Rationale and design of a pilot study to evaluate the acceptability and effectiveness of a revised protein sparing modified fast (rPSMF) for severe obesity in a pediatric tertiary care weight management clinic.

Aggressive dietary interventions may provide an accessible treatment option for children and adolescents with severe obesity who are not successful with traditional lifestyle behavioral interventions or do not want or qualify for weight loss surgery. One such intensive dietary option is the protein sparing modified fast (PSMF). The PSMF involves minimal carbohydrate intake to induce ketosis, while maintaining adequate or high protein intake to minimize catabolism. The PSMF, under medical supervision, can be an effective and safe intervention for children and adolescents, yet the PSMF diet is not regularly used in the treatment of pediatric severe obesity. This paper describes the rationale and design for a pilot study to evaluate the acceptability and effectiveness of a revised PSMF (rPSMF) implemented as a weight loss treatment option for children and adolescents with severe obesity in a pediatric tertiary care weight management clinic. The primary aim of the study is to evaluate the acceptability of the rPSMF as assessed by adherence, satisfaction with the intervention, and participation rate using quantitative and qualitative methods. The secondary aim is to investigate the effectiveness of the rPSMF on improving a) anthropometric measures (weight, body mass index [BMI], BMI z-score); b) metabolic measures (lipid profile, glycosylated hemoglobin, liver function tests); and c) quality of life. Results of this study will provide guidance for the standardization of a pediatric rPSMF protocol in a clinic setting, delineate which factors improve or hinder adherence and weight loss and provide preliminary data for a multicenter randomized controlled trial. CLINICALTRIALSGOV IDENTIFIER: NCT03899311.

Endogenously opsonized particles divert prostanoid action from lethal to protective in models of experimental endotoxemia.

We report that, in rats, the lethal consequences of high-dose endotoxin challenge are exacerbated by the intravascular administration of prostaglandin E1 but attenuated by the intravascular administration of endocytosable particles. This protection is mediated by opsonins. Nonopsonizable particles were unable to provide protection unless first pseudoopsonized with antibody directed against the CR3 (CD11b/CD18) phagocyte receptor. We show that endogenously opsonized particles can act in concert with prostaglandin E1 (putatively by elevation of neutrophil intracellular cAMP and the resultant downregulation of CR3) to completely rescue animals from the lethal late-stage sequelae of experimental endotoxemia. These data illustrate that the interaction of particles with cellular receptors can transform the overall systemic response to prostaglandin E1 from pro- to antiinflammatory. This suggests a role for multiple receptor engagement events in defining the systemic prostaglandin response and offers a rationale for developing new therapeutic modalities in the treatment of sepsis and other inflammatory diseases.

Role of thrombin in endothelial cell monolayer repair in vitro.

PURPOSE: We examined the effect of thrombin on human iliac artery endothelial cell monolayer repair and proliferation after denuding vascular injury. METHODS: Human iliac artery endothelial cell monolayer repair was determined by scrape wounding confluent monolayers and measuring the advancement of the cells into the wounded area for 3 days. Proliferation studies involved plating human iliac artery endothelial cells at one tenth confluence and counting the increase in cell number every 2 days for a 2-week period. Proliferation during monolayer repair was examined by determining bromodeoxyuridine uptake in cells located at the leading edge of a scrape-wounded monolayer. RESULTS: Thrombin (1 to 8 U/ml) inhibited human iliac artery endothelial cell monolayer repair in a concentration-related, reversible manner. The effect was augmented by decreasing serum concentration and was independent of the presence of endothelial cell growth supplement. Inactivation of thrombin's proteolytic site with diisopropylfluorophosphate eliminated its effect on monolayer repair. Thrombin (0.5 to 8 U/ml) inhibited human iliac artery endothelial cell proliferation in a dose-related manner. This effect was augmented by decreasing serum concentration. Finally, thrombin (4 U/ml) inhibited the proliferative response of cells located at the leading edge of wounded monolayers compared with control groups. CONCLUSION: Thrombin inhibits human arterial endothelial cell monolayer repair and proliferation after denuding vascular injury.

Enhanced adherence of human adult endothelial cells to plasma discharge modified polyethylene terephthalate.

Human adult aortic endothelial cell attachment to polyethyleneterephthalate (PET as mylar film) was examined in vitro. PET was examined in both the unmodified form (PET-) and in a modified form (PET+) that had undergone plasma discharge surface modification (PDSM). These surfaces were compared to unmodified tissue culture polystyrene (PS-). The kinetics of attachment and the force of attachment using the rotating disc were determined as a function of surface and substrate protein applied to the surface. Four proteins--fibronectin, collagen I/III, collagen IV/V, and laminin--were added and compared to saline pretreatment. The most significant variable affecting attachment was the time of incubation. When corrected for time, PET+ demonstrated significantly superior attachment kinetics when compared to PET- in most cases. These kinetics were similar to those seen on PS-. Fibronectin precoating of the surface greatly enhanced attachment kinetics on PET+ and PS- but to a much lesser degree on PET-. The fibronectin effect was synergistic with PDSM, suggesting that PDSM enhances protein adsorption on the surface. The force of attachment was generally independent of incubation time and surface/substrate combination except for laminin precoating. Taken together, these data indicate that human endothelial cell adherence to PET may be significantly enhanced by PDSM and surface precoating with fibronectin. Attachment occurs rapidly and, once attached, the cells demonstrate a very firm attachment force capable of resisting shear stresses up to 90 dynes/cm2.

