Immunohistochemical evaluation of endothelial nitric oxide synthase expression in primary breast cancer

To evaluate the clinical potential of endothelial nitric oxide synthase (e-NOS) in human breast cancer, we performed immunohistochemical (IHC) staining of paraffin-embedded primary breast cancer tissue of 163 patients for e-NOS using a monoclonal antibody. A correlation was found between e-NOS expression and both the classic prognostic factors and survival rates. Under half the patients were premenopausal (38.5%), 61.5% being postmenopausal. The median tumour size was 2 cm; in 41.7% of the patients there was involvement of the axillary lymph nodes. Most (84.1%) of the tumours were hormone receptor positive. e-NOS staining was positive in 62%, most of the positive tumours having weak (32.5%) or medium (21.5%) staining for e-NOS. The median follow-up time was 42 months, during which 46 (28%) patients had a local recurrence or metastatic disease. A positive correlation of e-NOS with the hormone receptor status was found (P=0.031). However, no impact on survival rates was observed.     

The Effects of Prolonged Water-Only Fasting and Refeeding on Markers of Cardiometabolic Risk

(1) Background: Cardiometabolic disease, including insulin resistance, hyperlipidemia, and hypertension, are major contributors to adverse health outcomes. Fasting has gained interest as a nonpharmacological therapeutic adjunct for these disorders. (2) Methods: We conducted a prospective, single-center study on the effects of prolonged water-only fasting followed by an exclusively whole-plant-food refeeding diet on accepted measures of cardiovascular risk and metabolic health. Participants were recruited from patients who had voluntarily elected to complete a water-only fast in order to improve their overall health according to an established protocol at an independent, residential medical center. Median fasting and refeed lengths were 17 and 8 days, respectively. The primary endpoint was to describe the mean glucose tolerance as indicated by Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) scores at baseline, end-of-fast (EOF), and end-of-refeed (EOR) visits. Secondary endpoints were to describe the mean weight, body mass index (BMI), abdominal circumference (AC), systolic blood pressure (SBP), diastolic blood pressure (DBP), lipid panel, and high-sensitivity C-reactive protein (hsCRP) at the same time points. (3) Results: The study enrolled 48 overweight/obese non-diabetic participants, of which 26 completed the full study protocol. At the EOF visit, the median SBP, AC, low-density lipoprotein (LDL), and hsCRP were decreased and triglycerides (TG) and HOMA-IR scores were increased. Conclusion: Prolonged water-only fasting and whole-plant-food refeeding holds potential as a clinical therapy for cardiometabolic disease but increased TG and HOMA-IR values after refeeding necessitate further inquiry.     

Genetic association signal near NTN4 in Tourette syndrome

Tourette syndrome (TS) is a neurodevelopmental disorder with a complex genetic etiology. Through an international collaboration, we genotyped 42 single nucleotide polymorphisms (p < 10⁻³) from the recent TS genomewide association study (GWAS) in 609 independent cases and 610 ancestry‐matched controls. Only rs2060546 on chromosome 12q22 (p = 3.3 × 10⁻⁴) remained significant after Bonferroni correction. Meta‐analysis with the original GWAS yielded the strongest association to date (p = 5.8 × 10⁻⁷). Although its functional significance is unclear, rs2060546 lies closest to NTN4, an axon guidance molecule expressed in developing striatum. Risk score analysis significantly predicted case–control status (p = 0.042), suggesting that many of these variants are true TS risk alleles. Ann Neurol 2014;76:310–315     

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury

The goal of this study was to establish an SPIO‐based cell‐tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri‐lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post‐injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease

Toll-like receptors (TLR) are signal molecules essential for the cellular response to bacterial cell wall components. Different functional effective polymorphisms for the TLR 4 gene (Asp299Gly; Thr399Ile) and for the TLR 2 gene (Arg677Trp, Arg753Gln) have recently been described that are associated with impaired lipopolysaccharide signal transduction. A total of 122 patients with chronic periodontal disease and 122 healthy unrelated controls were genotyped for the Asp299Gly and Thr399Ile polymorphism of the TLR 4 gene and the Arg677Trp and Arg753Gln mutation of the TLR 2 gene. The mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. The prevalence of the Asp299Gly and the Thr399Ile mutant allele was 4.1% (10/244) and 4.5% (11/244) among periodontitis patients. For the healthy controls the prevalence was 3.3% (8/244) for the Asp299Gly (P = 0.810) and 3.7% (9/244) for the Thr399Ile mutant allele (P = 0.819). The Arg753Gln mutant allele was found in 2.9% (7/244) of the periodontitis subjects as compared to 4.1% (10/244) in the control group (P = 0.622). The Arg677Trp mutant allele was not found in any of the study subjects. Unlike in ulcerative colitis there was not observed an association between chronic periodontitis and the various mutations of the TLR 2 and 4 gene.     

