Immunoreactive Substance Research Articles

Changes in Peptidergic Innervation in Chronic Pancreatitis

We sought to identify characteristics of peptidergic innervation that altered in patients with chronic pancreatitis. Pancreatic tissue removed from patients with chronic pancreatitis was analyzed by immunohistochemistry using antisera against neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, peptide histidine isoleucine, calcitonin gene-related peptide, and substance P, respectively. In accordance with recent findings, the number and diameter of intralobular and interlobular nerve bundles were found to be increased as compared with control pancreas from organ donors. The striking change in the peptidergic innervation pattern in chronic pancreatitis concerned these altered nerves. It consisted of an intensification of the immunostaining for calcitonin gene-related peptide and substance P in numerous fibers contained in these nerves. Adjacent sections showed that immunoreactive substance P and immunoreactive calcitonin gene-related peptide coexisted in these fibers. Because both of these peptides are generally regarded as pain transmitter candidates, our findings provide further evidence that changes in pancreatic nerves themselves might be responsible for the long-lasting pain syndrome in chronic pancreatitis.

Baclofen and the Release of Neuropeptides in the Cat Spinal Cord.

The antibody microprobe technique was used to study the effect of baclofen on the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of pentobarbitone-anaesthetized spinalized cats. Both peptides were released in the region of the substantia gelatinosa during ipsilateral noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of baclofen suppressed the excitation of lumbar dorsal horn neurons, but did not produce detectable alterations of the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial grey matter dorsal to these neurons. The results suggest that the antinociceptive action of baclofen does not involve a reduction of the intraspinal release of substance P or calcitonin gene-related peptide from the central terminals of nociceptive sensory fibres.

Influence of opioids on substance P release evoked by antidromic stimulation of primary afferent fibers in the hind instep of rats

The effect of opioids on the release of immunoreactive substance P (iSP) following simultaneous electrical stimulation of the sectioned sciatic and saphenous nerves was examined by perfusion of the subcutaneous space in the rat instep. Antidromic stimulation of both the nerves caused an increase in iSP release, which was dependent on the intensity of stimulation, and an approx. 200% increase in Evans blue extravasation. Stimulation-induced iSP release and extravasation were suppressed by pretreatment with capsaicin (50 mg/kg s.c.) and spantide (10 μmol/kg i.p.), respectively. Intra-arterial infusion of morphine (30 μmol/kg) or ethylketocyclazocine (30 μmol/kg) or [ d-Ala 2, d-Leu 5]enkephalin (30 μmol/kg) inhibited the increase in iSP release evoked by antidromic stimulation at 10 V. This inhibitory effect of morphine was antagonized by pretreatment with naloxone (2 mg/kg, i.p.). These results suggest existence of multiple types of opioid receptor on the peripheral endings of primary afferent fibers, that regulate SP release from the peripheral nerve endings into the extravascular space.

Involvement of lysosomes in substrate stabilization of tryptophan-2,3-dioxygenase in rat liver

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.

Are lymphocytes a target for substance P modulation in arthritis?

The contribution of the neuropeptide substance P to the pathogenesis of rheumatoid arthritis (RA) has recently been suggested. The presence of immunoreactive substance P in the serum and joint fluid of RA patients was significantly increased compared with age-matched control patients. To investigate the ability of substance P to alter lymphocyte activity during the disease, lymphocytes were isolated from the synovial fluid and blood of RA patients and their ability to respond to substance P as measured by [ 3H]thymidine uptake was characterized. Upon exposure of RA synovial fluid and peripheral blood lymphocytes to various concentrations of substance P in vitro, no increase in proliferation was witnessed. To the contrary, control peripheral blood lymphocyte proliferation was significantly enhanced by various concentrations of substance P. However, synoviocytes from the joints of RA patients were responsive to substance P stimulation. These data suggest that substance P receptors may be desensitized on systemic and local lymphocytes in RA, or the proinflammatory activities of substance P may be mediated via the synovial membrane during chronic inflammation.

Close association of peptidergic nerves with lymphocytes in canine and monkey ileal villi.

Intimate association of peptidergic nerves with lymphocytes of canine and monkey ileal villi was demonstrated by immunohistochemistry and transmission electron microscopy. A swollen, presumably terminal, portion of nerves containing large cored vesicles and small clear vesicles was in direct contact with a lymphocyte. The apposing membranes of the nerve and lymphocyte were thickened and darkened, being separated by a narrow uniform space. The lymphocyte-associated nerves contained immunoreactivity for substance P(SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP), localized in large-cored vessels. These result support the hypothesis that peptidergic nerves may play a regulatory role in mucosal immune responses.

