory response after arterial injury in C57BL/6 mice.

BACKGROUND AIMS: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. METHODS: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. RESULTS: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. DISCUSSION: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.

Differentiation of C57/BL6 mice bone marrow mononuclear cells into early endothelial progenitors cells in different culture conditions.

Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.

Circulating progenitor and mature endothelial cells in deep vein thrombosis.

INTRODUCTION: Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein thrombosis (DVT) has not been previously evaluated. PATIENTS AND METHODS: In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry. RESULTS: In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event. CONCLUSIONS: Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.

Vocal Changes of Men and Women from Different Age Decades: An Analysis from 30 Years of Age.

OBJECTIVE: To verify the modifications in auditory-perceptual and acoustic parameters of the fundamental frequency, short-term perturbation, noise, and cepstral measures of voice in young and elderly were distributed over the age of decades. METHODS: The study was conducted on 265 subjects (140 females and 125 males), aged 30 to 79 years, without a history of vocal complaints or dysphonia, distributed in five age groups for females and males (30-39 years until 70-79 years). Three speech therapists voice specialists performed the perceptual-auditory analysis using a 100-mm visual analog scale, based on the overall dysphonia grade (overall grade), roughness, and breathiness from the sustained vowel /a/. The smoothed cepstral peak prominence (CPPS), average fundamental frequency (F0), standard deviation of the fundamental frequency (std-F0), jitter percentage (%), shimmer percentage (%), noise-to-harmonic ratio (NHR), voice turbulence index (VTI), and soft phonation index (SPI) were extracted for the acoustic analysis RESULTS: In the case of the auditory-perceptual characteristics, the comparison between aged groups showed that, in men, the vocal quality did not show significant differences in the overall grade, roughness, and breathiness. However, women in the 3rd decade of life presented higher breathiness than those in the 5th, 6th, and 7th decades. Men had a higher overall grade and roughness than women. In the case of the acoustic data, the cepstral measure showed that in the 3rd decade, men presented higher CPPS than females. The noise measures, in the comparison by age groups, only VTI and SPI were different for women: VTI was higher in the 7th decade than in the 4th, whereas SPI was higher in the 4th decade than in older women. In the male population, the SPI in the 4th decade was also higher than that in the older population CONCLUSION: The analysis by age groups of life allowed the identification of breathiness as a sensitive parameter in the different stages of adult life in women. The CPPS analysis showed that the 7th age decade is the period of the appearance of vocal changes for females, characterized by higher CPPS. The results related to traditional short-term perturbation and noise measurements showed that changes in F0, jitter, shimmer, and NHR were not found. Among the other noise measurements, VTI and SPI demonstrated some changes in different decades of life.

Vocal Fatigue, Dysphonia Risk, and Associated Factors in Male Brazilian Evangelical Pastors.

OBJECTIVES: To identify the presence of vocal fatigue and dysphonia risk in evangelical pastors and to verify associations of vocal fatigue with dysphonia risk and other factors. STUDY DESIGN: Cross-sectional observational study. METHODS: Ninety-two evangelical pastors, professionally active, answered an on-line questionnaire divided in three steps. The first step consisted of data for the characterization of the sample. In the second stage, participants answered the vocal fatigue index questionnaire (VFI). Finally, they answered to the Brazilian Dysphonia Screening tool. A descriptive analysis of the data was performed along with Spearman's Correlation test between VFI factors and the three possible decision rules for the Brazilian Dysphonia Screening tool. A linear regression model was designed to verify predictive variables associated with the outcome "vocal fatigue". RESULTS: Ninety male individuals were included. The average sample was off the threshold scores of tiredness and voice impairment, physical discomfort, improvement of voice symptoms with rest, and the total score of VFI. The dysphonia risk was low and was weakly negatively correlated with the improvement of voice symptoms with rest. The linear regression indicated that the presence of a current vocal complaint increases about four points in the VFI. CONCLUSION: Evangelical pastors present symptomatology of vocal fatigue and low risk for dysphonia. However, the greater the improvement of voice symptoms with rest, the lower the risk for dysphonia. Furthermore, the presence of a current vocal complaint is associated with increased symptoms of vocal fatigue.

Hilab System Device in an Oncological Hospital: A New Clinical Approach for Point of Care CBC Test, Supported by the Internet of Things and Machine Learning.

