Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of vanA, -B, -C1, -C2-3, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%)>> PM-8 (5.2%)>> RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%)>> PM-8 (3.1%)>> QA (2.6%); and for VR, RBM = QA (3.0%)>> PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98. 6% for VR. In the RP, the percentages of results +/- 1 log2 dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.

Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, more than any described bacterium outside the order Bacteroidales. Here, we show that this organism also produces a high-molecular-weight extracellular polysaccharide (EPS). Expression of the EPS results in the formation of a large polysaccharide layer around the bacteria which prevents them from forming a tight pellet upon centrifugation and from entering a Percoll density gradient. Like expression of the capsular polysaccharides, expression of the EPS is phase variable and dictated by DNA inversion of its promoter. EPS expression is regulated at one level by the DNA invertase Tsr19, which is encoded by a gene immediately upstream of the EPS locus and inverts the EPS promoter, causing an on or off phenotype. Expression of the EPS is also regulated at another level, which dictates the amount of EPS produced. By analyzing a panel of tsr19 deletion mutants, we found that the number of inverted repeats (IRs) flanking the promoter is variable. Transcription into the EPS genes is greater in mutants with a single IR between the promoter and the downstream EPS genes than in mutants with more than one IR in this region, correlating with the synthesis of more EPS. By analyzing the relative orientations of the EPS promoter of bacteria obtained from human fecal samples, we showed that both DNA inversion and variation in the number of IRs are active processes of B. fragilis in the endogenous human intestinal ecosystem.

Protracted opiate withdrawal can extend for months of disrupted hormonal circadian rhythms. We examined rodent behaviors and these circadian disturbances in hormone and peptide levels as well as brain clock gene expression during 60 days of protracted withdrawal. Our behavioral tests included open field, elevated plus maze, and sucrose preference tests at 36 h, 10, 30, and 60 days after stopping chronic morphine. At these four assessment points, we collected samples every 4 h for 24 h to examine circadian rhythms in blood hormone and peptide levels and brain expression of rPER1, rPER2, and rPER3 clock genes. Decreased locomotor activity and elevated adrenocorticotropic hormone and melatonin levels persisted for 2 months after morphine withdrawal, but corticosterone was elevated only at 36 h and 10 days after withdrawal. Orexin levels were high at 36 h after withdrawal, but then reversed during protracted withdrawal to abnormally low levels. Beta-endorphin (β-EP) levels showed no differences from normal. However, circadian rhythms were blunted for all of these hormones. Corticosterone, adrenocorticotropic hormone, and orexin blunting persisted at least for 60 days. The blunted circadian rhythm of β-EP and melatonin recovered by day 60, but the peak phase of β-EP was delayed about 8 h. Blunted circadian rhythms and reduced expression of rPER1, rPER2, and rPER3 persisted at least for 60 days in the suprachiasmatic nucleus, prefrontal cortex, nucleus accumbens core, central nucleus of the amygdala, Hippocampus, and ventral tegmental area. Circadian rhythms of rPER1 in the nucleus accumbens shell and basolateral nucleus of the amygdala and of rPER2 in the central nucleus of the amygdala were reversed. Disrupted circadian rhythms of rPER1, rPER 2, and rPER3 expression in reward-related brain circuits and blunted circadian rhythms in peripheral hormones and peptides may play a role in protracted opiate withdrawal and contribute to relapse.

OBJECTIVE

To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized.

RESULTS

EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase.

CONCLUSIONS

Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides.

CONCLUSIONS

This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.

