Use of trimetasphere metallofullerene MRI contrast agent for the non-invasive longitudinal tracking of stem cells in the lung.

Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the ​in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a 'proof-of-concept' experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course.

Intestinal and systemic inflammatory responses are positively associated with sulfidogenic bacteria abundance in high-fat-fed male C57BL/6J mice.

Recent studies have highlighted the relation between high-fat (HF) diets, the gut microbiota, and inflammation. However, the role of sulfidogenic bacteria in mediating these effects has been explored only recently. Therefore, we tested the hypothesis that an HF diet rich in saturated fat stimulates sulfidogenic bacteria and that these increases correlate with intestinal and systemic inflammatory responses. Forty C57BL/6J male mice were fed a low-fat (LF; 10% of energy) or an HF lard-based (60% of energy) diet for 6 or 20 wk. Mucosa samples were collected from the ileum, cecum, and colon and used for measuring 16S ribosomal RNA and functional genes of sulfidogenic bacteria. Matching intestinal samples and visceral and subcutaneous white adipose tissue (WAT) depots were used to measure mRNA abundance for inflammatory genes. Mice fed the HF diet had greater (P < 0.05) abundance of 3 types of sulfidogenic bacteria, primarily in colonic mucosa, compared with LF-fed mice at week 20. Although HF feeding did not increase intestinal inflammation at week 6, ileal markers of macrophage infiltration and inflammation were upregulated (P < 0.05) 1- to 6-fold at week 20. HF feeding impaired the localization of the tight junction protein zonula occludens 1 at the apical area of the ileal epithelium at weeks 6 and 20. Mice fed the HF diet had 1- to 100-fold greater (P < 0.05) mRNA levels of markers of macrophage infiltration in visceral and subcutaneous WAT at week 20, but not at week 6, compared with LF-fed mice. These results provide evidence that chronic, but not acute, consumption of an HF lard-based diet may be linked with pathways of microbial metabolism that potentially contribute to chronic intestinal and systemic inflammation. Such linkage provides further support for reducing consumption of saturated fats.

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes.

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.

Conjugated linoleic acid reduces adiposity and increases markers of browning and inflammation in white adipose tissue of mice.

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.

Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.

Leucine is recognized as a nutrient signal; however, the long-term in vivo consequences of leucine signaling and the role of branched-chain amino acid (BCAA) metabolism in this signaling remain unclear. To investigate these questions, we disrupted the BCATm gene, which encodes the enzyme catalyzing the first step in peripheral BCAA metabolism. BCATm(-/-) mice exhibited elevated plasma BCAAs and decreased adiposity and body weight, despite eating more food, along with increased energy expenditure, remarkable improvements in glucose and insulin tolerance, and protection from diet-induced obesity. The increased energy expenditure did not seem to be due to altered locomotor activity, uncoupling proteins, sympathetic activity, or thyroid hormones but was strongly associated with food consumption and an active futile cycle of increased protein degradation and synthesis. These observations suggest that elevated BCAAs and/or loss of BCAA catabolism in peripheral tissues play an important role in regulating insulin sensitivity and energy expenditure.

Transocular entry of seasonal influenza-attenuated virus aerosols and the efficacy of n95 respirators, surgical masks, and eye protection in humans.

BACKGROUND: The efficacy of barrier precautions to prevent influenza transmission is unknown. METHODS: Twenty-eight participants were exposed to monodispersed live attenuated influenza vaccine (LAIV) particles (4.9 µm) in 6 groups: group 1, no precautions; group 2, ocular exposure only; group 3, surgical mask without eye protection; group 4, surgical mask with eye protection; group 5, fit-tested N95 respirator without eye protection; and group 6, fit-tested N95 respirator with eye protection. Influenza was detected by reverse-transcription polymerase chain reaction (RT-PCR) and culture in nasal washes. Exact 95% confidence intervals (CIs) were calculated. RESULTS: Influenza was detected in 4 of 4 participants in group 1 (95% CI, 0-.60), 3 of 4 in group 2 (95% CI, .006-.806]), 5 of 5 in group 3 (95% CI, 0-.522), 5 of 5 in group 4, (95% CI, 0-.522), 3 of 5 in group 5 (95% CI, .053-.853), and 1 of 5 in group 6 (95% CI, .05-.72). RT-PCR revealed significant differences between group 1 and all other groups except group 3. CONCLUSIONS: Transocular transmission of LAIV occured in most participants suggesting the necessity of eye protection. An N95 respirator provided the best guard further enhanced by eye protection.

