Toll-like receptor 2 activators modulate oral tolerance in mice.

BACKGROUND: Toll-like receptor 2 (TLR2) is a widely expressed pattern recognition receptor critical for innate immunity. TLR2 is also a key regulator of mucosal immunity implicated in the development of allergic disease. TLR2 activators are found in many common foods, but the role of TLR2 in oral tolerance and allergic sensitization to foods is not well understood. OBJECTIVE: The purpose of this study was to evaluate the impacts of TLR2 expression and TLR2 activation on oral tolerance to food antigens in a murine model. METHODS: Mice were fed ovalbumin (OVA) or peanut butter with or without the addition of low doses of TLR2 activators Pam3 CSK4 or FSL-1. Oral tolerance was assessed by analysing antibody responses after a systemic antigen challenge. OVA-specific Tregs were assessed in the Peyer's patches, mesenteric lymph nodes, and spleen in wild-type and TLR2(-/-) mice. Low-dose Pam3 CSK4 was also tested as an oral adjuvant. RESULTS: Oral tolerance was successfully induced in both wild-type and TLR2(-/-) recipient mice, with an associated regulatory T-cell response. Oral TLR2 activation, with low-dose Pam3 CSK4 or FSL-1, during oral antigen exposure was found to alter oral tolerance and was associated with the development of substantial IgE and IgA responses to foods upon systemic challenge. Low-dose oral Pam3 CSK4 treatment also selectively enhanced antigen-specific IgA responses to oral antigen exposure. CONCLUSIONS AND CLINICAL RELEVANCE: TLR2 is not necessary for oral tolerance induction, but oral TLR2 activation modulates humoral IgE and IgA responses during tolerance development. Low-dose Pam3 CSK4 is also an effective oral adjuvant that selectively enhances IgA production. These observations are pertinent to the optimization of oral allergen immunotherapy and oral vaccine development.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.

Prospective study of infantile haemangiomas: incidence, clinical characteristics and association with placental anomalies.

BACKGROUND: The aetiology and exact incidence of infantile haemangiomas (IHs) are unknown. Prior studies have noted immunohistochemical and biological characteristics shared by IHs and placental tissue. OBJECTIVES: We investigated the possible association between placental anomalies and the development of IHs, as well as the demographic characteristics and other risk factors for IHs. PATIENTS AND METHODS: Pregnant women (n = 578) were prospectively enrolled and their offspring followed for 9 months. Placental evaluations were performed and demographic data collected on all mother-infant pairs. RESULTS: We evaluated 594 infants: 34 haemangiomas [either IH or congenital (CH)] were identified in 29 infants, yielding an incidence of 4·5% for IH (27 infants) and 0·3% for CH (two infants). Placental anomalies were noted in almost 35% of haemangioma-related pregnancies, approximately twice the incidence noted in pregnancies with unaffected infants (P = 0·025). Other risk factors for IH included prematurity (P = 0·016) and low birth weight (P = 0·028). All IHs were present by 3 months of age, and cessation of growth had occurred in all by 9 months of age. Most occurred on the trunk. Of note, 20% of identified IHs were abortive or telangiectatic in nature, small focal lesions that did not proliferate beyond 3 months of age. Only one IH required intervention. CONCLUSIONS: This is the first prospective American study to document the incidence of IHs in infants followed from birth to early infancy. The association with placental anomalies was statistically significant. The overall incidence mirrors prior estimates, but the need for treatment was lower than previously reported.

Structural analysis of red blood cell aggregates under shear flow.

A set of measures of red blood cell (RBC) aggregates are developed and applied to examine the aggregate structure under plane shear and channel flows. Some of these measures are based on averages over the set of red blood cells which are in contact with each other at a given time. Other measures are developed by first fitting an ellipse to the planar projection of the aggregate, and then examining the area and aspect ratio of the fit ellipse as well as the orientations of constituent RBCs with respect to the fit ellipse axes. The aggregate structural measures are illustrated using a new mesoscale computational model for blood cell transport, collision and adhesion. The sensitivity of this model to change in adhesive surface energy density and shear rate on the aggregate structure is examined. It is found that the mesoscale model predictions exhibit reasonable agreement with experimental and theoretical data for blood flow in plane shear and channel flows. The new structural measures are used to examine the differences between predictions of two- and three-dimensional computations of the aggregate formation, showing that two-dimensional computations retain some of the important aspects of three-dimensional computations.

