Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism.

Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD.

Total cytokine immunoassay: a more accurate method of cytokine measurement?

BACKGROUND: Cytokines signal the normal processes of inflammation and repair in all organs, yet the aberrant expression of these peptide mediators is associated with significant organ dysfunction. The accurate measurement of cytokines is therefore critical. In this study, we sought to investigate the alterations in cytokine expression early after trauma in humans using a new competitive binding immunoassay that measures both free and bound cytokine and compare this with standard enzyme-linked immunosorbent assay (ELISA), which measures only free cytokine. METHODS: Peripheral blood was obtained from trauma patients at admission. Exclusion criteria were transfers, death within 24 hours, pregnancy, known acquired immunodeficiency syndrome, chemotherapy, transplant, or other chronic immune disorder. "Total" cytokine immunoassay was compared with ELISA for cytokines (interleukin [IL]-1, IL-6, and IL-10) measured in serum. RESULTS: Cytokine concentrations measured by total immunoassay were significantly higher (10- to 500-fold increase) than those measured by ELISA, and correlation between the two methods was poor (r2 = 0.193 for IL-10). No significant differences in mean serum cytokine concentrations were noted between trauma patients and normal controls for IL-1 (56 vs. 37 pg/mL), IL-6 (16 vs. 25 pg/mL), and IL-10 (4 vs. 26 pg/mL) using the ELISA method. In contrast, trauma patients had significantly higher serum concentrations of IL-1 (3,320 vs. 1,470 pg/mL, p < 0.05), IL-6 (2,415 vs. 1,048 pg/mL, p < 0.05), and IL-10 (2,307 vs. 1,480 pg/mL, p < 0.05) at admission compared with normal controls using total cytokine immunoassays. CONCLUSION: Cytokine measurements in peripheral blood in trauma patients and normal controls are significantly (10- to 500-fold) higher when using a total cytokine assay that measures both free and bound cytokine. Competitive immunoassays may be the method of choice when measuring endogenous cytokine levels in biologic fluids, and new normal ranges for cytokines must be established for future accurate research in critical care and trauma.

Calcitriol-mediated modulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-2.

Calcitriol-induced differentiation of U937 mononuclear phagocytes is known to have divergent effects on the synthesis of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). In this study, we sought to determine whether calcitriol affects the expression of these proteins by modulating intermediate signal transduction involving intracellular calcium and protein kinase C (PKC). U937 cells were stimulated with calcitriol (50 nM) for 6-72 hr, inducing a transient increase in specific binding of [3H]phorbol dibutyrate ([3H]PDBu), seen only after 24 hr. Staurosporine (2 nM), a PKC inhibitor, had no effect on calcitriol-induced secretion of plasminogen activator (PA) activity. However, staurosporine significantly (P less than 0.05) inhibited the ability of calcitriol to enhance phorbol myristate acetate (PMA)-induced secretion of PA inhibitor activity, indicating that this priming effect of calcitriol requires expression of PKC. The calcium ionophore A23187 (0.1 microM) induced a modest increase in secreted PA inhibitor activity, in contrast to the secretion of PA activity which is consistently seen in response to calcitriol. Northern blot analysis demonstrated that A23187 induced an increase in PAI-2 mRNA and a marked reduction in uPA mRNA, while calcitriol induced opposite changes in both mRNA species. We conclude that calcitriol modulates uPA and PAI-2 expression by multiple mechanisms that are both PKC dependent and PKC independent. Our studies also demonstrated that increased intracellular calcium alters the synthesis of both uPA and PAI-2 in a manner which favors expression of PA inhibitor activity.

Expression of heterogeneous profiles of plasminogen activators and plasminogen activator inhibitors by human glioma lines.

Expression of plasminogen activator (PA) enzyme activity is believed to be one of the mechanisms by which malignant cells cause pericellular proteolysis of stromal structures during implantation and tissue invasion. In this study, four cell lines derived from human gliomas were studied to ascertain which PA enzymes and PA inhibitors determine the level of secreted PA activity. A plasminogen-dependent esterolytic assay was used, and two lines (U251 and U373) were found to secrete high levels of PA activity, while PA activity was undetectable in the conditioned media from the remaining two lines (U138 and LM). The PA produced by U251 and U373 resolved as single bands comigrating with high molecular weight urokinase (Mr 54,000) on casein-plasminogen zymography. Northern blot analysis demonstrated high levels of mRNA for urokinase-type PA (uPA) in U251 and U373, as well as a considerably lower level of uPA message in LM. U251 and U373 also contained mRNA for tissue-type PA (tPA), although secreted tPA activity was not demonstrated by zymography. The U138 line contained essentially undetectable levels of mRNA for either uPA or tPA. U138 was also unique in secreting PA inhibitor activity and contained high levels of mRNA for PA inhibitor 2, which was not seen in any other line. All cell lines contained PA inhibitor 1 mRNA, with substantially more expression in the U138 and LM lines than in U251 and U373. None of the lines secreted measureable anti-plasmin activity. We conclude that there is considerable heterogeneity among human glioma cells in expression of PA enzymes and PA inhibitors. The coordinated regulation of these proteins likely determines secreted PA activity and the resultant role of plasminogen activation in tumor implantation and invasion.