Clofazimine in the treatment of multidrug-resistant tuberculosis

Clofazimine has shown activity against Mycobacterium tuberculosis, including multidrug-resistant strains in vitro and in animal studies. However, clinical experience with clofazimine in multidrug-resistant tuberculosis (MDR-TB) is scarce. We reported our clinical experience with 39 MDR-TB patients treated with combination regimens that included clofazimine. From January 2008 to March 2011, 39 patients received clofazimine for the treatment of MDR-TB in Shanghai Pulmonary Hospital. Patients had isolates resistant to a median of six drugs (range, 2–11 drugs). Of the 39 cases, 36 had cavitary changes noted on initial chest radiograph or chest computed tomography, and positive sputum-smear microscopy results at the time of MDR-TB diagnosis. At data censure, 15 of the 39 patients had successful therapy, with at least five consistently negative cultures documented for the final 12 months of treatment. Eleven continued to receive treatment. There were no deaths. Thirteen patients had a poor outcome, including four defaults and nine treatment failures. Culture conversion occurred in 22 cases at a median of 12 weeks. Side-effects occurred in 34 patients, mainly including skin discolouration, ichthyosis and gastrointestinal adverse events. No patients reported significant toxicity likely to be attributable to clofazimine therapy. Adverse events were managed by combinations of dose adjustment and symptom management. In our experience, clofazimine was well tolerated and may have efficacy in the treatment of MDR-TB.     

Evaluation of mast cell activation syndromes: impact of pathology and immunohistology

Mast cell activation syndromes (MCAS) are clinically defined disease states with a largely unknown morphological background. Since mastocytosis may be associated with MCAS, it is crucial in every patient to document or exclude mastocytosis by appropriate histological, molecular, and serological investigations of tissues/organs that are commonly involved in mastocytosis like skin, mucosa of the gastrointestinal tract and bone marrow. Accordingly, histopathological investigation including immunohistological stains is crucial to reach the final diagnosis in such patients and to classify MCAS into primary MCAS, which can present with or without evidence of overt mastocytosis, or secondary MCAS, where an underlying disease with or without tissue inflammation is detected. Cases without evidence of mastocytosis, monoclonal mast cells, or any underlying disease should be termed idiopathic MCAS. When the activating point mutant KIT D816V is detectable but criteria for diagnosis of mastocytosis are not completely met, a so-called (mono)clonal MCAS as a subvariant of primary MCAS should be diagnosed.     

Defining the roles of actigraphy and parent logs for assessing sleep variables in preschool children

Actigraphy provides a non-invasive objective means to assess sleep-wake cycles. In young children, parent logs can also be useful for obtaining sleep-wake information. The authors hypothesized that actigraphy and parent logs were both equally valid instruments in healthy preschool-aged children. The authors studied 59 children aged 3 to 5 years in full-time day care. Each child was screened for medical problems and developmental delays before being fitted with an actigraphy watch, which was worn for 1 week. Parents maintained logs of sleep and wakefulness during the same period, with input from day care workers. In general, parents overestimated the amount of nighttime sleep measured by actigraphy by 13% to 22% (all significant). Although there was no difference in sleep onset times, parents reported later rise times on the weekend and fewer nighttime awakenings. There was no significant difference between parent logs and actigraphy with regard to daytime napping. The authors conclude that parent logs are best utilized in assessing daytime sleep and sleep onset, whereas actigraphy should be used to assess nighttime sleep and sleep offset time.     

Prothrombin and factor V mutations in women with a history of thrombosis during pregnancy and the puerperium

BACKGROUND: Venous thromboembolism is a leading cause of morbidity and mortality during pregnancy and the puerperium. However, the role of mutations in the prothrombin and factor V genes and other thrombophilic abnormalities as risk factors for thromboembolism in women during pregnancy and the pueperium is not known. METHODS: In a study of 119 women with a history of venous thromboembolism during pregnancy and the puerperium and 233 age-matched normal women, we measured the activity of antithrombin, protein C, protein S, and lupus anticoagulant. We also performed genetic analyses to detect the G1691A mutation in the factor V gene (factor V Leiden), the G20210A mutation in the prothrombin gene, and the C677T mutation in the methylenetetrahydrofolate reductase gene. Blood samples were obtained at least three months post partum or after the cessation of lactation. RESULTS: Among the women with a history of venous thromboembolism, the prevalence of factor V Leiden was 43.7 percent, as compared with 7.7 percent among the normal women (relative risk of venous thromboembolism, 9.3; 95 percent confidence interval, 5.1 to 16.9); that of the G20210A prothrombin-gene mutation, 16.9 percent as compared with 1.3 percent (relative risk, 15.2; 95 percent confidence interval, 4.2 to 52.6); and that of both factor V Leiden and the G20210A prothrombin-gene mutation 9.3 percent as compared with 0 (estimated odds ratio, 107). Assuming an overall risk of 1 in 1500 pregnancies, the risk of thrombosis among carriers of factor V Leiden was 0.2 percent, among carriers of the G20210A prothrombin-gene mutation, 0.5 percent, and among carriers of both defects, 4.6 percent, as calculated in a multivariate analysis. CONCLUSIONS: The G20210A prothrombin-gene mutation and factor V Leiden individually are associated with an increased risk of venous thromboembolism during pregnancy and the puerperium, and the risk among women with both mutations is disproportionately higher than that among women with only one mutation.