Effect of particle collisions and aggregation on red blood cell passage through a bifurcation.

Blood flow through bifurcating vessels in the microvasculature leads to uneven distribution of red blood cells (RBC) in the downstream channels when the channels have different sizes or the flow rates through the channels are different. This phenomenon, known as plasma skimming, is responsible for the large variation in hematocrit throughout the circulatory system. Furthermore, the strong streamline curvature present within bifurcations leads to frequent collisions between the blood elements (red and white blood cells and platelets) and the vessel walls, as well as a rearrangement of the distribution of the blood elements in the channels downstream of the bifurcation. Computational models of bifurcation flows typically neglect collision and adhesion of RBCs with each other. In this paper, we use a new type of discrete-element model to investigate the effect of particle-particle collisions and RBC aggregation on modeling of plasma skimming in bifurcations. Cases are examined with and without RBC adhesion and for different hematocrit values, including validation against previous computational results. Results show significant plasma skimming in the low-flow daughter branch and an increase in fractional particle flux with increased hematocrit, as in experimental studies. Accounting for particle-particle collisions leads to a considerable increase in collision rate of particles with the vessel wall. Particles are found to approximately follow fluid velocity streamlines, and consequently particle-particle collisions and aggregation do not significantly affect plasma skimming.

Micro-scale dynamic simulation of erythrocyte-platelet interaction in blood flow.

Platelet activation, adhesion, and aggregation on the blood vessel and implants result in the formation of mural thrombi. Platelet dynamics in blood flow is influenced by the far more numerous erythrocytes (RBCs). This is particularly the case in the smaller blood vessels (arterioles) and in constricted regions of blood flow (such as in valve leakage and hinge regions) where the dimensions of formed elements of blood become comparable with that of the flow geometry. In such regions, models to predict platelet motion, activation, aggregation and adhesion must account for platelet-RBC interactions. This paper studies platelet-RBC interactions in shear flows by performing simulations of micro-scale dynamics using a computational fluid dynamics (CFD) model. A level-set sharp-interface immersed boundary method is employed in the computations in which RBC and platelet boundaries are tracked on a two-dimensional Cartesian grid. The RBCs are assumed to have an elliptical shape and to deform elastically under fluid forces while the platelets are assumed to behave as rigid particles of circular shape. Forces and torques between colliding blood cells are modeled using an extension of the soft-sphere model for elliptical particles. RBCs and platelets are transported under the forces and torques induced by fluid flow and cell-cell and cell-platelet collisions. The simulations show that platelet migration toward the wall is enhanced with increasing hematocrit, in agreement with past experimental observations. This margination is seen to occur due to hydrodynamic forces rather than collisional forces or volumetric exclusion effects. The effect of fluid shear forces on the platelets increases exponentially as a function of hematocrit for the range of parameters covered in this study. The micro-scale analysis can be potentially employed to obtain a deterministic relationship between fluid forces and platelet activation and aggregation in blood flow past cardiovascular implants.

Two-dimensional dynamic simulation of platelet activation during mechanical heart valve closure.

A major drawback in the operation of mechanical heart valve prostheses is thrombus formation in the near valve region. Detailed flow analysis in this region during the valve closure phase is of interest in understanding the relationship between shear stress and platelet activation. A fixed-grid Cartesian mesh flow solver is used to simulate the blood flow through a bi-leaflet mechanical valve employing a two-dimensional geometry of the leaflet with a pivot point representing the hinge region. A local mesh refinement algorithm allows efficient and fast flow computations with mesh adaptation based on the gradients of the flow field in the leaflet-housing gap at the instant of valve closure. Leaflet motion is calculated dynamically based on the fluid forces acting on it employing a fluid-structure interaction algorithm. Platelets are modeled and tracked as point particles by a Lagrangian particle tracking method which incorporates the hemodynamic forces on the particles. A platelet activation model is included to predict regions which are prone to platelet activation. Closure time of the leaflet is validated against experimental studies. Results show that the orientation of the jet flow through the gap between the housing and the leaflet causes the boundary layer from the valve housing to be drawn in by the shear layer separating from the leaflet. The interaction between the separating shear layers is seen to cause a region of intensely rotating flow with high shear stress and high residence time of particles leading to high likelihood of platelet activation in that region.

