Placebo effect in burning mouth syndrome: a systematic review.

Placebo controls play a critical role in the evaluation of any pharmacotherapy. This review surveys the placebo arm in 12 randomized controlled trials (RCTs) investigating burning mouth syndrome (BMS) and documents a positive placebo response in 6 of them. On average, treatment with placebos produced a response that was 72% as large as the response to active drugs. The lack of homogeneity in the use of placebos adds to the difficulty in comparing results and aggregating data. Future RCTs investigating BMS would benefit from larger sample sizes, adequate follow-up periods, and use of a standard placebo.

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3.

A total of 116,118 basepairs (bp) derived from three cosmids spanning the ERCC1 locus of human chromosome 19q13.3 have been sequenced with automated fluorescence-based sequencers and analysed by polymerase chain reaction amplification and computer methods. The assembled sequence forms two contigs totalling 105,831 bp, which contain a human fosB proto-oncogene, a gene encoding a protein phosphatase, two genes of unknown function and the previously-characterized ERCC1 DNA repair gene. This light band region has a high average density of 1.4 Alu repeats per kilobase. Human chromosome light bands could therefore contain up to 75,000 genes and 1.5 million Alu repeats.

Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.

A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.

Expressed genes, Alu repeats and polymorphisms in cosmids sequenced from chromosome 4p16.3.

The sequences of three cosmids (90 kilobases) from the Huntington's disease region in chromosome 4p16.3 have been determined. A 30,837 base overlap of DNA sequenced from two individuals was found to contain 72 DNA sequence polymorphisms, an average of 2.3 polymorphisms per kilobase (kb). The assembled 58 kb contig contains 62 Alu repeats, and eleven predicted exons representing at least three expressed genes that encode previously unidentified proteins. Each of these genes is associated with a CpG island. The structure of one of the new genes, hda1-1, has been determined by characterizing cDNAs from a placental library. This gene is expressed in a variety of tissues and may encode a novel housekeeping gene.

Genetic association of htSNPs across the major histocompatibility complex with rheumatoid arthritis in an African-American population.

Notwithstanding the well-established association of HLA-DRB1 shared epitope alleles, interest remains in identifying additional major histocompatibility complex (MHC) region variants associated with rheumatoid arthritis (RA). We used a panel of 1201 haplotype-tagging single nucleotide polymorphisms (SNPs) designed for African Americans to find genetic variants associated with RA in a 3.8-Mb region encompassing the MHC. Conditioning on seven covariates, including HLA-DRB1 risk alleles and population structure, we identified an SNP in HLA-DOA (rs9276977) significantly associated with RA; minor allele frequency (MAF) 0.27 in cases versus 0.21 in controls, odds ratio (+/-95% confidence interval)=2.86 (1.61, 5.31). Genotyping of rs9276977 in an independent sample of African-American RA patients and controls did not replicate the association (MAF 0.28 in cases versus 0.27 in controls). This study points to the potential association of a SNP in the HLA-DOA gene with RA in African Americans, but also underscores the importance of replication of findings in larger patient cohorts.

Complementary DNA sequencing: expressed sequence tags and human genome project.

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Evaluating linkage disequilibrium and recombination provides a haplotype-tagging SNP panel of the major histocompatibility complex in African Americans.

The major histocompatibility complex (MHC) (Chromosome 6p21.3) is a dynamic, immune gene-rich region that is associated with multiple diseases. Haplotype-tagging single-nucleotide polymorphism (htSNP) panels for the MHC can aid association studies but have only been reported for African, Asian and Caucasian populations to date. We genotyped 2154 SNPs spanning a 3.8-Mb region of the classical MHC in 94 healthy African Americans using Illumina BeadArray technology. We describe the haplotype structure of the MHC in African Americans, calculate the recombination rate (0.35 cM Mb(-1)) across the region, identify recombination hot spots and develop a panel of htSNPs for future genetic association studies in this population. We conclude that while patterns of LD and recombination are similar within the MHC to that reported in other populations, differences in minor allele frequency at specific markers necessitates an htSNP panel unique to African Americans, which we provide here for use in future genetic association studies.

