Acquisition of a second multi-drug resistance-encoding element by IncM1 plasmid pACM130 abolished conjugative transfer.

Within the IncM plasmid family there is a lineage that has a transposon Tn1721-based multiple-resistance island inserted in the backbone gene mucB. So far, this group includes R1215, p202c, pIGT15, pARM26, and pACM1, from Europe and the USA. A new member of this group, pACM130, was isolated at the same American hospital as pACM1 and has a similar resistance island, but also carries a copy of Tn1331 that interrupts the traY gene in the conjugation operon. The conjugative phenotype of this plasmid has been abolished, though pACM130 could be mobilized by an intact traY cloned into a laboratory vector and transformed into the same donor bacterium.

The complete nucleotide sequence of the multi-drug resistance-encoding IncL/M plasmid pACM1.

The 89,977 bp nucleotide sequence of pACM1, isolated from a 1993 outbreak strain of cephalosporin-resistant Klebsiella oxytoca, has been completed and assigned GenBank accession number KJ541681. The plasmid has a single 31,842 bp mosaic multi-drug resistance-encoding (MDR) region comprising the mer resistance module of Tn1696, two integrons with a total of seven cassettes, one complete copy each of IS1R and IS26, and the bla(SHV-5)-carrying Tn2003 (with defective IS26 termini), all within a Tn1721-like element inserted into the mucB gene of the IncL/M plasmid backbone. The Tn1721-Tn1696 combination resembles sequence found in the chromosomal MDR islands of some Acinetobacter baumannii isolates. Among the completely sequenced IncL/M resistance plasmids, the Tn1721-based MDR region is unique, but data from older studies suggest that this type of plasmid was widespread in the 1990s. Since resistance gene dosage is affected by plasmid copy number (PCN), we used a relatively simple new "efficiency-corrected" qPCR assay to measure the PCN of pACM1. There are approximately three copies per chromosome in an Escherichia coli DH5α host, and two in the original Klebsiella oxytoca isolate. We could not find similar PCN data for other medically important plasmids for comparison. The study of this plasmid property and its effect on resistance levels should be facilitated in the future by the availability of qPCR instruments and complete genome sequences.

Microarray analysis in B cells among siblings with/without MS - role for transcription factor TCF2.

BACKGROUND: We investigated if global gene expression and transcription networks in B-lymphocytes of siblings with multiple sclerosis (MS) were different from healthy siblings. RESULTS: Using virus-transformed immortalized B cells and human whole genome bioarrays with validation using RT-qPCR, we found that in siblings with MS, genes for CXCL10, serpin B1 and FUT4 were up regulated whereas CDC5L, TNFRSF19 and HLA-DR were down regulated, among others; transcription analysis showed two intersecting clusters of transcriptional factors - the larger, governed by the upregulated transcription factor 2 (TCF2) and the smaller network regulated by the downregulated CDC5L. CONCLUSION: No study has linked TCF2 to MS and to better understand the role of TCF2 in MS, studies in larger cohorts are required.

Global gene expression profiles associated with retinoic acid-induced differentiation of embryonal carcinoma cells.

We have evaluated the effects of retinoic acid (RA) treatment of F9 embryonal carcinoma (EC) cells, which induces differentiation into primitive endoderm, on gene expression patterns. F9 cells were exposed to RA in culture, and global expression patterns were examined with cDNA-based microarrays at early (8 hr) and later times (24 hr) after exposure. Of the 1,176 known transcripts examined, we identified 57 genes (4.8%) that were responsive to RA at 8 and/or 24 hr: 35 were induced, 20 were repressed, and 2 were differentially regulated at these time points. To determine if our results were dependent on the array technology employed, we also evaluated the response to RA at 24 hr with oligonucleotide-based arrays. With these more dense arrays (12,488 genes), we identified an additional 353 RA-regulated genes (2.8%): 173 were upregulated and 180 were downregulated. Thus, a total of 410 genes regulated by RA were identified with roughly equivalent numbers induced or repressed. Although the expression of many genes found on both array platforms was consistent, the results for some genes were disparate. Quantitative PCR studies on a subset of these genes supported the results obtained with the cDNA arrays. Our results confirmed the regulation of several known RA-responsive genes and we also identified a number of genes not previously known to be RA-responsive. Those novel genes that were induced presumably contribute to the cellular processes required for a shift from proliferation to differentiation, whereas those new genes that were downregulated may possibly contribute to the maintenance of cell proliferation.

