Diet quality, common genetic polymorphisms, and bladder cancer risk in a New England population-based study.

PURPOSE: We examined the interaction between common genetic bladder cancer variants, diet quality, and bladder cancer risk in a population-based case-control study conducted in New England. METHODS: At the time of enrollment, 806 bladder cancer cases and 974 controls provided a DNA sample and completed a diet history questionnaire. Diet quality was assessed using the 2010 Alternate Healthy Eating Index (AHEI-2010) score. Single nucleotide polymorphisms (SNPs) reported in genome-wide association studies to be associated with bladder cancer risk were combined into a polygenic risk score and also examined individually for interaction with the AHEI-2010. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. RESULTS: A 1-standard deviation increase in polygenic risk score was associated with higher bladder cancer risk (OR, 1.34; 95% CI 1.21-1.49). Adherence to the AHEI-2010 was not associated with bladder cancer risk (OR, 0.99; 95% CI 0.98-1.00) and the polygenic risk score did not appear to modify the association between the AHEI-2010 and bladder cancer risk. In single-SNP analyses, rs8102137 (bladder cancer risk allele, C) modified the association between the AHEI-2010 total score and bladder cancer risk, with the strongest evidence for the AHEI-2010 long chain fat guideline (OR for TT, 0.92; 95% CI 0.87-0.98; OR for CT, 1.02; 95% CI 0.96-1.08; OR for CC, 1.03; 95% CI 0.93-1.14; p for interaction, 0.02). CONCLUSIONS: In conclusion, rs8102137 near the cyclin E1 gene ( CCNE1 ) may be involved in gene-diet interactions for bladder cancer risk.

Diet Quality and Survival in a Population-Based Bladder Cancer Study.

Nutrition may impact bladder cancer survival. We examined the association between diet quality and overall and bladder cancer-specific survival. Bladder cancer cases from a population-based study reported pre-diagnosis diet. Diet quality was assessed using the 2010 Alternate Healthy Eating Index (AHEI-2010). Vital status was ascertained from the National Death Index. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated using proportional hazards and competing risks regression models. Overall AHEI-2010 adherence was not associated with overall or bladder cancer-specific survival among non-muscle invasive bladder cancer (NMIBC) cases (HR, 1.00; 95% CI, 0.98-1.01; HR, 1.00; 95% CI, 0.97-1.02) or muscle invasive bladder cancer (MIBC) cases (HR, 0.99; 95% CI, 0.96-1.03; HR, 1.01, 95% CI 0.97-1.06). AHEI-2010 sugar-sweetened beverages adherence was associated with poorer overall survival (HR, 1.04; 95% CI, 1.01-1.08) and AHEI-2010 sodium adherence was associated with better overall and bladder cancer-specific survival after NMIBC diagnosis (HR, 0.92, 95% CI, 0.85-1.00; HR, 0.82; 95% CI, 0.68-0.98). AHEI-2010 fruit adherence was associated with poorer overall and bladder cancer-specific survival after MIBC diagnosis (HR, 1.17; 95% CI, 1.02-1.33; HR, 1.26; 95% CI, 1.03-1.55). Consumption of sugar-sweetened beverages, sodium, and fruit, not overall AHEI-2010 adherence, may be associated with bladder cancer survival.

Neuronal Glutathione Content and Antioxidant Capacity can be Normalized In Situ by N-acetyl Cysteine Concentrations Attained in Human Cerebrospinal Fluid.

