Substance P- and calcitonin gene-related peptide immunoreactivity in primary afferent neurons of the cat's knee joint.

The distribution of substance P and calcitonin gene-related peptide was determined in primary afferent neurons of the medial and posterior articular nerve of the cat's knee joint. Perikarya of articular afferents were visualized by retrograde labelling with the fluorescent dye Fast Blue which was applied at the transected end of the peripheral nerves. Substance P was found in about 17% of labelled medial articular afferents and in about 16% of labelled posterior articular afferents, respectively, whereas calcitonin gene-related peptide was present in about 35 and 32% of the medial and posterior articular nerve cells, respectively. Taking into account that these neuropeptides are known to be co-localized, probably not more than one-third of the joint afferents contain substance P and/or calcitonin gene-related peptide. Quantification of cell diameters revealed that substance P was found only in small- or intermediate-sized perikarya (less than 50 microns) indicating that this peptide is predominantly found in unmyelinated neurons. Calcitonin gene-related peptide was present mainly in small- and intermediate- but also in some large-sized neurons (greater than 50 microns) providing evidence that this peptide is found in unmyelinated and to a lesser extent in myelinated neurons. This is consistent with previous studies that show that substance P and calcitonin gene-related peptide are present primarily in unmyelinated and thinly myelinated primary afferents. When the portion of substance P-positive neurons of the medial articular nerve is compared to the number of articular afferents displaying a nociceptive function as determined in earlier electrophysiological studies, it can be calculated that at most 30% of the nociceptive-specific articular afferents contain this neuropeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric comparison of synovium from patients with osteoarthritis and rheumatoid arthritis.

Synovium was collected from 15 patients who were undergoing joint surgery. Two groups were defined by clinical diagnosis: patients with primary osteoarthritis (n = 4); and those with rheumatoid arthritis (n = 11). The synovium was studied using histological and morphometric techniques. In agreement with previous studies, no histological features specific for either diagnosis were found. A previously validated morphometric method was used to estimate the cellular density of randomly picked fields within defined areas of synovium. The mean nuclear density of cellularity of comparable areas of synovium was significantly different between these two disease states, but the mean nuclear density between individual representative samples within each clinical group was homogeneous. The morphometric analysis of lymphocyte subsets showed that within the upper synovial region and cellular aggregates in osteoarthritis, the distribution of T cells expressing the CD4 and CD8 antigen was the same. In rheumatoid arthritis CD8 cells predominated in the upper synovial region and CD4 cells in the cellular aggregates. Plasma cells were rarely found in osteoarthritic synovia, but were common in rheumatoid arthritis, with IgG-producing plasma cells predominating. Morphometric studies of representative synovial samples may help to improve histological diagnosis and our understanding of pathological mechanisms.     

神经肽Y/降钙素基因相关肽在腰椎间盘中的分布与共表达

背景：研究发现,神经肽Y与降钙素基因相关肽在交感神经节中有共存现象。 目的：观察神经肽Y/降钙素基因相关肽在正常腰椎间盘的共分布及在突出腰椎间盘髓核组织中的共表达。 方法：从10例尸体中收集完整的腰椎间盘,在另10例尸体中收集腰椎间盘髓核组织作为对照组。收集30例有症状的腰椎间盘突出症患者,其中L4/5或L5/S1需行腰椎间盘髓核摘除,取出髓核组织作为实验组。 结果与结论：①正常椎间盘组织中神经肽Y/降钙素基因相关肽荧光双标染色阳性的神经纤维较多的分布于椎间盘纤维环外1/3,但在椎间盘内2/3及髓核中未见或少量分布。②髓核的共表达,神经肽Y/降钙素基因相关肽荧光双标染色神经纤维的阳性率实验组均高于对照组(P < 0.05)。提示在正常腰椎间盘组织中神经肽Y/降钙素基因相关肽分布于纤维环外1/3部分,在纤维环内2/3部分及髓核组织未分布,但在突出椎间盘髓核组织中有大量的神经肽Y/降钙素基因相关肽共表达。     

Immunoreactive tachykinins, calcitonin gene-related peptide and neuropeptide Y in human synovial fluid from inflamed knee joints

We have analysed the concentrations of substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in the synovial fluid from 5 patients suffering from arthritis with inflamed knee joints as well as from 5 healthy control subjects with an earlier traumatic meniscal or cruciate ligament injury. Competitive radioimmunoassay was done using antisera SP2 (SP), K12 (NKA), R8 (CGRP), and NPY1 (NPY). No SP-like immunoreactivity (-LI) was detected in any patient NKA-LI was found in all control patients but in none of the arthritis patients. CGRP-LI was seen in all arthritis patients as well as in control patients, a non-significant difference. NPY-LI was found in a significantly higher concentration in the arthritis group vs the control patients. The results support an involvement of neuropeptides in human joint inflammation.     