Interferon regulatory factor-5 is genetically associated with systemic lupus erythematosus in African Americans.

Increased expression of interferon (IFN)-inducible genes is implicated in the pathogenesis of systemic lupus erythematosus (SLE). One transcription factor responsible for regulating IFN, interferon regulatory factor-5 (IRF5), has been associated with SLE in genetic studies of Asian, Caucasian and Hispanic populations. We genotyped up to seven polymorphic loci in or near IRF5 in a total of 4870 African-American and Caucasian subjects (1829 SLE sporadic cases and 3041 controls) from two independent studies. Population-based case-control comparisons were performed using the Pearson's chi(2)-test statistics and haplotypes were inferred using HaploView. We observed significant novel associations with the IRF5 variants rs2004640 and rs3807306 in African Americans and replicated previously reported associations in Caucasians. While we identified risk haplotypes, the majority of haplotypic effects were accounted for by one SNP (rs3807306) in conditional analyses. We conclude that genetic variants of IRF5 associate with SLE in multiple populations, providing evidence that IRF5 is likely to be a crucial component in SLE pathogenesis among multiple ethnic groups.

Copy number variants in genetic susceptibility and severity of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder characterized by the presence of auto-antibodies to nuclear antigens, immune complex deposition, and subsequent tissue destruction. Early studies in twins suggested that SLE has, at least in part, a genetic basis, and a role for class II alleles in the major histocompatibility complex has been known for over 30 years. Through both linkage studies and candidate gene studies, numerous additional genetic risk factors have been identified. The recent publication of two SNP-based genome-wide association studies (GWAS) has resulted in the confirmation of a number of previously identified genetic risk loci and has identified new previously unappreciated loci conferring risk for development of SLE. A role for gene copy number variation (CNV) in SLE has also been appreciated through studies of the complement component 4 (C4) loci and more recent work in the IgG Fc receptor loci. The availability of large SNP-based GWAS datasets will undoubtedly lead to the genome-wide analysis and identification of copy number variants related to genetic susceptibility for development of SLE. We review current studies of CNV in SLE susceptibility that include reports of association between SLE and CNV in C4, IgG Fc receptors, TLR7, and CCL3L1.

High prevalence of fasciolosis and evaluation of drug efficacy against Fasciola hepatica in dairy cattle in the Maffra and Bairnsdale districts of Gippsland, Victoria, Australia.

Liver fluke (Fasciola hepatica) is a common parasite amongst grazing livestock in the south-eastern region of Australia and is responsible for significant production losses in the beef and dairy industries. Gippsland in Victoria is a major region for dairy production but no fluke prevalence data in livestock has been obtained in this region since the late 1970s prior to the introduction of Triclabendazole (TCBZ). TCBZ resistance is also now widespread in cattle in south east Australia. In this study, we evaluated the prevalence and intensity of liver fluke infections in dairy cattle in Gippsland and assessed the efficacy of TCBZ and other drenches against F. hepatica on one farm. We obtained 30 individual faecal samples from each of 15 different farms and, using the liver fluke coproantigen ELISA, tested bulk faecal samples pooled from each farm. Any farm that returned a positive bulk sample had all of the samples tested individually to assess the intra-herd prevalence. One farm in the Maffra district also had a coproantigen reduction test and faecal egg count reduction test to assess the efficacy of TCBZ, Clorsulon (CLOR) and Oxyclozanide (OXY). The coproantigen ELISA proved to be a highly sensitive test for liver fluke with a high correlation (R(2)=0.8849) observed between ELISA data from bulk samples and individual samples, suggesting that future larger scale screening on farms for fasciolosis could use the bulk analysis technique. The ELISA data revealed that animals on six of the 15 farms were infected with F. hepatica and the herd prevalence of the infected herds ranged from 47 to 100% (mean 81%) which exceeds the prevalence value for production losses of 25%. The intensity of fluke infection in cattle varied considerably both within and between herds with a proportion of animals exhibiting a positive control value in the coproantigen ELISA of 50-88%. We also confirmed that TCBZ resistance was present on one farm but that CLOR or OXY can be used to remove the adult stage of the TCBZ-resistant parasites. We conclude that fasciolosis is a significant disease and a likely cause of production losses in dairy cattle in the irrigation zones of Gippsland and that TCBZ resistance is a serious threat to fluke control. We suggest that more work needs to be performed in Gippsland to further define the extent of fasciolosis and drug resistance and to ensure that effective chemical and non-chemical methods of fluke control are incorporated on farms in order to improve animal welfare and reduce financial impacts on producers.

