Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease

BACKGROUND & AIMS: Most macrophages in the normal intestinal mucosa have a mature phenotype. In inflammatory bowel disease (IBD), a monocyte-like subset (CD14+ L1+) accumulates. The aim of this study was to characterize its potential with regard to cytokines. METHODS: Lamina propria mononuclear cells were adherence-separated, with or without depletion of CD14+ cells, and production of cytokines was investigated by bioassay, enzyme-linked immunosorbent assay, or immunocytochemistry. RESULTS: Tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-1 receptor antagonist were found mainly in cells positive for myelomonocytic L1. In undepleted IBD cultures, TNF-alpha, IL-1alpha and beta, and IL-10 were markedly up-regulated by pokeweed mitogen stimulation; IL-1alpha and beta and IL-10 were also up-regulated by stimulation of interferon gamma and lipopolysaccharide in combination. The latter stimulation had no effect on normal control or CD14-depleted IBD cultures. Indomethacin caused a marked increase of TNF-alpha, particularly in undepleted IBD cultures, whereas IL-10 and IL-4 decreased TNF-alpha and IL-1beta in both CD14+ and CD14 macrophages. CONCLUSIONS: In IBD mucosa, macrophages with a monocyte-like phenotype are primed for production of TNF-alpha and IL-1alpha/beta and may therefore be of significant pathogenic importance [corrected]. However, this CD14+ subset, as well as the mucosal resident macrophages, have preserved responsiveness to several down-regulatory factors such as the macrophage deactivators IL-10 and IL-4.     

Development of a cell culture system to study antibody convection in tumors

The convective transport of fluid and of a binding antibody through a cultured tumor cell layer was investigated with a mouse melanoma cell line (B16F10) grown on a microporous polycarbonate filter (Snapwell inserts). The inserts were precoated with Matrigel or collagen, or were uncoated. The cell layers were exposed to nominal pressure gradients from 5 to 25 cm H2O, and the volume flux was measured by collecting the effluent volume over time. The rate of convective transport of a binding monoclonal antibody that recognizes the murina transferrin receptor (a-TfR) was investigated at a nominal pressure gradient of 15 cm H2O and compared with that of an isotype matched, nonbinding control. The resistance, R, of the cell layer to fluid flow was quantified as the hydraulic conductivity, Lp (= 1/R); the ability of the cell layer to retard antibody transport was quantified as the reflection coefficient, sigma. The resulting Lp values decreased with increasing cell density, in a manner consistent with Poiseuille flow. Collagen or Matrigel precoating also decreased Lp values, with cells grown on Matrigel providing the greatest resistance. The sigma values were 0.67 (+/-0.08) for the a-TfR antibody and 0.51 (+/-0.06) for the control, indicating that the cell layer acts as a semipermeable barrier to convective transport of antibody that is less permeable to the binding antibody.     

Interaction between proteins containing homeodomains associated to leucine zippers from sunflower

A strategy based on the use of PCR with one degenerate oligonucleotide deduced from conserved sequences and lambda gt10 primers was used to isolate homeobox containing sequences from sunflower stem and root cDNA libraries. Six different partial cDNAs coding for the first 48 amino acids of homeodomains and amino terminal sequences were analyzed and found to be members of the HD-Zip superfamily, which contain a homeobox linked to a leucine zipper coding region. A full-length cDNA clone, Hahb-10, was isolated and characterized. The leucine zipper portions of Hahb-10 and of the previously reported Hahb-1 have been utilized to construct fusions with the N-terminal domain of the lambda repressor. These fusions were tested for their ability to bind to lambda promoters in vivo. The expression of a protein containing an active dimerization domain, but not capable of DNA binding, exerts a dominant negative effect on the ability of repressor-zipper fusions to bind to its target DNA. From these experiments, it was concluded that Hahb-1 and -10, when co-expressed, form preferentially homodimers. Exchange of conserved threonines and leucines at positions a1 and d1 of both zippers reduces dimerization efficiency and allows the formation of heterodimers, suggesting that these residues are, among others, determinants of the specificity of interaction, most likely through changes in hydrophobic packing interactions at the dimer interface. The results imply that a great number of interacting molecular entities compose this protein superfamily which is presumably involved in regulating plant developmental responses.     

Adulthood functioning: the joint effects of parental alcoholism, gender and childhood socio-economic stress

The popular literature has publicized the adjustment difficulties of adult children of an alcohol-dependent parent (ACOAs); however, empirical studies do not provide consistent support. We examined the impact of parental alcoholism, degree of childhood socio-economic stress and gender on three broad categories of adulthood functioning (psychopathology, socio-economic attainment and marital stability). These effects were investigated with a heterogeneous sample of 400 men and 226 women participating in studies at the University of Michigan Alcohol Research Center. Parental alcoholism and childhood socio-economic stress exerted significant independent effects on most adulthood functioning measures. Men and women differed substantially only on socio-economic attainment measures, and effects of parental alcoholism and childhood economic stress on men and women were generally similar. For marital stability, parental alcoholism and childhood socio-economic stress interacted. These results suggest that researchers who study the impact of family history for alcoholism on psychological functioning should consider other aspects of the family of origin that promote wellbeing. In addition, results of this study point to the need for more research on gender differences, protective factors that promote good adjustment and outcome measures reflecting general life adaptation.     

Scratching the (cell) surface: cytokine engineering for improved ligand/receptor trafficking dynamics

Cytokines can be engineered for greater potency in stimulating cellular functions. An obvious test criterion for an improved cytokine is receptor-binding affinity, but this does not always correlate with improved biological response. By combining protein-engineering techniques with studies of receptor trafficking and signaling, it might be possible to identify the ligand receptor-binding properties that should be sought.     

Red flags and religious coping: identifying some religious warning signs among people in crisis

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.     

Conservative operation for infective endocarditis of the mitral valve

BACKGROUND: Subvalvular preservation is necessary to maintain left ventricular function, but accidental retention of infected tissue could cause postoperative endocarditis. METHODS: We examined 71 consecutive patients who underwent operation for mitral endocarditis. Endocarditis was uncontrolled and active in 24 patients, partially treated (unfinished antibiotic course) in 17, and healed in 30. RESULTS: Valves were repaired in 17% versus 59% versus 63% and replaced with subvalvular preservation in 25% versus 6% versus 3% of the uncontrolled active, partially treated, and healed groups, respectively. Thirty-day mortality was 29% versus 0% versus 3.3% (p=0.003), total mortality was 46% versus 18% versus 17% (p=0.035), and complications-related mortality was 38% versus 11% versus 13% (p=0.054), respectively. There was a trend toward lower complications-related mortality with subvalvular preservation than without. Postoperative endocarditis occurred in 3 of 30 patients without and 1 of 41 patients with subvalvular preservation. CONCLUSIONS: Postoperative mortality in uncontrolled active mitral endocarditis remains high, but results are good with partially treated or healed endocarditis. Subvalvular preservation improves outcome, does not increase postoperative endocarditis rates, and should be performed whenever feasible.