Trogocytosis-based generation of suppressive NK cells

Trogocytosis is a fast uptake of membranes and associated molecules from one cell by another. Trogocytosis between natural killer (NK) cells and tumors is already described, but the functional relevance of NK-tumor targets material exchange is unclear. We investigated whether the immunosuppressive molecule HLA-G that is commonly expressed by tumors in vivo and known to block NK cytolytic function, could be transferred from tumor cells to NK cells, and if this transfer had functional consequences. We show that activated NK cells acquire HLA-G1 from tumor cells, and that upon this acquisition, NK cells stop proliferating, are no longer cytotoxic, and behave as suppressor cells. Such cells can inhibit other NK cells' cytotoxic function and protect NK-sensitive tumor cells from cytolysis. These data are the first demonstration that trogocytosis of HLA-G1 can be a major mechanism of immune escape that acts through effector cells made to act as suppressor cells locally, temporarily, but efficiently. The broader consequences of membrane sharing between immune and non-immune cells on the function of effectors and the outcome of immune responses are discussed.     

In vitro and in vivo characterization of alkyl hydroperoxide reductase mutant strains of Helicobacter hepaticus

Mutant strains in the tsaA gene encoding alkyl hydroperoxide reductase were more sensitive to O₂ and to oxidizing agents (paraquat, cumene hydroperoxide and t-butylhydroperoxide) than the wild type, but were markedly more resistant to hydrogen peroxide. The mutant strains resistance phenotype could be attributed to a 4-fold and 3-fold increase in the catalase protein amount and activity, respectively compared to the parent strain. The wild type did not show an increase in catalase expression in response to sequential increases in O₂ exposure or to oxidative stress reagents, so an adaptive compensatory mutation has probably occurred in the mutants. In support of this, chromosomal complementation of tsaA mutants restored alkyl hydroperoxide reductase, but catalase was still up-expressed in all complemented strains. The katA promoter sequence was the same in all mutant strains and the wild type. Like its Helicobacter pylori counterpart strain, a H. hepaticus tsaA mutant contained more lipid hydroperoxides than the wild type strain. Hepatic tissue from mice inoculated with a tsaA mutant had lesions similar to those inoculated with the wild type, and included coagulative necrosis of hepatocytes. The liver and cecum colonizing abilities of the wild type and tsaA mutant were comparable. Up-expression of catalase in the tsaA mutants likely permits the bacterium to compensate (in colonization and virulence attributes) for the loss of an otherwise important oxidative stress-combating enzyme, alkyl hydroperoxide reductase. The use of erythromycin resistance insertion as a facile way to screen for gene-targeted mutants, and the chromosomal complementation of those mutants are new genetic procedures for studying H. hepaticus.     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

A conserved sequence immediately N-terminal to the Bateman domains in AMP-activated protein kinase gamma subunits is required for the interaction with the beta subunits

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta, and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma1, gamma2, and gamma3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alphabetagamma complex. This region is required for the interaction with the beta subunits. The interaction between the alpha and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the alpha-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.     

Activation of 5-HT1A receptors in the paragigantocellularis lateralis decreases shivering during cooling in the conscious piglet

Activation of 5-HT(1A) receptors in the medullary raphé decreases sympathetic outflow to thermoregulatory mechanisms, including brown adipose tissue (BAT), thermogenesis, and peripheral vasoconstriction when these mechanisms are previously activated with leptin, prostaglandins, or cooling. These same mechanisms are also inhibited during rapid eye movement (REM) sleep. It is not known whether shivering is also modulated by medullary raphé neurons. We previously showed in the conscious piglet that activation of 5-HT(1A) receptors with 8-OH-DPAT (DPAT) in the paragigantocellularis lateralis (PGCL), a medullary region lateral to the midline raphé that contains 5-HT neurons, decreases heart rate, body temperature and muscle activity during non-rapid eye movement (NREM) sleep. We therefore hypothesized that activation of 5-HT(1A) receptors in the PGCL would also attenuate shivering and peripheral vasoconstriction during cooling. During REM sleep in a cool environment, shivering, carbon dioxide production, and body temperature decreased, and ear capillary blood flow and ear skin temperature increased. Shivering associated with rapid cooling was attenuated after dialysis of DPAT into the PGCL. In animals maintained in a continuously cool environment, dialysis of DPAT into the PGCL attenuated shivering and decreased body temperature, but there were no significant increases in ear capillary blood flow or ear skin temperature. We conclude that both naturally occurring REM sleep and exogenous activation of 5-HT(1A) receptors in the PGCL are associated with a suspension of shivering during cooling. Our data are consistent with the hypothesis that 5-HT neurons in the PGCL facilitate oscillating spinal motor circuits involved in shivering but are less involved in modulating sympathetically mediated thermoregulatory mechanisms.     