Concentrations of calcitonin gene-related peptide and neuropeptide Y in plasma increase during flushes in postmenopausal women

OBJECTIVE: To assess whether the plasma concentrations of calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), or neurokinin A (NKA) increase during hot flushes in postmenopausal women with vasomotor symptoms. DESIGN: Eight postmenopausal women (age range = 49-63 years) with vasomotor symptoms were included. During 1 day, repeated blood samples were taken between and during flushes; four samples were taken during each flush. The samples were analyzed for CGRP, NPY, and NKA using radioimmunoassay technique. RESULTS: The serum concentrations of CGRP and NPY increased significantly-73% and 34%, respectively-during the flushes (p = 0.018; p = 0.028), whereas the concentrations of NKA did not change significantly. CONCLUSIONS: CGRP and NPY may be involved in the mechanisms that cause vasomotor symptoms.     

Mycoplasma fermentans,but not M penetrans,detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders.

AIM/BACKGROUND: Mycoplasmas, especially Mycoplasma fermentans, were suggested more than 20 years ago as a possible cause of rheumatoid arthritis but this hypothesis was never substantiated. In view of the superior sensitivity of the polymerase chain reaction (PCR) assay over culture, the aim was to use this method to seek M fermentans and M penetrans in synovial samples from patients with various arthritides. METHODS: Synovial fluid samples (n = 154) and synovial biopsy specimens (n = 20) from 133 patients with various rheumatic disorders were stored at -80 degrees C for between one and 40 months. Aliquots (500 microliters) of the synovial fluid samples were centrifuged and the deposit, and also the synovial biopsy specimens (approximately 1 g) were placed in lysis buffer with proteinase K for DNA extraction. The DNA was tested by using a semi-nested PCR assay for M fermentans and a single-round PCR for M penetrans. RESULTS: M fermentans was detected in the joints of eight (21%) of 38 patients with rheumatoid arthritis, two (20%) of 10 patients with spondyloarthropathy with peripheral arthritis, one (20%) of five patients with psoriatic arthritis, and four (13%) of 31 patients with unclassified arthritis. M fermentans was not found in the joints of the seven patients with reactive arthritis, the 29 with osteoarthritis or post-traumatic hydrarthrosis, the nine with gouty arthritis, nor the four with chronic juvenile arthritis. M penetrans was not detected in any sample. CONCLUSIONS: These findings show that the presence of M fermentans in the joint is associated with inflammatory rheumatic disorders of unknown cause, including rheumatoid arthritis. However, whether this organism triggers or perpetuates disease of behaves as a passenger remains conjectural.     

Substance P and calcitonin gene-related peptide immunoreactive sensory DRG neurons innervating the lumbar intervertebral discs in rats.

The rat L5/6 disc is innervated from T13 to L6 dorsal root ganglia (DRGs) multisegmentally. Sensory fibers from T13, L1 and L2 DRGs have been reported to innervate through the paravertebral sympathetic trunks, whereas those from L3 to L6 DRGs innervate directly through sinuvertebral nerves on the posterior longitudinal ligament (PLL). The presence of substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (ir) nerve fibers has been demonstrated in the lumbar intervertebral discs, but their percentages in DRG neurons have not been studied. Fluoro-gold (F-G) labeled neurons innervating the L5/6 disc were distributed throughout DRGs from T13 to L6 levels. Of F-G labeled neurons innervating the L5/6 disc, the percentage of SP-ir T13 to L6 DRG neurons was 30%, and that of CGRP-ir neurons was 47%. The mean cross-sectional area of the cell of SP-ir neurons was 696+/-66 microm2 (mean +/- S. E.), and that of CGRP-ir neurons was 695+/-72 microm2 (mean +/- S. E.). SP- and CGRP-ir were mainly observed in small neurons. The percentages of SP- or CGRP-ir neurons in L1 and L2 DRGs innervating the L5/6 disc were not different from those in L3, L4 or L5 DRGs. In the physiological condition in rats, DRG neurons at all levels may have the same significant role in pain sensation of the disc.     