Optimization of Applied Biosystems 373 sequencer stretch modification.

We have optimized the conditions for using the Stretch modification for the Applied Biosystems 373 Automated DNA Sequencers for sequencing double-stranded DNA using 34-cm well-to-read and 48-cm well-to-read configurations. With the manufacturer's recommended settings, uneven spacing within the first 100 bases was observed, which led to miscalls, insertions and deletions in the analyzed data. A significant decrease in accuracy for reads greater than 400 bases was also observed. Various gel concentrations were tested to improve the base spacing for the first 100 bases while maintaining accuracy and usable length of data. A longer average usable length and better resolution of smaller fragments were achieved by increased acrylamide concentration coupled with increased wattage. Using the Applied Biosystems CATALYST 800 Molecular Biology LabStation, Taq dye primer cycle sequencing reactions were optimized for -21 M13 and M13RP1 primers to produce a more even distribution of dye-labeled fragments that increased the overall signal strengths and decreased background signal. These reaction products, run on the Stretch sequencers using the new gel conditions, provided longer reads with increased reliability and accuracy of the data.

Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions.

Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.

Possible relationship between degenerative cardiac valvular pathology and lyme disease.

We report an unusual clinical presentation of Lyme carditis in a previously healthy 20-year-old black woman without any epidemiologic history of Lyme disease, fulminant in nature, involving a heart valve necessitating emergent mitral valve replacement, and requiring further surgical intervention because of the development of pericardial effusion and tamponade. A dilated right ventricle with normal contractility and severe tricuspid regurgitation with increase in the right atrial size diagnosed later remains under close surveillance.

Global distribution of Pu isotopes and 237Np.

Inventories and compositions of Pu isotopes and 237Np in archived soil samples collected in the 1970s from 54 locations around the world were determined to provide regional baselines for recognizing possible future environmental inputs of non-fallout Pu and Np. As sample sizes used in this work were small (typically 1 g), inhomogeneities in Pu and Np concentrations were easily recognizable and, as a result, we were able to determine that atypical debris in South America, from French testing in the South Pacific, is more widely and uniformly distributed than previously supposed. From our results we conclude that fallout 237Np/239Pu atom ratios are generally lower in the Southern Hemisphere (approximately 0.35) than in the Northern Hemisphere (approximately 0.47.) Moreover, 237Np/239Pu atom ratios are more device-dependent, hence more variable, than counterpart 240Pu/239Pu atom ratios. Given predictable trends caused by sample inhomogeneities, with only two exceptions, the Pu results of this work are entirely consistent with (and in several instances improve on) results previously reported for these same samples. However, unlike earlier interpretations used to explain these results, we recommend that fallout isotopic signatures be represented by mixing lines, rather than averages, to better reflect regional variations of stratospheric fallout inventories relative to tropospheric fallout inventories, and provide the theoretical basis for doing so. Finally, the Np results of this work constitute one of the largest single compilations of such data reported to date.

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.

Size-fractionated plutonium isotopes in a coastal environment.

