Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates.

BACKGROUND: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. RESULTS: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. CONCLUSIONS: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.

Comparative genomic analyses of 17 clinical isolates of Gardnerella vaginalis provide evidence of multiple genetically isolated clades consistent with subspeciation into genovars.

Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.

Generation of genic diversity among Streptococcus pneumoniae strains via horizontal gene transfer during a chronic polyclonal pediatric infection.

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

Comparative supragenomic analyses among the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae using a modification of the finite supragenome model.

BACKGROUND: Staphylococcus aureus is associated with a spectrum of symbiotic relationships with its human host from carriage to sepsis and is frequently associated with nosocomial and community-acquired infections, thus the differential gene content among strains is of interest. RESULTS: We sequenced three clinical strains and combined these data with 13 publically available human isolates and one bovine strain for comparative genomic analyses. All genomes were annotated using RAST, and then their gene similarities and differences were delineated. Gene clustering yielded 3,155 orthologous gene clusters, of which 2,266 were core, 755 were distributed, and 134 were unique. Individual genomes contained between 2,524 and 2,648 genes. Gene-content comparisons among all possible S. aureus strain pairs (n = 136) revealed a mean difference of 296 genes and a maximum difference of 476 genes. We developed a revised version of our finite supragenome model to estimate the size of the S. aureus supragenome (3,221 genes, with 2,245 core genes), and compared it with those of Haemophilus influenzae and Streptococcus pneumoniae. There was excellent agreement between RAST's annotations and our CDS clustering procedure providing for high fidelity metabolomic subsystem analyses to extend our comparative genomic characterization of these strains. CONCLUSIONS: Using a multi-species comparative supragenomic analysis enabled by an improved version of our finite supragenome model we provide data and an interpretation explaining the relatively larger core genome of S. aureus compared to other opportunistic nasopharyngeal pathogens. In addition, we provide independent validation for the efficiency and effectiveness of our orthologous gene clustering algorithm.

Preliminary study: treatment with intramuscular interferon beta-1a results in increased levels of IL-12Rß2+ and decreased levels of IL23R+ CD4+ T - Lymphocytes in multiple sclerosis.

BACKGROUND: There are a lack of biomarkers which can be used to predict clinical outcomes for multiple sclerosis (MS) patients receiving interferon beta (IFN-ß). Thus the objective of this study was to characterize changes in CD4+ T-lymphocyte expression in an unbiased manner following initiation of intramuscular (IM) IFN-ß-1a treatment, and then to verify those findings using marker-specific assays. METHODS: Peripheral blood specimens were collected from twenty MS patients before and after treatment with intramuscular (IM) IFN-ß-1a and were used for isolation of mononuclear cells (PBMCs). mRNA expression patterns of negatively-selected CD4+ T-cells from the PBMCs were analyzed using microarray gene expression technology. IL-12 and IL-23 receptor levels on PBMC-derived CD4+ T-cells were analyzed by flow cytometry. The phosphorylation status of Stat4 was measured by performing densitometry on western blots. RESULTS: Microarray analyses demonstrated that mRNA expression of the IL-12Rß2 gene was uniformly up-regulated in response to IFN-ß-1a treatment and was associated with an increased number of IL-12Rß2+ CD4+ T-cells by flow cytometry in 4 of 6 patients. This finding was substantiated by demonstrating that Stat4 phosphorylation, a transcription factor for IL-12, was increased after treatment. Conversely, the number of IL-23R+ CD4+ T-cells was decreased following treatment. CONCLUSIONS: The IL-12 receptor shares a common subunit, the IL-12Rß2, with the IL-23 receptor. Both of these receptors have a probable role in regulating IL-17 and TH-17 cells, important mediators of inflammation in multiple sclerosis (MS). Thus, the changes in the numbers of CD4+ T-cells expressing these receptors in response to IFN-ß-1a treatment may point to an important mechanism of action for this drug, but further large scale studies are needed to confirm these preliminary observations.

Mechanical effects, antimicrobial efficacy and cytotoxicity of usnic acid as a biofilm prophylaxis in PMMA.

