β-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated β-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. β-Lapachone markedly induced cell death without caspase activation. β-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited β-lapachone-induced cell death and HMGB-1 release. In addition, β-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked β-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that β-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, β-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to β-lapachone. Taken together, our results demonstrate that β-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.
β-Lapachone; NQO1; ROS; RIP1; PARP1; AIF
Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors (LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.
thioridazine; TRAIL; Akt; Mcl-1; c-FLIP; ROS
Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset.
Adult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student’s t-test or one-way analysis of variance determined significance (P < 0.05).
CXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, were upregulated.
Collectively, our data characterize an endogenous neuroprotective phenotype that utilizes adaptive immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B cells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediate an adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.
Hypoxic preconditioning; B cells; CXCL13; Stroke; Neuroprotection; B10
At attended locations emotion and attention interact to benefit contrast sensitivity, a basic visual dimension. Whether there are associated costs at unattended locations is unknown. Furthermore, emotion and attention affect response time, and anxiety modulates these effects. We investigated how trait-anxiety influences the interaction of emotion and attention on contrast sensitivity. On each trial, non-predictive precues (neutral or fearful faces) directed exogenous attention to four contrast-varying, tilted stimuli (Gabor patches). Attention was cued toward the target (valid), a distracter (invalid), or distributed over all locations. Observers discriminated target orientation, and completed self-report measures of anxiety. Effects of fearful expressions were mediated by trait-anxiety. Only high-trait anxious individuals showed decreased target contrast sensitivity after attention was diverted to a distracter by a fearful cue, and anxiety score correlated with degree of impairment across participants. This indicates that increasing anxiety exacerbates threat-related attentional costs to visual perception, hampering processing at non-threat-related locations.
emotion; attention; contrast sensitivity; fear expression; anxiety
The objective of this study was to assess causative pathological factors associated with diffusion restriction on diffusion-weighted imaging (DWI) in patients who achieved pathological complete response (pCR) after treatment with neoadjuvant chemoradiation therapy (CRT) for locally advanced rectal cancer.
In total, 43 patients with locally advanced rectal cancer (≥T3 or lymph node positive) who underwent neoadjuvant CRT, subsequent surgery and ultimately achieved pCR were enrolled. All patients underwent pre- and post-CRT 3.0 T rectal MRI with DWI. Two radiologists blinded to pathological staging reviewed pre- and post-CRT 3.0 T rectal MRI for the presence of diffusion restriction in the corresponding tumour areas on post-CRT DWI, with a third radiologist arbitrating any disagreement. The consensus of these findings was then correlated with pathological data such as intramural mucin and the degree of proctitis and mural fibrosis seen on surgical specimen. Additionally, the pre-CRT tumour volume was measured to define the effect of this variable on the degree of radiation proctitis and fibrosis, as well as the presence of intramural mucin.
Diffusion restriction occurred in 18 subjects (41.9%), while 25 subjects remained diffusion restriction-free (58.1%). The diffusion restriction group tended to have more severe proctitis and mural fibrosis when compared with non-diffusion restriction group (p<0.001). Intramural mucin was also more common in the diffusion restriction group (p=0.052). Higher pre-CRT tumour volumes were significantly predictive of the degree of proctitis (p=0.0247) and fibrosis (p=0.0445), but not the presence of intramural mucin (p=0.0944). Proctitis and mural fibrosis severity were also identified as independent pathological risk factors for diffusion restriction on multivariate analysis (p=0.0073 and 0.0011, respectively).
Both radiation-induced proctitis and fibrosis were significant and independent predictors of diffusion restriction in patients achieving pCR after treatment with neoadjuvant CRT for locally advanced rectal cancer, and pre-CRT tumour volume significantly affects both variables.
