[Relationship between microsatellite instability and hepatocyte growth factor expression and their prognostic significance in colorectal cancer].

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF) and its relationship with microsatellite instability (MSI) and their influence on survival in patients with colorectal cancer. METHODS: Immunohistochemistry (IHC) was used to detect the expression of HGF and MSI in 98 specimens of colorectal cancer. Tumors lacking protein expression of any of the four mismatch repair genes (MLH1, PMS2, MSH2 or MSH6) were labelled as MSI, and the rest were considered as microsatellite stable (MSS). The associations between expression and clinicopathological factors were assessed using Chi-square tests. Kaplan-Meier curves, log-rank test, and Cox regression were used to analyze the association between biomarker expressions and overall survival. RESULTS: The incidence rate of MSI in 98 colorectal specimens was 32.7%, and was statistically significantly correlated with the location of tumor and differentiation degree (P<0.05). The HGF-expression rate was 71.4%. The patients with an MSI tumor had a significantly higher HGF expression, compared with the patients with an MSS tumor (P=0.048). The 5-year survival rate of MSI group and MSS group were 39.8% and 58.7%, respectively (P=0.009). The 5-year survival rate of HGF-positive group and HGF-negative group were 46.2% and 67.9% (P=0.035). The multivariate analysis showed that lymphocytic infiltration, TMN stage, MSI and HGF are independent prognostic factors in colorectal cancer (P<0.05 for all). CONCLUSIONS: HGF is highly expressed in colorectal cancer patients with microsatellite instability. Both microsatellite instability and HGF are independent factors affecting the prognosis in patient with colorectal cancer.

Reduction in greenhouse gas emissions from sewage sludge aerobic compost in China.

Sewage sludge is an important contributor to greenhouse gas (GHG) emissions and the carbon budget of organic solid waste treatment and disposal. In this case study, total GHG emissions from an auto-control sludge compost system, including direct and indirect emissions and replaceable reduction due to sludge compost being reused as fertilizer, were quantified. The results indicated that no methane generation needed to be considered in the carbon debit because of the advantages of auto-control for monitoring and maintenance of appropriate conditions during the composting process. Indirect emissions were mainly from electricity and fossil fuel consumption, including sludge transportation and mechanical equipment use. Overall, the total carbon replaceable emission reduction owing to sludge being treated by composting rather than landfill, and reuse of its compost as fertilizer instead of chemical fertilizer, were calculated to be 0.6204 tCO2e t(-1) relative to baseline. Auto-control compost can facilitate obtaining certified emission reduction warrants, which are essential to accessing financial support with the authentication by the Clean Development Mechanism.

Fas signaling promotes motility and metastasis through epithelial-mesenchymal transition in gastrointestinal cancer.

Fas signaling was reported to participate in cell apoptosis. However, this pathway has also been shown to promote tumor cell motility, leading to the hypothesis that Fas signaling may induce epithelial-mesenchymal transition (EMT) to promote metastasis. The effects of Fas-ligand (FasL) treatment and inhibition of Fas signaling on colorectal and gastric cancer cells were tested using motility assay, immunofluorescence, RT-PCR and immunoblot analyses. Fas signaling downregulated epithelial markers, upregulated mesenchymal markers and promoted motility in gastrointestinal (GI) cancer cells. FasL treatment also increased the expression of EMT transcriptional factors in the nucleus and induced a spindle shape cell morphology in these cells. Knockdown of Snail or Twist expression significantly decreased FasL-induced motility. The ERK1/2 pathway was activated by Fas signaling and is required for FasL-induced EMT and motility. Moreover, oxaliplatin, a chemotherapeutic agent, induced EMT partly through Fas signaling. Evaluation of human GI clinical specimens showed that FasL expression increased whereas E-cadherin expression decreased during GI cancer progression. Both markers were significantly inversely correlated. Tissue samples with a non-EMT phenotype were mainly distributed in patients with early cancer stages, whereas samples with an EMT phenotype were mostly distributed in patients with advanced cancer stages. A non-EMT phenotype significantly correlated with better prognosis. Altogether, these data indicate that Fas signaling may induce EMT to promote tumor motility and metastasis in GI cancer in vivo and in vitro.

Fabrication of engineered heart tissue grafts from alginate/collagen barium composite microbeads.

