Yolanda E. Valdez

TNF-α Promoter Polymorphisms and Susceptibility to Human Papillomavirus 16–Associated Cervical Cancer

BACKGROUND Polymorphisms in the TNF-alpha promoter region have recently been shown to be associated with susceptibility to cervical cancer. Some polymorphisms have been reported to influence transcription for this cytokine. Altered local levels in the cervix may influence an individuals immune response, thereby affecting persistence of human papillomavirus (HPV) 16 infection, a primary etiological factor for cervical cancer. METHODS AND RESULTS The association of 11 TNF-alpha single-nucleotide polymorphisms (SNPs) with susceptibility to HPV16-associated cervical cancer was investigated. Sequencing of the TNF-alpha promoter region confirmed 10 SNPs, and 1 previously unreported SNP (161 bp upstream of the transcriptional start site) was discovered. Microsphere-array flow cytometry-based genotyping was performed on 787 samples from Hispanic and non-Hispanic white women (241 from randomly selected control subjects, 205 from HPV16-positive control subjects, and 341 from HPV16-positive subjects with cervical cancer). The genotype distribution of 3 SNPs (-572, -857, and -863) was significantly different between case subjects and control subjects. Analysis of haplotypes, which were computationally inferred from genotype data, also revealed statistically significant differences in haplotype distribution between case subjects and control subjects. CONCLUSIONS We report new associations between several TNF-alpha SNPs and susceptibility to cervical cancer that support the involvement of the TNF- alpha promoter region in development of cervical cancer.

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

ABSTRACT A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

Flow Cytometric Characterization of Alveolar Macrophages

Phenotypes of lung free cells (FC) harvested from Fischer‐344 rats by episodic lavage were characterized by flow cytometry. Parameters evaluated included electronic volume (EV), axial light loss (ALL), 90° light scatter (LS), blue autofluorescence (BA), and green‐yellow autofluorescence (G‐YA). Three phenotypic populations, FC‐A, FC‐B, and FC‐C were identified by their differing LS characteristics. FC‐C represented 90% of the cells and were exclusively alveolar macrophages. Two subpopulations in FC‐C, FC‐CI and FC‐CII, were further distinguished by their unique ALL features. The morphologic appearances of these subpopulations by light microscopy clearly differed in sorted preparations. Based on their patterns of autofluorescence, these FC‐CI and FC‐CII phenotypes were found to be composed of eight subpopulations. In FC populations harvested during further lavage episodes of the same lungs, the relative contributions of FC‐CI to the FC‐C subpopulation decreased as FC‐CII correspondingly increased. This study demonstrates 1) that subpopulations of lavaged AM can be categorized according to their optical phenotypes by flow cytometry and 2) that the relative frequency of retrieval of some phenotypes depends on how exhaustively the lungs are lavaged. With regard to the latter, bronchoalveolar lavage does not randomly sample the underlying AM population in the alveolar compartment.

Lung and pleural “free‐cell responses” to the intrapulmonary deposition of particles in the rat

Deposition of a high lung burden of particulate carbon or latex in the mouse elicits a biphasic macrophagic response, with the early increase in the size of the population of the alveolar macrophages (AM) being mainly due to an influx of mononuclear phagocytes from the blood compartment and the later phase being due to the egression of interstitial macrophages (IM). In the present study, we investigated the lungs free-cell response in the rat to the intrapulmonary instillation of microspheres and determined the contributions of newly arrived blood monocytes to the macrophagic response over a 30-d period. As of 24 h after deposition, the lavageable population size increased approximately fivefold. Most of the increase in the free cell population was due to a recruitment of polymorphonucleated leukocytes (PMN), although the size of the AM population nearly doubled. Only about 13% of the AM on d 1 were positive for myeloperoxidase activity, suggesting that recruitment of mononuclear phagocytes from the blood compartment plays a limited role in the early expansion of the AM pool in the rat. As of 14 and 30 d after particle deposition, lavaged AM numbers continued to be elevated; the enlarged sizes of the AM populations at these times could be explained, in part, by a continuing influx of blood monocytes. In a parallel component of the study, we also investigated how the pleural cell population might be affected during the lungs free-cell response to the particulate burden. No acute influx of PMN into the pleural space (PS) accompanied the early free-cell response; the chemotactic factors initially involved in recruitment of the PMN into the lung apparently did not reach the PS in significant or biologically active concentrations, or they were inoperable in the pleural compartment. On the other hand, the numbers of macrophages in the PS were significantly elevated on d 1 and 14 after particle deposition in the lung. The macrophagic response in the PS subsided by d 30, but at this latter sacrifice time, pleural mast-cell and lymphocyte numbers were significantly increased and decreased, respectively. These results indicate that the deposition of particles into the lung can lead to numerical alterations in the cell types composing pleural cell populations.

Variation in HLA Class I Antigen-Processing Genes and Susceptibility to Human Papillomavirus Type 16—Associated Cervical Cancer

BACKGROUND Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. METHODS Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). RESULTS For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. CONCLUSIONS Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.

Migratory Behaviors of Alveolar Macrophages During the Alveolar Clearance of Light to Heavy Burdens of Particles

We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres. Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles. Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone. The lung retention kinetics of the particles were determined over an approximately 170 day period. On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition). The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations. The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process. Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance. The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time. The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57. On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs. Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres. The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro. Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.

Leukocytic responses to the intrapleural deposition of particles, particle-cell associations, and the clearance of particles from the pleural space compartment.

Polystyrene microspheres (2.02 micron diameter) were instilled in the pleural space compartment (PSC) of the Fischer 344 rat. Rats that were administered carrier vehicle only, and untreated rats served as controls. On d 1, 6, 14, and 28 following the instillations, the pleural free cells were harvested by pleural lavage and the major cell phenotypes retrieved were quantitated to determine how the pleural free cell population was altered by the particles. Concurrent with these studies, quantitative analyses were performed to determine the numbers of particles lavaged from the pleural space at the above sacrifice times, and these particle retention data were used to estimate particle translocation rates from the PSC. Deposition of the particles in the PSC brought about an early recruitment of polymorphonuclear leukocytes (PMN) and an enlargement in the size of the pleural mononuclear phagocyte (PMP) population. PMN numbers substantially decreased by d 6, but continued to remain elevated over the course of the study. The early increase in PMP appeared to subside by d 6, but again increased thereafter. Other notable changes were decreases in the size of the pleural eosinophil population at later times after particle deposition, and an approximate 200-fold increase in lymphocytes by d 28. The particles were cleared from the PSC with a biphasic pattern. The most rapid phase, which accounted for the clearance of greater than or equal to 80% of the particles, had a t1/2 = 0.3 d and the slower component had a t1/2 = 6 d. Most of the particles were translocated from the PSC to the retrosternal, caudal mediastinal tissue. The slower phase of particle clearance appeared to be macrophage-mediated, suggesting the t1/2 for macrophages in the PSC is also approximately 6 d.

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres †

BACKGROUND Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake. Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

Aim:  We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment.

Discovery of DNA operators for TetR and MarR family transcription factors from Burkholderia xenovorans

Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xenovorans, which are common to the genus Burkholderia. We also describe the development and application of a more streamlined version of the PBM technology that significantly reduced the experimental time. Despite the genus containing many pathogenically important species, only a handful of TF operator sites have been experimentally characterized for Burkholderia to date. Our study provides a significant addition to this knowledge base and illustrates some general challenges of discovering operators on a large scale for prokaryotes.