A prerequisite for the mechanistic simulation of a biochemical system is detailed knowledge of its kinetic parameters. Despite recent experimental advances, the estimation of unknown parameter values from observed data is still a bottleneck for obtaining accurate simulation results. Many methods exist for parameter estimation in deterministic biochemical systems; methods for discrete stochastic systems are less well developed. Given the probabilistic nature of stochastic biochemical models, a natural approach is to choose parameter values that maximize the probability of the observed data with respect to the unknown parameters, a.k.a. the maximum likelihood parameter estimates (MLEs). MLE computation for all but the simplest models requires the simulation of many system trajectories that are consistent with experimental data. For models with unknown parameters, this presents a computational challenge, as the generation of consistent trajectories can be an extremely rare occurrence.
We have developed Monte Carlo Expectation-Maximization with Modified Cross-Entropy Method (MCEM2): an accelerated method for calculating MLEs that combines advances in rare event simulation with a computationally efficient version of the Monte Carlo expectation-maximization (MCEM) algorithm. Our method requires no prior knowledge regarding parameter values, and it automatically provides a multivariate parameter uncertainty estimate. We applied the method to five stochastic systems of increasing complexity, progressing from an analytically tractable pure-birth model to a computationally demanding model of yeast-polarization. Our results demonstrate that MCEM2 substantially accelerates MLE computation on all tested models when compared to a stand-alone version of MCEM. Additionally, we show how our method identifies parameter values for certain classes of models more accurately than two recently proposed computationally efficient methods.
This work provides a novel, accelerated version of a likelihood-based parameter estimation method that can be readily applied to stochastic biochemical systems. In addition, our results suggest opportunities for added efficiency improvements that will further enhance our ability to mechanistically simulate biological processes.
A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.
Adult male SW/ND4, CCL2-null, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2-null and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.
Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2-null mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2-null mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.
The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.
Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.
The present study provides the first demonstration that central cannabinoids modulate the antinociceptive actions of metabotropic glutamate receptors (mGluRs) on formalin-induced temporomandibular joint (TMJ) nociception. Noxious scratching behavior induced by formalin injection in the TMJ was used as a model of pain. Intracisternal injection of 30 μg of WIN 55,212-2, a non subtype selective cannabinoid receptor agonist, attenuated the number of scratches by 75% as compared with the vehicle-treated group, whereas vehicle alone or 3 or 10 μg of WIN 55,212-2 had no effect. To explore the postulated interaction between central cannabinoid receptors and mGluRs, effects of combined administration of sub-analgesic doses of WIN 55,212-2 and group II or III mGluR agonists were tested. Group II or III mGluRs agonists were administered intracisternally 10 min after intracisternal administration of WIN 55,212-2. Neither 100 nmol APDC, a group II mGluRs agonist, nor L-AP4, a group III mGluR agonist, altered nociceptive behavior when given alone but significantly inhibited the formalin-induced nociceptive behavior in the presence of a sub-threshold dose (3 μg) of WIN 55,212-2. The ED50 value of APDC or L-AP4 was significantly reduced upon co-treatment with WIN 55,212-2, than in the vehicle-treated group, highlighting the important therapeutic potential of the combined administration of group II or III mGluR agonists with cannabinoids to effectively treat inflammatory pain associated with the TMJ. Potentiating effects of group II or III mGluRs agonists will likely permit administration of cannabinoids at doses that do not achieve significant accumulation to produce undesirable motor dysfunction.
Antinociception; Cannabinoid; Formalin; mGluRs; TMJ
Chronic cough is associated with increased sensitivity to inhaled capsaicin, and both tachykinins and their receptors play important roles in the cough reflex. However, associations between polymorphisms of the tachykinin receptor genes and cough sensitivity in patients with non‐productive chronic cough have not been reported.
Direct sequencing was used to identify single nucleotide polymorphisms (SNPs) in the genes for the neurokinin‐1 and neurokinin‐2 receptors (NK‐1R and NK‐2R, respectively). Informative non‐synonymous SNPs were scored using the single base extension method for 312 patients with chronic cough and for 100 age matched healthy controls. The cough response to capsaicin was recorded for 312 patients with chronic cough, and the potential genetic association between cough sensitivity to capsaicin and the NK‐1R and NK‐2R genotypes was evaluated.
