PGP 9.5, a new marker for human neuroendocrine tumours

PGP 9.5 is a soluble protein isolated from brain and is a general marker for neuronal and neuroendocrine tissue. Its function is not known. Until now neurone specific enolase (NSE) has been the only general marker for the paracrine system and tumours derived from it. Seventy-four neuroendocrine tumours, 17 melanocytic naevi, 51 melanomas and four granular tumours were stained immunohistochemically for PGP 9.5 and NSE. A variety of pulmonary and non-neuroendocrine tumours were also stained. Two so-called goblet cell carcinoids of the appendix were included in the series. Using NSE 59/74 neuroendocrine tumours were positive and 58/74 stained for PGP 9.5. In combination 63/74 of these tumours were positive for either NSE or PGP 9.5 or both. Staining for PGP 9.5 was better for demonstration of nerves in routinely processed material than was staining for NSE. Twenty-one out of 43 primary melanomas stained for PGP 9.5 and 36 showed staining for NSE. Only two of eight metastatic melanomas melanocytic stained for PGP 9.5 while seven of these eight stained for NSE. Six of 17 melanocytic naevi stained for PGP 9.5 and five stained for NSE. All four granular cell tumours stained for PGP 9.5 and NSE. Both "goblet cell carcinoids' of the appendix were negative for NSE and PGP 9.5. Fifteen out of 32 pulmonary cancers showed staining for either marker and no non endocrine tumour showed any specific staining. Staining for PGP 9.5 is a valuable additional probe in the exploration of the paracrine system and the diagnosis of tumours arising from it.     

Protein gene product (PGP) 9.5 as a reliable marker in primitive neuroectodermal tumours—an immunohistochemical study of 21 childhood cases

A number of antibodies to neural proteins have been used to demonstrate neuronal differentiation in primitive neuroectodermal tumours. One of them is protein gene product (PGP) 9.5, a neuronal protein isolated from brain, whose function is unknown at present. We have studied differentiation in 21 cases of primitive neuroectodermal tumours of the CNS in children. Immunocytochemical staining was performed for such neuronal markers as: PGP 9.5, neuron specific enolase and synaptophysin, a glycosylated protein associated with synaptic vesicles. Positive staining for PGP 9.5 was present in 16 cases (strong staining in 12), for neuron-specific enolase in 16 cases (strong staining in 10) and for synaptophysin in 10 cases (strong staining in six). Both PGP 9.5 and synaptophysin showed a clear staining pattern with less non-specific background than with neuron-specific enolase. Our findings demonstrate the value of using more than one antibody marker in assessing neuronal differentiation in tumours. The high incidence of positive staining with antibody to PGP 9.5 suggests that this is an essential marker in the panel of antibodies used for the identification of primitive neuroectodermal tumours.     

Distribution of GAP-43 nerve fibers in the skin of the adult human hand

Skin is an important region of somatic sensory input, and is one of the most innervated areas of the human body. In this study, we investigated in human hand skin the distribution of nervous structures immunoreactive for the growth-associated protein 43 (GAP-43) and the protein gene product 9.5 (PGP 9.5). GAP-43 is a neuronal presynaptic membrane protein that is generally considered to be a marker of neuronal plasticity. PGP 9.5 is a neuron-specific soluble protein that is widely used as general marker for the peripheral nervous system. The entire neural network of the dermis and epidermis was stained with antibody to PGP 9.5. In the dermis, there were fewer GAP-43-immunostained nerve fibers than PGP 9.5-immunostained nerve fibers, whereas in the epidermis the numbers were equal. Only some Merkel cells and Meissner corpuscles were GAP-43-immunoreactive. In conclusion, our results show that GAP-43 protein is expressed in a subset of PGP 9.5-immunoreactive nerve structures.     

