Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires.
We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene (IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms.
IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen–related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System.
A total of 97,610 IGH (including 8,135 IgE) sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes.
This is the first report that exploits next-generation sequencing to determine local and peripheral blood IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.
Next-generation sequencing; peripheral blood and nasal mucosal IgE repertoires; allergic rhinitis; AR, Allergic rhinitis; AR.IS, Allergic rhinitis inside the pollen season; AR.OS, Allergic rhinitis outside the pollen season; CDR, Complementarity-determining region; CSR, Class-switch recombination; GC, Germinal center; NA, Nonallergic healthy control subject; NGS, Next-generation sequencing; QC, Quality control; SHM, Somatic hypermutation
The aim of the present study was to estimate the wall effect of the self-made spherical graphite-walled cavity chamber with the Monte Carlo method for establishing the air-kerma primary standard of high-dose-rate (HDR) 192Ir brachytherapy sources at the Institute of Nuclear Energy Research (INER, Taiwan). The Monte Carlo method established in this paper was also employed to respectively simulate wall correction factors of the 192Ir air-kerma standard chambers used at the National Institute of Standards and Technology (NIST, USA) and the National Physical Laboratory (NPL, UK) for comparisons and verification. The chamber wall correction calculation results will be incorporated into INER's HDR 192Ir primary standard in the future. For the brachytherapy treatment in the esophagus or in the bronchi, the position of the isotope may have displacement in the cavity. Thus the delivered dose would differ from the prescribed dose in the treatment plan. We also tried assessing dose distribution due to the position displacement of HDR 192Ir brachytherapy source in a phantom with a central cavity by the Monte Carlo method. The calculated results could offer a clinical reference for the brachytherapy within the human organs with cavity.
A PCR for protein D (hpd#3) was used to differentiate nontypeable Haemophilus influenzae (NTHI) from Haemophilus haemolyticus. While 90% of nasopharyngeal specimens and 100% of lower-airway specimens from 84 Indigenous Australian children with bronchiectasis had phenotypic NTHI isolates confirmed as H. influenzae, only 39% of oropharyngeal specimens with phenotypic NTHI had H. influenzae. The nasopharynx is therefore the preferred site for NTHI colonization studies, and NTHI is confirmed as an important lower-airway pathogen.
To qualitatively and quantitatively assess the association of prehypertension with incident stroke through a meta-analysis of prospective cohort studies.
We searched Medline, Embase, the Cochrane Library, and bibliographies of retrieved articles. Prospective cohort studies were included if they reported multivariate-adjusted relative risks (RRs) and corresponding 95%confidence intervals (CI) of stroke with respect to baseline prehypertension.
Twelve studies with 518,520 participants were included. Prehypertension was associated with risk of stroke (RR 1.55, 95% CI 1.35–1.79; p < 0.001). Seven studies further distinguished a low prehypertensive population (systolic blood pressure [SBP] 120–129 mm Hg or diastolic blood pressure [DBP] 80–84 mm Hg) and a high prehypertensive population (SBP 130–139 mm Hg or DBP 85–89 mm Hg). Among persons with lower-range prehypertension, stroke risk was not significantly increased (RR 1.22, 0.95–1.57). However, for persons with higher values within the prehypertensive range, stroke risk was substantially increased (RR 1.79, 95% CI 1.49–2.16).
Prehypertension is associated with a higher risk of incident stroke. This risk is largely driven by higher values within the prehypertensive range and is especially relevant in nonelderly persons. Randomized trials to evaluate the efficacy of blood pressure reduction in persons with this designation are warranted.
Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3−/− mice and to further decipher its action in in-vitro systems.
We created Glis3−/− mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components.
In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2.
These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice.
GLIS3; Neonatal diabetes; Neurog3; Pancreatic islet differentiation
High-throughput immunoprecipitation (IP) studies of transcription factors and splicing factors have revolutionized the fields of transcription and splicing. Recent location studies on Nova1/2 and Fox2 have identified a set of cellular targets of these splicing factors. One problem with identifying binding sites for splicing factors arises from the transient role of RNA in gene expression. The primary role of most splicing factors is to bind pre-mRNA co-transcriptionally and participate in the extremely rapid process of splice site selection and catalysis. Pre-mRNA is a labile species with a steady state level that is three orders of magnitude less abundant than mRNA. As many splicing factors also bind mRNA to some degree, these substrates tend to dominate the output of location studies. Here we present an in-vitro method for screening RNA protein interactions that circumvents these problems. We screen approximately 4000 alternatively spliced exons and the entire Hepatitis C genome for binding of ASF/SF2, the only splicing factor demonstrated to function as an oncogene. From the pre-mRNA sequences returned in this screen we discovered physiologically relevant ASF recognition element motifs. ASF binds two motifs: a C-rich and a purine rich motif. Comparisons with similar data derived from the hnRNP protein PTB reveals little overlap between strong PTB and ASF/SF2 sites. We illustrate how this method could be employed to screen disease alleles with the set of small molecules that have been shown to alter splicing in search for therapies for splicing diseases.
