Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge

In this study, the potential of the bare skin as a non-invasive route for vaccination was examined. Following application of heat-labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti-LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes. In addition, LT enhanced the serum and mucosal antibody and proliferative T-cell responses to the model protein antigen beta-galactosidase (beta-gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect. When a peptide representing a T-helper epitope (aa 307-319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide- and virus-specific T cells, as measured in vitro by the interleukin-2 (IL-2) secretion assay. LT was shown to be as immunogenic as CT. Binding activity to GM1 gangliosides was essential for effective induction of anti-CT serum and mucosal antibody responses. Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin. These findings give further support to a growing body of evidence on the potential of skin as a non-invasive route for vaccine delivery. This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical.     

In vivo regulation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt) phosphorylation by acute and chronic morphine

In vitro evidence suggests that extracellular signal-regulated protein kinases (ERKs) and Akt (also referred to as protein kinase B) are among the myriad of intracellular signaling molecules regulated by opioid receptors. The present study examined the regulation of ERK and Akt activation in the nucleus accumbens and caudate putamen following acute and chronic morphine administration in the rat. ERK and Akt are activated by phosphorylation, hence the levels of phosphorylated ERK (pERK) and Akt (pAkt) as well as total levels of ERK and Akt protein were measured by Western blot analysis. Male Sprague-Dawley rats received either a single injection of morphine or twice daily injections of morphine for 6 or 10 days. Following acute morphine, pERK levels were significantly decreased in the nucleus accumbens but not in the caudate putamen. Phosphorylated Akt levels in the nucleus accumbens were significantly increased after a single morphine injection. Naltrexone pretreatment prevented both the morphine-induced pERK down-regulation and pAkt up-regulation. Although reductions in pERK levels were evident after 6 days of morphine administration, no differences were observed in pERK levels after 10 days. In contrast to the up-regulation seen after acute morphine, pAkt levels in the nucleus accumbens were significantly decreased after chronic morphine administration. Thus, the differential activation patterns of both ERK and Akt after acute and chronic morphine administration could have important implications for understanding additional pathways mediating opioid signaling in vivo.     

Glycine receptors and GABA receptor alpha 1 and gamma 2 subunits during the development of mouse hypoglossal nucleus

In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on the brain area studied, but their relative proportion to total synapses generally decreases with time. We have determined the sequential process of inhibitory synapse maturation in the hypoglossal nucleus in vivo. Immunocytochemistry and confocal microscopy were used for codetection of VIAAT, the common presynaptic vesicular transporter of glycine and GABA, GlyRs, GABA(A)R alpha1 and gamma2 subunits, and gephyrin, the scaffold protein implicated in the synaptic localization of inhibitory receptors. In E17 embryos, GlyRs were already clustered while GABA(A)R alpha1 and gamma2 subunit immunoreactivity (IR) displayed both diffuse and clustered patterns. Quantitative analysis at this stage revealed that the majority of GlyR clusters were apposed to VIAAT-IR accumulation and that 30% of them colocalized with gamma2GABA(A)R clusters. This proportion increased with age to 50% at P30. GlyR clusters that did not colocalize with gamma2GABA(A)R clusters were associated with GABA(A)R gamma2 diffuse IR. Interestingly, the percentage of GlyR clusters surrounded by GABA(A)R gamma2 diffuse IR decreased with age, while GlyR clusters colocalized with gamma2GABA(A)R clusters increased. The developmental coclustered pattern of gephyrin and GABA(A)R alpha1 and gamma2 subunits paralleled the coclustered pattern of GlyRs and GABA(A)R alpha1 and gamma2 subunits. Our results indicate that the proportion of GlyR-GABA(A)R coclusters increases until adulthood. A developmental sequence of the postsynaptic events is proposed in which diffuse extrasynaptic GABA(A)Rs accumulate at inhibitory synapses to form postsynaptic clusters, most of them being colocalized with GlyR clusters in the adult.     

Selenium diet-supplementation improves cocaine-induced myocardial oxidative stress and prevents cardiac dysfunction in rats

Chronic cocaine abuse causes cardiac dysfunction and induces oxidative stress. The goal of this study was to evaluate whether an enhanced antioxidant pool, induced by the administration of selenium, may prevent the myocardial dysfunction induced by cocaine. Cocaine was administered for 7 days (15 mg/kg/day, i.p.) to rats pretreated for 4 weeks with selenium (1.16 mg/L/day, p.o.). Cardiac function was evaluated by cardiac index and left ventricular (LV) fractional shortening (FS) measured by echocardiography. The redox ratio and enzymatic activities of glutathione peroxidase (GPX) and superoxide dismutase (SOD) were measured in the LV myocardium. Cocaine administration induced a cardiac dysfunction, as evidenced by a decrease in cardiac index and LV FS as well as by an increase in LV diameters. Moreover, antioxidant markers and redox ratio were altered in rats after cocaine exposure. Selenite supplementation induced a significant limitation of cardiac index and FS alterations observed after cocaine administration. This improvement in cardiac function was associated with a redox ratio recovery while SOD and GPX activities remained unchanged. Thus, selenite reversed both the oxidative stress and the contractile dysfunction induced by cocaine administration. These results suggest a major role of oxidative stress in the cocaine-induced cardiotoxicity.     

Phase I study to determine the safety, tolerability and immunostimulatory activity of thalidomide analogue CC-5013 in patients with metastatic malignant melanoma and other advanced cancers

We assessed the safety, tolerability and efficacy of the immunomodulatory drug, CC-5013 (REVIMID trade mark ), in the treatment of patients with metastatic malignant melanoma and other advanced cancers. A total of 20 heavily pretreated patients received a dose-escalating regimen of oral CC-5013. Maximal tolerated dose, toxicity and clinical responses were evaluated and analysis of peripheral T-cell surface markers and serum for cytokines and proangiogenic factors were performed. CC-5013 was well tolerated. In all, 87% of adverse effects were classified as grade 1 or grade 2 according to Common Toxicity Criteria and there were no serious adverse events attributable to CC-5013 treatment. Six patients failed to complete the study, three because of disease progression, two withdrew consent and one was entered inappropriately and withdrawn from the study. The remaining 14 patients completed treatment without dose reduction, with one patient achieving partial remission. Evidence of T-cell activation was indicated by significantly increased serum levels of sIL-2 receptor, granulocyte-macrophage colony-stimulating factor, interleukin-12 (IL-12), tumour necrosis factor-alpha and IL-8 in nine patients from whom serum was available. However, levels of proangiogenic factors vascular endothelial growth factor and basic foetal growth factor were not consistently affected. This study demonstrates the safety, tolerability and suggests the clinical activity of CC-5013 in the treatment of refractory malignant melanoma. Furthermore, this is the first report demonstrating T-cell stimulatory activity of this class of compound in patients with advanced cancer.