Hair Collection for Cortisol Among Youth Experiencing Homelessness.

BACKGROUND: Investigations of chronic physiological stress measured by hair cortisol are rapidly expanding among community samples of adolescents and adults. However, research examining physiological stress among youth experiencing homelessness is nascent despite the youth's increased risk for adverse exposures and subsequent impaired mental health. OBJECTIVE: This article aimed to examine the feasibility of collecting hair for measuring cortisol among diverse youth experiencing homelessness and gain an understanding of variation in participation. METHODS: Analysis of survey and hair participation data from three pilot studies among youth experiencing homelessness was conducted. Survey measures included sociodemographic characteristics (age, race and ethnicity, sex assigned at birth, and sexual orientation) and reasons for nonparticipation. Descriptive analysis examined participation rates in hair collection for cortisol measurement, including sociodemographic differences in participation. RESULTS: Participation in the hair sampling for cortisol was high for the combined sample (88.4%), with some variation across the three pilot studies. Insufficient hair for cutting was the most common reason for not participating; Black and multiracial youth, as well as male youth, had a higher prevalence of nonparticipation. DISCUSSION: The collection of hair for cortisol research among youth experiencing homelessness is feasible, and integration of physiological measures of stress into research with this vulnerable population should be considered, given their high risk for adversity and death by suicide and drug overdose. Methodological considerations and avenues for potential research are discussed.

Protocol to Measure Hair Cortisol in Low Mass Samples From Very Preterm Infants.

BACKGROUND: Hair cortisol is a measure of chronic or repeated hypothalamic-pituitary-adrenal axis activation in response to physical or psychological stressors. Hair cortisol has been successfully used as a measure of chronic stress in adults and children; however, its use as a valid measure in preterm infants has been limited by challenges in measuring cortisol in the low mass samples collectable from these infants. OBJECTIVES: The purpose of this report is to present a novel protocol for the measurement of hair cortisol in very low mass hair samples. METHODS: Small changes were made to previously published protocols. After washing and pulverizing the hair samples, a double methanol cortisol extraction was performed. Samples were spiked with a known quantity of cortisol and analyzed in duplicate using an enzyme-linked immunosorbent assay. RESULTS: Hair cortisol was detectable in samples weighing between 0.4 and 10.9 mg. The mean cortisol level was 23.74 pg/mg hair (SD = 26.38). DISCUSSION: With small changes to previously published laboratory protocols, cortisol is quantifiable in low mass hair samples from preterm infants. This technical advance is an important step toward quantifying the stress experiences of hospitalized preterm infants.

Collecting Hair Samples for Hair Cortisol Analysis in African Americans.

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Supplementation with eicosapentaenoic acid and docosahexaenoic acid reduces high levels of circulating proinflammatory cytokines in aging adults: A randomized, controlled study.

BACKGROUND: High levels of circulating proinflammatory cytokines are characteristic of inflammaging, a term coined to describe age-related chronic systemic inflammation involved in the etiology of many age-related disorders including nonhealing wounds. Some studies have shown that supplementing diets with n-3 polyunsaturated fatty acids (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) lowers systemic levels of key proinflammatory cytokines associated with inflammaging. However, findings from the few studies that have focused exclusively on older adults are inconclusive. As such, the objective of this randomized controlled study was to test the effects of EPA+DHA therapy on circulating levels of proinflammatory cytokines in adults in middle to late adulthood. METHODS: Plasma levels of fatty acids and interleukin (IL)-6, IL-1ß and tumor necrosis factor-α (TNF-α) were measured in 35 participants with chronic venous leg ulcers (mean age: 60.6 years) randomnly assigned to 8 weeks of EPA+DHA therapy (2.5 g/d) or placebo therapy. RESULTS: EPA+DHA therapy had a significant lowering effect on levels of IL-6, IL-1ß and TNF-α after 4 weeks of therapy and an even greater lowering effect after 8 weeks of therapy. Further, after adjusting for baseline difference, the treatment group had significantly lower levels of IL-6 (p = 0.008), IL-1ß (p < 0.001), and TNF-α (p < 0.001) at Week 4 and at Week 8 [IL-6 (p = 0.007), IL-1ß (p < 0.001), and TNF-α (p < 0.001)] compared to the control group. CONCLUSION: Adults in middle to late adulthood receiving EPA+DHA therapy demonstrated significantly greater reductions in circulating levels of proinflammatory cytokines compared with those receiving placebo therapy. EPA+DHA therapy may be an effective low-risk dietary intervention for assuaging the harmful effects of inflammaging.