Blind sterile 2 (bs2) is a spontaneous autosomal recessive mouse mutation exhibiting cataracts and male sterility. Detailed clinical and histological evaluation revealed that bs2 mice have cataracts resulting from severely disrupted lens fiber cells. Analysis of bs2 testes revealed the absence of mature sperm and the presence of large multinucleate cells within the lumens of seminiferous tubules. Linkage analysis mapped the bs2 locus to mouse chromosome 2, approximately 45cM distal from the centromere. Fine mapping established a 3.1Mb bs2 critical region containing 19 candidate genes. Sequence analysis of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. This mutation results in two abundantly expressed aberrantly spliced Agps transcripts: AgpsΔexon14 lacking exon 14 or AgpsexonΔ13–14 lacking both exons 13 and 14 as well as full-length Agps transcript. Agps is a peroxisomal enzyme which catalyzes the formation of the ether bond during the synthesis of ether lipids. Both aberrantly spliced AgpsΔexon14 and AgpsexonΔ13–14 transcripts led to frame shift, premature stop and putative proteins lacking the enzymatic FAD domain. We present evidence that bs2 mice have significantly decreased levels of ether lipids. Human mutations in Agps result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a disease for which bs2 is the only genetic model. Thus, bs2 is a hypomorphic mutation in Agps, and represents a useful model for investigation of the tissue specificity of ether lipid requirements which will be particularly valuable for elucidating the mechanism of disease phenotypes resulting from ether lipid depletion.
Agps; ether lipids; peroxisome; RCDP3; cataract; mouse model
Images in cardiology
Paediatricians rely on cough descriptors to direct them to the level of investigations needed for a child presenting with chronic cough, yet there is a lack of published data to support this approach. A study was undertaken to evaluate (1) whether historical cough pointers can predict which children have a specific cause for their cough and (2) the usefulness of chest radiography and spirometry as standard investigations in children with chronic cough.
This was a prospective cohort study of children referred to a tertiary hospital with a cough lasting >3 weeks between June 2002 and July 2004. All included children completed a detailed history and examination using a standardised data collection sheet and followed a pathway of investigation until a diagnosis was made.
In 100 consecutively recruited children of median age 2.8 years, the best predictor of specific cough observed was a moist cough at the time of consultation with an odds ratio (OR) of 9.34 (95% CI 3.49 to 25.03). Chest examination or chest radiographic abnormalities were also predictive with OR 3.60 (95% CI 1.31 to 9.90) and 3.16 (95% CI 1.32 to 7.62), respectively. The most significant historical pointer for predicting a specific cause of the cough was a parental history of moist cough (sensitivity 96%, specificity 26%, positive predictive value 74%).
The most useful clinical marker in predicting specific cough is the presence of a daily moist cough. Both chest examination and chest radiographic abnormalities are also useful in predicting whether children have a specific cause of their cough.
chronic cough; children; diagnosis
Images in cardiology
To evaluate the need for routine histopathological analysis of enucleated/eviscerated eyes and changes in indications for eye removal.
Retrospective review of all enucleation/evisceration histopathology reports over 20 years. Clinical history was correlated with pathological findings. Two 10 year periods (1984–93, 1994–2003) were compared to detect changes in indications for eye removal.
In total, 285 histopathology results were traced from 1984 to 2003; 161 and 124 were evisceration and enucleation specimens, respectively. Glaucoma, malignant melanoma, trauma, and retinal detachment were the most frequent diagnoses 1984–1993. Ocular trauma was the most frequent diagnosis 1994–2003, followed by phthisis bulbi and endophthalmitis. Three cases were diagnosed as metastatic carcinoma; all were suspected preoperatively. A fourth case was a diagnostic surprise: adenocarcinoma found in an eye removed for pain and phthisis. Comparison of two 10 year periods showed a decrease in the number of enucleations/eviscerations, perhaps reflecting a decrease in the number of specimens sent. A preference for eviscerations was evident over the 20 years.