The complete blood count (CBC) is a highly requested test that is generally restricted to centralized laboratories, which are limited by high cost, being maintenance-demanding, and requiring costly equipment. The Hilab System (HS) is a small, handheld hematological platform that uses microscopy and chromatography techniques, combined with machine learning (ML) and artificial intelligence (AI), to perform a CBC test. This platform uses ML and AI techniques to add higher accuracy and reliability to the results besides allowing for faster reporting. For clinical and flagging capability evaluation of the handheld device, the study analyzed 550 blood samples of patients from a reference institution for oncological diseases. The clinical analysis encompassed the data comparison between the Hilab System and a conventional hematological analyzer (Sysmex XE-2100) for all CBC analytes. The flagging capability study compared the microscopic findings from the Hilab System and the standard blood smear evaluation method. The study also assessed the sample collection source (venous or capillary) influences. The Pearson correlation, Student t-test, Bland-Altman, and Passing-Bablok plot of analytes were calculated and are shown. Data from both methodologies were similar (p > 0.05; r ≥ 0.9 for most parameters) for all CBC analytes and flagging parameters. Venous and capillary samples did not differ statistically (p > 0.05). The study indicates that the Hilab System provides humanized blood collection associated with fast and accurate data, essential features for patient wellbeing and quick physician decision making.

The first gene therapy for RPE65 biallelic dystrophy with voretigene neparvovec-rzyl in Brazil.

PURPOSE: To report the first Brazilian patient with RPE65 deficiency-inherited retinal dystrophy (RPE65-IRD) treated with voretigene neparvovec-rzyl (VN). METHODS: An adult patient with Leber congenital amaurosis-2 with a homozygous mutation in the RPE65 gene (p.Phe83Leu) was treated bilaterally with VN. The clinical and surgical aspects are described. The baseline and 4-month postoperative ophthalmologic examinations included measurement of the best-corrected visual acuity (BCVA), full-field stimulus threshold (FST) test, Octopus 900 semiautomated kinetic visual fields (VFs), and microperimetry. RESULTS: No complications developed in this patient. The BCVA remained stable. The full-field stimulus threshold test (FST) and VFs showed clinically significant improvements bilaterally. The patient reported significant improvements in the ability to perform daily activities, mainly for those requiring the VFs and vision in a low-luminescence environment. CONCLUSIONS: The treatments were beneficial for this patient who was homozygous for RPE65 p.Phe83Leu. The first VN treatments in an adult Brazilian patient in clinical practice showed measurable improvements in visual outcomes that were meaningful for the patient's daily activities. TRANSLATIONAL RELEVANCE: This case reinforces the clinical trial results and proves that the procedure is feasible in countries such as Brazil.

Matrix-assisted autologous chondrocyte transplantation for treatment of focal chondral lesions in the knee: the Hospital Israelita Albert Einstein experience.

OBJECTIVE: Phase 1 clinical trial to determine feasibility, safety, and efficacy of a new advanced cell therapy product for treatment of knee articular cartilage injuries. METHODS: Three participants with knee focal chondral lesions were included, with no signs of osteoarthritis. Chondrocytes were obtained through knee arthroscopy, cultured in collagen membrane for 3 weeks at the laboratory, subjected to tests to release the cell therapy product, and implanted. All patients underwent a specific 3-month rehabilitation protocol, followed by assessments using functional and imaging scales. The main outcome was the incidence of severe adverse events. RESULTS: Three participants were included and completed the 2-year follow-up. There was one severe adverse event, venous thrombosis of distal leg veins, which was no associated with therapy, was treated and left no sequelae. The clinical and radiological scales showed improvement in the three cases. CONCLUSION: The preliminary results, obtained with the described methodology, allow concluding that this product of advanced cell therapy is safe and feasible. ReBEC platform registration number: RBR-6fgy76.

Report of a clinical and laboratory management of cell therapy for knee cartilage in the face of mycoplasma contamination.

