Combined effect of low-penetrant SNPs on breast cancer risk.

BACKGROUND: Although many low-penetrant genetic risk factors for breast cancer have been discovered, knowledge about the effect of multiple risk alleles is limited, especially in women <50 years. We therefore investigated the association between multiple risk alleles and breast cancer risk as well as individual effects according to age-approximated pre- and post-menopausal status. METHODS: Ten previously described breast cancer-associated single-nucleotide polymorphisms (SNPs) were analysed in a joint European biobank-based study comprising 3584 breast cancer cases and 5063 cancer-free controls. Genotyping was performed using MALDI-TOF mass spectrometry, and odds ratios were estimated using logistic regression. RESULTS: Significant associations with breast cancer were confirmed for 7 of the 10 SNPs. Analysis of the joint effect of the original 10 as well as the statistically significant 7 SNPs (rs2981582, rs3803662, rs889312, rs13387042, rs13281615, rs3817198 and rs981782) found a highly significant trend for increasing breast cancer risk with increasing number of risk alleles (P-trend 5.6 × 10(-20) and 1.5 × 10(-25), respectively). Odds ratio for breast cancer of 1.84 (95% confidence interval (CI): 1.59-2.14; 10 SNPs) and 2.12 (95% CI: 1.80-2.50; 7 SNPs) was seen for the maximum vs the minimum number of risk alleles. Additionally, one of the examined SNPs (rs981782 in HCN1) had a protective effect that was significantly stronger in premenopausal women (P-value: 7.9 × 10(-4)). CONCLUSION: The strongly increasing risk seen when combining many low-penetrant risk alleles supports the polygenic inheritance model of breast cancer.

Local and systemic expressions of MMP-9, TIMP-1 and PAI-1 in patients undergoing surgery for clinically suspected appendicitis.

BACKGROUND/AIMS: To examine, compare and correlate the expressions of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) in appendiceal tissue and pre- and postoperative blood samples in patients undergoing surgery for clinically suspected appendicitis. METHODS: Fifty-seven patients with complete tissue and blood samples were included and divided into groups of noninflamed appendix/lymphadenitis (n = 7), phlegmonous appendicitis (n = 30), gangrenous appendicitis (n = 11) and perforated appendicitis (n = 9). The protein expressions were assessed with ELISAs. The local expressions of MMP-9, TIMP-1 and PAI-1 were correlated with the systemic expressions at the time of surgery while the systemic individual differences between surgery and recovery were compared. RESULTS: There was a positive correlation between tissue and plasma PAI-1 (p < 0.05). The individual differences for plasma MMP-9 and PAI-1 were statistically nonsignificant, while they were higher for TIMP-1 in patients with perforated appendicitis compared with phlegmonous (p < 0.0001) and gangrenous appendicitis (p < 0.01). CONCLUSIONS: Plasma PAI-1 reflected the levels in appendiceal tissue at the time of surgery. Systemic TIMP-1 could have the potential of distinguishing perforated from nonperforated appendicitis.

Effect of a DACC dressing on the growth properties and proliferation rate of cultured fibroblasts.

OBJECTIVE: To study the morphology and proliferation of cultured fibroblasts in combination with an experimental wound-healing model using cultured fibroblasts, with and without the presence of a hydrophobic, dialkyl carbamoyl chloride (DACC) dressing (Sorbact; Abigo Medical AB). METHOD: Human dermal fibroblasts were cultured and cell morphology and viability were investigated. Proliferation was investigated using an XTT assay. An experimental wound-healing model was employed, whereby mechanical damage was inflicted to the surface of cultured fibroblasts. The healing and closure of the wound was then monitored with and without the presence of the DACC dressing. RESULTS: Fibroblasts did not adhere easily to the dressing material. The presence of the DACC dressing increased the average proliferation rate of cultured fibroblasts by 50% compared with the untreated control medium (p<0.05). The DACC dressing significantly increased the healing rate by more than 100% after 72 hours (p<0.05) in the experimental model of wound healing, compared with the medium only. CONCLUSION: The enhanced wound healing observed in different types of wounds using the DACC dressing might be explained by an increase in cell growth and proliferation rate of cells in the wound area.

Similar collagen distribution in full-thickness skin grafts in intraperitoneal and onlay positions, an experimental mice-study.

