Inhibition of the cholesterol transporter ABCA1 by probucol decreases capacitation and tyrosine phosphorylation of dog spermatozoa, and is dose dependent.

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 µM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 µM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 µM), and 100, 250 and 500 µM PRO, but decreased with 1000 µM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 µM: 77.8 ± 10.6%, 100 µM: 63.7 ± 11.7%, 250 µM: 52.1 ± 12.9%, 500 µM: 37.7 ± 11.6%, 1000 µM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 µM: 34.6%, 1000 µM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 µM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.

Diagnostically challenging human papillomavirus-associated primary squamous cell carcinoma of the rectum with metastasis in both ovaries: a case report.

INTRODUCTION: Squamous cell carcinomas of the rectum are extremely rare and their pathogenesis is still under debate. Their proper diagnosis and treatment may thus be challenging. CASE PRESENTATION: A 52-year-old Caucasian woman was transferred to our department with a history of pelvic pain. Colonoscopy revealed a small tumorous lesion of the upper rectum and an endoscopic biopsy showed infiltration of the rectal mucosa by a squamous cell carcinoma. Afterward, tumorous lesions were found on imaging in both her ovaries. A laparoscopy with adnexectomy and anal mapping was performed and revealed tumor masses of squamous cell carcinoma in both ovaries. Based on the large size of the ovarian tumors and the concurrence of extensive, partly ciliated, macrocystic epithelium in one of the ovaries, a diagnosis of ovarian squamous cell carcinoma arising from a mature teratoma was rendered. However, human papillomavirus genotyping analyses were positive for human papillomavirus-16 in both the rectal tumor and ovarian tumors leading to a final diagnosis of a human papillomavirus-associated rectal squamous cell carcinoma metastatic to both ovaries. Neoadjuvant chemoradiation therapy of her rectum, total mesorectal excision, and hysterectomy were performed followed by adjuvant chemotherapy. CONCLUSION: Colorectal squamous cell carcinoma is a rare disease. In cases of colorectal squamous cell carcinoma, metastatic disease at any other location has to be excluded. Human papillomavirus genotyping is essential in this context. Discussion of the treatment strategies should be interdisciplinary and include chemoradiation therapy and radical surgery.

Intratympanic steroid use for idiopathic sudden sensorineural hearing loss: current otolaryngology practice in Germany and Austria.

AIMS: The frequency of the use of intratympanic steroids (ITS) as a treatment for idiopathic sudden sensorineural hearing loss (ISSNHL) in Europe is still unknown and remains a contentious issue amongst otolaryngologists. We undertook a survey of otolaryngologists in Germany and Austria to establish if there is any professional consensus with which to form a protocol for its use. METHODS: A survey of 21 questions was distributed electronically to otolaryngologists in Germany and Austria and data on demographics, indications for intratympanic treatment, procedure, follow-up, and outcomes were analysed. RESULTS: We received 908 responses. 49.1% of otolaryngologists used ITS for ISSNHL. Of those otolaryngologists who use ITS, 73.7% do not use it as primary treatment. 20.6% use ITS in conjunction with oral steroids for primary treatment and only 5.8% use ITS as monotherapy for primary treatment. 90.5% use ITS as salvage therapy. 81.1% do not consider the use of ITS after 2 weeks from the onset of symptoms. 8.3% used a tympanostomy tube and while the most commonly used steroid was dexamethasone at a concentration of 4 mg/ml (61%), a wide variety or other steroids and concentrations were used. CONCLUSIONS: This survey illustrates wide variation of current practice of intratympanic corticosteroid injection for ISSHL in Germany and Austria. In the absence of high-level evidence, knowing what current practice is allows clinicians to assess what they do against what their colleagues are doing, and if they do something very different, make them question their practice. Moreover, the obtained data will help to direct future clinical trials with the aim to compare the outcomes of more commonly used protocols.

17q21.31 duplication causes prominent tau-related dementia with increased MAPT expression.

