Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

A simple protocol for transfecting human mesenchymal stem cells.

OBJECTIVES AND RESULTS: Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment. CONCLUSION: MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.

Differences in bacterial composition between men's and women's restrooms and other common areas within a public building.

Humans distribute a wide range of microorganisms around building interiors, and some of these are potentially pathogenic. Recent research established that humans are the main drivers of the indoor microbiome and up to now significant literature has been produced about this topic. Here we analyzed differences in bacterial composition between men's and women's restrooms and other common areas within the same public building. Bacterial DNA samples were collected from restrooms and halls of a three-floor building from the Federal University of Pampa, RS, Brazil. The bacterial community was characterized by amplification of the V4 region of the 16S rRNA gene and sequencing. Throughout all samples, the most abundant phylum was Proteobacteria, followed by Actinobacteria, Bacteroidetes and Firmicutes. Beta diversity metrics showed that the structure of the bacterial communities were different among the areas and floors tested, however, only 6-9% of the variation in bacterial communities was explained by the area and floors sampled. A few microorganisms showed significantly differential abundance between men's and women's restrooms, but in general, the bacterial communities from both places were very similar. Finally, significant differences among the microbial community profile from different floors were reported, suggesting that the type of use and occupant demographic within the building may directly influence bacterial dispersion and establishment.

Comparison of human mesenchymal stromal cells from four neonatal tissues: Amniotic membrane, chorionic membrane, placental decidua and umbilical cord.

BACKGROUND: Mesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. This study was designed to compare the biological characteristics of MSCs isolated from amniotic membrane (A-MSCs), chorionic membrane (C-MSCs), placental decidua (D-MSCs) and umbilical cord (UC-MSCs) to ascertain whether any one of these sources is superior to the others for cellular therapy purposes. METHODS: MSCs were isolated from amniotic membrane, chorionic membrane, umbilical cord and placental decidua. Immunophenotype, differentiation ability, cell size, cell complexity, polarity index and growth kinetics of MSCs isolated from these four sources were analyzed. RESULTS: MSCs were successfully isolated from all four sources. Surface marker profile and differentiation ability were consistent with human MSCs. C-MSCs in suspension were the smallest cells, whereas UC-MSCs presented the greatest length and least width. A-MSCs had the lowest polarity index and UC-MSCs, as more elongated cells, the highest. C-MSCs, D-MSCs and UC-MSCs exhibited similar growth capacity until passage 8 (P8); C-MSCs presented better lifespan, whereas insignificant proliferation was observed in A-MSCs. DISCUSSION: Neonatal and maternal tissues can serve as sources of multipotent stem cells. Some characteristics of MSCs obtained from four neonatal tissues were compared and differences were observed. Amniotic membrane was the least useful source of MSCs, whereas chorionic membrane and umbilical cord were considered good options for future use in cell therapy because of the known advantages of immature cells.

Effects Of Hypoxia in Long-Term In Vitro Expansion of Human Bone Marrow Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are considered multipotent stromal, non-hematopoietic cells with properties of self-renovation and differentiation. Optimal conditions for culture of MSC have been under investigation. The oxygen tension used for cultivation has been studied and appears to play an important role in biological behavior of mesenchymal cells. The aim is characterize MSC in hypoxia and normoxia conditions comparing their morphological and functional characteristics. Bone marrow-derived mesenchymal stem cells obtained from 15 healthy donors and cultured. MSC obtained from each donor were separated into two cultivation conditions normoxia (21% O2 ) and hypoxia (three donors at 1%, three donors at 2%, five donors at 3%, and four donors at 4% O2 ) up to second passage. MSC were evaluated for proliferation, differentiation, immunophenotyping, size and cell complexity, oxidative stress, mitochondrial activity, and autophagy. Culture conditions applied did not seem to affect immunophenotypic features and cellular plasticity. However, cells subjected to hypoxia showed smaller size and greater cellular complexity, besides lower proliferation (P < 0.002). Furthermore, cells cultured in low O2 tension had lower mitochondrial activity (P < 0.03) and a reduced tendency to autophagy, although oxidative stress did not vary among groups (P < 0.39). Oxygen tension seems to be a key regulator of cellular adaptation in vitro, and metabolic effects underlying this variable remain undescribed. Heterogeneity or even lack of results on the impact of oxygen concentration used for expanding MSC highlights the need for further research, in order to optimize conditions of cultivation and expansion and achieve greater safety and therapeutic efficacy. J. Cell. Biochem. 118: 3072-3079, 2017. © 2017 Wiley Periodicals, Inc.