Cardiac tissue engineering holds great promise for the treatment of myocardial infarction. However, insufficient cell migration into the scaffolds used and inflammatory reactions due to scaffold biodegradation remain as issues to be addressed. Engineered heart tissue (EHT) grafts fabricated by means of a cell encapsulation technique provide cells with a tissue-like environment, thereby potentially enhancing cellular processes such as migration, proliferation, and differentiation, and tissue regeneration. This paper presents a study on the fabrication and characterization of EHT grafts from novel alginate/collagen composite microbeads by means of cell encapsulation. Specifically, the microbeads were fabricated from alginate and collagen by barium ion cross-linking, with neonatal rat cardiomyocytes encapsulated in the composite microbeads during the fabrication of the EHT grafts. To evaluate the suitablity of these EHT grafts for heart muscle repair, the growth of cardiac cells in the microbeads was examined by means of confocal microscopy and staining with DAPI and F-actin. The EHT grafts were analyzed by scanning electron microscopy and transmission electron microscopy, and the contractile function of the EHT grafts monitored using a digital video camera at different time points. The results show the proliferation of cardiac cells in the microbeads and formation of interconnected multilayer heart-like tissues, the presence of well-organized and dense cell structures, the presence of intercalated discs and spaced Z lines, and the spontaneous synchronized contractility of EHT grafts (at a rate of 20-30 beats min(-1) after two weeks in culture). Taken together, these observations demonstrate that the novel alginate/collagen composite microbeads can provide a tissue-like microenvironment for cardiomyocytes that is suitable for fabricating native heart-like tissues.

Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite.

The effects of single doses of intravenous (IV) ciprofloxacin and rifampin and of multiple doses of rifampin on glyburide exposure and blood glucose levels were investigated in nine healthy volunteers. A single IV dose of rifampin significantly increased the area under the concentration-time curve (AUC) of glyburide and its metabolite. Blood glucose levels were significantly lower than those observed after dosing with glyburide alone. Multiple doses of rifampin induced an increase in liver enzyme levels, leading to a marked decrease in glyburide exposure and blood glucose levels. When IV rifampin was administered after multiple doses of rifampin, the inhibition of hepatic uptake transporters masked the induction effect; however, the relative changes in AUC for glyburide and its hydroxyl metabolite were similar to those seen under noninduced conditions. The studies reported here demonstrate how measurements of the levels of both the parent drug and its primary metabolite are useful in unmasking simultaneous drug-drug induction and inhibition effects and in characterizing enzymatic vs. transporter mechanisms.

The impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients.

Persistent rejection in the face of treatment and multiple episodes of rejection are associated with the development of chronic rejection and graft loss in solid organ transplantation. The factors that create an environment for rejection that persists in the face of treatment are as yet not understood. The objective of this study was to evaluate the risk factors, including human multidrug resistance gene (MDR1), cytochrome P4503A5 (CYP3A5) and cytokine gene polymorphisms, associated with acute persistent rejection (APR) in lung transplant patients. One hundred and twenty-five adult lung transplant patients were studied. MDR1 G2677T, C3435T and CYP3A5 polymorphisms were assessed by direct sequencing of the polymorphic region in patient DNA. Cytokine genotyping for five cytokines was performed using the polymerase chain reaction-sequence specific primers (PCR-SSP) technique. Multivariate regression analysis was used to identify the predictors of acute persistent rejection. The dependent variable was the presence or absence of acute persistent rejection based on lung biopsies during the first postoperative year. The independent variables were MDR1 G2677T and C3435T, CYP4503A5 and cytokine polymorphisms, survival status, age, gender, survival days and HLA mismatches. The MDR1 C3435T polymorphism and age were independently associated with acute persistent rejection (p = 0.025, odds ratio = 0.29, 95% CI 0.1-0.86 and p = 0.016, odds ratio = 0.94, 95% CI 0.89-0.98, respectively). For the MDR1 C3435T polymorphism, 72% of patients with the C allele had acute persistent rejection in comparison to 52% for TT patients (p = 0.04). For age, a significant difference was found between the nonrejection group and the rejection group (mean+/-S.D. 52.1+/-11.2 vs. 44.4+/-12.3, p = 0.01). This is the first report of the association of a drug disposition genotype with drug-resistant acute rejection in organ transplant patients. The major predictor of acute persistent rejection in the first postoperative year for lung transplant patients was the MDR1 C3435T genotype. This association could be due to drug resistance, altered drug disposition or other immunologic effects associated with P-glycoprotein (P-gp) function. Future prospective treatment algorithms should be developed that will incorporate the knowledge of gene polymorphisms into treatment regimens to improve the outcome following lung transplantation.

The impact of pharmacogenomic factors on steroid dependency in pediatric heart transplant patients using logistic regression analysis.

