Engineering of Escherichia coli strains for plasmid biopharmaceutical production: Scale-up challenges

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.     

Overlapping phenotypes of multidrug resistance among panels of human cancer-cell lines

In addition to P-glycoprotein (Pgp), 2 proteins related to multidrug resistance (MDR) have recently been described. The Multidrug-Resistance-associated protein (MRP) is one of the ATP-binding-cassette (ABC) transporters. The Lung-Resistance Protein (LRP) is the major component of human vaults, which are newly described cellular organelles and thought to mediate intracellular transport processes. Using immunocytochemical methods, we have examined the expression of MRP and LRP among panels of human cancer-cell lines not selected for drug resistance which have been previously characterized for expression of Pgp, and in vitro response to a variety of anti-cancer drugs. Expression of MRP and LRP was observed in 47/55 (87%) and 46/59 (78%) cell lines, respectively. Statistically significant correlations were observed between expression of each of these 3 proteins and in vitro sensitivity to at least one drug classically associated with MDR. LRP showed the greatest individual predictive value, which also applied to several non-classical MDR drugs. Co-expression of 2–3 MDR-related proteins was observed in 64% of the lines and was, in general, associated with high relative levels of drug resistance. Previously identified “classic” MDR lines as well as “pan-resistant” lines concurrently expressed all 3 MDR-related proteins. Some highly drug-resistant cell lines without detectable MDRI/Pgp were found to express relatively high levels of MRP and LRP. The high prevalence of MRP and LRP expression observed in this large set of cell lines, which have not been subjected to laboratory drug selection, suggests that MDR mechanisms associated with these proteins may be widespread in human malignancies. Moreover, the overlapping of these more recently recognized MDR phenotypes with Pgp-type MDR results in a complex phenotype, the understanding of which may be of importance in the development of new drugs and design of clinical treatment protocols, particularly those seeking to employ strategies to reverse the MDR phenotype. © 1996 Wiley-Liss, Inc.     

Severity of bulimia: Relationship with depression and other psychopathology

There were 53 women (35 bulimics and 18 normal controls) who were compared on the Minnesota Multiphasic Personality inventory, the Symptom Checklist-90R, and the Beck Depression Inventory. The bulimics were separated into two groups, high (N = 18) and low (N = 17) frequency purgers, based upon a median split self-monitored purging data. Bulimics who purged less than 3 times per week (M = 1.71) were classified as low frequency purgers, and those who purged more than 3 times per week (M = 9.94) were classified as high frequency purgers. Comparisons of the three groups showed that both groups of bulimics differed from normals on measures of depression, but on other measres, i. e., anxiety interpersonal sensitivity and sociopathic trait, only the high frequency purgers differed from normals. Correlational analyses indicated that these same variables, and especially depression, were positively correlated with purging, suggesting that severity of bulimia may be associated with additional psychopathology. These findings were discussed in terms of the existing literature concerning the psychopathology of bulimia.     

Psychopathology associated with bulimia,binge eating,and obesity

Bulimic binge-purgers, bulimic binge-eaters, nonbulimic obese presenting for treatment for obesity, obese not in treatment, and normal control subjects were compared using the Minnesota Multiphasic Personality Inventory (MMPI), Symptom Checklist-90-Revised (SCL-90-R), and Beck Depression Inventory (BDI). Results suggested a continuum of severity, with the binge-purger group showing the highest levels of psychopathology, and the binge-eaters and clinical obese showing significantly more distress than the two control groups. Analysis of the frequency of clinically elevated scores on each scale of the MMPI and SCL-90R as well as the BDI as a function of group indicated that the clinical groups evidenced significantly greater psychopathology compared with their appropriate control groups. These results clarify some of the conflicting results of previous investigations of obesity which did not differentiate obese patients and nonpatients. The implications of these findings for treatment research on obesity are that perhaps a more broadspectrum form of treatment may be required in order to address the psychological problems of the chronically obese.     

Neurocysticercosis in radiographically imaged seizure patients in U.S. emergency departments

Neurocysticercosis appears to be on the rise in the United States, based on immigration patterns and published cases series, including reports of domestic acquisition. We used a collaborative network of U.S. emergency departments to characterize the epidemiology of neurocysticercosis in seizure patients. Data were collected prospectively at 11 university-affiliated, geographically diverse, urban U.S. emergency departments from July 1996 to September 1998. Patients with a seizure who underwent neuroimaging were included. Of the 1,801 patients enrolled in the study, 38 (2.1%) had seizures attributable to neurocysticercosis. The disease was detected in 9 of the 11 sites and was associated with Hispanic ethnicity, immigrant status, and exposure to areas where neurocysticercosis is endemic. This disease appears to be widely distributed and highly prevalent in certain populations (e.g., Hispanic patients) and areas (e.g., Southwest).