We have examined the distribution of individual Pu isotopes (239Pu, 240Pu, and 241Pu) in seawater from the Gulf of Maine (GOM). Samples were size-fractionated with a 1 kD cross-flow ultrafiltration (CFF) membrane. Subfractioned samples were radiochemically purified and Pu isotopes were analyzed using a three-stage thermal ionization mass spectrometer (TIMS). To our knowledge, this is the first time that both size class and Pu isotopic data have been obtained for seawater samples. Within measurement uncertainties a single 240Pu/239Pu atom ratio of 0.18 was found for all sample collection depths and sample size fractions. This signifies a current, single Pu source in GOM waters, namely global fallout, and suggests that no measurable isotopic fractionation occurred during CFF processing. The majority of Pu was found in the low molecular weight fraction (< 1 kD). Colloidal Pu varied from 8% of the total in surface waters to < 1% in the deepest (250 m) seawater sample. Evidence suggests that the vertical distribution of Pu in GOM is primarily controlled by conservative mixing processes. The high Pu fraction found in the low molecular size fraction implies that most of the Pu is in the non-particle-reactive oxidized fraction, and is consistent with the conservative Pu behavior. The activity levels are in agreement with other studies which show a slow decrease in Pu with time due to continued mixing and relatively slow particle removal.

Serum correlates of the placebo effect in irritable bowel syndrome.

BACKGROUND In diseases defined primarily by the subjective nature of patient self-report, placebo effects can overwhelm the capacity of randomized controlled trials to detect medication-placebo differences. Moreover, it is unclear whether such placebo effects represent genuine psychobiological phenomena or just shifts in selective attention. Knowledge of predictors of the placebo response could improve the design of clinical trials and the delivery of personalized medical care. METHODS In patients with irritable bowel syndrome (IBS), a subset of our previous study that were randomized to placebo treatment (sham acupuncture) or no-treatment group (waitlist), we tested an enriched panel of 10 serum biomarkers at the enrolment and the 3rd week of intervention, using a multiplex electrochemiluminescent immunoassay. KEY RESULTS More pronounced changes overtime in serum levels of osteoprotegerin (OPG) have been found in patients who received placebo treatment compared with the waitlist group (P = 0.039). Moreover, serum levels of OPG at baseline were found to be higher (P = 0.0167) in patients who subsequently achieved adequate relief (AR) of their IBS symptoms, independently of their treatment group. Besides, serum levels of TNF-related weak inducer of apoptosis (TWEAK) at baseline were also higher (P = 0.0144) in patients who reported AR and in particular in those who received the placebo treatment. CONCLUSIONS & INFERENCES These two measurable biological parameters associated with placebo, namely serum OPG and TWEAK, provide a proof of principle for discovering putative molecular signatures of placebo response in IBS and perhaps in other illnesses with patient self-reported outcomes.

Meta-analysis: the effects of placebo treatment on gastro-oesophageal reflux disease.

BACKGROUND: There appears to be a significant placebo response rate in clinical trials for gastro-oesophageal reflux disease. Little is known about the determinants and the circumstances associated with placebo response in the treatment of gastro-oesophageal reflux disease (GERD). AIMS: To estimate the magnitude of the placebo response rate in randomized controlled trials for GERD and to identify factors that influence this response. METHODS: A meta-analysis of randomized, double-blind, placebo-controlled trials, published in English language, which included >20 patients with GERD, treated with either a proton pump inhibitor or H(2)-receptor antagonist for at least 2 weeks. Medline, Cochrane and EMBASE databases were searched, considering only studies that reported a global response for 'heartburn'. RESULTS: A total of 24 studies included 9989 patients with GERD. The pooled odds ratio (OR) for response to active treatment vs. placebo was 3.71 (95% CI: 2.78-4.96). The pooled estimate of the overall placebo response was 18.85% (range 2.94%-47.06%). Patients with erosive oesophagitis had a non-significantly lower placebo response rate than patients without it (11.87% and 18.31%, respectively; P = 0.246). Placebo response was significantly lower in studies of PPI therapy vs. studies of H(2) RAs (14.51% vs. 24.69%, respectively; P = 0.05). CONCLUSIONS: The placebo response rate in randomized controlled trials for GERD is substantial. A lower placebo response was associated with the testing of PPIs, but not the presence of erosive oesophagitis.