A genome-wide association study of alcohol-dependence symptom counts in extended pedigrees identifies C15orf53.

Several studies have identified genes associated with alcohol-use disorders (AUDs), but the variation in each of these genes explains only a small portion of the genetic vulnerability. The goal of the present study was to perform a genome-wide association study (GWAS) in extended families from the Collaborative Study on the Genetics of Alcoholism to identify novel genes affecting risk for alcohol dependence (AD). To maximize the power of the extended family design, we used a quantitative endophenotype, measured in all individuals: number of alcohol-dependence symptoms endorsed (symptom count (SC)). Secondary analyses were performed to determine if the single nucleotide polymorphisms (SNPs) associated with SC were also associated with the dichotomous phenotype, DSM-IV AD. This family-based GWAS identified SNPs in C15orf53 that are strongly associated with DSM-IV alcohol-dependence symptom counts (P=4.5 × 10(-8), inflation-corrected P=9.4 × 10(-7)). Results with DSM-IV AD in the regions of interest support our findings with SC, although the associations were less significant. Attempted replications of the most promising association results were conducted in two independent samples: nonoverlapping subjects from the Study of Addiction: Genes and Environment (SAGE) and the Australian Twin Family Study of AUDs (OZALC). Nominal association of C15orf53 with SC was observed in SAGE. The variant that showed strongest association with SC, rs12912251 and its highly correlated variants (D'=1, r(2) 0.95), have previously been associated with risk for bipolar disorder.

Genome-wide association analysis implicates the involvement of eight loci with response to tocilizumab for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease affecting the joints. A heterogeneous response to available therapies demonstrates the need to identify those patients likely to benefit from a particular therapy. Our objective was to identify genetic factors associated with response to tocilizumab, a humanized monoclonal antibody targeting the interleukin (IL)-6 receptor, recently approved for treating RA. We report the first genome-wide association study on the response to tocilizumab in 1683 subjects with RA from six clinical studies. Putative associations were identified with eight loci, previously unrecognized as linked to the IL-6 pathway or associated with RA risk. This study suggests that it is unlikely that a major genetic determinant of response exists, and it illustrates the complexity of performing genome-wide association scans in clinical trials.

Aseptic meningitis and adult respiratory distress syndrome caused by Borrelia persica.

INTRODUCTION: The occurrence of some of the clinical complications of tickborne relapsing fever varies with Borrelia species. For example, adult respiratory distress syndrome (ARDS), a newly reported complication, was described so far only with B. hermsii infection. MATERIALS AND METHODS: A previously healthy young Israeli man was admitted for fever and headache and was diagnosed as aseptic meningitis. Shortly before the lumbar puncture was performed he started to experience shortness of breath and developed acute respiratory insufficiency necessitating mechanical ventilation. Radiography, which was normal on admission, demonstrated bilateral lung infiltrates consistent with ARDS. Spirochetes suggestive of Borrelia were seen on a thick blood smear preparation, and polymerase chain reaction was positive for B. persica. CONCLUSION: This is the first reported case of ARDS in association with Borrelia spp. occurring outside the U.S.A. and the first one due to B. persica infection.

A bacterial biosensor encoding a genetically modified LuxR receptor exhibits improved detection of Pseudomonas aeruginosa's biomarker molecule 2-aminoacetophenone.

2-Aminoacetophneone (2-AA) is a volatile molecule produced in high amounts by the opportunistic pathogen Pseudomonas aeruginosa. We have previously shown that 2-AA activates the quorum sensing (QS) LuxR receptor of Aliivibrio fischeri. In the present study we were able to improve LuxR's affinity and detection limit for 2-AA by genetic modification of three amino acids within the binding pocket of the receptor. Expression of the modified LuxR receptor in a luminescent bacterial biosensor provided an efficient detection assay of 2-AA in clinical P. aeruginosa strains isolated from blood and lung infections, as well as in phlegm samples obtained from subjects suffering from lung infections.

High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter's syndrome.

With the introduction of intracytoplasmic sperm injection (ICSI) as a practical successful treatment for male infertility, we are able to offer the procedure to a group of patients who probably could never father a child of their own. From a patient with mosaic Klinefelter's syndrome, sufficient motile sperm for intracytoplasmic sperm injection were obtained from a fresh ejaculate estimated to contain < 100 motile sperm. In the first IVF-ICSI attempt, out of seven oocytes that were collected from the wife, four were mature and were injected by ICSI. Fertilization occurred in all four oocytes but only one cleaved and was transferred to the uterus. Pregnancy test was negative 16 days after ET. In the second treatment cycle four out of eight oocytes were selected for ICSI. All four fertilized, three cleaved at the right time, and two were transferred into the wife's uterus. One embryo was frozen. Pregnancy test 16 days after ET was negative. The high fertilization rate achieved in this case indicates the potential of ICSI to treat extreme male infertility. Its use offers hope to those patients with conditions previously considered to be untreatable.

Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility.

OBJECTIVE: To determine the success of intracytoplasmic sperm injection for severe male infertility. DESIGN: A retrospective survey. SETTING: A tertiary infertility service. PATIENTS AND INTERVENTIONS: One hundred fourteen couples had 119 intracytoplasmic sperm injection treatments because of previous failure of standard IVF, poor results with subzonal insemination, sperm concentration < 2 x 10(6)/mL, other sperm defects, or male genital tract obstruction. MAIN OUTCOME MEASURES: Fertilization, implantation, and pregnancy rates. RESULTS: Of 1,185 oocytes treated by intracytoplasmic sperm injection, normal fertilization and cleavage occurred in 717 of 1,073 that survived (67% normal fertilization rate). Abnormal fertilization occurred in 113 oocytes (11% abnormal fertilization rate) and 112 oocytes did not survive the procedure (survival rate of 90%). In 117 couples, 251 embryos were transferred fresh, 409 embryos were cryopreserved, and 224 were transferred after thawing. The implantation rate was 7.4% (fetal heart per embryo transferred). To date 36 clinical pregnancies have been achieved (12% per fresh transfer, 20% per frozen transfer, and 30% overall), 24 are ongoing or delivered (6% per fresh transfer, 14% per frozen transfer, and 20% per intracytoplasmic sperm injection). The fertilization rates were the same (65%) with various sperm defects but higher with genital tract obstructions (75%). CONCLUSION: Intracytoplasmic sperm injection has improved the prognosis of severe male infertility.

Sperm preparation for intracytoplasmic injection: methods and relationship to fertilization results.

Sperm preparation for intracytoplasmic sperm injection (ICSI) is described and the effect of high speed centrifugation during preparation on fertilization rate is evaluated. No significant differences were found in the 2-pronuclear or abnormal fertilization rates between sibling oocytes injected with sperm prepared by swim-up or mini-Percoll combined with high speed centrifugation. The high fertilization rate obtained with both methods indicates that high speed centrifugation is not necessary to prepare sperm for ICSI. Fertilization rates were also compared for sperm obtained from ejaculates, fresh and frozen epididymal aspirates, and testicular biopsies. High fertilization rates were obtained from all groups but they were significantly higher in those oocytes injected with epididymal sperm (78% per oocyte surviving injection). The high fertilization rate with epididymal sperm may reflect sperm quality or may result from the method of sperm preparation for injection. Fertilization after the injection of sperm from which the tail was dislodged during immobilization was compared with that obtained using intact sperm. A significantly lower rate of 2-pronuclear fertilization was found in those oocytes injected with sperm heads only (55%) compared with intact sperm (68%), although cleavage rates between the two groups were similar. The use of hypo-osmotic medium to select potentially live sperm from an immotile sample is also described and fertilization was obtained after the injection of sperm with a structural defect which were selected using this technique. These results indicate that high fertilization rates can be obtained with ejaculated, epididymal and testicular sperm without special treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility.

The outcome of treatment by intracytoplasmic sperm injection (ICSI) is described for patients with severe male infertility. In 296 consecutive cycles, a normal fertilization rate of 69% was achieved with 288 cycles (97%) resulting in embryos suitable for transfer. A total of 32 clinical pregnancies were achieved from the transfer of fresh embryos (clinical pregnancy rate of 12% per transfer) and an additional 44 clinical pregnancies were obtained after the transfer of frozen-thawed embryos (clinical pregnancy rate of 16% per transfer). Overall, 57 of the 76 pregnancies were ongoing or delivered. An analysis of outcome in 5 male factor subgroups revealed no significant differences in pregnancy and implantation rates between the categories. However, the fertilization rate was significantly lower in patients with oligoasthenoteratozoospermia and significantly higher in those patients for whom epididymal sperm were used for insemination. The treatment of patients with extreme male infertility is also described; normal fertilization and embryo development were obtained using ICSI in patients with mosaic Klinefelter's syndrome, severe sperm autoimmunity, round-headed acrosomeless sperm (globozoospermia), completely immotile sperm selected by hypo-osmotic swelling and sperm isolated from testicular biopsies. Three ongoing pregnancies were obtained from 6 patients for whom testicular sperm were used. These results demonstrate the value of ICSI in the management of severe male infertility, however, the treatment of some types of extreme male infertility using ICSI may be limited.

