A double-blinded placebo-controlled evaluation of adipose-derived mesenchymal stem cells in treatment of canine atopic dermatitis.

Mesenchymal stem cells (MSCs) have emerged as a new therapy for various immune-mediated inflammatory diseases. In this study we perform the first double-blinded, placebo-controlled evaluation of the efficacy of adipose-derived allogenic canine MSCs for the treatment of canine atopic dermatitis (cAD). Enrolled canine patients were randomly divided into placebo (PBS saline), low-dose (5 × 105 cells/kg), and high-dose (5 × 106 cells/kg) treatment groups. Each patient received three subcutaneous MSCs treatments or PBS saline at four-week intervals with injections at five sites. Patients were monitored by physical exams, pruritus visual analog scales (PVAS) signed by the primary caretaker, canine atopic dermatitis extent and severity index-4 (CADESI-4) scores by two veterinarians, and complete blood count and serum chemistry analysis along with laboratory analysis for potential biomarkers. Patients were kept off any immune-modulating drugs during the study period, and oral antibiotics and topicals were used for managing pruritus and secondary infections. The PVAS scores and the serum miR-483 levels were significantly lower in the high dose group compared to the placebo group at day90 post first-treatment. The CADESI-4 scores of the high dose group also showed downward trends. No severe adverse effects were observed in any patient in this study. The high dose MSC treatment is efficacious in alleviating the clinical signs of cAD until 30 days after the last subcutaneous administration of MSCs, and miRNA-483 may be a reliable prognostic biomarker for cAD. The MSCs efficacy and potential biomarkers should be further explored by a larger scale clinical trial.

Intra-articular Administration of Allogeneic Adipose Derived MSCs Reduces Pain and Lameness in Dogs With Hip Osteoarthritis: A Double Blinded, Randomized, Placebo Controlled Pilot Study.

This study was conducted to investigate the therapeutic effect of allogeneic adipose-derived MSCs on dogs with hip osteoarthritis (OA). Twenty dogs with bilateral osteoarthritis of the coxofemoral (hip) joint, diagnosed by a veterinarian through physical examination and radiographs were randomly allocated into four groups. Group 1 served as a placebo control and were injected with 0.9% sodium chloride (saline) (n = 4). Group 2 were injected with a single dose of 5 million MSCs (n = 5). Group 3 received a single dose of 25 million MSCs (n = 6) and Group 4 received a single dose of 50 million MSCs (n = 5). Intra-articular administration of allogeneic MSCs into multiple joints did not result in any serious adverse events. The average lameness score of the dogs in the placebo control group (-0.31) did not show improvement after 90 days of intra-articular saline administration. However, the average lameness score of the all MSC-treated dogs was improved 2.11 grade at this time point (P < 0.001). Overall, sixty five percent (65%) of the dogs that received various doses of MSCs showed improvement in lameness scores 90 days after intra-articular MSC administration. Our results showed that intra-articular administration of allogeneic adipose derived MSCs was well-tolerated and improved lameness scores and reduced pain in dogs associated with hip OA. All doses of MSCs were effective. Subsequent studies with more animals per group are needed to make a conclusion about the dose response. The improved lameness effect was present up to 90 days post-injection. Serum interleukin 10 was increased in a majority of the dogs that received MSCs and that also had improved lameness.

Intra-Ethnic Racial Differences in Waterpipe Tobacco Smoking among Latinos?

BACKGROUND AND OBJECTIVES: This study examined differences in waterpipe smoking (both lifetime and current) by race and ethnicity. More specifically, we evaluated intra-ethnic racial differences among Latinos using a nationally representative sample. METHODS: Pooled data from the National Adult Tobacco Survey (NATS) [2012-2014] was used, in which Log-Poisson multivariable regression models were deployed to determine the prevalence of waterpipe smoking behavior. Models were stratified by gender and we further investigated acculturation, controlling for relevant sociodemographic characteristics. RESULTS: In fully-adjusted models assessing lifetime WTS, Black Latinos and White Latinos exhibited an increase prevalence of lifetime WTS compared to their non-Hispanic white counterparts. Once stratifying by gender, Black Latino men (PR = 1.49; 95% CI = 1.16, 1.90) exhibited increased prevalence of lifetime WTS compared to their non-Hispanic white men counterparts; although white Latino men (PR = 0.88; 95% CI = 0.80, 0.98) exhibited decreased prevalence compared to their non-Hispanic white male counterparts. Similar trends were found for current WTS among men. In fully adjusted models assessing lifetime WTS, among women, only white Latina's (PR = 1.23; 95% CI = 1.04, 1.46) exhibited increased prevalence compared to their non-Hispanic white women counterparts. When evaluating current WTS, Black Latina's (PR = 2.19; 95% CI = 1.32, 3.65) and white Latinas (PR = 1.28; 95% CI = 1.00, 1.63) exhibited increased prevalence of WTS compared to their non-Hispanic white women counterparts. Conclusions/Importance: Among the U.S. general adult population, intra-ethnic racial differences in WTS behaviors exist among Latinos; and is shaped by gender. Future efforts to eliminate racial disparities in WTS should be attentive intra-ethnic racial differences among Latinos.

