Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Sugar transport systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Fatty Acids Induce Chloride Permeation in Rat Liver Mitochondria by Activation of the Inner Membrane Anion Channel (IMAC)

The inner membrane of freshly isolated mammalian mitochondria is poorly permeable to Cl(-). Low, nonlytic concentrations (< or =30 microM) of long-chain fatty acids or their branched-chain derivatives increase permeation of Cl(-) as indicated from rapid large-scale swelling of mitochondria suspended in slightly alkaline KCl medium (supplemented with valinomycin). Myristic, palmitic, or 5-doxylstearic acid are powerful inducers of Cl(-) permeation, whereas lauric, phytanic, stearic, or 16-doxylstearic acid stimulate Cl(-) permeation in a lesser extent. Fatty acid-induced Cl(-) permeation across the inner membrane correlates well with the property of nonesterified fatty acids to release endogenous Mg(2+) from mitochondria. Myristic acid stimulates anion permeation in a selective manner, similar as was described for A23187, an activator of the inner membrane anion channel (IMAC). Myristic acid-induced Cl(-) permeation is blocked by low concentrations of tributyltin chloride (IC(50) approximately 1.5 nmol/mg protein). Moreover, myristic acid activates a transmembrane ion current in patch-clamped mitoplasts (mitochondria with the outer membrane removed) exposed to alkaline KCl medium. This current is best ascribed to the opening of an ion channel with a single-channel conductance of 108 pS. We propose that long-chain fatty acids can activate IMAC by withdrawal of Mg(2+) from intrinsic binding sites.     

Is pulse oximetry a reliable tool for detection of aspiration?

This study was designed to determine whether significant differences in saturation levels existed among patients with aspiration and patients without and wether pulse oximetry can reliably detect aspiration in patients with dysphagia. We also examined the effects of gender and disease (neurologic versus non neurologic) on saturation levels. We studied 38 patients. They all underwent a videofluoroscopic study of swallowing (VFSS). Twenty patients aspirated on videofluoroscopic study of swallowing: ten patients were solid aspirators, ten patients were liquid aspirators. In each group (liquid aspirators, solid aspirators or non aspirators) we found no significant difference in saturation levels. We found however a significant difference in saturation levels between each group before, during and after videofluoroscopic study of swallowing. Both gender and disease had an effect on saturation levels. We conclude that pulse oximetry can not serve as a screening tool for detection of aspiration as saturation levels are dependent on many factors. Therefore one can not reliably predict aspiration with a single saturation screening.