Candidate genetic risk factors of stroke: results of a multilocus genotyping assay

BACKGROUND: Epidemiological studies indicate that genetic factors play a role in the risk of stroke, particularly in younger individuals, but the role of single-nucleotide polymorphisms (SNPs) is controversial. We tested the possible association of a number of previously described SNPs with stroke risk. METHODS: We investigated the prevalence of 60 polymorphisms located in 35 genes in 450 white patients who suffered an acute stroke or transient ischemic attack before the age of 60 years and in 817 healthy control individuals by a multilocus PCR-based assay. The controls were randomly selected from attendees of a health service program. Genetic variations were detected by hybridization to nylon strips (Roche Molecular Systems) containing detection oligonucleotides for the SNPs. We used P values of <0.05 for confirmatory analysis of the SNPs in the genes for APOE (allele 4), angiotensin converting enzyme, factor V, prothrombin, and methylenetetrahydrofolate reductase. To account for multiple testing we defined a P value of <0.001 as statistically significant for all exploratory tests. The genes represented in the test panel by more than 1 SNP were also evaluated by haplotype analysis. RESULTS: Frequencies of all 60 tested SNPs among patients and controls were very similar. No SNP reached an odds ratio of 2, and no association with stroke risk was statistically significant. CONCLUSIONS: Our results do not indicate a clinically relevant role of any of the investigated SNPs for stroke risk in individuals hospitalized for ischemic stroke/transient ischemic attack before or at 60 years of age. These results are in accordance with previous metaanalyses showing at most a very modest or no significant effect of these SNPs on stroke risk.     

Teriparatide Treatment of Severe Osteoporosis Reduces the Risk of Vertebral Fractures Compared with Standard Care in Routine Clinical Practice

Teriparatide (TPTD) is often used for the treatment of patients with severe osteoporosis, but its effectiveness in this patient group has not been specifically studied. Here, we report upon the results of an observational study involving 323 patients with severe osteoporosis (bone density T-score of ?4 or less) who were treated at a specialist osteoporosis clinic with TPTD (n = 217) or standard care (n = 106) over a 5.5-year period. The standard care group did not receive TPTD because they declined to self-inject (59.4 %), had a contraindication (7.5 %), or were already stabilized on oral bisphosphonates (33 %). The two groups were matched for the severity of osteoporosis, fracture risk, and most other clinical variables. The annual percentage change in lumbar spine bone mineral density (BMD) was greater in the TPTD group (8.2 ± 6.0 vs. 5.0 ± 8.4, p = 0.002), but there was no difference in response of hip BMD. During follow-up, 3/217 (1.38 %) TPTD-treated patients had new vertebral fractures compared with 7/106 (6.6 %) receiving standard care (p = 0.011), but there was no difference between the groups in the rate of nonvertebral fractures (11.1 vs. 8.5 %, p = 0.47). Logistic regression analysis adjusting for baseline characteristics showed that the risk of vertebral fractures in TPTD-treated patients was significantly reduced compared with standard care (odds ratio = 0.12, 95 % confidence interval 0.03–0.55, p = 0.007). Treatment of severe spinal osteoporosis with TPTD substantially reduces the risk of vertebral fractures compared with standard care and may be the preferred treatment in this patient group.     

Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor

We have analyzed several cases of Beckwith-Wiedemann syndrome (BWS) with Wilms' tumor in a familial setting, which give insight into the complex controls of imprinting and gene expression in the chromosome 11p15 region. We describe a 2.2-kbp microdeletion in the H19/insulin-like growth factor 2 (IGF2)-imprinting center eliminating three target sites of the chromatin insulator protein CTCF that we believe here is necessary, but not sufficient, to cause BWS and Wilms' tumor. Maternal inheritance of the deletion is associated with IGF2 loss of imprinting and up-regulation of IGF2 mRNA. However, in at least one affected family member a second genetic lesion (a duplication of maternal 11p15) was identified and accompanied by a further increase in IGF2 mRNA levels 35-fold higher than control values. Our results suggest that the combined effects of the H19/IGF2-imprinting center microdeletion and 11p15 chromosome duplication were necessary for manifestation of BWS.     

Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers.

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.     