Standardized cryopreservation of human primary cells.

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel.

Developing a clinical-grade cryopreservation protocol for human testicular tissue and cells.

Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients.

Are black Hispanics black or Hispanic? Exploring disparities at the intersection of race and ethnicity.

BACKGROUND: Disparities in health among blacks and Hispanics compared to whites individuals exist for a number of health measures; however, the health profile of individuals who are both black and Hispanic is not well known. We sought to determine whether race and ethnicity have synchronous or independent effects on health-related outcomes. METHODS: We combined the National Health Interview Survey for 2000-2007 to identify 896 black Hispanics. We selected health-related outcomes where white Hispanics and non-Hispanic blacks significantly differed. We computed adjusted prevalence estimates for black Hispanics and compared them to determine whether their health-related outcomes more closely resemble white Hispanics or non-Hispanic blacks. All prevalence estimates were adjusted for age, sex, education, marital status, income and survey year. RESULTS: Black Hispanics' health behaviours resembled white Hispanics or were similar to both white Hispanics and non-Hispanic blacks. For health services outcomes, they resembled non-Hispanic blacks. However, their health status was influenced by both race and ethnicity, with black Hispanics resembling both white Hispanic and non-Hispanic black people. CONCLUSION: We conclude that health behaviour interventions incorporating knowledge of Hispanic cultures may be sufficient to reach black Hispanics. However, health services or health status, interventions targeted broadly to Hispanic people may not be sufficient. In some respects black Hispanic people comprise a distinct subgroup that may require targeted attention in public health interventions.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.

BACKGROUND: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Human adipose-derived side population stem cells cultured on demineralized bone matrix for bone tissue engineering.

BACKGROUND: Tissue engineering of new bone relies on the combination and application of osteoconductive, osteoinductive, and osteogenic elements. Natural scaffolds, such as demineralized bone matrix (DBM), contain collagenous networks with growth factors such as bone morphogenetic protein-2. Stem cells from readily available sources, including discarded adipose tissue, have the propensity to differentiate into bone. The present study examines a multi-component technique consisting of a novel side population of adipose stem cells cultured on DBM for tissue engineering applications. METHODS: Adipose-derived side population stem cells were cultured on DBM for up to 14 days. Cell proliferation, alkaline phosphatase activity, extracellular matrix protein production, and calcium-containing mineral deposit formation were assayed. Ectopic bone formation in a rat model was also evaluated. RESULTS: Side population stem cells attached to and proliferated on DBM while generating markers of new bone formation. When these cell/substrate composites were implanted into an ectopic model, newly formed bone was 30% greater than that of DBM alone. CONCLUSIONS: Novel populations of adipose-derived stem cells cultured on DBM compose a system that develops new bone matrix in vitro and in vivo. This strategy provides a novel approach using naturally occurring materials for bone repair in tissue engineering applications.

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary.

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.

Adipose stem cell side population in the mouse.

Adipose tissue has become a reliable source of adult stem cells, which appear to possess a yet-undetermined degree of plasticity. With the difficulties associated with harvesting adult bone marrow stem cells, adipose tissue may represent a valuable and easily acquired source of stem cells. Stem cells have been identified using the DNA binding dye Hoechst 33342 and flow cytometry in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population stem cells in adult adipose tissues. Flow cytometric identification and isolation of this subpopulation of stem cells revealed that in the mouse there are 2.5% of adipose SP cells within the stromal vascular fraction of adipose tissue. In culture, mouse adipose SP cells showed the capacity to undergo in vitro differentiation into osteogenic, chondrogenic and adipogenic lineages. In NOD/SCID mice, freshly sorted mouse adipose SP cells were able to engraft and assist in wound healing. This animal model study showed that adipose SP cells were able to regenerate epithelial layers and connective tissue with minor scar formation. The ability of this novel cell population within adipose tissue to undergo directional differentiation in vitro and to regenerate skin in vivo has potential impact for uses in surgical dermal applications.

