Equivalent pore modeling: vesicles and channels

The methods used to estimate equivalent pores in capillary walls using lymph protein flux analyses are presented. The capillary walls in several different organs can be described by a two-pore model that incorporates pores of radii 50-80 and 200-250 A. The most likely ultrastructural correlates of these physiological pores appear to be the transendothelial channels. In addition, the positive charge associated with endogenous protein transport in intestinal and lung capillaries may reflect the positive charge located on the inner walls of the transendothelial channel.     

Cortisol diurnal patterns,associations with depressive symptoms,and the impact of intervention in older adults: Results using modern robust methods aimed at dealing with low power due to violations of standard assumptions

Advances in salivary bioscience enable the widespread integration of biological measures into the behavioral and social sciences. While theoretical integration has progressed, much less attention has focused on analytical strategies and tactics. The statistical literature warns that common methods for comparing groups and studying associations can have relatively poor power compared to more modern robust techniques. Here we illustrate, in secondary data analyses using the USC Well Elderly II study (n = 460, age 60–95, 66% female), that modern robust methods make a substantial difference when analyzing relations between salivary analyte and behavioral data. Analyses that deal with the diurnal pattern of cortisol and the association of the cortisol awakening response with depressive symptoms and physical well-being are reported. Non-significant results become significant when using improved methods for dealing with skewed distributions and outliers. Analytical strategies and tactics that employ modern robust methods have the potential to reduce the probability of both Type I and Type II errors in studies that compare salivary analytes between groups, across time, or examine associations with salivary analyte levels.     

Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Oxidized Biopterins in Pediatric Falciparum Malaria: Association with Disease Severity

Decreased bioavailability of nitric oxide (NO) is a major contributor to the pathophysiology of severe falciparum malaria. Tetrahydrobiopterin (BH₄) is an enzyme cofactor required for NO synthesis from L-arginine. We hypothesized that systemic levels of BH4 would be decreased in children with cerebral malaria, contributing to low NO bioavailability. In an observational study in Tanzania, we measured urine levels of biopterin in its various redox states (fully reduced [BH₄] and the oxidized metabolites, dihydrobiopterin [BH₂] and biopterin [B₀]) in children with uncomplicated malaria (UM, n = 55), cerebral malaria (CM, n = 45), non-malaria central nervous system conditions (NMC, n = 48), and in 111 healthy controls (HC). Median urine BH4 concentration in CM (1.10 [IQR:0.55–2.18] μmol/mmol creatinine) was significantly lower compared to each of the other three groups — UM (2.10 [IQR:1.32–3.14];p<0.001), NMC (1.52 [IQR:1.01–2.71];p = 0.002), and HC (1.60 [IQR:1.15–2.23];p = 0.005). Oxidized biopterins were increased, and the BH4:BH2 ratio markedly decreased in CM. In a multivariate logistic regression model, each Log10-unit decrease in urine BH4 was independently associated with a 3.85-fold (95% CI:1.89–7.61) increase in odds of CM (p<0.001). Low systemic BH4 levels and increased oxidized biopterins contribute to the low NO bioavailability observed in CM. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.     

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.     

In vitro lymphocyte cytotoxicity. I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT₂, LT₂ and LT₃, respectively. They are secreted by cells from a variety of lympboid tissues, i.e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT₁ and LT₂ persist in these cultures through day 5, LT₃ is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT₁, LT₂ and LT₃ activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).     

Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets.

A myocardial glycolipid (P3) mixed with protein antigens in oil-in-water emulsion induced lasting delayed hypersensitivity (DH) and granulomatous inflammation after intradermal injection into guinea pigs. This did not occur when P3 and bovine serum albumin (BSA) were given in Freund's incomplete adjuvant. The oil-in-water emulsions consisted of microscopic oil droplets suspended in aqueous medium. By separating oil and aqueous phases from BSA + P3 emulsion it was shown that antigen retained with oil droplets led to DH and granuloma formation. The association of antigen with oil droplets was P3 dependent and was quantitated with 125I-labeled BSA. The same phenomenon occurred with 125I-labeled rabbit gamma-globulin (RGG) + P3 emulsion. Fluorescein-conjugated RGG was observed in a particulate state within or on oil droplets in emulsion containing P3. These physical characteristics of antigen + P3 emulsion appeared to be important for immunogenicity.     

Relationship of CIF, LT, and PIF released in vitro by activated human lymphocytes. II. A further functional comparison of LT and PIF activities on HeLa and L-929 target cells.

Lymphotoxin (LT), and proliferation inhibition factor (PIF) activities found in 5-day supernatants of mitogen-activated human lymphocytes (SAL) were further compared. In agreement with previous results, the activities could not be distinguished functionally. Quantitative differences in the amount of activity detected in the SAL could be accounted for on the basis of target cell differences, concentration of the lymphocyte effector molecules in the supernatant, and the parameter employed to assess cell function. Growth inhibitory activity detected at high supernatant dilutions was completely reversible, whereas the cytotoxic activity detected at low supernatant dilutions was irreversible. When the active medium was fractionated on DEAE, two peaks of inhibitory activity were detected. Depending upon the amount of activity and target cell, both peaks of activity were growth inhibitory or cytotoxic. Since both peaks of material affected HeLa and L-929 cells, the materials were not species specific. Thus, it appears that cloning inhibitor factor, LT, and PIF activities may actually be measures of the same stable materials found in 5-day activated lymphocyte supernatants.