Enantioselective analysis of lansoprazole in rat plasma by LC-MS/MS: Application to a stereoselective pharmacokinetic study.

A rapid, sensitive and enantioselective method was developed and fully validated for the separation and determination of lansoprazole enantiomers in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes and the internal standard (esomeprazole) were both extracted from plasma samples by liquid-liquid extraction with diethyl ether-dichloromethane (70:30; v/v). Satisfactory resolution (Rs = 2.0) was achieved within 7.3 min on a Chiralpak ID column (250 × 4.6 mm, 5 µm) employing acetonitrile-water (60:40, v/v) as the mobile phase at a flow rate of 0.6 mL/min. The acquisition of mass spectrometric data was performed in the multiple reaction monitoring mode coupled with a positive electrospray ionization source. A comprehensive validation of this method was rigorously conducted over the concentration range of 1.00-500.0 ng/mL for both enantiomers. All of the validation data demonstrated that the desirable linearity, sensitivity, accuracy, precision, recovery and stability were attained from the proposed approach. The established method was successfully applied to a stereoselective pharmacokinetic study of lansoprazole enantiomers in rat plasma after oral administration of 3 mg/kg racemic lansoprazole or dexlansoprazole. No chiral inversion was observed during the experimental procedure.

The Efficacy and Safety of Once-daily Fluticasone Furoate/Umeclidinium/Vilanterol Versus Twice-daily Budesonide/Formoterol in a Subgroup of Patients from China with Symptomatic COPD at Risk of Exacerbations (FULFIL Trial).

The FULFIL study evaluated once-daily fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100 µg/62.5 µg/25 µg versus twice-daily budesonide/formoterol (BUD/FOR) 400 µg/12 µg in patients with symptomatic COPD at risk of exacerbations. FULFIL demonstrated clinically meaningful and statistically significant improvements at Week 24 in trough forced expiratory volume in 1 second (FEV1), St George's Respiratory Questionnaire (SGRQ) Total scores and reduced exacerbation frequency. Predefined analyses were performed to evaluate treatment effects in a subgroup of patients recruited in China (China subgroup; FF/UMEC/VI, n = 32; BUD/FOR, n = 29). Analyses included treatment by region (China versus non-China) to allow estimated treatment effects in patients from China to be compared with those of the non-China subgroup and the overall FULFIL intent-to-treat (ITT) population. In the China subgroup at Week 24: the mean change from baseline in trough FEV1 was 125 mL (95% confidence interval [CI] 36, 214) for FF/UMEC/VI and -70 mL (95% CI -163, 23) BUD/FOR (between-treatment difference: 195 mL [95% CI 67, 323]; p = 0.003) and in SGRQ Total score was -5.6 units (95% CI -10.5, -0.7) and -0.3 units (95% CI -5.4, 4.7), respectively (between-treatment difference: -5.3 [95% CI -12.3, 1.7]; p = 0.140). Fewer moderate/severe exacerbations occurred with FF/UMEC/VI than BUD/FOR (16% and 28%, respectively). The overall incidence of adverse events was similar between arms (FF/UMEC/VI: 38%; BUD/FOR: 31%). This prespecified subgroup analysis of patients recruited in China to FULFIL demonstrated comparable efficacy and safety to that observed in the non-China and in the overall ITT populations, for FF/UMEC/VI versus BUD/FOR.

Enantioselective separation and determination of miconazole in rat plasma by chiral LC-MS/MS: application in a stereoselective pharmacokinetic study.