The number of eyes removed and histologically analysed decreased in the period 1994 to 2003, perhaps because of better treatment options, allowing globe preservation. There was a significant shift in the diagnosis in the two time periods, and a preference for evisceration in both. Only one diagnostic surprise was discovered (0.35%). This study does not support the need to send all globes/contents for histopathological examination. However, because of the one unexpected finding, it is recommended where the examination is incomplete or the history of visual loss is unclear.
enucleations; eviscerations; pathology; surprises
Images in cardiology
We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1 (“r”, “rd”, or “rodless”) mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (β-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for β-PDE immunoreactivity starting at about the same time as wild type (P10), though signal averaged less than 40% of wild type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.
mouse model; retinal degeneration; retinitis pigmentosa; rd1; rd10; nmf137; PDE6b; betaphosphodiesterase; rod photoreceptor; cGMP-PDE; beta-subunit of rod cGMP phosphodiesterase gene
IL1-RN is an important anti-inflammatory cytokine that modulate the inflammation response by binding to IL1 receptors, and as a consequence inhibits the action of proinflammatory cytokines IL1α and IL1β. In this study, we hypothesise that sequence variants in the IL1-RN gene are associated with prostate cancer risk. The study population, a population-based case–control study in Sweden, consisted of 1383 prostate cancer case patients and 779 control subjects. We first selected 18 sequence variants covering the IL1-RN gene and genotyped these single-nucleotide polymorphisms (SNPs) in 96 control subjects. Gene-specific haplotypes of IL1-RN were constructed and four haplotype-tagging single-nucleotide polymorphisms (htSNPs) were identified (rs878972, rs315934, rs3087263 and rs315951) that could uniquely describe >95% of the haplotypes. All study subjects were genotyped for the four htSNPs. No significant difference in genotype frequencies between cases and controls were observed for any of the four SNPs based on a multiplicative genetic model. Overall there was no significant difference in haplotype frequencies between cases and controls; however, the prevalence of the most common haplotype (ATGC) was significantly higher among cases (38.7%) compared to controls (33.5%) (haplotype-specific P=0.009). Evaluation of the prostate cancer risk associated with carrying the ‘ATGC' haplotype revealed that homozygous carriers were at significantly increased risk (odds ratio (OR)=1.6, 95% confidence interval (CI)=1.2–2.2), compared to noncarriers, while no significant association was found among subjects heterozygous for the haplotype (OR=1.0, 95% CI=0.8–1.2). Restricting analyses to advanced prostate cancer strengthened the association between the ‘ATGC' haplotype and disease risk (OR for homozygous carriers vs noncarriers 1.8, 95% CI=1.3–2.5). In conclusion, the results from this study support the hypothesis that inflammation has a role of in the development of prostate cancer, but further studies are needed to identify the causal variants in this region and to elucidate the biological mechanism for this association.
prostate cancer; inflammation; IL1-RN; association; SNPs
Objective To evaluate the efficacy of treatment for gastro-oesophageal reflux disease (GORD) on chronic cough in children and adults without an underlying respiratory disease.
Design Systematic review and meta-analysis.
Data sources Cochrane, Medline, and Embase databases, references from review articles.
Included studies Randomised controlled trials on GORD treatment for cough in children and adults without primary lung disease. Two reviewers independently selected studies and extracted paediatric and adult data on primary (clinical failure) and secondary outcomes.
Results 11 studies were included. Meta-analysis was limited to five studies in adults that compared proton pump inhibitors with placebo. All outcomes favoured proton pump inhibitors: the odds ratio for clinical failure (primary outcome) was 0.24 (95% confidence interval 0.04 to 1.27); number needed to treat (NNT) was 5 (harm 50 to ∞ to benefit 2.5). For secondary outcomes, the standardised mean difference between proton pump inhibitors and placebo was -0.51 (-1.02 to 0.01) for mean cough score at the end of the trial and -0.29 (-0.62 to 0.04) for change in cough score at the end of the trial. Subgroup analysis with generic inverse variance analysis showed a significant mean change in cough (-0.41 SD units, -0.75 to -0.07).
Conclusion Use of a proton pump inhibitor to treat cough associated with GORD has some effect in some adults. The effect, however, is less universal than suggested in consensus guidelines on chronic cough and its magnitude of effect is uncertain.
Objectives: To examine factors associated with compliance and with perceived readiness for the new Washington State booster seat law, and to identify perceived barriers to compliance among licensed childcare centers.
Design/methods: Surveys were mailed to a random sample of 550 licensed childcare centers in Washington State, approximately nine months before the law was to go into effect.
Results: Only 18% of centers reported being compliant with the law at the time of the survey. Factors associated with current compliance included awareness and knowledge of the law, and being comfortable asking staff and parents to use booster seats. A lack of center-owned booster seats was associated with a lower likelihood of compliance. Only 43% of centers had already started preparing for the law, and only 48% believed they would definitely be ready in time.
