An improved autoradiographic technique for the detection of antibody-forming cells

An autoradiographic technique for the detection of antibody-forming cells has been developed for the assay of anti-DNP responses. The lymphoid cells suspension to be assayed was allowed to sediment on to a glass slide coated with DNP-conjugated gelatin to which the secreted antibody bound during subsequent incubation. The bound antibody and its Ig class was revealed by a second incubation using ¹²⁵I-anti-immunoglobulin reagents followed by autoradiography. Studies on the sensitivity and specificity of the method are presented and its advantages over other techniques described. The technique should be readily applicable to other haptens.     

Co-storage of enkephalins and adrenaline in the bovine adrenal medulla

Fluorescence histochemical and immunohistochemical techniques have been used to examine the distribution of catecholamine-containing and enkephalin-containing cells in sections of adult bovine adrenal medulla. Noradrenaline-containing cells were identified by fluorescence microscopy following perfusion fixation with 4% paraformaldehyde (formaldehyde-induced fluorescence, technique of Era¨nko¨⁸). Adrenaline-containing cells did not fluoresce under these conditions. Adrenaline-synthesizing cells were identified by immunofluorescence with an antiserum to bovinephenyl-N-methyl transferase. An antiserum to bovine dopamine-β-hydroxylase was used to identify noradrenaline plus adrenaline cells in the same section. Leu- and met-enkephalin-containing cells were identified immunohistochemically with their respective antisera. To determine whether there was a preferential association of leu- or met-enkephalin with adrenaline or noradrenaline cells, these various antisera were used singly or sequentially on sections treated with formaldehyde in which the localization of endogenous noradrenaline fluorescence had been recorded and then the fluorescence removed by washing overnight.Immunoreactive leu- and met-enkephalin were found to be associated exclusively with adrenaline-synthesizing cells. The finding that both enkephalins are localized in the one cell type (adrenaline cells) in the bovine adrenal medulla is consistent with the proposed common precursor model for synthesis of the two opioid pentapeptides. These findings on co-storage of enkephalins with adrenaline in the adrenal medulla may have implications for other areas of the peripheral and central nervous system where co-storage of catecholamines and enkephalins is known to occur.     

Establishment,characterization and fibre outgrowth of isolated bovine adrenal medullary cells in long-term cultures

Medullary cells were enzymatically dissociated from adult bovine adrenal medullae by a perfusion technique using collagenase and maintained in culture for up to 4 weeks. The perfusion technique was a simplified modification of that described by Livett & Co-Workers (Fenwick, Fajdiga, Howe & Livett, 1978) and included cyclical perfusions of cortex-free medullae via the central vein at 37 C for up to 90 min with three 30 ml batches of 0.5% collagenase-containing phosphate buffered saline or Hanks's balanced salt solution and a final trituration step of the minced tissue. The cells were cultured on collagen-coated glass coverslips in modified Rose chambers, on collagen-coated Petri dishes or in Falcon flasks, studied by phase contrast and electron microscopy, catecholamine cytochemistry and assayed for adrenaline and noradrenaline.When grown on collagen about 50% of the catecholamine-storing cells extended short, broad processes as judged by catecholamine cytochemistry using the glyoxylic acid method. These processes rarely grew longer than 60 μm and many of them were seen to retract after 1 week in culture. Process formation was more pronounced and increased with time, when cells were grown on plastic surfaces. Fifteen to 20% of these cells formed long varicose axons after 18 days in culture. Fibre outgrowth from bovine chromaffin cells was also observed, when pieces of medullary tissue were transplanted on to the sympathetically denervated iris in the anterior eye chamber of Nunu mice.Addition of either nerve growth factor (10–1000 ng/ml 2.5S nerve growth factor) or antibodies to nerve growth factor (1.5 μg/ml) did not affect fibre outgrowth in vitro.Ultrastructural examinations revealed that the vast majority of cells in our cultures were typical chromaffin cells. Using the different densities of primary and secondary amine storing granular vesicles as a criterion for distinguishing noradrenaline- and adrenaline-containing cells we found a significant decrease in the number of adrenaline-containing cells with time. By 3 weeks virtually all chromaffin cells contained storage vesicles typical of noradrenaline. Chromaffin storage vesicles appeared to be more densely packed and the amount of rough endoplasmic reticulum was increased in cells treated with nerve growth factor.Results obtained by biochemical analyses indicated that chromaffin cells lost about 50% of their original amine content and stored equal amounts of noradrenaline and adrenaline from day 9 onwards compared to an initial noradrenaline/adrenaline ratio of approximately 1:6. Administration of nerve growth factor did not significantly alter the proportion and levels of adrenaline and noradrenaline in cultured bovine chromaffin cells.This investigation reveals that the capacity of bovine catecholamine-storing cells to form neurite-like processes differs considerably from that which corresponding cells from young rat adrenal medullae exhibit in vitro (Unsicker, Krisch, Otten & Thoenen, 1978 b). Chromaffin cells from adult bovine adrenal medullae kept in culture retain a large number of features typical of differentiated cells over a considerable length of time. Thus they may be profitably used to study long-term effects of drugs and interactions with other cells at morphological and biochemical levels.     