Many pharmacogenomic predictors of drug response are now available, and include both drug metabolism-disposition factors and drug targets. Information on statistical approaches to analyzing large clinical data sets in relation to genetic polymorphisms is limited. The objective of this study was to evaluate whether logistic regression could identify pharmacogenomic predictors of outcome in a large data set in a complex transplant patient population. Seventy pediatric heart transplant patients were studied. Patients were followed for at least 1 yr post-transplantation as outpatients, and weaned from corticosteroids if clinically appropriate. Logistic regression analysis was used to identify the predictors of steroid dependency. The dependent variable was the presence or absence of steroid therapy at 1 yr post-transplantation. The independent variables were the patients' transplant age, gender, MDR1 C3435T and G2677T, CYP3A53B and cytokine polymorphisms. By chi-square test for the MDR1 C3435T polymorphism, 12 of 18 (67%) patients in the CC group were still on prednisone, whereas only 18 of 47 (38%) of the CT/TT group were still receiving prednisone (p = 0.04). For the IL-10 groups, two of 15 patients with the high producer genotype (13.3%) remained on prednisone, in comparison with 16 of 28 patients with the intermediate producer genotype (57.1%) and 15 of 26 patients with the low producer genotype (57.7%, p = 0.01). Logistic regression analysis confirmed MDR1 C3435T (p = 0.021), and IL-10 polymorphisms (intermediate producer genotype p = 0.015; low producer genotype p = 0.013) as independent risk factors for steroid dependency at 1 yr after transplantation. This approach identifies pharmacogenomic factors, which can be studied more extensively in larger data sets, and used in prospective studies to individualize immunosuppressive therapy following solid organ transplantation.

Localization of Barrington's nucleus in the pontine dorsolateral tegmentum of the rabbit.

The localization of Barrington's nucleus in the dorsolateral pons of the rabbit and its projections to the sacral spinal cord were examined by using retrograde and anterograde labeling methods combined with immunohistochemistry. After injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or a fluorescence tracer, tetramethylrhodamine-dextran amine (TMR), into the sacral spinal cord segments, a cluster of neurons labeled with WGA-HRP or TMR were seen in the pontine dorsolateral tegmentum. To identify whether the retrogradely labeled neurons were situated within the locus coeruleus, the sections containing TMR-labeled neurons through the pons were incubated with anti-tyrosine hydroxylase (TH) antibody and observed under epifluorescence microscope. It was shown that the cluster of TMR-labeled neurons in the dorsolateral tegmentum were surrounded by TH-positive neurons, but they were negatively immunostained with TH-like immunoreactivity. In anterograde experiment, injection of WGA-HRP into the dorsolateral tegmentum resulted in many anterogradely labeled nerve fibers and terminals in the sacral spinal cord, including the sacral parasympathetic nucleus. The present results suggest that the cluster of neurons in the dorsolateral tegmentum of the rabbit may correspond to Barrington's nucleus revealed in the rat and cat, and thus may be involved in micturtion reflex of the rabbit.

The distribution of substance P receptor (NK1)-like immunoreactive neurons in the newborn and adult human spinal cord.

Substance P receptor (i.e. NK1)-like immunoreactive (SPR-LI) neurons were observed in the newborn and adult human spinal cord. Substance P receptor-like immunoreactive neuronal cell bodies were seen most frequently in lamina I, and were scattered throughout the remaining laminae of the dorsal horn and the area around the central canal. Some neurons in the intermediolateral nucleus also showed weak immunoreactivity. The pattern of distribution of SPR-LI neurons in the adult spinal cord was essentially the same as that in the newborn spinal cord. However, SPR-LI neurons cell bodies were seen much more frequently in the newborn than in the adult dorsal horn, especially in lamina II.

Evaluation of tinnitus patients by peroral multi-drug treatment.

To evaluate patients complaining of subjective tinnitus, this study examined their response to peroral betahistine mesilate, vitamin B complex and diazepam in combination. Because three drugs were used together, it remains to be seen whether a single drug or a combination of drugs was effective. We issued questionnaires to 67 patients with tinnitus associated with sensorineural hearing loss of unknown etiology or tinnitus, despite normal hearing in pure tone audiometry and lack of distinct systemic disorders. Our original questionnaire contained seven items and allotted points for each item to facilitate evaluation. After prescribing the above drugs and observing patients' progress for 5 weeks, 50 of the 67 subjects were evaluated again by the same questionnaire. The present study evaluates tinnitus of patients as an example of clinical applications; this was not a controlled double blind study. It was found that, after patients took the prescribed medication, the total number of points were significantly reduced (paired t-test, P < 0.001). After medication, cases of bilateral tinnitus were significantly reduced from 27 to 14, and cases of two types of tinnitus sound, were significantly decreased from 22 to 11 (chi 2-test, P < 0.05). After 5 weeks of administration, 54% of patients felt treatment had been effective. Preliminary results suggest this peroral multi-drug treatment may provide relief for some patients with subjective tinnitus. However, long-term efficacy of the treatment was not investigated.

