Effect of polyethylene glycol on the rate of immune complex formation by non-precipitating antibody

A kinetic study formation of large size complexes of non-precipitating pig-Dnp anti-Dnp antibody and multivalent dinitrophenylated serum albumin was performed using light scattering and absorption spectroscopy of the Dnp-group. A very rapid phase of the process resulted in the formation of complexes having molecular weight of about 2 × 10⁶. Further increase of the complex size was much slower. Addition of PEG affected positively the rate of complex growth even in concentrations below 1%. The spectroscopic kinetic curves also showed a rapid and a slow phase, sensitive to the presence of PEG. The character of the kinetic data does not support the simple view that polymers enhance precipitate-formation by the steric exclusion of complexes from the polymer domains. It can be assumed that the interaction of the polymer with the antigen-antibody system consists of a subtle temporary attachment of the polymer to the antibody molecule resulting in a change of the shape and/or flexibility of the antibody molecule, favouring its cross-linking capacity.     

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.     

Sensory substance P-innervation of the urinary bladder: possible site of action of capsaicin in causing urine retention in rats

In rats treated neonatally with capsaicin there is, in later life, a tendency tendency towards urine retention. Since capsaicin is known to cause irreversible loss of certain primary sensory neurons, notably those containing substance P, we have studied the sensory innervation of the bladder in capsaicin-treated and control rats using retrograde tracing methods and immunohistochemistry; in addition, the motor function of the bladder was assessed in in vitro experiments, using electrical field stimulation. Five days after injection of the fluorescent tracer True Blue into the wall of the bladder, numerous labelled cells were identified in dorsal root ganglia T13, L1, L2, L6, and S1 and smaller numbers of cells were found in T12 and L3. In capsaicin-treated rats the numbers of labelled cells were reduced by over 50% in L1, L6 and S1. In control rats, 10-16% of True Blue labelled cells also contained substance P as demonstrated by indirect immunofluorescence, but in capsaicin-treated rats substance P cells were virtually absent. In in vitro studies, contractions of the detrusor muscle to electrical field stimulation, both before and after atropine, were similar in control and capsaicin-treated rats. We suggest that capsaicin causes urine retention in rats due to an impairment of sensory transmission from the bladder (that could involve substance P) and a consequent failure in the normal micturition reflexes.     

Enkephalin-like immunoreactivity in the mossy fiber pathway of the hippocampal formation of the tree shrew (Tupaia glis)

The distribution of enkephalin-like immunoreactivity within the hippocampal formation of the tree shrew (Tupaia glis) has been investigated. By far the most conspicuous immunoreactive structures within the hippocampal formation were components of the mossy fiber pathway. Immunoreactive granule cells within the dentate gyrus could often be seen sending dendrites into the molecular layer and axons into the hilus of the dentate gyrus. Within the regio inferior of Ammon's horn, massive immunoreactive terminal swellings associated with thin fibers were located in the zone just above the pyramidal cell layer. While the mossy fiber pathway was the most intensely labeled portion of the hippocampal formation, occasionally sparse terminals and lightly immunoreactive cell bodies were noted in regio superior.These results indicate that the mossy fiber system may be an important pathway for opiate modulation of hippocampal activity.     

The distribution of enkephalin immunoreactive fibers and terminals in the monkey central nervous system: An immunohistochemical study

Using immunohistochemical techniques, the distribution of met-enkephalin fibers and terminals was studied in the central nervous system of adult old-world monkeys. Areas which showed the greatest density of immunoreactivity included substantia gelatinosa, nucleus tractus solitarius, nucleus parabrachialis, substantia nigra, median eminence, globus pallidus (external segment), patches within the striatum and the region of nucleus accumbens and the olfactory area. Striking and discrete zones of enkephalin immunoreactive fibers and terminals which did not conform to known nuclear boundaries were observed in the latter areas.The distribution of enkephalin in the monkey is compared to what has been described in the rat central nervous system. In general, the two species are similar, however, differences were observed in some areas including the hypoglossal nucleus, substantia nigra and in the region of the nucleus accumbens and olfactory area. The results are discussed with regard to the possible functional significance of enkephalin localization in regions related to regulation of pain, mood, and autonomie function.     

Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum

Substance P-like immunoreactivity has been localized in whole mount preparations of the isolated layers of the guinea-pig ileum. Axons containing substance P formed dense networks around the nerve cells and ran in the primary, secondary and tertiary nerve bundles of the myenteric plexus. 3.6% of the nerve cell bodies of the myenteric plexus and 11.3% of the cell bodies in the submucous plexus showed immunoreactivity for substance P. Axons ran in fine nerve bundles parallel to the longitudinal muscle, between this muscle and the myenteric plexus. Axons containing substance P also ran in small nerve trunks parallel to the circular muscle throughout its thickness and in the deep muscular plexus at the base of this muscle coat. In the submucosa, these axons ramified amongst ganglion cells of the plexus and ran in the internodal strands. In addition they formed a perivascular network around submucous arteries and contributed to the paravascular nerves following these arteries. Axons containing substance P formed a delicate plexus in the mucosa. After extrinsic denervation the nerves containing substance P that were associated with submucous arteries, and some in the submucous plexus, disappeared. The nerves in the other areas were not detectably different from normal.Comparison with the distribution of somatostatin, enkephalin and vasoactive intestinal polypeptide indicated the neurons containing substance P constitute a separate population within the enteric nervous system.     

Measurement of antibody and antigen concentrations with a sodium sulphate glutaraldehyde technique

Gamma-globulins can be crosslinked at low concentrations by simultaneous treatment with sodium sulphate and glutaraldehyde (SSG-method). This precipitate can be used for the assay of antibody or antigens. Examples are given for the measurement of (1) antidinitrophenyl antibody by reaction of the precipitate with ¹²⁵I-dinitrophenylovalbumin, (2) IgA by inhibition of the reaction of an anti-IgA precipitate with an ¹²⁵I-labelled IgA myeloma protein, (3) idiotypic antibody by inhibition of the binding of a ¹²⁵I-labelled myeloma protein by an anti-idiotype precipitate.     

D2-protein in PC12 pheochromocytoma cells after nerve growth factor stimulation

The rat brain D2-protein has previously been shown to play a role in interneuronal adhesion Monospecific antisera against this protein reacted with adrenal medulla cells and with PC12 pheochromocytoma cells as demonstrated by indirect immunofluorescence. Upon stimulation by nerve growth factor the PC12 cells extend neurites. These neurites were shown to contain D2-protein both by immunofluorescence and by crossed immunoelectrophoresis. The findings substantiate the close relationship between neurons from the central nervous system and both the PC12 cells line, as well as adrenal medulla cells.     

The origins,pathways and terminations of neurons with VIP-like immunoreactivity in the guinea-pig small intestine

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9–12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles.It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.     

Somatostatin is contained in and released from cholinergic nerves in the heart of the toad Bufo marinus

The heart of the toad Bufo marinus contained a substance with somatostatin-like immunoreactivity which eluted with somatostatin on reverse phase high pressure liquid chromatography. Immunoreactivity to somatostatin was localised histochemically to nerve fibers in muscle bundles of the sinus venosus, atria and ventricles and to nerve cell bodies in the sinus venosus and inter-atrial septum. Nerve cell bodies were localised both by interference contrast microscopy and immunohistochemistry; all detectable intracardiac neurons were immunoreactive. Synthetic somatostatin inhibited the rate and force of beat of atrial preparations, but did not affect the driven ventricle. Vagal stimulation caused inhibition of all cardiac chambers. After muscarinic blockade with hyoscine, vagal stimulation with 3 Hz or more still caused inhibition of the pacemaker and atrium, but not of the ventricle. The hyoscine-resistant vagal effects were diminished by about 60% after induction of tachyphylaxis to somatostatin. When when the vagus nerves were stimulated intermittently for 1 h at 10 Hz, in the presence or absence of hyoscine, the effect of somatostatin was reduced by about 60%. It is concluded that the cholinergic postganglionic neurons of the cardiac vagus contain somatostatin. When the vagus is stimulated at 3 Hz or more, the neurons release sufficient somatostatin to inhibit the pacemaker and atrial muscle.     

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.     

Experimental and immunohistochemical studies on the cerebellar substance P of the rat: localization, postnatal ontogeny and ways of entry to the cerebellum

With the indirect immunofluorescence technique, the localization (including the postnatal ontogeny) of substance P in the cerebellum, and the ways of entry of substance P-containing fibers into the cerebellum were explored. In the newborn rat cerebellum, dense fiber bands of axons with substance P-like immunoreactivity which can be traced to the lower brain stem are found. These fibers are also traceable to the developing granular cell layer. Two weeks after birth, however, substance P-containing structures seen in the cerebellum begin to decrease progressively and in the cerebellum of the adult rats, only a small amount of substance P-containing structures is observed. The present study established that substance P-containing fibers are mostly derived from extracerebellar substance P-containing cells and demonstrated the presence of three sites of entry of these substance P-containing fibers to the cerebellum, via (1) the inferior cerebellar peduncle, (2) the fasciculus uncinatus and (3) the middle cerebellar peduncle, respectively. Following deafferentation of the cerebellum, substance P-accumulating fibers are observed only ventral to the lesion (i.e. on the brain stem side), while in the cerebellum a remarkable decrease of substance P-containing fibers is seen and no substance P-accumulating fibers are found dorsal to the lesion (cerebellar side).     