Experiments were performed to test the null hypothesis that the addition of a natural occurring antibiotic would not alter mechanical properties of polymethylmethacrylate (PMMA). Compression and four-point bending tests were used to assess mechanical properties of zirconium dioxide bearing bone cement (Type Zr) and barium sulfate bearing bone cement (Type Ba), mixed with the antibiotic usnic acid ("usnic"), used to create a surface resistant to biofilm formation. Addition of usnic had a statistically significant effect on the material properties. Compressive and bending strengths decreased as usnic was added and Type Zr was stronger than Type Ba although material properties remained above recommended minima. With implications of liver toxicity with large doses of usnic taken as a dietary supplement, cytotoxicity tests using bone cement coupons were performed and showed very little or no toxicity in primary cultures of rabbit skin derived fibroblasts. A simple test of usnic's efficacy as a biofilm prophylaxis in PMMA was also conducted. Bone cement coupons with usnic were tested for their effectiveness against methicillin resistant Staphylococcus aureus. Diminished biofilm formation on usnic-containing coupons indicated that usnic can be an effective anti-microbial agent.

Objective determination of peripheral edema in heart failure patients using short-wave infrared molecular chemical imaging.

SIGNIFICANCE: Peripheral pitting edema is a clinician-administered measure for grading edema. Peripheral edema is graded 0, 1 + , 2 + , 3 + , or 4 + , but subjectivity is a major limitation of this technique. A pilot clinical study for short-wave infrared (SWIR) molecular chemical imaging (MCI) effectiveness as an objective, non-contact quantitative peripheral edema measure is underway. AIM: We explore if SWIR MCI can differentiate populations with and without peripheral edema. Further, we evaluate the technology for correctly stratifying subjects with peripheral edema. APPROACH: SWIR MCI of shins from healthy subjects and heart failure (HF) patients was performed. Partial least squares discriminant analysis (PLS-DA) was used to discriminate the two populations. PLS regression (PLSR) was applied to assess the ability of MCI to grade edema. RESULTS: Average spectra from edema exhibited higher water absorption than non-edema spectra. SWIR MCI differentiated healthy volunteers from a population representing all pitting edema grades with 97.1% accuracy (N = 103 shins). Additionally, SWIR MCI correctly classified shin pitting edema levels in patients with 81.6% accuracy. CONCLUSIONS: Our study successfully achieved the two primary endpoints. Application of SWIR MCI to monitor patients while actively receiving HF treatment is necessary to validate SWIR MCI as an HF monitoring technology.

Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.

OBJECTIVES: We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. METHODS: Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. RESULTS: Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. CONCLUSIONS: Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

Direct demonstration of Staphylococcus biofilm in an external ventricular drain in a patient with a history of recurrent ventriculoperitoneal shunt failure.

External ventricular drains (EVD) are associated with a high infection rate. Early detection of infection is frequently problematic due to a lack of clinical signs and the time period required for culturing. Bacterial biofilms have been suggested to play an important role in the infection of EVD, but direct evidence is as yet lacking. We report the case of a 17- year-old male with Dandy-Walker malformation who presented with headache, nausea and drowsiness; a CT scan revealed enlarged ventricles. The patient had a history of ventriculoperitoneal shunt revision 3 weeks prior to admission. The shunt was removed on suspicion of infection and an EVD placed. Daily surveillance cultures through the EVD were negative and the EVD was replaced on day 5. Examination of the initial EVD by confocal microscopy demonstrated clear intraluminal biofilm formation; molecular analysis by PCR identified Staphylococcus aureus resident on the catheter. To our knowledge, this is the first direct demonstration of an intraluminal biofilm compromising an EVD. Despite the presence of biofilm on this catheter, the patient demonstrated no clinical signs of infection, and the routine surveillance culture was negative. Undetected biofilm may pose a latent risk on EVD and other neurosurgical catheters.

Expression of receptor for activated C kinase 1 in healing skin and mucosal wounds.

Postnatal (adult) mammalian wound healing results in the formation of scar, whereas fetal mammals are able to effect wound repair without scar. We have investigated the expression pattern of the receptor of activated C kinase 1 (RACK1), a pleiotropic G-protein-like molecule, in healing skin and mucosal wounds in a rabbit model after obtaining a full-length clone of the rabbit RACK1 cDNA. In both adult skin and mucosal wounds, RACK1 mRNA expression is decreased relative to unwounded controls. In contrast, in fetal skin wounds RACK1 expression is unaltered from fetal control. Fibroblasts derived from adult skin tissue express more RACK1 message than fetal skin fibroblasts. These observations suggest that RACK1 may play an important role in distinguishing scarless fetal wound healing from its scirrhous counterpart in adults.

Biofilms in pediatric respiratory and related infections.

