Analysis of unconventional approaches for the rapid detection of surface lectin binding ligands on human cell lines

For over a decade our laboratory has developed and used a novel histochemical assay using derivatized agarose beads to examine the surface properties of various cell types. Most recently, we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity of these lectins in culture, as a possible approach in the design of more specific anti-cancer drugs. Although we have examined the validity of the bead-binding assay in sea urchin cell systems, we have not previously validated this technique for mammalian cells. Here the binding results of the bead assay are compared with conventional fluorescence assays, using lectins from three species (Triticum vulgaris, Phaseolus vulgaris, and Lens culinaris) on the two colon cell lines. These lectins were chosen because they seemed to interact with the two cell lines differently.

Binding results obtained using both assays were compared for frozen, thawed and fixed; cultured and fixed; and live cells. Both qualitative and quantitative fluorescence results generally correlated with those using the bead assay. Similar results were also obtained with all of the three different cell preparation protocols. The fluorescence assay was able to detect lower lectin binding ligand levels than the bead assay, while the bead assay, because it can so rapidly detect cells with large numbers of lectin binding ligands, is ideal for initial screening studies that seek to identify cells that are rich in surface binders for specific molecules. The direct use of frozen, thawed and fixed cells allows rapid mass screening for surface molecules, without the requirement for costly and time consuming cell culture.     

Bacterial indicators of pollution of the Douala lagoon, Cameroon: Public health implications

Background

Indiscriminate disposal of untreated wastes which are often heavily laden with sewage microorganisms some of which are pathogenic to humans into aquatic environments near cities could serve as potential dangers to human health.

Objective

A prospective study was undertaken to investigate the scope of potential bacterial pathogens and to assess the extent of pollution of the Douala lagoon.

Methods

A total of eighty water samples were collected fortnightly from the lagoon at five stations from March to October 2005 and analysed for heterotrophic bacterial densities, coliform counts, faecal coliform and faecal streptococcal counts. Bacteria were isolated and identified using standard microbiology and biochemical techniques.

Results

High heterotrophic bacterial counts (33 × 10⁵ − 161 × 10⁵ CFU/ mL), total coliform counts (1.8 × 10² − 2.4 × 10² CFU/100 mL), faecal coliform counts (2.2 × 10² − 2.4 × 10² CFU/ 100 mL) and faecal streptococcal counts (2.1 × 102 − 2.3 x 10² CFU/100mL were observed in all sampling stations. Eleven species of bacteria: Bacteroides fragilis, Proteus vulgaris, Klebsiella pneumoniae, E. coli, Enterococcus faecalis, Enterobacter aerogenes, Citrobacter freundii, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus mycoides and Serratia marcesens, were frequently isolated.

Conclusion

The presence of potential bacterial agents such as Bacteroides fragilis, Pseudomonas aeruginosa, Aeromonas hydrophila, Klebsiella pneumoniae and E. coli in the lagoon may pose a serious threat to the health and well being of users of the Lagoon and calls for urgent intervention.     

The effect of maternal smoking and drinking during pregnancy upon (3)H-nicotine receptor brainstem binding in infants dying of the sudden infant death syndrome: initial observations in a high risk population.

The high rate of the sudden infant death syndrome (SIDS) in American Indians in the Northern Plains (3.5/1000) may reflect the high incidence of cigarette smoking and alcohol consumption during pregnancy. Nicotine, a neurotoxic component of cigarettes, and alcohol adversely affect nicotinic receptor binding and subsequent cholinergic development in animals. We measured (3)H-nicotine receptor binding in 16 brainstem nuclei in American Indian SIDS (n = 27) and controls (n = 6). In five nuclei related to cardiorespiratory control, (3)H-nicotinic binding decreased with increasing number of drinks (P < 0.03). There were no differences in binding in SIDS compared with controls, except upon stratification of prenatal exposures. In three mesopontine nuclei critical for arousal there were reductions (P < 0.04) in binding in controls exposed to cigarette smoke compared with controls without exposure; there was no difference between SIDS cases with or without exposure. This study suggests that maternal smoking and alcohol affects (3)H-nicotinic binding in the infant brainstem irrespective of the cause of death. It also suggests that SIDS cases are unable to respond to maternal smoking with the "normal" reduction seen in controls. Future studies are needed to establish the role of adverse prenatal exposures in altered brainstem neurochemistry in SIDS.     