Splenic erythroblasts of mice infected with the anemia-inducing strain of Friend virus can be isolated in large numbers with less than 5% contamination with other cell types. In short-term culture, the isolated cells will initiate globin synthesis and undergo other aspects of terminal differentiation only if erythropoietin (EP) is added to the medium. An early effect of the hormone on these cells is stimulation of total RNA synthesis. EP also causes initiation of transcription of the beta-globin genes after a lag period of 4 to 6 h. By 6 h, the transcription rate of beta-globin RNA is enhanced threefold, and by 12 h, it is nearly maximal at ca. 20 times the level of control cells which received no EP. Transcription rates of alpha and beta-globin genes are approximately equal to each other throughout the period of terminal differentiation. In the splenic erythroblasts, the chromatin structure in the vicinity of the beta-major globin gene was analyzed with two nucleases during these transcription rate changes. No S1 nuclease-hypersensitive site is detectable near the gene. The beta-major gene is quite sensitive to DNase I in comparison with the albumin gene; however, the level of sensitivity is the same before EP addition as it is during maximal gene transcription after EP addition. Also, a hypersensitive site near the 5' cap site of the beta-major gene is quantitatively equivalent both before and after EP addition. Analysis of cytosine methylation at two sites upstream from the gene showed no changes upon induction of beta-globin gene transcription by EP. Thus, the initiation of beta-globin transcription by EP appears to be at some step after chromatin structural alteration such as synthesis, release, or activation of a specific transcription initiation factor.

Behavioral, hormonal and neuronal responses to prolonged exposure to the volatile components of essential oil (EO) extracted from citrus lemon were investigated in male and female rats. Animals were exposed to the lemon essence for 2 weeks while in their cage. Anxiety was then determined with the elevated plus-maze apparatus while nociception was evaluated with a phasic thermal pain stimulus (plantar test) and with a chemical pain stimulus (formalin test). At the end of the experimental sessions, brain areas were dissected to measure beta-endorphin (beta-EP) concentrations in the hypothalamus and periaqueductal gray matter (PAG). Blood samples were collected to determine corticosterone plasma levels. In both sexes, prolonged EO exposure decreased the time spent in the open arms of the plus-maze apparatus. EO-exposed males and females showed higher thermal nociceptive thresholds than controls when tested with the plantar test apparatus. EO exposure induced female-specific decreases in formalin-induced pain behaviors during the formalin test. beta-EP concentrations in the hypothalamus and PAG were affected by EO. Corticosterone was lower in EO-exposed animals of both sexes than in their controls. These results suggest that long-term exposure to lemon EO can induce significant, at times sex-specific, changes in neuronal circuits involved in anxiety and pain.

Pro-opiomelanocortin (POMC) is a prohormone of various neuropeptides, including corticotropin, alpha-melanocyte-stimulating hormone (alpha-MSH), and beta-endorphin (beta-EP). POMC neuropeptides are potent inflammation inhibitors and immunosuppressants and may exert opposite influences during tumorigenesis. However, the role of POMC expression in carcinogenesis remains elusive. We evaluated the antineoplastic potential of POMC gene delivery in a syngenic B16-F10 melanoma model. Adenovirus-mediated POMC gene delivery in B16-F10 cells increased the release of POMC neuropeptides in cultured media, which differentially regulated the secretion of pro- and anti-inflammatory cytokines in lymphocytes. POMC gene transfer significantly reduced the anchorage-independent growth of melanoma cells. Moreover, pre- or post-treatment with POMC gene delivery effectively retarded the melanoma growth in mice. Intravenous injection of POMC-transduced B16-F10 cells resulted in reduced foci formation in lung by 60 to 70% of control. The reduced metastasis of POMC-transduced B16-F10 cells could be attributed to their attenuated migratory and adhesive capabilities. POMC gene delivery reduced the cyclooxygenase-2 (COX-2) expression and prostaglandin (PG) E(2) synthesis in melanoma cells and tumor tissues. In addition, application of NS-398, a selective COX-2 inhibitor, mimicked the antineoplastic functions of POMC gene transfer in melanoma. The POMC-mediated COX-2 down-regulation was correlated with its inhibition of nuclear factor kappaB (NFkappaB) activities. Exogenous supply of alpha-MSH inhibited NFkappaB activities, whereas application of the alpha-MSH antagonist growth hormone-releasing peptide-6 (GHRP-6) abolished the POMC-induced inhibition of NFkappaB activities and melanoma growth in mice. In summary, POMC gene delivery suppresses melanoma via alpha-MSH-induced inhibition of NFkappaB/COX-2 pathway, thereby constituting a novel therapy for melanoma.

