Mesenchymal Stromal Cells from Perinatal Tissues as an Alternative for Ex Vivo Expansion of Hematopoietic Progenitor and Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) serves as a source of hematopoietic stem and progenitor cells (HSPCs) utilized in the regeneration of hematopoietic and immune systems, forming a crucial part of the treatment for various benign and malignant hematological diseases. UCB has been utilized as an alternative HSPC source to bone marrow (BM). Although the use of UCB has extended transplantation access to many individuals, it still encounters significant challenges in selecting a histocompatible UCB unit with an adequate cell dose for a substantial proportion of adults with malignant hematological diseases. Consequently, recent research has focused on developing ex vivo expansion strategies for UCB HSPCs. Our results demonstrate that co-cultures with the investigated mesenchymal stromal cells (MSCs) enable a 10- to 15-fold increase in the cellular dose of UCB HSPCs while partially regulating the proliferation capacity when compared to HSPCs expanded with early acting cytokines. Furthermore, the secretory profile of UCB-derived MSCs closely resembles that of BM-derived MSCs. Moreover, both co-cultures exhibit alterations in cytokine secretion, which could potentially impact HSPC proliferation during the expansion process. This study underscores the fact that UCB-derived MSCs possess a remarkably similar supportive capacity to BM-derived MSCs, implying their potential use as feeder layers in the ex vivo expansion process of HSPCs.

Rapid sealing of porcine renal blood vessels, ex vivo, using a high power, 1470-nm laser, and laparoscopic prototype.

Energy-based, radiofrequency (RF) and ultrasonic (US) devices currently provide rapid sealing of blood vessels during laparoscopic procedures. We are exploring infrared lasers as an alternate energy modality for vessel sealing, capable of generating less collateral thermal damage. Previous studies demonstrated feasibility of sealing vessels in an in vivo porcine model using a 1470-nm laser. However, the initial prototype was designed for testing in open surgery and featured tissue clasping and light delivery mechanisms incompatible with laparoscopic surgery. In this study, a laparoscopic prototype similar to devices currently in surgical use was developed, and performance tests were conducted on porcine renal blood vessels, ex vivo. The 5-mm outer-diameter laparoscopic prototype featured a traditional Maryland jaw configuration that enables tissue manipulation and blunt dissection. Laser energy was delivered through a 550 - ? m -core-diameter optical fiber with side-delivery from the lower jaw and beam dimensions of 18 - mm ? length × 1.2 - mm ? width . The 1470-nm diode laser delivered 68 W with 3-s activation time, consistent with vessel seal times associated with RF and US-based devices. A total of 69 fresh porcine renal vessels with mean diameter of 3.3 ± 1.7 ?? mm were tested, ex vivo. Vessels smaller than 5-mm diameter were consistently sealed (48/51) with burst pressures greater than malignant hypertension blood pressure (180 mmHg), averaging 1038 ± 474 ?? mmHg . Vessels larger than 5 mm were not consistently sealed (6/18), yielding burst pressures of only 174 ± 221 ?? mmHg . Seal width, thermal damage zone, and thermal spread averaged 1.7 ± 0.8 , 3.4 ± 0.7 , and 1.0 ±

Thulium fiber laser recanalization of occluded ventricular catheters in an ex vivo tissue model.

Hydrocephalus is a chronic medical condition that occurs in individuals who are unable to reabsorb cerebrospinal fluid (CSF) created within the ventricles of the brain. Treatment requires excess CSF to be diverted from the ventricles to another part of the body, where it can be returned to the vascular system via a shunt system beginning with a catheter within the ventricle. Catheter failures due to occlusion by brain tissues commonly occur and require surgical replacement of the catheter. In this preliminary study, minimally invasive clearance of occlusions is explored using an experimental thulium fiber laser (TFL), with comparison to a conventional holmium: yttrium aluminium garnet (YAG) laser. The TFL utilizes smaller optical fibers ( < 200 - ? m OD) compared with holmium laser ( > 450 - ? m OD), providing critical extra cross-sectional space within the 1.2-mm-inner-diameter ventricular catheter for simultaneous application of an endoscope for image guidance and a saline irrigation tube for visibility and safety. TFL ablation rates using 100 - ? m core fiber, 33-mJ pulse energy, 500 - ? s pulse duration, and 20- to 200-Hz pulse rates were compared to holmium laser using a 270 - ? m core fiber, 325-mJ, 300 - ? s , and 10 Hz. A tissue occluded catheter model was prepared using coagulated egg white within clear silicone tubing. An optimal TFL pulse rate of 50 Hz was determined, with an ablation rate of 150 ?? ? m / s and temperature rise outside the catheter of ? 10 ° C . High-speed camera images were used to explore the

