Subsequent Larger Studies Research Articles

Abstract 2354: Prospective Determination of %NODS (Not consistent with Organic Deficit of Stroke) Likelihood with Resultant rt-PA Safety and Functional Outcome

Objective: To assess safety and functional outcomes of rt-PA patients prospectively assigned a high %NODS score. NODS include mimics, functional overlay and complex presentations of mild symptoms. Background: Evidence exists that mimics may be safe to treat but the act of giving rt-PA often results in a stroke diagnosis. Additionally, patients with ICH are unlikely to be coded as non-strokes. Attempts to assess mimics are usually done retrospectively, however after treatment there is often uncertainty in classification of patients as ‘NODS’ or ‘True Stroke.’ Prospective designations of such may not be recorded. To our knowledge, this is the first outcome study for patients prospectively labeled with a high %NODS score prior to rt-PA treatment. Methods: We reviewed the UCSD SPOTRIAS database, (8/09-Present) for patients prospectively defined as NODS (% likelihood that deficits are Not consistent with Organic Deficits of Stroke) ≥75% and NODS≥50%. Baseline characteristics, safety (SxICH), and outcome (90-day mRS(0-2)) were compared to ‘True Strokes’ (NODS≤25%). Continuous variables were compared using Wilcoxon-Rank Sum. Categorical variables were compared with Fisher's Exact. Results: There were 114 'True Strokes', 7 NODS≥75%, and 12 NODS≥50%. Mean age was 70.7, 51.9 (p=0.0064) and 51.1 years (p<0.001) respectively. Pre-stroke mRS(0-2) was 85.1%, 100% (p=0.59) and 91.7% (p>0.999). Baseline NIHSS was 11.5, 7.7 (p=0.36) and 6.6 (p=0.08). Outcomes were adjusted for baseline mRS. Home d/c was 40.95%, 71.4% and 66.7%. 90-day mRS(0-2) was 50%, 66.7% and 70%. Ambulance use was 85.1%, 57.1% and 58.3%. SxICH was 6.2%, 0%, 0%. Timelines were similar except for “Onset to Arrival” (74min, 42min, 61min), and “CT to decision” (22min, 43min, 38min). Conclusions: Small numbers precluded statistical significance, but absolute numbers were of clinical interest. NODS patients were younger, had less prior deficit, had milder strokes, activated EMS less frequently (possibly due to mild or less “real” deficit), had better 90-day mRS, less ICH, shorter 'onset-arrival' (perhaps arriving earlier due to improved recognition, increased fear of true or perceived deficit, or even EMS issues) but longer 'CT-decision' (perhaps requiring more time to consider pros and cons of rt-PA therapy). This intriguing exploratory analysis serves to generate hypotheses for our subsequent larger studies. If verified, high %NODS score patients may be considered safe for rt-PA therapy.

Thermal precipitation fluorescence assay for protein stability screening

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies.