We describe for the first time a short sequence of the CD11c promoter (700 bp, named as CD11cS) which induces selective antigen expression in dendritic cells (DCs). It showed a stronger promoter activity than the hitherto used long CD11c promoter (5.5 kb, named as CD11cL), which had been reported inefficient to induce immune responses. After application to the ear pinna and electroporation (EP), CD11cS based DNA vaccines induced specific B- and T-cell responses, including IFN-gamma secretion. Such vaccines achieved induction of anti-tumor immunity comparable in strength to vaccines having the strong tissue non-specific CMV promoter, in both prophylactic and therapeutic settings of mouse tumor models. This short CD11c promoter appears to be a safe and a practical tool for DC-specific gene targeting and for vaccination.

OBJECTIVE

The combination of etoposide (E) and cisplatin (P) is an accepted standard therapy for small-cell lung cancer (SCLC); however, the optimal sequencing and administration schedule has not been defined. This study was designed to evaluate different sequencing and administration schedules of E and P in the treatment of SCLC.

METHODS

Five hundred fifty-two eligible patients with limited-(LD) and extensive-stage (ED) SCLC were randomized to receive one of the following regimens: arm A, P 30 mg/m2 by intravenous (IV) bolus followed by E 130 mg/m2 bolus; arm B, E 130 mg/m2 bolus followed by P 30 mg/m2 bolus; arm C, E 130 mg/m2 by 24-hour infusion and P 30 mg/m2 bolus at the end of each 24-hour infusion of E; arm D, E 130 mg/m2 by 24-hour infusion and P 45 mg/m2 by 24-hour infusion on day 2 and 3 only. Two 3-day induction cycles of IV EP were administered 4 weeks apart. Subsequent therapy was the same for all arms, consisting of four cycles of cyclophosphamide, doxorubicin, and vincristine (CAV) at 4-week intervals. Consolidative thoracic radiation therapy (TRT) and prophylactic cranial irradiation (PCI) were administered to responders.

RESULTS

The overall response rate (84%) was similar in all treatment arms. Treatment arm A was associated with the best complete response (CR) rate (52%), the most favorable median survival time (MST) of 15 months, and a 26% 2-year survival rate. Patients with LD on arm A had a MST of 20 months and a 42% 2-year survival rate. Multivariate analysis indicated that extent of disease, performance status, arm of therapy, and sex were significant independent factors influencing survival. Toxicity of the four regimens was similar, except for greater thrombocytopenia on arm D.

CONCLUSIONS

The bolus administration of EP with E following P for the first two cycles of chemotherapy was the most effective regimen, with especially encouraging survival for LD patients.

OBJECTIVE

A previous study has shown age- and sex-related differences in abdominal aortic compliance. In that study blood pressure determined by auscultation in the brachial artery was assumed to be equal to blood pressure in the abdominal aorta. To validate our findings we investigated the pressure-diameter (P-D) relationship of the abdominal aorta.

METHODS

Diameter and pulsatile diameter change of the abdominal aorta were determined noninvasively by an ultrasound phase-locked echo-tracking system with simultaneous measurement of aortic pressure resulting in P-D curves in 27 healthy male and female volunteers 23 to 72 years of age. The degree of error in aortic compliance as calculated from blood pressure determined by auscultation of the brachial artery rather than from direct measurement of aortic pressure was evaluated. Compliance was defined as the inverse of pressure strain elastic modulus (Ep) or of stiffness (beta).

RESULTS

There was no significant difference in the systolic pressure at the two sites, but the diastolic pressure was systematically overestimated by approximately 10 mm Hg when determined by the auscultatory method (p < 0.01) leading to a 15% to 20% underestimation of Ep and stiffness (beta). The individual P-D curves exhibited hysteresis, were nonlinear, and revealed that the aorta is more distensible at lower than at higher pressures. The steepness of the P-D curve decreased with increasing age and this occurred at an earlier age in men than in women.

CONCLUSIONS

This investigation demonstrates a decrease in abdominal aortic wall distensibility with age, which occurs at an earlier age in men, and confirms earlier results by use of the indexes Ep and stiffness (beta). This implies that the abdominal aorta in men is more prone to degenerative changes, which may be one of the factors responsible for the sex difference in aortic vascular disease.