N-acetyl cysteine (NAC) supports the synthesis of glutathione (GSH), an essential substrate for fast, enzymatically catalyzed oxidant scavenging and protein repair processes. NAC is entering clinical trials for adrenoleukodystrophy, Parkinson's disease, schizophrenia, and other disorders in which oxidative stress may contribute to disease progression. However, these trials are hampered by uncertainty about the dose of NAC required to achieve biological effects in human brain. Here we describe an approach to this issue in which mice are used to establish the levels of NAC in cerebrospinal fluid (CSF) required to affect brain neurons. NAC dosing in humans can then be calibrated to achieve these NAC levels in human CSF. The mice were treated with NAC over a range of doses, followed by assessments of neuronal GSH levels and neuronal antioxidant capacity in ex vivo brain slices. Neuronal GSH levels and antioxidant capacity were augmented at NAC doses that produced peak CSF NAC concentrations of ≥50 nM. Oral NAC administration to humans produced CSF concentrations of up to 10 µM, thus demonstrating that oral NAC administration can surpass the levels required for biological activity in brain. Variations of this approach may similarly facilitate and rationalize drug dosing for other agents targeting central nervous system disorders.

Surgical menopause initiates molecular changes that do not result in mechanical changes in normal and healing ligaments.

OBJECTIVES: Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model. METHODS: Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress. RESULTS: Surgical menopause in normal medial collateral ligaments initiated molecular changes in all the categories evaluated. In early healing medial collateral ligaments, surgical menopause resulted in downregulation of specific collagens, proteinases and inflammatory mediators at 6 weeks of healing, and proteoglycans, growth factors and hormone receptors at 14 weeks of healing. Surgical menopause did not produce mechanical changes in normal or early healing medial collateral ligaments. With or without surgical menopause, healing ligaments exhibited increased total creep strain and decreased failure stress compared with uninjured ligaments. CONCLUSIONS: Surgical menopause did not affect the mechanical properties of normal or early healing medial collateral ligaments in a rabbit model. The results in this preclinical model suggest that menopause may result in no further impairment to the ligament healing process. Cite this article: Bone Joint Res 2015;4:38-44.

Activation of neuronal NMDA receptors induces superoxide-mediated oxidative stress in neighboring neurons and astrocytes.

Excitotoxic neuronal death is mediated in part by NMDA receptor-induced activation of NOX2, an enzyme that produces superoxide and resultant oxidative stress. It is not known, however, whether the superoxide is generated in the intracellular space, producing oxidative stress in the neurons responding to NMDA receptor activation, or in the extracellular space, producing oxidative stress in neighboring cells. We evaluated these alternatives by preparing cortical neuron cultures from p47(phox-/-) mice, which are unable to form a functional NOX2 complex, and transfecting the cultures at low density with GFP-tagged p47(phox) to reconstitute NOX2 activity in widely scattered neurons. NMDA exposure did not induce oxidative stress or cell death in the nontransfected, p47-phox(-/-) cultures, but did produce oxidative stress and neuronal death in neurons surrounding the transfected, NOX2-competent neurons. This cell-to-cell spread of NMDA-induced oxidative injury was blocked by coincubation with either superoxide dismutase or the anion channel blocker 4'-diisothiocyanostilbene-2,2'-disulphonate, confirming superoxide anion as the mediating oxidant. In neurons plated on a preexisting astrocyte layer, NMDA induced oxidative stress in both the neurons and the astrocytes, and this was also prevented by superoxide dismutase. These findings show that activation of NMDA receptors on one neuron can lead to oxidative stress and cell death in neighboring neurons and astrocytes by a process involving the extracellular release of superoxide by NOX2.

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes.

Astroglial excitability operates through increases in Ca2+cyt (cytosolic Ca2+), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na+cyt (cytosolic Na+) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca2+-ATPase), NCX (plasma membrane Na+/Ca2+ exchanger) and NKA (Na+/K+-ATPase) in complex and coordinated regulation of Ca2+cyt and Na+cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na+ and Ca2+ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca2+ and Na+ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca2+ and Na+ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca2+-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca2+-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca2+ release from the ER (endoplasmic reticulum).

N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse.

