T cells reactive to a single immunodominant self-restricted allopeptide induce skin graft rejection in mice.

Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.

Atropine dissociates complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein in heart membranes.

Complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein (G protein) are formed in the presence of the agonist carbachol. The complexes remain stable after removal of agonist, and survive subsequent solubilization from cardiac membranes and purification. Dissociation of the receptor from the G protein occurs when the antagonist atropine is added following removal of agonist. This is the first direct demonstration of destabilization of receptor-G protein complexes by the binding of an antagonist.

High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.

BACKGROUND: Whether alloreactive T cells in a naive host derive from naive or memory T cells remains unclear. It is also unclear whether graft rejection alters the phenotype of these T cells. Proliferation assays and cytokine enzyme-linked immunosorbent assays performed on culture supernatants do not differentiate primary T-cell alloreactivity from recall responses in allograft-primed mice, suggesting that these methods are inadequate measures of the alloreactive immune repertoire. METHODS: To better characterize alloreactivity in naive and skin allograft-primed mice, we used a modified, high-resolution cytokine enzyme-linked immunosorbent spot assay capable of detecting cytokine production over short time periods. RESULTS: Twenty-four-hour analysis of alloreactivity in mice that rejected fully MHC-disparate skin allografts revealed a high frequency of interferon (IFN)-gamma- and interleukin (IL)-4-producing, L-selectin-negative T cells, consistent with a memory phenotype. In contrast, 24-hr allostimulation of T cells from naive mice resulted in IL-2 production with minimal secretion of IFN-gamma or IL-4. The frequency of IL-2 producers was low and their phenotype was L-selectin positive, suggesting that they were naive and not memory T cells. When maintained in culture for 48 hr, however, the T cells from the primary mixed lymphocyte reaction began producing IFN-gamma, consistent with in vitro priming. CONCLUSIONS: The primary alloresponse does not seem to involve clones that have been preprimed by environmental antigens, but instead behaves similarly to self-MHC-restricted immunity directed toward prototypic protein antigens: T cells with a naive phenotype are specifically induced to differentiate into high-frequency memory populations. These findings may have important implications for therapeutic induction of allograft tolerance.

Characterization and manipulation of T cell immunity to skin grafts expressing a transgenic minor antigen.

BACKGROUND: Minor histocompatibility antigens play a significant role in allograft rejection when donor and recipient are matched at MHC loci. An improved understanding of T cell immunity directed toward a model minor antigen may provide new approaches for preventing graft rejection. METHODS: C57BL/6 (B6) recipient mice were engrafted with skin from B6 beta-galactosidase transgenic (beta-gal tg) donors and the induced T cell immune responses were characterized by cytokine ELISA spot assay. beta-gal-specific immunity was manipulated prior to transplant through preinjection with beta-gal in complete Freund's adjuvant (CFA) or through preinjection with soluble beta-gal i.v. RESULTS: B6 mice rejected beta-gal tg skin by day 25. Rejection was associated with a low frequency of predominantly CD8+, interferon-gamma-producing T cells capable of directly recognizing both beta-gal tg cells and an immunodominant major histocompatibility complex I-restricted peptide derived from the beta-gal protein. Rejection of multiple minor antigen disparate skin and major histocompatibility complex-disparate skin occurred significantly faster, and was associated with a 10- to 30-fold higher frequency of alloreactive T cells, than rejection of beta-gal tg skin. Prepriming of recipients with beta-gal in complete Freund's adjuvant resulted in an increased frequency of beta-gal-specific T cells and accelerated rejection of beta-gal tg skin. Intravenous injection of soluble beta-gal-induced graft tolerance and a lack of detectable beta-gal-specific immunity. CONCLUSIONS: The findings reveal that transgenically expressed beta-gal behaves as a minor transplantation antigen and that manipulation of the beta-gal-specific T cell repertoire can dramatically affect rejection of beta-gal tg skin grafts. The work provides the foundation for mechanistic studies of tolerogenesis to minor antigenic determinants.

Induction of T helper 2 immunity to an immunodominant allopeptide.

Neonatal tolerance to alloantigens and autoantigens in mice is mediated by T helper (Th)2 immunity. If a strong and pure Th2 response could be engaged to alloantigens in adult mice, it might result in allograft tolerance. In an attempt to induce Th2 immunity in adults, we studied the T-cell response to peptide I-A beta(k)58-71 (I-Ap), a dominant indirect pathway determinant during rejection of B10.A skin by BALB/c mice. Our data show that the naturally occurring response to this peptide during rejection is Th1, consistent with the notion that Th1 immunity is central to destruction of the allograft. In contrast, vigorous and unipolar Th2-type immunity to this peptide can be readily induced by intraperitoneal immunization with incomplete Freund's adjuvant, a protocol previously thought to induce T-cell unresponsiveness. Thus, adjuvant can be used to Th2-guide the indirect pathway alloresponse in an effort to antagonize naturally occurring Th1 alloimmunity.