Conclusion: This study suggests that Washington State childcare centers need support and assistance to increase their knowledge of booster seats and reduce the financial costs of compliance.
prostate cancer; linkage analysis; hereditary cancer syndromes
prostate cancer; association; hereditary; haplotype; CYP1B1
AIMS—This study investigated the effect of peribulbar and retrobulbar local anaesthesia on intraocular pressure (IOP) and pulsatile ocular blood flow (POBF), as such anaesthetic techniques may adversely affect these parameters.
METHODS—20 eyes of 20 patients who were to undergo phacoemulsification cataract surgery were prospectively randomised to receive peribulbar or retrobulbar anaesthesia. The OBF tonometer (OBF Labs, Wiltshire, UK) was used to simultaneously measure IOP and POBF before anaesthesia and 1 minute and 10 minutes after anaesthesia. Between group comparisons of age, baseline IOP, and baseline POBF were performed using the non-parametric Mann-Whitney test. Within group comparisons of IOP and POBF measured preanaesthesia and post-anaesthesia were performed using the non-parametric Wilcoxon signed ranks test for both groups.
RESULTS—There was no statistically significant IOP increase post-anaesthesia in either group. In the group receiving peribulbar anaesthesia, there was a significant reduction in POBF initially post-anaesthesia which recovered after 10 minutes. In the group receiving retrobulbar anaesthesia, there was a persistent statistically significant reduction in POBF.
CONCLUSIONS—Retrobulbar and peribulbar injections have little effect on IOP. Ocular compression is not needed for IOP reduction when using local anaesthesia for cataract surgery. Conversely, POBF falls, at least for a short time, when anaesthesia for ophthalmic surgery is administered via a retrobulbar route or a peribulbar route. This reduction may be mediated by pharmacologically altered orbital vascular tone. It may be safer to use other anaesthetic techniques in patients with ocular vascular compromise.
BACKGROUND/AIMS—In search of a way to prevent postoperative adhesion after strabismus surgery, an animal study was performed to assess the effect of a gel consisting of a polyglycan ester in a gelatin matrix (ADCON-L).
METHODS—Bilateral recessions of superior rectus muscle (SR) were performed on 16 rabbits. ADCON-L was applied beneath and over the SR in the right eyes of all rabbits, while the operative fields in the left eyes were irrigated with a balanced salt solution (BSS). The adjustment was performed on each SR at 4 and 7 days postoperatively on the same eye. The length and force of the adjustment and the degree of adhesion were recorded. At 3 weeks postoperatively, disinsertional force was measured in several of the eyes, and the other eyes were enucleated.
RESULTS—The length of the adjustment was longer and the force of the adjustment was less in the ADCON-L group than in the BSS treated group at 4 and 7 days postoperatively (p=0.00). A significant reduction (p=0.00) in the degree of adhesion was noted in eyes treated with ADCON-L. There was no significant difference in disinsertional force between the two groups. Histopathological evaluation of the muscle revealed decreased fibrosis of perimuscular connective tissue in eyes treated with ADCON-L at 3 weeks postoperatively.
CONCLUSION—This study suggests that ADCON-L helps to prevent postoperative adhesion in rabbits and enables adjustment twice within 7 days postoperatively without complications.
AIM—To compare pulsatile ocular blood flow (POBF) and intraocular pressure (IOP) between eyes of patients receiving either peribulbar (with and without balloon compression) or subconjunctival local anaesthesia (LA).
METHODS—30 eyes of 30 patients undergoing cataract surgery by phacoemulsification were investigated in a study of parallel group design. Ten patients had peribulbar LA and 10 minutes compression with a Honan's balloon (group A). A further 10 patients who received peribulbar LA alone (group B) acted as controls for the effects of balloon compression. Ten other patients were given subconjunctival LA (group C). POBF and IOP were measured using a modified Langham pneumatonometer. Three measurements were made in each eye, the first recording immediately before LA, the second 1 minute after, and the third 10 minutes after LA.
RESULTS—No significant change in POBF or IOP was recorded in eyes receiving subconjunctival LA. In the peribulbar groups (A and B), there was a drop in median POBF of 252 and 138 µl/min respectively 1 minute after LA, which was statistically significant in both groups (p<0.01). By 10 minutes, POBF tended to return to baseline levels, but remained significantly reduced in group B (p<0.05). In addition, there was a significant (p<0.05) reduction in IOP (mean drop of 4.82 mm Hg) in group A following peribulbar LA with balloon compression.
CONCLUSIONS—POBF was significantly reduced after peribulbar LA but was unchanged after subconjunctival LA. Balloon compression reduced IOP and improved POBF following peribulbar LA. The findings may have clinical implications in patients with compromised ocular circulation or significant glaucomatous optic neuropathy.