Isolation and Flow Cytometric Analysis of the Stromal Vascular Fraction Isolated from Mouse Adipose Tissue.

Evidence from preclinical research and clinical trials demonstrates the use of the stromal vascular fraction (SVF) as therapy for numerous indications. These results demonstrate that autologous SVF is not only safe and effective but provides robust anti-inflammatory, immunomodulatory, and reparative effects in vivo. The potency of the SVF is attributed to the cellular composition which includes adipose-derived stem cells (ASCs), adipocytes, endothelial cells, and various immune cells. As the name would suggest, these SVF cells are derived from the stromal compartment of adipose, or fat. Once digested, the cells that constitute adipose are released and collected as the SVF. The cellular frequencies within the SVF can then be assessed using a fluorescent antibody-based technique known as flow cytometry. The following chapter provides a standard operating protocol that describes the procedures from harvesting the fat tissue from experimental mice to isolating and characterizing the SVF.

Cognitive assessment of refugee children: Effects of trauma and new language acquisition.

Each year, approximately 60,000 children of refugee background are resettled in Western countries. This paper reviews the effects of the refugee experience on cognitive functioning. The distinctive influences for these children include exposure to traumatic events and the need to acquire a new language, factors that need to be considered to avoid overdiagnosis of learning disorders and inappropriate educational placements. Prearrival trauma, psychological sequelae of traumatic events, developmental impact of trauma, and the quality of family functioning have been found to influence cognitive functioning, learning, and academic performance. In addition, the refugee child may be semiproficient in several languages, but proficient in none, whilst also trying to learn a new language. The influence that the child's limited English proficiency, literacy, and school experience may have on academic and test performance is demonstrated by drawing on the research on refugees' English language acquisition, as well as the more extensive literature on bilingual English language learners. Implications for interventions are drawn at the level of government policy, schools, and the individual. The paper concludes with the observation that there is a major need for longitudinal research on refugee children's learning and academic performance and on interventions that will close the academic gap, thereby enabling refugee children to reach their educational potential.

Wide-ranging cognitive deficits in adolescents following early life maltreatment.

OBJECTIVE: Studies of adolescents with histories of maltreatment typically report specific cognitive deficits in higher order functioning and attention. Emerging evidence suggests that the cognitive difficulties seen in maltreated adolescents are much broader, and go beyond executive functioning impairments. This study examined whether maltreated adolescents exhibited cognitive deficits across a number of cognitive domains, in addition to executive functioning. METHOD: A group of 39 adolescents with documented histories of severe maltreatment were compared with 43 controls on measures of learning and memory, executive function, processing speed, working memory, visuoperceptual function, and language. Groups were matched demographically and on the Wechsler Intelligence Scale for Children-IV (WISC-IV) Full Scale Intelligence Quotient (FSIQ; Wechsler, 2003). RESULTS: Using multivariate analyses, the maltreated group showed significant impairments on measures of executive function and attention, working memory, learning, visuospatial function and visual processing speed. Effect sizes ranged from medium to large. CONCLUSIONS: The FSIQ indicated that these adolescents were performing comparably with their nonmaltreated peers, though this was not the case when specific cognitive functions were measured. This demonstrates that maltreated adolescents are more likely to have a range of cognitive deficits that can only be identified with thorough neuropsychological assessment. Such deficits have the potential to significantly impair adaptive, social, emotional, and academic functioning, explaining many of the typical difficulties seen in maltreated adolescents.

Drosha regulates hMSCs cell cycle progression through a miRNA independent mechanism.

Recently we demonstrated that the miRNA regulate human mesenchymal stem cells (hMSCs) differentiation. To determine the role of the miRNA pathway in hMSCs proliferation, Drosha and Dicer knockdown hMSCs were generated using a lentiviral based tetracycline inducible shRNA. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSCs proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.

Executive control among adolescent inhalant and cannabis users.

INTRODUCTION AND AIMS: Inhalants are frequently among the first drugs abused by adolescents; however, little is known about how chronic inhalant abuse affects cognition (e.g. executive functioning). Several studies have examined cognitive deficits among inhalant users; however, no study has thoroughly addressed the confounding issues frequently associated with inhalant users (e.g. polysubstance use). The aim of the current study was to examine possible deficits in cognitive control among young, regular inhalant users and explore the relationship between inhalant use and executive functioning. DESIGN AND METHODS: Three groups (n = 19) of young people (aged 14-24) were recruited: an inhalant-using group, a drug-using control group and a community control group. The inhalant and drug-using controls were matched on demographic, clinical and substance use measures. All three groups were matched on age, sex and education. Cognitive control was assessed using Stroop and Go/No-Go tasks. RESULTS: There were no significant differences in performance between the groups on any measure. However, three measures (incongruent reaction times and congruent errors for the Stroop and omission errors for the Go/No-Go) were significantly correlated with inhalant use measures, suggesting inhalant use was associated with poorer performance. DISCUSSION AND CONCLUSIONS: The lack of significant differences between the groups is surprising; however, it raises important questions regarding cognitive deficits among chronic inhalant users. Further longitudinal studies using well-matched control participants are required to delineate the nature and timing of cognitive and neurobiological pathology among adolescent inhalant users.

Verbal memory, learning, and executive functioning among adolescent inhalant and cannabis users.

OBJECTIVE: Inhalant use is a common form of drug misuse among young adolescents. However, very little is known about how chronic inhalant misuse affects cognition. Several studies have examined cognitive deficits among inhalant users, but no study has thoroughly addressed the confounding issues frequently associated with inhalant users (e.g., polysubstance use). The aim of the current study was to examine possible deficits in memory, learning, and executive components of memory (interference susceptibility) among young, regular inhalant users relative to a statistically equivalent drug-using control group (primarily cannabis users) and a community control group. METHOD: Three groups of 21 young people (aged 13-24 years) were recruited: an inhalant- using group, a drug-using control group, and a community control group. The inhalant and drug-using controls were matched at the group level on demographic, clinical, and substance use measures. All three groups were statistically equivalent on age, sex, and education. The Rey Auditory Verbal Learning Test was used to assess memory, learning, and interference susceptibility. RESULTS: Community controls performed significantly better than both drug-using groups, while inhalant users were more susceptible to proactive interference relative to drug-using controls. CONCLUSIONS: Difficulty in successful proactive interference resolution demonstrated by the inhalant group may relate to inhalant-specific deficits in executive functioning. These findings raise important questions regarding the hypothesized toxicity of inhalants and of substance-specific cognitive deficits among regular adolescent substance users. Future studies should consider using more specific, experimental probes of cognitive functioning to identify potentially subtle changes among substance-using adolescents.

Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.

Bone marrow-derived stromal cells (BMSCs) are defined by their ability to self-renew and differentiate into at least three mesenchymal cell types (bone, adipose, and cartilage). The inability to isolate a reliably efficacious and homogeneous population of early progenitor cells has limited efforts to increase their therapeutic potential. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured BMSC population. Markers of progenitor status were identified by comparing BMSCs at early and late passage, donor-matched skin fibroblasts, and commercially available dermal fibroblast cell lines. Differentiation potential was determined according to in vitro assays of osteogenesis, adipogenesis, and chondrogenesis. Early-passage BMSCs differentiated into all three lineages, whereas late-passage BMSCs and both fibroblast preparations did not. To identify novel markers of early progenitors, microarray transcript analysis between early-passage BMSCs and fibroblasts was performed. Messenger RNA encoding the cytokine leukemia inhibitory factor (LIF) was identified as differentially expressed. Enzyme-linked immunosorbent assay on conditioned media confirmed that LIF secretion was much higher from early progenitor BMSCs than donor-matched or commercial lines of fibroblasts and dropped with extensive expansion or induction of differentiation. In clonally expanded BMSCs, colonies that retained progenitor status expressed significantly higher levels of LIF than those that failed to differentiate. Our results indicate that LIF expression may represent a marker to quantify the differentiation potential of BMSCs and may be especially suited for the rapid, noninvasive quality control of clinical preparations.

Differentiation and characterization of human MSCs.

One of the hallmark characteristics of human MSCs (hMSCs) is their ability to differentiate into adipocytes, chondrocytes and osteocytes in culture. The default fate for hMSCs appears to be bone: if late-passage cultures are left in basic culture medium, the hMSCs will become confluent and produce mineral, an indication of bone formation. However, when grown under certain culture conditions or in media containing specific components, the cells can be driven to become a number of other specific cell types including neural cells, myocytes, and cardiomyocytes. The protocols given here are the basic differentiation procedures for inducing osteogenesis, adipogenesis, and chondrogenesis in cultures of hMSCs. Although there is still no clear consensus on the antigen expression pattern that will define hMSCs, a protocol is also presented for the flow cytometric analysis using a series of antibody panels. The analysis of these surface epitope patterns can aide in the isolation and characterization of hMSCs.

Acute hypobaric hypoxia (5486 m) induces greater pulmonary HIF-1 activation in hilltop compared to madison rats.