Two informative SNPs were identified in NK‐2R (Gly231Glu and Arg375His), whereas no informative SNP was found in NK‐1R. After adjusting for atopy, sex, age, and smoking, the prevalence of enhanced cough sensitivity to capsaicin was higher in the chronic cough patients with the 231Glu allele (p = 0.004; OR 1.69 (95% CI 1.18 to 2.42)) and the 231Glu_375Arg haplotype (p = 0.003; OR 1.71 (95% CI 1.20 to 2.24)]. Moreover, the lowest capsaicin concentration to cause five consecutive coughs (C5) was significantly lower in patients with 231Glu (mean (SD) 44.1 (53.2) v 60.9 (55.8) μM/l, p = 0.04) and those with 231Glu_375Arg (43.2 (52.7) v 69.6 (52.0) μM/l, p = 0.03).
The results of this study suggest that NK‐2R gene polymorphisms are involved in the enhanced cough sensitivity to capsaicin of patients with chronic cough.
chronic cough; neurokinin receptors; polymorphism; capsaicin sensitivity
Background and aim: It is known that bile acids can induce mucosal injury, stimulate cell proliferation, and promote tumorigenesis. A large body of genetic and biochemical evidence indicate that the biosynthetic pathway of prostaglandin E2 (PGE2) may play an important role in human and rodent tumours. Therefore, we examined the expression pattern of cyclooxygenase 1 (COX-1), COX-2, and microsomal prostaglandin E synthase 1 (mPGES-1), as well as EP receptor subtypes in rat oesophageal lesions induced by duodenal contents reflux.
Methods: Oesophagoduodenal anastomosis was performed in rats to induce duodenal contents reflux. We examined histological changes and expression of COX-1, COX-2, mPGES-1, and EP receptor subtypes in the oesophagus by immunohistochemistry and reverse transcription-polymerase chain reaction.
Results: Normal control oesophageal tissues showed COX-1 expression in subepithelial stromal cells, including endothelial cells and muscular cells, and did not reveal expression of COX-2 or mPGES-1. In the case of squamous cell lesions, immunoreactivity of COX-1 was similar to that of normal lesions, and COX-2 was maximally expressed around the vascular papillae of tissues showing dysplasia and surrounding epithelial layer and basal layer. mPGES-1 was highly expressed in stromal cells with COX-2 expression. In the case of Barrett’s oesophagus, COX-2 and mPGES-1 were predominantly in subepithelial stromal cells. mRNA levels of COX-2, mPGES-1, EP2, EP3, and EP4 were higher in the experimental groups than in controls.
Conclusions: We suggest that the biosynthetic pathway of PGE2 may play an important role in oesophageal squamous cell dysplasia and glandular metaplasia induced by duodenal contents reflux.
cyclooxygenase 2; microsomal prostaglandin E synthase 1; EP receptors; squamous cell dysplasia; Barrett’s oesophagus
The apolipoprotein E (APOE) epsilon4 allele is a known risk factor for the development of Alzheimer's disease, however, an association of the APOE genotype with schizophrenia is controversial. We investigated the association in 60 Korean schizophrenic patients and 60 healthy controls. APOE genotypes were identified by reverse hybridization-based line probe assay. There were significant differences in the distribution of APOE genotypes between schizophrenic patients and controls. APOE epsilon2 and epsilon3 allele frequencies in schizophrenic patients were significantly different from those in controls. Our results suggest that APOE alleles seem to be operative in the pathogenesis of schizophrenic disorders.