Posterior Superior Mesenteric Artery (SMA) First Approach vs. Standard Pancreaticoduodenectomy in Patients with Resectable Periampullary Cancers: a Prospective Comparison Focusing on Circumferential Resection Margins

Background

The ‘SMA-first’ (P-SMA) pancreatoduodenectomy (PD) allows dissection directly on the right lateral aspect of superior mesenteric artery (SMA) which may decrease circumferential resection margin (CRM) positivity. This comparative study between standard PD (sPD) and P-SMA approach was planned focusing on CRM involvement.

Methods

This was a prospective study comparing consecutive patients with resectable periampullary cancers (PACA) undergoing PD using the standard or P-SMA approach. The perioperative outcomes and the CRM positivity rates (specimens analysed according to the standardized Leeds pathology protocol (LEEPP)) were compared.

Results

Overall, 39 patients (28 men; mean age 54 years; sPD 21, P-SMA 18) were included. Both groups were comparable with regard to demographic/tumour characteristics and perioperative outcomes. The P-SMA technique was significantly faster (321.1 ± 54.0 vs. 357.6 ± 55.8 min; p = 0.05). Though the mean tumour size (2.2 vs. 2.1 cm; p = 0.84) and T stage (T2 and T3) distribution were similar in both groups, lymph node yield was significantly higher in the P-SMA group (10.7 vs. 5.95; p = 0.001; mean 8 (2–21)). Though CRM positivity (margin <1 mm) occurred in 8 (21.1%), we did not find the P-SMA PD to yield significantly lower CRM positivity rates compared to the sPD (3/17 (17.6%) vs. 5/21(23.8%); p = 0.71). At a median follow-up of 28 months, fewer patients in the P-SMA PD group developed recurrence (2/15 vs. 5/19; p = 0.3) or died (3/15 vs. 7/19; p = 0.19), though this difference was not significant.

Conclusions

In patients with resectable PACA, P-SMA PD was significantly faster and yielded higher lymph node counts in the specimen but did not lower the rate of CRM positivity as determined by the LEEPP.

     

Risk factors for coronary artery disease in Indians

Background: A case control study was carried out to study the emerging risk factors for coronary artery disease in Indians.     

Left ventricular mass and incident hypertension in individuals with initial optimal blood pressure: the Strong Heart Study

OBJECTIVE: Metabolic abnormalities have been shown to predict 8-year incident arterial hypertension in individuals with optimal blood pressure. As echocardiographic left ventricular mass has also been reported to predict incident hypertension in individuals with baseline blood pressure of less than 140/90 mmHg, we determined whether left ventricular mass predicts 4-year incident hypertension also in individuals with initial optimal blood pressure (<120/80 mmHg), independent of metabolic factors influencing blood pressure. METHODS: We studied 777 of 3257 members of the American Indian population-based Strong Heart Study cohort with optimal blood pressure (34% men, 45% obese, and 35% diabetic), aged 57 +/- 7 years, and without prevalent cardiovascular disease. RESULTS: Over 4 years, 159 individuals (20%, group H) developed hypertension (blood pressure >/=140/90 mmHg). They had a greater baseline BMI, waist girth, and blood pressure (112/69 vs. 109/68 mmHg, all P < 0.03) than those remaining normotensive (group N), with similar lipid profile and renal function. At baseline, left ventricular mass was significantly greater in group H than in group N (P < 0.004). The difference in left ventricular mass was confirmed after controlling for initial BMI, systolic blood pressure, homeostatic model assessment index, and diabetes. The probability of incident hypertension increased by 36% for each standard deviation of left ventricular mass index (P = 0.006), independent of covariates. Participants with left ventricular mass of more than 159 g (75th percentile of distribution) had 2.5-fold (95% confidence interval, 1.4-3.6; P < 0.001) higher adjusted risk of incident hypertension than those below this value. CONCLUSION: Left ventricular mass predicts incident arterial hypertension in individuals with initially optimal blood pressure. This association is independent of body build, prevalent diabetes, and initial blood pressure.     