The Role of Chronic Helicobacter Infection in Hepatobiliary Diseases

Purpose: It is known that Enterohepatic Helicobacter species were identified in the liver tissue, the wall of the bile ducts, and bile. Clinical significance of the presence of bile-resistant Helicobacter spp. in the biliary tract and liver is unclear. We studied a specific pool of patients without any apparent evidence of liver disease, but with chronic gastrointestinal inflammation associated with Helicobacter infection with symptoms that presented for more than 6 months. Methods: A total of 89 patients out of 536 with chronic gastroduodenitis associated with H. Pylori infection were selected. Patients with known liver diseases were excluded from the study. Antibody responses to H. Pylori were evaluated with enzyme-linked immunoassay. Other studies included upper endoscopy with histological study for H. Pylori; abdominal ultrasound; complete biochemical panel; and hepatitis panel. Results: It was found that patients with chronic Helicobacter gastroduodenitis had relatively frequent changes in clinical and laboratory data, specifically increased liver echogenicity, wall thickness of extrahepatic biliary ducts and changes in hepatic transaminases. Most of the patients had a minimal to moderate elevation of transaminases. Majority of the patients (82.5 percent) had elevated levels of anti-Helicobacter IgG and 24.4 percent of the patients had elevated levels of anti-Helicobacter IgM. Total 44 percent of the patients had high anti-Helicobacter IgG levels with concomitant alanine aminotrasferase elevation. However, only a small number of patients with low anti-Helicobacter IgG levels (2.5 percent) had elevation of transminases. Non-specific ultrasound findings included thickening of the gall bladder wall in 89.8 percent of the patients and common bile duct in 33.7 percent. About half of the patients with chronic Helicobacter gastroduodenitis had increased liver echogenicity and elevated specific IgG levels at the same time. Conclusion: Patients with chronic Helicobacter infection that was confirmed by histological and immunological studies were found to have elevation of specific anti-Helicobacter IgG levels that were associated with ALT elevation and increased liver echogenicity as well as sonographic evidence of thickening of the gall bladder wall and extrahepatic bile ducts, indicating possible involvement of hepatobiliary system into the pathological process. There is no clear explanation of these findings, thus, subsequent larger studies are warranted in the future to explore the significance of anti-Helicobacter spp. antibody levels or a role of particular enterohepatic Helicobacter sp. in the pathogenesis of chronic changes in the hepatobiliary system.Table: [238] Overview of nonspecific findings on ultrasound and liver function panel in patients with chronic Helicobacter gastroduodenitis ( n =89)Table: [238] Frequency of alanine aminotransferase elevation in patients with different levels of anti-Helicobacter IgG, IgA, and IgM levels, n/percent

Maternal mineral and vitamin supplementation in pregnancy

Deficiency of vitamins and minerals, collectively known as micronutrients, during pregnancy can have important adverse effects on maternal and birth outcomes. Evidence-based nutrition interventions can make a difference and potentially avert these outcomes. Iron supplementation has been shown to improve maternal mean hemoglobin concentration at term and reduce the risk of anemia. Zinc supplementation has been shown to result in a small but significant reduction in preterm births. A cluster-randomized study in Nepal showed a 40% reduction in maternal mortality up to 12 weeks postpartum with weekly vitamin A and 49% biweekly β-carotene supplementation but subsequent large studies in Bangladesh and Ghana have failed to demonstrate any impact on mortality. Maternal vitamin A supplementation has no role in preventing mother-to-child transmission of HIV in HIV-infected pregnant women. Periconceptional folic acid supplementation reduces the risk of neural tube defects, while supplementation with vitamin D reduces the incidence of neonatal hypocalcemia with no impact on craniotabes. Iodine supplementation during pregnancy has also been suggested to reduce the risk of perinatal and infant mortality, and the risk of endemic cretinism at 4 years of age. Calcium supplementation reduced the risk of preeclampsia in women with low baseline calcium dietary intake, while magnesium supplementation has been associated with a lower frequency of preterm births and adverse neurodevelopmental outcomes in childhood. Other vitamins and minerals, such as vitamins B, C and E, copper and selenium, have been associated with fetal development, but their impact on pregnancy outcomes is not clear. Given such widespread maternal vitamin and mineral deficiencies, it is logical to consider supplementation with multiple micronutrient preparations in pregnancy. The clinical benefits of such an approach over single-nutrient supplements are unclear, and this article explores the current concepts, evidence and limitations of maternal multiple-micronutrient supplementation.

