The role of the estrogen receptor β (ERβ) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERα and ERβ knockout (αERKO and βERKO) mice were injected with azoxymethane (AOM), a colon chemical carcinogen. Fourteen weeks after AOM exposure the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. βERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 ± 2.5) as compared to αERKO (3.4 ± 1.0) and WT (4.6 ± 1.0) mice. The colons in several βERKO mice had increased thickness and loss of normal morphology. It has been reported that ERβ plays a role in maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of βERKO mice. The presence of mucin depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, in order to surpass the limitations with ACF scoring, we performed alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the βERKO mice colons (38.3 ± 4.0; P < 0.001), comparing to WT and αERKO mice (6.6 ± 1.5, and 10.0 ± 1.9, respectively), and served to confirm the ACF results. Together these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERβ in colon carcinogenesis.
Estrogen receptors; colon cancer; aberrant crypt foci; mucin depleted foci
Many chronic inflammatory diseases are associated with increased risk of developing cancer. In the colon, strong support for a link between chronic inflammation and cancer extends, in part, from population-based studies of persons with inflammatory bowel disease (IBD). Patients with IBD are at increased risk of developing colorectal cancer (CRC). The general consensus is that IBD results from the combined effects of genetics and environment factors known to affect the immune system. Vitamin D, an important regulator of the immune system, has been linked to IBD. Despite the strong potential reported for 1,25-dihydroxyvitamin D (1,25-OH)2D), its strong effects on calcium metabolism limits its potential value. Recently, less active vitamin D metabolites, cholecalciferol and 25-hydroxyvitamin D (25(OH)D), have gained considerable attention as promising agents against IBD-related colon cancer. Yet, their anti-proliferative properties and mechanism of action remain to be better defined. We present several signaling pathways commonly regulated by vitamin D compounds and highlight their regulation on TLR4. The efficacy of 25(OH)D and 1α-hydroxyviatmin D5 are defined using the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced IBD-related colon carcinogenesis model. In summary, vitamin D supplementation may provide a cost-effective approach to reduce IBD related colon cancer.
Vitamin D; inflammation; colon cancer
This study was to investigate the association between tCho and the clinical characteristics and biomarker status of breast cancer. Sixty-two patients with breast cancer which was 1.5 cm or larger in size on MR images were studied. The tCho concentration was correlated with the MR imaging features, the contrast enhancement kinetics, clinical variables, and biomarkers. Pair-wise two-tailed Spearman’s non-parametric test was used for the statistical analysis. The tCho was higher in high grade than moderate/low grade tumor (p=0.04) and in tumors with higher Ktrans and kep (p<0.001 for both). The association of tCho with age (p=0.05) and triple negative biomarker (p=0.09) approached significance. tCho was not detected in 17 patients, including 15 invasive ductal cancer and 2 infiltrating lobular cancer. Fifteen of the 17 patients had moderate to low grade cancers, and 11 had HER-2 negative cancer, suggesting these two factors might lead to false negative choline. Higher tCho in high grade tumors and tumors with higher Ktrans and kep indicates choline is associated with cell proliferation and tumor angiogenesis. Higher choline level in younger women may be due to their more aggressive tumor type. The results presented here may aid in better interpretation of 1H MRS for diagnosis of breast lesions.
We describe the changes in the prevalence of overweight and obesity in 4606 students that applied to a Mexican University during 1994 to 2008. The mean (± standard deviation [SD]) age was 17.7 ± 1.2 years-old. Progressive and significant increments of bodyweight (female [F] = 2.6, P = 0.03), body mass index (BMI) (F = 4.4, P = 0.001), and waist circumference (F = 30.08, P < 0.0001) in women, and bodyweight (male [M] = 8.9, P < 0.001), BMI (M = 10.4, P < 0.001), and waist circumference (M = 13.01, P < 0.001) in men were observed. A significant increment (P < 0.05) in the prevalence of overweight since 1994 (n = 87, 12.1%) throughout 1997 (n = 102, 14.1%), 1998 (n = 133, 18.4%), 1999 (n = 1993, 26.8%), and 2008 (n = 206, 19.9%) was documented. Similarly, the prevalence of obesity had a significant increment in all students evaluated (P < 0.0001) since 1994 (n = 29, 13.2%) through 1997 (n = 11, 5.0%), 1998 (n = 45, 20.5%), 1999 (n = 53, 24.1%), and 2008 (n = 82, 37.3%). The increment was significant in both women (P = 0.02) and men (P < 0.001). In summary, we report a significant increment in the prevalence of overweight and obesity in Mexican students living in an urban setting over a time period of 14 years.
body mass index; adolescents; weight problems; obese
Clinical and experimental studies showing lung damage associated with biologically active neutrophil derivatives suggests the possibility that intrauterine neutrophil activation may predispose to the development of pulmonary haemorrhage in extremely low birthweight infants early after birth.