Compared to Madison strain Sprague-Dawley rats, the Hilltop strain is resistant to acute hypoxic pulmonary vasoconstriction and pulmonary leak, a pathology resembling high altitude pulmonary edema (HAPE) in humans. Hypoxia inducible transcription factor-1 (HIF-1) mediates transcription of proteins that can "rescue" tissue from hypoxia, including vasoactive and angiogenic proteins such as inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF). Because these proteins have theoretical relevance to the etiology of HAPE, we hypothesized that hypoxia-resistant Hilltop rats acutely exposed to high altitude would have greater HIF-1 activity and expression of iNOS and VEGF as compared to hypoxia-sensitive Madison rats. Animals were exposed to normobaric normoxia or hypobaric hypoxia (18 h at 5486 m). The presence of nuclear HIF-1 heterodimer subunits, HIF-1-DNA binding, and iNOS and VEGF protein expression were determined in lung tissue. Hypoxic HIF-1beta expression, HIF-1-DNA binding, and iNOS and VEGF expression were greater in Hilltop than in Madison rats. After 18-h hypobaric hypoxia, HIF-1 activity and HIF-mediated protein expression were elevated in Hilltop rats, but not in Madison rats. To our knowledge, this is the first report of differing HIF-1 activation between two strains of animals with clearly divergent physiological responses to identical hypoxic conditions.

Neutral endopeptidase null mice are less susceptible to high altitude-induced pulmonary vascular leak.

Hypoxia increases pulmonary vascular leak, which is regulated in part by neutral endopeptidase (NEP). NEP is a cell-surface metalloprotease that degrades several vasoactive peptides, including endothelin-1 (ET-1) and atrial natriuretic peptide (ANP). We therefore hypothesized that NEP attenuates high altitude-induced pulmonary vascular leak. Wild-type and NEP null mice were exposed to a simulated high altitude (HA) of 6,728 m (22,000 ft; P(B) = 328 mmHg) or remained at the relatively low altitude (LA) of 1,500 m (4,920 ft; P(B) = 640 mmHg) for 24 h. Plasma ANP and ET-1 concentrations, right ventricular pressure (P(RV)), and indexes of lung injury were recorded. At HA, lung wet weight-to-body weight increased in all animals, but was greatest in the NEP wild-type mice. Vascular leak, as measured by Evans blue dye, increased only in the NEP wild-type mice at HA. P(RV) increased in both genotypes at HA. Plasma ANP concentrations increased at HA in both genotypes, but plasma ET-1 concentrations were elevated only in the NEP null mice at HA. Correlations between lung wet weight-to-body weight versus P(RV) (r = 0.56; p = 0.0136) and ANP versus P(RV) (r = -0.54; p = 0.02) were noted. We conclude that NEP null mice exposed to HA have a greater rise in ANP versus ET-1 plasma concentration, decreased pulmonary vascular pressure, and reduced high altitude-induced pulmonary vascular leak.

Airway fibroblasts exhibit a synthetic phenotype in severe asthma.

BACKGROUND: Platelet-derived growth factor (PDGF) may have a significant role in airway remodeling in asthma, because it is a powerful inductor of many airway fibroblast activities such as collagen synthesis. OBJECTIVE: To determine whether PDGF is a significant contributor to airway remodeling in patients with asthma by enhancing airway fibroblast procollagen I expression. METHODS: Six normal controls without asthma, 10 subjects with mild to moderate asthma, and 5 subjects with severe asthma underwent bronchoscopy with endobronchial biopsy. Biopsies were placed in Dulbecco modified Eagle medium and fibroblasts cultured in the presence and absence of PDGF isoforms -AA, -BB, and -AB (1, 5, 10, 100 ng/mL) and insulin-like growth factor 1 (100 ng/mL). Fibroblast procollagen I and PDGF receptors (PDGFRs) alpha and beta expression were determined by ELISA. RESULTS: Platelet-derived growth factor BB significantly enhanced fibroblast procollagen I expression in patients with severe asthma compared with patients with mild/moderate asthma and normal controls. Furthermore, the baseline fibroblast expression of PDGFR-beta was significantly greater in patients with severe asthma compared with the other groups. CONCLUSION: This pilot study suggests that airway fibroblasts from patients with severe asthma exhibit a synthetic phenotype, which may be driven by the overexpression of PDGFR-beta.

Neuropsychological diversity in Apert syndrome: a comparison of cognitive profiles.

Apert syndrome is characterized by craniosynostosis, central nervous system anomalies, midface hypoplasia, and syndactyly. Current research has focused on genetic and neurologic correlates. Cognitive assessment has been primarily limited to global intellectual evaluations, which can fail to detect the diverse cognitive attributes of these children at an individual level. This report describes in detail the neuropsychological profiles of 2 children with Apert syndrome, incorporating clinical, radiographic, molecular and surgical data. One child showed intellectual deficits consistent with a moderate intellectual disability. The second child, while of normal intelligence, displayed neuropsychological deficits associated with anterior-brain-region cognitive functions. These data highlight the diversity of neuropsychological outcomes in Apert syndrome in the same genetic mutation and underline the importance of detailed neuropsychological evaluations as integral to the management protocols of affected individuals.