A subpopulation of mesencephalic dopamine neurons projecting to limbic areas contains a cholecystokinin-like peptide: Evidence from immunohistochemistry combined with retrograde tracing

Using indirect immunofluorescence histochemistry combined with the elution and restaining technique ofTramu, Pillez &Leonardelli (1978), evidence has been obtained that at least some mesencephalic dopamine neurons contain a cholecystokinin (CCK)-like peptide, mainly the C-terminal octapeptide (CCK-8). These cholecystokinin-dopamine cells were present predominantly in the ventral tegmental area (A10), but were also found in the pars lateralis of the substantia nigra and, mainly at a rostral level, in the zona compacta. Terminal nerve fibre networks containing a cholecystokinin-like peptide and tyrosine hydroxylase (the marker for dopamine neurons), respectively, were found on adjacent sections with overlapping distribution patterns in the caudal-medial parts of the nucleus accumbens, the medial tuberculum olfactorium, the bed nucleus of the stria terminalis, the caudal periventricular caudate nucleus and the central amygdaloid nucleus. After injection of 6-hydroxy-dopamine into the lateral hypothalamus or into the ventral mesencephalon, or a frontal hemisection at the hypothalamic level, a marked reduction of both types of fibres was observed in these areas. By combining the indirect immunofluorescence technique with the retrograde tracing of fluorescent dyes, it could be established that cholecystokinin-dopamine cell bodies in the ventral tegmental area project to the caudal, medial nucleus accumbens. Colchicine injected into the lateral hypothalamus caused overlapping accumulations of cholecystokinin- and tyrosine hydroxylase-like immunoreactivity caudal to the injection site. Similar findings were seen caudal to a frontal hypothalamic hemitransection.Cholecystokinin immunoreactive neuronal structures were observed in many other brain areas and some systems of interest in relation to the ascending mesencephalic dopamine neurons are described, such as cholecystokinin fibres present in the dorsal aspects of the zona compacta and in patches in the caudate nucleus. No evidence was obtained that these two latter and some other cholecystokinin immunoreactive nerve terminal systems contained dopamine.The findings demonstrate the coexistence of a cholecystokinin-like peptide and dopamine in a population of mesencephalic neurons projecting to limbic areas.     

Simultaneous demonstration of phenylethanolamine N-methyltransferase immunofluorescent and catecholamine fluorescent nerve cell bodies in the rat medulla oblongata

The distribution of phenylethanolamine N-methlytransferase-immunoreactive nerve cell bodies was investigated in the rat medulla using an antiserum to bovine phenylethanolamine N-methyltransferase raised in rabbits. A procedure that combines immunohistochemistry and catecholamine fluorescence histochemistry was developed with a formaldehyde/glutaraldehyde mixture as a fixative. Three groups of immunoreactive nerve cell bodies were found in the medulla: a ventrolateral group, C1, a dorsal group, C2, in the nucleus of the tractus solitarius and a smaller medial group of cells, C3, scattered in the medial longitudinal fasciculus. Most of the phenylethanolamine N-methyltransferase positive nerve cells did not show catecholamine fluorescence and did not correspond to the catecholamine cell groups A1 and A2. Both groups C1 and C2 of immunoreactive nerve cells extended further rostrally than A1 and A2. Group C3 has not previously been described as a distinct group of catecholamine fluorescent nerve cell bodies.Inhibition of phenylethanolamineN-methyltransferase and monoamine oxidase results in the appearance of catecholamine fluorescence in the immunoreactive cell bodies suggesting that they usually store adrenaline which reacts poorly with the formaldehyde/glutaraldehyde mixture or other aldehydes which induce catecholamine fluorescence and it is for this reason that they are not normally identified in maps of catecholamine fluorescent cells.     