To assess neutrophil functional activity in cord blood from extremely low birthweight infants who subsequently developed severe pulmonary haemorrhage.
Neutrophil functional activity was evaluated in the cord blood of preterm neonates (gestational age <28 weeks and weight <800 g) who developed pulmonary haemorrhage (n = 6) and in controls who did not (n = 6). Infants with congenital abnormalities and noteworthy maternal complications such as diabetes, pre‐eclampsia, haemorrhagic disorders, and connective tissue disease were excluded. Neutrophils isolated from cord blood immediately after delivery were tested to evaluate neutrophil chemotaxis, random motility, and chemiluminescence. Standard methodology was used.
Chemotaxis and chemiluminescence of neutrophils from cord blood of infants with pulmonary haemorrhage was higher than in the controls (34.8 (2.0) v 28.6 (0.8) μm, p<0.001, and 95.8 (8.5) v 73.2 (3.6) cpm ×103, p<0.001 respectively). Random motility of cord blood neutrophils was comparable in the two groups (28.6 (1.2) v 25.3 (1.2) μm, p<0.09).
Intrauterine induction of chemotactic activity and metabolic oxygenation of neutrophils may predispose to the development of pulmonary haemorrhage in extremely low birthweight infants with respiratory distress early after birth.
preterm; neutrophil functional activity; pulmonary haemorrhage; lungs
To identify in a prospective observational study the cardiac structural and functional abnormalities and mortality in patients with end stage renal disease (ESRD) with a raised cardiac troponin T (cTnT) concentration.
126 renal transplant candidates were studied over a two year period. Clinical, biochemical, echocardiographic, coronary angiographic, and dobutamine stress echocardiographic (DSE) data were examined in comparison with cTnT concentrations dichotomised at cut off concentrations of < 0.04 μg/l and < 0.10 μg/l.
Left ventricular (LV) size and filling pressure were significantly raised and LV systolic and diastolic function parameters significantly impaired in patients with raised cTnT, irrespective of the cut off concentration. The proportions of patients with diabetes and on dialysis were higher in both groups with raised cTnT. With a cut off cTnT concentration of 0.04 μg/l but not 0.10 μg/l, significantly more patients had severe coronary artery disease and a positive DSE result. The total ischaemic burden during DSE was similar in cTnT positive and negative patients, irrespective of the cut off concentration used. LV end systolic diameter index and E:Ea ratio were independent predictors of cTnT rises ⩾ 0.04 μg/l and ⩾ 0.10 μg/l, respectively. Diabetes was independently associated with cTnT at both cut off concentrations. Mortality was higher in all patients with raised cTnT.
Patients with ESRD with raised cTnT concentrations have increased mortality. Raised concentrations are strongly associated with diabetes, LV dilatation, and impaired LV systolic and diastolic function, but not with severe coronary artery disease.
end stage renal disease; cardiac troponin T; renal failure; cardiac structure
A simple, specific, accurate and stability indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of atorvastatin calcium and amlodipine besylate in tablet dosage forms. A phenomenex Gemini C-18, 5 μm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing 0.02 M potassium dihydrogen phosphate:acetonitrile:methanol (30:10:60, v/v/v) adjusted to pH 4 using ortho phosphoric acid was used. The flow rate was 1.0 ml/min and effluents were monitored at 240 nm. The retention times of atorvastatin calcium and amlodipine besylate were 11.6 min and 4.5 min, respectively. The calibration curves were linear in the concentration range of 0.08-20 μg/ml for atorvastatin calcium and 0.1-20 μg/ml for amlodipine besylate. Atorvastatin calcium and amlodipine besylate stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. The proposed method was validated and successfully applied to the estimation of atorvastatin calcium and amlodipine besylate in combined tablet dosage forms.