To describe a case of autologous chondrocyte implantation after cell culture contamination by Mycoplasma pneumoniae and the measures taken to successfully complete cell therapy in a patient with focal chondral lesion. A 45-year-old male patient, complaining of chronic pain on the knee and no history of trauma. He had a chondral lesion in the trochlear region of the femur and clinical tests compatible with pain in the anterior compartment of the knee. Conservative treatment failed to alleviate symptoms. Surgical treatment was indicated, but due to the size of the lesion, membrane-assisted autologous chondrocyte implantation was the technique of choice. Cartilage biopsies were collected from the intercondylar region of the distal femur. After isolation, chondrocytes were expanded ex vivo in a trained laboratory, for three weeks, and seeded onto a commercially available collagen membrane prior to implantation in the knee. Two days before surgery, a cell culture sample tested positive for Mycoplasma pneumoniae. The source of contamination was found to be autologous blood serum, extracted from the patient´s peripheral vein, and used to supplement the cell culture medium. After treating the patient with antibiotics, all procedures were repeated and the new final cell product, free from contaminants, was successfully implanted. We discuss the strategies available to deal with this situation, and describe the results of this particular case, which led to modifications in the autologous chondrocyte implant protocol.

Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use.

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by ß-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient's MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients' cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient's cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient's cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.

Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5.

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

Dermatan sulfate and bone marrow mononuclear cells used as a new therapeutic strategy after arterial injury in mice.

BACKGROUND AIMS: Previously, we have demonstrated that administration of dermatan sulfate (DS) suppresses neointima formation in the mouse carotid artery by activating heparin co-factor II. A similar suppressive effect was observed by increasing the number of progenitor cells in circulation. In this study, we investigated the combination of DS and bone marrow mononuclear cells (MNC), which includes potential endothelial progenitors, in neointima formation after arterial injury. METHODS: Arterial injury was induced by mechanical dilation of the left common carotid artery. We analyzed the extension of endothelial lesion, thrombus formation, P-selectin expression and CD45(+) cell accumulation 1 and 3 days post-injury, and neointima formation 21 days post-injury. Animals were injected with MNC with or without DS during the first 48 h after injury. RESULTS: The extension of endothelial lesion was similar in all groups 1 day after surgery; however, in injured animals treated with MNC and DS the endothelium recovery seemed to be more efficient 21 days after lesion. Treatment with DS inhibited thrombosis, decreased CD45(+) cell accumulation and P-selectin expression at the site of injury, and reduced the neointimal area by 56%. Treatment with MNC reduced the neointimal area by 54%. The combination of DS and MNC reduced neointima formation by more than 91%. In addition, DS promoted a greater accumulation of MNC at the site of injury. CONCLUSIONS: DS inhibits the initial thrombotic and inflammatory processes after arterial injury and promotes migration of MNC to the site of the lesion, where they may assist in the recovery of the injured endothelium.

Tongue measurements and pharyngeal residue in amyotrophic lateral sclerosis.

Aim: We aimed to analyze the relationship between tongue measurements and vallecular residue in patients with amyotrophic lateral sclerosis (ALS). Materials & methods: Twenty-one patients with ALS were assessed for posterior maximum tongue isometric pressure (PMTIP) and posterior tongue isometric endurance (PTIE) by the Iowa Oral Performance Instrument; vallecular residue after 10 ml of moderately thickened consistency by Fiberoptic Endoscopic Evaluation of Swallowing; and tongue thickness (TT) by ultrasonography. Results: PMTIP, PTIE and TT were decreased compared with the reference values for healthy individuals and were not different between patients with and without vallecular residue. Conclusion: In ALS, PMTIP, PTIE and TT are not good predictors of vallecular residue in the tested volume and food consistency.

Lay abstract This study aimed to analyze the relationship of measurable characteristics of the tongue in patients with amyotrophic lateral sclerosis (ALS) and the accumulation of residue after swallowing in an area located just after the tongue. This accumulation of residue provides a sensation of bolus stuck in the throat and increases the risk of weight loss, choking and pulmonary complications. Twenty-one ALS patients were assessed for tongue pressure, tongue endurance, tongue thickness and residue after swallowing of the 10 ml of moderately thickened consistency. This study concluded that all tongue measurements were low in ALS patients with and without residue. Therefore, these tongue measurements are not good predictors of vallecular residue in the tested volume and food consistency.

Effectiveness of Vocal Therapy for the Elderly When Applying Conventional and Intensive Approaches: A Randomized Clinical Trial.