Miconazole has one chiral center, and consists of two enantiomers. In this study, a novel chiral liquid chromatography-tandem mass spectrometry method was developed for enantioselective separation and determination of miconazole in rat plasma. For the first time, the enantioselective pharmacokinetics of miconazole was investigated by the current method. Firstly, attempts were made to separate the enantiomers in reversed-phase mode with a mobile phase that was mass spectrometry compatible. Baseline separation was achieved on a Chiralpak IC column with a mobile phase composed of acetonitrile and aqueous ammonium hydrogen carbonate (5 mM; 80:20, v/v). Data were acquired in multiple reaction monitoring mode with positive electrospray ionization by triple-quadrupole mass spectrometry. Then, overall method validation regarding the linearity, accuracy, precision, extraction recovery, matrix effect, and stability of each enantiomer was performed, and acceptable results were obtained for all of these. Finally, the method developed was applied in an enantioselective pharmacokinetic study of miconazole enantiomers in rats after oral administration of racemic miconazole at doses of 5 and 10 mg/kg. The results demonstrated that (-)-(R)-miconazole had a higher concentration than (+)-(S)-miconazole in plasma, with a ratio of 1.3-1.7 for both doses. This is the first experimental evidence of enantioselective behavior of miconazole in vivo, and provides a reference for clinical practice and encourages further research into miconazole enantioselective metabolism and drug interactions. Graphical Abstract A stereoselective pharmacokinetic study of the miconazole enantiomers was investigated using a novel chiral liquid chromatography-tandem mass spectrometry method. Baseline separation was achieved on Chiralpak IC column, and Chiralcel OJ column was used to collect single enantiomer. A significant difference between the two enantiomers was observed in view of the plasma concentration.

Comparison of three S-ß-CDs with different degrees of substitution for the chiral separation of 12 drugs in capillary electrophoresis.

Three kinds of sulfated ß-cyclodextrin (S-ß-CD), including a single isomer, heptakis-6-sulfato-ß-cyclodextrin (HS-ß-CD), degree of substitution (DS) of 7, which was synthesized in our laboratory and another two commercialized randomly substituted mixtures, a sulfated ß-cyclodextrin with DS of 7 to 11, as well as a highly sulfated-ß-cyclodextrin with DS of 12 to 15, were used for the enantioresolution of 12 drugs (the ß-blockers, phenethylamines, and anticholinergic agents) in capillary electrophoresis. The enantioseparation under varying concentrations of S-ß-CD and background electrolyte pH were systematically investigated and compared. Based on the experimental results, the effect of the nature of S-ß-CD and analyte structure on the enantioseparation is discussed.

Carboxymethyl ß-cyclodextrin as chiral selector in capillary electrophoresis: Enantioseparation of 16 basic chiral drugs and its chiral recognition mechanism associated with drugs' structural features.

Herein we present the enantioseparation of 10 cardiovascular agents and six bronchiectasis drugs including propranolol, carteolol, metoprolol, atenolol, pindolol, esmolol, bisoprolol, bevantolol, arotinolol, sotalol, clenbuterol, procaterol, bambuterol, tranterol, salbutamol and terbutaline sulfate using carboxymethyl-ß-cyclodextrin (CM-ß-CD) as chiral selector. To our knowledge, there is no literature about using CM-ß-CD for separating carteolol, esmolol, bisoprolol, bevantolol, arotinolol, procaterol, bambuterol and tranterol. During the course of work, changes in pH, CM-ß-CD concentration, buffer type and concentration were studied in relation to chiral resolution. Excellent enantiomeric separations were obtained for all 16 compounds, especially for procaterol. An impressive resolution value, up to 17.10, was obtained. In particular, most of them achieved rapid separations within 20 min. Given the fact that enantioseparation results rely on analytes' structural characters, the possible separation mechanisms were discussed. In addition, in order to obtain faster separation for propranolol enantiomers in practical application, the effective length of capillary was innovatively shortened from 45 to 30 cm. After the validation, the method was successfully applied to the enantiomeric purity determination of propranolol in the formulation of drug substances.

A novel UPLC-MS/MS method for simultaneous determination of 10 effective constituents in the Jixingshizhen preparation.