OBJECTIVE: Dopaminergic neuronal death in Parkinson's disease (PD) is accompanied by oxidative stress and preceded by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of animal models that mimic these features and demonstrate an age-related progression. The EAAC1(-/-) mouse may be useful in this regard, because EAAC1(-/-) mouse neurons have impaired neuronal cysteine uptake, resulting in reduced neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes in nigral dopaminergic neurons in the EAAC1(-/-) mouse, and (2) use the EAAC1(-/-) mouse to evaluate N-acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD. METHODS: Wild-type mice, EAAC1(-/-) mice, and EAAC1(-/-) mice chronically treated with N-acetylcysteine were evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities. RESULTS: EAAC1(-/-) mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by increased nitrotyrosine formation, nitrosylated α-synuclein, and microglial activation. These changes were substantially reduced in mice that received N-acetylcysteine. INTERPRETATION: These findings suggest that the EAAC1(-/-) mouse may be a useful model of the chronic neuronal oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus for clinical evaluation of glutathione repletion in PD.

Habitat diversity and adaptation to environmental stress in encysted embryos of the crustacean Artemia.

Encysted embryos (cysts) of the brine shrimp, Artemia, provide excellent opportunities for the study of biochemical and biophysical adaptation to extremes of environmental stress in animals. Among other virtues, this organism is found in a wide variety of hypersaline habitats, ranging from deserts, to tropics, to mountains. One adaptation implicated in the ecological success of Artemia is p26, a small heat shock protein that previous evidence indicates plays the role of a molecular chaperone in these embryos. We add to that evidence here. We summarize recently published work on thermal tolerance and stress protein levels in embryos from the San Francisco Bay (SFB) of California inoculated into experimental ponds in southern Vietnam where water temperatures are much higher. New results on the relative contents of three stress proteins (hsp70, artemin and p26) will be presented along with data on cysts of A. tibetiana collected from the high plateau of Tibet about 4.5 km above sea level. Unpublished results on the stress protein artemin are discussed briefly in the context of this paper, and its potential role as an RNA chaperone. Interestingly, we show that the substantial tolerance of A. franciscana embryos to ultraviolet (UV) light does not seem to result from intracellular biochemistry but, rather, from their surrounding thick shell, a biophysical adaptation of considerable importance since these embryos receive heavy doses of UV in nature.

Assessment of mRNA levels for matrix molecules and TGF- beta1 in rabbit flexor and peroneus tendons reveals regional differences in steady-state expression.

This study analysed the differences on a molecular level between two segments of the deep flexor tendon, and compared the intrasynovial flexor tendon with the tendon sheath and the extrasynovial peroneus tendon in a rabbit model. The TRIspin method of RNA extraction was combined with the reverse transcription polymerase chain reaction to assess mRNA levels in the tissue segments. Significant differences were detected for all genes studied. mRNA levels for aggrecan, biglycan and collagen III were significantly higher in the fibrocartilaginous proximal segment of the flexor tendon. Collagen I was higher in the flexor tendon than the sheath and the peroneus tendon, and TGF-beta1 was significantly lower in the peroneus tendon. This study demonstrates differences at the mRNA level between different segments of tendon, indicating that the tendon tissue may be adapted to its environment.

The pig as a model for excisional skin wound healing: characterization of the molecular and cellular biology, and bacteriology of the healing process.

A pig model of wound healing was developed by excision of 2-cm-diameter full thickness skin in young Yorkshire pigs. The results indicated that wound re-epithelialization in this animal model took an average of 20 days. Analysis of cellular change was assessed by use of DNA quantification and determination of apoptotic cells in tissue sections. The results indicate that RNA and DNA contents paralleled each other throughout the healing process, and observed changes in the pattern of RNA and DNA content of the scar tissues were consistent with cell loss due to apoptosis in this model. Expression of mRNA for relevant genes was assessed by use of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, using porcine specific primer sets and RNA isolated from normal skin and specimens obtained at various times after wounding. The mRNA values for tumor necrosis factor-alpha (TNF-alpha), connective tissue growth factor (CTGF), insulin-like growth factor II (IGF-II), and decorin were significantly high at specific times after wounding, but mRNA values for the transcription factors (c-fos and c-jun) were significantly decreased. Quantitative bacteriologic results indicated that the total bacterial count in this animal model reached 10(9) colony-forming units (CFU)/g, with the highest value at post-wounding day 7, and Pseudomonas aeruginosa and Staphylocococci aureus were the most common bacteria detected in this model. Further definition of this model should identify unique points in the healing process, and such information could lead to development of therapeutic interventions to improve skin wound healing.

Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy.

Hsp47 (also termed "colligin") is a 47 kDa protein that is localized in the ER and cis-Golgi vesicles of fibrocytes, chondrocytes, and other collagen-secreting cells. Under stress conditions, Hsp47 expression is upregulated as part of the heat shock/stress response that mitigates cell damage from noxious stimuli such as elevated temperature, heavy metals, and oxidative stress. Under non-stress conditions, Hsp47 functions as a collagen-specific molecular chaperone that facilitates intracellular procollagen polypeptide synthesis, and triple helix assembly in connective tissues. Previously it has been shown that levels of collagen-specific gene expression are significantly altered in ligaments, menisci, and other connective tissues of the rabbit following surgically induced injuries (increased), and during pregnancy (decreased). The present study was undertaken to determine whether expression of mRNA for the Hsp47 collagen-binding stress protein was also influenced in these experimental models. Since no sequence information was available on the rabbit Hsp47 gene, a partial cDNA for rabbit Hsp47 was first isolated and cloned using reverse transcriptase PCR (RT-PCR) with degenerate oligonucleotide primers. Rabbit Hsp47 sequence-specific primers then designed enabled analysis of Hsp47 mRNA expression in rabbit connective tissues using semiquantitative RT-PCR. It was found that Hsp47 expression is affected in a complex, tissue-specific manner by injury and pregnancy. Hsp47 transcript levels were elevated in the medial collateral ligament (MCL) of the rabbit knee following surgical induction of a gap injury. Transection of the anterior cruciate ligament (ACL), which leads to chronic progressive damage to menisci of the rabbit knee joint, was accompanied by an upregulation of Hsp47 expression in the medial and lateral menisci. Hsp47 mRNA levels were depressed during pregnancy in the kidney and ACL of primigravid adolescent rabbits, but were not altered in corneal tissue during pregnancy or in the ACL of skeletally mature multiparous females. The changes in Hsp47 transcript levels within these connective tissues following injury/pregnancy often, but not always, paralleled changes in collagen-specific gene expression.

Heterogenous response of knee cartilage to pregnancy in the rabbit: assessment of specific mRNA levels.

OBJECTIVE: The purpose of this study was to evaluate the effect of pregnancy on mRNA levels for several relevant molecules between five articular cartilage surfaces of the rabbit knee joint. DESIGN: Total RNA was extracted from the following five knee joint articular surfaces: the lateral and medial femoral condyles (LFC and MFC); the lateral and medial tibial plateau (LTP and MTP); and the femoral groove (G) from pregnant and age-matched non-pregnant skeletally immature New Zealand White rabbits. The RNA was analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets. Two types of comparisons were performed: (i) comparison of mRNA levels between cartilage surfaces and (ii) comparison of mRNA levels between pregnant and non-pregnant rabbits within the same articular surfaces. RESULTS: (i) Total RNA yield from the MFC and G represented 53 and 58% of the total RNA amount from the five cartilage surfaces in both non-pregnant and pregnant rabbits, respectively. Transcript levels for progesterone receptor (PR), aggrecan and biglycan were similar in all of the cartilage surfaces. In contrast, the cartilage surfaces exhibited significantly different transcript levels with a similar pattern for the estrogen receptor (ER), collagenase and urokinase (i.e., MTP

Gene expression in rabbit menisci during pregnancy.

