Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome.

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health.

Cytokine-dependent bidirectional connection between impaired social behavior and susceptibility to seizures associated with maternal immune activation in mice.

Maternal immune activation (MIA) results in the development of autism in the offspring via hyperactivation of IL-6 signaling. Furthermore, experimental studies showed that the MIA-associated activation of interleukin-1ß (IL-1ß) concurrently with IL-6 increases the rate and the severity of hippocampal kindling in mice, thus, offering an explanation for autism-epilepsy comorbidity. We examined whether epileptic phenotype triggered by prenatal exposure to IL-6 and IL-1ß combination is restricted to kindling or whether it is reproducible in another model of epilepsy, whereby spontaneous seizures develop following kainic acid (KA)-induced status epilepticus. We also examined whether in mice prenatally exposed to IL-6 and IL-6+IL-1ß, the presence of spontaneous seizures would exacerbate autism-like features. Between days 12 and 16 of pregnancy, C57BL/6J mice received daily injections of IL-6, IL-1ß, or IL-6+IL-1ß combination. At postnatal day 40, male offspring were examined for the presence of social behavioral deficit, and status epilepticus was induced by intrahippocampal KA injection. After 6weeks of monitoring for spontaneous seizures, sociability was tested again. Both IL-6 and IL-6+IL-1ß offspring presented with social behavioral deficit. Prenatal exposure to IL-6 alleviated, while such exposure to IL-6+IL-1ß exacerbated, the severity of KA-induced epilepsy. Increased severity of epilepsy in the IL-6+IL-1ß mice correlated with the improvement of autism-like behavior. We conclude that complex and not necessarily agonistic relationships exist between epileptic and autism-like phenotypes in an animal model of MIA coupled with KA-induced epilepsy and that the nature of these relationships depends on components of MIA involved.

Evaluating non-adherence to immunosuppressant medications in pediatric liver transplant recipients.

Non-adherence with recommended immunosuppressant medications is common post-pediatric liver transplant and is the most important reason for organ rejection in long-term survivors. However, there is currently no validated, standard method to measure adherence, with a well-defined threshold, making it extremely difficult to evaluate interventions to improve adherence. Previous studies have suggested that the degree of fluctuation of medication blood levels over time can provide an idea about how regularly the medication is being taken. The present study, conducted at UCLA medical center, sought to identify a specific threshold value of the s.d. of individual tacrolimus blood levels in pediatric liver transplant recipients which would be associated with rejection episodes in these patients. A threshold of 3.0 has been identified in other studies, and was supported by the analysis of retrospective data from 96 subjects. However, further analysis found that a s.d. of 2.5 appeared to have a better fit with the data. These findings suggest the utility of monitoring the s.d. of routine tacrolimus blood levels in pediatric liver transplant recipients for detecting non-adherence to immunosuppressant medication prior to clinical rejection, allowing earlier interventions.

Utilisation of a Bioreactor for Culture and Expansion of Epithelial Cells without the use of Trypsin or Enzymes.

OBJECTIVE: To develop a bioreactor for automated culture, maintenance, and collection of normal human keratinocytes using an enzyme-free propagation method. METHODS: The culture of normal human epithelial keratinocytes was compared in two culture methods - a study team-developed automated bioreactor utilising an enzyme-free passage method, and a manual culture method. Cell size, glucose utilisation, and the proliferative capacity of the two cultures were evaluated. RESULTS: An automated bioreactor, not using enzymes for passage, but instead using the novel Epithelial Pop Up Keratinocytes (ePUK)1 culture technique, resulted in an extended culture longevity and proliferative capacity in normal primary human keratinocytes. Daughter cells were collected up to three times per day utilising the bioreactor. The daughter cells produced by the bioreactor were smaller than daughter cells produced by the manual culture method. The proliferative capacity and health of the parent monolayer within both the bioreactor and the manual culture flask was dependent upon sufficient glucose availability. Due to the contact inhibition nature of epithelial keratinocytes, the bioreactor enabled the study of an adherent cell type soon after cytokinesis and before the cell has integrated as part of an adherent matrix. CONCLUSION: The study demonstrates that increasing the number of media changes per day as necessary, based on glucose utilisation, is necessary for prolonged longevity and functional productivity of ePUK cultures.

