Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse.

The intrapancreatic cellular distribution and effects on basal and stimulated insulin secretion of the 37-amino-acid polypeptide, calcitonin gene-related peptide (CGRP), were investigated in the mouse. The cellular localization of CGRP was also studied in the rat pancreas. In both species, CGRP was demonstrated in pancreatic islet cells and nerve fibers. Immunocytochemical double staining experiments revealed the CGRP-immunoreactive cells in the mouse to be identical with a majority population of the insulin cells. In the rat, on the other hand, CGRP-immunoreactive cells were identical with somatostatin cells. CGRP-immunoreactive nerve fibers were observed, in both species, running in the exocrine parenchyma, particularly around blood vessels, and they were occasionally seen also within the islets. In in vivo experiments, CGRP was found to inhibit both basal and stimulated insulin secretion in the mouse. Thus, 6 min after the iv injection of CGRP (0.85 nmol/kg), plasma insulin levels were 13 +/- 2 (SE) microU/ml compared to 30 +/- 4 microU/ml in controls (P less than 0.01). At this dose level, CGRP inhibited the insulin secretory response to carbachol, leaving that to glucose unaffected. However, at a higher dose level (4.25 nmol/kg), CGRP inhibited glucose-induced insulin secretion as well. We conclude that CGRP occurs in islet cells and in intrapancreatic nerve fibers of both the mouse and the rat, and inhibits both basal and stimulated insulin secretion in vivo in the mouse.

Peptide YY: a neuropeptide in the gut. Immunocytochemical and immunochemical evidence.

Peptide YY immunoreactivity was detected in neuronal elements in the upper digestive tract of the rat, cat, ferret and pig by immunocytochemistry and radioimmunoassay combined with high-performance liquid chromatography. The two peptide YY antisera used do not recognize the related peptides neuropeptide Y and pancreatic polypeptide. In the rat peptide YY-immunoreactive nerve fibres were virtually restricted to the stomach smooth muscle of the minor curvature where they were numerous. Peptide YY-immunoreactive nerve cell bodies occurred in small ganglia on the serosal surface of the minor curvature. In the cat and ferret peptide YY-immunoreactive nerve fibres occurred in the circular smooth muscle layer of both the minor and major curvatures of the stomach and also in the upper small intestine; such fibres were numerous also in myenteric ganglia in these regions. In the pig, they were few but had roughly the same distribution as in the cat and ferret, except that they were quite numerous in thick muscle bundles close to the oesophagogastric junction. The presence of peptide YY-immunoreactive nerve cell bodies within the myenteric ganglia along the upper digestive tract of cat, ferret and pig, and in serosal ganglia of the rat stomach, indicates that at least some of the peptide YY-immunoreactive fibres demonstrated originate in or close to the gut wall. Double-staining experiments revealed that virtually all peptide YY-containing neurons and nerve fibres were distinct from those storing neuropeptide Y. Peptide YY-immunoreactive endocrine cells were encountered not only in the lower intestines but also in the stomach of the four species studied. In the antrum such cells were numerous and constituted a subpopulation of the gastrin-containing cells. In the oxyntic mucosa they were few and contained somatostatin. Radioimmunoassay revealed peptide YY-like peptides in gastric mucosa and smooth muscle from the upper digestive tract of all four species examined. The results of high-performance liquid chromatography suggest that the peptide YY-like material in the upper digestive tract is distinct from neuropeptide Y and pancreatic polypeptide and identical with authentic peptide YY except in the antral mucosa where only a small proportion of the peptide YY-immunoreactive material eluted like authentic peptide YY.

Calcitonin, calcitonin gene-related peptide, and gastrin-releasing peptide in familial thyroid medullary carcinoma.

We examined immunocytochemically the occurrence of the three peptides calcitonin, calcitonin gene-related peptide (CGRP), and gastrin-releasing peptide (GRP) in medullary carcinoma of the thyroid. We also sought to determine whether the plasma levels of these peptides were increased when stimulated with calcium and pentagastrin in familial medullary carcinoma of the thyroid (MCT). The tumor tissue from all 17 cases examined was found to exhibit calcitonin and CGRP immunoreactivity, and in 15 of the 17 cases the tumor tissue also contained GRP immunoreactivity. In 7 of the cases selected at random, an intravenous injection of calcium carbonate (2 mg/kg body weight) and pentagastrin (0.6 microgram/kg body weight) produced marked elevation in plasma levels of calcitonin but did not significantly alter the plasma levels of CGRP or GRP. We conclude that most MCT tumors contain CGRP and GRP immunoreactive cells but that the plasma levels of CGRP and GRP are not altered on stimulation. This finding is clearly in contrast to the markedly elevated calcitonin levels. Hence, determination of plasma calcitonin levels still seems to be the most appropriate diagnostic test for MCT.

