A minimally invasive tool to study immune response and skin barrier in children with atopic dermatitis

Atopic dermatitis (AD, atopic eczema) is a very common skin condition affecting 10-20% of children. It affects children of all skin colours and seems to occur more often in Asian children and children with dark skin types. However, most research is performed on children with light skin types. This study, performed in Amsterdam, the Netherlands, aimed to investigate differences between AD in children with dark and light skin types. To study this, the investigators took tape strips from 53 AD children aged 0-12 years and 50 healthy children as control (comparison). Tape stripping is a painless procedure which is ideal to perform in children, in which a small round sticker is attached to the skin. When removing this special sticker, a thin layer of skin cells remains attached to the sticker, allowing the investigators to study several aspects of skin inflammation and skin barrier. The authors found that AD skin from children with light and dark skin have similar levels and types of skin inflammation. However, they found differences in skin barrier markers between these two groups. In light skinned children, markers of good skin barrier were lower in AD skin when compared to healthy children's skin, while in dark AD skin these skin barrier markers were not significantly different from healthy dark skin. This study showed that dark-skinned and light-skinned AD children are similar when it concerns skin inflammation, but in light skinned AD children the skin barrier dysfunction may play an additional role in the development of AD. This suggests that AD in light and dark skin has different mechanisms of development.     

Supraoptic nucleus afferents from the main olfactory bulb--II. Intracellularly recorded responses to lateral olfactory tract stimulation in rat brain slices

To establish the functional nature of the anatomically demonstrated main olfactory bulb inputs to the supraoptic nucleus, electrophysiological responses of intracellularly recorded supraoptic neurons to lateral olfactory tract stimulation were recorded in horizontal slices of basal forebrain and hypothalamus. A total of 71 synaptically influenced neurons were studied in slices from adult rats of both sexes. Of these, 60 cells (84%) were monosynaptically activated by olfactory tract stimulation; seven cells (10%) were activated via polysynaptic pathways; and four cells (6%) were characterized by long latency inhibitory responses. Lucifer Yellow was injected into 64 cells and subsequent immunocytochemical identification of 44 of these neurons showed that both oxytocin and vasopressin cells, in approximately equal numbers, were excited by olfactory stimulation. Polysynaptically mediated excitation, however, was only associated with oxytocin cells (six of the six identified cells). These results corroborate anatomical tract tracing data showing main olfactory bulb efferents to both supraotic neurons and to neurons of the perinuclear zone. Also supported are earlier speculations of olfactory participation in release of oxytocin and vasopressin during various physiological states.     

Recombinant human bactericidal/permeability-increasing protein (rBPI23) is a universal lipopolysaccharide-binding ligand.

A recombinant 23-kDa protein (rBPI23) derived from human bactericidal/permeability-increasing protein (BPI) possesses potent endotoxin-neutralizing abilities in vitro and in vivo. Binding of rBPI23 to those endotoxins (lipopolysaccharides [LPSs]) encountered clinically would be a prerequisite for efficacy in decreasing mortality among patients suffering from gram-negative sepsis and shock, a disease state in which an etiological role for LPS has been implicated. rBPI23 binds well to lipid A (n = 7), to rough-mutant O-chain-deficient LPS (n = 18, Re to Ra chemotypes), to lipid A-core covalently linked to the O chain, to LPSs from clinically relevant serotypes (n = 100), and to bacterial cells (n = 88) of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, the species most often implicated in clinical gram-negative sepsis and shock. Significant binding of rBPI23 to these antigens took place at rBPI23 concentrations of 1 to 500 ng/ml (median, 16 to 32 ng/ml). Binding did not involve 3-deoxy-D-manno-octulosonate of the inner core. Determining the exact epitope recognized by rBPI23 would require further studies with synthetic lipid A substructures. The demonstrated ability of rBPI23 to universally bind LPS provides a sound basis for further testing of its endotoxin-neutralizing abilities, including clinical trials.     

The involvement of nitric oxide synthase in the effect of histamine on guinea-pig airway smooth musclein vitro

The influence of histamine on nitric oxide synthase (NOS) in the development of airway smooth muscle hyperresponsiveness to histamine was investigatedin vitro. In the absence of histamine,N ^G-nitro-l-arginine methyl ester (l-NAME, 100 M) had no significant effect on the basal tone. However, precontraction of the tissues with histamine (0.3 M) resulted in a significant contractile response tol-NAME in the preparations with intact epithelium. Removal of the epithelial layer decreased the responses tol-NAME.l-arginine could partially reverse the contraction produced byl-NAME.l-NAME enhanced the maximal response to histamine, but the sensitivity of the tracheal smooth muscle to histamine was not affected. These results suggest that, in the airway, histamine can activate NOS, resulting in the release of nitric oxide. The latter may be regarded as a local negative modulator to maintain the tissue in a physiological homeostasis.     

Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response

Six overlapping genomic regions of capsid proteins VP1 and VP3 of hepatitis A virus (HAV) inserted into the expression vectors pBD or pUR respectively expressed beta-galactosidase-HAV fusion proteins. The recombinant proteins were poorly soluble so they were difficult to detect by human anti-HAV sera in radioimmunoassay, but the fusion proteins dissolved in sodium dodecyl sulfate reacted with human and rabbit anti-HAV-positive sera in immunoblots. Antisera against VP1 and VP3 recombinant proteins reacted with the respective structural proteins of HAV in immunoblots. Two recombinant proteins, one including the first 120 amino acids of the N-terminus of VP1 and the other containing all of VP1 except for the first 60 N-terminal amino acids, induced a transient neutralizing antibody response in rabbits. Antisera directed against other regions of VP1 and VP3 neither neutralized viral infectivity nor recognized native virus in a competitive radioimmunoassay. However, when immunized animals were challenged with a sub-immunogenic dose of HAV, all animals responded with stable virus-neutralizing antibodies.     

Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system

Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer's patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4(+) T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal, Morganella morganii, to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability of M. morganii to translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4(+), CD45RBlow T cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization with M. morganii was able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation of M. morganii in the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.     

Mycoplasma pulmonis Inhibits Electrogenic Ion Transport across Murine Tracheal Epithelial Cell Monolayers

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by Mycoplasma pulmonis, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of M. pulmonis upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers. M. pulmonis-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na⁺ absorption, cyclic AMP, and cholinergic-stimulated Cl⁻ secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to M. pulmonis was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na⁺ and Cl⁻, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 Ω/cm²; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in M. pulmonis-infected animals. Furthermore, the ability of M. pulmonis to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-M. pulmonis model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport.Mycoplasmas, the smallest free-living prokaryotes, continue to be a significant cause of respiratory infections in a variety of animals, including humans (44). Their limited biosynthetic capability dictates that these organisms must both colonize and parasitize epithelial cell surfaces. It is therefore surprising that mycoplasmas produce diseases that are slowly progressing and chronic and yet often clinically inconspicuous. The molecular mechanisms responsible for this tenuous truce between the pathogen and the host cell have not yet been identified. Murine respiratory mycoplasmosis, a naturally occurring respiratory disease in laboratory rats and mice caused by Mycoplasma pulmonis (6, 8) and characterized by chronic, often lifelong tracheitis, bronchopneumonia, and bronchiectasis (7, 23, 28), would seem to be an ideal model with which to study these mechanisms. Although M. pulmonis-infected animals generate intense local and systemic immune responses (24, 33, 42, 43), they are incapable of eliminating the organism from the respiratory epithelium. Remodeling of the airways typically observed in infected animals, impairment of pulmonary clearance, and accumulation of mucus (23) suggest that M. pulmonis may compromise the ability of the epithelial cells to absorb and secrete fluid and electrolytes.Airway epithelial cells possess two major active transport processes, Na⁺ absorption and Cl⁻ section. Water, in turn, osmotically follows the transepithelial movement of these ions, thereby providing a fluid film between the mucus layer and the epithelial cell surface. The depth and composition of this fluid microenvironment must be carefully regulated to allow the exchange of gases and the humidification of the airway epithelium and to ensure proper mucociliary clearance (47, 49). The consequences of impaired fluid and electrolyte transport in the airways are strikingly apparent in cases of cystic fibrosis, a recessive genetic disease resulting from the loss of epithelial chloride channels (31, 48). The studies reported here were designed to determine whether M. pulmonis infection alters electrolyte transport across the murine tracheal epithelium.The effect of bacteria upon mammalian epithelia in the absence of immune and inflammatory cells can be systematically evaluated by using the Ussing chamber model. Short-circuit current (I_sc) studies have helped determine the mechanism by which cholera toxin (18) and other enterotoxins affect the intestinal mucosa (17, 35). Study of various types of epithelial cells by using the Ussing chamber model has also resulted in the identification of numerous microbial substances capable of directly altering the ion transport capacity of the airway epithelium (3, 20, 41).In the present study, control and M. pulmonis-infected mouse tracheal epithelial cells (MTEs) were evaluated in Ussing chambers. The results clearly show that M. pulmonis infection directly alters epithelial ion transport and that this alteration is species specific, dose and time related, and dependent upon the association of viable organisms with the apical cell surface.