Oxytocin Levels in Community-Collected Saliva Samples Transported by Dry Versus Wet Ice.

Oxytocin (OT), a neuropeptide produced primarily in the hypothalamus, is associated with both critical physiological and psychological processes, particularly stress and feelings of affiliation. Increasingly, researchers are seeking ways to reliably incorporate OT as an outcome biomarker in clinical research. Previously, OT levels were measured in plasma or urine. Recently, researchers have measured this biomarker in saliva, particularly when conducting research in clinical and community settings. In spite of increased interest in the use of salivary OT in clinical research, procedures for handling, transport, and analysis of specimens vary. It is not known if significant OT protein degradation occurs if samples are initially transported on wet ice before being frozen. The aim of this study is to evaluate the effect of transport media (wet vs. dry ice) on OT levels derived from saliva collected from 12 postpartum women residing in the community. Saliva collected from each participant was divided between two microcentrifuge tubes (MIDSCI, Valley Park, MO), one placed on wet ice and one on dry ice for transport from the participant's home to the laboratory freezer. Time from collection to storage freezer was recorded. Laboratory personnel, blinded to method of transport, batch processed the samples. No significant differences in OT levels were found by transport method. Despite large interperson variations in OT levels, there were negligible intraperson variations. Although further research is required to identify factors (including transport time) related to interperson variation, this study supports the use of wet ice as a means of transporting salivary OT specimens in community-based research.

Manipulation of oxygenation and flow-induced shear stress can increase the in vitro yield of platelets from cord blood.

A method to produce clinically useful platelets in vitro would help overcome the frequent shortages, donor deferrals, disease transmission, and alloimmunization with volunteer donor-derived platelets. Using CD34 positively selected cord blood cells, we investigated ways to increase platelet quality and yield in a three-dimensional modular perfusion bioreactor system. We found a two- to threefold increase in platelet numbers produced only when the early phases of the culture process were carried out at 5% oxygen, versus when 20% oxygen was used throughout the culture period (p<0.05), and much more than when 5% oxygen was used throughout. When the medium was routed through the cell-scaffold construct, versus when it flowed under and over the construct, or just intermittent feeding was used, the number of platelets increased two- to threefold (p<0.05), and enhanced collagen-induced aggregation. The 5% oxygen early in the culture process mimics the marrow adjacent to the bone where early progenitors proliferate. Flow through the cell-scaffold construct creates shear forces that mimic the flow in central venous sinuses of the marrow and enhances platelet production from proplatelets. The use of altered oxygen levels and cross flow enhanced platelet numbers and quality, and will contribute to eventual in vitro platelet production for clinical use.

Effect of increased HoxB4 on human megakaryocytic development.

In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.

Prolonged continuous in vitro human platelet production using three-dimensional scaffolds.

OBJECTIVE: Methods producing human platelets using growth on plastic, on feeder layers, or in suspension have been described. We hypothesized that growth of hematopoietic progenitors in a three-dimensional (3D) scaffold would enhance platelet production sans feeder layer. MATERIALS AND METHODS: We grew CD34 positively selected human cord blood cells in surgical-grade woven polyester fabric or purpose-built hydrogel scaffolds using a cocktail of cytokines. RESULTS: We found production of functional platelets over 10 days with two-dimensional (2D), 24 days with 3D scaffolds in wells, and more than 32 days in a single-pass 3D perfusion bioreactor system. Platelet numbers produced daily were higher in 3D than 2D, and much higher in the 3D perfusion bioreactor system. Platelet output increased in hydrogel scaffolds coated with thrombopoietin and/or fibronectin, although this effect was largely obviated with markedly increased production caused by changes in added cytokines. In response to thrombin, the platelets produced aggregated and displayed increased surface CD62 and CD63. CONCLUSION: Use of 3D scaffolds, especially in a bioreactor-maintained milieu, may allow construction of devices for clinical platelet production without cellular feeder layers.

Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells.

OBJECTIVE: The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells. MATERIALS AND METHODS: A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents. RESULTS: Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor. CONCLUSION: To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.