Microbiological Profile, Prevalence, and Characterization of Salmonella enterica in Peanuts, Pecans, Raisins, Sun-Dried Tomatoes, and Chocolate Sprinkles Sold in Bulk in Markets in Querétaro, Mexico.

ABSTRACT: In Mexico, the prevalence of Salmonella enterica in low-water-activity foods and its link to outbreaks are unknown. The aim of this study was to determine the microbiological profile and the prevalence of S. enterica in several low-water-activity foods, including peanuts, pecans, raisins, sun-dried tomatoes, and chocolate sprinkles, purchased in retail establishments in Querétaro, Mexico. Seventy samples of each food item sold in bulk were purchased. Aerobic plate count, molds, yeasts, total coliforms, Escherichia coli, and Staphylococcus aureus were quantified in 10-g samples. The prevalence of S. enterica in 25-g samples was determined. From positive samples, S. enterica isolates (60) were characterized based on their antimicrobial susceptibility to 14 antibiotics, the presence-absence of 13 virulence genes, and serotype. The concentration of aerobic plate count, molds, yeasts, total coliforms, and E. coli ranged from 3.1 to 5.2 log CFU g-1, from 2.0 to 2.4 log CFU g-1, from 2.0 to 3.0 log CFU g-1, from 0.6 to 1.1 log most probable number (MPN) g-1, and from 0.5 to 0.9 log MPN g-1, respectively. S. aureus was not detected in any sample (<10 CFU g-1). The prevalence of S. enterica in chocolate sprinkles, raisins, peanuts, pecans, and sun-dried tomatoes was 26, 29, 31, 40, and 52%, respectively. Most isolates (68.3%) were resistant to at least one antibiotic. Chromosome-associated virulence genes were found in all isolates, and only one strain had sopE, and 98.3% of the isolates were grouped in the same virulotype. Among the isolates, the most frequent serotype was Tennessee (51 of 60). According to the characteristics evaluated, we grouped the isolates into 24 clusters. The elevated prevalence of S. enterica highlights the role of low-water-activity food items sold in bulk at markets as potential vehicles for pathogen transmission. Regardless of the low variability among S. enterica isolates, their characterization could be helpful to elucidate which strains are circulating in these foods for improving epidemiological surveillance.

Immune reactivity to type VII collagen: implications for gene therapy of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genodermatosis caused by loss-of-function mutations in COL7A1 encoding type VII collagen, the component of anchoring fibrils. As exogenous type VII collagen may elicit a deleterious immune response in RDEB patients during upcoming clinical trials of gene therapies or protein replacement therapies, we developed enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assays to analyze B- and T-cell responses, to the full-length type VII collagen. The ELISA was highly sensitive and specific when tested against sera from 41 patients with epidermolysis bullosa acquisita (EBA), and the IFN-gamma ELISPOT detected a cellular response that correlated with ongoing EBA manifestations. Both tests were next applied to assess the risk of an immune response to type VII collagen in seven RDEB patients with a range of type VII collagen expression profiles. Immune responses against type VII collagen were dependent on the expression of type VII collagen protein, and consequently on the nature and position of the respective COL7A1 mutations. These immunologic tests will be helpful for the selection of RDEB patients for future clinical trials aiming at restoring type VII collagen expression, and in monitoring their immune response to type VII collagen after treatment.

Functional complementation of a genetic deficiency with human artificial chromosomes.

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.

The assembly of large BACs by in vivo recombination.

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.

Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.

Retrofitting vectors for Escherichia coli-based artificial chromosomes (PACs and BACs) with markers for transfection studies.

P1-based artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have significantly expanded the size of fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules. Functional characterization of a gene within a given PAC or BAC clone often requires transferring the DNA into eukaryotic cells for transient or long-term expression. To facilitate transfection studies, we have developed protocols using the Notl restriction sites of any PAC or BAC clone to introduce a transfection reporter gene, lacZ, together with a selectable marker, neo. This enables transfected cells to be detected by X-Gal staining to verify DNA uptake, and clones of stably transformed cells may be selected for in the presence of the antibiotic G418. The same retrofitting protocols may be applied with other markers of interest to extend the functionality of PAC and BAC libraries, and specialized aspects of such manipulation of E. coli-based artificial chromosomes are outlined.

Oxysterol-induced apoptosis in human monocytic cell lines.

Oxysterols constitute a large family of natural compounds, endowed with various biological activities including cholesterol regulation, immunosuppression and antitumoral potency. In the present study, we examine and compare the cytotoxic effects of two representative members of this family: 7 beta-hydroxycholesterol (7 beta-OH) and 25-hydroxycholesterol (25-OH), in two human monocytic cell lines, U-937 and HL-60. In both cell lines 7 beta-OH at 30 mu M induces cell death by apoptosis within the first hours of treatment. Under the same conditions and in contrast with results previously obtained with lymphoma cells, 25-OH is cytostatic only. It is interesting to note that the simultaneous treatment of U-937 cells by equimolar concentrations of 7 beta-OH and 25-OH leads to a considerably decreased induction of apoptosis. Such an effect is not observed with HL-60 cells. Taken together, these results indicate for the first time that: 1) oxysterols hydroxylated on the sterol nucleus are also able to induce apoptosis, 2) apoptosis can be induced by these substances in cells belonging to the myeloid lineage and 3) as far as apoptosis is concerned, a combined treatment with 7 beta-OH and 25-OH can lead to opposite effects depending on the cell type.

DNA methylation and the origin of complement factor B polymorphism.

BF is a polymorphic complement component encoded in the MHC. In each of two frequent alleles of BF, BF*FA and BF*FB, the difference in relation to the major allele BF*S has been shown to consist in the nonsynonymous substitution of only one base of the coding sequence. Both substitutions occur within the same codon and affect contiguous positions, corresponding to the dinucleotide CpG in BF*S. We propose here that BF*FA and BF*FB arose independently from BF*S by the frequently described transition mutations associated with cytosine methylation at CpG sites. By probing sperm DNA with methylation-sensitive restriction enzymes, we obtained experimental evidence of germ line methylation of the CpG site considered. The dinucleotide of the BF gene probably constitutes a site for recurrent mutation, and this is of relevance for the use of BF as a genetic marker, and the origin of forms of the protein with altered functional properties.

Genomic analysis of the F subtypes of human complement factor B.

Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.

Human factor B. Complete cDNA sequence of the BF*S allele.

The gene of human complement factor B (BF) is located within the class III region of the major histocompatibility complex. The knowledge of the coding sequence of the BF gene rests on a set of partial sequence studies reported by various sources, and full-length sequences ascribed to specific alleles of this polymorphic complement component have not yet been published. Now, we have isolated and sequenced a collection of cDNA clones derived from BF*S, the major BF allele. We present an uninterrupted, allele-specific sequence of the entire coding region and the 3' untranslated segment of the cDNA. Extensive comparison of this and previously available sequence data was carried out, and a number of base substitutions were observed in relation to some of the earlier sequences. The possibility that these differences arise from polymorphism in the BF gene is discussed.

Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes.

Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed.