Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the "default" T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2-3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-gamma by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing- associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor alpha or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.     

Laparoscopic visual field. Voice vs foot pedal interfaces for control of the AESOP robot

BACKGROUND: In order for robotic devices to be introduced successfully into surgical practice, the development of transparent surgeon/machine interfaces is critical. METHODS: This study evaluated the standard foot pedal for the AESOP robot compared to a voice control interface. Speed, accuracy, learning curves, durability of learning at 2 weeks, and operator-interface failures were analyzed in an ex vivo model. RESULTS: Foot control was faster and had less operator-interface failures. Voice control was more accurate as measured by "pass points." The foot control learning curve reached a plateau at the third trial, while the voice control did not fully plateau. Durability of learning favored the foot control but was not significantly different. CONCLUSIONS: Currently, the voice control is more accurate and has the advantage of not requiring the surgeon to look away from the operative field. However, it is slower and may require more attention as an interface. As voice recognition software continues to advance, speed and transparency are anticipated to improve.     

Compensatory neutral mutations and the evolution of RNA

There are many examples of RNA molecules in which the secondary structure has been strongly conserved during evolution, but the base sequence is much less conserved, e.g., transfer RNA, ribosomal RNA, and ribonuclease P. A model of compensatory neutral mutations is used here to describe the evolution of the base sequence in RNA helices. There are two loci (i.e., the two sides of the pair) with four alleles at each locus (corresponding to A, C, G, U). Watson-Crick base pairs (AU, CG, GC, and UA) are each assigned a fitness 1, whilst all other pairs are treated as mismatches and assigned fitness 1-s. A population of N diploid individuals is considered with a mutation rate of u per base. For biologically reasonable parameter values, the frequency of mismatches is always small but the frequency of the four matching pairs can vary over a wide range. Using a diffusion model, the stationary distribution for the frequency x of any of the four matching pairs is calculated. The shape depends on the combination of variables beta = 8Nu2/9s. For small beta, the distribution diverges at the two extremes, x = 0 and x = 1-z, where z is the mean frequency of mismatches. The population typically consists almost entirely of one of the four types of matching pairs, but occasionally makes shifts between the four possible states. The mean rate at which these shifts occur is calculated here. The effect of recombination between the two loci is to decrease the probability density at intermediate x, and to increase the weight at the extremes. The rate of transition between the four states is slowed by recombination (as originally shown by Kimura in a two-allele model with irreversible mutation). A very small recombination rate r approximately u2/s is sufficient to increase the mean time between transitions dramatically. In addition to its application to RNA, this model is also relevant to the 'shifting balance' theory describing the drift of populations between alternative equilibria separated by low fitness valleys. Equilibrium values for the frequencies of the different allele combinations in an infinite population are also calculated. It is shown that for low recombination rates the equilibrium is symmetric, but there is a critical recombination rate above which alternative asymmetric equilibria become stable.     

Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species

Macrophage foam cells of atherosclerotic lesions store lipid in lysosomes and cytoplasmic inclusions. Oxidized low density lipoprotein (oxLDL) has been proposed to be the atherogenic particle responsible for the free and esterified cholesterol stores in macrophages. Currently, however, there is a paucity of data showing that oxLDL can induce much cholesterol accumulation in cells. The present studies compare the ability of mildly oxLDL (TBARS = 5 to 10 nmols/mg LDL protein) with acetylated LDL to induce free cholesterol (FC) and esterified cholesterol (EC) accumulation in pigeon, THP-1, and mouse macrophages. Mildly oxLDL stimulated high levels of loading comparable to acLDL where the cellular cholesterol concentrations ranged from 160 to 420 microg/mg cell protein with EC accounting for 52-80% of the cholesterol. Pigeon and THP-1 macrophages stored most (60-90%) of oxLDL cholesterol (both FC and EC) in lysosomes, and the bulk (64-88%) of acLDL cholesterol in cytoplasmic inclusions. Consistent with lysosomal accumulation, cholesterol esterification was 75% less in THP-1 macrophages enriched with oxLDL cholesterol compared with acLDL. Furthermore, addition of an acyl-CoA:cholesterol acyltransferase inhibitor did not significantly affect either cholesterol loading or the percent distribution of FC and EC in THP-1 and pigeon cells incubated with oxLDL. Surprisingly, mouse macrophages stored most of oxLDL (71%) and acLDL (83%) cholesterol within cytoplasmic inclusions. Also, in mouse macrophages, esterification paralleled cholesterol loading, and was 3-fold more in oxLDL treated cells compared with acLDL treated cells. Inhibition of ACAT led to a 62% and 90% reduction in the %EC in oxLDL and acLDL treated mouse macrophages, respectively. The results demonstrate that mildly oxidized low density lipoprotein (oxLDL) stimulates macrophage foam cell formation and lipid engorgement of lysosomes. However, the fate of oxLDL cholesterol markedly differs in macrophages of different species.     