Changes in NGF Receptor-Like Immunoreactive Substance in the Suprachiasmatic Nucleus of the Hypothalamus in Blind Rats.

In mammals, the hypothalamic suprachiasmatic nucleus (SCN), a circadian oscillator, receives the retinohypothalamic tract (RHT), which is essential for the synchronization of circadian rhythms by light-dark cycle; and nerve growth factor (NGF) receptors with low affinity have been detected in the SCN. Thus, we examined whether low-affinity NGF receptors (LNGFR) are present in the terminals of the RHT by studies on the LNGFR-like immunoreactive substance (LNGFRLIS) in the SCN of blind rats. LNGFRLIS in the SCN gradually decreased with age in control rats, though it could be observed until 16-20 weeks of age. After bilateral orbital enucleation of 4-week-old rats, the density of the LNGFRLIS gradually decreased on both sides of the SCN from week 4 after the operation, disappearing 7 weeks after the operation. In congenitally blind, hereditary microphthalmic rats, LNGFRLIS was not detectable in the SCN, but in half-blind hereditary microphthalmic rats LNGFRLIS was detectable on both sides of the SCN. 125I-NGF injected into the SCN was found in the optic nerve and the retina. Since NGF is suggested to be retrogradely transported and to support survival of neurons, these findings suggest that LNGFR is present at the terminal of the RHT, and raise the possibility that NGF or other growth factors such as brain-derived neurotrophic factor in or around the SCN plays a role in maintaining the retinal ganglion cells through LNGFR.

Enzyme immunoassay of thyrotropin releasing hormone (TRH).

A sensitive and specific double-antibody enzyme immunoassay (EIA) for a thyrotropin releasing hormone (TRH)-like immunoreactive substance has been developed. In order to synthesize TRH-labeled beta-D-galactosidase (beta-gal), a newly devised TRH derivative, pGlu-His-Pro-NH-(CH2)6-NH2 (TRH-Hex), was employed. TRH-Hex was linked to beta-gal by the N-(epsilon-maleimidocaproyloxy) succinimide coupling procedure. For competitive reactions, the TRH antibody was incubated with standard TRH and TRH-Hex-beta-gal (delayed addition). Free and antibody-bound enzyme hapten were separated by using an anti-rabbit immunoglobulin G coated immunoplate. Activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows detection of 0.8 to 100 pmol/well of TRH.

Human urinary trypsin inhibitor (urinastatin)-like substance in mouse liver

Mouse liver contains a human urinary trypsin inhibitor (urinastatin, UT)-like immunoreactive substance with trypsin inhibitory activity. Northern blot analysis demonstrates the presence of the appropriate 1.3 kb mRNA band in liver tissue but not in kidney or other tissues examined. Administration of hydrocortisone, which is known to increase the urinary excretion of the UT-like substance, increased the levels of UT-like substance in serum and in the liver tissue. In contrast, deoxycorticosterone acetate did not have such an effect. These results suggest that the gene encoding UT-like substance is primarily expressed in the liver of the mouse, and that glucocorticoids play an important role in regulating the hepatic synthesis of UT-like substance. Furthermore, these findings indicate that the mouse is a suitable species for research on the biological function of UT or UT-like substances.

Existence of a Human Urinary Trypsin Inhibitor (Urinastatin)-Like Substance in the Rat Brain

A human urinary trypsin inhibitor, urinastatin (UT)-like immunoreactive substance with trypsin inhibitory activity, was demonstrated in certain brain regions in rats, especially the cerebral cortex, hippocampus and hypothalamus. Although this UT-like substance in the rat brain displayed an N-terminal amino acid sequence similar to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), it did not show any GAPDH activity. These results indicate that the UT-like substance in the rat brain is a protein different from GAPDH and indicates a localized distribution within certain brain regions partly related to learning and memory.

Light and electron microscopic immunohistochemistry of (Met)-Enkephalin-Arg6-Gly7-Leu8-like immunoreactive neurons in the rat cerebellum.

