Component-resolved diagnosis with commercially available D. pteronyssinus Der p 1, Der p 2 and Der p 10: relevant markers for house dust mite allergy.

OBJECTIVE: To establish the prevalence and serum levels of IgE to commercial Der p 1, Der p 2, Der p 10 and the carbohydrate MUXF3 in house dust-mite allergic patients. To compare individual vs. allergen microarray methods. METHODS: Prevalence and serum levels of IgE to Dermatophagoides pteronyssinus extract and components Der p 1, Der p 2, Der p 10 and MUXF3, specific IgG4 to D. pteronyssinus, total serum IgE levels, and clinical features (age, asthma, rhinitis and atopic dermatitis) were determined in 123 patients (64 children) with the ImmunoCAP® method. ImmunoCAP ISAC® was performed in 24 patients. RESULTS: All patients had serum IgE to D. pteronyssinus. Prevalences of serum IgE to commercial components were Der p 1 93%, Der p 2 77% (Der p 1 or Der p 2 94%), Der p 10 28% and MUXF3 25%. Levels of D. pteronyssinus IgE strongly correlated with Der p 1 and Der p 2 IgE (r = 0.89 and 0.85 respectively), but not Der p 10 and MUXF3. ImmunoCAP® and ImmunoCAP ISAC® were concordant, but the quantitative correlation was poor. No clinical implication for the prevalence, levels, or molecular IgE reactivity profile to house dust mite components was found. CONCLUSIONS AND CLINICAL RELEVANCE: Commercially available Der p 1 and Der p 2 strongly correlate with IgE D. pteronyssinus. The lack of Der p 1 and Der p 2 IgE may help with differential diagnosis. Der p 10 serum IgE prevalence and levels suggest different patterns in food and mite-related tropomyosin sensitization. Serum IgE to carbohydrate MUXF3, although unexpectedly prevalent, were low and did not modify D. pteronyssinus IgE levels. Follow-up may be best carried out with individual rather than microarrayed components.

Depth matters: cells grown on nano-porous anodic alumina respond to pore depth.

Recent experiments have shown unambiguously that living cells respond to the nano-topography of surfaces they grow on-specifically, the fate of stem cells grown on nano-porous titania or alumina have been shown to be decided by the pore size. However, most experiments have focused on pore size or pitch. Here we show that in addition to pore size and pitch, the depth of the pores has a profound effect on cell morphology and the arrangement of the actin cytoskeleton.

Jet exhaust particles alter human dendritic cell maturation.

OBJECTIVE AND DESIGN: Among combustion-derived air pollutants, little is known about jet kerosene characteristics and effects. MATERIALS AND METHODS: Particles yielded by experimental kerosene combustion in a jet engine were characterized with electron microscopy and X-ray energy dispersive spectroscopy. Immature human monocyte-derived dendritic cells were exposed for 18 h to 10, 25 or 100 µg/mL jet exhaust particles and/or Escherichia coli-derived endotoxin. Antigen-presenting and costimulation molecules (HLA DR, CD40, CD80, CD86, CD11c), tumor necrosis factor-α and interleukin-10 production were measured. RESULTS: The primary particles of jet exhaust are spherical (9.9 nm), carbonaceous and exert an adjuvant effect on human monocyte-derived dendritic cell maturation in vitro. Concomitant particle and endotoxin stimulation induced a high cytokine production with low antigen-presenting molecules; particle contact prior to endotoxin contact led to an opposite phenotype. Finally, low cytokine production and high costimulation molecules were present when particle adjunction followed endotoxin contact. CONCLUSIONS: Jet exhaust particles act as adjuvants to endotoxin-induced dendritic cell maturation, suggesting possible implications for human health and a role for the time pattern of infectious and pollutant interplay.

[Immune profile of elderly patients admitted in a geriatric short care unit]. / Étude du profil immunitaire de sujets âgés hospitalisés en unité de court séjour gériatrique.