Genomic Analysis of Left Ventricular Remodeling

The left ventricle (LV) of the heart remodels in response to hemodynamic load through the processes of physiological or pathophysiological hypertrophy.1–3 Despite extensive study of the molecular basis of cardiac hypertrophy and LV remodeling,1,4 the mainstay of medical treatment of maladaptive remodeling remains based on therapies initially devised for treating hypertension. The limited progress in the development of specific therapies for LV remodeling has led some to refer to “the impossible task of developing a new treatment for heart failure” and is the reason why, in part, device therapies have received such recent attention.5–8 To gain a better understanding of the biology of maladaptive LV remodeling, with a view to identifying therapeutic targets, new paradigms and experimental approaches are needed, and genomics provides one such methodology. Genomics is the study of genome-scale data sets, at the DNA or RNA level, and when combined with studies of physiological traits or disease phenotypes, genomics can be used to infer molecular insights. Unlike single-gene studies, which are the favored hypothesis-driven design,4 genomic approaches examine variation in up to gigabases of sequences to find statistical associations between transcripts and traits in a hypothesis-free system. These hypothesis-free studies have often been criticized as unfocused, but following the successes of genome-wide association studies (GWAS) and the recent achievements of integrated genetic and genomic analyses, a new era of genomic experimentation presents itself.9–14 It is notable that GWAS and integrated genomic studies in humans often identify regions associated with disease rather than specific genes and that effect sizes are small; nevertheless, these data remain highly informative.15–17 In this article, we review the genomic analysis of LV remodeling in the context of the recent past, the state of the art, and the imminent use of next-generation sequencing. A glossary of terms is …

Obesity and the Emergence of Sleep-Wake Gonadotropin Secretion in Girls during Early Pubertal Development

It has been clearly established that obesity is associated with reduced gonadotropin secretion in men and women. In normal women, early studies involving relatively few subjects had shown that serum LH levels were significantly reduced in obese individuals compared with levels of normal-weight controls (1, 2). Subsequent larger studies that examined the relationship of LH secretory dynamics to body weight included women with polycystic ovary syndrome (PCOS) as well as normal women (3, 4). In particular, LH secretion in both groups of obese women was characterized by reduced basal levels and decreased pulse amplitude, whereas LH frequency appeared unaffected. Recently, it was demonstrated that morbidly obese women with normal menstrual function exhibited a similar pattern of LH suppression compared with that of normal women (5). In adolescent girls, there are essentially no data concerning the influence of obesity on the rate and amplitude of LH secretion. However, during puberty the progressive maturation of gonadotropin secretion in normal girls through various stages of development has been well documented (6). Given the observed impact of obesity on LH secretion in adults, it seems logical to investigate this relationship at a time when activation of hypothalamic-pituitary interaction is emerging. This notion is underscored by the association of obesity with an earlier onset of puberty and precocity, increased risk of chronic anovulation with menstrual irregularities, and greater likelihood of PCOS. The last of these conditions commonly manifests clinical symptomatology during or soon after puberty. In this issue of JCEM, McCartney et al. (7) have extended their efforts to define the role of obesity on gonadotropin secretion during pubertal development. In previous studies using a cross-sectional design, they showed that during early puberty (Tanner 1–3), obese girls had significantly increased serum total and free testosterone levels compared with nonobese controls accompanied by corresponding decreases of circulating LH concentrations (8, 9). In addition, obese girls exhibited hyperinsulinemia and increased homeostasis model of assessment values compared with that of normal-weight controls, which may have accounted, in part, for hyperandrogenemia in the presence of lowered serum LH. In early pubertal girls, diurnal variation was observed for both testosterone and progesterone with levels lower at night and rising in the early morning hours. The incremental change in both hormones was significant in nonobese girls but failed to achieve statistical significance in obese girls likely because of too few numbers. Nevertheless, the heightened level of progesterone in obese girls may have contributed to reduced LH levels in early puberty. This notion relates to the insensitivity of the GnRH pulse generator to progesterone negative feedback in women with PCOS. In normal women, progesterone administration inhibits LH secretion, e.g. pulse frequency. Prior studies by this group have shown that androgen may interfere with this feedback mechanism as suggested by reduction of rapid LH release in response to progesterone administration in women with PCOS treated with an antiandrogen, which implies that hyperandrogenemia may contribute to increased LH secretion in this disorder. This proposed mechanism is suggested in young adolescents because some girls with PCOS treated with progesterone failed to exhibit a reduced rate of LH release (10). Because their earlier studies had been restricted to basal serum LH measurements, McCartney et al. (7) proceeded to conduct an extensive frequent sampling study of obese [body mass index (BMI) 95th percentile] and nonobese (BMI 95th percentile) girls at Tanner stages 1–5 and found differences in LH secretion within and between groups during early puberty (11). The experimental design employed a sampling interval of 1900–2300 h to denote daytime secretion and 2300–0300 h and 0300– 0700 h to reflect nighttime release. As expected, LH pulse frequency, amplitude, and mean concentrations in nonobese Tanner 1–2 girls were, in general, greater with sleep than values observed in awake subjects. In Tanner 1–2 obese girls, there was little change in LH secretion over the same intervals of study.