BACKGROUND

There is little information about mechanical properties of large arteries in patients (pts) with aortic stenosis (AS).

METHODS

Nineteen patients with AS (aortic valve area: 0.88 +/- 0.29 cm(2)) and 24 control subjects without AS but with a similar distribution of risk factors were recruited. beta index, pressure-strain elastic modulus (Ep), arterial compliance (AC), augmentation index (AIx), and local pulse-wave velocity (PWV) were obtained at the level of right common carotid artery (CCA) by a real time echo-tracking system. Time to dominant peak of carotid diameter change waveform, corrected for heart rate (tDPc), and maximum rate of rise of carotid diameter (dD/dt) were measured. Systemic arterial compliance (SAC) was also calculated. Parameters of AS severity (mean gradient, valve area, stroke work loss [SWL]) were determined.

RESULTS

tDPc was higher in patients with AS than in controls (7.9 +/- 0.6 vs. 6.6 +/- 0.7, P < 0.0001) while dD/dt was lower (5.3 +/- 3.6 mm/s vs. 7.8 +/- 2.8 mm/s, P = 0.01). AIx was significantly higher in AS group (32.5 +/- 13.6% vs. 20.6 +/- 12.2%, P = 0.005) and had a linear correlation both with tDPc (r = 0.63, P < 0.0001) and with dD/dt (r =-0.38, P = 0.01). There was a significant correlation between carotid AC and SAC (r = 0.49, P = 0.03), but only carotid AC was related to SWL (r = 0.51, P = 0.02), while SAC was not (P = 0.26).

CONCLUSIONS

AIx was the only parameter of arterial rigidity found to be higher in patients with AS than in controls. Carotid AC showed a significant correlation with SAC and it seemed to be more closely related to AS severity than to SAC.

Evidence that gamma-aminobutyric acid (GABA) and benzodiazepine receptors play a role in the inhibition of ACTH-cortisol secretion in humans has until now been drawn only from data indicating that sodium valproate, a GABA mimetic, and diazepam, a benzodiazepine, decrease hypothalamus-pituitary-adrenal (HPA) axis secretion in patients affected by pathological hypersecretion of the axis. Therefore, the present study investigated the effects, in the same healthy subjects, of sodium valproate or diazepam, on both basal and stress-stimulated concentrations of beta-endorphin (beta-EP), beta-lipotropin (beta-LPH) and cortisol. A single maximal dose of sodium valproate (400 mg) or diazepam (10 mg) did not significantly modify basal concentrations of beta-EP, beta-LPH and cortisol. On the other hand, in the same subjects, pretreatment with sodium valproate (20 mg X 3) or diazepam (10 mg X 2) blocked the increases in these hormones produced by hypoglycemic stress in all patients tested (p less than 0.01 vs. placebo at 45, 60 and 90 min after insulin injection), without affecting the decrease in blood glucose levels. The present data show that sodium valproate and diazepam inhibit stress-induced beta-EP, beta-LPH and cortisol secretion in humans, suggesting that endogenous GABA and benzodiazepine receptors participate in physiological mechanisms regulating the activity of the HPA axis.

BACKGROUND

The prevalence of epilepsy with onset in adulthood increases with age, mainly due to the accumulation of brain damage. However, a significant proportion of patients experience seizures of unknown cause. Alzheimer's disease (AD) is associated with an increased risk of seizures. Seizure activity is interpreted as a secondary event related to hyperexcitability caused by amyloid-β aggregation.

OBJECTIVE

Since neurodegenerative processes begin several years before clinical symptoms, epilepsy could be more frequent in the presymptomatic stages of dementia.

METHODS

We retrospectively reviewed the prevalence of epilepsy of unknown origin with adult onset before cognitive decline in a large cohort of AD patients (EPS-AD) recruited based on clinical and neuropsychological data. Data of patients with epilepsy followed by AD were compared with two control groups: patients with AD without seizures (no EPS-AD) and a large reference population (RP).