Effective Design of Multifunctional Peptides by Combining Compatible Functions.

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK rearranged lung cancer.

INTRODUCTION: The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK. METHODS: We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 µM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases. RESULTS: All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as "bypass" tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib+erlotinib (reversible EGFR TKI) and crizotinib+afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib. CONCLUSIONS: We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.

Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing.

PURPOSE: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). METHODS: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. RESULTS: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173, 55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). CONCLUSIONS: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets.

Life course association of maternal smoking during pregnancy and offspring's height: data from the 1993 Pelotas (Brazil) birth cohort.

PURPOSE: To evaluate the effect of (1) maternal smoking during pregnancy; and (2) partner smoking on offspring's height in infancy, childhood, and adolescence. METHODS: All hospital live births from 1993 (5,249) were identified, and these infants were followed up at several ages. Height for age, expressed as z-scores using the World Health Organization growth curves, was measured at all follow-up visits. Maternal smoking during pregnancy was collected retrospectively at birth and analyzed as number of cigarettes/day smoked categorized in four categories (never smoked, <10, 10-19, and ≥ 20 cigarettes/day). Partner smoking was analyzed as a dichotomous variable (No/Yes). Unadjusted and adjusted analyses were performed by use of linear regression. RESULTS: The prevalence of self-reported maternal smoking during pregnancy was 33.5%. In the crude analysis, the number of cigarettes/day smoked by the mother during pregnancy negatively affected offspring's height in infancy, childhood, and adolescence. After adjustment for confounders and mediators, this association remained statistically significant, although the magnitude of the regression coefficients was reduced. Paternal smoking was not associated with offspring's height in the adjusted analyses. CONCLUSIONS: In addition to the well-known harmful effects of smoking, maternal smoking during pregnancy negatively affects offspring's height. Public health policies aimed at continuing to reduce the prevalence of maternal smoking during pregnancy must be encouraged.

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element.

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

The impact of sociodemographic conditions on quality of life among adolescents in a Brazilian birth cohort: a longitudinal study / La repercusión de las condiciones sociodemográficas sobre la calidad de vida de los adolescentes en una cohorte de nacimiento en el Brasil: un estudio longitudinal

OBJECTIVE: To examine associations between certain aspects of self-reported Quality of Life (QoL) in early adolescence, and gender, skin color, maternal education at birth, and changes in socioeconomic position (SEP) occurring from 0-11 years of age. METHODS: A longitudinal study of a birth cohort composed of 5 249 individuals born in 1993 in Pelotas, Brazil, who were followed periodically from birth to age 11. Socioeconomic variables were collected at birth in 1993, and again, in 2004. The following eight variables were analyzed: relationship with mother, relationship with father, family conflicts, physical punishment by parents, family relationship problems, discrimination, academic failure, and fear of neighborhood of residence. Adjustments were made using a hierarchical model. RESULTS: Skin color was related to all the variables (more so for nonwhites than for whites), except in family conflicts. Physical punishment and academic failure were more frequent in males. Discrimination and fear of neighborhood were more prevalent in females. Maternal education at birth was inversely associated, especially with failing/repeating a grade in school(19.5 times more frequent in the lower maternal education group than in the highest). A similar pattern was found with the SEP change: worse QoL in the group that was always poor (at birth and at 11 years of age) than in the group that was never poor. Fear of neighborhood was not related to maternal education or SEP change. CONCLUSIONS: Gender, skin color, maternal education, and SEP change were related to various measured aspects of QoL. These results should be considered in policymaking that seeks to minimize inequities at birth, across the life-course, and for future generations.