Pancreatic function in the essential fatty acid deficient rat.

The influence of essential fatty acid (EFA) deficiency on pancreatic endocrine and exocrine function was studied in 120-day-old rats. The plasma insulin response was determined after in vivo administration of glucose and arginine. The plasma glucagon response was assessed after infusion of arginine. Islet peptides were examined by immunocytochemistry. The exocrine function of pancreas was studied by amylase secretion in isolated pancreatic acinar cells after stimulation with the cholinergic agonist carbacholine chloride. The EFA-deficient (EFAD) rats showed higher basal plasma insulin concentrations and lower basal glucose levels than control rats (P less than .01 and P less than .01, respectively). The plasma insulin response to glucose was potentiated in the EFAD rats (P less than .001). Both insulin and glucagon responses to arginine were normal. The isolated pancreatic acinar cells showed a low basal amylase secretion, but a normal response to carbacholine chloride. There were no overt morphological changes seen in the pancreas and the immunocytochemical staining pattern of insulin, glucagon, somatostatin, and pancreatic polypeptide cells did not differ from controls. The results of the study show that the secretory function of the endocrine and exocrine pancreas is operational in EFA deficiency. The EFA deficiency was accompanied by a basal hyperinsulinemia and hypoglycemia and an exaggerated insulin response to glucose, the pathophysiology of which has to be further studied.

PYY-like peptides in the central and peripheral nervous system of a frog and a lizard.

PYY-immunoreactive material was detected in endocrine cells in the gut of a lizard, Lacerta vivipara, and a frog, Rana temporaria. The findings are consistent with other reports on reptiles, amphibians and higher species. In addition, however, PYY-like material was found in neuronal elements both in the gut and in the brain. High performance liquid chromatography of frog brain extracts showed the PYY-like material to elute in one minor fraction (eluting position similar but not identical to that of porcine synthetic PYY) and two major fractions (distinct from PYY and probably representing smaller and more hydrophobic PYY-like peptides).

Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study.

Electron microscopic immunocytochemistry using protein A-gold labelling of ultrathin sections revealed immunoreactive glicentin (gut-type glucagon) and peptide YY (PYY) in virtually all secretory granules in a population of L-type endocrine cells in feline colon and human rectum. The granules of the human glicentin/PYY cells were considerably smaller in size than those in the cat. In both species the results indicate co-existence of glicentin and PYY in the same secretory granules, despite the probable derivation of the two peptides from two different precursors.

Peptide YY in the mammalian pancreas: immunocytochemical localization and immunochemical characterization.

Peptide YY (PYY) was demonstrated by immunochemical and/or immunocytochemical methods in endocrine cells in the pancreas of adult mice, rats, guinea-pigs, cats, dogs, pigs and cows. In the pancreas of mouse and rat, immunoreactive PYY was observed in a major subpopulation of the glucagon cells (splenic lobe of the pancreas); immunoreactive PYY also occurred in a subpopulation of the pancreatic polypeptide (PP) cells (duodenal lobe), and in a few extra-insular endocrine cells dispersed throughout the pancreatic parenchyma. In the pancreas of cat, dog and pig immunoreactive PYY was found to coexist with PP, but not with glucagon. Radioimmunoassay (RIA) revealed PYY-like material in extracts of pancreas (and colon) of all the species examined. The highest concentrations were found in the pancreas of cat and mouse; moderate amounts were found in the rat and only small amounts were detected in guinea-pig and pig. The concentrations in the pancreas were uniformly much lower than those in the colon. Analysis by high performance liquid chromatography (HPLC) showed that the PYY-immunoreactive material from pancreas (and rat colon) had an elution profile very similar to that of synthetic PYY, and distinct from that of PP and neuropeptide Y.

Immunocytochemical localisation of the icosapeptide fragment of the PP precursor: a marker for 'true' PP cells?