Immunohistochemical demonstration of neuron-specific enolase and microtubule-associated protein 2 in reactive astrocytes after injury in the adult forebrain.

Transformation of normal resting astrocytes to reactive astrocytes in the adult brain after injury has been well documented. Using double immunofluorescent labeling methods, we report that astrocytes in both the ischemically damaged and the retrogradely/anterogradely degenerating forebrain nuclei express not only the glial cell markers glial fibrillary acidic protein and vimentin, but also the neuronal markers neuron-specific enolase and microtubule-associated protein 2. Since these neuronal markers are expressed in glial precursor cells, these results suggest that one of the characteristic responses of astrocytes in the adult brain after injury may be re-expression of fetal trait(s) of early differentiating glial cells/neurons.

Stimulation of cell proliferation and inhibition of gap junctional intercellular communication by linoleic acid.

The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01-3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.

Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures.

The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

Expression of gamma-aminobutyric acid immunoreactivity in reactive astrocytes after ischemia-induced injury in the adult forebrain.

Transient ischemia induces an increase in glial fibrillary acidic protein (GFAP) immunoreactivity which can be detected in specific forebrain regions of the adult gerbil as early as day 2, becomes prominent by day 4-7 and persists for at least 3 months. These forebrain areas include layers 2/3 of the somatosensory and auditory cortices, the CA1 and CA4 sectors of the hippocampus, the dorsolateral region of the striatum, and the dorsolateral subregion of the medial septal nucleus. In addition, astrocytes in the ischemically lesioned areas stain with gamma aminobutyric acid (GABA) antiserum. These GABA-immunoreactive astrocytes are not found in non-damaged areas. The time-course of expression of GABA immunoreactivity is similar to that of GFAP immunoreactivity. Using a double immunofluorescent staining method, reactive astrocytes which express GABA immunoreactivity were also found to immunostain with either GFAP or vimentin. On the other hand, astrocytes were not found to be immunoreactive with antibodies to glutamic acid decarboxylase or glutamate. Our present finding demonstrates, in an in vivo model, an aberrant expression of GABA immunoreactivity by astrocytes which is not observed in non-ischemic adult animals.

Neuroprotective activity of dimer of 16,16'-dimethyl-15-dehydroprostaglandin B1 (di-Calciphor) in cerebral ischemia.

Post-ischemic treatment of di-Calciphor (16,16'-dimethyl-15- dehydroprostaglandin B1) significantly improves animal survival and prevents ischemia-induced neurodegeneration of vulnerable forebrain regions assessed with histochemical and biochemical techniques in gerbils. Neuronal degeneration seen by Cresyl violet staining and silver impregnation in the CA1 sector of the hippocampus and the dorso-lateral sector of the striatum was significantly reduced in animals treated with di-Calciphor. In addition, the early onset of selective degradation of calpain I substrates spectrin and microtubule-associated protein (MAP2) in these same vulnerable regions was prevented. The lack of adverse side effects may facilitate the potential therapeutic use of this drug in preventing neuronal damage caused by stroke.

Effects of nimodipine, felodipine and amlodipine on electroconvulsive shock-induced amnesia in the rat.

The effects of various doses (0.03, 0.1, 0.3 or 1.0 mg/kg) of the Ca2+ channel blockers nimodipine, felodipine and amlodipine on the learning ability of rats exposed to electroconvulsive shock were examined. The animals were trained in a passive avoidance procedure. The drugs tested were injected 30 min before the learning trial started. The electroconvulsive shock was given immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 h later. It was found that electroconvulsive shock strongly impaired the retention of the passive avoidance response. Nimodipine, felodipine and amlodipine did not influence the passive avoidance behavior in the sham electroconvulsive shock group, but significantly improved the retention deficits in the animals exposed to electroconvulsive shock. These findings support the hypothesis that perturbations in Ca2+ homeostasis can contribute to the memory deficits associated with electroconvulsive shock. The antiamnestic effects of the substances tested make them interesting candidates for clinical trials in patients with cognitive impairment caused by electroconvulsive shock therapy.

Naphazoline nasal drops intoxication in children.

Naphazoline, a sympathomimetic and an imidazoline derivative, is used as 0.05-0.1% solution for local decongestion of the nasal and ocular mucosa. In excessive dosage, or if ingested by accident, may cause depression of the central nervous system (disturbances of consciousness progressing to coma), hypothermia, bradycardia and sweating. These naphazoline effects are particularly strongly pronounced in children. Anglo-Saxon pharmacotherapy excludes the application of naphazoline nasal drops in children younger than six years, whereas the Croatian pharmacotherapeutic literature (and practice) allows its use even in infancy. At the Kantrida Paediatric Clinic, Clinical Hospital Centre in Rijeka, 11 children with signs of intoxication with naphazoline nasal drops were hospitalized from 1990 to 1992. The symptoms pertaining to the central nervous system i.e. disturbances of consciousness in the form of somnolence were clearly marked in all children. Some children developed skin pallor, bradycardia, bradypnoea and hypothermia. Resolution occurred within 24 hours and the findings returned to normal values. Clinical picture followed by rapid resolution and normal findings, with a personal history of drug taking, is a safe indication for diagnosis. There are several reasons to account for intoxication (drops difficult to use with children, containers inadequate for proper dosage), but the major factor is the age of the patient--all hospitalized children were younger than six years. It is pointed out that administration of naphazoline drops at an early age is not advisable.