The distribution and fine structure of [Met] -Enkephalin-Arg6-Gly7-Leu8-like immunoreactive (MEAGL-LI) neurons in the rat cerebellum were examined by light and electron microscopic immunohistochemistry. Immunoreactive neurons which were designated to be Golgi cells were found in the granular layer. These MEAGL-LI Golgi cells were distributed heterogeneously in the cerebellar cortex; the densest distribution was seen in the ventral and anterior vermis, moderate distribution in the pedunculofloccular lobe (PFL) and less in the dorsal and posterior parts of the cerebellar hemispheres. Immunoreactive Golgi cells showed well-developed organelles, such as rough-surfaced endoplasmic reticulum (r-ER) and Golgi complex as well as immunoreactive substances throughout the cytoplasm. On the surfaces of immunoreactive neurons non-immunoreactive axons made axo-somatic synapses. MEAGL-LI neuronal elements were distributed as components of the cerebellar glomeruli in the granular layer. Some of them were presynaptic axons which made axo-dendritic synapses with non-immunoreactive dendrites, and others were postsynaptic dendrites with which non-immunoreactive axons made axo-dendritic synapses. MEAGL-LI neuronal elements were also seen to make intimate contact with Purkinje cells in the Purkinje cell layer.

Hepatoblastomas: an ultrastructural and immunohistochemical study.

Seven hepatoblastomas were studied by electron microscopy, and four of these were studied by immunohistochemistry. Five tumors were purely epithelial, and two were mixed epithelial-mesenchymal. They showed a spectrum of cellular differentiation ranging from primitive epithelial cells to differentiated cells resembling adult hepatocytes. Glycogen, lipid, basal lamina, and canaliculi were present in all cases. Mitochondria with large, membrane-bound, amorphous inclusions were present in one tumor, and large, complex, basal cell processes were present in two tumors. Ultrastructural features most characteristic of hepatocytes were most common in fetal type hepatoblastomas. Immunoreactive chromogranin cells were present in two tumors, one of which also contained immunoreactive somatostatin cells. The somatostatin-positive tumor had cells with granules resembling those seen in somatostatin-containing cells of normal pancreas and somatostatin-containing neuroendocrine carcinomas. Other immunoreactive substances were present, including alpha 1-antitrypsin (four cases), vimentin (embryonal cells in four cases; fetal cells in three cases), low-molecular weight cytokeratin (embryonal cells in three cases; fetal cells in four cases), and high-molecular weight cytokeratin (embryonal cells in one case; fetal cells in two cases). Osteoidlike material was positive for epithelial membrane antigen, vimentin, and S-100 protein.

Dynamics and synthesis of new placental tissue protein PP19

Summary Placental protein 19 (PP19) is a new placental tissue protein identified in extracts from the human term placenta by Bohn and Winkler (1985) . We measured PP19 concentrations in body fluids and placental tissue culture media by radioimmunoassay. The minimum detectable dose of standard PP19 was 15 ng/ml. HCG and PP19 concentrations in media from placental tissue culture rose markedly at 144 hours, but were not increased by adding 0.1 mM cycloheximide. The circulating serum PP19 concentration was 4.5±1.1 ng/ml (mean±standard deviation) in the proliferative phase (n=8) and 5.1±1.6 ng/ml in the secretory phase (n=7) for nonpregnant women and 4.6±2.2 ng/ml from mean (n=12). Seminal plasma (n=8) contained 212.3±99.7 ng/ml. The maternal serum PP19 concentration in 291 normal pregnancies increased from 6.2 ng/ml (median) at 6–7 weeks of gestation to 34.1 ng/ml at 38–39 weeks. The mean PP19 concentration was higher in amniotic fluid and retroplacental blood but lower in umbilical cord blood than in circulating maternal serum. In hydatidiform mole, vesicular fluid contained high PP19 concentrations (1154.6±659.5 ng/ml), although these were not statistically higher than the normal range. The immunological identity of standard PP19 and PP19-like immunoreactive substances in the maternal serum, retroplacental blood, vesicular fluid from hydatidiform mole, and seminal plasma was indicated by parallelism between their dose-response curves in serial dilutions. An elevated (>10 ng/ml) serum PP19 concentration was observed in endometriosis (2 of 2 samples), squamous cell carcinoma of uterine cervix (2 of 11), ovarian adenocarcinoma (11 of 19), choriocarcinoma (1 of 5), invasive mole (3 of 11) and gastric adenocarcinoma (3 of 4). The level was low in myoma uteri (6 of 6), benign ovarian cyst (3 of 3), and endometrial carcinoma stage I (7 of 7). Ascites (2 of 5) and ovarian tumor fluid content (6 of 8) showed high PP19 concentrations. A metastatic gestational choriocarcinoma case showed elevated serum PP19 level in accordance with occurrence of DIC. In blood cell fractions separated by the Ficoll-Paque/Macrodex method, PMN fraction contained the highest PP19 concentration. These results indicate that PP19 has an extraplacental source even though the placental villous trophoblast may be the main source throughout pregnancy, and that PP19 may be associated with polymorphonuclear leukocyte function although the exact biological function has yet to be clarified.