Amnion membrane hydrogel and amnion membrane powder accelerate wound healing in a full thickness porcine skin wound model.

There is a need for effective wound treatments that retain the bioactivity of a cellular treatment, but without the high costs and complexities associated with manufacturing, storing, and applying living biological products. Previously, we developed an amnion membrane-derived hydrogel and evaluated its wound healing properties using a mouse wound model. In this study, we used a full thickness porcine skin wound model to evaluate the wound-healing efficacy of the amnion hydrogel and a less-processed amnion product comprising a lyophilized amnion membrane powder. These products were compared with commercially available amnion and nonamnion wound healing products. We found that the amnion hydrogel and amnion powder treatments demonstrated significant and rapid wound healing, driven primarily by new epithelialization versus closure by contraction. Histological analysis demonstrated that these treatments promote the formation of a mature epidermis and dermis with similar composition to healthy skin. The positive skin regenerative outcomes using amnion hydrogel and amnion powder treatments in a large animal model further demonstrate their potential translational value for human wound treatments.

Low level of trans-10, cis-12 conjugated linoleic acid decreases adiposity and increases browning independent of inflammatory signaling in overweight Sv129 mice.

The objective of this study was to determine the extent to which a low level of trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) decreases adiposity and increases browning in overweight mice, its dependence on inflammatory signaling and potential synergistic effects of daily exercise. Young, Sv129 male mice were fed a high-fat diet for 5 weeks to make them fat and glucose intolerant and then switch them to a low-fat diet with or without 0.1% 10,12 CLA, sodium salicylate or exercise for another 7 weeks. 10,12 CLA decreased white adipose tissue (WAT) and brown adipose tissue mass, and increased the messenger RNA and protein levels, and activities of enzymes associated with thermogenesis or fatty acid oxidation in WAT. Mice fed 10,12 CLA had lower body temperatures compared to controls during cold exposure, which coincided with decreased adiposity. Although sodium salicylate decreased 10,12 CLA-mediated increases in markers of inflammation in WAT, it did not affect other outcomes. Exercise had no further effect on the outcomes measured. Collectively, these data indicate that 10,12 CLA-mediated reduction of adiposity is independent of inflammatory signaling, and possibly due to up-regulation of fatty acid oxidation and heat production in order to regulate body temperature. Although this low level of 10,12 CLA reduced adiposity in overweight mice, hepatomegaly and inflammation are major health concerns.

Isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success. hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Oleic acid attenuates trans-10,cis-12 conjugated linoleic acid-mediated inflammatory gene expression in human adipocytes.

The weight loss supplement conjugated linoleic acid (CLA) consists of an equal mixture of trans-10,cis-12 (10,12) and cis-9,trans-11 (9,11) isomers. However, high levels of mixed CLA isomers, or the 10,12 isomer, causes chronic inflammation, lipodystrophy, or insulin resistance. We previously demonstrated that 10,12 CLA decreases de novo lipid synthesis along with the abundance and activity of stearoyl-CoA desaturase (SCD)-1, a δ-9 desaturase essential for the synthesis of monounsaturated fatty acids (MUFA). Thus, we hypothesized that the 10,12 CLA-mediated decrease in SCD-1, with the subsequent decrease in MUFA, was responsible for the observed effects. To test this hypothesis, 10,12 CLA-treated human adipocytes were supplemented with oleic acid for 12 h to 7 days, and inflammatory gene expression, insulin-stimulated glucose uptake, and lipid content were measured. Oleic acid reduced inflammatory gene expression in a dose-dependent manner, and restored the lipid content of 10,12 CLA-treated adipocytes without improving insulin-stimulated glucose uptake. In contrast, supplementation with stearic acid, a substrate for SCD-1, or 9,11 CLA did not prevent inflammatory gene expression by 10,12 CLA. Notably, 10,12 CLA impacted the expression of several G-protein coupled receptors that was attenuated by oleic acid. Collectively, these data show that oleic acid attenuates 10,12 CLA-induced inflammatory gene expression and lipid content, possibly by alleviating cell stress caused by the inhibition of MUFA needed for phospholipid and neutral lipid synthesis.

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism.

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.