Brian Schofield

A murine model of IgE-mediated cow's milk hypersensitivity.

BACKGROUND Cows milk allergy (CMA) is one of the leading causes of food allergy in children. Understanding the mechanisms involved in the development of CMA has been hampered by the lack of suitable animal models. OBJECTIVE We sought to develop a mouse model of IgE-mediated cows milk hypersensitivity (CMH) that mimics the clinical features of immediate CMA in humans. METHODS Three-week-old C3H/HeJ mice were sensitized by intragastric administration of cows milk (CM) plus cholera toxin and boosted 5 times at weekly intervals. RESULTS CM-specific IgE antibody levels were significantly increased at 3 weeks and peaked at 6 weeks after the initial feeding. Intragastric challenge with CM at week 6 elicited systemic anaphylaxis accompanied by vascular leakage, significantly increased plasma histamine, and increased intestinal permeability to casein. Histologic examination of intestinal tissue revealed marked vascular congestion, edema, and sloughing of enterocytes. The role of IgE in mediating CMH was confirmed by abrogation of passive cutaneous anaphylaxis reactions by heat inactivation of immune sera. Development of IgE-mediated CMH in this model is likely to be TH2 cell mediated because in vitro stimulation of spleen cells from mice allergic to CM induced significant increases in the levels of IL-4 and IL-5, but not IFN-gamma. CONCLUSION This model should provide a useful tool for evaluating the immunopathogenic mechanisms involved in CMA and for exploring new therapeutic approaches.

Experimental Autoimmune Myocarditis in A/J mice Is an Interleukin-4-Dependent Disease with a Th2 Phenotype

Myocarditis in humans is often associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen. Experimental autoimmune myocarditis (EAM) is induced in mice by immunization with CM. We found that EAM in A/J mice exhibits a Th2-like phenotype demonstrated by the histological picture of the heart lesions (eosinophils and giant cells) and by the humoral response (association of IgG1 response with disease and up-regulation of total IgE). Blocking interleukin (IL)-4 with anti-IL-4 monoclonal antibody (mAb) reduced the severity of EAM. This reduction in severity was associated with a shift from a Th2-like to a Th1-like phenotype represented by a reduction in CM-specific IgG1; an increase in CM-specific IgG2a; an abrogation of total IgE response; a decrease in IL-4, IL-5, and IL-13; as well as a dramatic increase in interferon (IFN)-gamma production in vitro. Based on the latter finding, we hypothesized that IFN-gamma limits disease. Indeed, IFN-gamma blockade with a mAb exacerbated disease. The ameliorating effect of IL-4 blockade was abrogated by co-administration of anti-IFN-gamma mAb. Thus, EAM represents a model of an organ-specific autoimmune disease associated with a Th2 phenotype, in which IL-4 promotes the disease and IFN-gamma limits it. Suppression of IFN-gamma represents at least one of the mechanisms by which IL-4 promotes EAM.

CpG Oligodeoxynucleotides Can Reverse Th2-Associated Allergic Airway Responses and Alter the B7.1/B7.2 Expression in a Murine Model of Asthma

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-γ concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.

Pathogenesis of Skin Lesions Caused by Sulfur Mustard

Sulfur mustard (SM) (di-2-chlorethyl sulfide), used for chemical warfare in World War I, is a highly reactive radiomimetic alkylating agent. When applied to the skin of rabbits and guinea pigs, it produced vascular leakage, leukocyte infiltration, and slow death of basal epidermal cells. Thirty to sixty minutes after exposure to SM, injury to the superficial microvasculature (beneath the SM application site) was detected by measuring vascular leakage with Evans blue dye and also with horseradish peroxidase. At this same time, injury to the superficial fibroblasts was observed ultrastructurally; and an unexpectedly high percentage of basophils was found among the early infiltrating granulocytes. At 2 to 4 hr, the vascular leakage ceased, and had resumed by 8 hr in a more diffuse form. At this time, the basal epidermal cells showed pyknotic nuclei, an increase in their lysosomal enzymes (observed histochemically), and autophagic vacuoles (observed ultrastructurally). Leukocyte infiltration was marked, consisting mostly of heterophils (PMN) with a reduced percentage of basophils. During the next 24 to 72 hr, the entire inflammatory reaction reached its peak; and a superficial, crust-covered ulcer developed. Then, over the next 10 days, the lesion gradually subsided with concomitant repair and healing. Glucocorticosteroids decreased the early edematous phase, but did not affect the rate of healing. These findings suggest that the skin response to sulfur mustard has an immediate and a delayed phase. The immediate phase, i.e., within the first hour, was characterized by injury to the superficial fibroblasts and to the endothelium of superficial capillaries and venules, possibly because of direct damage to their cell membranes. At this time, a restricted vascular leakage and a selective granulocyte infiltration containing many basophils occurred. The delayed phase, which became evident after 8 hr, was characterized by the death of basal epidermal cells, probably because of DNA damage. This phase was accompanied by generalized vascular leakage, by massive heterophil immigration, and eventually by ulceration.