Papillary immature metaplasia (PIM) is a distinctive exophytic lesion of the uterine cervix and shares some histologic and cytologic features with ordinary squamous metaplasia (SM), atypical immature squamous metaplasia (AIM), high-grade squamous intraepithelial neoplasia (HSIL) and papillary squamous cell carcinoma (PSC). PIM has been suggested to be a subset of condyloma associated with low-risk type human papilloma virus (HPV), however, the etiologic role of HPV and biologic behavior of the disease are still elusive. We compared the clinical and histopathological findings, immunohistochemical expression of Ki-67 and p53 protein, and HPV typing of 5 cases of PIM with SM (n=9), HSIL (n=6), and PSC (n=4) to know the helpful features for the differential diagnosis. Histologically, all 5 cases showed a papillary proliferation of immature metaplastic cells involving the proximal transformation zone and endocervix. On HPV typing by polymerase chain reaction-restriction fragment length polymorphism, 2 out of 5 PIM were confirmed to have HPV 6 or HPV 11, while 2 out of 4 PSC were proved having HPV 31 and HPV 16 each. Ki-67 labeling index and mitotic index of PIM were significantly lower than those of HSIL or PSC. There were no significant differences of Ki-67 labeling index and mitotic index between PIM and SM. The expression of p53 varied among the groups and thus it was not helpful for the differential diagnosis.
We report a case of renin-secreting juxtaglomerular cell tumor which developed in a hypertensive 47-yr-old Korean man. Presumptive clinical diagnosis was made before surgery based on the high level of plasma renin and the radiologic evidence of renal mass. Grossly, a round, bulging, well-encapsulated mass of 3 x 3 cm was located in the mid-portion of the right kidney. On microscopic examination, the tumor was composed of ovoid to polyhedral cells with bland nuclei, indistinct nucleoli and light eosinophilic cytoplasm. The immunostaining for renin showed strong positivity in the cytoplasm of tumor cells. The characteristic rhomboid shaped renin protogranules were observed in ultrastructural analysis.
Citrus red mite (CRM) is known as the most common sensitizing allergen in subjects with asthma and rhinitis working on citrus farms. The aim of this study is to evaluate the role of specific IgG1 (slgG1) and specific IgG4 (slgG4) to CRM in citrus farmers. Questionnaire survey and skin prick test including CRM antigen was done by 136 workers. Specific IgE (slgE), slgG1 and slgG4 to CRM were detected by enzyme-linked immunosorbent assay (ELISA). CRM-sensitive-asthma was diagnosed upon presence of asthmatic symptoms by questionnaire, airway hyperresponsiveness to methacholine and slgE to CRM. CRM-sensitive rhinitis was diagnosed upon presence of rhinitis symptoms and slgE to CRM. Eleven (8.1%) had CRM-sensitive asthma and 25 (18.4%) had CRM-sensitive rhinitis. Significant association was noted between presence of asthmatic symptoms and slgE or slgG4 (p<0.05, respectively), while no significant association was noted in slgG1 (p>0.05). Significant association was noted in the prevalence between slgG4 and slgE (p<0.05), while no significant association was noted between slgG1 and slgG4 or slgE (p<0.05, respectively). There was a significant correlation between slgE and slgG4 level (r=0.39, p<0.05). These findings suggest that the presence of slgG1 to CRM is response to CRM exposure, and further studies will be needed to evaluate the role of slgG4.
Fibrosing cholestatic hepatitis is an aggressive and usually fatal form of viral hepatitis in immunosuppressed patients. We report three cases of fibrosing cholestatic hepatitis in various clinical situations. Case 1 was a 50-year-old man who underwent a liver transplant for hepatitis B virus (HBV)-associated liver cirrhosis. Two and a half years after the transplant, he complained of fever and jaundice, and liver enzymes were slightly elevated. Serum HBsAg was positive. Case 2 was a 30-year-old man in an immunosuppressed state after chemotherapy for acute lymphoblastic leukemia. He was a HBV carrier. Liver enzymes and total bilirubin were markedly elevated. Case 3 was a 50-year-old man who underwent renal transplantation as a known HBV carrier. One year after the transplant, jaundice developed abruptly, but liver enzymes were not significantly elevated. Microscopically lobules were markedly disarrayed, showing ballooning degeneration of hepatocytes, prominent pericellular fibrosis, and marked canalicular or intracytoplasmic cholestasis. Portal inflammation was mild, but interphase activity was definite and cholangiolar proliferation was prominent. Hepatocytes were diffusely positive for HBsAg and HBcAg in various patterns. Patients died of liver failure within 1 to 3 months after liver biopsy in spite of anti-viral treatment.