FOR DEBATE: IS THE EVIDENCE FOR THE EFFICACY OF CHOLINESTERASE INHIBITORS IN THE SYMPTOMATIC TREATMENT OF ALZHEIMER'S DISEASE CONVINCING OR NOT?

Over 20 years ago the publication of an optimistic report by Summers and colleagues (1986) on the use of the cholinesterase inhibitor tetrahydroaminoacridine (THA/tacrine) to treat patients affected by Alzheimer's disease (AD) started an avalanche of research publications on the use of cholinesterase inhibitors for the symptomatic treatment of the cognitive deficits seen in people with AD. Despite the fact that the Summers et al. study turned out to be very deeply flawed indeed (Division of Neuropharmacological Drug Products, 1991), the juggernaut whose movement it initiated has not yet ceased to roll. Subsequent large studies of tacrine performed on people with carefully diagnosed AD established a plausible methodology for the conduct of clinical studies of drug efficacy in the treatment of AD (Ritchie et al., 2007). These studies suggested that the drug had modest beneficial effects in some patients (Ritchie et al., 2007), and revealed that it had a high rate of adverse effects on the gut (Brodaty, 1996) and liver (Ames et al., 1990).

Tyrosine Hydroxylase

Investigations of the genetic underpinnings of hypertension have had a long history that includes twin studies, adoption studies, and family studies. Subsequent large pedigree studies showed substantial heritability for many traits related to hypertension.1,2 Guided by these high heritabilities, segregation analysis provided evidence that common mendelian genes had moderate to large effects on multiple intermediate phenotypes for blood pressure and for blood pressure itself.3 Subsequent linkage analyses suggested that these large common gene effects were not as strong as originally thought because few linkage signals reached the strength seen for known monogenic traits.4,5 The excitement surrounding the potential of whole-genome linkage scans seemed to diminish because few scans agreed on the same chromosome regions. However, as more scans have been published and more meta-analyses performed, increasing consensus exists for particular chromosomal regions containing genes contributing to blood pressure control (see the National Heart, Lung, and Blood Institute GENELINK meta-analysis Web site, https://genelink.nhlbi.nih.gov/index.jsp). Recently, a number of genes have been discovered by fine mapping regions under linkage peaks ( SLC4A5, ATP1B1, SELE, RGS5 ), suggesting that despite the lack of universal agreement of multiple scans on linked regions and only moderately sized linkage statistics, it is still possible to find disease genes under linkage signals.6,7 Article p 993 In addition to the recent gene-finding successes of family studies, whole-genome association studies have become the new hope to discover additional genes with small but important effects on blood pressure. Genome-wide association studies have now identified genes or specific chromosomal locations influencing many diseases such as diabetes mellitus,8–11 obesity,12 and coronary heart disease.13 Additional association scans now in progress will surely identify other disease genes, including new blood pressure genes. A genome-wide association study consisting of 2000 subjects for each of 7 diseases and 3000 control subjects suggested …