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve terminals in the whole mount preparations of the dog urethra.

To visualize the entire shape of the intraepithelial nerve fibers, whole mount preparations of the dog urethra were produced and immunostained with an antiserum against CGRP, one of the predominant substances contained in the nerves. The immunoreactive nerves in the lamina propria were smooth (non-beaded) in appearance and weak in immunoreaction. Within the epithelium, they displayed typical beaded profiles and were intense in immunoreaction. The intraepithelial fibers branched and wound into an extensive network with wide meshes ("reticular terminal"). The bead-like swellings included large ones resembling Herring bodies in hypophyseal neurosecretory fibers. Another type of nerve terminal, consisting of fine and weakly immunopositive fibers, was also found in the epithelium. These branched in dendritic or in dense bouquet-like fashion, occupying smaller areas ("bouquet-like terminals). Vesicular swellings often characterized these terminals, though they were smaller and more uniform in size and far less in their amount of immunoreactive substance than were the swellings in the reticular terminals. Both types of nerve terminals originated from the same nerve trunk. The connection between the reticular and bouquet-like terminals, which may presumably represent secretory and receptive parts, respectively, morphologically supports the possible occurrence of an axon reflex in the urethral CGRP neurons. Our whole mount preparations, when doubly stained with CGRP and serotonin antibodies, further revealed the CGRP-positive reticular terminals being closely associated with serotonin- or CGRP-immunoreactive paraneurons dispersed in the epithelium.

Immunohistochemical identification of substance P in cutaneous sensory organs (electroreceptors) of gymnotid teleost fish

The distribution of the neuropeptide substance P, which is considered to be a neurotransmitter or neuromodulator of the central nervous system, has been studied in the cutaneous electroreceptor organs (tuberous and ampullary organs) of 3 species of gymnotid fish: Apteronotus leptorhynchus, Eigenmannia virescens and Sternopygus sp. Immunohistochemical data have revealed that substance P is never present in the afferent fibers but is specifically localized in the electroreceptors of the three species examined. Substane P immunoreactivity is strictly localized in the sensory cells of the ampullary organs of all three species and in those of the tuberous organs of Apteronotus leptorhynchus and Sternopygus sp. In contrast, weak substance P immunoreactivity was observed only in certain tuberous sensory cells of Eigenmannia. Substance P immunoreactivity was also found in the accessory cells of certain organs: it was detected in the two types of accessory cells of the tuberous organs of Eigemmannia virescens, in the accessory cells type 2 of the tuberous organs of Sternopygus sp., and in all accessory cells of ampullary organs of Sternopygus sp. and Apteronotus leptorhynchus. In Sternopygus sp., positive staining was only evident if the substance P antibody was used at low concentration. Immunoreactivity for substance P in the sensory cells suggests that it has a transmitter or modulator function in these electroreceptors; the presence of substance P in the accessory cells remains to be explained.

Digoxin-like immunoreactivity in cord blood plasma extracts is not only due to endogenous corticosteroids

Methods for quantitative extraction and enrichment of digoxin-like immunoreactive substances (DLIS) from human cord blood plasma (CBP) using organic solvents combined with a solid state absorbent are presented. Sephadex LH-20 column chromatography is more suitable than sephadex G-25 for DLIS separation and purification from CBP extract. The elution pattern of sephadex LH-20 chromatography of deproteinized and desalted CBP shows two distinct DLIS peaks. The first peak coelutes with the steroids aldosterone, cortisol, progesterone, 17-OH-progesterone, testosterone and estradiol, while the second DLIS peak coelutes with dehydroepiandrosterone sulfate (DHEA-S). The Na+,K(+)-ATPase inhibitory activity peak partially overlaps with the first DLIS peak. Coeluted steroids could account only partially for digoxin-like immunoreactivity in the first DLIS peak; however, they did not contribute to the Na+,K(+)-ATPase inhibitory activity, whereas the second DLIS peak could be ascribed to endogenous DHEA-S.