PURPOSE: Autologous full-thickness skin grafting (FTSG) has the potential to become an option in abdominal wall repair. An understanding of tissue remodelling in the extracellular matrix (ECM) is crucial as this interplay determines such parameters as tissue strength and flexibility. This cross-sectional preclinical laboratory study in mice provides information on the distribution of collagen types and matrix metalloproteinases (MMPs) in the ECM of FTSGs in the intraperitoneal and onlay positions compared with internal controls. The aim was to evaluate morphologic changes after tissue remodelling and repair in FTSGs applied in the two positions and to detect any adverse host response. METHODS: ECM components were evaluated as follows: qualitative examination of collagen bundle thickness using Picrosirius Red staining (collagen types I, III and IV); and evaluation of collagen types IV and V, as well as MMPs 1, 8 and 9 using immunohistochemical staining. Full-thickness grafts transplanted between female twin mice were examined as this best mimics autologous transplantation. RESULTS: At 8 weeks, FTSGs in the intraperitoneal position did not show any noticeable differences in morphologic appearance to those in the onlay position. Both intraperitoneal and onlay FTSGs showed increases in the amount of thick collagen bundles compared to internal controls. No correlation was seen between distribution of MMPs 1, 8 or 9 and distribution of collagen types I, III, IV or V. CONCLUSION: This preclinical study shows that FTSGs in both intraperitoneal and onlay positions are possible application site options and, by extension, promising application site options for abdominal wall reinforcement in hernia surgery. Clinical studies in humans are required to confirm these findings.

A hidden Markov model to assess drug-induced sleep fragmentation in the telemetered rat.

Drug-induced sleep fragmentation can cause sleep disturbances either via their intended pharmacological action or as a side effect. Examples of disturbances include excessive daytime sleepiness, insomnia and nightmares. Developing drugs without these side effects requires insight into the mechanisms leading to sleep disturbance. The characterization of the circadian sleep pattern by EEG following drug exposure has improved our understanding of these mechanisms and their translatability across species. The EEG shows frequent transitions between specific sleep states leading to multiple correlated sojourns in these states. We have developed a Markov model to consider the high correlation in the data and quantitatively compared sleep disturbance in telemetered rats induced by methylphenidate, which is known to disturb sleep, and of a new chemical entity (NCE). It was assumed that these drugs could either accelerate or decelerate the transitions between the sleep states. The difference in sleep disturbance of methylphenidate and the NCE were quantitated and different mechanisms of action on rebound sleep were identified. The estimated effect showed that both compounds induce sleep fragmentation with methylphenidate being fivefold more potent compared to the NCE.

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1alpha and beta.

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1alpha and beta. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1alpha or beta in presence or absence of TGF-beta1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin.

Results at 3-year follow-up of totally extraperitoneal (TEP) hernia surgery with long-term resorbable mesh.

INTRODUCTION: Synthetic non-resorbable mesh is almost standard in hernia surgery. However, several studies have showed negative effects of permanent implants such as chronic inflammation and complications involving different organs bordering the mesh. Such complications can raise the risk of chronic post-operative pain (CPP). Recently promising results regarding CPP have been published in patients with Lateral Inguinal Hernia (LIH) using a slowly resorbable mesh in Lichtenstein technique. For this reason the aim of the present study was to find the effect of a slowly resorbable implant on the long-term rate of hernia recurrence and chronic post-operative pain in patients with LIH repaired with TEP procedure. METHODS: Prospective pilot study of TEP repair using TIGR® Matrix Surgical Mesh in 35 primary LIH. At 3-year follow-up the Visual Analogue Scale (VAS) and the Inguinal Pain Questionnaire were employed to assess pain. Recurrence was determined by ultrasound and clinical examination. RESULTS: All patients completed the pain questionnaires but one patient did not attend the planned clinical examination for the 3-year follow-up. No patients had CPP, as defined in the World Guidelines for Groin Hernia Management. Almost all patients had lower VAS score in any activity 3 years following surgery in comparison to the preoperative period. Three patients (8.8%) suffered symptomatic recurrence during the 3-year follow-up. CONCLUSION: TEP repair in patients with LIH using a synthetic long-term resorbable mesh was found to be encouraging respecting chronic post-operative pain at 3-year follow-up but at the cost of an increased risk of recurrence.

Characterisation of the effects of caffeine on sleep in the rat: a potential model of sleep disruption.