Bacteria can grow as free-floating, planktonic bacteria or complex communities called biofilms. Biofilms promote bacterial growth and diversity and offer bacteria unique environments, including aerobic and anaerobic layers, that facilitate resistance to antimicrobial therapies. Respiratory and related structures provide ideal environments for the development of bacterial biofilms, which predispose patients to recurrent and chronic infections. Biofilms are important for the persistence of chronic rhinosinusitis, pulmonary infections in cystic fibrosis, chronic otitis media, and device-related infections. Antimicrobial therapy that is proven effective against planktonic bacteria is often insufficiently effective against the defenses of biofilms. Furthermore, biofilms modify themselves following exposure to antimicrobial therapy, thus developing increased resistance. Understanding the nature of biofilms in common pediatric infections is essential to comprehending the expected course of bacterial illness and identifying treatments that are most likely to be beneficial against more resistant biofilms.

Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates.

BACKGROUND: Streptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro. RESULTS: Multiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains. CONCLUSION: All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing.

Integumentary wound healing in early fetal life is regenerative and proceeds without scar formation. Expressomic analysis of this phenomenon by differential display has previously determined that the eta subunit of the cytosolic chaperonin containing T-complex polypeptide (CCT) is downregulated in the healing fetal wound milieu. We now report that no other CCT subunit shares this distinct pattern of gene regulation as determined by limiting dilution reverse transcriptase polymerase chain reaction (RT-PCR); all seven of the remaining CCT subunits demonstrate no change in messenger RNA (mRNA) expression in healing fetal wounds compared to unwounded control tissue. The alpha subunit, however, did evidence reduced message levels in healing adult wound tissue. We herein report on the cloning and sequence of the complementary DNA (cDNA) for rabbit CCT-alpha and confirm its wound specific decrease in adult tissues through quantitative real-time RT-PCR assay. We also confirm that quantitative evaluation of CCT-alpha and CCT-zeta mRNA expression shows no change in healing fetal wounds.

Comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal supragenome.

The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strain's genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters ( approximately 3,000) that are represented in the S. pneumoniae population at frequencies of >or=0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.

Virulence phenotypes of low-passage clinical isolates of nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media.

BACKGROUND: The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease. RESULTS: The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA - PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME. CONCLUSION: Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

The role of biofilms in otolaryngologic infections: update 2007.

PURPOSE OF REVIEW: Biofilms have been shown to play a role in otitis media, sinusitis, cholesteatoma, tonsillitis, adenoiditis, and device infections. This article is written to review recent advances in the field. RECENT FINDINGS: The role of biofilms in the persistence of chronic, mucosal-based ENT-related infections was first recognized in otitis media. Definitive proof was lacking until the demonstration of bacterial biofilms on the middle-ear mucosa of children, not only with chronic otitis media with effusion, but also with recurrent otitis media. Strains of Pseudomonas aeruginosa isolated from cholesteatoma are avid biofilm formers. Biofilms have been reported in the adenoids of children with chronic rhinosinusitis, helping to explain the clinical observation that adenoidectomy can be beneficial to children with chronic otitis or chronic rhinosinusiti. Additional studies have confirmed the presence of biofilms in chronic tonsillitis. Biofilms have also been shown to be involved in infected cochlear implants and tracheotomy tubes. SUMMARY: The recognition that chronic otolaryngologic bacterial infections are biofilm related has been the impetus for the development of new technologies for the study of biofilms and their prevention and treatment. Understanding that chronic bacterial infections are biofilm related is fundamental to developing rationale strategies for treatment and prevention.

Codon usage comparison of novel genes in clinical isolates of Haemophilus influenzae.

A similarity statistic for codon usage was developed and used to compare novel gene sequences found in clinical isolates of Haemophilus influenzae with a reference set of 80 prokaryotic, eukaryotic and viral genomes. These analyses were performed to obtain an indication as to whether individual genes were Haemophilus-like in nature, or if they probably had more recently entered the H.influenzae gene pool via horizontal gene transfer from other species. The average and SD values were calculated for the similarity statistics from a study of the set of all genes in the H.influenzae Rd reference genome that encoded proteins of 100 amino acids or longer. Approximately 80% of Rd genes gave a statistic indicating that they were most like other Rd genes. Genes displaying codon usage statistics >1 SD above this range were either considered part of the highly expressed group of H.influenzae genes, or were considered of foreign origin. An alternative determinant for identifying genes of foreign origin was when the similarity statistics produced a value that was much closer to a non-H.influenzae reference organism than to any of the Haemophilus species contained in the reference set. Approximately 65% of the novel sequences identified in the H.influenzae clinical isolates displayed codon usages most similar to Haemophilus sp. The remaining novel sequences produced similarity statistics closer to one of the other reference genomes thereby suggesting that these sequences may have entered the H.influenzae gene pool more recently via horizontal transfer.