RESULTS

In AD patients, the prevalence of epilepsy of unknown origin, with onset in the adulthood before cognitive decline is 17.1 times higher compared with the RP (95% CI: 10.3-28.3). In EPS-AD, seizures begin on average 4.6 years (median 2.0) before the onset of cognitive symptoms and cognitive decline starts 3.6 years earlier compared with noEPS-AD.

CONCLUSIONS

Neurodegenerative processes of dementia could play a key role in the pathogenesis of epilepsy in a subgroup of individuals intended to develop cognitive decline. Adult-onset epilepsy of undefined cause could thus represent a risk factor for the ongoing neurodegenerative damage, even preceding by years the onset of clinical symptoms of dementia.

Transforming growth factor-beta (TGF beta), a protein known to antagonize many of the functions of the epidermal growth factor-receptor system, was localized immunohistochemically in unruptured ectopic pregnancies (EP) removed by salpingectomy (n = 8), uterine decidua from EP (n = 4), and decidua and trophoblast from electively terminated first trimester pregnancies (ETP; n = 8). Two rabbit polyclonal antisera that recognize both TGF beta 1 and beta 2 were used. Immunostaining for TGF beta was identified in all three forms of trophoblast, cytotrophoblasts, intermediate trophoblasts, and syncytiotrophoblasts, which were differentiated histologically and immunohistochemically. Moderate cytoplasmic immunostaining was found in villous cytotrophoblasts in both EP and ETP. Nonvillous (anchoring) cytotrophoblasts in these same tissues demonstrated moderate immunostaining adjacent to the villous and light immunostaining distal to the villous. In intermediate trophoblasts, moderate to intense immunostaining was seen in EP and ETP. Syncytiotrophoblasts demonstrated moderate cytoplasmic immunostaining in EP and ETP as well as moderate to intense staining of plasma membranes and microvilli. Nuclear staining was not evident in any form of trophoblast. TGF beta immunostaining was demonstrated in both glands and stroma of decidua from both EP and ETP; however, staining was more intense in decidua from ETP. With the known presence of TGF beta receptors and mRNA in placenta, these results suggest an autocrine/paracrine role for TGF beta regulation of endometrial-trophoblast function during human implantation.

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPSB. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPSEPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

To investigate age-related changes in the oestrous cycle and reproductive hormone levels in senescence-accelerated mouse (SAM), we examined these parameters in 3-, 5-, 7-, 9- and 11-month-old female SAM-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) strains. Levels of beta-endorphin (beta-EP) and substance P (SP) in the hypothalamus were also measured. The oestrous cycle and dioestrus of 9-month-old SAMP8 mice were significantly prolonged compared with age-matched SAMR1 mice. Furthermore, the concentration of serum oestradiol was lower and the level of pituitary luteinising hormone was higher in SAMP8 mice compared with SAMR1 mice. This characterises the hypothalamus-pituitary-ovary (HPO) axis of the SAMP8 strain as hypergonadotropic-hypogonad. The levels of beta-EP and SP in the SAMP8 hypothalamus were lower than in the SAMR1 hypothalamus. These results indicate that the function of the HPO axis in SAMP8 mice declines early and this may be attributed, in part, to the decline in beta-EP and SP concentrations in the hypothalamus.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-β, Myxovirus resistance gene A (MxA) and RNaseL.