Photodynamic therapy of diseased bone

Photodynamic therapy (PDT) defines the light activation of a photosensitizing compound, leading to the generation of cytotoxic, reactive oxygen species. We are investigating the unique application of PDT for diseased bone. Bioluminescent, human breast cancer cells (MT-1) were injected intracardially into athymic rats. At 2-3 weeks following injection rats developed diffuse bone metastases that were visible using an in vivo bioluminescence imaging system. Metastatic lesions within vertebrae and long bones were treated with differing regimens of 690nm laser light (25-150J) following injection of benzoporphyrin-derivative monoacid (BPD-MA) and the tumour response assayed histologically upon sacrifice. Subsequent large animal studies involved light propagation studies in porcine vertebrae and BPD-MA-PDT treatment of large primary osteosarcomas in canine. Canine were subject to high dose PDT (500J/cm). A second rat model involved bacterial infection in bone. Bioluminescent Staphylococcus aureus (S. aureus) were grown as biofilms onto wires, implanted into rat tibia and exposed to 5-aminolevulinic acid (ALA)-PDT delivered percutaneously with 635nm laser light (75J/cm(2)). Response to therapy was monitored as changes in bioluminescence signal and colony forming assay upon sacrifice. PDT (150J) proved effective in ablating metastatic lesions within rat femur and lumbar vertebrae. Porcine studies confirmed the average depth of light penetration into trabecular bone as 0.16±0.04cm with an average incident fluence rate of 4.3mW/cm(2), however, the necrotic/non-necrotic interface extended 0.6cm out from the treatment fiber. In canine a single, high dose treatment created well-circumscribed margins of effect encompassing the entire 3-4cm osteosracoma. Finally, ALA-PDT proved effective against S. aureus biofilms in bone, although recurrent infection did occur in some. Results support the application of PDT to the treatment of primary or metastatic lesions within bone. Secondly, ALA-PDT may be useful as a treatment for osteomyelitis with repeat or chronic treatment regimens.

Nationwide Efficacy-Safety Study of Nebivolol in Mildly Hypertensive Patients

Nebivolol has been adequately tested in clinical efficacy trials of patients with mild hypertension. Clinical efficacy trials or their meta-analyses did not accurately predict the outcome of subsequent large studies. The primary objective was to assess the efficacy/safety of nebivolol 5-10 mg daily in a nationwide study of patients with mild hypertension. Secondary objectives were (1) to compare efficacy/safety as monotherapy versus add-on therapy and (2) to assess the effect of nebivolol on ISH. This was an open-label, 6-week follow-up study of 6,356 patients with mild hypertension or ISH, as defined by the 1999 World Health Organization guidelines, recruited from 2,700 facilities. Previous monotherapies were continued except for beta-blockers. Results are reported as means+/-SDs. Intention-to-treat analysis is given. A total of 5,740 patients completed the study; of the withdrawals, 90% were lost for follow-up or were noncompliant, 38% were untreated before, 23% had beta-blockers. In the accumulated data, mean systolic and diastolic blood pressures fell by 24+/-14 and 13+/-9 mm Hg (both P<0.001). The differences between the blood pressure-reducing effects of nebivolol monotherapy and add-on therapy were not statistically significant: 28+/-16 and 22+/-14 mm Hg for systolic and 15+/-11 and 11+/-8 mm Hg for diastolic blood pressures. Adverse events were limited to 0.5% of the patients, no serious adverse events were observed. In the ISH patients, diastolic blood pressure fell by 4+/-6 mm Hg compared with 15+/-10 mm Hg in the no-ISH patients (P<0.01). Efficacy-safety effects of nebivolol in patients with mild hypertension can be generalized in a nationwide assessment. The efficacy of nebivolol as monotherapy and as the efficacy as add-on therapy are very similar. Nebivolol is highly efficacious in patients with ISH.

Urine Markers as Possible Tools for Prostate Cancer Screening: Review of Performance Characteristics and Practicality

In recent years, an increasing number of urine-based tests have been proposed as potential screening tests for prostate cancer. The goal of this review was to summarize the current status of evidence regarding performance characteristics of the proposed tests and their practicality under screening conditions. Relevant articles published up to and including May 2005 were identified in the PubMed database. At least 10 cases and 10 controls had to be analyzed for a study to be included in the review. Data concerning the study population, performance characteristics, and the collection and processing of urine samples were extracted from the reviewed articles. In all, 34 retrospective studies evaluating 21 different markers complied with the inclusion criteria. Most of the studies were rather small and included heterogeneous clinical study populations. Promising results were reported for a few markers in single studies, but they have often not been replicated in subsequent larger studies. Some of the more promising results were obtained with 24-h urines or with specimen-handling procedures that might be difficult to perform under screening conditions. Larger studies with a prospective design are required to confirm promising findings regarding performance characteristics of some novel markers recently reported in mostly small studies. Future studies should also pay particular attention to the practicality of the markers under screening conditions.