Recent studies have indicated that pregnancy can affect cellular activity in connective tissues such as cartilage, ligaments, and tendons. However, the impact of pregnancy on cellular activity in the menisci, a critical component in joint function, has not been reported. Therefore,the purpose of this study was to evaluate mRNA levels for several relevant molecules in both medial and lateral menisci from the knees of first-time pregnant immature rabbits (primigravida), third-time pregnant mature rabbits (multiparous), and nonpregnant rabbits (age-matched immature and mature controls) by the sensitive molecular technique of semiquantitative reverse transcription--polymerase chain reaction. Total RNA yields from the medial meniscus of multiparous rabbits were reduced to 66% of age-matched control values; however, yields from medial and lateral menisci from primigravida animals or the lateral meniscus of multiparous animals were not significantly depressed. DNA yields were not affected by pregnancy. Type I collagen mRNA levels were significantly depressed in both menisci only in primigravida rabbits. Versican mRNA levels were significantly elevated in both menisci only in multiparous rabbits. None of the transcripts for the other matrix molecules assessed were influenced by pregnancy. Collagenase mRNA levels were unaffected by pregnancy, but TIMP-1 mRNA levels were significantly decreased in the medial meniscus of primigravida rabbits and in the lateral meniscus of multiparous rabbits. Complex changes in the pattern of mRNA expression were observed for growth factors (TGF-beta bFGF, and IGF2). Inducible nitric oxide synthase mRNA expression was not altered during pregnancy. Transcripts for cyclooxygenase-2 were 61 and 50% of control values in the medial meniscus of primigravida and multiparous animals, respectively, while cyclooxygenase-2 mRNA levels were 150 and 188% of control values in the lateral meniscus from primigravida and multiparous animals, respectively. Such results indicate that pregnancy-induced changes in the pattern of mRNA expression are meniscus specific. Furthermore, the results support the hypothesis that different connective tissues of the knee respond to pregnancy in a unique manner.

Influence of pregnancy on gene expression in rabbit articular cartilage.

OBJECTIVE: Articular cartilage is known to be influenced by estrogen and the pregnancy-associated hormone, relaxin, in vitro. Such observations have raised the possibility that articular cartilage in females may be subjected to unique regulatory influences by such hormones in vivo. The purpose of this study was to evaluate mRNA levels for several relevant molecules in the articular cartilage of pregnant and non-pregnant rabbits. DESIGN: Total RNA was extracted from New Zealand White rabbit knee articular cartilage using the TRIspin method. The total RNA was reverse transcribed and analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets. RESULTS: Total RNA yield from articular cartilage from primigravida rabbits was reduced to 65% of age-matched control values (P = 0.0003); however the yield from multiparous animals was not significantly depressed. In both cases, DNA yields were not affected by pregnancy. There was a general tendency for depressed mRNA levels for most genes investigated in cartilage from pregnant animals. Articular cartilage from multiparous rabbits showed a significant decrease in mRNA levels for relevant molecules such as type II collagen, biglycan, collagenase and tissue inhibitors of metalloproteinases (TIMP)-1, as well as necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2 (COX-2). Transcripts for collagenase and lumican were significantly lower in cartilage from primigravida rabbits. Transforming growth factor beta 1 (TGF-beta 1) transcript levels were significantly decreased in both pregnant groups. In contrast, basic fibroblast growth factor (bFGF) and insulin-like growth factor-2 (IGF-2) mRNA levels were significantly decreased in cartilage from primigravida rabbits, whereas transcripts for these molecules were upregulated in the cartilage of multiparous rabbits. CONCLUSIONS: The present study demonstrates that regulation of RNA levels in articular cartilage during pregnancy is complex and is influenced by the parity and/or the skeletal maturity of the animals.

Transcatheter laser dissolution of human atherosclerotic plaques: a model for testing catheters and techniques.

We constructed a model of the human arterial circulation that can be used to test laser angioplasty catheters and techniques on obstructed human coronary arteries under simulated physiologic conditions of blood pressure and flow. In this model system, a balloon-tipped catheter with a central, 0.02-cm light fiber was used to deliver 4 W of laser energy to two obstructed human cadaver coronary artery segments and four normal dog femoral artery segments. Flushing the catheter with saline during the lases minimized lateral thermal tissue damage. Channels were created in the obstructed arteries that were twice the diameter of the light fiber used to lase. One of four dog arteries perforated, underscoring the potential hazards of the procedure and the need for laser catheters that are flexible and capable of precise alignment.