Caffeine is known to disrupt sleep and its administration to human subjects has been used to model sleep disruption. We previously showed that its effects on sleep onset latency are comparable between rats and humans. This study evaluated the potential use of caffeine as a model of sleep disruption in the rat, by assessing its effects on sleep architecture and electroencephalogram (EEG) frequency spectrum, and using sleep-promoting drugs to reverse these effects. Rats were implanted with radiotelemetry devices for body temperature, EEG, electromyogram and locomotor activity. Following recovery, animals were dosed with caffeine (10 mg/kg) alone or in combination with zolpidem (10 mg/kg) or trazodone (20 mg/kg). Sleep was scored for the subsequent 12 h using automated analysis software. Caffeine dose-dependently disrupted sleep: it increased WAKE time, decreased NREM (non-REM) sleep time and NREM bout duration (but not bout number), and decreased delta activity in NREM sleep. It also dose-dependently increased locomotor activity and body temperature. When given alone, zolpidem suppressed REM whilst trazodone increased NREM sleep time at the expense of WAKE, increased NREM bout duration, increased delta activity in NREM sleep and reduced body temperature. In combination, zolpidem attenuated caffeine's effects on WAKE, whilst trazodone attenuated its effects on NREM sleep, NREM bout duration, delta activity, body temperature and locomotor activity. Caffeine administration produced many of the signs of insomnia that were improved by two of its most successful current treatments. This model may therefore be useful in the study of new drugs for the treatment of sleep disturbance.

Effects on sleep stages and microarchitecture of caffeine and its combination with zolpidem or trazodone in healthy volunteers.

Caffeine is the world's most popular stimulant and is known to disrupt sleep. Administration of caffeine can therefore be used in healthy volunteers to mimic the effects of insomnia and thus to test the hypnotic effects of medication. This study assessed the effects of caffeine on sleep architecture and electroencephalography (EEG) spectrum alone and in combination with two different sleep-promoting medications. Home polysomnography was performed in 12 healthy male volunteers in a double-blind study whereby subjects received placebo, caffeine (150 mg), caffeine plus zolpidem (10 mg) and caffeine plus trazodone (100 mg) at bedtime in a randomised crossover design. In addition to delaying sleep onset, caffeine decreased total sleep time (TST), sleep efficiency (SE) and stage 2 sleep without significantly altering wake after sleep onset or the number of awakenings. Zolpidem attenuated the caffeine-induced decrease in SE and increased spindle density in the caffeine plus zolpidem combination compared with placebo. Trazodone attenuated the decrease in SE and TST, and it also increased stage 3 sleep, decreased the number of awakenings and decreased the spindle density. No significant changes in rapid eye movement (REM) sleep were observed, neither was any significant alteration in slow wave activity nor other EEG spectral measures, although the direction of change was similar to that previously reported for caffeine and appeared to 'normalise' after trazodone. These data suggest that caffeine mimics some, but not all of the sleep disruption seen in insomnia and that its disruptive effects are differentially attenuated by the actions of sleep-promoting compounds with distinct mechanisms of action.

Paleo-Rock-Hosted Life on Earth and the Search on Mars: A Review and Strategy for Exploration.

Here we review published studies on the abundance and diversity of terrestrial rock-hosted life, the environments it inhabits, the evolution of its metabolisms, and its fossil biomarkers to provide guidance in the search for life on Mars. Key findings are (1) much terrestrial deep subsurface metabolic activity relies on abiotic energy-yielding fluxes and in situ abiotic and biotic recycling of metabolic waste products rather than on buried organic products of photosynthesis; (2) subsurface microbial cell concentrations are highest at interfaces with pronounced chemical redox gradients or permeability variations and do not correlate with bulk host rock organic carbon; (3) metabolic pathways for chemolithoautotrophic microorganisms evolved earlier in Earth's history than those of surface-dwelling phototrophic microorganisms; (4) the emergence of the former occurred at a time when Mars was habitable, whereas the emergence of the latter occurred at a time when the martian surface was not continually habitable; (5) the terrestrial rock record has biomarkers of subsurface life at least back hundreds of millions of years and likely to 3.45 Ga with several examples of excellent preservation in rock types that are quite different from those preserving the photosphere-supported biosphere. These findings suggest that rock-hosted life would have been more likely to emerge and be preserved in a martian context. Consequently, we outline a Mars exploration strategy that targets subsurface life and scales spatially, focusing initially on identifying rocks with evidence for groundwater flow and low-temperature mineralization, then identifying redox and permeability interfaces preserved within rock outcrops, and finally focusing on finding minerals associated with redox reactions and associated traces of carbon and diagnostic chemical and isotopic biosignatures. Using this strategy on Earth yields ancient rock-hosted life, preserved in the fossil record and confirmable via a suite of morphologic, organic, mineralogical, and isotopic fingerprints at micrometer scale. We expect an emphasis on rock-hosted life and this scale-dependent strategy to be crucial in the search for life on Mars.

Learned movements elicited by direct stimulation of cerebellar mossy fiber afferents.