The distribution of cholecystokinin octapeptide-like structures in the lower brain stem of the rat: an immunohistochemical analysis

The distribution of immunoreactive cholecystokininoctapeptide (CCK-8)-like structures in the lower brain stem of the rat was investigated using indirect immunofluorescence. In addition to the well known immunoreactive CCK-8-like containing cell groups such as those in the ventral tegmental area, substantia grisea centralis of the mesencephalon, and n. linealis rostralis, the present study demonstrated a much wider distribution of immunoreactive CCK-8-like cells in the lower brain stem, i.e. those in the inferior colliculus, n. parabrachialis colliculi posterioris, lateral lemniscus, lateral parabrachial area, n. centralis superior, nucleus of group O, pontine substantia grisea centralis, n. tractus solitarii, area postrema, n. tractus spinalis nervi trigemini and reticular formation just dorsal to the inferior olivary complex. We also demonstrated an extensive network of immunoreactive CCK-8-like fibers in various areas of the lower brain stem, including the auditory system, visual system, viscerosensory area, parabrachial nucleus, dorsal and ventral tegmental nuclei, and interpeduncular nucleus. The possible importance of CCK is briefly discussed.     

Heterogeneity in anticatalytic activity of antibody specific for horseradish peroxidase

Rabbit antibodies specific for horseradish peroxidase are heterogeneous in their ability to inhibit enzyme activity. Heterogeneity was demonstrated by fractionation of the total antiperoxidase pool by differential ammonium sulfate precipitation and differential elution of antibody from enzyme affinity columns. Both fractionation methods yielded antibody subpopulations that differed in anticatalytic activity. Some antibody subpopulations decreased enzyme activity almost completely at low molar ratios of antibody to peroxidase. Other subpopulations were not effective inhibitors even at great molar excess. Admixture experiments demonstrated that inefficient antibody pools decreased the anticatalytic effect of highly inhibitory antibody. The degree of inhibition observed with unfractionated antiserum is a reflection of the interaction of various antibody subpopulations with the enzyme. No correlation was found between the immunoglobulin class of antiperoxidase and anticatalytic efficiency in analyses of numerous antisera. The determinant specificity of an antiperoxidase molecule determines its anticatalytic ability. A peptide fragment (mol. wt 22,500) of peroxidase prepared by partial tryptic digestion bount 60 to 70% of the total antiperoxidase in a number of antisera. However, the peptide did not bind inhibitory antibodies.     

Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons

The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, It could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.     

Reticulo-facial enkephalinergic pathway in the rat: an experimental immunohistochemical study

Enkephalins have been detected in the cranial motor nuclei and may play a role in the motor system. The afferent source of leucine-enkephalin-like immunoreactive fibers in the facial nucleus was investigated using experimental immunohistochemistry. These leucine-enkephalin-containing fibers were markedly reduced on the operated side after the destruction of the ventrolateral part of the caudal medullary reticular formation, where a number of leucine-enkephalin-positive cell bodies were observed. This fact strongly suggests that these leucine-enkephalin-positive cells project ipsilaterally to the facial nucleus and so could have some effects on facial motoneurons.     

Long-term development of mesencephalic dopaminergic neurons of mouse embryos in dissociated primary cultures: morphological and histochemical characteristics

In 13 and 15 day-old mouse embryos mesencephalic dopaminergic neurons could already be visualized at the level of the mesencephalic flexure by tyrosine hydroxylase immunocytochemistry at day 13. At this time, noradrenergic cells in the locus coeruleus area were not detectable. In most in vitro experiments, dissociated mesencephalic cells of 13 day-old embryos were grown in presence of serum. Four approaches were used to identify the dopaminergic neurons in vitro: fluorescence histochemistry of newly taken up exogenous norepinephrine, radioautography after labelling with (3H) dopamine, tyrosine hydroxylase-like immunoreactivity and fluorescence histochemistry of endogenous stores of catecholamines. Control experiments performed at various times in vitro with selective inhibitors of amine transport into dopaminergic, noradrenergic and serotoninergic neurons indicated that only dopaminergic neurons were detected by these various approaches, noradrenergic neurons being virtually absent from the cultures. The uptake of exogenous norepinephrine was detected already 24 h after plating and preceded the appearance of tyrosine hydroxylase-like immunoreactivity (48 h). The number of neurons revealed by these two techniques increased up to 4 and 10 days, respectively. Endogenous stores of dopamine were only seen after three weeks in vitro by fluorescence histochemistry. At this time, the same number of neurons was revealed whatever the method used. The presence of striatal target cells (co-cultures) affected neither the sequential appearance of the markers nor the number of dopaminergic cells. The two main types of dopaminergic neurons (fusiform and multipolar) described in vivo both in the substantia nigra (A9) and the ventral tegmental area (A10) of adult animals were identified in vitro and their development into well-differentiated neurons can be followed for up to six weeks. This in vitro system seems, therefore, to be particularly suitable for biochemical and electrophysiological studies of these dopaminergic neurons.     

