A case of an atypical femoral fracture associated with bacterial biofilm--pathogen or bystander?

We report a case of an 86-year-old woman with an atypical femoral fracture (AFF) who was treated with intramedullary nailing followed by lateral femoral plating. She developed a second femoral shaft fracture distal to the intramedullary nail which required a second operation. Biopsy of the periosteum overlying the site of the initial proximal AFF was sent for pathogen analysis. Using the Ibis T5000 platform and the BAC plate assay, a polymicrobial infection was diagnosed consisting of Bifidobacterium subtile and Pseudomonas mendocina. This raises the possibility that bacterial infections may play some role in atypical fractures of the femur.

Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Medical treatment of craniosynostosis: recombinant Noggin inhibits coronal suture closure in the rat craniosynostosis model.

INTRODUCTION - The mechanisms underlying craniosynostosis remains unknown. However, mutations in FGFR2 are associated with craniosynostotic syndromes. We previously compared gene expression patterns of patent and synostosing coronal sutures in the nude rat and demonstrated down regulation of Noggin in synostosing sutures. Noggin expression is also suppressed by FGF2 and constitutive FGFR2 signaling [Warren et al. (2003) Nature, vol. 422, pp. 625-9; McMahon et al. (1998) Genes Dev, vol. 12, pp. 1438-52]. Thus, we therefore hypothesized that the addition of rhNoggin to prematurely fusing sutures should prevent synostosis. MATERIALS AND METHODS - Cohorts of nude rats were subjected to: 1) surgical elevation of the coronal suture (shams); 2) surgical elevation and placement of normal or FGFR2 mutant human osteoblasts onto the underlying dura (xenotransplants); or 3) xenotransplantation with co-application of heparin acrylic beads soaked with recombinant human (rh) Noggin. Eleven days post-surgery the sutures were harvested, stained, and histologically examined. RESULTS - Animals that received control osteoblasts, sham surgery, or no surgery demonstrated normal skull growth and coronal suture histology, whereas animals transplanted only with FGFR2 mutant osteoblasts showed evidence of bridging synostosis on the calvarial dural surface. Sutures treated with FGFR2 mutant osteoblasts and rhNoggin remained patent. CONCLUSION - The chimeric nude rate model is a viable model of craniosynostosis. FGFR2 mutations in osteoblasts induce bridging osteosynthesis demonstrating one of the mechanisms for premature suture fusion. Topical application of rhNoggin protein prevents craniosynostosis in the weanling nude rat xenotransplantation model of syndromic craniosynostosis.

Gene expression changes in peripheral blood mononuclear cells from multiple sclerosis patients undergoing beta-interferon therapy.

OBJECTIVE: Multiple sclerosis (MS) is a disabling idiopathic inflammatory disorder with evidence of immune dysfunction. Current therapies for MS include preparations of beta-interferon (beta IFN). We studied the gene expression patterns in peripheral blood mononuclear cells from relapsing-remitting MS patients undergoing weekly beta IFN-1a therapy (Avonex; 30 mg intramuscular) to identify biomarkers for beta IFN responsiveness. METHODS: Oligonucleotide microarrays were used for the comparative analysis of gene expression patterns from longitudinal PBMC samples taken from five patients undergoing beta IFN therapy. RESULTS: On the basis of two-fold changes in expression levels and statistical analyses we selected a candidate diagnostic set of 136 genes that were differentially expressed between pretreatment and IFN-beta-1a-treated MS patients. When we applied this gene set to cluster the specimens according to their expression profiles, the pretreatment samples clustered in one branch, and acute and chronic samples following treatment clustered in another branch. However, the chronic samples from the single clinical non-responder clustered with the pretreatment branch, suggesting that a possible reversal of beta IFN-induced gene expression may be contributing to the poor clinical response. CONCLUSIONS: These 136 genes represent potential targets for new MS therapeutics and the basis for lack of beta IFN response.

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.

Mapping of an autosomal dominant gene for Dupuytren's contracture to chromosome 16q in a Swedish family.Dupuytren's contracture (DC) (OMIM 126900) is the most common connective tissue disease of mankind and has both heritable and sporadic forms. The inherited form is most frequently observed among the xanthochroi peoples of Northern Europe where its most common manifestations are thickening of the palmar fascia and contracture of the fingers. We ascertained a five-generation Swedish family in which DC is inherited in an autosomal dominant manner with high, but incomplete, penetrance by the end of the fifth decade. Blood was collected from all affected and informative unaffected family members for the performance of a genome-wide scan at a resolution of approximately 8 cM for all autosomes. Linkage was established to a single 6 cM region between markers D16S419 and D16S3032 on chromosome 16. A maximal two-point logarithm of odds (LOD) score of 3.18 was achieved at microsatellite marker D16S415 with four other markers in the region producing LODs of >1.5.

Century of Jackson-Weiss syndrome: further definition of clinical and radiographic findings in "lost" descendants of the original kindred.