To assess the role of rare copy number variations in Alzheimer's disease (AD), we conducted a case-control study using whole-exome sequencing data from 522 early-onset cases and 584 controls. The most recurrent rearrangement was a 17q21.31 microduplication, overlapping the CRHR1, MAPT, STH and KANSL1 genes that was found in four cases, including one de novo rearrangement, and was absent in controls. The increased MAPT gene dosage led to a 1.6-1.9-fold expression of the MAPT messenger RNA. Clinical signs, neuroimaging and cerebrospinal fluid biomarker profiles were consistent with an AD diagnosis in MAPT duplication carriers. However, amyloid positon emission tomography (PET) imaging, performed in three patients, was negative. Analysis of an additional case with neuropathological examination confirmed that the MAPT duplication causes a complex tauopathy, including prominent neurofibrillary tangle pathology in the medial temporal lobe without amyloid-ß deposits. 17q21.31 duplication is the genetic basis of a novel entity marked by prominent tauopathy, leading to early-onset dementia with an AD clinical phenotype. This entity could account for a proportion of probable AD cases with negative amyloid PET imaging recently identified in large clinical series.

Association between circadian genes, bipolar disorders and chronotypes.

Abnormalities in circadian rhythms play an important role in the pathogenesis of bipolar disorders (BD). Previous genetic studies have reported discrepant results regarding associations between circadian genes and susceptibility to BD. Furthermore, plausible behavioral consequences of at-risk variants remain unclear since there is a paucity of correlates with phenotypic biomarkers such as chronotypes. Here, we combined association studies with a genotype/phenotype correlation in order to determine which circadian genes variants may be associated with the circadian phenotypes observed in patients with BD. First, we compared the allele frequencies of 353 single nucleotide polymorphisms spanning 21 circadian genes in two independent samples of patients with BD and controls. The meta-analysis combining both samples showed a significant association between rs774045 in TIMELESS (OR = 1.49 95%CI[1.18-1.88]; p = 0.0008) and rs782931 in RORA (OR = 1.31 95%CI[1.12-1.54]; p = 0.0006) and BD. Then we used a "reverse phenotyping approach" to look for association between these two polymorphisms and circadian phenotypes in a subsample of patients and controls. We found that rs774045 was associated with eveningness (p = 0.04) and languid circadian type (p = 0.01), whereas rs782931 was associated with rigid circadian type (p = 0.01). Altogether, these findings suggest that these variants in the TIMELESS and RORA genes may confer susceptibility to BD and impact on circadian phenotypes in carriers who thus had lower ability to properly adapt to external cues.

[Pathophysiology of chemotherapy-associated thrombosis]. / Pathophysiologie der Chemotherapie-assoziierten Thrombose.

Epidemiological studies revealed that most antineoplastic agents and regimes enhance the risk of venous and arterial thromboembolic events in cancer patients. The purpose of this article is to review clinical and pathophysiological data related to chemotherapy-associated thromboembolism under special consideration of newer treatment strategies, such as angiogenesis inhibitors and immunmodulatory agents. Despite numerous clinical and experimental studies it has to be concluded that we are far from a comprehensive understanding of the pathogenesis of chemotherapy-associated thrombosis.

Comparison between idiopathic deep vein thrombosis of the upper and lower extremity regarding risk factors and recurrence.

BACKGROUND: The pathogenesis and natural course of idiopathic upper extremity deep vein thrombosis (UEDVT) are unclear. OBJECTIVE: To compare patients with UEDVT and with idiopathic lower extremity deep vein thrombosis (LEDVT) regarding risk factors and recurrence. METHODS: We followed 50 patients with first idiopathic UEDVT and 841 patients with first idiopathic LEDVT for an average of 59 and 46 months, respectively. We excluded patients with natural inhibitor deficiency, lupus anticoagulant, cancer, pregnancy, isolated pulmonary embolism (PE), or long-term antithrombotic treatment. The endpoint was recurrent venous thromboembolism (VTE). RESULTS: In comparison to LEDVT patients, UEDVT patients were younger (38 +/- 13 years vs. 49 +/- 16 years, P < 0.001), slimmer (body mass index: 24 +/- 4 vs. 27 +/- 5, P < 0.001), less frequently had a family history of VTE (18% vs. 31%, P = 0.06) or concomitant PE (8% vs. 31%, P =0.001), were less frequently carriers of factor V Leiden (12% vs. 30%, P = 0.009), and had lower thrombin generation marker levels (D-dimer, 283 +/- 361 ng mL(-1) vs. 456 +/- 446 ng mL(-1), P < 0.001; peak thrombin, 298 +/- 101 nm vs. 363 +/- 111 nm, P = 0.001). Recurrence occurred in two of 50 patients with UEDVT (4%) and in 129 of 841 patients with LEDVT (15%). After 5 years, the likelihood of recurrence was 2% [95% confidence interval (CI) 0-6] among UEDVT patients and 19% (95% CI 16-22; P = 0.02) among LEDVT patients. As compared to LEDVT patients, the adjusted risk of recurrence was 0.26 (95% CI 0.06-1.05; P = 0.059) in UEDVT patients. CONCLUSION: The pathogenesis and natural course of the disease differ between patients with idiopathic UEDVT and LEDVT.