INTRODUCTION: Immunosenescence embraces the whole of age-induced changes observed in the immunomodulatory functions of a living organism, and is mostly characterized by a decrease in cell-mediated immunity and important modifications of the immunological repertoire. The impact of the pathology on ageing immunity is poorly understood and few data are available on the immunological status of old polypathological patients. METHODS: We report the results of a prospective study aiming at characterizing several established immunological parameters in patients of 75 years old or more, and admitted for diverse pathologies in a unit of acute geriatric ward. RESULTS: Among the 51 included patients (35 women and 16 men), 90% displayed poly-pathologies. We found a prevalence of 86% of immunological abnormalities, with lymphopenia among 41% of the patients (<1500/mm(3)) and abnormal lymphocytes phenotypes among 95% of the oldest patients (>85 years). A strong skewing towards memory T lymphocytes (CD45RO+) over naive T lymphocytes (CD45RA+) was found in 80% of the cases and inverted CD4/CD8 T cells ratio was observed in 12% of our patients. Vitamin D insufficiency (<30ng/ml), which is frequent among the patients (94%), is a predictive factor for T and B cell lymphopenia. CONCLUSION: Immunological abnormalities are frequent in this frail population and lymphopenia, in particular, could constitute a reinforcing factor of fragility. Vitamin D deficiency could also affect elderly patients' immunity.

[Immunosenescence and infections, myth or reality?]. / Immunosénescence et infections, mythe ou réalité?

The susceptibility of elderly people to infectious diseases is usually associated to increasing risk factors found in young adults. However, the role of immune function ageing is associated with the decline of immune function but this decline is not homogenous. Some functions such as the cellular immune system are altered but others are enhanced such as innate immunity. The important events of immune ageing are modifications of lymphocyte subsets with accumulation of memory cells, decrease in proliferative response, and a chronic inflammatory state. The chronic antigenic load throughout life is responsible for gaps in the antigenic system with a greater sensitivity to new antigens. These immune system changes are all the more important that diseases are severe and that denutrition is associated. These diseases will speed up the ageing process. The interaction between immunosenescence and pathology is an important phenomenon to consider. This review outlines the immune system changes due to ageing, their relationship with diseases of the aged patient, and the consequences of these modifications on vaccination effectiveness.

Early contacts between T lymphocytes and activating surfaces.

Cells continually probe their environment to adapt their behaviour. A current challenge is to determine how they analyse nearby surfaces and how they process information to take decisions. We addressed this problem by monitoring human T lymphocyte attachment to surfaces coated with activating anti-CD3 or control anti-HLA antibodies. Interference reflection microscopy allowed us to monitor cell-to-surface apposition with a few nanometre vertical resolution during the first minutes following contact. We found that (i) when a cell fell on a surface, contact extension was preceded by a lag of several tens of seconds. (ii) During this lag, vertical membrane undulations seemed to generate transient contacts with underlying surfaces. (iii) After the lag period, the contact area started increasing linearly with a rate of about 1.5 µm(2) s(-1) on activating surfaces and about 0.2 µm(2) s(-1) on control surfaces. (iv) Concomitantly with lateral surface extension, the apparent distance between cell membranes and surfaces steadily decreased. These results are consistent with the hypothesis that the cell decision to spread rapidly on activating surfaces resulted from the integration of information yielded by transient contacts with these surfaces generated by membrane undulations during a period of about 1 min.

[Fatal ewe's milk-induced anaphylaxis: laboratory work-up]. / Anaphylaxie fatale au lait de brebis: place de la biologie.

Selective ewe's or goat's milk allergy without cow's milk allergy often presents as a severe picture. Diagnosis and follow-up are facilitated by the use of laboratory tests. Serum tryptase is elevated after mast cell degranulation. Combined determination of serum tryptase and immunoglobulin-E levels are particularly useful in the work-up of anaphylaxis, should it have been fatal. A case of ewe's milk allergy without cow's milk allergy and its laboratory work-up are reported.

Is there a predictable relationship between surface physical-chemical properties and cell behaviour at the interface?

There is much interest in predicting and controlling the outcome of interaction between artificial surfaces and living cells. However, although there is an impressive amount of information on the behaviour of many cell populations deposited on a variety of surfaces, there is presently no available theory to explain or even summarize these data. Indeed, it is not even obvious that such a theory may exist. The aim of the present review is to emphasize the problems encountered when one attempts to build such a theory. Three sequential steps of cell surface interactions are considered: 1) protein adsorption is a preliminary step liable to involve irreversible interaction between the surface and several hundreds of molecular species occurring in blood or plasma. 2) the second step is the formation of adhesive bonds. Several theoretical frameworks were suggested to account for this step, including DLVO theory, physical chemistry of surfaces, and formation of specific ligand-receptor bonds. It is concluded that present evidence supports the latter approach, although this involves serious difficulties. 3) The last step is the triggering of a specific cell program such as apoptosis, proliferation, migration, differentiation or activation. Recent evidence suggests that in addition to the nature and amount of stimulated surface receptors, additional cues such as substratum mechanical or topographical properties may significantly affect cell behaviour.