Effects of in vitro aging on human endothelial cell adherence to dacron vascular graft material.

The adherence and growth characteristics of cultured human adult large vessel endothelial cells (EC) maintained on substrate-coated polyethylene terephthalate in the form of woven dacron vascular graft were examined. Two different populations of EC, a low passage (EC (low] and a high passage (EC (high] population, were incubated at cell densities from 10(3) to 10(5) EC/cm2 for 24 hr. Cell counts were performed at 24 hr and after 14 days in tissue culture. At 24 hr on collagen I/III-coated Dacron, EC adherence was independent of the number of passages or the incubation density. When examined after 14 days in culture only EC (low) incubated at 10(5) EC/cm2 maintained initial cell numbers. Human plasma precipitated upon Dacron was necessary before significant cell growth occurred. We conclude that increasing in vitro EC age is associated with decreasing attachment and growth on Dacron. Growth on this important vascular replacement surface requires low passage EC incubated at a high density and the presence of plasma proteins in the substrate coating.

Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure.

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

Cilofungin (LY121019), an antifungal agent with specific activity against Candida albicans and Candida tropicalis.

Cilofungin (LY121019) is an antifungal agent that interferes with beta-glucan synthesis in the cells walls of fungi. The activity of this agent against 256 clinical isolates of yeasts was determined. It was found to be very active in vitro against Candida albicans (MIC for 90% of isolates [MIC90], less than or equal to 0.31 microgram/ml; minimal fungicidal concentration for 90% of isolates [MFC90], less than or equal to 0.31 micrograms/ml) and C. tropicalis (MIC90, less than or equal to 0.31 microgram/ml; MFC90, less than or equal to 0.31 microgram/ml) and moderately active against Torulopsis glabrata (MIC90 and MFC90, less than or equal to 20 micrograms/ml). All C. parapsilosis, Cryptococcus, and Saccharomyces cerevisiae strains were resistant. The activity of cilofungin was affected by medium and inoculum size. Antibiotic medium no. 3 was used as the standard medium. Isolates of C. albicans and C. tropicalis demonstrated a paradoxical effect in Sabouraud dextrose broth and yeast nitrogen base broth in that growth was partially inhibited at MICs equivalent to those in antibiotic medium no. 3, but growth continued, in many instances, throughout all concentrations tested. There was decreased activity of cilofungin with inocula greater than 10(5) CFU/ml. The temperature and duration of incubation did not affect its activity.

Kinetics of endothelial cell-surface attachment forces.

Physical and biochemical forces exist that are necessary for the persistent attachment and function of ECs on native and prosthetic blood vessels. The optimization of conditions that permit regeneration of these attachment forces may allow rapid establishment of a durable, biocompatible EC monolayer. We examined the effects of three major factors, protein substrate, EC incubation time, and shear stress, on the attachment kinetics of human adult ECs to two different polymers. ECs were incubated up to 30 minutes on polymers (PS or PET) coated with extracellular matrix proteins: collagen I/III, fibronectin, collagen IV/V, laminin, gelatin, or saline control. After incubation, continued attachment in the presence of shear stress (created in a rotating disc device) between zero and 90 dynes/cm2 for 30 minutes was evaluated. Maximal adherence was observed on all substrates by 30 minutes. Therefore, after a 30-minute incubation, the percentage of cells attached (postshear ECs/preshear ECs/preshear ECs X 100) was measured as a function of shear stress. ECs attached to a matrix of fibronectin or collagen I/III demonstrated shear-resistant adherence after as little as 5 minutes of static incubation before initial shear exposure. By 30 minutes, more than 90% of the ECs on both matrices demonstrated the ability to remain attached in the presence of 90 dynes/cm2 of shear stress. We conclude that forces that attach ECs to surfaces are affected by temporal factors (incubation time) and substrate composition and may be quantified with a defined shear stress detachment assay. Understanding and manipulating these temporal physiochemical parameters should allow one to re-create an optimal EC monolayer on a blood-contacting surface.

Detection of methicillin-resistant Staphylococcus epidermidis.

To determine whether methods suggested for detecting methicillin-resistant Staphylococcus aureus apply equally to methicillin-resistant Staphylococcus epidermidis, 135 S. epidermidis isolates were tested by the Vitek AMS gram-positive susceptibility card (Vitek Systems, Inc., Hazelwood, Mo.) and by modifications of agar screen, disk diffusion, and microdilution methods. Modifications included 24- versus 48-h incubation, unsupplemented versus 2% NaCl-supplemented broth, and standard versus direct inoculum. At 24 h, the highest number of resistant strains, 59, was detected by oxacillin (1 microgram) disk diffusion. At 48 h, three additional strains were judged resistant. With one exception, results for oxacillin disk diffusion and agar screen were equivalent at 24 and 48 h. Vitek detected 50 resistant strains. Significantly fewer resistant strains were detected at 24 h by methicillin disk diffusion (5 micrograms) and methicillin microdilution with 2% NaCl. For oxacillin microdilution, neither 2% NaCl supplementation nor the method of inoculum preparation significantly affected the results. Oxacillin microdilution with cation- rather than non-cation-supplemented broth detected significantly fewer (n = 33) resistant strains at 24 h; 51 were resistant at 48 h. To detect methicillin-resistant S. epidermidis, a direct inoculum with either 24-h oxacillin disk diffusion and reincubation of intermediate strains for an additional 24 h or 24-h oxacillin agar screen and reincubation of strains with no growth for a total of 48 h is recommended.