Model-Based Optimal Design and Execution of the First-Inpatient Trial of the Anti-IL-6, Olokizumab.

The first-in-patient study for olokizumab (OKZ) employed model-based, optimal design and adaptive execution to define the concentration-C-reactive protein (CRP) suppression response. Modeling and exploratory statistics activities involved: reverse engineering of first-in-class (tocilizumab) pharmacokinetic/pharmacodynamic (PK/PD) models, adaptation of models to OKZ with a priori knowledge and preclinical data translation, application of multidimensional Desirability Index for optimal study design, sample size reestimation based on new information, optimization of second study part via Bayesian analysis of interim data, and interim and final analysis for PK/PD objective attainment. Design work defined a dose window (0.1-3 mg/kg) for CRP suppression exploration and suggested 72 patients in five single-dose levels would suffice. During execution, new information resulted in reestimating the study size to half. Halting the first part and conducting interim analysis for second part optimization followed. Second interim and final analyses confirmed attainment of study objective, illustrating efficiency and optimality of the study.

The lab test: a tale of quality.

Customers expect a minimum standard of quality from the goods and services they purchase. When they judge the total quality, however, they demand a whole lot more.

Attributional biases among clinicians and nonclinicians.

Presented 27 randomly chosen clinicians (psychiatrists, psychologists, and social workers) and 18 nonclinicians with a series of statements that describe deviant and nondeviant actor behavior and employ McArthur's (1972) social psychological constructs of emotion, action, and opinion. Ss made a series of causal attributions of actor behaviors. Data were subjected to both univariate and multivariate analyses of variance. The data showed a clear dispositional attribution bias among both clinicians and nonclinicians across all stimulus statements. Further specific tests revealed that clinicians did not differ from each other or from nonclinicians in their attributions and that a significantly greater dispositional bias occurred with deviant actor behavior and in situations that featured actor actions over opinions over emotions. These results were discussed in terms of their theoretical implications and in terms of their applicability to clinical practice.

Moderate stress protects female mice against bacterial infection of the bladder by eliciting uroepithelial shedding.

We have previously shown (M. Aronson, O. Medalia, D. Amichay, and O. Nativ, Infect. Immun. 56:1615-1617, 1988) that shedding of viable uroepithelial cells (elicited by invading microorganisms) constitutes an antimicrobial defense mechanism. The present study deals with two different stress-involving procedures, in which increased uroepithelial shedding rendered female mice resistant to vesical infection. Moderate stress was induced in female mice by exposing the animals either to constant illumination for 96 h or to 37 degrees C heat for 24 h. In both cases, the rate of infection was considerably reduced as a result of increased epithelial shedding (P < 0.0001). Stress was manifested by both reduced thymic weight and increased blood corticosterone levels. Shedding was also elicited by intraperitoneal injection of norepinephrine together with hydrocortisone or by intravesical injection of corticosterone. Constant illumination as well as heat enormously facilitated the migration of polymorphonuclear cells into the bladder following the action of chemotactic stimuli. Male mice subjected to identical stress-generating conditions did not display considerable epithelial shedding or increased migration of polymorphonuclear cells, and they were not protected from intravesical infection.

TNF-alpha and IL-1 sequentially induce endothelial ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice.

Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-alpha and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-alpha by 14 wk, with levels of circulating IL-1alpha and IL-1beta being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-alpha antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-alpha and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-alpha is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.

Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity in mice: evidence for a dominant role of TNF-alpha.

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.

Limited endothelial E- and P-selectin expression in MRL/lpr lupus-prone mice.

OBJECTIVE: Inflammation in MRL/lpr mice may involve dysfunctional leucocyte-endothelial cell (EC) interactions. Previously, we have shown that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) increase with age in a tumour necrosis factor alpha (TNF alpha)- and interleukin-1 (IL-1)-dependent manner. The object of this study was to determine the expression of E- and P-selectin. METHODS: Selectin expression was quantified in MRL/lpr mice and BALB/c controls by intravenous injection of differentially radio-labelled antibodies. RESULTS: E-selectin, but not P-selectin, was up-regulated in the kidneys of older mice. Neither was up-regulated elsewhere. There was no defect in selectin inducibility, as a further inflammatory stimulus (intraperitoneal lipopolysaccharide) resulted in up-regulation. Serum from older MRL/lpr did not induce selectin expression by EC in vitro. CONCLUSION: The increase in E-selectin in the kidney may contribute to the development of glomerulonephritis. However, the lack of systemic E- and P-selectin expression may represent a protective mechanism which limits the interaction between leucocytes and the endothelium in the chronic inflammatory context.