Substance P and calcitonin gene-related peptide in the ureter of chicken and guinea-pig: distribution, binding sites and possible functions.

To elucidate the possible functional significance of sensory neuropeptides in visceral organs of mammals and birds the distribution, binding sites and the effects on ureteric peristalsis of substance P and calcitonin gene-related peptide (CGRP) were investigated in the ureter of guinea-pigs and chickens. In the guinea-pig numerous substance P and CGRP-immunoreactive fibres were located in the adventitia, smooth muscle layer, submucosa and occasionally in the epithelium. Varicose peptidergic fibres were often found on blood vessels. Binding sites for substance P were associated with blood vessels and epithelium in the following density order: venules greater than epithelium greater than arterioles. The highest density of CGRP binding sites was detected on the smooth muscle; venules and arterioles expressed moderate binding. The peristalsis frequency of the isolated ureter of the guinea-pig was increased by neurokinin A and substance P, whereas CGRP inhibited ureteric motility. In the chicken the immunoreactivity to substance P and CGRP was less pronounced. Immunoreactive fibres were found in the submucosa close to the epithelium and around ureteric ganglion cells. Correspondingly, substance P binding sites were located in the epithelium and in ureteric ganglia; however, specific CGRP binding was restricted to large blood vessels. In the chicken none of the sensory neuropeptides affected ureteric motility. Only high doses of the sensory neurotoxin capsaicin (greater than 10 microM) repeatedly produced a non-specific inhibitory effect, similar to that found in a capsaicin-desensitized guinea-pig ureter preparation. The data suggest that in the guinea-pig ureter sensory neuropeptides play a modulatory role in the regulation of ureteric motility and might have vascular and epithelial functions. In the chicken, substance P might be involved in the regulation of epithelial function and modulation of ganglionic transmission. The physiological or pathophysiological role of sensory neuropeptides and the efferent functions of afferent fibres appears to be much better developed in the guinea-pig than in the chicken.     

Studies on rat and human thymus to demonstrate immunoreactivity of calcitonin gene-related peptide, tyrosine hydroxylase and neuropeptide Y

The peptidergic and noradrenergic innervation of rat and human thymus was investigated by immunohistochemistry at the light and electron microscopical level (avidin-biotin-complex, sucrose-phosphate-glyoxylic-acid, and immunogold techniques). The distribution of noradrenergic neural profiles, and positive immunoreactivity for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) is described in female rats during ageing, and in human children. In the neonatal rat thymus, the arteries and septa are well supplied by fine varicose nerves. In older animals (2 wk–1 y) the number of septa and blood vessels increase and consequently also the innervation. No nerves were found in the cortex. Apart from the innervation of the septal areas, immunoreactivity for CGRP and TH was present in thymic cells. Except for the young rats (neonatal–14 d), all rats showed CGRP positivity in subcapsular/perivascular epithelial cells (type 1 cells). All rat thymuses also contained a few TH positive cells in the medulla, which could only be confirmed as epithelial cells (type 6 cells) in children. Type 1 cells in the human thymus were not CGRP positive, but as in the rat, there were similar TH positive cells in the medulla. It was concluded that in addition to nerves containing CGRP, noradrenaline or dopamine, epithelial cells also contain these transmitters. They could therefore act on different cells (compared with neural targets) in a paracrine manner.     

Substance P- and calcitonin gene-related peptide-like immunoreactive neurons in the rat trigeminal ganglion--with special reference to meningeal and pineal innervation.

The distribution of perikarya showing substance P- (SP) or calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the rat trigeminal ganglion (TG) were investigated by means of immunohistochemical methods. Approximately 50% of the perikarya contain CGRP while SP-LI was observed in 1/3 of the cells. IR fibres were seen to leave the ganglion via the ophthalmic, maxillary, and mandibular nerves. The combination of peptide histochemistry and retrograde labelling of cells in the ganglion following injection of a fluorescent tracer into the pineal gland reveals that few SP- or CGRP-LI trigeminal neurons innervate the pineal gland. In contrast, the vast majority of perikarya in the TG were labelled upon application of the tracer to the meningeal surface supporting the view that meninges and meningeal arteries in rodents are heavily innervated by SP- and CGRP-LI trigeminal neurons.     