The myeloid differentiation factor 88 is dispensable for the development of a delayed host response to Pseudomonas aeruginosa lung infection in mice.

Because MyD88 transduces a core set of Toll-like receptor (TLR)-induced signals, microbial-induced host responses can be divided broadly into the MyD88-dependent and MyD88-independent pathways. A specific pathogen induces a distinct pattern of host response dependent upon the signalling pathways employed. Recently, we demonstrated that a MyD88-dependent pathway is essential for the development of early (4-8 h) host response to Pseudomonas aeruginosa lung infection. Here, we show that the development of a delayed (24-48 h) host response to P. aeruginosa is independent of MyD88. Using MyD88-deficient mice, the production of macrophage inflammatory protein 2, tumour necrosis factor and interleukin 1alpha in the airway was observed following P. aeruginosa lung infection for 24 or 48 h. Moreover, the MyD88-deficient mice recruited sufficient neutrophils in the lung and cleared the bacteria efficiently from the lung after 48 h. Thus, the full development of host responses to P. aeruginosa lung infection involves, in a sequential, stepwise fashion, a MyD88-dependent early response and a MyD88-independent delayed mechanism.

A Th1-inducing adjuvant, MPL, enhances antibody profiles in experimental animals suggesting it has the potential to improve the efficacy of allergy vaccines.

BACKGROUND: Monophosphoryl lipid A (MPL) is a detoxified derivative of the lipopolysaccharide (LPS) moiety of Salmonella minnesota R595, which has retained immunostimulatory activities. MPL has been administered to many subjects in clinical trials as an adjuvant component of infectious disease vaccines and is currently a component of a licensed cancer vaccine, Melacine (Corixa Inc., Schering Plough). MPL has, in particular, been shown to promote Th1-type antigen specific responses. L-tyrosine is a depot adjuvant which is fully metabolisable and has been successfully employed in allergy vaccines for a number of years. METHODS: Mice were immunised with MPL adjuvant in conjunction with separate preparations of either ovalbumin or glutaraldehyde-modified ragweed pollen extract both coprecipitated with L-tyrosine. The specific antibody isotypes IgG1, IgG2a, IgG2b and also IgE were measured. Rats received booster injections of keyhole limpet haemocyanin (KLH) in conjunction with MPL adjuvant following priming with KLH in alum alone. KLH-specific antibody responses were measured. RESULTS: It was shown that a combination of L-tyrosine and MPL were synergistic in enhancing murine antigen specific IgG antibody responses without enhancing antigen specific IgE responses. Furthermore, this adjuvant combination promoted strong IgG2 antigen specific responses indicative of a Th1 directed response. In KLH sensitised rats, treatment with MPL was shown to prevent a secondary IgE antibody response when injected with booster injections of antigen. CONCLUSIONS: Immunisation of mice with two different antigens adsorbed to L-tyrosine induced a Th1-skewed primary response when in conjunction with MPL adjuvant which also generally enhances a specific IgG response. Incorporation of MPL adjuvant in the immunisation of rats prevented a secondary specific IgE response. These results suggest that the employment of this new adjuvant in clinical allergy vaccination formulations may result in an improved efficacy which could be utilised in various ways to improve compliance.

Structure and expression of the genes encoding proteins resident in large peripheral vesicles of Phytophthora cinnamomi zoospores.

Zoospores of Phytophthora spp. contain several characteristic types of peripheral vesicles. One of these, large peripheral vesicles, has been proposed to act as a nutrient store and in P. cinnamomi has been shown to contain three immunologically related high-molecular-weight proteins, designated LPVs. We have used antibodies directed against P. cinnamomi zoospores and cysts to isolate several cDNA clones which are products of the Lpv genes and encode one or more of the LPV proteins present in large peripheral vesicles. Northern blot analysis demonstrated the presence of three large Lpv transcripts (11-14 kb) in RNA isolated from hyphae which had been induced to form sporangia. Coordinate accumulation of the three transcripts occurred after induction of sporangium formation: no transcript was observed in uninduced hyphae and maximum transcript levels of all three transcripts were seen 4-6 h after induction. Genomic Southern blots indicated that P. cinnamomi contains three Lpv genes, presumably corresponding to the three transcripts and proteins seen in Northern and Western blots, respectively. Partial genomic clones representing two of the Lpv genes were isolated and characterized by restriction mapping and partial DNA sequencing. In the regions sequenced, the genes were > 99% identical, the high degree of conservation extending at least 415 bp downstream of their polyadenylation sites. The Lpv coding regions contained a variable number (approximately 12-18) of highly conserved 534 bp repeats, flanked by apparently unique sequences. Variation in the number of repeats in the Lpv genes was responsible for the different sizes of the three transcripts and proteins. Database searches using the Lpv nucleotide and deduced amino acid sequences failed to detect any similar sequences. We discuss the molecular events which may have been involved in the evolution of the Lpv genes and the nature of the products of these genes.