OBJETIVO: Evaluar la correlación que existe entre ciertos aspectos autonotificados de la calidad de vida en la adolescencia temprana y el género, el color de la piel, la escolaridad materna en el momento del nacimiento y los cambios de situación socioeconómica que ocurren entre el nacimiento y los 11 años. MÉTODOS: Se llevó a cabo un estudio longitudinal de una cohorte de nacimiento compuesta por 5 249 personas nacidas en 1993 en Pelotas (Brasil) a las cuales se les realizó un seguimiento periódico desde el nacimiento hasta los 11 años. Las variables socioeconómicas se recogieron al momento del nacimiento en 1993 y nuevamente en el 2004. Se analizaron las siguientes ocho variables: la relación con la madre, la relación con el padre, los conflictos familiares, el castigo físico infligido por los padres, los problemas de las relaciones familiares, la discriminación, el fracaso académico y el temor al vecindario de residencia. Se realizaron ajustes mediante el uso de un modelo jerárquico. RESULTADOS: El color de la piel se relacionó con todas las variables (aún más en las personas que no eran blancas que en las blancas), con la excepción de los conflictos familiares. El castigo físico y el fracaso académico fueron más frecuentes en los hombres. La discriminación y el temor al vecindario fueron más prevalentes en las mujeres. La escolaridad materna en el momento del nacimiento presentó una relación inversa, especialmente con respecto a la pérdida o la repetición de un año escolar (19,5 veces más frecuente en el grupo con escolaridad materna más baja que en el grupo con la mayor escolaridad). Se encontró un perfil similar con los cambios de situación socioeconómica: una calidad de vida inferior en el grupo que siempre fue pobre (al nacimiento y a los 11 años de edad), en comparación con la del grupo que nunca lo fue. El temor al vecindario no se relacionó con la escolaridad materna ni con la variación de la situación socioeconómica. CONCLUSIONES: El sexo, el color de la piel, la escolaridad de la madre y los cambios de situación socioeconómica se relacionaron con varios aspectos de la calidad de vida que se evaluaron. Es importante considerar estos resultados cuando se formulen las políticas que tienen por objeto reducir al mínimo la falta de equidad al nacimiento, a lo largo de la vida y en las generaciones futuras.

Cryptosporidium and giardia recoveries in natural waters by using environmental protection agency method 1623.

Relatively few studies have examined recoveries from source waters by using Environmental Protection Agency method 1623 with organism spike doses that are environmentally realistic and at turbidity levels commonly found in surface waters. In this study, we evaluated the filtration capacities and recovery efficiencies of the Gelman Envirochek (standard filter) and the Gelman Envirochek high-volume (HV) sampling capsules under environmental conditions. We also examined the performance of method 1623 under ambient conditions with matrix spike experiments using 10 organisms/liter. Under turbid conditions, the HV capsule filtered approximately twice the volume filtered by the standard filter, but neither could filter 10 liters without clogging. In low-turbidity waters, oocyst, but not cyst, recoveries were significantly higher when the HV capsule was used. In turbid waters, organism recoveries were lower than those in nonturbid waters and were not significantly different for the different filters. When the HV capsule was used, Cryptosporidium recoveries ranged from 36 to 75%, and Giardia recoveries ranged from 0.5 to 53%. For both organisms, recoveries varied significantly by site. Turbidity could explain variation in Giardia recoveries (r(2) = 0.80) but not variation in Cryptosporidium recoveries (r(2) = 0.16). The inconsistent recoveries across sites suggested that the background matrix of the ambient water affected recovery by method 1623. A control sample collected at the height of the winter rainy season detected one organism, highlighting the difficulty of using this method to accurately measure pathogen abundance under natural conditions. Our findings support the use of the HV filter under field conditions but suggest that designing a cost-effective and statistically valid monitoring program to evaluate sources and loads of protozoan pathogens may be difficult.

Transgenic targeting with regulatory elements of the human CD34 gene.

The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5' flanking region and 26 kb of the 3' flanking region of the CD34 gene directs expression of the human CD34 gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.