Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study.

OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.

A note on allelic tests in case-control association studies.

This paper reconsiders the relevant contribution of Sasieni in the validity of allele-based tests in case-control genetic association studies. In particular, the author clearly demonstrates that the classical chi-square test applied to allelic contingency tables is biased when the combined case-control population is not in Hardy-Weinberg equilibrium. As an alternative, he suggests using the Cochran-Armitage test for trends by basing his argument on the fact that these two tests are asymptotically equivalent at the Hardy-Weinberg equilibrium. However he only demonstrates the equality of the statistics when the observed genotypic proportions are strictly in equilibrium--which does not formally imply the suggested, and often accepted, asymptotic behavior. In this short communication, we complement this contribution by providing the proof that allelic and trend statistics are asymptotically equivalent under the conditions mentioned above. In addition, since the 'biased' allelic test is still widely used in the literature, we briefly discuss the different alternatives that have been subsequently developed, based on Sasieni's conclusions.

Associations between genetic factors, tobacco smoking and autoantibodies in familial and sporadic rheumatoid arthritis.

OBJECTIVES: The objective of this study was to investigate the association between genes (HLA-DRB1 and PTPN22) and tobacco smoking, separately as well as combined, and serological markers of rheumatoid arthritis (RA) in a French population with RA. METHODS: 274 patients with RA with half of them belonging to RA multicase families, were genotyped for HLA-DRB1 allele and for PTPN22-1858 polymorphism. IgM rheumatoid factor and anti-cyclic citrullinated peptide (anti-CCP) antibodies were determined by ELISA method. The search for association relied on chi(2) test and odds ratio with 95% confidence interval calculation. The interaction study relied on the departure-from-additivity-based method. RESULTS: The presence of at least one shared epitope (SE) allele was associated with anti-CCP antibodies presence (82.5% vs. 68.4%, p = 0.02), particularly with HLA-DRB1*0401 allele (28.0% vs. 16.4%, p = 0.01). Tobacco exposure was associated with anti-CCP antibodies, but only in presence of SE. A tendency toward an interaction was found between tobacco, the presence of at least one HLA-DRB1*0401 allele and anti-CCP antibodies (attributable proportion due to interaction = +0.24 (-0.21+0.76)). The cumulative dose of cigarette smoking was correlated with anti-CCP antibody titres (r = 0.19, p = 0.04). The presence of both SE and 1858T alleles was associated with a higher, but not significantly different, risk for anti-CCP antibodies presence than for each separately. No association was found between PTPN22-1858T allele and tobacco smoking for autoantibody positivity. CONCLUSIONS: Our findings suggest an association between SE alleles and tobacco smoking for anti-CCP positivity and a tendency toward an interaction between the HLA-DRB1*0401 allele and smoking for anti-CCP positivity in this sample of RA.

Inverse complementary homologues of short cysteine signatures.

Inversions of short genomic sequences may play a central role in the generation of protein complexity. We report here the existence of an heterogeneous group of proteins (the trefoil precursors MUC-1 and MUA-1, six preproendothelins, and five classes of zinc finger knot proteins) having both cysteine signatures (Cs) and their inverse complementary sequences (Cs) in the same polypeptide chain. We have also found cases in which the (Cs) of a given signature is not present in the same protein, but elsewhere. TGEKPYK, a cysteine-free motif of the human transcription factor, Krab, coexists with its inverse complementary sequence in 31 proteins; the inverse complementary alone is present in a great number of proteins. Our findings suggest that short DNA inversions are a widespread feature of the genome.

The triangle test statistic (TTS): a test of genetic homogeneity using departure from the triangle constraints in IBD distribution among affected sib-pairs.

The proportions of affected sibs sharing 2, 1 or 0 identical by descent parental marker alleles have been shown to conform to the 'triangle constraints' (Suarez, 1978; Holmans, 1993). It has also been shown (Dudoit & Speed, 1999) that the constraints are verified provided certain assumptions hold. In this study we explore a realistic situation in which the constraints fail due to the presence of a factor in which the sibs differ, a factor on which penetrance depends. This factor may be a characteristic of the trait (severe vs. mild form), or the presence/absence of an associated trait or an environmental factor. We show that under such situations, using the triangle constraints may lead to important loss of power to detect linkage by the MLS test. We propose here an alternative approach in order to detect both linkage and heterogeneity.

A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis.

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.

Rapid preparation of small-volume autologous fibrinogen concentrate and its same day use in bleb leaks after glaucoma filtration surgery.