Definitive evidence is presented that the conditioned stimulus (CS) in classical conditioning reaches the cerebellum via the mossy fiber system. Decerebrate ferrets received paired forelimb and periocular stimulation until they responded with blinks to the forelimb stimulus. When direct mossy fiber stimulation was then given, the animals responded with conditioned blinks immediately, that is, without ever having been trained to the mossy fiber stimulation. Antidromic activation was prevented by blocking mossy fibers with lignocaine ventral to the stimulation site. It could be excluded that cerebellar output functioned as the CS. Analysis of latencies suggests that conditioned responses (CRs) are not generated by mossy fiber collaterals to the deep nuclei. Hence, the memory trace is probably located in the cerebellar cortex.

Effect of the beta3 adrenoceptor agonist CL 316243 on hypothalamic 5-HT synthesis and suppression of REM sleep in the rat.

Beta3 adrenoceptor agonists show an antidepressant-like profile in preclinical rodent assays and improve mood in clinically-obese patients. These observations suggest a possible antidepressant utility for beta3 adrenoceptor agonists. The present study examined the effects of acute and chronic administration of the beta3 adrenoceptor agonist CL 316243 on two physiological indicators of antidepressant activity in the rat: hypothalamic 5-HT synthesis and suppression of REM sleep. 5-HT synthesis was estimated by the accumulation of 5-hydroxytryptophan (5-HTP) after treatment with the L-aromatic acid decarboxylase inhibitor NSD 1015. Sleep-wake patterns were monitored using electroencephalogram and electromyogram signals collected by radiotelemetry. Rats were administered CL 316243 acutely or once daily for 11 days. Acute administration of CL 316243 significantly increased hypothalamic 5-HT synthesis, as indicated by increased levels of 5-HTP, and reduced the amount of REM sleep. However, chronic administration produced no changes in 5-HTP or REM compared with vehicle treatment. The present observations suggest that acute administration of CL 316243 causes antidepressant-like effects on REM sleep, possibly mediated by increased central 5-HT synthesis. However, these effects are not maintained with repeated dosing.

Composition and dynamics of the uterine NK cell KIR repertoire in menstrual blood.

Uterine natural killer (NK) cells are abundantly present in endometrium and decidua. Their function is governed by interactions between killer cell immunoglobulin-like receptors (KIRs) and cognate human leukocyte antigen (HLA) class I ligands. These interactions have implications for reproductive success. Whereas most uterine NK cells are known to express KIRs, little information is available about KIR repertoire formation and stability over time. This is primarily due to inherent difficulties in gaining access to human uterine tissue. As endometrial immune cells are shed during menstruation, menstrual blood may serve as a source for studies of KIRs on uterine NK cells. Here, we performed a combined assessment of six inhibitory and activating KIRs on uterine NK cells from paired menstrual and peripheral blood. Menstrual blood contained a high frequency of uterine NK cells expressing KIRs. The uterine NK cell KIR repertoires were markedly different from those in peripheral blood NK cells, biased toward KIR2D-receptor expression, and formed independently of selection conferred by cognate HLA class I molecules. Moreover, uterine NKG2C+self-KIR+ NK cell expansions were detected. Finally, the distinct KIR repertoires of uterine NK cells were stable over multiple menstrual cycles. Our results provide novel insight into KIR repertoire formation on human uterine NK cells.

Advantages of doubly polished thin sections for the study of microfossils in volcanic rock.

Doubly polished thin sections, originally prepared for fluid inclusion studies, present great advantages in the study of microfossils in volcanic rocks. Better visibility and light conditions, variation in thickness of the thin sections and the possibility to combine fluid inclusion studies with microfossil studies lead to a wide range of advantages over ordinary thin sections. This includes the study of morphology, internal microstructures, colonies, association with the substrate that microfossils are attached to and geological and environmental context in which the microfossil once lived. When meeting the criteria of microfossil recognition the advantages of doubly polished thin sections are substantial and can be crucial in distinguishing between biogenic microfossils and abiotically formed abiomorphs.

Cytokines produced by T cells in adenoid surface secretion are mainly downregulatory or of Th1 type.

CONCLUSIONS: T cells in adenoid surface secretion (AdSS) possess the property of cytokine production and are mainly downregulatory or of Th1 type. These results strengthen the hypothesis of an active immunological defense in AdSS. OBJECTIVES: Recently, we demonstrated the presence of activated T cells in AdSS. The aims of this study were to elucidate the ability of these T cells to produce cytokines and to reveal their cytokine profile. MATERIAL AND METHODS: Eight children subjected to adenoidectomy were enrolled. Samples of AdSS, adenoid tissue and peripheral blood were obtained, as well as a nasopharyngeal culture. T cells obtained from AdSS, adenoid tissue and peripheral blood were then cultured and stimulated with anti-CD3 and -CD28 for 5 days. The production of interferon (IFN)-gamma, tumor necrosis factor (TNF)-a and IL-2, -4, -5 and -10 was then analyzed using flow cytometry. RESULTS: All children had T cells in AdSS capable of cytokine production. T cells in AdSS, adenoid tissue and peripheral blood samples from all children produced IFN-gamma. Of the cytokines analyzed, IFN-gamma was produced in the highest concentrations. In 6/8 children, T cells in AdSS also produced TNF-a and IL-10.