A novel, reliable and reproducible UPLC-MS/MS method was developed and validated to determine simultaneously the 10 bioactive constituents (baicalin, baicalein, wogonoside, wogonin, luteoloside, dictamnine, fraxinellone, obacunone, geniposidic acid and glycyrrhizic acid) in Jixingshizhen (JXSZ) preparation. Briefly, chromatographic separation was achieved on a Waters BEH C18 column with gradient elution employing a mobile phase composed of acetonitrile and 0.1% formic acid at a flow rate of 0.3 mL/min. All analytes containing internal standard (verapamil) were detected without interference in the multiple reaction monitoring mode with positive electrospray ionization. Further, a comprehensive validation of the method was rigorously executed according to the guidelines of the International Conference on Harmonization and Chinese Pharmacopoeia (2015 Edition). The results indicated that the validated method afforded desired linearity, precision, accuracy, sensitivity and stability. At length, the newly established method was successfully applied to quantify the 10 effective ingredients in JXSZ granules from different production batches, indicating the proposed method in this paper was particularly preferable for the routine analysis of JXSZ preparation as well as the quality control, particularly in situations where high sample throughput and fast analytical speed are required.

Tumor-infiltrating CD8(+) T lymphocytes associated with clinical outcome in anal squamous cell carcinoma.

BACKGROUND: The prognostic significance of tumor-infiltrating CD8(+) T lymphocytes in anal squamous cell carcinoma (SCC) remains unclear. We designed the study to investigate the association between CD8(+) T cells and clinical prognosis among anal SCC patients. METHODS: The density of CD8(+) T cells was assessed by immunohistochemistry. The numbers of CD8(+) T cells were counted and their relationship with clinicopathological factors and survival was explored. RESULTS: A strong positive correlation was noted between intratumoral and peritumoral CD8(+) T cells (r = 0.77, P < 0.001). High intratumoral and peritumoral CD8(+) T cells was associated with well tumor differentiation, early-stage diagnosis, and better prognosis (P < 0.05). Better disease-free survival rates were demonstrated in patients with high CD8(+) T cell density in intratumoral nest (P = 0.01); peritumoral stroma (P = 0.004); and both in combination (P = 0.01). High peritumoral CD8(+) T cell was associated with overall survival (P = 0.025). In HIV-infected patients, high CD8(+) T cell density also had association with disease-free survival (P < 0.05). CONCLUSIONS: High tumor-infiltrating CD8(+) T cell density showed the potential to indicate a favorable effect on prognosis and survival for anal SCC patients.

Molecular Signatures for Microbe-Associated Colorectal Cancers.

Background: Genetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. Methods: A Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum ( Fn ), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice ( CPC-APC Min+/- ) and patients (FAP, Lynch Syndrome, PJS, and JPS). Results: The MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APC Min+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. Conclusions: Computational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

John Cunningham virus T-antigen expression in anal carcinoma.

BACKGROUND: Anal carcinoma is thought to be driven by human papillomavirus (HPV) infection through interrupting function of cell regulatory proteins such as p53 and pRb. John Cunningham virus (JCV) expresses a T-antigen that causes malignant transformation through development of aneuploidy and interaction with some of the same regulatory proteins as HPV. JCV T-antigen is present in brain, gastric, and colon malignancies, but has not been evaluated in anal cancers. The authors examined a cohort of anal cancers for JCV T-antigen and correlated this with clinicopathologic data. METHODS: Archived anal carcinomas were analyzed for JCV T-antigen expression. DNA from tumor and normal tissue was sequenced for JCV with viral copies determined by quantitative polymerase chain reaction and Southern blotting. HPV and microsatellite instability (MSI) status was correlated with JCV T-antigen expression. RESULTS: Of 21 cases of anal cancer (mean age 49 years, 38% female), 12 (57%) were in human immunodeficiency virus (HIV)-positive individuals. All 21 cancers expressed JCV T-antigen, including 9 HPV-negative specimens. More JCV copies were present in cancer versus surrounding normal tissue (mean 32.54 copies/µg DNA vs 2.98 copies/µg DNA, P = .0267). There was no correlation between disease stage and viral copies, nor between viral copies and HIV-positive or -negative status (28.7 vs 36.34 copies/µg DNA, respectively, P = .7804). In subset analysis, no association was found between JCV T-antigen expression and HPV or MSI status. CONCLUSIONS: Anal carcinomas uniformly express JCV T-antigen and contain more viral copies compared with surrounding normal tissue. JCV and its T-antigen oncogenic protein, presumably through interruption of cell regulatory proteins, may play a role in anal cancer pathogenesis.