Basic Fibroblast Growth Factor-Like Substance in Nuclei of Male Germ Cells Undergoing Meiosis

The presence of a basic fibroblast growth factor-like immunoreactive substance was demonstrated in the nuclei of germ cells at stages from spermatocyte to spermatid in adult rat testis by using immunohistochemistry with an antibody raised against a synthetic peptide corresponding to residues 1-10 of bovine basic fibroblast growth factor [1-146]. The fluorescence was very weak in the nuclei and cytoplasm of spermatogonia, Sertoli cells, and most of the interstitial compartments, except for capillary endothelial cells. This is the first study to demonstrate the presence of basic fibroblast growth factor-like immunoreactive material in the nuclei of haploid cells in vivo.

Immunocytochemical characterization of the suprachiasmatic nucleus and the intergeniculate leaflet in the diurnal ground squirrel, Spermophilus lateralis

The suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are retinorecipient structures that play important roles in the expression of circadian rhythmicity. We examined these two structures in a diurnal ground squirrel, Spermophilus lateralis, using i immunohistochemical techniques, and cholera toxin-bound horseradish peroxidase. A number of immunoreactive substances are distributed within the ground squirrel SCN in a pattern similar to that reported in many other mammals. These include vasopressin, vasoactive intestinal polypeptide, serotonin, neuropeptide Y (NPY), and glial fibrillary acidic protein. The squirrel SCN differs from that of most other species examined to date in two respects. First, a dense cluster of cells containing immunoreactive l-enkephalin ( l-ENK-IR) is observed in the center of the SCN. Second, there is a contralateral, but no ipsilateral, projection from the retina to the SCN. In the lateral geniculate region there is a substantial region that contains NPY-immunoreactive cells and receives a bilateral retinal projection. This region is assumed to be homologous with the IGL described in other mammals. Cells containing l-ENK-IR are distributed throughout the LGN in groups that overlap, but which have a distinctly different distribution than the more extensive groups of NPY-IR cells.

Interference of Digoxin-Like Immunoreactive Substances with TDx Digoxin II Assay in Different Patients

The TDx digoxin II immunoassay was used in controls and in patients not taking digoxin or any other cardiac glycoside. We report the frequency of interference thought to be caused by digoxin-like factors (DLFs). We found a high incidence of false-positive results in 15 patients with severe hepatic disease (60% false-positive results). In newborn infants we found false-positive results both when their blood was drawn from a peripheral vein (89% false-positive results) and, more strikingly (100% false-positive results), when it was obtained from an umbilical cord vein (p less than 0.001). Compared to a control group, no statistically significant false-positive results were found in patients with mild to moderate chronic renal failure (n = 21) or in pregnant women (n = 15). In patients with chronic renal failure undergoing hemodialysis six of 25 had false-positive results. These results suggest that digoxin levels must be interpreted carefully in patients with chronic liver disease and chronic renal failure and in newborns until new methods that eliminate the interference caused by DLFs become readily available.

Calcitonin gene-related peptide and substance P decrease in the rabbit colon during colitis: A time study

The sensory neuropeptides, substance P and calcitonin gene-related peptide, have been implicated in inflammatory reactions in several tissues. An immune-complex model of colitis was used in rabbits to determine the colonic content (nmol/g protein) of immunoreactive substance P and calcitonin gene-related peptide at various times after induction of inflammation to assess changes in these neuropeptides during the inflammatory response. Calcitonin gene-related peptide content was decreased by 66% 4 hours after induction of inflammation and reached a maximum of 80% at 48 hours. The substance P content was decreased at 8 hours, with a maximum decrease of 64% at 48 hours. Substance P decrease was detected in the muscle layer. The amounts of substance P in the mucosal/submucosal layer extracts were too low to allow accurate measurements. Calcitonin gene-related peptide decreased both in the muscle and the mucosal-submucosal layers. Immunohistochemical analysis showed that calcitonin gene-related peptide and substance P innervation patterns were comparable in normal and inflamed colon, even though there appeared to be a decrease in density and intensity of the staining, particularly for calcitonin gene-related peptide at 48 hours. The early decrease of calcitonin gene-related peptide and substance P during the time course of colitis might be due to release from nerve terminals of the gut during the inflammatory response. The profound changes in colonic calcitonin gene-related peptide and substance P content during colitis may have important implications during inflammation and subsequent tissue repair and may also lead to disturbances in gut motility.