Assessment of T lymphocyte cytokine production in induced sputum from asthmatics: a flow cytometry study.

BACKGROUND: Asthma results from a bronchial inflammation in which Th2 lymphocytes play a pivotal role, as shown in invasive bronchial biopsies and broncho-alveolar lavages. Induced sputum (IS) is a non-invasive method of recovery of bronchial cells, which can be repeated in the same patients. However, lymphocyte activation has not been studied in IS to date, because of the low number of T cells recovered. Herein we took advantage of flow cytometry, a method suitable for the study of small cell populations, to assess T cell cytokine production in IS. OBJECTIVES: (1) To assess induced sputum T cell cytokine production by flow cytometry in asthmatic subjects and controls. (2) To compare the T cell cytokine production between symptomatic and non-symptomatic asthmatics. METHODS: Thirteen asthmatics and 19 controls were included. Sputum was induced by a hypertonic saline. Sputum cells were stimulated and intracellular IL-13 and IFN-gamma were detected in T cells by flow cytometry. RESULTS: Stimulation induced an increase of IL-13 and IFN-gamma production by T cells. This increase was higher in asthmatics. IL-13-producing T cells were increased in asthmatics after stimulation. In symptomatic asthma, IFN-gamma-producing T cells were in higher proportion than in controlled asthma. CONCLUSION: IS T cell cytokine production indicates a basic Th2 bias in asthma, accompanied during symptoms by a Th1-like activation. These results open the field for longitudinal studies of the variation of T cell activation in asthma.

Increased soluble p55 and p75 tumour necrosis factor-alpha receptors in patients with hepatitis C-associated mixed cryoglobulinaemia.

To investigate whether tumour necrosis factor alpha (TNFalpha) plays a role in the pathogenesis of hepatitis C virus-associated mixed cryoglobulinaemia (HCV-MC), we measured soluble TNFalpha and its soluble p55 (sTNFR1) and p75 (sTNFR2) receptors in the serum of patients with HCV-MC. TNFalpha, sTNFR1 and sTNFR2 were measured in the serum of 32 patients with HCV-MC, 18 patients with hepatitis C without MC (HCV) and 18 healthy volunteers, using specific immunoassays. Correlations between clinical and biological parameters and the concentrations of TNFalpha and sTNFRs were established by studying detailed clinical records of the 32 HCV-MC patients. Although higher, TNFalpha levels were not significantly different in HCV-MC patients compared with healthy or HCV controls. sTNFR1 and sTNFR2, however, were significantly higher in HCV-MC compared with controls or with HCV patients, and higher concentrations of sTNFR1 and sTNFR2 were observed in patients with severe visceral vasculitis, compared with patients with limited purpura. sTNFR1 concentrations positively correlated with fibrinogen levels but TNFalpha, sTNFR1 and sTNFR2 did not correlate with other biological parameters such as rheumatoid factor concentrations, CH50 or C4 values. These data suggest a role for TNFalpha in the pathogenesis of the immune complex-mediated vasculitis associated with HCV-MC.

Cell fitting to adhesive surfaces: A prerequisite to firm attachment and subsequent events.

Cell adhesion usually involves extensive shape reorganization. This process is important because i) it is required for efficient cross-linking of interacting surfaces by adhesion receptors the length of which does not exceed several tens of nanometers and ii) it influences subsequent cell differentiation and activation. This review focuses on the initial phase of cell deformation, preceding the extensive reorganization process known as spreading. This first phase includes local flattening at the micrometer scale and membrane alignment at the nanometer level, resulting in fitting of the cell to an adhesive surface. Three main points are considered. First, experimental methods available to study cell apposition to a surface are described, with an emphasis on interference reflection microscopy. Second, selected experimental evidence is presented to show that there is a quantitative relationship between "adhesiveness" and "contact extension", and some theoretical models aimed at relating these parameters are briefly sketched. Third, experimental data on the kinetics of initial contact extension are described and possible mechanisms for driving this extension are discussed, including nonspecific forces, receptor-mediated interactions, active cell movements or passive membrane fluctuations. It is concluded that both passive physical phenomena and random active cell movements are possible candidates for the initial triggering of contact extension.

