Beneficial effects of xenon inhalation on behavioral changes in a valproic acid-induced model of autism in rats.

BACKGROUND: Xenon (Xe) is a noble gas that has been used for the last several decades as an anesthetic during surgery. Its antagonistic effect on glutamate subtype of NMDA (N-methyl-D-aspartate) receptors resulted in evaluation of this gas for treatment of CNS pathologies, including psychoemotional disorders. The aim of this study was to assess the behavioral effects of acute inhalation of subanesthetic concentrations of Xe and to study the outcomes of Xe exposure in valproic acid (VPA)-induced rodent model of autism. METHODS: We have conducted two series of experiments with a battery of behavioral tests aimed to evaluate locomotion, anxiety- and depression-like behavior, and social behavior in healthy, VPA-treated and Xe-exposed young rats. RESULTS: We have shown that in healthy animals Xe exposure resulted in acute and delayed decrease of exploratory motivation, partial decrease in risk-taking and depressive-like behavior as well as improved sensorimotor integration during the negative geotaxis test. Acute inhalations of Xe in VPA-exposed animals led to improvement in social behavior, decrease in exploratory motivation, and normalization of behavior in forced-swim test. CONCLUSION: Behavioral modulatory effects of Xe are probably related to its generalized action on excitatory/inhibitory balance within the CNS. Our data suggest that subanesthetic short-term exposures to Xe have beneficial effect on several behavioral modalities and deserves further investigation.

Comparing atmospheric and hypoxic cultured mesenchymal stem cell transcriptome: implication for stem cell therapies targeting intervertebral discs.

BACKGROUND: Mesenchymal stem cells (MSCs) represent an attractive avenue for cellular therapies targeting degenerative diseases. MSC in vitro expansion is required in order to obtain therapeutic numbers during the manufacturing process. It is known that culture conditions impact cellular properties and behavior after in vivo transplantation. In this study, we aimed at evaluating the benefit of hypoxic culturing of human bone marrow derived mesenchymal stem cells on cell fitness and whole genome expression and discussed its implication on cellular therapies targeting orthopedic diseases such as chronic lower back pain. METHODS: Human bone marrow mesenchymal stem cells (hBMMSCs) were isolated from fresh human anticoagulated whole bone marrow and were cultured side by side in atmospheric (20% O2) and hypoxic (5% O2) oxygen partial pressure for up to 3 passages. Stem cell fitness was assessed by clonogenic assay, cell surface marker expression and differentiation potential. Whole genome expression was performed by mRNA sequencing. Data from clonogenic assays, cell surface marker by flow cytometry and gene expression by quantitative PCR were analyzed by two-tailed paired Student's t-test. Data from mRNA sequencing were aligned to hg19 using Tophat-2.0.13 and analyzed using Cufflinks-2.1.1. RESULTS: Hypoxic culturing of hBMMSCs had positive effects on cell fitness, as evidenced by an increased clonogenicity and improved differentiation potential towards adipocyte and chondrocyte lineages. No difference in osteoblast differentiation or in cell surface markers were observed. Only a small subset of genes (34) were identified by mRNA sequencing to be significantly dysregulated by hypoxia. When clustered by biological function, these genes were associated with chondrogenesis and cartilage metabolism, inflammation and immunomodulation, cellular survival, migration and proliferation, vasculogenesis and angiogenesis. CONCLUSIONS: Hypoxic culturing positively impacted hBMMSCs fitness and transcriptome, potentially improving inherent properties of these cells that are critical for the development of successful cellular therapies. Hypoxic culturing should be considered for the in vitro expansion of hBMMSCs during manufacturing of cellular therapies targeting orthopedic disorders such as lower back pain.

Induction of alloimmune tolerance in heart transplantation through gene silencing of TLR adaptors.

Toll-like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4(+) CD25(+) FoxP3(+) regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.

Targeted gene silencing of TLR4 using liposomal nanoparticles for preventing liver ischemia reperfusion injury.

RNAi-based therapy is a promising strategy for the prevention of ischemia-reperfusion injury (IRI). However, systemic administration of small interfering RNA (siRNA) may cause globally nonspecific targeting of all tissues, which impedes clinical use. Here we report a hepatocyte-specific delivery system for the treatment of liver IRI, using galactose-conjugated liposome nanoparticles (Gal-LipoNP). Heptocyte-specific targeting was validated by selective in vivo delivery as observed by increased Gal-LipoNP accumulation and gene silencing in the liver. Gal-LipoNP TLR4 siRNA treatment resulted in a significant decrease of serum alanine transferase (ALT) and aspartate transaminase (AST) in a hepatic IRI model. Histopathology displayed an overall reduction of the injury area in the Gal-LipoNP TLR4 siRNA treated mice. Additionally, neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by MPO, MDA and ROS respectively, were attenuated after Gal-LipoNP TLR4 siRNA treatment. Moreover, therapeutic effects of Gal-LipoNP TLR4 siRNA were associated with suppression of the inflammatory cytokines IL-1 and TNF-α. Taken together, this study is the first demonstration of liver IRI treatment using liver-specific siRNA delivery.

Outstanding survival and regeneration process by the use of intelligent acellular dermal matrices and mesenchymal stem cells in a burn pig model.