Microdosing Assessment to Evaluate Pharmacokinetics and Drug Metabolism in Rats Using Liquid Chromatography-Tandem Mass Spectrometry

PURPOSE: To evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to support human microdosing study with sub-pharmacological dose, investigate proportionality of pharmacokinetics from the microdose to therapeutic human equivalent doses in rats and characterize circulating metabolites in rats administered with the microdose. MATERIALS AND METHODS: Five drugs of antipyrine, metoprolol, carbamazepine, digoxin and atenolol were administered orally to male Sprague-Dawley rats at 0.167, 1.67, 16.7, 167 and 1,670 microg/kg doses. Plasma samples were extracted using either solid phase extraction or liquid-liquid extraction, and analyzed using LC-MS/MS. RESULTS: Using 100 microl of plasma sample, the lower limit of quantitation for antipyrine (10 pg/ml), carbamazepine (1 pg/ml), metoprolol (5 pg/ml), atenolol (20 pg/ml), and digoxin (5 pg/ml) were achieved using an API 5000. Proportional pharmacokinetics were observed from 0.167 microg/kg to 1,670 microg/kg for antipyrine and carbamazepine and from 1.67 to 1,670 microg/kg for atenolol and digoxin, while metoprolol exhibited a non-proportional pharmacokinetics relationship. Several metabolites of carbamazepine were characterized in plasma from rats dosed at 1.67 mug/kg using LC-MS/MS. CONCLUSIONS: This study has shown the promise of sensitive LC-MS/MS method to support microdose pharmacokinetics and drug metabolism studies in human.     

Glycemic control in diabetic American Indians. Longitudinal data from the Strong Heart Study.

OBJECTIVE: To describe glycemic control and identify correlates of elevated HbA1c levels in diabetic American Indians participating in the Strong Heart Study, which is a longitudinal study of cardiovascular disease in American Indians in Arizona, Oklahoma, South Dakota, and North Dakota. RESEARCH DESIGN AND METHODS: This analysis is based on data from the baseline (1989-1992) and first follow-up (1994-1995) examinations of the Strong Heart Study. The 1,581 diabetic participants included in this analysis were aged 45-74 years at baseline, were diagnosed with diabetes before and at baseline, and had their HbA1c levels measured at follow-up. HbA1c was used as the index of glycemic control. Characteristics that may affect glycemic control were evaluated for cross-sectional and longitudinal relationships by analysis of covariance and multiple regression. RESULTS: There was no significant difference between median HbA1c at baseline (8.4%) and at follow-up (8.5%). Sex, age (inversely), and insulin and oral hypoglycemic agent therapy were significantly related to HbA1c levels in both the cross-sectional and longitudinal analyses. Current smoking, prior use of alcohol, and duration of diabetes were significant only for the cross-sectional data. Baseline HbA1c significantly and positively predicted HbA1c levels at follow-up. Comparison of HbA1c by therapy type shows that insulin therapy produced a significant decrease in HbA1c between the baseline and follow-up examinations. CONCLUSIONS: Glycemic control was poor among diabetic American Indians participating in the Strong Heart Study. Women, patients taking insulin or oral hypoglycemic agents, and younger individuals had the worst control of all the participants. Baseline HbA1c, and weight loss predicted worsening of control, whereas insulin therapy predicted improvement in control. Additional therapies and/or approaches are needed to improve glycemic control in this population.