OBJECTIVES: The aim of this study was to verify the effects of the method Vocal Therapy for the Elderly and the differences in treatment efficacy when it was administered intensively or in the conventional way. METHODS: Twenty-seven elderly individuals were randomized into two groups and referred for 16 sessions of vocal therapy. The Intensive Group (IG) had therapy four times a week, whereas the Conventional Group had it twice a week. The effects of the therapy were assessed by auditory-perceptual analysis, the Voice-Related Quality of Life protocol, and visual-perceptive analysis of laryngoscopy examinations. The first stage consisted of evaluating the vocal quality and self-assessment of 15 subjects before and after a time period equal to that which they would undergo in vocal therapy. The second stage consisted of comparing the assessments of all participants in the week preceding the beginning of treatment, in the week following the end of treatment, and 1 month after that. RESULTS: There was no difference between perceptual voice parameters and self-assessment when the subjects were not undergoing therapy. When comparing the periods immediately before and after therapy, there was improvement in vocal quality and Voice-Related Quality of Life. One month later, the benefits that had been revealed through the self-assessment protocol, and some of the improvements in vocal parameters were maintained. There was no difference between the IG and Conventional Group with the exception of vocal fold bowing, which decreased in the IG group. CONCLUSIONS: The Vocal Therapy for the Elderly program is effective for treating voice presbyphonia. An intensive approach may be superior with regard to vocal fold bowing.

Clinical Translation of Mesenchymal Stromal Cell Therapy for Graft Versus Host Disease.

Graft versus host disease (GVHD) is a common condition in patients subjected to allogeneic hematopoietic stem cell transplantation (HSCT). The immune cells derived from the grafted stem cells attack recipient's tissues, including those from the skin, liver, eyes, mouth, lungs, gastrointestinal tract, neuromuscular system, and genitourinary tract, may lead to severe morbidity and mortality. Acute GVHD can occur within few weeks after the allogeneic cells have engrafted in the recipient while chronic GVHD may occur any time after transplant, typically within months. Although treatable by systemic corticosteroid administration, effective responses are not achieved for a significant proportion of patients, a condition associated with poor prognosis. The use of multipotent mesenchymal stromal cells (MSCs) as an alternative to treat steroid-refractory GVHD had improved last decade, but the results are still controversial. Some studies have shown improvement in the life quality of patients after MSCs treatment, while others have found no significant benefits. In addition to variations in trial design, discrepancies in protocols for MSCs isolation, characterization, and ex vivo manipulation, account for inconsistent clinical results. In this review, we discuss the immunomodulatory properties supporting the therapeutic use of MSCs in GVHD and contextualize the main clinical findings of recent trials using these cells. Critical parameters for the clinical translation of MSCs, including consistent production of MSCs according to Good Manufacturing Practices (GMPs) and informative potency assays for product quality control (QC), are addressed.

Cytogenetics of four species of Spirostreptidae (Diplopoda, Spirostreptida).

Considering an estimated number of millipedes of approximately 80,000, cytogenetic studies on these animals are rare, as only a total of 70 species have their karyotypes described. The present study reports on the chromosomal number of four Brazilian diplopods of the family Spirostreptidae: Urostreptus atrobrunneus with 2n=24, XY; Gymnostreptus olivaceus 2n=12, XY and Alloporus araraquarensis and A. principes, 2n=18, XY. The C-banding pattern and NOR staining of U. atrobrunneus, G. olivaceus and A. araraquarensis are described.

Improving the Transduction of Bone Marrow-Derived Cells with an Integrase-Defective Lentiviral Vector.

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow-derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells.

Matrix-assisted autologous chondrocyte transplantation for treatment of focal chondral lesions in the knee: the Hospital Israelita Albert Einstein experience

ABSTRACT Objective Phase 1 clinical trial to determine feasibility, safety, and efficacy of a new advanced cell therapy product for treatment of knee articular cartilage injuries. Methods Three participants with knee focal chondral lesions were included, with no signs of osteoarthritis. Chondrocytes were obtained through knee arthroscopy, cultured in collagen membrane for 3 weeks at the laboratory, subjected to tests to release the cell therapy product, and implanted. All patients underwent a specific 3-month rehabilitation protocol, followed by assessments using functional and imaging scales. The main outcome was the incidence of severe adverse events. Results Three participants were included and completed the 2-year follow-up. There was one severe adverse event, venous thrombosis of distal leg veins, which was no associated with therapy, was treated and left no sequelae. The clinical and radiological scales showed improvement in the three cases. Conclusion The preliminary results, obtained with the described methodology, allow concluding that this product of advanced cell therapy is safe and feasible. ReBEC platform registration number: RBR-6fgy76