Toll-like receptor 4-mediated activation of murine mast cells.

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are critical for cellular responses to a variety of bacterial, viral, and fungal products. Mast cells are important to host survival in a number of models of bacterial infection and might act as sentinel cells in host defense. We therefore examined the expression of TLRs and associated molecules by murine bone marrow-derived mast cells (BMMCs). BMMCs and the murine mast cell line MC/9 expressed mRNA for TLR2, TLR4, and TLR6 but not TLR5 and for both adapter molecule MD-2 and signaling molecule MyD88 but lacked surface CD14. After activation with the TLR2- and TLR4-dependent stimuli Staphylococcus aureus-derived peptidoglycan and Escherichia coli-derived lipopolysaccharide (LPS), respectively, mast cells produced significant levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). To determine whether mast cells require TLR4 for cellular responses to LPS, mast cells were derived from the bone marrow cells of C3H/HeJ and C57Bl/10ScNCr mice containing a point mutation and a null mutation, respectively, in TLR4. Using these models, we demonstrated that the BMMC IL-6 and TNF-alpha responses to LPS were completely dependent on functional TLR4 with no significant LPS response observed in its absence. These findings have important implications for the mechanism of mast cell responses to pathogens and their products and suggest that different TLR4-expressing cells might have different thresholds for activation with LPS.

Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi.

The oomycete genus Phytophthora contains some of the world's most devastating plant pathogens. We report here the existence in P. cinnamomi of four genes encoding the pyrophosphate-utilizing glycolytic/gluconeogenic enzyme pyruvate, phosphate dikinase (PPDK). The coding regions of the four genes are >99% identical. At least three of the genes comprise a small gene cluster, which may have arisen through recent gene duplication and inversion events. Levels of Pdk mRNA are low in vegetative hyphae, but increase rapidly and transiently upon transfer of cultures to nutrient-free media, conditions that trigger asexual sporulation. PPDK protein and enzyme activity levels do not show a similar increase during sporulation. Assays of PPDK activity in P. cinnamomi hyphal extracts suggest that the majority of glycolytic flux in sporulating hyphae probably occurs via PPDK, rather than pyruvate kinase. This finding, combined with the existence of Phytophthora-expressed sequence tags encoding two other pyrophosphate-utilizing enzymes, indicates that pyrophosphate-based metabolism may be important in Phytophthora. The possibility that PPDK and other enzymes of pyrophosphate-based metabolism may provide targets for the development of novel control measures for Phytophthora and other oomycete pathogens is discussed.

SDF-1 induces IL-8 production and transendothelial migration of human cord blood-derived mast cells.

BACKGROUND: Mast cell numbers and expression of chemokines are known to increase in the context of angiogenesis and inflammation, but the mechanisms by which this occurs are not understood. Stromal-derived factor-1 (SDF-1) is an important chemokine in angiogenesis and cell migration. The effects of SDF-1 on human mast cells were examined. METHODS: Expression of the SDF-1 receptor CXC chemokine receptor 4 (CXCR4) on mast cells was examined by RT-PCR and flow cytometry. The ability of labeled cord blood-derived mast cells to migrate across HUVEC monolayers in response to SDF-1 was determined. The cytokine and chemokine responses of cord blood-derived mast cells to SDF-1 treatment over 24 h were examined by ELISA. RESULTS: Cord blood-derived human mast cells expressed the CXCR4 receptor for SDF-1 and migrated across HUVEC monolayers in response to this chemokine. Treatment of cord blood-derived mast cells with SDF-1 did not induce degranulation or the production of several cytokines but did induce a highly selective IL-8 response. CONCLUSION: Human mast cells can both migrate across vascular endothelium and produce the pro-angiogenic chemokine IL-8 in response to SDF-1. These responses may be important in angiogenic processes.