The authors evaluated small-volume preparation of autologous fibrin glue (AFG) and same day use in postglaucoma filtration surgery patients with Seidel positive bleb leaks and determined fibrinogen concentrations in autologous fibrinogen concentrates (AFCs) from 10 volunteers. Thirty milliliters of blood was centrifuged (5 min, 2400 x g); plasma was frozen (5 min-ethanol and ice), thawed (1-6 C, 30-60 min), and centrifuged (10 min, 5 C, 2800 x g); and the precipitate was transferred to a 1.0-ml tuberculosis syringe. Thrombin (1000 U) was dissolved (0.8 sterile water, 0.2 ml aminocaproic acid) and warmed (37 C). Average preparation time was 90 minutes. Alternating drops of AFC and thrombin were applied to bleb leaks until AFC clotted. Seidel testing with fluorescein determined success. AFC was prepared from 10 volunteers and fibrinogen was measured. AFG was initially successful with two (Seidel negative) eyes; one eye remained negative. AFG was unsuccessful in one briskly Seidel-positive leak. Mean +/- SD fibrinogen concentration in AFCs from the 10 volunteers was 2314 +/- 643 mg/dl (range 1608-3431 mg/dl). AFG may successfully close bleb leaks in outpatient settings. Brisk aqueous flow may impair effectiveness of AFG. Fibrinogen concentrations were comparable with previous reports.

Caution in the interpretation of MLS.

We study the statistical properties of the maximum likelihood score (MLS) test. We show that the criteria for reaching conclusions about linkage are not the same for single point analysis as for multipoint, where the maximization is performed over an additional parameter, the position in the marker interval where the MLS is computed. In addition, this test is shown to be very sensitive to errors in allele frequencies and recombination fraction.

Comparison of methods to evaluate the optic nerve head and nerve fiber layer for glaucomatous change.

PURPOSE: To compare the rates of optic nerve damage in early human glaucoma as measured by four methods to evaluate change in the optic nerve and nerve fiber layer. METHODS: Four techniques were used to detect progressive glaucomatous damage in a prospective, longitudinal study: (1) qualitative evaluation of stereoscopic color optic disk photographs, (2) qualitative evaluation of monochromatic nerve fiber layer photographs, (3) manual stereoplanimetric measurements of disk rim area, and (4) computerized measurement of peripapillary nerve fiber layer height. One eye of each patient with glaucoma or ocular hypertension was evaluated at the beginning and end of a follow-up period of not less than one year. The rates of structural change measured by these techniques and the rate of visual field change measured with threshold automated perimetry were determined. RESULTS: We followed up 193 patients for a mean (+/- S.D.) of 3.3 +/- 1.0 years (range, one to six years). Twenty-nine (15%) of 193 eyes progressed by qualitative optic disk evaluation, 14 (7.2%) of 193 eyes progressed by qualitative nerve fiber layer evaluation, seven (3.6%) of 193 eyes progressed by stereoplanimetry, and 24 (13.2%) of 182 eyes progressed by measurement of nerve fiber layer height. Visual field deterioration was detected in 12 (5.2%) of 193 patients and correlated best with qualitative optic disk and nerve fiber layer evaluations. Evaluation by stereoplanimetry and nerve fiber layer height measurement detected change in eyes with primarily diffuse structural damage, a pattern not well detected by qualitative methods. CONCLUSION: Both qualitative and quantitative methods of optic disk and nerve fiber layer evaluation contribute to the identification of progressive damage, depending on the stage of disease and the characteristics of optic nerve cupping.

Exceptional motifs in different Markov chain models for a statistical analysis of DNA sequences.

Identifying exceptional motifs is often used for extracting information from long DNA sequences. The two difficulties of the method are the choice of the model that defines the expected frequencies of words and the approximation of the variance of the difference T(W) between the number of occurrences of a word W and its estimation. We consider here different Markov chain models, either with stationary or periodic transition probabilities. We estimate the variance of the difference T(W) by the conditional variance of the number of occurrences of W given the oligonucleotides counts that define the model. Two applications show how to use asymptotically standard normal statistics associated with the counts to describe a given sequence in terms of its outlying words. Sequences of Escherichia coli and of Bacillus subtilis are compared with respect to their exceptional tri- and tetranucleotides. For both bacteria, exceptional 3-words are mainly found in the coding frame. E. coli palindrome counts are analyzed in different models, showing that many overabundant words are one-letter mutations of avoided palindromes.

Double-blind comparison of an acetaminophen 400 mg-codeine 25 mg combination versus aspirin 1000 mg and placebo in acute migraine attack.