Expression of Prominin 1/CD133 is associated with poor prognosis and chemoresistance in several types of solid tumors. The aim of the present study was, therefore, to evaluate Prominin 1/CD133 expression in colorectal carcinoma after short-term preoperative treatment by non-steroidal anti-inflammatory drugs (NSAIDs). Patients aimed at curative operation for colon cancer were randomized to receive NSAIDs (indomethacin 50 mg x2 or celecoxib 100 mg x2) three days preoperatively. Antisecretory prophylaxis (esomeprasol 40 mg x1) was provided to all patients and served as sham intake. CD133 expression in tumor tissue was also assessed in tumors from Dukes' B patients selected for either long or short postoperative survival. No patients received perioperative chemoradiotherapy. Tumor tissue was collected at surgery for quantification of mRNAs (Prom1 and Wnt4) by qPCR. Immunohistochemistry stained for CD133, Ep-CAM, CD34 and CD45. PGE2 content in tumor tissue was determined. Transcript of CD133 in tumor tissue was lower in patients treated with NSAIDs (0.28 ± 0.07 vs. 0.51 ± 0.08; p<0.03) as well as some other stem cell-related transcripts. In treated patients 36% of all tumors stained positive for CD133 compared to 71% in sham-treated control patients (p<0.05). Short survivors with Dukes' B tumors displayed 78% CD133 expression as compared to 33% of tumors in long-term survivors (p<0.002). Tumor tissue PGE(2) content was negatively related to patient survival. Our results show that short-term preoperative NSAID treatment downregulates colon cancer tissue expression of Prominin 1/CD133, a stem cell marker indicative of survival prognosis as confirmed.

The pathogenesis of polycystic liver disease is not well understood. The putative function of the associated proteins, hepatocystin and Sec63p, do not give insight in their role in cystogenesis and their tissue-wide expression does not fit with the liver-specific phenotype of the disease. We designed this study with the specific aim to dissect whether pathways involved in polycystic kidney diseases are also implicated in polycystic liver disease. Therefore, we immunohistochemically stained cyst tissue specimen with antibodies directed against markers for apoptosis, proliferation, growth receptors, signaling and adhesion. We analyzed genotyped polycystic liver disease cyst tissue (n=21) compared with normal liver tissue (n=13). None of the cysts showed proliferation of epithelial cells. In addition, anti-apoptosis marker Bcl-2 revealed slight increase in expression, with variable increase of apoptosis marker active caspase 3. Growth factor receptors, EGFR and c-erbB-2, were overexpressed and mislocalized. We found EGFR staining in the nuclei of cyst epithelial cells regardless of mutational state of the patient. Further, in hepatocystin-mutant polycystic liver disease patients, apical membranous staining of c-erbB-2 and adhesion markers, MUC1 and CEA, was lost and the proteins appeared to be retained in cytoplasm of cyst epithelia. Finally, we found loss of adhesion molecules E-cadherin and Ep-CAM in cyst epithelium of all patients. Nevertheless, we observed normal beta-catenin expression. Our results show that polycystic liver disease cystogenesis is different from renal cystogenesis. Polycystic liver disease involves overexpression of growth factor receptors and loss of adhesion. In contrast, proliferation or deregulated apoptosis do not seem to be implicated. Moreover differential findings for PRKCSH- and SEC63-associated polycystic liver disease suggest a divergent mechanism for cystogenesis in these two groups.

BACKGROUND

A significant population of patients with severe asthma and chronic obstructive pulmonary disease is less responsive to beta(2)-adrenoceptor agonists and corticosteroids, and there are possible safety issues concerning long-term use of these drugs. Inhaled prostaglandin E(2) (PGE(2)) is antiinflammatory and a bronchodilator in patients with asthma, but it also causes cough.

OBJECTIVE

We aimed to identify the receptor involved in PGE(2)-induced sensory nerve activation and cough using a range of in vitro and in vivo techniques.

METHODS

Depolarization of vagal sensory nerves (human, mouse, and guinea pig) was assessed as an indicator of sensory nerve acitivity. Cough was measured in a conscious guinea pig model.

RESULTS

Using an extensive range of pharmacological tools, we identified that the EP(3) receptor mediates PGE(2)-induced depolarization of sensory nerves in human, mouse, and guinea pig. Further supporting evidence comes from data showing that responses to PGE(2) are virtually abolished in isolated vagus nerves from EP(3)-deficient mice (Ptger3(-/-)). Finally, we demonstrated the role of the EP(3) receptor in vivo using a selective EP(3) antagonist to attenuate PGE(2)-induced cough.

CONCLUSIONS

Identification of the receptor mediating PGE(2)-induced cough represents a key step in developing a drug that is antiinflammatory and a bronchodilator but without unwanted side effects.