Jackson-Weiss syndrome (JWS) is a condition consisting of craniosynostosis characterized by premature fusion of the cranial sutures and/or characteristic radiographic anomalies of the feet. The condition is inherited as an autosomal dominant trait with high penetrance and variable expressivity. Six different mutations in the fibroblast growth factor receptor 2 have been identified in patients with the clinical diagnosis of JWS. Jabs et al. [1994: Nat Genet 8:275-279] identified an Ala344Gly substitution in two branches of the family in which the clinical syndrome was originally described. This is the only publication to document this mutation in a family with the clinical diagnosis of JWS. In this study, we have identified a previously unrecognized branch of the original family with individuals that meet the clinical criteria for the diagnosis of JWS. We demonstrate that a mutation that produces the Ala344Gly substitution is present in affected members. This family illustrates the widely variable expression of the mutation, including a novel phenotype in the proband with a leg-length discrepancy and unilateral absence of the fifth digital ray in her right foot. We identify the clinical and detailed radiographic features of each affected individual and offer considerations when making the diagnosis of JWS.

Inhibition of apoptosis: a potential mechanism for syndromic craniosynostosis.

The biologic pathogenesis of syndromic craniosynostosis remains unknown. The purpose of this investigation was to determine whether specific biologic differences exist between normal calvarial osteoblasts and osteoblasts derived from patients with syndromic craniosynostosis. This study (1) examined the apoptotic rate and cell cycle of osteoblasts derived from patients with syndromic craniosynostosis, and (2) investigated for the presence of soluble factors released from syndrome-derived osteoblasts. Osteoblast cell lines were established from calvarial specimens of patients with clinically diagnosed syndromic synostosis and from normal controls. A co-culture technique was used to investigate for the presence of elaborated soluble factors. Apoptotic rate and cell cycle analyses were performed by using flow cytometry after staining with annexin V-fluorescein isothiocyanate and propidiumiodide, respectively. The apoptotic rate was significantly reduced in syndrome-derived osteoblasts as compared with control osteoblasts. Control osteoblasts co-cultured with syndromic osteoblasts demonstrated a dramatic reduction in their apoptotic rate as compared with those co-cultured with control osteoblasts. These results indicate that osteoblasts derived from patients with syndromic craniosynostosis display a lower apoptotic rate, a normal DNA synthetic rate, and the capability to reduce the apoptotic rate in normal calvarial osteoblasts through the elaboration of soluble factors.

Up-regulation of prostaglandin EP4 receptor messenger RNA in fetal rabbit skin wound.

BACKGROUND: Scar formation and subglottic stenosis often cause health problems in surgical otolaryngology. However, fetal wounds demonstrate scarless healing. The underlying mechanism remains poorly understood. We isolated differentially expressed genes by comparison between nonwounded with wounded skin of fetal and adult rabbits. METHODS: Skin incisional wounds were made in fetal (21 to 23 days' gestation) and adult rabbits. Nonwounded and wounded skin were harvested 12 hours after surgery. Total RNA was extracted. By means of messenger RNA differential display, differentially expressed complementary DNA fragments were isolated, cloned, and sequenced. The expressed transcripts were verified by reverse RNA dot blot and semiquantitative reverse transcription and polymerase chain reaction. RESULTS: One complementary DNA tag that was induced in fetal skin wounds and repressed in adult skin wounds was isolated. The sequence of this complementary DNA (352 base pairs) encodes the messenger RNA for the E-prostanoid (EP) 4 receptor for prostaglandin E(2) (PGE(2)). The truly differential expression of the transcript was confirmed. In normal skin, the EP4 receptor messenger RNA levels were higher in adults than in fetuses. Twelve hours after wounding, the EP4 receptor transcript was remarkably induced in fetal skin wounds but repressed in adult skin wounds. CONCLUSIONS: Our study demonstrates the differential expression of the EP4 receptor messenger RNA in fetal and adult skin before and 12 hours after wounding. Our results suggest that prostaglandin E(2) is involved in the differential cellular responses and in the regulation of the intracellular signal transduction through its binding to EP4 receptor during fetal wound repair.

Mapping of a gene for severe pediatric gastroesophageal reflux to chromosome 13q14.

CONTEXT: Gastroesophageal reflux (GER) has not previously been widely regarded as a hereditary disease. A few reports have suggested, however, that a genetic component may contribute to the incidence of GER, especially in its severe or chronic forms. OBJECTIVE: To identify a genetic locus that cosegregates with a severe pediatric GER phenotype in families with multiple affected members. DESIGN: A genome-wide scan of families affected by severe pediatric GER using polymorphic microsatellite markers spaced at an average of 8 centimorgans (cM), followed by haplotyping and by pairwise and multipoint linkage analyses. SETTING: General US community, with research performed in a university tertiary care hospital. SUBJECTS: Affected and unaffected family members from 5 families having multiple individuals affected by severe pediatric GER, identified through a patient support group. MAIN OUTCOME MEASURES: Determination of inheritance patterns and linkage of a genetic locus with the severe pediatric GER phenotype by logarithm-of-odds (lod) score analysis, considering a lod score of 3 or greater as evidence of linkage. RESULTS: In these families, severe pediatric GER followed an autosomal dominant hereditary pattern with high penetrance. A gene for severe pediatric GER was mapped to a 13-cM region on chromosome 13q between microsatellite markers D13S171 and D13S263. A maximum multifamily 2-point lod score of 5.58 and a maximum multifamily multipoint lod score of 7.15 were obtained for marker D13S1253 at map position 35 cM when presumptively affected persons were modeled as unknown (a maximum multipoint score of 4.88 was obtained when presumptively affected persons were modeled as unaffected). CONCLUSION: These data suggest that a gene for severe pediatric GER maps to chromosome 13q14. JAMA. 2000;284:325-334

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Refined localization of a gene for pediatric gastroesophageal reflux makes HTR2A an unlikely candidate gene.