Perioperative factors as predictors of operative mortality and morbidity in pneumonectomy.

Pneumonectomy for lung cancer is associated with significant morbidity and mortality. Risk factors for the morbidity and mortality have been reported, but consistent conclusive data are undetermined. Current accepted 30-day mortality rates for pneumonectomy range from 7 to 11 per cent. The objective of this study is to determine whether various perioperative factors can serve as predictors of morbidity and mortality in pneumonectomy patients and to review outcome data on patients undergoing pneumonectomy for lung cancer. A total of 105 patients undergoing pneumonectomy for lung cancer from 1988 through 1998 are studied in a retrospective chart review. The main outcome measure is the 30-day operative mortality and morbidity. Complications occurring in 10 per cent or more of the patients included atrial fibrillation (33.3%), respiratory failure (23.8%), pneumonia (21.9%), and bronchopleural fistula (12.4%). The 30-day mortality rate was 10.5 per cent (11 deaths). By Fisher's exact test for Chi-square only three statistically significant mortality factors were identified: respiratory failure (P < 0.021), sepsis (P < 0.008), and male sex (P < 0.031); respiratory failure, sepsis, and sex were predictors of death. Significant correlation could not be made to predict postoperative morbidity. Overall long-term clinical outcome for pneumonectomy as lung cancer treatment was poor. Clinical judgment remains an essential factor when considering pneumonectomy as an option for lung cancer treatment.

CpG-containing oligodeoxynucleotides induce TNF-alpha and IL-6 production but not degranulation from murine bone marrow-derived mast cells.

Mast cells are sentinel cells critical to the initiation of innate immune and inflammatory responses, particularly at mucosal surfaces. To fulfill this function they can be activated by several pathogen-associated stimuli to produce cytokines with or without concurrent degranulation. We examined the ability of immunostimulatory DNA sequences including CpG motifs, which are found in increased quantities in bacterial DNA, to activate mouse bone marrow-derived mast cells (mBMMC). Mast cells were treated with a range of doses of CpG-containing oligodeoxynucleotides or control oligodeoxynucleotides without CpG within their sequence. There was a dose-dependent increase in the production of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by mast cells treated with the CpG-containing oligodeoxynucleotides. The cytokine levels induced were directly related to the number of CpG within a given length of sequence. Treatment with oligonucleotides containing 3CpG induced an eightfold increase in TNF production over control incubated mast cells. Other cytokines, including granulocyte-macrophage colony-stimulating factor, IL-4, interferon-gamma, and IL-12 were not induced by oligonucleotide treatment. Neither CpG containing oligodeoxynucleotides nor control oligodeoxynucleotides induced degranulation of mast cells. Bacterial DNA from Escherichia coli also induced IL-6 from mBMMC but neither calf thymus DNA nor methylase-treated E. coli DNA had such an effect. Examination of the uptake of Texas red-labeled CpG and non-CpG-containing oligodeoxynucleotides revealed that they were both similarly taken up by the mBMMC. These results have important implications for the mechanism by which mast cells respond to bacteria and for the potential role of mast cells in DNA vaccination.

Blockade of either alpha-4 or beta-7 integrins selectively inhibits intestinal mast cell hyperplasia and worm expulsion in response to Nippostrongylus brasiliensis infection.

Mast cells are known to express high levels of alpha4 integrins including alpha4beta7 and are found in increased numbers in mucosal inflammation. Mast cell accumulation is particularly prominent in the intestine following nematode infection. The adhesion molecule requirements for this process have not yet been defined. The role of alpha4 and beta7 integrin chains in the intestinal mast cell hyperplasia following infection of rats with the nematode parasite Nippostrongylus brasiliensis was examined in this study. Rats were infected with N. brasiliensis larvae and treated with either anti-alpha4 (TA-2), anti-beta7 or isotype-matched control antibodies. The initial mast cell hyperplasia in response to N. brasiliensis infection was significantly inhibited by either anti-alpha4 or anti-beta7 treatment. In contrast, the intestinal eosinophil response to N. brasiliensis infection was not reduced at day 14 or day 16. Elevations in serum IgE levels due to N. brasiliensis infection were also not inhibited by anti-alpha4 or anti-beta7 antibody treatment. Anti-alpha4 antibody but not anti-beta7 antibody treatment also induced a small but significant decrease in the numbers of mast cells in tongue tissue. These data suggest a role for alpha4 integrins, in particular alpha4beta7, in the regulation of mast cell precursor migration to the intestine.

