A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration

Klebsiella pneumoniae Multiresistance Plasmid pMET1: Similarity with the Yersinia pestis Plasmid pCRY and Integrative Conjugative Elements

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31).The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains

The eck fistula in animals and humans

In all species so far studied, including man, portacaval shunt causes the same changes in liver morphology, including hepatocyte atrophy, fatty infiltration, deglycogenation, depletion and disorganization of the rough endoplasmic reticulum (RER) and its lining polyribosomes and variable but less specific damage to other organelles. Many, perhaps all, biosynthetic processes are quickly depressed, largely secondary to the selective damage to the RER, which is the "factory" of the cell. These structural and metabolic changes in the liver after portal diversion are caused by the diversion around the liver of the hepatotrophic substances in portal venous blood, of which endogenous insulin is the most important. In experimental animals, the injury of Eck's fistula can be prevented by infusing insulin into the tied-off hilar portal vein. The subtle but far-reaching changes in hepatic function after portal diversion have made it possible to use this procedure in palliating three inborn errors of metabolism: glycogen storage disease, familial hypercholesterolemia, and α1-antitrypsin deficiency In these three diseases, the abnormalities caused by portal diversion have counteracted abnormalities in the patients that were caused by the inborn errors. In these diseases, amelioration of the inborn errors depends on the completeness of the portal diversion. In contrast, total portal diversion to treat complications of portal hypertension is undesirable and always will degrade hepatic function if a significant amount of hepatopetal portal venous blood is taken from the liver. When total portal diversion is achieved (and this is to be expected after all conventional shunts), the incidence of hepatic failure and hepatic encephalopathy is increased. If portal diversion must be done for the control of variceal hemorrhage, a selective procedure such as the Warren procedure is theoretically superior to the completely diverting shunt. In practice, better patient survival has not been achieved after selective shunts than after conventional shunts, but the incidence of hepatic encephalopathy has been less. © 1983 Year Book Medical Publishers, Inc

Modelling of early viral kinetics and pegylated interferon-α2b pharmacokinetics in patients with HBeAg-positive chronic hepatitis B

Background: Pegylated interferon α2b (PEG-IFN-α2b) is effective for the treatment of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, although its mechanism of action remains unclear. HBeAg loss is achieved in 36% of patients after one year of PEG-IFN-α2b treatment and combination therapy with lamivudine is not superior to PEG-IFN-α2b monotherapy. Methods: Early pharmacokinetics and viral kinetics were analysed in patients treated for 52 weeks with PEG-IFN-α2b with or without lamivudine. Results: After 4 weeks of treatment, there was a median viral decline of 2.94 log 10 copies/ml in those treated with PEG-IFN-α2b and lamivudine and only 0.45 log10 copies/ml in the PEG-IFN-α2b monotherapy group. Peak PEG-IFN-α2b levels were reached approximately one day after administration and subsequently declined exponentially, consistent with a viral load rebound near to baseline levels at the end of the dosing period in most patients receiving PEG-IFN-α2b monotherapy. Modelling of pharmacokinetics and viral kinetics data in this group revealed that viral load was minimal 3.6 days after PEG-IFN-α2b administration, the mean maximal and mean antiviral effectiveness was 70% and 48% with a mean infected cell loss rate of 0.07 per day, while no significant biphasic decline was observed. Conclusions: PEG-IFN-α2b induces a sustained response in a considerable number of patients despite limited direct antiviral activity during the first weeks of antiviral therapy.</p

Modelling of early viral kinetics and pegylated interferon-α2b pharmacokinetics in patients with HBeAg-positive chronic hepatitis B

Background: Pegylated interferon α2b (PEG-IFN-α2b) is effective for the treatment of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, although its mechanism of action remains unclear. HBeAg loss is achieved in 36% of patients after one year of PEG-IFN-α2b treatment and combination therapy with lamivudine is not superior to PEG-IFN-α2b monotherapy. Methods: Early pharmacokinetics and viral kinetics were analysed in patients treated for 52 weeks with PEG-IFN-α2b with or without lamivudine. Results: After 4 weeks of treatment, there was a median viral decline of 2.94 log 10 copies/ml in those treated with PEG-IFN-α2b and lamivudine and only 0.45 log10 copies/ml in the PEG-IFN-α2b monotherapy group. Peak PEG-IFN-α2b levels were reached approximately one day after administration and subsequently declined exponentially, consistent with a viral load rebound near to baseline levels at the end of the dosing period in most patients receiving PEG-IFN-α2b monotherapy. Modelling of pharmacokinetics and viral kinetics data in this group revealed that viral load was minimal 3.6 days after PEG-IFN-α2b administration, the mean maximal and mean antiviral effectiveness was 70% and 48% with a mean infected cell loss rate of 0.07 per day, while no significant biphasic decline was observed. Conclusions: PEG-IFN-α2b induces a sustained response in a considerable number of patients despite limited direct antiviral activity during the first weeks of antiviral therapy.</p

Corneofungimetry bioassay on Malassezia spp. under ketoconazole and desonide influences.

BACKGROUND: Glucocorticoids can boost some Malassezia-driven dermatoses. However, both antifungals and topical corticosteroids improve lesions of seborrheic dermatitis. OBJECTIVE: To revisit the topical activity of the antifungal ketoconazole and the corticosteroid desonide on Malassezia growth on human stratum corneum. Material and Methods: The computer-assisted corneofungimetry bioassay was used to compare the growth of M. furfur, M. globosa and M. restricta on human stratum corneum coated with olive oil. Four blinded gel formulations were tested. They contained either 2% ketoconazole, 0.05% desonide or a combination of 2% ketoconazole and 0.05% desonide; one gel was unmedicated. Untreated stratum corneum and specimens coated with a 2% ketoconazole cream were used as negative and positive comparators, respectively. A total of 45 samples (15 M. furfur, 15 M. globosa, and 15 M. restricta) were used for each test formulation in this randomized, double-blind study. RESULTS: The 2% ketoconazole gel and cream and the combination of 2% ketoconazole and 0.05% desonide formulation abated similarly and significantly the M. furfur, M. globosa and M. restricta growth. The 3 species were similarly sensitive to these formulations. By contrast, no significant inhibitory effect was yielded by the 0.05% desonide gel and the vehicle. CONCLUSION: The presence of 0.05% desonide does not impair or improve the Malassezia susceptibility to 2% ketoconazole when growing on lipid-enriched human stratum corneum