Chemotherapy-induced thrombin generation via procoagulant endothelial microparticles is independent of tissue factor activity.

BACKGROUND: Cisplatin-based chemotherapy predisposes cancer patients to thromboembolic events. OBJECTIVES: To investigate whether endothelial damage, via formation of procoagulant endothelial microparticles (EMPs), contributes to cisplatin-related hypercoagulability. METHODS: Cell viability and caspase-3/7 activities were assessed in two endothelial cell (EC) lines [human umbilical vein ECs (HUVECs) and human pulmonary microvascular ECs (HMVEC-Ls)] after exposure to cisplatin (1, 2.5, 5, 10 and 20 microm) for up to 120 h. Counts and procoagulant activity of EMPs were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen and TF-dependent procoagulant activity of EMP were determined by enzyme-linked immunosorbent assay and a novel functional assay. RESULTS: By inducing apoptosis, cisplatin dose- and time-dependently decreased the viability of confluent HUVECs and HMVEC-Ls. Progression of EC death was accompanied by an increased release of EMPs (relative increase at 20 microm cisplatin for 48 h vs. control: HUVECs 6.5-fold, P < 0.001; HMVEC-Ls 18.4-fold, P < 0.001). EMPs were highly procoagulant (relative increase at 20 microm cisplatin for 48 h vs. control: HUVECs 2.5-fold, P < 0.001; HMVEC-Ls 5.9-fold, P < 0.001). EMP-driven thrombin generation, however, was not dependent on TF: TF expression and TF procoagulant activity levels on microparticles were only marginal and EMP-associated thrombin generation remained unchanged when the extrinsic pathway was blocked by omission of factor VIIa and/or incubation with an anti-human TF antibody. In contrast, blocking of phospholipids by annexin V markedly diminished EMP-associated procoagulant activity. CONCLUSIONS: In vitro, cisplatin induced the release of EMPs that showed TF-independent procoagulant activity.

A high-resolution linkage map for the Z chromosome in chicken reveals hot spots for recombination.

A comprehensive linkage map for chicken chromosome Z was constructed as the result of a large-scale screening of single nucleotide polymorphisms (SNPs). A total of 308 SNPs were assigned to Z based on the genotype distribution among 182 birds representing several populations. A linkage map comprising 210 markers and spanning 200.9 cM was established by analyzing a small Red junglefowl/White Leghorn intercross. There was excellent agreement between the linkage map for Z and a recently released assembly of the chicken genome (May 2006). Almost all SNPs assigned to chromosome Z in the present study are on Z in the new genome assembly. The remaining 12 loci are all found on unassigned contigs that can now be assigned to Z. The average recombination rate was estimated at 2.7 cM/Mb but there was a very uneven distribution of recombination events with both cold and hot spots of recombination. The existence of one of the major hot spots of recombination, located around position 39.4 Mb, was supported by the observed pattern of linkage disequilibrium. Thirteen markers from unassigned contigs were shown to be located on chromosome W. Three of these contigs included genes that have homologues on chromosome Z. The preliminary assignment of three more genes to the gene-poor W chromosome may be important for studies on the mechanism of sex determination and dosage compensation in birds.

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay.

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.

A novel procedure for efficient genotyping of single nucleotide polymorphisms.

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.

Detecting sincerity of effort: a summary of methods and approaches.

Despite the widespread use of methods that are supposed to detect the sincerity of patients' efforts in clinical assessment, little has been written summarizing the literature that addresses the reliability and validity of measurements obtained with these methods. The purpose of this article is to review the literature on the reliability and validity of scores for Waddell's nonorganic signs, descriptions of pain behavior and symptom magnification, coefficients of variation, correlations between musculoskeletal evaluation and function, grip measurements, and the relationship between heart rate and pain intensity. The authors of the articles reviewed conclude that none of these methods have been examined adequately. Some of these methods, such as Waddell's nonorganic signs, were not developed for the purpose of detecting sincerity of effort. Clinicians are encouraged to critically read the literature addressing these methods. With further research, some of the discussed methods may prove useful. Until such research is reported in the peer-reviewed literature, however, clinicians should avoid basing evaluation of sincerity of effort on these tests. Therapists are encouraged, instead, to use a biobehavioral approach to better understand and address the complex factors underlying delayed recovery.