To compare the mediator releasability between atopic and nonatopic asthmatics, we measured basophil histamine releasability (BaHR) using a calcium-ionophore A23187 and anti-IgE in 137 subjects who were treated at Seoul National University Hospital. Subjects were categorized into atopic (group AA, n=77) or nonatopic asthmatics (group NA, n=32), or normal controls (group NC, n=28). Serum total IgE levels were determined and correlation with BaHR was assessed. Anti-IgE-induced maximal BaHR in groups AA, NA, and NC was 41.0+/-3.2, 23.1+/-4.5, and 16.8+/-3.8, respectively (mean+/-SE, %). Anti-IgE-induced BaHR in group AA was significantly higher than that in groups NA and NC (p<0.05). Calcium ionophore A23187-induced maximal BaHR was 43.1+/-2.8, 40.8+/-4.4, and 50.5+/-5.2, respectively (mean+/-SE, %), and there was no significant difference among the groups. Serum total IgE level correlated significantly with anti-IgE-induced maximal BaHR (r=0.281, p<0.01) but not with that induced by calcium ionophore A23187. In conclusion, IgE receptor-related BaHR is higher in atopic asthmatics than in nonatopic asthmatics, and this increased BaHR in atopics is significantly associated with increased serum total IgE level.
Nuclear DNA content was analysed by means of flow cytometric measurements in 103 patients with gastric carcinomas, using paraffin-embedded archival tissue. DNA aneuploidy was found in 40 cases (38.8%). The mean DNA index of aneuploid tumors was 1.45(range 1.13 to 2.37). No significant association between ploidy and either age, sex, tumor location, size, stage, growth pattern, or histologic type was found. However, the incidence of aneuploidy was higher in high grade carcinomas than in low grade carcinomas; the incidence of aneuploidy was 10%, 68.8%, and 45.8% for Grade II, III, and IV carcinomas, respectively, as compared with Grade I carcinomas which were all diploid. On statistical analysis, Abnormal cellular DNA content was significantly correlated with high histologic grade (P < 0.005). Patients with aneuploid cancer (39.2%) had a poorer prognosis than those with diploid cancer (70.0%) based on (P < 0.01). The 2-year survival rate for advanced gastric carcinoma. Therefore, DNA ploidy might be a useful prognostic factor in cases of advanced gastric cancer.
Intravesical bacillus Calmette-Guerin (BCG) administration has been used as an adjuvant therapy after transurethral resection for superficial bladder cancer, but the exact mechanisms of its antitumor activity are not yet known. The aim of this study was to characterize the immunologic aspects of antitumor activity of BCG using an animal model. C3H/He inbred mice and murine bladder tumor cell line, MBT-2 were used. The changes in immune cells such as helper T cells, suppressor T cells, macrophages and natural killer cells in the bladder and spleen were analysed by immunohistochemical method in intravesical BCG instilled in normal bladder, MBT-2 implanted after electrocauterization of the bladder mucosa and MBT-2 implanted and intravesical BCG treated group. The changes in natural killer cell activity of the splenocytes and peritoneal lymphocytes were evaluated using 51chromium release assay at regular time intervals following intraperitoneal BCG instillation. The prophylactic anticancer effect was evaluated by observing the tumor growth in the intravesically BCG treated group after intravesical MBT-2 implantation. In immunohistochemical examination, a remarkable infiltration of macrophage and helper T cell was observed in the lamina propria of the bladder, and the helper and suppressor T cells ratio (Th/Ts ratio) was increased after intravesical BCG therapy. In 51chromium release assay, enhanced natural killer cell activity of the splenocytes and peritoneal lymphocytes was observed after intraperitoneal BCG inoculation. The growth of implanted tumor was suppressed following intravesical instillation of BCG. These results suggest that the antitumor activity of BCG is not related to the simple inflammatory reaction but to the local and systemic immune response in which helper T lymphocytes and mononuclear cells play an important role.
