Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.     

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs

The aim of this work was to identify which proteins in horse dander extracts are allergens and to characterise them. Two-dimensional PAGE showed that horse dander preparations are composed of up to 50 proteins, all having acidic isoelectric points in the pH range 3-4.5. Immunoblots of two-dimensional PAGE were used to compare the reactivity of the proteins with IgE from 23 allergic patients. Patient sera were divided into two main groups recognising either allergens of 18.5 kDa or proteins of 27-29 and 31 kDa. The proteins of 27-29 kDa and 31 kDa were all N-glycosylated and their glycan chains seem to play a role in the binding of IgE from allergic patients. The sugar composition of their carbohydrate moiety was determined and lectin-binding experiments indicated presence of terminal sialic acid linked alpha-(2-->6) to galactose, galactose linked beta-(1-->4) to N-acetylglucosamine, and possibly presence of sialic acid linked alpha-(2-->3) to galactose. The 27-29-kDa glycoproteins had heterogeneous isoelectric points, most probably due to different degrees of sialylation in their oligosaccharide chains. The two 18.5-kDa allergens exhibited slightly different isoelectric points and their N-terminal sequences were identical, showing that they most likely were isoforms of the same protein. Sequence analyses revealed that their N-terminal sequences are similar to proteins belonging to the lipocalin family. We named the two 18.5-kDa proteins Equ c 2.0101 and Equ c 2.0102, according to International Allergen Nomenclature recommendations [King, T. P., Hoffman, D., Lowenstein, H., Marsh, D. G., Platts-Mills, T. A. E. & Thomas, W. (1995) J. Allergy Clin. Immunol. 96, 5-14]. The N-terminal of the allergens of 27-29 kDa were blocked and their sequences were not determined. Their amino acid compositions were determined and comparison with acidic mammalian proteins in the Swiss-Prot database revealed high scores with lipocalin proteins. This suggests that the glycosylated horse dander allergens belong to the lipocalin family, like Equ c 2.0101 and Equ c 2.0102.     

Assessment of sperm count in rural population of central India

26 clinician trainees' recollections of experiences in a diagnostic preschool program were analyzed in terms of strength and weaknesses of the program.     

Large-scale functional connectivity in associative learning: interrelations of the rat auditory, visual, and limbic systems

Large-scale functional connectivity in associative learning: interrelations of the rat auditory, visual, and limbic systems. J. Neurophysiol. 80: 3148-3162, 1998. Functional relations between specialized parts of the brain may be important determinants of learned behaviors. To study this, we examined the interrelations of the auditory system with several extraauditory structures in two groups of rats having different behavioral histories. Both groups were trained to associate a tone conditional stimulus (CS) with an aversive unconditional stimulus (US). For one group, a light presented with the tone predicted the absence of the US (group TL-). In the other group, the light was a neutral stimulus (group TL0). Fluorodeoxyglucose (FDG) incorporation was measured in the presence of the tone-light compound. Because the tone-light compound was physically identical for both groups, neural differences between groups reflected differences in the learned associative properties of the stimuli. Covariances of FDG uptake in the auditory system and extraauditory structures were examined using partial least squares. Three strong covariance or functional connectivity patterns were identified. The first pattern mainly reflected similarities between groups, with strong interrelations between the subcortical auditory system and the thalamocortical visual system, cerebellum, deep cerebellar nuclei, and midline thalamus. This pattern of interactions may represent part of a common circuit for relaying the associative value of the tone CS to the cerebellum and the midline thalamus. The external nucleus of the inferior colliculus and medial division of the medial geniculate nucleus were associated more strongly with this pattern for group TL-, which was interpreted as representing the change of the associative value of the tone by the light, mediated through extraauditory influences on these two regions. A second pattern involved midbrain auditory regions, superior colliculus, zona incerta, and subiculum and was stronger for group TL0. The relations between midbrain structures may represent the excitatory conditioned response (CR) evoked by the tone in this group. The final pattern was strongest in group TL- and involved interrelations of the thalamocortical auditory system with hippocampus, basolateral amygdala, and hypothalamus. This pattern may represent the learned inhibition of the CR to the tone in the presence of the light. These findings are consistent with behavioral studies suggesting that at least two types of associations are formed during associative learning. One is the sensory relation of the stimuli and another is the relation between the CS and the affective components of the US. These behavioral associations are mapped to the patterns of functional connectivity between auditory and extraauditory regions.     

Childhood cancer incidence and trends in Minnesota, 1988-1994

Childhood cancer incidence patterns for Minnesota, obtained from the Minnesota Cancer Surveillance System, were compared with national rates as well as with historic data from eight Minnesota counties. In total, 1,140 neoplasms were diagnosed in children (ages 0 to 14) between 1988 and 1994. Leukemias were the most common diagnosis for boys (30.3%) and girls (29.6%), followed by central nervous system tumors. The average annual age-adjusted incidence rates for all cancer sites were 167.2 and 136.2 per million for boys and girls, respectively. These rates were somewhat higher than national rates. In particular, the incidence rate for astrocytoma in boys was significantly elevated. Childhood cancer incidence, particularly brain tumors, has increased in the eight-county region from 1969 to 1994. This analysis demonstrated the Minnesota's childhood cancer incidence patterns are similar to national patterns.     

Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.     

Red nucleus stimulation inhibits within the inferior olive

Red nucleus stimulation inhibits within the inferior olive. J. Neurophysiol. 80: 3127-3136, 1998. In the anesthetized cat, electrical stimulation of the magnocellular red nucleus (RNm) inhibits responses of rostral dorsal accessory olive (rDAO) neurons to cutaneous stimulation. We tested the hypothesis that RNm-mediated inhibition occurs within the inferior olive by using stimulation of the ventral funiculus (VF) of the spinal cord in place of cutaneous stimulation of the hindlimb. Fibers in the VF terminate on hindlimb rDAO neurons, so inhibition of this input would have to occur within the olive. rDAO responses elicited by VF stimulation were inhibited by prior stimulation of the RNm, indicating that inhibition occurs within the olive. In contrast, evoked potentials recorded from the VF or dorsal columns following hindlimb stimulation were not affected by prior stimulation of RNm, indicating that stimulation of the RNm does not inhibit olivary afferents at spinal levels. RNm stimulation that inhibited rDAO responses had little effect on evoked somatosensory responses in thalamus, indicating that inhibition generated by activity in RNm may be specific to rDAO. To test limb specificity of RNm-mediated inhibition, conditioning stimulation was applied to the dorsolateral funiculus at thoracic levels, which selectively activates RNm neurons projecting to the lumbar cord. Stimulation at thoracic levels inhibited evoked responses from hindlimb but not forelimb regions of rDAO, suggesting that inhibitory effects of RNm activity are limb specific. Several studies have reported that olivary neurons have reduced sensitivity to peripheral stimulation during movement; it is likely that RNm-mediated inhibition occurring within the olive contributes to this reduction of sensitivity. Inhibition of rDAO responses by descending motor pathways appears to be a salient feature of olivary function.