A pig model with a deep large burn was used to study the regeneration process induced by mesenchymal stem cells (MSCs) and acellular pig dermal matrices, made intelligent by the combination with biodegradable nanofibers loaded with growth factors (granulocyte-macrophage colony-stimulating factor and epidermal growth factor) and coated with the anti-CD44 monoclonal antibody (intelligent acellular dermal matrices, IADMs). These IADMs are specially designed to integrate in the wound bed as new biological scaffolds as well as to specifically recruit and attach circulating and/or externally applied MSCs through the anti-CD44 antibody while delivering precise amounts of growth factors. In this way, the reparative process as well as the aesthetic and functional results were enhanced in our burn model. The animal survived, the wound was completely closed, and total regeneration of the skin was obtained without much scarring. Surprisingly, hair follicles and other skin appendages developed despite the severity and deepness of the burn. Even burned muscles and ribs seemed to have undergone a regenerative process by the end of the study. Based on these findings, we have proposed the use of IADMs and autologous, allogeneic or xenogeneic MSCs, as a new paradigm for the future treatment of large burns and probably other dermatological and cosmetic human conditions.

Circulating endothelial progenitor cells and erectile dysfunction: possibility of nutritional intervention?

To provide an overview of molecular and cellular processes involved in erectile dysfunction (ED) with emphasis on circulating endothelial progenitor cells (EPC) and discuss possible nutraceutical means of intervention. A review of literature on Pubmed related to EPC and ED was conducted. Patients with ED appear to possess a reduced number of circulating EPC, which is associated with poor endothelial function possibly as a result of underlying low-grade inflammation. Several studies support the possibility of improving erectile function by inhibition of inflammation as well as administration of various stem cell types. One particularly interesting approach is nutraceutical supplementation to increase circulating EPC, as demonstrated in the product Stem-Kine. Interventions aimed at increasing circulating EPC may have potential in treatment of vascular ED.

Inherent and iatrogenic immune defects in hairy cell leukemia: revisited.

IMPORTANCE OF THE FIELD: T- and NK-cell abnormalities, decreased number and function of monocytes, neutrophils and dendritic cells, decreased production of certain cytokines and increased incidence of certain autoimmune conditions have been identified in untreated hairy cell leukemia (HCL) patients. These alterations are responsible for an increased rate of infections and additional malignancies in HCL. AREAS COVERED IN THIS REVIEW: The authors offer a focused review of the most relevant preclinical and clinical studies exploring the immune abnormalities in both untreated and treated HCL. WHAT THE READER WILL GAIN: The use of potent immunosuppressive agents such as cladribine and pentostatin in HCL therapeutics has generated increasing concerns about the likelihood of additional immune impairments in these patients. While the NK cells, monocytes and neutrophils were shown to recover shortly after complete responses are achieved with these agents, the CD4(+) T-cell counts may require in excess of 2 - 3 years in order to re-enter the normal range. TAKE HOME MESSAGE: Given the advent of new molecular, genetic and immunologic techniques, a comprehensive characterization of the immune abnormalities in untreated and treated HCL represents a reachable goal and could translate into improved outcomes in clinical practice.

Inhibition of allergic responses by CD40 gene silencing.

BACKGROUND: Gene silencing using small interfering RNA (siRNA) is a potent method of specifically knocking down molecular targets. Small interfering RNA is therapeutically promising, however, treatment of allergic diseases with siRNA has not been explored in vivo. The aim of this study was to evaluate therapeutic effects of CD40 siRNA on inhibition of allergic responses. METHODS: Mice sensitized with ovalbumin (OVA) and alum were treated with CD40 siRNA, scrambled siRNA, or phosphate buffer saline (PBS) alone, and then challenged intranasally with OVA. RESULTS: A significant reduction in nasal allergic symptoms was observed in the CD40 siRNA treated OVA-allergic mice compared to the controls of scrambled siRNA and PBS alone, which is correlated with the decrease of local eosinophil accumulation. CD40 siRNA treatment knocked down CD40 expression on dendritic cells (DCs) in vivo and impaired their antigen presenting function. Treatment with CD40 siRNA resulted in inhibition of OVA-specific T cell response and decrease of interleukin-4 (IL-4), IL-5, and interferon-gamma production from T cells stimulated with OVA. Administration of CD40 siRNA also suppressed CD40 expression on B cells, resulting in down-regulation of OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a levels. Additionally, increased regulatory T cells were observed in the CD40 siRNA treated mice. CONCLUSIONS: The present study demonstrates a novel therapeutic use for siRNA in allergy. CD40 siRNA attenuated allergy through inhibition of DC and B cell functions and generation of regulatory T (Treg) cells.

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice.

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen.

Prevention of bladder tumor formation in mice by a novel bone marrow-derived factor, reptimed.

BACKGROUND: Reptimed is a novel, species-conserved, bone marrow-derived molecule which possesses anti-neoplastic activity. Previously, we established an orthotopic murine bladder tumor (MBT-2) model and reported accurate documentation of the presence and the extent of intravesical involvement of bladder tumor implants using magnetic resonance imaging (MRI) (1). Herein, we investigated the activity of exogenously administered Reptimed in the MBT-2 model. MATERIALS AND METHODS: Intravesicular and intraperitoneal administration of Reptimed concurrently with and following transurethral tumor cell implantation was performed and MBT-2 tumor response was assessed at several time points post tumor implant. RESULTS: Serial MRI scans of Reptimed-treated mice at days 14 to 33 post tumor transplant revealed significant inhibition of bladder tumor growth with no significant tumor growth observed by MRI on day 33 post-implant. The corresponding histological examination of the whole mount bladder sections revealed similar inhibitory effects of Reptimed with respect to the topography and depth of intravesical tumor involvement. In contrast, control, untreated bladders revealed extensive exophytic tumors with deeply invasive transitional cell carcinoma. CONCLUSIONS: These studies demonstrate the anti-tumor effect of Reptimed and highlight its importance as a potential therapy for cancer.