Our objective in this study was to determine whether mutations in the gene for the 5-hydroxytryptamine receptor 2A (HTR2A) cause the autosomal dominant form of severe pediatric gastroesophageal reflux (GER), which we had previously mapped to a 21-cM region at chromosome 13q14. Direct sequencing of the HTR2A gene was carried out on DNA from affected and unaffected members of families with severe pediatric GER displaying genetic linkage to the HTR2A locus. In addition, we performed high-resolution linkage mapping within the GER gene region using additional polymorphic markers closely linked to HTR2A. Several previously reported polymorphisms in the HTR2A gene were identified in three families affected with GER. In addition, we identified a novel polymorphism at nucleotide -1273 in the HTR2A promoter. No mutant allele cosegregated exclusively with the GER phenotype in any family. Linkage analysis using additional polymorphic markers narrowed the region of the GER gene to a 9 cM interval between markers D13S263 and CAGR1, formally excluding HTR2A as a candidate gene. In conclusion, sequence analysis of HTR2A and linkage analysis argue against mutations in HTR2A being a cause of severe pediatric GER.

Development of a simplex polymerase chain reaction-based assay for the detection of Neisseria meningitidis.

BACKGROUND: This study was undertaken to develop a sensitive and specific polymerase chain reaction (PCR)-based assay for Neisseria meningitidis and Neisseria gonorrhoeae that could ultimately be incorporated into a multiplex assay designed to screen cerebrospinal fluid (CSF) for a panel of pyogenic bacterial species associated with bacterial meningitis. METHODS: N.meningitidis-specific primers were designed from porA gene sequences with the aid of a commercial software program and were used to develop and validate a clinical assay using a liquid hybridization-gel retardation detection system. The analytic sensitivity of the assay was determined using CSF spiked with N. meningitidis and comparing the PCR-based results with culture. RESULTS: Analytic sensitivity experiments showed the assay's limit of detection to be 100 fg purified input target DNA and 0.0125 colony-forming units of N. meningitidis spiked into CSF. Specificity experiments showed the assay could detect all strains of N. meningitidis and N. gonorrhoeae tested, but did not support amplification of the commensal neisserial species or a panel of other human bacterial pathogens. CONCLUSIONS: This PCR-based assay for pathogenic neisserial species is sensitive and specific and suitable for incorporation into a multiplex assay for the clinical differentiation of aseptic and septic meningitis.

Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion.

Split-hand/split-foot malformation (SHFM, ectrodactyly, or lobster-claw deformity) is a human limb malformation characterized by aberrant development of central digital rays with absence of fingers and toes, a deep median cleft, and fusion of remaining digits. SHFM is clinically heterogeneous, presenting both in an isolated form and in combination with additional abnormalities affecting the tibia and/or other organ systems, including the genitourinary, craniofacial, and ectodermal structures. Three SHFM disease loci have been genetically mapped to chromosomes 7q21 (SHFM1), Xq26 (SHFM2), and 10q24 (SHFM3). We mapped data from a large Turkish family with isolated SHFM to chromosome 10q24 and have narrowed the SHFM3 region from 9 cM to an approximately 2-cM critical interval between genetic markers D10S1147 and D10S1240. In several instances we found evidence for a more severe phenotype in offspring of a mildly affected parent, suggesting anticipation. Finally, data from this family, combined with those from six other pedigrees, mapped to 10q24, demonstrate biased transmission of SHFM3 alleles from affected fathers to offspring. The degree of this segregation distortion is obvious in male offspring and is possibly of the same magnitude for female offspring.

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family.

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.

Lack of R117H mutation in the cationic trypsinogen gene in patients with tropical pancreatitis from Bangladesh.

The etiology of nonalcoholic chronic pancreatitis, occurring in tropical regions, is unknown. Although environmental factors may play a role in its pathogenesis, a specific genetic predisposition may be necessary. The genetic mutation responsible for hereditary pancreatitis was described recently. Unlike in patients with hereditary pancreatitis, we found a lack of the R117H mutation in the cationic trypsinogen gene in all patients with tropical pancreatitis from Bangladesh.

Correlation between presence of viable bacteria and presence of endotoxin in middle-ear effusions.

The presence of endotoxin (detected by the Limulus amebocyte lysate assay) was compared to the presence of viable Haemophilus influenzae and Moraxella catarrhalis (detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria H. influenzae and M. catarrhalis. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.

Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method.

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Comparative evaluation of culture and PCR for the detection and determination of persistence of bacterial strains and DNAs in the Chinchilla laniger model of otitis media.

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.