New cytochrome P450 2D6*56 allele identified by genotype/phenotype analysis of cryopreserved human hepatocytes.

Genotype/phenotype analysis with human hepatocytes has identified a new inactive CYP2D6 allele, CYP2D6*56. Cryopreserved human hepatocytes from 51 livers were evaluated for CYP2D6 activity with dextromethorphan as the probe substrate. Hepatocyte lots that lacked CYP2D6 activity were further evaluated for CYP2D6 expression and known genetic variations, including CYP2D6*2, *3, *4, *5, *6, *7, *8, *9, *10, *11, *14, *15, *17, *18, *19, *20, *25, *26, *29, *30, *35, *40, *41, *43, and various multiple copy CYP2D6 alleles (*1xn, *2xn, and *4xn) by the AmpliChip CYP450 prototype microarray (Roche Molecular Systems, Inc., Branchburg, NJ). Two discrepancies were uncovered between the CYP2D6 genotype and activity by this approach. In one sample, a previously unreported 3201C 224 T transition in exon 7 resulted in Arg344(CGA) being replaced by a stop codon (TGA), resulting in a CYP2D6 enzyme lacking the terminal 153 amino acids. This allele was given the designation of CYP2D6*56 and the GenBank accession number DQ282162. The lack of CYP2D6 activity in cryopreserved hepatocytes and microsomes found in the second sample, despite a normal level of CYP2D6 expression and a genotype (*10/*1) predictive of normal CYP2D6 activity, was attributed to enzyme inactivation by an unknown metabolite. The identification and characterization of the CYP2D6*56 allele indicates that commercial cryopreserved human hepatocytes may provide a valuable means to rapidly identify genetic variations with functional relevance. This integrated approach of identifying alleles and examining allele relationships to gene expression and function could be of tremendous value to understanding the mechanism responsible for functional differences in gene variation. The commercial availability of human cryopreserved hepatocytes also makes this potential readily available to any who are interested in it, not just those with access to private liver banks.

Enhanced multiepitope-based vaccines elicit CD8+ cytotoxic T cells against both immunodominant and cryptic epitopes.

A frequent issue in vaccinology is to elicit balanced T cell responses against both immunodominant and cryptic T cell epitopes, from one or several antigens presented at the same time to the immune system. Using HLA-A2.1.1 restricted epitopes from the Melan A/MART-1 or gp 100 melanoma-associated antigens as a model, we engineered a series of constructs in the ALVAC canarypox vector system: T cell epitopes were expressed either as linear polyepitopes (with or without spacers), or as minigenes encoding a single epitope. The latter were found to allow the best processing and presentation of most T cell epitopes, following infection by ALVAC recombinants of the HLA A2+ bladder carcinoma cell line and stimulation of epitope-specific human TIL lines. These various constructs were also used to immunize HLA-A2.1.1 HHD transgenic mice to compare their capacity to elicit T cells responses. Polyepitopes but also minigenes encoding wild-type epitopes could not elicit in a reliable manner balanced CTL responses against all target epitopes from gp100. We could rescue T cells responses against poorly immunogenic epitopes after introducing appropriate point mutations to enhance their interaction with MHC Class I molecules. Epitope enhancement within either polyepitope, multiepitopes (i.e. minigenes expressed under the control of separate promoters) or full length immunogens should be systematically considered when designing vaccines containing both cryptic and immunodominant target epitopes.

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen.

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-gamma) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/10(6) PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-gamma responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.