The neurotransmitter role of calcitonin gene-related peptide in the rat and guinea-pig ureter: effect of a calcitonin gene-related peptide antagonist and species-related differences in the action of omega conotoxin on calcitonin gene-related peptide release from primary afferents.

In the rat and guinea-pig isolated ureter electrical field stimulation of intrinsic nerves (10 Hz for 10 s) produces transient inhibition of evoked (20 mM KCl or 0.1-1 microM neurokinin A) rhythmic contractions by releasing transmitter(s) from peripheral endings of capsaicin-sensitive primary afferents. The C-terminal fragment of human calcitonin gene-related peptide (8-37) blocked the inhibitory effect of electrical field stimulation as well as that produced by exogenous calcitonin gene-related peptide, while leaving unaffected the inhibitory response to isoprenaline. Human calcitonin gene-related peptide (8-37) was devoid of any inhibitory activity of its own but enhanced the amplitude and frequency of KCl-evoked rhythmic contractions in the rat ureter, probably by antagonizing the inhibitory effect of endogenous calcitonin gene-related peptide released by KCl. Omega conotoxin fraction GVIA, a peptide which possesses a potent blocking activity of N-type voltage-sensitive calcium channels, prevented the inhibitory response to electrical stimulation in the guinea-pig ureter, while leaving the response unaffected in the rat ureter. Conotoxin had no effect toward the inhibition produced by exogenous calcitonin gene-related peptide indicating its prejunctional site of action, demonstrated previously in the guinea-pig ureter [Maggi et al. (1990) Neurosci, Lett. 114, 203-206]. Dermorphin, an amphibian peptide with potent agonist activity on mu-type opioid receptors, inhibited the response to electrical stimulation in the guinea-pig ureter but had no effect in the rat ureter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential release of calcitonin gene-related peptide and neuropeptide Y from the isolated heart by capsaicin, ischaemia, nicotine, bradykinin and ouabain

The influence of various drugs as well as total ischaemia on the outflow of calcitonin gene-related peptide (CGRP), which is present in sensory nerves, and neuropeptide Y (NPY), which is co-stored with noradrenaline (NA), from the isolated guinea-pig heart, was studied in vitro. Capsaicin exposure and total ischaemia for 5-30 min induced a Ca2+-dependent increase in the outflow, suggesting release, of CGRP- but not NPY-like immunoreactivity (LI) from the heart. When characterized by high performance liquid chromatography (HPLC), the CGRP-LI present in heart extracts and the released CGRP-LI by capsaicin eluted in a major peak corresponding to synthetic CGRP. Incubation with morphine, indomethacin or reserpine pretreatment did not influence the capsaicin-evoked release of CGRP-LI. Capsaicin pretreatment depleted the cardiac content of CGRP-LI but not NPY-LI. The increase in perfusate volume observed after 30 min ischaemia in controls was reduced after capsaicin pretreatment. Nicotine exposure induced release of CGRP- as well as NPY-LI in a concentration- and Ca2+-dependent manner. The increased outflow of NPY-LI was not influenced by capsaicin pretreatment. Among other agents tested, bradykinin and ouabain caused increased outflow of CGRP but not of NPY-LI. Noradrenaline, tyramine, histamine, vasopressin, alpha,beta methylene ATP, ATP or adenosine induced changes in cardiac contractility or flow but did not evoke any detectable release of CGRP- or NPY-LI. In conclusion, the release of multiple neuropeptides can be studied in combination with contractile recordings using the isolated perfused guinea-pig whole heart preparation. Activation of cardiac sensory nerves by capsaicin, nicotine, bradykinin and ouabain, as well as ischaemia, induced release of CGRP while nicotine also evoked NPY release.     