<A<em>b</em>stractText>To correlate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters to tumor grading and to assess their relia<em>b</em>ility in predicting pathological complete response (pCR) <em>b</em>efore neoadjuvant chemoradiotherapy (CRT) in patients with locally advanced rectal cancer (LARC).</A<em>b</em>stractText><A<em>b</em>stractText>Forty patients (24 male; mean age, 67.3 ± 8.1 years) with histologically proven LARC who had undergone 3-Tesla DCE-MRI <em>b</em>efore (MRI_1) and after CRT (MRI_2) <em>b</em>etween August 2015 and Fe<em>b</em>ruary 2016 were included in this retrospective study. DCE-MRI parameters at MRI_1 and MRI_2 were extracted <em>b</em>y two <em>b</em>oard certified radiologists in consensus reading with Olea Sphere 2.3 software using the extended Tofts model. Based on DCE-MRI results, patients were divided in complete responders (CR) and non-complete responders (nCR) and the perfusion parameters were correlated to tumor grading and pCR.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Wash-out and K<su<em>b</em>)<em>ep</em></su<em>b</em>) at MRI_1 showed significant correlation with LARC grading (P = 0.004 and 0.01, respectively). V<su<em>b</em>)e</su<em>b</em>) showed a significant increase <em>b</em>etween MRI_1 (0.47 ± 0.27) and MRI_2 (0.63 ± 0.23; P = 0.007). K<su<em>b</em>)trans</su<em>b</em>) measured at MRI_1 was significantly higher in CR (0.66 ± 0.48) compared to nCR (0.53 ± 0.34, P = 0.02).</p><p><div>(<em>b</em>)CONCLUSION</<em>b</em>)</div>Wash-out and K<su<em>b</em>)<em>ep</em></su<em>b</em>) measured <em>b</em>efore CRT correlate with LARC grading. V<su<em>b</em>)e</su<em>b</em>) changes during CRT, while K<su<em>b</em>)trans</su<em>b</em>) measured <em>b</em>efore CRT may predict the response to therapy. Therefore, DCE-MRI parameters can predict tumor aggressiveness and CRT efficacy, playing a role as imaging <em>b</em>iomarkers in patients with LARC.</p>

Studies show that estrogens can influence alcohol consumption; however, findings are variable and an etiology remains unknown. Furthermore, estrogen administration can alter several neurotransmitter systems implicated in alcohol consumption, including the beta-endorphin (beta-EP) system. The present studies investigate (a) whether estradiol valerate (EV) alters voluntary alcohol consumption in Wistar and Lewis rats, (b) if an effect of EV on drinking is associated with changes in hypothalamic or pituitary beta-EP content, and (c) whether differences in alcohol drinking between treatment and rat groups are related to locomotor or defensive behavior/anxiety scores. Of 30 Wistar and 30 Lewis rats used in this study, half were injected with 2 mg EV in 0.2 ml sesame oil, while the remainder were injected with the vehicle only. After 8 weeks, all animals were tested in the open field and elevated plus maze. A week later, 4-6 animals in each group were sacrificed. The remaining animals were tested for voluntary alcohol drinking for 24 days prior to being sacrificed on the last day. Radioimmunoassay was used to estimate hypothalamic and pituitary beta-EP content. Wistar and Lewis rats injected with EV showed an increase in alcohol drinking, but their behavior scores and beta-EP levels remained unaltered. This result suggests that any EV effect on drinking is unrelated to changes in beta-EP or behavioral performance. Furthermore, Wistar rats show higher alcohol drinking, locomotor and defensive behavior scores, and hypothalamic beta-EP than Lewis rats. Higher alcohol drinking by Wistar rats might be due to higher behavioral scores or endogenous opioid activity/sensitivity.