Cerebellar cortical AMPA-kainate receptor blockade prevents performance of classically conditioned nictitating membrane responses.

Classical conditioning of the nictitating membrane-eye blink response of rabbits is a simple form of associative motor learning. Lesion studies have shown that performance of learned responses is dependent on the cerebellum, but they have not shown whether there is storage of memories within the cerebellum or distinguished the roles of the cerebellar cortex and nuclei. Reversible inactivations of the cerebellar nuclei have directly implicated the cerebellum in the acquisition of nictitating membrane conditioning, but previously the cerebellar cortex has not been reversibly inactivated to assess its contribution to the performance or acquisition of conditioned responses. Here we use the water-soluble disodium salt of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly to block cerebellar cortical AMPA-kainate receptors in lobule HVI and quantitative autoradiography to map its distribution. Conditioned responses are completely, but reversibly, abolished for 10-60 min depending on the concentration of the CNQX infusion and its location within HVI. Zebrin immunohistochemistry was used to define the optimal cortical infusion site that, we suggest, corresponds to the location of the eye blink control regions. We confirm that areas in HVI are essential for the expression of classically conditioned nictitating membrane responses, and we establish a method to analyze the role of cerebellar cortex in the acquisition of this form of motor learning.

Selective inhibition of apoptosis by TPA-induced differentiation of U937 leukemic cells.

U937 leukemic cells treated for 24 h with 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), that induces their macrophagic terminal differentiation, become resistant to etoposide-induced apoptosis. Exposure of undifferentiated U937 cells to 50 microM etoposide for 6 h, that triggers apoptosis in 80% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Mcl-1 expression without modifying Bcl-2, Bcl-xL and Bax protein levels. All these events are inhibited in TPA-differentiated U937 cells that are also resistant to vinblastine-induced and Fas-mediated cell death. Interestingly, these cells are not inherently resistant to apoptosis induction. Exposure of TPA-differentiated U937 cells to 0.8 microg/ml cycloheximide for 24 h, that triggers apoptosis in 50% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Bcl-xL expression without modifying Bcl-2, Mcl-1 and Bax protein levels. All these events are not observed in undifferentiated cells treated in similar conditions. These results indicate that the apoptotic pathway that involves the release of cytochrome c from mitochondria and the cleavage of procaspases remains functional in TPA-differentiated cells.

Biogenicity of an Early Quaternary iron formation, Milos Island, Greece.

A ~2.0-million-year-old shallow-submarine sedimentary deposit on Milos Island, Greece, harbours an unmetamorphosed fossiliferous iron formation (IF) comparable to Precambrian banded iron formations (BIFs). This Milos IF holds the potential to provide clues to the origin of Precambrian BIFs, relative to biotic and abiotic processes. Here, we combine field stratigraphic observations, stable isotopes of C, S and Si, rock petrography and microfossil evidence from a ~5-m-thick outcrop to track potential biogeochemical processes that may have contributed to the formation of the BIF-type rocks and the abrupt transition to an overlying conglomerate-hosted IF (CIF). Bulk δ(13) C isotopic compositions lower than -25‰ provide evidence for biological contribution by the Calvin and reductive acetyl-CoA carbon fixation cycles to the origin of both the BIF-type and CIF strata. Low S levels of ~0.04 wt.% combined with δ(34) S estimates of up to ~18‰ point to a non-sulphidic depository. Positive δ(30) Si records of up to +0.53‰ in the finely laminated BIF-type rocks indicate chemical deposition on the seafloor during weak periods of arc magmatism. Negative δ(30) Si data are consistent with geological observations suggesting a sudden change to intense arc volcanism potentially terminated the deposition of the BIF-type layer. The typical Precambrian rhythmic rocks of alternating Fe- and Si-rich bands are associated with abundant and spatially distinct microbial fossil assemblages. Together with previously proposed anoxygenic photoferrotrophic iron cycling and low sedimentary N and C potentially connected to diagenetic denitrification, the Milos IF is a biogenic submarine volcano-sedimentary IF showing depositional conditions analogous to Archaean Algoma-type BIFs.