Hypothermia Augments Neuroprotective Activity of Mesenchymal Stem Cells for Neonatal Hypoxic-Ischemic Encephalopathy

Though hypothermia is the only clinically available treatment for neonatal hypoxic-ischemic encephalopathy (HIE), it is not completely effective in severe cases. We hypothesized that combined treatment with hypothermia and transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) would synergistically attenuate severe HIE compared to stand-alone therapy. To induce hypoxia-ischemia (HI), male Sprague-Dawley rats were subjected to 8% oxygen for 120 min after unilateral carotid artery ligation on postnatal day (P) 7. After confirmation of severe HIE involving >50% of the ipsilateral hemisphere volume as determined by diffusion-weighted brain magnetic resonance imaging (MRI) within 2 h after HI, intraventricular MSC transplantation (1 × 105 cells) and/or hypothermia with target temperature at 32°C for 24 h were administered 6 h after induction of HI. Follow-up brain MRI at P12 and P42, sensorimotor function tests at P40–42, evaluation of cytokines in the cerebrospinal fluid (CSF) at P42, and histologic analysis of peri-infarct tissues at P42 were performed. Severe HI resulted in progressively increased brain infarction over time as assessed by serial MRI, increased number of cells positive for terminal deoxynucleotidyl transferase nick-end labeling, microgliosis and astrocytosis, increased CSF cytokine levels, and impaired function in behavioral tests such as rotarod and cylinder tests. All of the abnormalities observed in severe HIE showed greater improvement after combined treatment with hypothermia and MSC transplantation than with either therapy alone. Overall, these findings suggest that combined treatment with hypothermia and human UCB-derived MSC transplantation might be a novel therapeutic modality to improve the prognosis of severe HIE, an intractable disease that currently has no effective treatment.     

Structural Brain Changes after Traditional and Robot-Assisted Multi-Domain Cognitive Training in Community-Dwelling Healthy Elderly

The purpose of this study was to investigate if multi-domain cognitive training, especially robot-assisted training, alters cortical thickness in the brains of elderly participants. A controlled trial was conducted with 85 volunteers without cognitive impairment who were 60 years old or older. Participants were first randomized into two groups. One group consisted of 48 participants who would receive cognitive training and 37 who would not receive training. The cognitive training group was randomly divided into two groups, 24 who received traditional cognitive training and 24 who received robot-assisted cognitive training. The training for both groups consisted of daily 90-min-session, five days a week for a total of 12 weeks. The primary outcome was the changes in cortical thickness. When compared to the control group, both groups who underwent cognitive training demonstrated attenuation of age related cortical thinning in the frontotemporal association cortices. When the robot and the traditional interventions were directly compared, the robot group showed less cortical thinning in the anterior cingulate cortices. Our results suggest that cognitive training can mitigate age-associated structural brain changes in the elderly.

Trial Registration

ClnicalTrials.gov NCT01596205     

Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Strain Biofilms

Background

Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo.

Methods and Results

Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms.

Conclusion/Major Finding

This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may be used to control or eradicate S. aureus biofilm-related infections.     

Cellular Size as a Means of Tracking mTOR Activity and Cell Fate of CD4+ T Cells upon Antigen Recognition

mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTOR^hi and mTOR^lo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTOR^hi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTOR^lo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTOR^lo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.     

Adoptive Transfer of Treg Cells Combined with Mesenchymal Stem Cells Facilitates Repopulation of Endogenous Treg Cells in a Murine Acute GVHD Model

Therapeutic effects of combined cell therapy with mesenchymal stem cells (MSCs) and regulatory T cells (Treg cells) have recently been studied in acute graft-versus-host-disease (aGVHD) models. However, the underlying, seemingly synergistic mechanism behind combined cell therapy has not been determined. We investigated the origin of Foxp3⁺ Treg cells and interleukin 17 (IL-17⁺) cells in recipients following allogeneic bone marrow transplantation (allo-BMT) to identify the immunological effects of combined cell therapy. Treg cells were generated from eGFP-expressing C57BL/6 mice (Treg^egfp cells) to distinguish the transferred Treg cells; recipients were then examined at different time points after BMT. Systemic infusion of MSCs and Treg cells improved survival and GVHD scores, effectively downregulating pro-inflammatory Th×and Th17 cells. These therapeutic effects of combined cell therapy resulted in an increased Foxp3⁺ Treg cell population. Compared to single cell therapy, adoptively transferred Treg^egfp cells only showed prolonged survival in the combined cell therapy group on day 21 after allogeneic BMT. In addition, Foxp3⁺ Treg cells, generated endogenously from recipients, significantly increased. Significantly higher levels of Treg^egfp cells were also detected in aGVHD target organs in the combined cell therapy group compared to the Treg cells group. Thus, our data indicate that MSCs may induce the long-term survival of transferred Treg cells, particularly in aGVHD target organs, and may increase the repopulation of endogenous Treg cells in recipients after BMT. Together, these results support the potential of combined cell therapy using MSCs and Treg cells for preventing aGVHD.     

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02 µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (V_mem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only V_mem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and V_mem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.