Diversity and composition of vaginal microbiota of pregnant women at risk for transmitting Group B Streptococcus treated with intrapartum penicillin.

BACKGROUND: Administering intravenous antibiotics during labor to women at risk for transmitting Group B Streptococcus (GBS) can prevent infections in newborns. However, the impact of intrapartum antibiotic prophylaxis on mothers' microbial community composition is largely unknown. We compared vaginal microbial composition in pregnant women experiencing preterm birth at ≤ 32 weeks gestation that received intrapartum antibiotic prophylaxis with that in controls. METHODS: Microbiota in vaginal swabs collected shortly before delivery from GBS positive women that received penicillin intravenously during labor or after premature rupture of membranes was compared to controls. Microbiota was analyzed by 16S rRNA sequencing using the PGM Ion Torrent to determine the effects of penicillin use during hospitalization and GBS status on its composition. RESULTS: Penicillin administration was associated with an altered vaginal microbial community composition characterized by increased microbial diversity. Lactobacillus sp. contributed only 13.1% of the total community in the women that received penicillin compared to 88.1% in the controls. Streptococcus sp. were present in higher abundance in GBS positive woman compared to controls, with 60% of the total vaginal microbiota in severe cases identified as Streptococcus sp. CONCLUSIONS: Vaginal communities of healthy pregnant women were dominated by Lactobacillus sp. and contained low diversity, while Group B Streptococcus positive women receiving intrapartum antibiotic prophylaxis had a modified vaginal microbiota composition with low abundance of Lactobacillus but higher microbial diversity.

Natural killer cell adoptive immunotherapy: Coming of age.

Cell therapy is a promising alternative to harsh chemotherapy and radiation therapy for cancer. Natural killer (NK) cells in particular have great potential for direct use in adoptive immunotherapy (AI) for cancer and to improve the graft-vs-leukemia (GVL) effect of hematopoietic stem cell transplants (HSCTs). NK cell number and function are associated with a strong GVL effect without inducing graft-versus-host disease in most settings. Clinical trials demonstrating the therapeutic role of NK cells in HSCT recipients or testing the safety and efficacy of AI with NK cells have been primarily directed at treating acute myeloid leukemia, although investigators have used NK cells for treatment of other hematological diseases, sarcomas, carcinomas, and brain tumors. Major challenges must be overcome in making NK cell-based therapy cost-effective, the most important being the need to collect or generate an adequate number of effector cells. In this review, we discuss protocols for isolation, expansion, and in vitro propagation of large quantities of functional NK cells that meet the criteria for clinical applications. Among the methods described are the use of bioreactors for scaling up production and expansion of NK cells in the presence of interleukins and feeder cells. We also discuss novel methodologies that optimize the generation of clinical grade NK-cell products for AI.

Mesenchymal stem cell therapy and acute graft-versus-host disease: a review.

Mesenchymal stem cells (MSCs) are being widely studied as potential cell therapy agents due to their immunomodulatory properties, which have been established by in vitro studies and in several clinical trials. Within this context, mesenchymal stem cell therapy appears to hold substantial promise, particularly in the treatment of conditions involving autoimmune and inflammatory components. Nevertheless, many research findings are still contradictory, mostly due to difficulties in characterization of the effects of MSCs in vivo. The purpose of this review is to report the mechanisms underlying mesenchymal stem cell therapy for acute graft-versus-host disease, particularly with respect to immunomodulation, migration, and homing, as well as report clinical applications described in the literature.