The inflammatory response in transgastric surgery: gastric content leak leads to localized inflammatory response and higher adhesive disease.

BACKGROUND: Risk of gastric spillage during transgastric surgery is a potential complication of NOTES procedures. The aim of this study was to determine risk outcomes from gastric spillage in a rat survival model by measuring local and systemic inflammatory markers, adhesive disease, and morbidity. METHODS: We performed a minilaparotomy with needle aspiration of 2 ml of gastric contents mixed with 2 ml of sterile saline (study group, SG) or 4 ml of sterile saline (control group, CG) injected into the peritoneal cavity of 60 male rats. Inflammatory markers (TNFalpha, IL-6, and IL-10) were analyzed at 1, 3, 6, and 24 h postoperatively by obtaining plasma levels and peritoneal washings. At necropsy, the peritoneal cavity was examined grossly for adhesions. RESULTS: Adhesions were seen more frequently in the SG versus the CG (100% vs. 33.3%, p < 0.014). There was a significant difference in the peritoneal TNFalpha levels in the SG compared with the CG, which peaked 1 h after surgery (p < 0.02). Both peritoneal IL-6 and IL-10 levels were higher in the SG versus the CG, which peaked 3 h after surgery (p < 0.005 and p < 0.001, respectively). All peritoneal inflammatory markers returned to undetectable levels at 24 h for both groups. Plasma cytokines were undetectable at all time intervals. CONCLUSION: The inflammatory response was found to be a localized and not systemic event, with plasma cytokine levels remaining normal while peritoneal washings revealed a brisk, short-lived localized inflammatory response. There was a significantly higher rate of adhesive disease in the SG compared with the CG; this, however did not translate into a difference in apparent clinical outcome. We conclude that gastric leakage in this NOTES rodent model induces a localized inflammatory response, followed by mild to moderate adhesive disease. This may be important in human NOTES.

Development of Human-Derived Cell Culture Lines for Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is mainly caused by human papillomavirus (HPV) 6 and 11. While various adjuvant therapies have been reported, no effective therapy has been documented to universally "cure" this disease. In the era of precision medicine, it would be valuable to identify effective intervention based on drug sensitivity testing and/or molecular analysis. It is essential to be able to successfully carry out in vitro culture and expand tumor cells directly from patients to accomplish this goal. Here we report the result of successful culture of HPV-infected cell lines (success rate 70%, 9/13) that express the E6/E7 RNA transcript, using pathologic tissue biopsies from patients treated at our institution. The availability of such a system would enable ex vivo therapeutic testing and disease modeling.

Layer-by-layer self-assembly of gold nanoparticles/thiols ß-cyclodextrin coating as the stationary phase for enhanced chiral differentiation in open tubular capillary electrochromatography.

The popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. Herein, a novel, simple, and economical method for the preparation of chiral stationary phases for enantioselective open tubular capillary electrochromatography (OTCEC) was reported for the first time. This novel capillary column was fabricated through layer-by-layer self-assembly of GNPs on a 3-mercaptopropyl-trimethoxysilane (MPTMS)-modified fused-silica capillary and subsequent surface functionalization of the GNPs through self-assembly of thiols ß-cyclodextrin (SH-ß-CD). With this method, monolayer and multilayer GNPs film capillary columns were prepared and evaluated using meptazinol and its three intermediate enantiomers as templates. Consequently, the three-layer GNPs capillary column was found to exhibit favorable enantioseparation performance. Factors that influence the chiral separation resolution were examined. Under the optimized conditions, enantioselectivity was obtained for meptazinol and its three intermediate enantiomers (intermediates Ⅱ, Ⅲ and Ⅳ) in less than 20min with resolutions of 2.05, 0.36, 1.67, 1.53, respectively. The proposed column revealed adequate repeatability concerning run-to-run and day-to-day. In brief, these results confirmed the use of GNPs as the electrochromatographic support can enhance the phase ratio of OTC column in capillary electrochromatography (CEC). Then, this proposed method was well validated with good linearity (≥0.999), recovery (92.0-94.5%) and repeatability, and was successfully used for enantioseparation of meptazinol in spiked urine samples, which indicated the new column's potential usage in biological analysis.

Comparison of fluticasone propionate with budesonide administered via nebulizer: a randomized controlled trial in patients with severe persistent asthma.

BACKGROUND: This study compared the efficacy and safety of fluticasone propionate (FP) inhalation n solution with budesonide (BUD) suspension for inhalation administered via nebulizer, in Chinese adult patients with severe, persistent asthma. METHODS: This was a multicenter, randomized, active-controlled, single-blind, parallel-group study, conducted at 26 clinical sites in China. Participants were randomized 1:1 to FP nebules 1 mg twice daily or BUD 2 mg twice daily via nebulizer for 12 weeks. RESULTS: A total of 317 adult patients were randomized. The primary endpoint was mean change in morning peak expiratory flow (PEF) over weeks 1-12 from baseline, and analyzed in the ITT (n=315) and PP populations (n=283). Week 12 PEF increase from baseline was 26.7 L/min (14.1%) and 28.0 L/min (15.3%) in the ITT population, and 29.1 L/min (15.7%) and 30.1 L/min (16.2%) in the PP population, in the FP and BUD groups, respectively; all improvements were of clinical significance. Lower limits of the two-sided 95% CIs for the least squares (LS) mean treatment difference (FP minus BUD) were -12.19 L/min (ITT) and -12.95 L/min (PP), both above the pre-specified non-inferiority criteria -12.00 L/min and not clinically meaningful. There was no significant difference in the week 12 mean FEV1 increase between the FP and BUD groups (0.237 L/16.79% vs. 0.236 L/17.73%). Lower limits of the 95% CIs for LS mean treatment difference in morning PEF change from baseline over weeks 1-4 in a post hoc analysis were -10.41 and -11.96 L/min in the ITT and PP populations respectively; both above -12.00 L/min. A review of safety data indicated that rates of AEs, SAEs, and drug-related AEs were similar between two groups. CONCLUSIONS: The 12-week treatment of FP inhalation solution administered via nebulizer is safe and effectively for treating severe, persistent asthma in Chinese patients over 12 week.

Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm.

AIMS: The macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC). METHODS: AIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined. RESULTS: macroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours. CONCLUSIONS: Loss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC.

Pharmacokinetics and tolerability of inhaled umeclidinium and vilanterol alone and in combination in healthy Chinese subjects: a randomized, open-label, crossover trial.