Autism-Like Behavior in BTBR Mice Is Improved by Electroconvulsive Therapy.

Autism is a developmental disorder characterized by impairments in social and communication abilities, as well as by restricted and repetitive behaviors. Incidence of autism is higher than earlier estimates, and treatments have limited efficacy and are costly. Limited clinical and experimental evidence suggest that patients with autism may benefit from electroconvulsive therapy (ECT). We examined the therapeutic potential of ECT in BTBR T+ tf/j mice, which represent a validated model of autism. A series of 13 electroconvulsive shocks (ECS) delivered twice a day over 7 days reversed core autism-like behavioral abnormalities-impaired sociability, social novelty, and repetitive behavior-when the animals were tested 24 h after the last ECS. The effect lasted up to 2 weeks after ECT. Neither single ECS nor a series of 6 ECS modified animals' behavior. Chronic infusion into the lateral brain ventricle of a preferential oxytocin receptor blocker (2S)-2-Amino-N-[(1S,2S,4R)-7,7-dimethyl-1-[[[4-(2-methylphenyl)-1-piperazinyl]sulfonyl]methyl]bicyclo[2.2.1]hept-2-yl]-4-(methylsulfonyl)butanamide hydrochloride abolished ECT-induced improvement of sociability and mitigated improvement of social novelty but did not affect ECT-induced reversal of repetitive behavior. These proof-of-principle experiments suggest that ECT may, indeed, be useful in the treatment of autism, and that its therapeutic effects may be mediated, in part, by central oxytocin signaling.

Fabrication of Large Size Ex Vivo-Produced Oral Mucosal Equivalents for Clinical Application.

The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 µm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 µm) to the thinnest (228.6-330.2 µm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME graft for reconstructing large mucosa defects.

The effects of exogenous p53 overexpression on HPV-immortalized and carcinogen transformed oral keratinocytes.

BACKGROUND: Overexpression of p53 in head and neck carcinoma cells has demonstrated tumor growth suppression using in vitro and in vivo models. The effects of exogenous overexpression of wild-type p53 on human papilloma virus (HPV)-immortalized and carcinogen transformed oral keratinocytes were determined. METHODS: The p53 gene was overexpressed in IHGK (immortalized human gingival keratinocyte), IHGKN [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)]-carcinogen transformed keratinocytes, and two head and neck squamous carcinoma (HNSCC) cell lines, HN30 and HN12. The transfection efficiency, growth suppression, and inhibition of the cell cycle along with the induction of apoptosis were measured. RESULTS: Transfections with adenoviruses were more efficient for IHGK cells than for IHGKN, HN12, and HN30 cells. Inhibition of proliferation in all cell lines was proportional to the viral particle to cell (VPC) ratios. IHGK cells were more sensitive to p53 than IHGKN cells. HN12 cells were more suppressed than HN30 cells. HN12 were the most suppressed at 72 hours whereas HN30 cells were most suppressed at 24 hours. Expression of exogenous p53-induced G1 cell cycle arrest and p21 expression as VPC ratios increased in IHGK and IHGKN cell lines. Apoptosis also was induced in these cells by p53 as VPC increased. IHGK cells were more sensitive to p53-induced growth inhibition, cell cycle regulation, p21 expression and apoptosis than IHGKN cells. HN12 (mutated p53) cells were more sensitive to p53 overexpression than HN30 (wild-type p53) cells. Gene transfer and expression of exogenous p53 by using Ad-p53 demonstrates suppressive effects on HPV immortalized and carcinogen transformed oral keratinocytes. CONCLUSIONS: Cell cycle regulation by gene transfer is feasible in immortalized oral keratinocytes. Carcinogen transformed cells are less susceptible to the effects of p53 overexpression. Expression of exogenous p53 through p53 gene transfer can suppress HPV immortalization and carcinogen transformation in oral keratinocytes. The sensitivity of HNSCC cell lines to p53-induced cell cycle regulation and apoptosis is variable and dependent on the cell line and duration of exposure. In vitro results using p53 gene transfer must be validated in clinical studies with patients at risk for HNSCC.