Atorvastatin calcium; amlodipine besylate; degradation; reversed phase liquid chromatography; stability indicating; validation
A simple, specific, accurate and stability indicating reversed phase liquid chromatographic method was developed for the determination of nebivolol hydrochloride in tablet dosage forms. A phenomenex Gemini C-18, 5 μm column having 250×4.6 mm i.d., with mobile phase containing methanol: acetonitrile: 0.02 M potassium dihydrogen phosphate (60:30:10, v/v/v; pH 4.0) was used. The retention time of nebivolol hydrochloride was 2.6 min. The linearity for nebivolol hydrochloride was in the range of 0.2-10 μg/ml. The recovery was found to be in the range of 98.68-100.86%. The detection limit and quantification limit were found to be 0.06 μg/ml and 0.2 μg/ml, respectively. Nebivolol stock solutions were subjected to acid, alkali and neutral hydrolysis, chemical oxidation and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. The proposed method was validated and successfully applied to the estimation of nebivolol hydrochloride in tablet formulations.
Nebivolol hydrochloride (NEB); degradation; reversed phase liquid chromatography; stability indicating; validation
A simple, specific, accurate, and precise reverse phase liquid chromatographic method was developed and validated for the estimation of risperidone in tablet dosage forms. A Phenomenex Gemini C-18, 5 μm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing methanol: acetonitrile: 50 mM potassium dihydrogen orthophosphate (80:10:10 v/v) was used. The flow rate was 1.3 ml/min and effluents were monitored at 234 nm. Clozapine was used as an internal standard. The retention time of risperidone and clozapine were 2.5 min and 3.3 min, respectively. The method was validated for linearity, accuracy, precision, specificity, limit of quantification, limit of detection, robustness and stability. The limit of detection and limit of quantification for estimation of risperidone was found to be 500 ng/ml and 990 ng/ml, respectively. Recovery of risperidone was found to be in the range of 99.02-101.68%. Proposed method was successfully applied for the quantitative determination of risperidone in tablet formulations.
Reverse phase liquid chromatography; risperidone; RP-HPLC; specificity; validation
A simple, specific, accurate, and precise reverse phase high performance liquid chromatographic method was developed and validated for the estimation of venlafaxine hydrochloride in tablet dosage forms. A Phenomenex Gemini C-18, 5 μm column having 250 × 4.6 mm i.d. in isocratic mode, with mobile phase containing methanol: 0.05 M potassium dihydrogen orthophosphate (70:30, v/v; pH 6.2) was used. The flow rate was 1.0 ml/min and effluents were monitored at 226 nm. Carbamazepine was used as an internal standard. The retention time of venlafaxine hydrochloride and carbamazepine were 3.7 min and 5.3 min, respectively. The method was validated for specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness and solution stability. Limit of detection and limit of quantification for estimation of venlafaxine hydrochloride were found to be 100 ng/ml and 300 ng/ml, respectively. Recoveries of venlafaxine hydrochloride in tablet formulations were found to be in the range of 99.02-101.68%. Proposed method was successfully applied for the quantitative determination of venlafaxine hydrochloride in tablet dosage forms.
Venlafaxine hydrochloride; carbamazepine; antidepressant; RP-HPLC; validation
We describe a simple technique that utilises local skin flaps of closure of a large tracheocutaneous-fistula with a adequate support to the tracheal wall by double breasting of local flaps, thus avoiding the post-operative respiratory problems that can occur due to a single flap projecting into the tracheal lumen during inspiration.
This technique avoids the complications associated with primary closure of fistulae & also the morbidity of waiting for secondary closure to occur. It also precludes the need for a more extensive operation in the form of muscle flap rotation. The fistulous tract does not need to be excised but forms a component of the repair.
invasion; MAPK; MMP-9; PD 098059
Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.