Antisera were raised against the icosapeptide fragment of the pancreatic polypeptide (PP) isolated from the canine pancreas. They were used for the immunocytochemical study of the cellular localisation and distribution of the icosapeptide in the gut and pancreas of various mammals. The results indicate that PP and the icosapeptide coexist in the majority of the PP-immunoreactive cells in the pancreas of cat, dog, pig, monkey and man and in all the PP-immunoreactive cells in the stomach of the cat and dog. The icosapeptide does not seem to occur in cells or nerves containing PP-related peptides, such as peptide YY or neuropeptide Y. PP-immunoreactive cells devoid of the icosapeptide could be demonstrated in the large intestine. These cells are probably distinct from the pancreatic PP cell type, and the PP-immunoreactive material probably represents the homologous peptide YY rather than PP. The present findings support the view that the icosapeptide is part of the PP precursor and hence, only the cells containing immunoreactive icosapeptide in addition to immunoreactive PP are to be considered 'true' PP cells. The icosapeptide antisera did not stain PP cells in mouse, rat and guinea-pig, suggesting marked species variation in the amino acid sequence of the icosapeptide portion of the PP precursor.

Peptide YY-like immunoreactivity in the central nervous system of the rat.

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.

The presence and actions of NPY in the canine endocrine pancreas.

Immunofluorescent staining for neuropeptide Y (NPY) in canine pancreatic tissue was performed together with an evaluation of the effects of synthetic NPY on the release of insulin (IRI), glucagon (IRG) and somatostatin (SLI) from the duodenal lobe of the canine pancreas in situ. NPY-like immunoreactivity was localized in perivascular nerve fibers throughout the acinar tissue. NPY-immunoreactive fibers were also demonstrated in the islets, usually surrounding blood vessels but also occasionally in fibers associated with endocrine cells, primarily at the periphery of islets. In addition, the ganglia dispersed in the pancreatic parenchyma were densely innervated by NPY-immunoreactive fibers, and these ganglia regularly contained cell bodies staining for NPY. Direct infusion of NPY into the pancreatic artery (p.a.) produced a dose-dependent decrease of pancreatic SLI output and of pancreatic venous blood flow. Low-dose p.a. infusion of NPY (50 pmol/min) had no effect on basal IRI or IRG output or on the islet response to glucose (5-g bolus, i.v.). High-dose p.a. infusion of NPY (500 pmol/min) transiently stimulated IRI output and modestly increased IRG output. However, the comparatively sparse innervation of canine islets with NPY-like immunoreactive fibers and the relatively minor effects of large doses of synthetic NPY on pancreatic hormone release lead us to conclude that this peptide is not an important neuromodulator of islet function in the dog.

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut.

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.

Peptide YY: intrapancreatic localization and effects on insulin and glucagon secretion in the mouse.

We studied the intrapancreatic localization of peptide YY (PYY) and the effects of PYY on insulin and glucagon secretion in the mouse. Immunofluorescence staining of mouse pancreatic tissue showed that PYY occurred within islet cells. These cells were located preferentially at the periphery of the islets. Sequential and simultaneous double immunostaining revealed that most PYY cells also displayed glucagon immunoreactivity; some PYY cells contained immunoreactive pancreatic polypeptide (PP). At the electromicroscopic level, PYY immunoreactivity was demonstrated within the secretory granules of both glucagon cells and of a small granular cell type, which showed structural similarities to PP cells. In in vivo experiments, PYY at dose levels between 0.53 and 8.5 nmol/kg had no influence on basal plasma levels of insulin, glucagon, or glucose. In contrast, insulin secretion stimulated by glucose or the cholinergic agonist carbachol was inhibited by PYY (by 33 and 26%, respectively, at 4.25 nmol/kg). Similarly, carbachol-induced glucagon secretion was inhibited by PYY (by 47% at 4.25 nmol/kg). We conclude that PYY occurs in islet cells of the mouse pancreas, most of which are glucagon cells, and that PYY inhibits stimulated insulin and glucagon secretion in vivo in the mouse.

GLP-1 and GLP-1(7-36) amide: influences on basal and stimulated insulin and glucagon secretion in the mouse.