We developed a new approach that couples Southwestern <em>b</em>lotting and mass spectrometry to discover proteins that <em>b</em>ind extracellular DNA (eDNA) in <em>b</em>acterial <em>b</em>iofilms. Using <i>Staphylococcus aureus</i> as a model pathogen, we identified proteins with known DNA-<em>b</em>inding activity and uncovered a series of lipoproteins with previously unrecognized DNA-<em>b</em>inding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent <em>b</em>iofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on <em>b</em>iofilm accumulation, these lipoprotein mutations increased <em>b</em>iofilm porosity, suggesting that lipoproteins and their associated interactions contri<em>b</em>ute to <em>b</em>iofilm structure. For one of the lipoproteins, SaeP, we showed that the <em>b</em>iofilm phenotype requires the lipoprotein to <em>b</em>e anchored to the outside of the cellular mem<em>b</em>rane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the <em>b</em>acterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact <em>b</em>iofilm development. It has <em>b</em>een reported that <i>S. aureus</i> <em>b</em>iofilms are sta<em>b</em>ilized <em>b</em>y positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favora<em>b</em>le electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-<em>b</em>inding proteins and mem<em>b</em>rane-attached lipoproteins that can function as anchor points <em>b</em>etween eDNA in the <em>b</em>iofilm matrix and the <em>b</em>acterial cell surface.(<em>b</em>)IMPORTANCE</<em>b</em>) Many <em>b</em>acteria are capa<em>b</em>le of forming <em>b</em>iofilms encased in a matrix of self-produced extracellular polymeric su<em>b</em>stances (<em>EPS</em>) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, <em>b</em>acterial <em>b</em>iofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions <em>b</em>etween the <em>b</em>acterial cell surface and <em>EPS</em> that sta<em>b</em>ilize <em>b</em>iofilms. Extracellular DNA (eDNA) has <em>b</em>een recognized as an <em>EPS</em> constituent for many <em>b</em>acterial species and has <em>b</em>een shown to <em>b</em>e important in promoting <em>b</em>iofilm formation. Using <i>Staphylococcus aureus</i> <em>b</em>iofilms, we show that mem<em>b</em>rane-attached lipoproteins can interact with the eDNA in the <em>b</em>iofilm matrix and promote <em>b</em>iofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting <em>b</em>acterial <em>b</em>iofilms.

B. longum has been reported to exert an alleviative effect on colitis, but the results also suggested significant differences among strains. Here in this study, we compared the effect of B. longum subsp. longum strains with different properties in EPS production on DSS-induced colitis. To investigate the alleviative effect of a ropy-exopolysaccharide (EPS) producing strain, Bifidobacterium longum subsp. longum YS108R, on experimental colitis, C57BL/6J mice (male, 6-8 weeks old) were randomly assigned to six groups (n = 8): normal control, DSS colitis and four DSS colitis groups orally administered with three B. longum subsp. longum strains (YS108R, C11A10B and HAN4-25) and B. animalis subsp. lactis BBEPS, C11A10B produced non-ropy-EPS, HAN4-25 did not produce EPS and BBEPS producing strain YS108R could alleviate the symptoms and remit inflammation induced by DSS, in which YS108R could decrease the pro-inflammatory cytokine IL-6 and IL-17A levels after DSS challenge (from 102 ± 45.22 to 37.95 ± 20.33 pg mL-1 and from 22.14 ± 5.43 to 12.58 ± 2.74, p < 0.05), but another non-ropy-EPS producing strain C11A10B did not decrease the levels of these pro-inflammatory cytokines. Furthermore, YS108R could maintain the expression levels of genes related to the mucosal barrier, but strain HAN4-25, a non-EPS producer, was not able to maintain the expression levels of these genes after DSS challenge. Analysis of gut microbiota showed that DSS treatment significantly increased the relative abundance of Enterobacteriaceae and Peptostreptococcaceae (0.2623 ± 0.162 and 0.0512 ± 0.0361) and decreased the relative abundance of S24-7 (0.042 ± 0.0326); however, YS108R administration could decrease the relative abundance of Enterobacteriaceae and Peptostreptococcaceae to 0.0848 ± 0.0399 and 0.0032 ± 0.0047 and increase the relative abundance of S24-7 to 0.2625 ± 0.0566 (p < 0.05). The results showed that B. longum subsp. longum YS108R could alleviate DSS-induced colitis by modulating the inflammation related cytokines, maintenance of the normal mucosal barrier and reverting the change of microbiota.