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism.

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.

Modulation of rat uterine contractility by mast cells and their mediators.

OBJECTIVE: This study was designed to test the possibility that mast cells play a role in the regulation of uterine contractility. STUDY DESIGN: Histamine and rat mast cell protease II levels were determined by radioenzymatic assay and standard radial immunodiffusion techniques, respectively, in uterine tissues from Wistar rats with timed pregnancies. Isolated uterine strips from nonsensitized and ovalbumin-sensitized nonpregnant and pregnant Wistar rats were used for isometric tension recording. Contractile responses to compound 48/80, carbachol, ovalbumin, normal rabbit serum, antirat immunoglobulin E, and 5-hydroxytryptamine were obtained. Antagonists methysergide, ketanserin, 5,8,11, 14-eicosatetraynoic acid, diphenhydramine, and sodium meclofenamate were also used. RESULTS: Tissue levels of rat mast cell protease II and histamine were decreased during delivery compared with prepartum and postpartum levels. Carbachol and compound 48/80 stimulated uterine contractility, and responses were highest during late gestation (day 16 to term). Responses to ovalbumin of uterine tissues in rats sensitized to the antigen were highest at midpregnancy and decreased during the last 10 days of gestation. Ovalbumin challenge in vitro increased the frequency and magnitude of contractions in tissues from ovalbumin-sensitized rats. Compound 48/80 and antirat immunoglobulin E stimulated contractility in both control and sensitized rats. None of the antagonists prevented the contractile responses. CONCLUSIONS: Activation of mast cells is an effective mechanism for stimulation of uterine contractility and may play an important role in the control of term and preterm parturition.

Release of vasoactive cytokines by antibody-enhanced dengue virus infection of a human mast cell/basophil line.

We report here the first demonstration of dengue virus infection and vasoactive cytokine response of a cell of the mast cell/basophil lineage. Infection of KU812 cells was dependent on dengue-specific antibody and gave rise to infectious virions. This antibody-enhanced dengue virus infection triggered a four- to fivefold increase in the release of interleukin-1beta (IL-1beta) and a modest increase for IL-6 but not for an alternate cytokine, granulocyte-macrophage colony-stimulating factor. The results suggest a potential role for mast cells/basophils in the pathogenesis of dengue virus-induced disease.

TNF-alpha dysregulation in asthma: relationship to ongoing corticosteroidtherapy.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is a major proinflammatory cytokine that is thought to be important in the pathogenesis of asthma. However, alterations in systemic regulation of this cytokine in asthma have not been examined in the context of corticosteroid therapy. OBJECTIVES: To examine the ability of peripheral blood mononuclear cells (PBMC) from three different groups of patients with asthma requiring varying amounts of inhaled corticosteroids (ICS) for clinical control, and to examine cells from age- and sex-matched nonasthmatic patients to produce TNF-alpha. DESIGN: All patients with asthma had a positive methacholine challenge test. 'High dose' ICS patients with asthma required ICS greater than or equal to 800 microg/day. 'Medium dose' patients with asthma were on less than or equal to 500 microg/day of ICS, whereas 'no ICS' patients with asthma had received no ICS for at least three months. Each patient with asthma was examined in parallel with an age- and sex-matched, nonasthmatic, nonatopic control subject. Cells were cultured (with or without the addition of potential stimulators phytohemagglutinin, lipopolysaccharide, formyl-methionine-leucine-phenylalanine or antihuman CD3), and TNF-alpha production was assessed by ELISA. MAIN RESULTS: PBMC from both high dose ICS (n=8) and no ICS (n=11) patients with asthma produced more than twice the amount of TNF-alpha than cells from matched nonasthmatic control patients (P<0.01) when cultured alone or in the presence of each stimulus (P<0.05). In contrast, there was no significant difference in TNF-alpha production between medium dose ICS patients with asthma and control patients. A group of asymptomatic atopic patients (n=6) did not have an increased level of TNF-alpha production. CONCLUSIONS: Increases in TNF-a production within the PBMC compartment can be observed in both patients with asthma receiving high dose ICS and in a group of patients with mild asthma receiving no ICS therapy, but not in patients with asthma receiving a medium dose of ICS or atopic patients.