We studied the cellular distribution of glucagon-like peptide-1 (GLP-1) in the pancreas and gut and the effects of GLP-1 and its truncated form, GLP-1(7-36) amide, on basal and stimulated insulin and glucagon secretion in the mouse. Immunofluorescence staining showed that GLP-1 immunoreactivity occurred within peripheral islet cells and in cells located mainly distally in the small intestine and in the entire large intestine. Double-immunostaining revealed that the GLP-1-immunoreactive cells were identical to the glucagon/glicentin cells. Experiments in vivo revealed that basal insulin secretion was stimulated by GLP-1(7-36) amide at the dose levels of 8 and 32 nmol/kg, and by GLP-1 at 32 nmol/kg. Furthermore, GLP-1(7-36) amide showed additive stimulatory influence with glucose (2.8 mmol/kg), the cholinergic agonist carbachol (0.16 mumol/kg), and the C-terminal octapeptide of cholecystokinin (CCK-8, 5.3 nmol/kg), when injected at 8 or 32 nmol/kg. In contrast, stimulated insulin secretion was unaffected by GLP-1. Moreover, the glucagon secretory responses to carbachol and CCK-8 were inhibited by GLP-1(7-36) amide but were unaffected by the entire GLP-1. We conclude that GLP-1(7-36) has the potential for being a modulator of islet hormone secretion.

Pancreatic peptide YY in alloxan diabetic mice.

Peptide YY (PYY) immunoreactive material was detected in the splenic and duodenal portions of the adult mouse pancreas, using immunocytochemical and immunochemical methods. Cells displaying PYY immunoreactivity generally occurred at the islet periphery. Double immunostaining enabled localization of PYY to a major subpopulation of the glucagon cells and to subpopulations of the pancreatic polypeptide (PP) cells and the somatostatin cells. In contrast, no PYY immunoreactivity occurred in the insulin cells. In alloxan-treated hyperglycemic mice, PYY immunoreactive cells were increased in number and distributed throughout the islets, in parallel with the glucagon, PP, and somatostatin cells. Analysis by radioimmunoassay indicated a significant increase in the concentration of pancreatic PYY after alloxan treatment in the splenic portion of the pancreas, but not in the duodenal portion. Pancreatic glucagon concentrations were not significantly changed. It is concluded that the islet content of PYY increases in alloxan diabetes, which might contribute to the accompanying alterations in islet function.

[The new Spinal Cord Injuries Centre of the Hamburg Industrial Injuries Insurance Institutes Accident Hospital--report on its 1st year]. / Das neue Querschnittgelähmten-Zentrum des Berufsgenossenschaftlichen Unfallkrankenhauses Hamburg--Bericht über das erste Jahr der Tätigkeit.

The former Spinal Cord Injuries Department within the Hamburg Accident Hospital of the Industrial Injuries Insurance Institutes has been expanded from 33 to 100 beds. 60 beds started operation on Febr. 1, 1981, another 20 on Aug. 1, 1981 and the remaining 20 beds on Jan. 1, 1982. The present report is based on admissions during the first twelve months of the Centre's operation, i.e. 136 recent cases and 98 re-admissions, 92% of which had come from Northern Germany. 35% of the recent cases had been admitted during the first 48 hours post-onset. 39% of the patients were tetraplegics, 57% had complete paralysis, 13% non-traumatic affections. 60% of the patients had associated injuries. 60% were covered by statutory sickness insurance. The average length of stay was 113 days for tetraplegic, and 112 days for paraplegic patients. In 60% the extent of paralysis remained unchanged. Of 10 deaths, three were due to pulmonary embolism. Decubitus ulcers had been the indication for re-admission in 36%, of whom only 20% were tetraplegics. Average stay for decubitus ulcers was three times as long as in other re-admission causes. The services rendered by the various professional departments as well as by medical consultants are reported in tabular form. The expansion responded to an urgent need, and has, among others, contributed to easing many avoidable social problems (such as long distances from home).

Localization and peptide content of endocrine pancreatic tumors.

Endocrine pancreatic tumors contain and frequently secret neurohormonal peptides. This phenomenon can be used as a diagnostic and classifying tool. This study analyzes 31 patients operated on because of an endocrine pancreatic tumor, including the diagnostic procedures and the localization methods. In 15 insulinoma cases only 6 patients had a positive arteriography, while all 11 selective pancreatic vein samplings were positive. The immunoreactivity showed that, besides insulin, most tumors also contained other peptides. Of four gastrinoma cases the arteriography was positive in three, but the selective vein sampling localized the tumor in all. The tumor's content of peptides showed mixed patterns. In the four glucagonomas, the arteriography was positive in all and the venous sampling performed in three of the cases also was positive. In five pancreatic polypeptide-containing tumors (PP-omas) the arteriography was positive in four and sampling performed in two was positive in both. In the PP-omas the peptide pattern showed that these tumors frequently contain several peptides. We used selective pancreatic vein sampling in 21 cases with positive result in all. In the cases in which arteriography was negative, the sampling results helped the surgeon to find the tumor. The peptide pattern in the tumors varied greatly and most tumors were multihormonal.