Constance Oliver

Cockroach allergens and asthma in Brazil: Identification of tropomyosin as a major allergen with potential cross- reactivity with mite and shrimp allergens

BACKGROUND Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil. OBJECTIVE The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens. METHODS Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence. RESULTS Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates. P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.

Mast Cell Function: A New Vision of an Old Cell

Since first described by Paul Ehrlich in 1878, mast cells have been mostly viewed as effectors of allergy. It has been only in the past two decades that mast cells have gained recognition for their involvement in other physiological and pathological processes. Mast cells have a widespread distribution and are found predominantly at the interface between the host and the external environment. Mast cell maturation, phenotype and function are a direct consequence of the local microenvironment and have a marked influence on their ability to specifically recognize and respond to various stimuli through the release of an array of biologically active mediators. These features enable mast cells to act as both first responders in harmful situations as well as to respond to changes in their environment by communicating with a variety of other cells implicated in physiological and immunological responses. Therefore, the critical role of mast cells in both innate and adaptive immunity, including immune tolerance, has gained increased prominence. Conversely, mast cell dysfunction has pointed to these cells as the main offenders in several chronic allergic/inflammatory disorders, cancer and autoimmune diseases. This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role.

Transcytosis of Protein through the Mammalian Cerebral Epithelium and Endothelium: III. Receptor-Mediated Transcytosis through the Blood–Brain Barrier of Blood-Borne Transferrin and Antibody against the Transferrin Receptor

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.

Permeabilization of cell membranes.

In order to detect intracellular antigens, cells must first be permeabilized especially after fixation with cross-linking agents such as formaldehyde and glutaraldehyde. Permeabilization provides access to intracellular or intraorganellar antigens. Two general types of reagents are commonly used: organic solvents, such as methanol and acetone, and detergents such as saponin, Triton X-100 and Tween-20. The organic solvents dissolve lipids from cell membranes making them permeable to antibodies. Because the organic solvents also coagulate proteins, they can be used to fix and permeabilize cells at the same time. Saponin interacts with membrane cholesterol, selectively removing it and leaving holes in the membrane. The disadvantage of detergents such as Triton X-100 and Tween-20 is that they are non-selective in nature and may extract proteins along with the lipids. This chapter provides methods for the use of organic solvents and detergents to permeabilize cell membranes.

INORGANIC TRIMETAPHOSPHATASE AS A HISTOCHEMICAL MARKER FOR LYSOSOMES IN LIGHT AND ELECTRON MICROSCOPY

A new cytochemical method is presented for the light and electron microscopic localization of lysosomes in mineralized and soft tissues. Inorganic trimetaphosphate is used as substrate in a lead chelate incubation medium at pH 3.9. Lysosomes in several tissues are strongly reactive, and reaction product is frequently present in Golgi saccules and GERL. The reaction can be differentiated from acid glycerophosphatase activity, is relatively insensitive to fixation and demineralization procedures, and the reaction is often complete after short incubation times.

Distribution of anomalous lysosomes in the beige mouse: a homologue of Chediak-Higashi syndrome.

The beige mouse has been considered as a homologue of Chediak-Higashi syndrome in man. A light microscopic survey of 23 beige mouse tissues, using acid phosphatase as a lysosomal marker, revealed the presence of enlarged (anomalous) lysosomes, often in the form of aggregates, in 15 of the tissues examined. The degree of anomaly varied from one tissue to another and within cells of a given tissue. The most striking alterations occurred in liver parenchymal cells, kidney proximal tubule cells, Purkinje cells and granulocytes. Electron microscopy of liver and kidney revealed enlarged lysosomes containing numerous lipid-like inclusions. The widespread occurrence of anomalous lysosomes strengthens the homology between the beige mouse and Chediak-Higashi syndrome and further supports the concept that lysosomes are intimately involved in this disorder.

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells.

Synopsisthe structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebners), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach

BACKGROUND Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma. OBJECTIVE To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides. METHODS Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb 1A6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program. RESULTS A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb 1A6. CONCLUSION Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Growth of exocrine acinar cells on a reconstituted basement membrane gel

SummaryMethods have been developed for culturing a dividing population of morphologically differentiated rat parotid, lacrimal, and pancreatic acinar cells in vitro. Isolated acinar cells were plated onto tissue culture dishes coated with a three-dimensional, reconstituted basement membrane gel. After attachment in Ham’s nutrient mixture F12, the cells were cultured at 35°C in F12 supplemented with 10% heat inactivated rat serum, epidermal growth factor, dexamethasone, insulin, transferrin, selenium, putrescine, reduced glutathione, ascorbate, penicillin, streptomycin, and the appropriate secretagogue. Under these conditions, the cells attached rapidly and DNA synthesis was initiated within 2 to 3 d. Although the cells flattened on the substratum, they continued to maintain their differentiated morphology. The cells contained secretory granules, and the secretory enzymes peroxidase and amylase could be detected. The use of a reconstituted basement membrane gel proved critical for the attachment and growth of exocrine acinar cells.

Isolation and maintenance of differentiated exocrine gland acinar cells in vitro

SummaryMethods have been developed for isolating and maintaining differentiated rat exorbital lacrimal, parotid, and pancreatic acinar cells for up to 1 month in culture. The dissociated cells retained their differentiated morphology when cultured as suspension cultures at 35°C with the appropriate secretagogue (exorbital lacrimal, 10−6M carbamyl choline; pancreas 10−5M carbamyl choline; parotid, 10−6M isoproterenol). Under these conditions the cells remained viable and differentiated for up to 4 weeks in culture and continued to incorporate3H-leucine at rates similar to those of freshly isolated cells. If secretagogue was omitted from the medium, the cells rapidly degenerated. These results indicate that differentiated from the medium, the cells rapidly degenerated. These results indicate that differentiated exocrine gland acinar cells may be maintained in vitro and utilized as a model system for the study of secretory processes.