Direct projections from the lumbosacral spinal cord to Barrington's nucleus in the rat: a special reference to micturition reflex.

In the present study, direct projections from the lumbosacral cord to Barrington's nucleus in the rat were investigated by using retrograde and anterograde tracing techniques. After injection of cholera toxin B subunit (CTb) into Barrington's nucleus, a number of moderately CTb-labeled neurons were observed in the lumbosacral cord, with a slight ipsilateral dominance; most were located in the spinal parasympathetic and dorsal commissural nuclei of the lumbosacral cord. In addition, some retrogradely labeled neurons were found in the periaqueductal gray (PAG). These findings were confirmed by an anterograde labeling experiment. After biotinylated dextran amine (BDA) was injected into the lumbosacral cord, dense BDA-labeled axon terminals were found in Barrington's nucleus as well as in the PAG. Injection of BDA into the PAG resulted in many BDA-labeled terminals in Barrington's nucleus. The present results provided clear evidence for a direct projection from the spinal parasympathetic and dorsal commissural nuclei to Barrington's nucleus that could subserve conveying bladder-filling information from the lumbosacral cord to Barrington's nucleus in the micturition reflex of the rat.

GABAergic synapses on mu-opioid receptor-expressing neurons in the superficial dorsal horn: an electron microscope study in the cat spinal cord.

A double-immunocytochemical electron microscope study was performed in the cat to examine whether GABAergic axons might be in synaptic contact with spinal neurons expressing mu-opioid receptor (MOR) in laminae I and II of the spinal dorsal horn at the lumbar cord segments. Structures showing MOR-like immunoreactivity (-LI) and those showing GABA-LI were labeled, respectively, with diaminobenzidine/peroxidase-reaction products and immunogold particles. Approximately one-third of dendritic profiles with MOR-LI in laminae I and II were postsynaptic to axon terminals with GABA-LI; about one-fourth of somatic profiles with MOR-LI were also postsynaptic to axon terminals with GABA-LI. The results suggest that activation of MOR on postsynaptic neurons may modulate effects which are induced by GABA released from presynaptic neurons.

Coexistence of mu-opioid receptor-like and substance P-like immunoreactivities in the cat dorsal root ganglionic neurons.

Coexistence of mu-opioid receptor (MOR)-like immunoreactivity (LI) and substance P (SP)-LI in the neurons of the cat dorsal root ganglia (DRG) was examined by a double immunofluorescence histochemical method. Approximately 91% of SP-LI neurons in the DRG showed MOR-LI. However, SP-LI was exhibited in approximately 28% of the neurons labeled with MOR-LI. These morphological findings indicated that the MOR exist on most of the primary afferent SP-containing terminals, and suggest that MOR may regulate SP release from the primary afferent terminals in the cat dorsal horn.

Central origins of substance P-like immunoreactive fibers and terminals in the spinal trigeminal caudal subnucleus in the rat.

After trigeminal rhizotomy, some substance P-like immunoreactive (SP-LI) fibers and terminals in the spinal trigeminal caudal subnucleus (Vc), specially in its superficial laminae (laminae I and II), still remained in the rat. Employing a combination of Fluoro-Gold retrograde tracing and immunofluorescence histochemical staining for SP, we found that the main central origins of these SP-LI fibers and terminals were midbrain periaqueductal gray (PAG), nucleus raphe magnus (NRM) and other raphe nuclei, and nucleus reticularis gigantocellularis pars alpha; all of them are important structures of the endogenous pain control system. The present results provided morphological evidence for PAG or NRM stimulation could inhibit neuronal activities in the Vc evoked by orofacial nociceptive stimulation and also suggested that SP might be an important neurotransmitter or neuromodulator for endogenous pain control system.

Reproducibility of pitch-matching test for tinnitus. Using a heptatonic scale.

We evaluated the reproducibility of measured values obtained using a heptachord pitch-matching test (PMT). Investigation of the measured values by considering the minimum unit width limits of the testing scale revealed that the coincidence ratios of the measured values of intra-daily variations, intra-weekly variations and eight values measured consecutively in one test were as high as 100%, 94.8% and 83.3%, respectively. We therefore concluded that the reproducibility of this method is satisfactory. The measured values obtained by both ordinary one-octave-interval and heptachord PMT are compared in this article.