The paraventricular nucleus of hypothalamus mediates the pressor response to noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats

The medial prefrontal cortex (MPFC) is a structure that is also involved in cardiovascular modulation. The injection of norepinephrine (NE) into the prelimbic (PL) area of the MPFC of unanesthetized rats evokes a pressor response which is mediated by acute vasopressin release. Vasopressin is synthesized by magnocellular cells of the paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the present study, we endeavored to determine which vasopressin-synthesizing hypothalamic nucleus is involved in the pressor pathway activated after NE injection into the PL area of the MPFC. We report here that lidocaine microinjection into the SON did not change the pressor response evoked by NE injection into the PL. However, the response to NE was blocked by prior injection of lidocaine or CoCl₂ into the PVN, indicating that this area is responsible for the mediation of this pressor response. A neuroanatomic experiment in which the neuronal tracer biotinylated dextran amine (BDA) was microinjected into the MPFC showed a lack of axons or neuronal cell bodies in the PVN, indicating that there are no direct connections between the PL area of the MPFC and the PVN. The results suggest that the PVN is involved in the mediation of the pressor response to NE in the PL area and that this pathway must relay in other brain structures before reaching the PVN.     

Effect of influenza A virus on leukocyte histamine release

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.     

Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones

The afterhyperpolarization (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca²⁺-sensitive K⁺ conductances following Ca²⁺ influx via Ca²⁺ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05±3.76% of those evoked at 90-s stimulus intervals (n=35). Since there was no consistent effect of ryanodine (5 M), ruthenium red, (300 M) or dantrolene Na (35 M) on this frequency dependence, it is unlikely to result from release of Ca²⁺ from intracellular stores. Ca²⁺ currents (I _Ca, studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca²⁺-induced I _Ca inactivation and I _Ca run-down. There was excellent correlation (r=0.964) between the estimated changes in Ca²⁺ influx and the expected activation of the Ca²⁺-sensitive K⁺ current, I _AHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of I _Ca.     

The radioimmunoassay of histamine release from circulating basophils in an in vitro study as support for the in vivo active systemic anaphylaxis test in the guinea pig

An in vitro anaphylaxis test is described which explores the ability of compound re-challenge to induce histamine release from polynuclear basophils in whole blood of guinea pigs that had previously been sensitised. This test is used in drug safety, in support of the in vivo active systemic anaphylaxis test, which is sometimes required by regulatory authorities, when the observed clinical signs are not conclusive. This sensitive and discriminative test is inexpensive and reduces animal utilisation.     

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.     

Activity of Allergenic Proteins from Dermatophagoides pteronyssinus

Two purified allergens from Dermatophagoides pteronyssinus, Dp 42 (identical to P1) and Dp X were studied for their ability to induce histamine release from washed leukocytes and to bind to IgE antibodies from the serum of 27 mite-sensitive children. Almost all patients were demonstrated to be sensitive to both proteins by both assays. Dp 42 was found to have the highest allergenic activity, releasing histamine from leukocytes at a median concentration 10 times lower than for Dp X. There was a positive correlation between basophil sensitivity to both proteins and allergen specific serum IgE concentrations.     

Flow-Cytometric Analysis of Human Basophil Degranulation

Quantification of human basophils and their degranulation were performed using a flow-cytometric system. It was shown that the number of basophils among total leukocytes, before and after the effect of degranulating agents, can be obtained rapidly and reproducibly, following alcian blue, staining. Dose-response curves of degranulation by anti-IgE and anti-IgG₄ were studied, and it was shown that these antibodies can induce basophil degranulation in a Ca⁺⁺ dependent manner. It was also shown that degranulation is completed within 10 min.
Values obtained by flow-cytometry and microscopic evaluation, as well as comparison made between percent basophil degranulation and percent histamine release, agreed well with each other in our system. Flow-cytometric analysis of human basophil degranulation provides a new reliable method to assess in vitro the morphological basis of immediate hyper-sensitivity.     

Development of allergy in children. I. Association with virus infections.

Children born into allergic families, with two allergic parents, are at high risk of developing allergy within the first 5 years of life. In order to observe possible external factors in the sensitization process, a prospective study of 13 such children was done, in which serial clinical and immunologic observations were made at 3- to 6-month intervals over a period of 1 to 4 yr. Eleven of these children are now clinically allergic; 5 have asthma. Immunologic evidence for allergic sensitization was observed in these 11 children by RAST, antigen-induced leukocyte histamine release, lymphoblastogenesis, and rise in serum IgE. Upper respiratory infections (URI) occurred in these 11 allergic children 1 to 2 months prior to the onset of allergic sensitization. In 10 of these 11 URI children, complement-fixing antibodies to viruses (parainfluenza, RSV, CMV) increased in the same blood samples in which immunologic allergic sensitization was first evidenced. This coincidence suggests that certain viruses may contribute to the allergic sensitization process.     

Allergoprints in Horse Allergic Children

In order to determine the allergenic activity of five purified horse allergens, 22 children allergic to horses according to history, skin test, and leukocyte histamine release were evaluated. Washed leukocytes from all patients were tested for allergen-induced histamine release utilizing four epidermal horse allergens (Ags 6, 9, 11 , and 15) and horse serum albumin. Crossed radioimmunoelectrophoresis was carried out with a standardized horse dander extract and serum from each patient.
The results showed considerable variation in the individual allergoprints. Ag 11 had the highest mean allergenic activity. Sensitivity to horse serum albumin was demonstrated three times. Our data show that the amount of serum IgE antibodies bound by horse allergens correlates significantly with the capacity of the allergens to induce histamine release from washed leukocytes.     

A new method for histamine release from purified peripheral blood basophils using monoclonal antibody-coated magnetic beads

A new method for evaluating histamine release from purified basophils was developed. Basophil-containing leukocytes were directly purified from a small amount of peripheral blood using monoclonal antibody BA312-coated magnetic beads. The purified basophils still rosetted to magnetic beads maintained a normal response to anti-IgE and to dust mite allergen in comparison with the conventional method using washed leukocytes. This methodology facilitates the purification of basophils, anti-IgE- and allergen-induced histamine release, and subsequent histamine determination within only 3 h. The released histamine was analyzed by an enzyme-linked immunosorbent assay (ELISA) with a characteristic detection profile. Since all steps were performed in 96-well microplates, many clinical samples could be analyzed at the same time, permitting easy applications in routine laboratories.