The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing

BACKGROUND: Epithelial junctional networks assume packing geometries characterized by different cell shapes, neighbor number distributions and areas. The development of specific packing geometries is tightly controlled; in the Drosophila wing epithelium, cells convert from an irregular to a hexagonal array shortly before hair formation. Packing geometry is determined by developmental mechanisms that likely control the biophysical properties of cells and their interactions. RESULTS: To understand how physical cellular properties and proliferation determine cell-packing geometries, we use a vertex model for the epithelial junctional network in which cell packing geometries correspond to stable and stationary network configurations. The model takes into account cell elasticity and junctional forces arising from cortical contractility and adhesion. By numerically simulating proliferation, we generate different network morphologies that depend on physical parameters. These networks differ in polygon class distribution, cell area variation, and the rate of T1 and T2 transitions during growth. Comparing theoretical results to observed cell morphologies reveals regions of parameter space where calculated network morphologies match observed ones. We independently estimate parameter values by quantifying network deformations caused by laser ablating individual cell boundaries. CONCLUSIONS: The vertex model accounts qualitatively and quantitatively for the observed packing geometry in the wing disc and its response to perturbation by laser ablation. Epithelial packing geometry is a consequence of both physical cellular properties and the disordering influence of proliferation. The occurrence of T2 transitions during network growth suggests that elimination of cells from the proliferating disc epithelium may be the result of junctional force balances.     

Challenges of microsatellite isolation in fungi

Although they represent powerful genetic markers in many fields of biology, microsatellites have been isolated in few fungal species. The aim of this study was to assess whether obtaining microsatellite markers with an acceptable level of polymorphism is generally harder from fungi than in other organisms. We therefore surveyed the number, nature and polymorphism level of published microsatellite markers in fungi from the literature and from our own data on seventeen fungal microsatellite-enriched libraries, and in five other phylogroups (angiosperms, insects, fishes, birds and mammals). Fungal microsatellites indeed appeared both harder to isolate and to exhibit lower polymorphism than in other organisms. This appeared to be due, at least in part, to genomic specificities, such as scarcity and shortness of fungal microsatellite loci. A correlation was observed between mean repeat number and mean allele number in the published fungal microsatellite loci. The cross-species transferability of fungal microsatellites also appeared lower than in other phylogroups. However, microsatellites have been useful in some fungal species. Thus, the considerable advantages of these markers make their development worthwhile, and this study provides some guidelines for their isolation.     

Immunological determinants of ventilatory changes induced in mice by dermal sensitization and respiratory challenge with toluene diisocyanate

The objective of the study was to characterize better the immunologic mechanisms underlying a previously developed animal model of chemical-induced asthma. BALB/c and severe combined immunodeficiency disease (SCID) mice received toluene diisocyanate (TDI) or vehicle on each ear on day 1 and/or day 7. On day 10, they were intranasally challenged with TDI or vehicle. Ventilatory function was monitored by whole body plethysmography for 40 min after challenge. Reactivity to methacholine was measured 23 h later: enhanced pause and actual resistance measurements. Pulmonary inflammation was assessed 1, 6, and 24 h after challenge by bronchoalveolar lavage (BAL). Tumor necrosis factor-alpha and macrophage inflammatory protein (MIP)-2 levels were measured in BAL. Immunological parameters included total IgE, IgG1, and IgG2a in serum, lymphocyte populations in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells, cultured with or without concanavalin A. Ventilatory changes suggestive of airway obstruction and increased methacholine reactivity were observed in all TDI-sensitized and TDI intranasally instilled mice, except in SCID mice. A neutrophil influx, accompanied by an increase in MIP-2 levels, was found in BAL of all responding groups 6 and 24 h after intranasal challenge. In BALB/c mice an increased level of CD19+ B cells was found in the auricular lymph nodes. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular lymph node cells from BALB/c mice completely treated with TDI. These results indicate that our model is dependent on the presence of lymphocytes, but it is not characterized by a preferential stimulation of Th1 or Th2 lymphocytes.