A standardized weight-based heparin protocol. Improving clinical outcomes.

A 3-year study evaluated various heparin dosing regimens. The clinical outcomes of a traditional non-weight-based method were compared with a newly designed standard protocol that related the heparin dose to patient weight. The relative time to achieve therapeutic anticoagulation, the number of additional intravenous heparin injections required and the length of hospitalization were all evaluated. Overall, it was found that the individualized weight-based protocol improved clinical outcomes and lowered medical costs.

The effects of lumbosacral support belts and abdominal muscle strength on functional lifting ability in healthy women.

STUDY DESIGN: This study focused on lumbosacral support belts, abdominal muscle strength, and lifting ability in healthy women. Subjects underwent manual muscle testing to determine muscle strength and performed lifting procedures to determine lifting capacity. OBJECTIVES: The purpose of this study is threefold: 1) to determine the effectiveness of lumbosacral support belts in improving lifting ability in healthy women, 2) to determine if lumbosacral support belts are more effective for those with weak abdominals than those with strong abdominals, and 3) to determine if the maximum amount of weight varies with abdominal muscle strength. SUMMARY OF BACKGROUND DATA: In a review of published literature, one study has addressed the relationship of lumbosacral support belts and lifting capacity. However, no study has examined the use of lumbosacral support belts and lifting capacity in a female population. METHODS: A convenient sample of 69 healthy women, aged 20 to 40 years, participated in this study. Subjects were categorized into one of three groups based on lower and upper abdominal muscle strength. Each subject then performed two lifting procedures, one with a lumbosacral support belt and one without, to determine two maximum lifts. RESULTS: Women between the ages of 20 and 40 years could lift approximately 1.0 kg more weight from the floor to waist height with the lumbosacral support belt. The maximum weight lifted varied with abdominal strength. Lumbosacral support belts were not more effective for those with weak abdominals than those with strong abdominals. CONCLUSIONS: When applied properly and used in conjunction with proper lifting technique, lumbosacral support belts slightly improved lifting ability in healthy women. The magnitude of the increase, although statistically significant, is not sufficient to advocate the use of lumbosacral support belts to increase lifting capacity.

Reliability and validity of a newly developed test of physical work performance.

This study examined the interrater reliability and validity of a newly developed test of physical work abilities, the Physical Work Performance Evaluation. Eleven physical therapists were trained to administer and score this evaluation. From this group, two therapists at a time simultaneously and independently evaluated 50 patients with musculoskeletal disorders as they performed the tasks of the Physical Work Performance Evaluation. At the conclusion of the evaluation, each therapist determined the safe level of physical work for each patient. A comparison of the two independent evaluations was used to determine reliability. To determine validity, the predicted level of work was compared with the actual level of work. Kappa coefficient between the two therapists on the level of work was .83. Spearman rho correlations between the predicted and actual levels of work ranged from .41 to .55. Only 14 to 18% were working above the level predicted by the Physical Work Performance Evaluation. These results indicate high interrater reliability. Given the lack of a perfect standard for validity comparisons, these results also provide evidence in support of convergent validity. The test can be used in making decisions regarding return to work after injury, preemployment placement, and vocational exploration.

Work hardening and work conditioning interventions: do they affect disability?

The purpose of this article is to review the research on the effectiveness of work hardening and work conditioning programs. Twelve studies of work hardening and work conditioning programs in the United States and abroad were reviewed. One study produced convincing evidence in a randomized study that a work conditioning program was useful in producing a higher percentage of return to work and an earlier return to work in a group of patients off work for at least 2 months. Another study demonstrated that a work hardening program increased the rate of return to work by 52% in patients off work for greater than 4 months. Most of the other studies reviewed suggested positive results, but more carefully documented, randomized, and controlled studies are needed to support the efficacy of these programs and to determine the optimum and most cost-effective work hardening and work conditioning interventions.