Inactivation of lysosomal function in normal cultured human fibroblasts by chloroquine

Abstract The lysosomal function of mucopolysaccharide degradation in normal cultured, living human fibroblasts can specifically be inhibited without affecting over-all cell viability or growth. In an earlier report we have shown that an increase in medium pH will progressively inhibit this function. The present paper demonstrates that chloroquine in concentrations around 1–2 × 10−5 M strongly interferes with mucopolysaccharide degradation in the living cell. Electron microscopy shows that a morphological picture resembling a lysosomal storage disease develops in these cells after less than 2 days exposure to the drug. We propose that the antimalarial effect of chloroquine (and preliminary results would include quinacrine and quinine) and at least some of the major side effects of the drug are due to the same basic mechanism: an inhibition of normal lysosomal activities in the parasite as well as in the human cell.

Electron microscopic localization of hydrolytic enzymes in osteoclasts

SynopsisAcid glycerophosphatase activity (pH optimum, 5.0) has been found within osteoclasts by numerous workers but relatively few studies have been concerned with the neutral hydrolytic enzymes that have pH optima around 7.2. Evidence is presented in this paper to show that neutral enzyme activity can be demonstrated withp-nitrophenyl phosphate, ATP andp-chloranilidophosphonate as substrates. Activity against β-glycerophosphate, inorganic trimetaphosphate orp-nitrocatachol sulphate as substrates was found to be limited to an acid pH range.Electron microscopic evidence indicated that many of the hydrolytic enzymes were present within single membrane-bound bodies, vacuoles, Golgi elements, and the agranular endoplasmic reticulum of the osteoclast. This reticulum was dilated to form large lysosomes. Such activity was distingly different from the function of the Golgi membrane in its formation of primary lysosomes.The relationships between lysosomes, cytoplasmic vacuoles, and extracellular release of enzyme through the specialized ‘ruffled’ border are shown and discussed.

Histochemical demonstration of enzyme activities in plastic and paraffin embedded tissue sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, beta-galactosidase, and cytochrome oxidase in plastic embedded and ruotine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polysciences JB-4) and cut at 2 micrometers, were far superior to frozen sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.

Presence of nicotinic acetylcholine receptors in cat carotid body afferent system.

With immunocytochemical techniques using a monoclonal antibody for alpha7 subunits of neuronal nicotinic acetylcholine receptors, we have found these subunits to be exclusively expressed in nerve fibers in the carotid body. Double-immunostaining showed that alpha7 subunit-positive nerve endings enveloped tyrosine hydroxylase-positive glomus cells. Some carotid sinus nerve fibers and tyrosine hydroxylase-positive petrosal ganglion neurons also expressed alpha7 subunits. These data support a role for acetylcholine in carotid body neurotransmission.

Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentration principle

SynopsisThe critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0m MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH.Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material was the same in tissue in which Alcian Blue was added to the fixative and tissue stained after fixation in the absence of MgCl2. This stained material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones.A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5m MgCl2. In these cases, however, the stained material exhibited a ribbonlike configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0m resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size.The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associeated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.

Effect of ageing on pulmonary inflammation, airway hyperresponsiveness and T and B cell responses in antigen‐sensitized and ‐challenged mice

Background The effect of ageing on several pathologic features of allergic asthma (pulmonary inflammation, eosinophilia, mucus hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.