To study the characteristics and histogenesis of the malignant lymphomas derived from the gastrointestinal mucosa, histologic and immunohistochemical analyses were performed on a series of 28 malignant lymphomas of the gastrointestinal tract. By cytomorphologic classification, there were two small lymphocytic lymphomas, one small cleaved cell lymphoma, two mixed small cleaved and large cell lymphomas, 17 large cell lymphomas, one small noncleaved cell lymphoma, three immunoblastic lymphomas, and two lymphoblastic lymphomas. This distribution of histologic types was compatible with that of nodal lymphoma. The lymphomas with poor prognostic histology (23 cases) outnumbered those with favorable prognosis (five cases). Three of 28 cases (one in the stomach and two in the small intestine) had cytologic features consistent with centrocytoid cell lymphoma of the mucosa associated lymphoid tissue and were large cell lymphomas. Immunophenotypically, 23 cases expressed B-cell markers (82.1%) and three cases reacted with T-cell markers. Two cases did not react with either T-cell or B-cell markers. True histiocytic lymphomas were not identified. Gastric lymphomas (nine cases) and colorectal lymphomas (three cases) were of B-lymphocyte origin whereas T-cell lymphomas were noted in the small intestine (two cases) and ileocecal region (one case). Three cases of centrocytoid lymphoma were of B-lymphocyte origin. Histologically B-cell lineage lymphomas were evenly distributed on various histologic subtypes but all T-lineage lymphomas belonged to the large cell type. The two cases with undetermined phenotype were lymphoblastic lymphomas histologically. This study showed that the primary GIT lymphomas, mostly of B-cell lineage, were not cytomorphologically distinctive from the nodal lymphomas.(ABSTRACT TRUNCATED AT 250 WORDS)
sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B. subtilis and Escherichia coli. However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable. It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor. We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus. We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F). The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable. By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein. When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.
To assess the efficacy and safety of once-daily lixisenatide versus placebo in Asian patients with type 2 diabetes insufficiently controlled on basal insulin ± sulfonylurea.
In this 24-week, randomized, double-blind, placebo-controlled, parallel-group, multicentre study, participants (mean baseline HbA1c 8.53%) from Japan, Republic of Korea, Taiwan and the Philippines received lixisenatide (n = 154) or placebo (n = 157) in a stepwise dose increase to 20 µg once daily. The primary endpoint was HbA1c change from baseline to week 24.
Once-daily lixisenatide significantly improved HbA1c versus placebo (LS mean difference vs. placebo = −0.88% [95%CI= −1.116, −0.650]; p < 0.0001), and allowed more patients to achieve HbA1c <7.0% (35.6 vs. 5.2%) and ≤6.5% (17.8 vs. 1.3%). Lixisenatide also significantly improved 2-h postprandial plasma glucose and glucose excursion, average 7-point self-monitored blood glucose and fasting plasma glucose. Lixisenatide was well tolerated; 86% of patients on lixisenatide completed the study versus 92% on placebo. Ten (6.5%) lixisenatide and 9 (5.7%) placebo patients experienced serious adverse events. More lixisenatide patients [14 (9.1%)] discontinued for adverse events versus placebo [5 (3.2%)], mainly with gastrointestinal causes. Nausea and vomiting were reported in 39.6 and 18.2% of patients on lixisenatide versus 4.5 and 1.9% on placebo. Symptomatic hypoglycaemia was more frequent with lixisenatide (42.9%) versus placebo (23.6%), but was similar between groups (32.6 vs. 28.3%, respectively), in those not receiving sulfonylureas. No severe hypoglycaemia was reported.
In an Asian type 2 diabetes population insufficiently controlled by basal insulin ± sulfonylurea, once-daily lixisenatide significantly improved glycaemic control, with a pronounced postprandial effect, and was well tolerated.
Asian patients; basal insulin; GLP-1; lixisenatide; type 2 diabetes
Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.