Stimulation of cardiac receptors with veratrum alkaloids inhibits ADH secretion

The purpose of this study was to determine whether cardiac receptors that are stimulated by veratrum alkaloids exert an inhibitory influence on the secretion of vasopressin (ADH). In six chloralose-anesthetized dogs, injection of cryptenamine (2 microgram/kg) into the circumflex coronary artery resulted in a significant (P < 0.05) fall in arterial pressure (-45 +/- 5 mmHg). Despite this hypotension and the presence of intact arterial baroreflexes, there was no change in plasma ADH (measured in the superior vena cava). In eight dogs with sinoaortic baroreceptor denervation, hemorrhage of 10 ml/kg decreased arterial pressure 20 +/- 5 mmHg (P < 0.05) and increased plasma ADH from 14 +/- 5 to 38 +/- 13 microU/ml (P < 0.05). Intracoronary injection of cryptenamine (1 microgram/kg) decreased plasma ADH to 23 +/- 7 microU/ml during the 5 min immediately after cryptenamine injection and to 16 +/- 4 microU/ml 20 min after injection. In eight dogs with sinoaortic denervation, hemorrhage and injection of vehicle resulted in a progressive increase in plasma ADH over the same time period. Vagotomy abolished the inhibitory response to cryptenamine injection. These data show that stimulation of cardiac receptors with vagal afferents by intracoronary injection of a veratrum alkaloid inhibits ADH secretion. Activation of these receptors can prevent arterial baroreflex-induced increases in ADH.     

Ratings of the performances of practicing internists by hospital-based registered nurses

PURPOSE: To examine the feasibility and reliability of ratings completed by hospital-based registered nurses of the humanistic qualities, communication skills, and selected aspects of the clinical skills of practicing internists. METHOD: In 1988-1989, registered nurses who worked in the same 175 hospitals as 232 internists with admitting privileges at these hospitals rated the internists' performances. The nurses were selected from medicine floors, specialty floors, and intensive care units and/or critical care units, using lists provided by head nurses. A total of 1,877 rating questionnaires with 13 performance categories were collected (with a mean of 8.01 nurses per internist). The ratings were analyzed to determine measurement characteristics and the relationships of the nurses' demographic characteristics to the ratings. In addition, for each of ten performance categories for 152 of the internists, the average rating each internist received from nurses was compared with the average rating each internist received from peer physicians. Statistical analysis used Pearson correlations, canonical correlations, factor analyses, Student's t-tests, analysis of variance, and stepwise multiple regression. Finally, the internists themselves, including physicians who were not actually rated by the nurses, were asked to complete a brief questionnaire that included questions about their opinions of the use of nurses' ratings. RESULTS: The nurses' ratings correlated moderately strongly with the peer physicians' ratings and had a common structure. However, the nurses' ratings were lower for several humanistic qualities, including respect, integrity, and responsibility, and their ratings were higher for medical knowledge and verbal communications. Across the 13 performance categories, approximately 10-15 ratings from nurses were needed to obtain a reliable assessment of an internist's humanistic qualities and communication skills. Many internists felt that nurses' ratings should be used equally with, or at least as a lesser contribution to, ratings by peer physicians of humanistic qualities and communication skills. CONCLUSION: Nurses' ratings appear to provide a feasible and reliable method of evaluating the internists' communication skills and humanistic qualities, when used in conjunction with ratings by peer physicians.     

Structure and organization of the human alpha class glutathione S-transferase genes and related pseudogenes

We have isolated and characterized genomic DNA encoding several human Alpha class glutathione S-transferase genes and pseudogenes. All the genes are composed of seven exons with boundaries identical to those of the Alpha class genes in rats. The GSTA1 gene is approximately 12 kb in length and is closely flanked by other Alpha class gene sequences. The complete sequence of the 1.7-kb intergenic region between exon 7 of an upstream pseudogene and exon 1 of the GSTA1 gene has been determined. An additional gene that encodes an uncharacterized Alpha class glutathione S-transferase has been identified. The protein derived from this gene would have 19 amino acid substitutions compared with the GSTA1 isoenzyme. Several pseudogenes with single-base and/or complete exon deletions have been identified, but no reverse-transcribed pseudogenes have been detected. The occurrence of multiple genes and pseudogenes on a single fragment of cloned genomic DNA and the prior identification of a single chromosomal region (6p12) of hybridization (Board and Webb, 1987, Proc. Natl. Acad. Sci. USA 84:2377-2381) suggest that all the Alpha class genes are members of a closely linked gene family that has evolved by duplication and gene conversion events.     

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.