类风湿关节炎滑膜组织及关节液中DcR3表达的研究

目的:观察研究人诱骗受体3(DcR3)在类风湿关节炎(RA)关节液及滑膜组织中的表达及其与滑膜炎性病变程度的相关性.方法:用免疫组织化学的方法对12例RA患者、6例骨关节炎(OA)患者及4例无关节病变的骨折患者滑膜组织中DcR3的表达进行描述分析,并对DcR3表达情况与RA患者滑膜炎性病变程度相关性进行分析.同时用ELISA的方法检测了26例RA及19例OA患者关节液中DcR3的表达.结果:DcR3在RA滑膜组织主要分布于血管翳周围炎性细胞及部分滑膜细胞中,DcR3阳性细胞数约为72％.OA滑膜组织细胞中DcR3也呈现少量的阳性表达,但与RA比较阳性程度较弱,阳性细胞数量较少:对DcR3表达与RA滑膜炎性程度的相关性分析发现,DcR3与RA滑膜炎性程度呈正相关(r=0.832,P＜0.01),同时发现RA患者关节液中表达也明显高于OA患者.结论:DcR3在RA滑膜组织炎性细胞及滑膜细胞上的异常表达可能是其参与RA滑膜炎及滑膜组织侵袭等病理过程,从而导致滑膜损伤的一个重要机制.     

Tachykinins, calcitonin gene-related peptide and neuropeptide Y in nerves of the mammalian thymus: interactions with mast cells in autonomic and sensory neuroimmunomodulation?

By the use of light microscopic (LM) immunohistochemistry the distribution of tachykinin (TK)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivity in nerves supplying the mammalian (rat, mouse, guinea-pig, cat) thymus gland has been determined. There were no interspecies variations. Fibres staining for TK and CGRP completely overlapped indicating coexistence. They were present in the capsule, in interlobular septa and in the corticomedullary boundary and occurred in perivascular and paravascular plexus supplying arteries, veins and the microvasculature. Some TK/CGRP-immunoreactive (ir) fibres travelled between lymphoid cells and close contacts with mast cells were frequent. NPY-ir fibres were different from those staining for TK/CGRP and predominated in the perivascular plexus of arterial blood vessels. Only very rarely they coursed in the lymphoid parenchyma. Intimate contacts of NPY-ir fibres with mast cells were less frequent than those of TK/CGRP-ir fibres. We conclude that the NPY innervation is mainly sympathetic noradrenergic while thymic nerves coding for TK and CGRP are most likely of sensory origin. These pathways may play a differential neuroimmunomodulatory role in the thymus, possibly via interaction with mast cells.     

Detection of calcitonin and calcitonin gene-related peptide mRNA in human medullary thyroid carcinoma. A retrospective study

In Situ hybridization finds many applications in modern pathology. In many cases, special attention is paid to the processing of the tissues prior to in situ hybridization. In order to investigate the value of RNA in situ hybridization (RISH) in retrospective studies, we performed RISH for calcitonin and calcitonin gene-related peptide (CGRP)-I and HomRNA in eight medullary thyroid carcinomas processed in 1981–1983. RISH was successful with radioactive calcitonin and CGRP-1 probes. With biotinylated probes, only calcitcnin-specific probes gave adequate results. The concentrations of CGRP mRNA were probably too low to be detected by non-radioactive RISH. The results of RISH were correlated with the immunohistochemical localization of the polypeptides. The results matched in all cases except one, where hybridization for calcitonin mRNA was found, but no immunoreactive calcitonin polypeptide. We con-clude that RISH can be successfully used for retrospective analysis, even after long storage of tissue embedded in poraffin.     

Immunohistochemical comparison of chromogranins A and B and secretogranin II with calcitonin and calcitonin gene-related peptide expression in normal, hyperplastic and neoplastic C-cells of the human thyroid

Normal and hyperplastic thyroid C-cells and 14 cases of medullary thyroid carcinoma were investigated immunohistochemically with antibodies against chromogranins A and B, secretogranin II, calcitonin and calcitonin gene-related peptide (CGRP). Normal and hyperplastic C-cells showed strong calcitonin and chromogranin A immunoreactivity whereas CGRP, chromogranin B and secretogranin II expression was less intense. Strong calcitonin and chromogranin A immunoreactivity was also found in the majority of tumour cells in medullary thyroid carcinoma. The CGRP, chromogranin B and secretogranin II staining observed was present in variable patterns. In some cases CGRP, chromogranin B and secretogranin II could only be demonstrated in isolated tumour cells with elongated processes suggestive of neuronal differentiation of these cells. The biological function(s) of the chromogranins/secretogranins remain(s) still unclear. There is evidence that these proteins are pro-peptides which give rise to functionally active compounds. Studies on normal C-cells and medullary thyroid carcinoma may elucidate the role of chromogranins/secretogranins in endocrine and neuronal cells.