ABSTRACT
Phagocytosis plays a pivotal and essential role in host immune defense, both as a focal constituent of the innate immune response and a bridging element linking innate and adaptive immunity. Phagocytosis has been demonstrated to be critical in development, tissue remodeling, wound healing and resolution of inflammation through clearance of foreign organisms, apoptotic cells and the production of anti-inflammatory mediators. During pre-clinical investigations, therapeutic drug candidates may alter host resistance to infectious agents by modulating the immune system. The assessment of phagocytic function can be a critical parameter of immunotoxicology for this adverse effect. Utilizing pH-sensitive pHrodo™ BioParticles®, a flow cytometric phagocytosis method was developed and validated in rodent and non-human primate (NHP) species under rigorous GLP compliant procedures. Using species-specific granulocyte markers as well as appropriate temperature and pharmacologic controls, we have developed an ex vivo assay to measure phagocytic function. The method has been optimized to utilize minimal sample volume of whole blood. The assay represents a rapid and reliable tool that can be implemented to evaluate the immunotoxic and immunomodulatory effects of therapeutic candidates.
Subject(s)
Flow Cytometry/methods , Granulocytes/immunology , Phagocytosis/immunology , Animals , Cytochalasin D/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Escherichia coli/immunology , Female , Granulocytes/metabolism , Macaca fascicularis , Macrolides/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nucleic Acid Synthesis Inhibitors/pharmacology , Phagocytosis/drug effects , Reproducibility of ResultsABSTRACT
Studies centered on understanding how molecular structure affects biological function have historically focused on proteins. Circular dichroism (CD) is commonly used to analyze protein secondary structure, yet its application to other molecules is far less explored. In fact, little is known about how glycan conformation might affect function, likely because of a lack of tools for measuring dynamic structural changes of carbohydrates. In the present study, we developed a method based on CD to monitor conformational changes in the zwitterionic T-cell-activating glycoantigen polysaccharide A1 (PSA). We found that PSA helical structure produces a CD spectrum that is strikingly similar to proteins rich in alpha-helical content and is equally sensitive to nonpolar solvents. Like conventional T-cell-dependent proteins, PSA requires processing before major histocompatibility complex class II (MHCII) binding. CD spectra of PSA fragments of varying sizes indicated that fragments smaller than three repeating units lack helical content and are incapable of MHCII binding. Likewise, neutralization of charged groups in the repeating unit resulted in major conformational changes as measured by CD, which correlated with a lack of MHCII presentation. These data represent two significant findings: CD can be used to measure conformational changes in carbohydrates and the functional epitope from PSA is dependent on a specific conformation that is stabilized by adjacent repeating units and a zwitterionic charge motif. As a result, this work demonstrates that CD is a valuable tool for use in functional glycomics efforts that seek to align chemical and conformational structure with biological activity.
Subject(s)
Circular Dichroism/methods , Polysaccharides/chemistry , Polysaccharides/immunology , T-Lymphocytes/immunology , HLA-DR1 Antigen/metabolism , Models, Biological , Models, Molecular , Molecular Weight , Protein Binding , Protein Conformation , Protein Denaturation/physiology , Solvents/pharmacology , Structure-Activity Relationship , Thermodynamics , Water/chemistryABSTRACT
OBJECTIVE: To determine predictors of survival and of weaning off parenteral nutrition (PN) in pediatric short bowel syndrome (SBS) patients. SUMMARY BACKGROUND DATA: Pediatric SBS carries extensive morbidity and high mortality, but factors believed to predict survival or weaning from PN have been based on limited studies. This study reviews outcomes of a large number of SBS infants and identifies predictors of success. METHODS: Multivariate Cox proportional hazards analysis was conducted on 80 pediatric SBS patients. Primary outcome was survival; secondary outcome was ability to wean off PN. Nonsignificant covariates were eliminated. P < 0.05 was considered significant. RESULTS: Over a mean of 5.1 years of follow-up, survival was 58 of 80 (72.5%) and 51 weaned off PN (63.8%). Cholestasis (conjugated bilirubin > or =2.5 mg/dL) was the strongest predictor of mortality (relative risk [RR] 22.7, P = 0.005). Although absolute small bowel length was only slightly predictive, percentage of normal bowel length (for a given infant's gestational age) was strongly predictive of mortality (if <10% of normal length, RR of death was 5.7, P = 0.003) and of weaning PN (if > or =10% of normal, RR of weaning PN was 11.8, P = 0.001). Presence of the ileocecal valve (ICV) also strongly predicted weaning PN (RR 3.9, P < 0.0005); however, ICV was not predictive of survival. CONCLUSIONS: Cholestasis and age-adjusted small bowel length are the major predictors of mortality in pediatric SBS. Age-adjusted small bowel length and ICV are the major predictors of weaning from PN. These data permit better prediction of outcomes of pediatric SBS, which may help to direct future management of these challenging patients.