TNF-alpha, IL-4, and IFN-gamma regulate differential expression of P- and E-selectin expression by porcine aortic endothelial cells.

P- and E-selectin are surface glycoproteins that mediate leukocyte rolling on the surface of endothelium in inflammation. We have cloned porcine P-selectin cDNA and generated a mAb, 12C5, with which to examine P-selectin expression by porcine aortic endothelial cells (PAEC) in comparison with that of E-selectin. Basal expression by PAEC of P-selectin was greater than that of E-selectin, whereas E-selectin expression was more prominently enhanced than that of P-selectin by stimulation with TNF-alpha or IL-1alpha. Both human or porcine IL-4 led to an increase in P-selectin expression, with kinetics that were delayed compared with those seen following stimulation with TNF-alpha or IL-1alpha, but IL-4 did not stimulate expression of E-selectin. When cells were stimulated with TNF-alpha in the presence of IL-4, we observed enhanced P-selectin expression with a parallel reduction in E-selectin expression. Finally, the increase in P-selectin expression due to human IL-4 was reduced in the presence of porcine but not human IFN-gamma. These observations show that E-selectin and P-selectin expression are differentially regulated in PAEC, and that IL-4 leads to a shift in the relative surface density of the two molecules toward P-selectin. The ability of porcine IFN-gamma to inhibit IL-4-induced P-selectin expression suggests that the balance between Th1 and Th2 cytokine production may determine the relative densities of the two selectins in chronic immune-mediated inflammation. Because the increased expression of P-selectin induced by human IL-4 was not inhibited by human IFN-gamma, this balance may be shifted toward P-selectin expression in porcine xenografts infiltrated by human lymphocytes.

Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence.

The selectins are adhesion molecules that mediate the tethering and rolling of leukocytes on vascular endothelium. Although E-selectin and P-selectin are known to be expressed by endothelial cells (EC) in response to proinflammatory stimuli, their pattern and mechanisms of expression in immune-mediated inflammation remain poorly understood. By quantifying luminal endothelial selectin expression via i.v. administration of radiolabeled mAb, we detected constitutive expression of P-selectin, but not E-selectin, in mouse skin. Both selectins were transiently up-regulated after intradermal TNF-alpha, IL-1alpha, or IL-1beta. In contrast, during a contact sensitivity response to oxazolone, expression of both selectins was prolonged, with distinct peaks at 6 and 48 h. Experiments with P-selectin gene-targeted mice showed that the P-selectin measured was exclusively expressed by EC rather than platelets. The early and late phases of selectin expression in contact sensitivity were differentiated in terms of their requirement for prior sensitization, and the action of IL-1. Whereas the early phase was a nonspecific 'irritant' response to oxazolone, the late phase was Ag specific and was partially IL-1 dependent. Therefore, persistence of both E- and P-selectin expression in vivo can occur as a result of sequential and distinct EC activation processes that appear to be at least partially different from those previously reported as stimulating ICAM-1 and VCAM-1 expression. The further elucidation of mechanisms of EC activation in this model may help determine the relative roles of selectins and ligands for leukocyte integrins in the sequential recruitment of T cells and other leukocyte subsets during ongoing immune-mediated inflammatory responses.

Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin.

We have aimed at selective gene delivery to vascular endothelial cells (EC) at sites of inflammation, by targeting E-selectin, a surface adhesion molecule that is only expressed by activated EC. An anti-E-selectin mAb, 1.2B6, was complexed with the adenovirus vector AdZ.FLAG (expressing the FLAG peptide) by conjugating it to an anti-FLAG mAb. Gene transduction of cultured EC was increased 20-fold compared with AdZ.FLAG complexed with a control bsAb providing EC were activated by cytokines. The anti-E-selectin-complexed vector transduced 29 +/- 9% of intimal EC in segments of pig aorta cultured with cytokines ex vivo, compared with less than 0.1% transduced with the control construct (P < 0.05). This strategy could be developed to target endothelium in inflammation with genes capable of modifying the inflammatory response.