Phenotypic and molecular characterization of spermatogonial stem cells in adult primate testes.

BACKGROUND: Knowledge about the identity and characteristics of spermatogonial stem cells (SSCs) in human is very limited. Here, Rhesus monkey was used as an animal model to investigate molecular and phenotypic characteristics of SSCs in the adult testes. METHODS: A variety of immunohistological, molecular biological and functional assays were used to study different populations of SSCs in the adult testes. RESULTS: In adult primate testes, there are distinct populations of CD90+ CD49f+ CD117- (Triple Stained) cells and a small population of stage-specific embryonic antigen-4 (SSEA-4)+ cells which both localized at the basement membrane of seminiferous tubules. Both SSEA-4+ and Triple Stained cells express germ cell and SSC-specific markers and show high telomerase activity; however, only adult Rhesus monkey SSEA-4+ testis cells appear to contain functional and actively dividing SSCs that can repopulate recipient mouse testes following spermatogonial transplantation. DNA analysis of these populations showed that SSEA-4+ cells contain a DNA profile similar to the actively dividing cells, whereas Triple Stained cells showed an accumulated number of cells arrested in the S phase of the cell cycle. SSEA-4+ cells also showed significantly higher proliferation activity, as shown by proliferating cell nuclear antigen staining, than Triple Stained cells (P < 0.01). Interestingly, SSEA-4+ cells expressed a significantly higher level of promyelocytic leukemia zinc finger, a factor required for SSC self-renewal, than Triple Stained cells (P < 0.001). CONCLUSIONS: Our data indicate that Triple Stained cells may represent a quiescent population of SSCs, whereas SSEA-4 might be expressed on a subpopulation of actively dividing SSCs.

Generation of multipotent cell lines from a distinct population of male germ line stem cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Local and systemic B cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosine-phosphate-guanine adjuvant.

Vaccine strategies that stimulate the ocular mucosal immune system (OMIS), the immune barrier that protects the surface of the eye are needed. However, most vaccines fail to induce local ocular immune responses and, in the absence of adjuvant, may induce a state of immunological tolerance. In this study, we present a new vaccine strategy that consists of ocular mucosal (OM) delivery of peptide epitopes, selected from the herpes simplex virus (HSV-1) glycoprotein D (gD) mixed with synthetic immunostimulatory oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG2007). Repeated topical ocular application of gD peptide epitopes and CpG2007 induced peptide-specific and virus-neutralizing IgA/IgG in tears as well as in serum. As a second marker, generation of local and systemic peptide- and virus-specific T cells confirmed the potent immunogenicity of peptides-CpG2007 formulation when applied through the OM route. Moreover, OM delivery of peptides-CpG2007 induced local IFN-gamma and IL-2 responses and low IL-4 production, demonstrating the polarization towards a Th1 response. Immunization, using free CpG2007 ODNs or peptides alone did not produce OMIS stimulation. This novel vaccine strategy may be key for ocular infectious pathogens, such as HSV-1, that require both secretory antibody and the Th1 responses. The results suggest the clinical feasibility of developing an OM delivery system using epitope-based vaccines.

Lipopeptide epitopes extended by an Nepsilon-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity.

Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV-1) gD(1-23) peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either Nepsilon-palmitoyl-lysine (palmitoyl-lipidated peptide, palmitoyl-LP) or cholesterol-lysine (cholesterol-lipidated peptide, cholesterol-LP). Immunization of mice with the palmitoyl-LP, but not with its cholesterol-LP analog, induced a strong T cell-dependent protective immunity against lethal HSV-1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1-type immune response was stimulated by the palmitoyl-LP, as opposed to a Th2 response generated by its cholesterol-LP analog. The palmitoyl-LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time- and dose-dependent manner, and induced phenotypic maturation and production of pro-inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl-LP induced antigen-specific T cell responses through the Toll-like receptor-2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

Development of T cells expressing an altered TCR complex.

Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.