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells.

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 micromol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a "hyperphosphorylated" connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.

Microtubule-associated protein 2 as an early indicator of ischemia-induced neurodegeneration in the gerbil forebrain.

Microtubule-associated protein 2 (MAP-2) was studied in the gerbil hippocampus and striatum after transient ischemia. Western immunoblot analysis shows that there is a significant decrease of MAP-2 in the dorsolateral sector of the striatum and a slight decrease of MAP-2 in the CA1 region of the hippocampus 6-12 h after ischemia in the gerbil forebrain. The immunohistochemical staining pattern of MAP-2 in these two regions also shows a loss of immunostaining of MAP-2. In particular, a beaded MAP-2 immunostaining pattern at the apical dendritic region of the CA1 neurons of the hippocampus was found within 12 h after ischemia compared with the smooth dendritic immunostaining of MAP-2 in normal CA1 neurons. In vitro assays of MAP-2 degradation suggest that dendritic loss of immunoreactivity after ischemia seen on western blots may be due to calpain I degradation of MAP-2. Loss of MAP-2 in both the striatum and hippocampus was found to occur earlier than spectrin degradation by western blot analysis. These results suggest that loss of MAP-2 may participate in the initial phase of neuronal dysfunction and that dendritic breakdown may be a first sign of neurodegeneration.

cGMP-dependent cation channel of retinal rod outer segments.

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.

Identification of a 34,000-dalton mitogenic protein associated with plasma membranes from human A431 epidermoid carcinoma cells.

We present evidence for a discrete 34,000-Da polypeptide with mitogenic activity, associated with plasma membranes from human A431 carcinoma cells. Plasma membranes from A431 cells are highly mitogenic for quiescent fibroblasts. A significant fraction of the membrane-associated activity can be released by treatment with a high concentration of salt and is relatively acid stable. Incubation of 125I-labeled salt-extracted proteins with target fibroblasts results in preferential binding of a 34,000-Da protein--i.e., greater than 90% of the cell-bound radioactivity is associated with the 34,000-Da polypeptide. Studies correlating the mitogenic activity with the cell-binding 34,000-Da protein indicate that this protein is the acid-stable, peripherally attached, mitogenically active component of A431 membranes. Available data suggest that this protein may be mitogenically active in the nanomolar concentration range. Some properties of this protein are described.

Incorporation of serine into Paramecium ethanolamine phospholipid and phosphonolipid head groups.

Ethanolamine phospholipid head groups in Paramecium were synthesized directly from ethanolamine. As in other cell types, radioactivity from ethanolamine failed to incorporate significantly into head groups of ethanolamine phosphonolipids, indicating that the phosphonolipids are not derived from their phospholipid analogues. Unlike other systems previously examined, radioactivity from serine is incorporated into both ethanolamine phospholipid and phosphonolipid head groups of glycerolipids and sphingolipids in this ciliate. These observations suggest that synthesis of ethanolamine phosphonolipids involves synthesis de novo of free phosphonoserine, which is then incorporated into lipids, and then lipid-bound phosphonoserine intermediates (glycerolipids or sphingolipids) undergo decarboxylation, forming lipidbound phosphonoethanolamine compounds.

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.

Characterizations of six ethanolamine sphingophospholipids from Paramecium cells and cilia.

Six ethanolamine sphingophospholipids from axenically cultured Paramecium tetraurelia were isolated from cells and purified ciliary fractions, and were characterized. The sphingolipids comprised 10.7% of whole cell and 32.5% of ciliary ethanolamine phospholipid fractions purified by ion exchange column chromatography. The individual sphingolipids were characterized by thin-layer chromatographic analyses of parent compounds and the polar head group and long chain base moieties, gas-liquid chromatography, and mass spectrometry of amide-linked fatty acids and long chain bases, and nuclear magnetic resonance of the compounds. Colorimetric assays of differential hydrolysis products and 31P nuclear magnetic resonance were used to determine the nature of phosphorus linkages. The sphingolipids were identified as N-acyl-trans-4-hydroxy-sphinganine-1- phosphonoethanolamine , N-acyl-trans-4-hydroxy-sphinganine-1-phosphoethanolamine, N-acyl-sphingenine-1- phosphonoethanolamine , N-acyl-sphingenine-1-phosphoethanolamine, N-acyl-sphinganine-1- phosphonoethanolamine and N-acyl-sphinganine-1-phosphoethanolamine. All six had greater than 90% saturated fatty acids. These sphingolipids were quantified by radioisotope methods and plate densitometry of thin-layer chromatograms. Changes in the relative amounts of each species were detected in cells grown in different culture media as well as in cells at different culture ages.