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

Venom immunotherapy induces monocyte activation.

BACKGROUND: Venom immunotherapy (VIT) is an efficient treatment of hymenoptera venom allergy. The mechanism of VIT is based on the induction of tolerance of allergen-specific Th2 cells. The mechanisms of this T cell modulation are unknown, and could depend on cytokines produced by other cell types such as interleukin (IL)-12, tumour necrosis factor (TNF)-alpha and IL-10 by monocytes. OBJECTIVE: To assess if VIT modifies the monocyte production of IL-12, TNF-alpha and IL-10 during the 45 first days of treatment. METHODS: Fourteen patients and seven controls were included. Blood samples were taken once in controls and at day (D)1, D30 and D45 of VIT in patients. Monocytes were isolated, cultured with and without lipopolysaccharide (LPS), and the culture supernatant was harvested. IL-10, IL-12 and TNF-alpha were assayed in supernatants by ELISA. RESULTS: Baseline cytokine levels were not statistically different between patients and controls. During treatment, an increase of spontaneous monocyte production of IL-12 and TNF-alpha was observed at D15 and D45. The production of IL-10 increased at D15 and D45 but not significantly. After LPS-stimulation, IL-12, TNF-alpha and IL-10 monocyte production was not modified by VIT. CONCLUSION: VIT induces a monocyte activation characterized by a delayed overproduction of IL-12 and TNF-alpha. These cytokines could be relevant to the inhibition of Th2 cells during VIT. Therefore, VIT-induced tolerance could depend not only on the specific action of venom antigens on T cells, but also on a secondary non-specific action of monocytes.

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.

Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells.

Endothelial cells play a key role in prominent immunological and pathological processes such as leukocyte trafficking, inflammation, atheroma or cancer cell metastasis. Umbilical veins are probably the most widely used source for human endothelial cells, since they are more easily available than many other vessels, they are free from any pathological process and they are physiologically more relevant than many established cell lines. Here, we describe a standard protocol for preparation, maintenance and quality control of these cells.

Comparative CD43 behavior on monocytes and lymphocytes in kidney transplants.

In human cultured monocytic cells stimulated by cytokines, CD43 was demonstrated to exhibit a modification of sialylated epitopes (dys-sialylation) [Soler et al: Leukoc Biol 1997;61:609-618]. Therefore, we chose to investigate CD43 behavior on patients who present pathological status implicating monocytes after renal graft (KTR). We performed flow cytometry after immune staining using monoclonal antibodies to CD43 sialic acid-dependent (L60) and -independent (L10) epitopes. Compared to normal controls, mean fluorescence intensity was never altered on lymphocytes. Conversely, on monocytes, we found different profiles with L60: 26% of patients having normal CD43 expression, 54% displayed decreased values and 20% had a double population of monocytes, the major one being normal and the minor one with a very low staining. Decreased values were more frequent among KTR during the first 3 months following transplantation. L10 immunostaining was not altered on monocytes in patients with low values of CD43 staining by L60, confirming that the mechanism involved was a CD43 dys-sialylation. We investigated a possible role of cyclosporin (CsA) on human monocytic (THP-1) and lymphoid (Jurkat) cell lines. CsA decreases CD43 expression in monocytic and not in lymphoid cell lines and could be responsible for the specific dys-sialylation of KTR monocytes. Whatever, CD43 dys-sialylation might lead to functional abnormalities of monocytes in KTR, possibly involving the adhesion process.

Effect of dietary lipid (soybean lecithin and triacylglycerol) on hepatic F-actin microfilaments in cyclosporine A-treated rats: image analysis by confocal laser scanning microscopy.

We studied and quantified the effect of cyclosporine A on hepatic F-actin on bile canalicular and basolateral membranes in rats fed either soybean lecithin, triacylglycerol-enriched diet, or low-fat diet by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was quite normal in the lecithin-cyclosporine A group but decreased significantly in the other cyclosporine A-treated groups (by 40% and 25% of control in triacylglycerol-cyclosporine A and cyclosporine A groups, respectively). The alteration of F-actin by cyclosporine A, related to cholestasis evidenced by a decrease in bile salt secretion, was prevented by dietary soybean lecithin and amplified by dietary soybean triacylglycerol.

Assessment of the Th1/Th2 paradigm in whole blood in atopy and asthma. Increased IFN-gamma-producing CD8(+) T cells in asthma.

Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (31 atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. Interleukin-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.