UNLABELLED: Inhaled umeclidinium (UMEC) and the combination of inhaled UMEC with vilanterol (UMEC/VI) are approved maintenance treatments for chronic obstructive pulmonary disease in the US and EU. This was a randomized, open-label, three-period crossover, single- and repeat-dose study to assess the pharmacokinetics (PK), safety, and tolerability of inhaled UMEC/VI 62.5/25 µg (delivering 55/22 µg) and UMEC/VI 125/25 µg (delivering 113/22 µg) compared with their monotherapy components (UMEC 62.5 µg, UMEC 125 µg and, VI 25 µg [delivering 55, 113, and 22 µg, respectively]) in healthy Chinese subjects (n=20). UMEC and VI were rapidly absorbed following single and repeat dosing (time to maximum plasma concentration [tmax]: UMEC = 5 min; VI = 5 min). The median tlast was 2­4 h for UMEC and 1­2 h for VI following single doses of UMEC/VI and UMEC monotherapy (both doses). UMEC reached steady-state prior to Day 10; steady-state for VI could not be assessed. UMEC accumulation following repeat dosing was 11­34% based on Cmax and 19­59% based on area under the concentration-time curve from time zero to 2 h (AUC(0-2)). VI accumulation following repeat dosing was 25­66% based on Cmax and 17­43% based on AUC(0-2). The evidence was not sufficient to suggest that systemic exposure was substantially different between UMEC/VI combination therapy and the constituent monotherapies following single or repeat dosing. Following both single- and repeat-dose administration, the inter-subject coefficient of variation for all UMEC PK parameter estimates ranged from 12% to 165% for all treatments, indicating a wide range of variability in inhaled PK parameters. Twelve subjects experienced ≥1 adverse event (AE). Six subjects experienced ≥1 treatment-related AE; the most commonly reported treatment-related AE was chest discomfort (n=3 [15%]). No clinically important changes in vital signs or electrocardiogram parameters were reported. These data suggest that single- and repeat-dose administration of UMEC/VI combination therapy in healthy Chinese subjects did not result in substantial differences in systemic exposure compared with UMEC and VI as monotherapies. TRIAL REGISTRATION: Clinicaltrials.gov NCT01899638 NCT01899638.

Detection of multiple human papillomavirus genotypes in anal carcinoma.

UNLABELLED: Infection with human papillomavirus (HPV) is a major risk factor for development of anal squamous cell carcinoma. Despite over 100 genotypes of the virus, HPV 16 and 18 are considered pathogenic as they are seen in the majority of cervical and anal cancers. We have employed a custom microarray to examine DNA for several HPV genotypes. We aimed to determine the accuracy of our microarray in anal cancer DNA for HPV genotypes compared to the DNA sequencing gold standard. METHODS: We utilized a sensitive microarray platform to classify 37 types of mucosal HPVs including 14 known high-risk and 23 low-risk types based on cervical cancer data. We utilized DNA from pathologically confirmed cases of anal squamous cell carcinoma. All samples underwent microarray HPV genotyping and PCR analysis. RESULTS: HPV was detected in 18/20 (90%) anal cancers. HPV genotypes 16 and 18 were present in the majority of specimens, with HPV 16 being the most common. Eighty percent of anal cancers had at least two HPV types. Ten percent of cases (2/20) tested negative using our microarray; DNA sequencing confirmed the lack of presence of HPV DNA in these samples. CONCLUSIONS: Microarray technology is an accurate way to screen for various genotypes of HPV in anal cancer, with 100% correlation with genomic DNA detection of HPV. The majority of anal cancers in our study associated with pathogenic HPV 16 and/or 18. Other HPV genotypes are present simultaneously with HPV 16 and 18, and might contribute to its pathogenesis.

Relationship of EMAST and microsatellite instability among patients with rectal cancer.