ELAC2/HPC2 polymorphisms, prostate-specific antigen levels, and prostate cancer.

The ELAC2 gene has been proposed to be a prostate cancer susceptibility gene and is being referred to as HPC2, in part because three case-control studies suggested that two common polymorphisms (Ser217Leu and Ala541Thr) are associated with risk. However, four subsequent larger studies have not confirmed this association. In five of the seven total studies, subject selection was influenced by prostate-specific antigen (PSA) levels. We examined the association and possible effect of subject selection in a larger study and a meta-analysis. In a population-based study in Australia, 825 case patients and 732 control subjects were genotyped for the Ser217Leu and Ala541Thr polymorphisms of ELAC2. Odds ratios (ORs) for prostate cancer were estimated by unconditional logistic and polytomous regression. A meta-analysis was conducted combining our data with those from seven published studies. The association of genotype with the logarithm of plasma PSA levels in control subjects was analyzed by linear regression. The ORs for prostate cancer were 0.74 (95% confidence interval [CI] = 0.50 to 1.09) for Leu217 homozygotes and 1.01 (95% CI = 0.68 to 1.50) for Thr541 heterozygotes and homozygotes compared with Ser217 and Ala541 homozygotes, respectively. ORs were not changed by excluding control subjects with elevated PSA levels. Among control subjects, there were no statistically significant associations between genotype frequencies and PSA level for either polymorphism (both P>.4). The meta-analysis gave pooled OR estimates of 1.04 (95% CI = 0.85 to 1.26) for Leu217 homozygotes and 1.18 (OR = 0.98 to 1.42) for Thr541 homozygotes and heterozygotes. There is no evidence that either ELAC2 polymorphism is associated with prostate cancer or PSA level.

HLA Studies in Fibromyalgia

Objective: Fibromyalgia Syndrome [FMS] is a prevalent rheumatic disorder whose etiology and pathogenesis remains largely unknown. Two previous studies have reported familial clustering of FMS cases and patients with history of rheumatism and/or arthritis in first degree relatives. Another study showed the association of HLA-DR4 antigens in FMS patients, a finding not confirmed in subsequent larger studies. The objective of this study was to investigate the possible association of FMS with individual class I [loci A, B and C] and class II [DR] human leukocyte antigen [HLA] antigens. Methods: HLA-typing was determined in fifty-two FMS patients [49 females; mean age 46.7 years ± 8.9]. All FMS patients met the American College of Rheumatology 1990 classification criteria. Patients were compared with a control group of 869 normal individuals. Loci A and B were determined in all the control population, locus C in 832 and locus DR in 344 of those individuals. All loci were studied by National Institutes of Hea...

Underestimation of Helicobacter pylori Infection Rate by Biopsy Urea Broth Test

To the Editor.— I share the views of Greenberg and Bank 1 that when considering the etiopathological role of Helicobacter pylori in nonulcer dyspepsia one needs to take into account the age of the patients surveyed. However, I fear that the authors might have underestimated the true prevalence of H pylori colonization in their study population by using a single antral biopsy urea broth test. The biopsy urea broth test has recently been compared in a pilot study 2 with a modified technique, cytological brushing urea broth test, and histologic examination used in our unit and has been shown to underestimate H pylori colonization by 15% (85% sensitivity of biopsy urea broth test and 100% sensitivity of cytologic brushing urea broth test, respectively). The false-negative rate has increased to about 20% in a subsequent larger study (unpublished results). No mention has been made in the article by Greenberg and Bank