Assessment of regulatory T cells in childhood immune thrombocytopenic purpura.

This study had the objective to assess the frequency of Tregs in children newly diagnosed with ITP and ascertain whether an association exists between Tregs and platelet counts, by means of a comparison with healthy controls. This case-control study included 19 patients newly diagnosed with ITP-whose blood samples were collected at four points in time: before any therapy and 1, 3, and 6 months after diagnosis-and 19 healthy controls. Tregs (CD4(+) CD25(+)Foxp3 T cells) were evaluated by flow cytometry. There was a statistically significant difference in platelet count between the case and control groups. There were no significant differences in Treg counts between cases and controls at any point during the course of the study and no difference in Treg counts between the chronic and nonchronic groups and no significant correlation between Tregs and platelet counts in the case and control groups. The findings of this study did not show any statistically significant correlation between Tregs and number of platelets in the case and control groups. Treg cells did not play a role in the regulation of autoimmunity in children with ITP.

Validation of the EBMT Risk Score for South Brazilian Patients Submitted to Allogeneic Hematopoietic Stem Cell Transplantation.

Background. Allogeneic hematopoietic stem cell transplantation (HSCT) is still associated with a high transplant-related mortality rate. In 2009, the EBMT risk score was validated as a simple tool to predict the outcome after allogeneic HSCT for acquired hematological disorders. Objectives. The aim of this study was to validate the applicability of the EBMT risk score for allogeneic HSCT on South Brazilian patients. Methods. A retrospective observational study was performed based on patients' records and data base at Hospital de Clínicas de Porto Alegre, including all allogeneic transplants for malignant and severe aplastic anemia from 1994 to 2010. Patients were categorized according to EBMT risk score and overall survival (OS). Nonrelapse mortality (NRM) and relapse rate (RR) were analyzed. Results. There were 278 evaluable patients. OS, NRM, and RR at five years median followup were 48.7%, 40.7%, and 30.7%, respectively. The OS was 81.8% for risk score 0 and 0% for score 6 (P < 0.001), and NRM was 13.6% and 80% for risk scores 0 and 6, respectively (P = 0.001). Conclusion. The EBMT risk score can be utilized as a tool for clinical decision making before allogeneic HSCT for malignant hematological diseases and severe aplastic anemia at a single center in Brazil.

REST is a novel prognostic factor and therapeutic target for medulloblastoma.

Medulloblastoma is a malignant pediatric brain tumor. Current treatment following patient stratification into standard and high-risk groups using clinical features has improved survival. However, a subset of patients with standard risk features have unanticipated aggressive disease, underscoring the need for a better understanding of tumor biology and the development of novel treatments. Poor differentiation, a hallmark of medulloblastomas is associated with elevated expression levels of the repressor of neuronal differentiation called repressor element 1-silencing transcription factor (REST). Here, we assessed whether elevated REST expression levels had prognostic significance and whether its pharmacologic manipulation would promote neurogenesis and block tumor cell growth. REST levels in patient tumors were measured by immunohistochemistry and stratified into negative, low/moderate- (+/++/+++), and high-REST (+++++) groups. Kaplan-Meier curves revealed that patients with high-REST tumors had worse overall and event-free survival compared with patients with REST-negative or REST-low tumors. Because histone deacetylases (HDAC) are required for REST-dependent repression of neurogenesis, we evaluated a panel of HDAC inhibitors (HDACI) for their effects on growth and differentiation of established and primary REST-positive cell lines. MS-275, trichostatin-A (TSA), valproic acid (VPA), and suberoylanilide hydroxamic acid (SAHA) upregulated expression of the REST-target neuronal differentiation gene, Syn1, suggesting a potential effect of these HDACIs on REST function. Interestingly, VPA and TSA substantially increased histone acetylation at the REST promoter and activated its transcription, whereas SAHA unexpectedly promoted its proteasomal degradation. A REST-dependent decrease in cell growth was also observed following SAHA treatment. Thus, our studies suggest that HDACIs may have therapeutic potential for patients with REST-positive tumors. This warrants further investigation.