Antibody producing human-human hybridomas. I. Technical aspects

Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested.     

Dysregulation of dopamine signaling in the dorsal striatum inhibits feeding

Dopamine signaling is an important component of many goal-directed behaviors, such as feeding. Acute disruption of dopamine signaling using pharmacological agents tends to inhibit normal feeding behaviors in rodents. Likewise, genetically engineered dopamine-deficient (DD) mice are unable to initiate sufficient feeding and will starve by approximately 3 weeks of age if untreated. Adequate feeding by DD mice can be achieved by daily administration of L-3,4-dihydroxyphenylalanine (L-dopa), a precursor of dopamine, which can be taken up by dopaminergic neurons, converted to dopamine, and released in a regulated manner. In contrast, adequate feeding cannot be restored with apomorphine (APO), a mixed agonist that activates D1 and D2 receptors. Viral restoration of dopamine production in neurons that project to the dorsal striatum also restores feeding in DD mice. Administration of amphetamine (AMPH) or nomifensine (NOM), drugs which increase synaptic dopamine concentration, inhibits food intake in virally rescued DD mice (vrDD) as in control animals. These results indicate that the dysregulation of dopamine signaling in the dorsal striatum is sufficient to induce hypophagia and suggest that regulated release of dopamine in that brain region is essential for normal feeding and, probably, many other goal-directed behaviors.     

Properties of HTLV-I transformed CD8 T-cells in response to HIV-1 infection

HIV-1 infection studies of primary CD8⁺ T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8⁺ T-cells in the context of HIV-1 infection. In this study, a set of CD8⁺ T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4⁺ T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8⁺ T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8⁺ T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8⁺ T-cell dysfunction.     

Electro-acupuncture protects against hypoxic–ischemic brain-damaged immature rat via hydrogen sulfide as a possible mediator

We investigated whether hydrogen sulfide (H₂S) may be a mediator of electro-acupuncture (EA) stimulation treatment for hypoxic–ischemic brain-damage (HIBD). We studied a HIBD 7-day-old rat model with 4 types of treatments: (1) 14 sessions of EA; (2) hydroxylamine (HA), an inhibitor of cystathionine-β-synthase (CBS), the key enzyme of H₂S generation; (3) both EA and HA; or (4) no treatment. Sham-treated rats with or without EA were also studied. Regional cerebral blood flow (rCBF) was monitored before, during and after EA at different periods of treatment (d1, 7 and 14 sessions). We evaluated motor function, H₂S levels and CBS expression in the cerebral cortex and prepared cerebral pathomorphological images after 14 sessions of treatment. EA stimulation could increase local blood circulation and improve motor function in HIBD rats. HIBD significantly increased H₂S levels of brain tissue as compared with sham treatment, and EA treatment could decrease the H₂S generation. Rats with HIBD receiving both EA and HA therapy showed greatly recovered motor function and brain morphology. H₂S might be a mediator of EA treatment of HIBD in rats.     

The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling

Although the function of c-Myc has been clarified in many tissues, until now its expression and role in hair follicle morphogenesis and the hair cycle remains unknown. In this study we detected c-Myc expression pattern in the process of mouse hair follicle development and normal cycle. We found that during hair follicle morphogenesis, the stage-specific expression of c-Myc was detected in mouse skin and was predominantly localized to the hair follicle epithelium. c-Myc expression was also consistently found in mouse skin throughout the hair follicle cycle. Through the in vivo injection of c-Myc inhibitory peptide and c-Myc expression plasmid, we also investigated the direct effects of c-Myc on the hair follicle structures during the hair follicle cycle. Our results showed that c-Myc inhibitory peptide significantly restrained the development of anagen hair follicles, while the injection of plasmid DNA encoding c-Myc in vivo clearly promoted anagen development. Our data indicate that c-Myc may play an important role in the proliferation and differentiation of the hair follicle keratinocytes during hair follicle development. c-Myc also was shown to participate in the regulation of the mouse hair growth cycle and could promote the proliferation of the hair matrix keratinocytes as well as the differentiation of the inner root sheath.