The purpose of this study was to compare the efficacy and tolerance of a single dose of the acetaminophen 400 mg-codeine 25 mg combination (ACC) aspirin 1000 mg (A) and a placebo (P) for the treatment of acute migraine attack. The study design was randomized, multicentre, double-blind and double dummy with cross-over on three periods. Of the 198 patients who had three attacks 29.8%, 52.3% and 49.7% had recorded the complete or almost complete disappearance of the pain at 2 h after P, A and ACC respectively. When compared with the placebo, the difference was significant for the A and ACC. When complete disappearance of pain at 2 h was used as a criterion, no significant difference was observed. These results enabled the sensitivity of the evaluation criteria suggested for clinical trials of migraine attack to be discussed.

Videographic and histologic comparison of Nd:YAG and diode laser contact transscleral cyclophotocoagulation.

We performed contact transscleral cyclophotocoagulation in two human autopsy eyes with continuous-wave Nd:YAG and diode lasers. Duration of exposure was two seconds, and powers were 4 to 7.3 W with the Nd:YAG laser and 1.75 to 2.5 W with the diode laser. In both procedures, we used hand-held quartz fiberoptic contact probes for energy delivery. Tissue responses were viewed with high-magnification videographic recording technique to analyze the real-time laser effects. The treated tissues were then studied by light microscopy. We observed different tissue responses both videographically and histologically. Nd:YAG laser lesions were characterized by prominent tissue whitening and contraction of the ciliary epithelium, while the diode laser lesions had less whitening and the tissue contraction appeared to be deeper in the ciliary body. The histologic correlate was coagulation and disruption of the ciliary epithelium and little effect on the underlying ciliary muscle with the Nd:YAG laser, while the diode laser had less effect on the ciliary epithelium but caused a significant coagulative response in the ciliary muscle. Comparative trials are needed to establish the clinical significance of these videographic and histologic observations.

The influence of exposure duration in transscleral Nd:YAG laser cyclophotocoagulation.

Transscleral cyclophotocoagulation was performed in human autopsy eyes by using three Nd:YAG lasers with different durations of exposure: a pulsed, contact laser with a duration of 0.75 millisecond and a range of one to ten pulses per burst (GLase 106, Sunrise Technologies, Fremont, California); a pulsed, noncontact laser with a duration of 20 milliseconds (Microruptor 2, Lasag Medical Lasers, Thun, Switzerland); and a continuous-wave, contact laser with durations of 700 and 2,000 milliseconds (Microruptor 3, Lasag Medical Lasers, Thun, Switzerland). Tissue responses were observed with a high-magnification videographic recording technique to analyze the immediate, real-time laser effects, and by light microscopy to characterize the laser-induced lesions further. Videographically, both pulsed lasers were noted to cause mild whitening of the pigment epithelium with frequent vaporization and explosive tissue disintegration. Histologically, the 0.75-millisecond pulse typically produced the most marked epithelial disruption, referred to as an explosive-like lesion, whereas the 20-millisecond pulse more often caused moderate tissue disruption with elevation of the epithelial layers in a blister-like lesion. In contrast, the continuous-wave laser was observed videographically to produce prominent tissue whitening and puckering, seen histologically as convolution of the epithelium and coagulation of stroma, which was called a shrinkage-like lesion. Our study suggests that exposure duration influences in vitro tissue response to transscleral Nd:YAG cyclophotocoagulation, although in vivo studies and clinical trials are needed to determine which tissue response is optimum for clinical use.

In vitro videographic comparison of argon and Nd:YAG laser iridotomy.

We used an in vitro technique with high-magnification video recording to evaluate from the posterior side of the iris the immediate sequence of events during argon and Nd:YAG laser peripheral iridotomy. The observed effects differed strikingly. The argon laser caused a gradual mounding up of iris pigment epithelium with each successive energy application before final penetration. This effect was reduced but not eliminated with higher power levels. The Nd:YAG laser caused complete disruption and dispersal of the pigment epithelium with a single pulse of energy. Additionally, a multiple focal point configuration of the Nd:YAG laser was observed to produce a significantly larger iridotomy than a single focal point configuration for comparable energy settings. These observations may in part explain the observed clinical advantage of the Nd:YAG laser over the argon laser for creation of a patent iridotomy.

A new method to test genetic models in HLA associated diseases: the MASC method.

We propose a new method to analyse data on HLA associated diseases. The method uses the simultaneous information on the marker associations and segregation with the disease. It may also take into account the differential risk of being affected for specific relatives of a patient as well as the differential HLA haplotype sharing according to the marker genotype of the patient. It is based on the principle of minimization of a sum of independent chi-squares. It allows us to test the goodness-of-fit of various models in an easy and economical way. The method is applied to a sample of 269 French IDDM patients and their relatives leading to the rejection of models with one locus closely linked to HLA with two and three alleles.

Morphology of the exocrine glands of the frog skin.

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.

Reevaluation of brush border motility: calcium induces core filament solution and microvillar vesiculation.

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this "contractile ring," and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.