Human susceptibility to viral infection: the search for HIV-protective alleles among Africans by means of genome-wide studies.

Human immunodeficiency virus (HIV) infection represents a major global health problem, with HIV now recognized as the fourth leading cause of death on a worldwide basis. One approach to developing effective anti- HIV interventions is to identify and understand the molecular mechanisms by which natural genetic variations provide protection from infection or disease progression. This approach can be used to identify human gene alleles that confer resistance or increased susceptibility to HIV infection. To date, however, this approach has been underutilized in the African population and all HIV-resistance alleles that have been described have been identified by evaluating candidate genes. This limited approach is based upon a researcher's assumption that those genes that will provide the host with a benefit can be predicted, a priori, but it does not provide for a large scale systematic screen of all possible candidate genes. Nonetheless, this method has met with some success in identifying HIV-resistance genes, mostly among the white population. The lack of a comprehensive genetic approach, both in terms of the populations studied and the percentage of the genome investigated, likely explains why all of the HIV-restriction alleles identified to date fall within two gene families, and why no resistance genes have been identified among black Africans. It is likely, as with any complex trait, that most protective alleles will provide only partial HIV resistance. Thus, HIV resistance in most persons likely arises through a QTL (quantitative trait loci) mechanism meaning that protection is a polygenic trait. This feature coupled with interpopulation genetic heterogeneity makes the candidate gene mapping approach a daunting task. A comprehensive genome-wide case-control allelic association study in the African population will maximize our chances of identifying new targets for the development of new therapeutics that have the promise of benefiting all persons infected with HIV.

Unique challenges of obtaining regulatory approval for a multicenter protocol to study the genetics of RRP and suggested remedies.

OBJECTIVE: Investigations that seek to generalize findings or to understand uncommon diseases must be conducted at multiple centers. This study describes the process of obtaining regulatory approval for a minimal risk genetic study in a multi-center setting as undertaken by the Recurrent Respiratory Papillomatosis (RRP) Task Force. STUDY DESIGN AND SETTING: Sequential cohort of American children's hospitals. A single protocol was submitted to each Institutional Review Board (IRB). RESULTS: Documentation was prepared for 14 IRBs over 2.5 years. The median time between enlistment and approval at the first 8 sites was 15 months. Institutions varied considerably in their requirements and in the issues that were raised. Protocols were submitted sequentially and accumulated experience was used in the preparation of applications to subsequent IRBs. Nevertheless, there was no correlation between the accumulated experience and the number of issues that were raised. CONCLUSION: Despite uniform federal standards, all local IRBs required unique and individualized submissions. For multicenter studies, investigators should seriously consider the establishment of cooperative authorization agreements. On a simpler level, a standardized format for applications needs to be adopted nationwide. EBM RATING: B-3b.

Mucin gene cDNA sequence characterization in chinchilla middle ear epithelium.

OBJECTIVES: To identify mucin genes in chinchilla middle ear epithelium and characterize complimentary deoxyribonucleic acid (cDNA) sequences to facilitate further investigations into mucin physiology and pathophysiology on a molecular level using the chinchilla model. METHODS: Chinchilla mucin gene exploration and cDNA characterization was accomplished using reverse transcriptase-polymerase chain reactions (RT-PCR). Forward and reverse primer pairs were designed using consensus sequences available for human and rodent species. Chinchilla middle ear epithelium was harvested and primary cell cultures (CMEEC) were established. The CMEEC were explored for the expression of chinchilla mucin genes 1, 2, 4 and 5AC (cMuc1, cMuc2, cMuc4 and cMuc5AC). Identified cDNA amplicons for each of these genes was sequenced and homology compared to previously published human and rodent sequences. RESULTS: CMEEC express all four of the mucin genes cMuc1, cMuc2, cMuc4 and cMuc5AC. cDNA amplicons for each of the genes were able to be sequenced with lengths ranging from 66 to 362 base pairs. Each of the chinchilla cDNA sequences expressed significant homology with published human and rodent cDNA for these mucin genes. A cDNA sequence for the housekeeping gene, beta-actin, was also identified. CONCLUSIONS: Chinchilla middle ear epithelium grown in culture expresses the mucin genes 1, 2, 4 and 5AC, which have been identified as important in mucin regulation in the middle ear. cDNA sequences corresponding to these mucin genes were identified and may serve as important molecular tools in future studies of otitis media using the chinchilla model.