BACKGROUND: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature identified in 60% of sporadic colon cancers and may be linked with heterogeneous expression of the DNA mismatch repair (MMR) protein hMSH3. Unlike microsatellite instability-high (MSI-H) in which hypermethylation of hMLH1 occurs followed by multiple susceptible gene mutations, EMAST may be associated with inflammation and subsequent relaxation of MMR function with the biological consequences not known. We evaluated the prevalence of EMAST and MSI in a population-based cohort of rectal cancers, as EMAST has not been previously determined in rectal cancers. METHODS: We analyzed 147 sporadic cases of rectal cancer using five tetranucleotide microsatellite markers and National-Cancer-Institute-recommended MSI (mononucleotide and dinucleotide) markers. EMAST and MSI determinations were made on analysis of DNA sequences of the polymerase chain reaction products and determined positive if at least two loci were found to have frame-shifted repeats upon comparison between normal and cancer samples from the same patient. We correlated EMAST data with race, gender, and tumor stage and examined the samples for lymphocyte infiltration. RESULTS: Among this cohort of patients with rectal cancer (mean age 62.2 ± 10.3 years, 36% female, 24% African American), 3/147 (2%) showed MSI (three males, two African American) and 49/147 (33%) demonstrated EMAST. Rectal tumors from African Americans were more likely to show EMAST than Caucasians (18/37, 49% vs. 27/104, 26%, p = 0.014) and were associated with advanced stage (18/29, 62% EMAST vs. 18/53, 37%, non-EMAST p = 0.02). There was no association between EMAST and gender. EMAST was more prevalent in rectal tumors that showed peri-tumoral infiltration compared to those without (30/49, 60% EMAST vs. 24/98, 25% non-EMAST, p = 0.0001). CONCLUSIONS: EMAST in rectal cancer is common and MSI is rare. EMAST is associated with African-American race and may be more commonly seen with metastatic disease. The etiology and consequences of EMAST are under investigation, but its association with immune cell infiltration suggests that inflammation may play a role for its development.

Tobacco smoking and risk of recurrence for squamous cell cancer of the anus.

OBJECTIVE: Squamous cell cancer of the anus is associated with multiple risk factors, including infection with human papillomavirus, immunosuppression, chronic inflammation, and tobacco smoking, although there is little data on these factors for the prediction of recurrent disease. Here, we evaluated the risk of recurrence and mortality of anal carcinoma in association with tobacco smoking. METHODS: We conducted a retrospective review of cases of anal carcinoma from two local hospitals. We obtained information on treatment response and cancer recurrence, as well as tobacco usage from medical records. RESULTS: We identified 64 patients with squamous cell cancer of the anus, and 34 of these (53%) had a tobacco smoking history. Current smokers had higher carcinoma recurrence rates (11/34, 32%) than non-smokers (6/30, 20%). Overall mortality was 33% (21/64), and cancer-related mortality was 23% (15/64). Smokers were more likely to die from recurrence than non-smokers, with 45% of smokers dead compared to only 20% of non-smokers by 5 years after treatment. CONCLUSION: Tobacco smoking appears to be associated with anal carcinoma disease recurrence, and is related to increased mortality. This data suggests that patients should be cautioned about tobacco smoking once a diagnosis of anal carcinoma is made in attempt to improve their long-term outcome.

TNF-alpha downregulates the leukotriene C4 synthase gene in mononuclear phagocytes.

We studied the effect of tumor necrosis factor (TNF)-alpha exposure on cysteinyl leukotriene (LT) synthesis by cells of monocyte/macrophage lineage. TNF-alpha conditioning of monocytic THP-1 cells and primary human monocytes resulted in a decreased capacity for LTC(4) release. TNF-alpha exposure (for 16-24 h) decreased LTC(4) synthase mRNA in THP-1 cells, primary mouse bone marrow-derived macrophages, and eosinophilic AML14.3D10 cells. TNF-alpha downregulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with downregulation observed as early as 4 h. The effect of TNF-alpha on LTC(4) synthase mRNA expression was mediated via the MEK/ERK pathway, but not via cyclooxygenase or nitric oxide synthase pathways. Conditioning of actinomycin D-treated cells with TNF-alpha did not accelerate degradation of LTC(4) synthase mRNA. TNF-alpha produced sustained activation of p50 and p65, which were previously reported by our group to decrease LTC(4) synthase promoter activity. In transiently transfected THP-1 cells, TNF-alpha decreased promoter activity via an element located within the first 620 bp of the promoter. We conclude that TNF-alpha exposure downregulates the synthetic capacity for cysteinyl LT release and LTC(4) synthase gene expression in monocytes/macrophages via a transcriptional mechanism.