Immunohistological demonstration of terminal transferase (TdT) in the age-involuted human thymus

By using a sensitive immunoperoxidase technique, serological determinants of terminal deoxynucleotidyl transferase (TdT, E. C. 2.7.7.31) were found in cortical lymphocytes of thymus biopsies of adult and aged persons (21-70 years) of both sexes. In contrast to biochemical determinations, specific immunohistological studies showed that TdT is continuously present in very large numbers of cortical thymic lymphocytes during human life, despite subtotal physiological involution of the thymus.     

Immunology of DNA. II. The effect of size and structure of the antigen on the Farr assay.

The Farr assay for the detecton of antibodies to double stranded (ds) DNA is influenced by the DNA preparations used as antigen. To elucidate this the molecular weight of the antigen preparation, contamination with proteins and presence or absence of single stranded (ss) regions were studied with the following conclusions: 1) The degree of DNA binding by antibodies is linearly dependent on the molecular weight of the DNA, provided that this does not exceed 10 X 10(6). 2) Deproteinization of E. coli DNA by chromatography on methylated albumin-kieselguhr(MAK) columns results in lower binding by most sera. 3) ds DNA preparations sometimes contain ss regions which bind antibodies to ss DNA. The difference in behaviour of different ds DNA preparations may be ascribed entirely to these factors and not to differences in antigenic determinants. We have standardized the Farr assay and enhanced its specificity by the use of circular DNA isolated from bacteriophage PM2.     

Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: Morphological evidence for neuroendocrine γ -aminobutyrate (GABA)

GABAergic cells and axon terminals were localized in the basal hypothalamus of different species (rat, mouse and cat), by means of an immunocytochemical approach using a specific and well-characterized antiserum to the GABA biosynthetic enzyme, glutamate decarboxylase. Lightmicroscopic visualization was performed with an indirect immunofluorescence method and electron-microscopic observations were made on material with pre-embedding staining and use of the peroxidase-antiperoxidase procedure.At the light-microscopic level, a dense immunofluorescent plexus was observed over both the medial and lateral parts of the external layer of the median eminence. The labelling extended from the rostral part of the median eminence up to the pituitary stalk. Over the subependymal and internal layers only a few immunoreactive dots were visible, except around the blood vessels where they appeared more concentrated. Immunoreactive varicosities could be found following the outlines of the capillary loops and lining tanycyte processes, especially in the median eminance midportion.At the electron-microscopic level, the immunolabelling was exclusively found over neuronal profiles in the median eminence. The latter represented a small fraction of the total number of varicosities visible on the same section. Labelled profiles typically contained numerous small clear synaptic vesicles and only a few or no dense-core vesicles. In the subependymal and internal layers, rare labelled endings were found close to ependymal cells or among transversally cut fibers, respectively. In the palisadic zone, elongated positive boutons were visible intermingled with bundles of unlabelled axons and glial or ependymal processes. In the neurohemal contact zone, immunoreactive endings were observed among unlabelled neurosecretory endings in close vicinity to fenestrated capillary perivascular space.Small moderately intense immunofluorescent varicosities were observed all over the hypothalamus. The density of the glutamate decarboxylase-positive network was higher than in most diencephalic regions. Intraventricular or topical injection of colchicine allowed the visualization of small lightly immunoreactive cells in the diffusion area of colchicine. In the arcuate nucleus labelled axonal endings containing small pleomorphic synaptic vesicles and sometimes a few dense-core vesicles were observed at the electron-microscopic level. Typical synaptic junctions were commonly found between positive endings and unlabelled perikarya, or more frequently, unlabelled dendrites.These findings show that glutamate decarboxylase-containing endings are localized in several strategic sites for potential GABAergic neuroendocrine regulations. The GABAergic endings found among neurosecretory endings in the neurohemal contact zone may provide the morphological support for the release of γ -aminobutyrate into the portal blood flow as an hypothalamic hypophysiotropic hormone. Alternatively, neurosecretory cells might be under GABAergic control expressed either at their terminal level within the median eminence or the cell body level within the parvicellular hypothalamic nuclei.