The therapeutic efficacy of liposomal clofazimine (L-CLF) against Mycobacterium avium complex (MAC) was evaluated in the acute and chronic infection models of the beige mouse (C57BL/6J bgj bgj). The maximum tolerated dose of L-CLF was inversely proportional to the infection level. L-CLF showed higher antibacterial activity than free clofazimine. Treatment with 25 mg of L-CLF per kg of body weight (intravenously) was started at days 1, 8, 15, and 22 postinfection and was studied at three levels of MAC infection (10(4), 10(5), and 10(6) bacilli/mouse). L-CLF treatment caused a significant (P < 0.05 to 0.001) reduction in the numbers of viable bacteria in lung, liver, and spleen at all infection levels, irrespective of time of treatment. However, the best results were obtained when an already established infection was treated (day 22). The organ-related differences in response to the treatment were also affected by the level of infection. A marked reduction in the numbers of CFU was observed in the lungs of mice with lower infection levels, whereas liver and spleen were treated more efficiently at higher infection levels. These studies might help in evaluations of host responses to therapy.
The activities of free and liposomal resorcinomycin A against Mycobacterium avium-Mycobacterium intracellulare complex (MAC) grown in broth and in murine peritoneal macrophages were evaluated. Liposomal resorcinomycin A was composed of dimyristoyl phosphatidylcholine and phosphatidylinositol at a molar ratio of 9:1. Both free resorcinomycin A and liposomal resorcinomycin A showed no toxicity to macrophages at concentrations up to 50 micrograms/ml, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Minimal inhibitory concentrations of free resorcinomycin A and liposomal resorcinomycin A in broth were 6 and 12 micrograms/ml, respectively, as determined by the MTT colorimetric microassay. In macrophages, liposomal resorcinomycin A caused significantly higher intramacrophage antimycobacterial activity than the free form of the drug. At doses ranging from 6 to 50 micrograms/ml, liposomal resorcinomycin A caused 50 to 93% MAC growth inhibition, respectively (as determined by CFU), while free resorcinomycin A was associated with 33 to 62% MAC growth inhibition, respectively, 3 days after drug treatment. In addition, antimycobacterial activity of liposomal resorcinomycin A in macrophages was maintained 7 days after treatment, whereas the activity of free resorcinomycin A was reduced to negligible 3 days after treatment. In summary, liposome encapsulation of resorcinomycin A resulted in significant enhancement of antibacterial activity against intramacrophagic MAC infection.
Disseminated infections caused by the Mycobacterium avium-M. intracellulare complex (MAC) are the most frequent opportunistic bacterial infections in patients with AIDS. MAC isolates are resistant to many of the standard antituberculous drugs. Failure to obtain significant activities of certain drugs is due to difficulty in achieving high concentrations at the sites where the infections reside. New and improved agents for the treatment of mycobacterial infections are therefore required. Earlier, the anti-MAC activities of various agents in free or liposomal form were studied; liposomes were used as drug carriers to ultimately target the drugs to macrophages where mycobacterial infections reside. Clofazimine was chosen for further studies because it could be effectively encapsulated and its activity was well maintained in liposomal form. The present studies with both erythrocytes and macrophages as the model systems show that liposomal drug is far less toxic in vitro than the free drug. The in vivo toxicity of clofazimine was also significantly reduced after liposome encapsulation. The therapeutic efficacies of free and liposomal drugs were compared in a beige mouse model of disseminated MAC infection. An equivalent dose of liposomal drug (10 mg/kg of body weight) was more effective in eliminating the bacterial from the various organs studied, particularly from the liver. Moreover, because of the reduced toxicity of liposomal drug, higher doses could be administered, resulting in a significant reduction in the numbers of CFU in the liver, spleen, and kidneys. The data demonstrate that liposomal clofazimine is highly effective in the treatment of MAC infections, even if the treatment is initiated after a disseminated infection has been established. The present studies thus suggest the potential usefulness of liposomal clofazimine for the treatment of disseminated MAC infections.
We established a panel of 17 xenografts from primary human breast carcinomas. We examined which characteristics of the original tumours and the xenografts facilitate growth in animals. Tumours expressing medium or strong immunoreactivity for p53 protein had significantly (P < 0.05) higher incidence (92%) of in vivo tumour take than those showing weak or negative immunoreactivity (9.1%). No such association was observed between either c-erbB-2 or epidermal growth factor receptor (EGFR) expression in the original tumours and their in vivo tumour take. Following subcutaneous (s.c.) transplantation of original breast tumours or established xenografts, 7/17 tumours showed metastatic disease spread to distant sites (mainly lungs). This study suggests that selective growth of highly aggressive tumours occurs during in vivo propagation of malignant tumours, and these tumours will be of particular interest in evaluating various chemotherapeutic agents for breast cancer management.