Massive Schmerzen und livide Verfärbung des linken Beins nach intravaskulärer Heroininjektion bei einem 32-jährigen Patienten

Acute leg ischemia after intra-arterial drug injection represents a critical vascular emergency scenario. Due to lack of evidence-based standards therapeutic strategies are oriented to the underlying pathomechanisms. For a sufficient therapy a close clinical monitoring and laboratory analyses as well as treatment with analgesics, anticoagulants, anti-inflammatory and spasmolytic agents are of utmost importance. This article reports on the diagnostic and therapeutic approaches in a 32-year-old patient with acute leg ischemia after intra-arterial administration of heroin and secondary infection with Peptostreptococcus and Peptoniphilus species.     

[11C]5-HTP and microPET are not suitable for pharmacodynamic studies in the rodent brain

The PET tracer [¹¹C]5-hydroxytryptophan ([¹¹C]5-HTP), which is converted to [¹¹C]5-hydroxytryptamine ([¹¹C]5-HT) by aromatic amino acid decarboxylase (AADC), is thought to measure 5-HT synthesis rates. But can we measure these synthesis rates by kinetic modeling of [¹¹C]5-HTP in rat? Male rats were scanned with [¹¹C]5-HTP (60 minutes) after different treatments. Scans included arterial blood sampling and metabolite analysis. 5-HT synthesis rates were calculated by a two-tissue compartment model (2TCM) with irreversible tracer trapping or Patlak analysis. Carbidopa (inhibitor peripheral AADC) dose-dependently increased [¹¹C]5-HTP brain uptake, but did not influence 2TCM parameters. Therefore, 10 mg/kg carbidopa was applied in all subsequent study groups. These groups included treatment with NSD 1015 (general AADC inhibitor) or p-chlorophenylalanine (PCPA, inhibitor of tryptophan hydroxylase, TPH). In addition, the effect of a low-tryptophan (Trp) diet was investigated. NSD 1015 or Trp depletion did not affect any model parameters, but PCPA reduced [¹¹C]5-HTP uptake, and the k₃. This was unexpected as NSD 1015 directly inhibits the enzyme converting [¹¹C]5-HTP to [¹¹C]5-HT, suggesting that trapping of radioactivity does not distinguish between parent tracer and its metabolites. As different results have been acquired in monkeys and humans, [¹¹C]5-HTP-PET may be suitable for measuring 5-HT synthesis in primates, but not in rodents.     

Implementation of Community Health Fund in Tanzania: why do some districts perform better than others?

In early 1990s, Tanzania, like other African countries, introduced user fees in public health systems. Although user fees were considered important in promoting health, they appear to reduce people's access to health services. To counteract the detrimental effects of the user fees, various types of health insurances were introduced, including the Community Health Fund (CHF). Drawing from the review of minutes, health facility visits and key informant interviews, this study explored why implementation of the CHF in Tanzania has been more successful in some districts than in others. The findings indicate that in Lindi district, the enrolment rate for the CHF was very low. This was attributed to high premium rates, frequent drug stock‐out, lack of trust by the community members to the health providers, low incentives and local politics. In contrast, in Iramba district, the performance was better. Availability of drugs in the health facilities, effective supervision, commitment of the top district‐level officials and incentives to the health facility committees were the main factors that facilitated good performance of the fund in Iramba district. The focus of the implementation needs to be placed on the active engagement of the local‐level leaders and politicians who are responsible for the implementation of the policy. Equally important is the availability of quality health services in the health facilities. Copyright © 2013 John Wiley & Sons, Ltd.     

Breakdown of Phosphatidylserine Asymmetry Following Treatment of Erythrocytes with Lumefantrine

Background: Lumefantrine, a commonly used antimalarial drug, inhibits hemozoin formation in parasites. Several other antimalarial substances counteract parasitemia by triggering suicidal death or eryptosis of infected erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), formation of ceramide, oxidative stress and/or activation of p38 kinase, protein kinase C (PKC), or caspases. The present study explored, whether lumefantrine stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species from 2'',7''-dichlorodihydrofluorescein-diacetate fluorescence, content of reduced glutathione (GSH) from mercury orange fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to lumefantrine (3 µg/mL) was followed by a significant increase of annexin-V-binding without significantly altering forward scatter, [Ca²⁺]_i, ROS formation, reduced GSH, or ceramide abundance. The annexin-V-binding following lumefantrine treatment was not significantly modified by p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM), PKC inhibitor staurosporine (1 µM) or pancaspase inhibitor zVAD (1 or 10 µM). Conclusions: Lumefantrine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca²⁺, ceramide formation, ROS formation, glutathione content, p38 kinase, PKC or caspases.