Neuroepithelial bodies not connected to pulmonary slowly adapting stretch receptors

Neuroepithelial bodies (NEBs) are believed to be connected with one of the known types of airway receptors. The present studies determined whether NEB afferents are pulmonary slowly adapting stretch receptors (SARs). NEBs are immunoreactive with antibodies against protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP), whereas SARs are reactive with antibody to Na(+)/K(+)-ATPase. Using histochemical staining in combination with confocal microscopy, we compared the morphology of NEBs and SARs in the rat. Our results show that NEBs and SARs are different in location, size, and shape. Double staining of airway tissues for PGP (or CGRP) plus Na(+)/K(+)-ATPase shows that NEBs and SARs do not co-localize. In addition, we electrophysiologically recorded single-unit activity of SARs from the cervical vagus nerve, identified their receptive fields, dissected them into blocks, and then double-stained and examined the receptor structures. We found that the blocks contain the SAR, but not NEB structures. Thus, we conclude that NEBs are not connected to SARs.     

低氧对肺降钙素阳性神经内分泌细胞影响的定量研究

用光镜免疫组织化学和原位包埋免疫电镜技术观察了低氧对成年大鼠肺降钙素阳性神经内分泌细胞的影响，计数了单位面积内单个神经内分泌细胞、神经上皮小体以及ＮＥＣ与ＮＥＢ细胞个数之和的阳性细胞总数。统计分析结果显示，低氧导致ＴＮＥ、ＮＥＢ和组成ＮＥＢ的细胞数目增多。原位包埋免疫电镜法发现，降钙素免疫反应物定位于神经分泌颗粒内。     

Histamine induces the neuronal hypertrophy and increases the mast cell density in gastrointestinal tract

Histamine is an endogenous biogenic amine that is synthesized from the basic amino acid histidine. Ability to mimic anaphylaxis is one of the first described functions of histamine and it has been demonstrated that histamine plays a significant role in the regulation of immune system and neuronal function, influences neuronal morphology and is involved in mast cells (MCs) chemotaxis. MCs as histamine releasers, may thus also interact with neuronal function. In the present study, we aimed to evaluate the role of histamine on mast cell density and neuronal morphology in the gastrointestinal tract of the mouse.Ten mice were daily injected intraperitoneally for 7 days with 20 mg/kg of histamine diluted in 0.5 ml physiological serum. After 7 days, mice were euthanised and samples from stomach, small bowel, colon and appendix were processed for histological examination. Immunohistochemistry was performed employing primary antibodies directed against triptase for mast cells and PGP 9.5 antigen for neuronal structures. The density of triptase and PGP 9.5 positive cells and the morphology of the ganglia were quantitatively evaluated by digital image analysis.The number of ganglia was higher in stomach, small bowel, colon and appendices of the histamine group when compared with the control group. Only in appendices and colon, the number of Schwann cells was significantly higher than that of the control group. The PGP 9.5 expression and the mean area of ganglia showed a significant increase only in appendices. In histamine group the MCs were clustered especially in the lamina propria. Mast cell density (MCD) was significantly higher than the control group in the small bowel, colon and appendices tissues.The intraperitoneally injection histamine increases the MCD and induces the neuronal hypertrophy and after the comparison of the organs in the gastrointestinal tract the results indicated the most effected organ as the appendices.     

UCHL1 (PGP 9.5): Neuronal biomarker and ubiquitin system protein

UCHL1/PGP 9.5 (also known as UCHL1 and PGP 9.5) was first detected as a “brain-specific protein” over 28 years ago. The protein is highly conserved and localized in neurones and neuroendocrine cells in vertebrates, forming an estimated 5–10% of cytoplasmic protein. A minor proportion in brain is tightly membrane-bound and the protein is also found in human oocytes and spermatogonia. A few specialised neurones lack UCHL1/PGP 9.5 and possibly replaceable neurones have low levels of the protein. UCHL1/PGP 9.5 shows sequence homology with UCHL3 (ubiquitin carboxyl-terminal hydrolase L3) and will similarly hydrolyse C-terminal adducts of ubiquitin. Both proteins show an unusual highly knotted structure with five “crossovers” but there are differences in substrate specificity, amino-acid sequence, and tissue distribution between them. There is no convincing evidence that UCHL1/PGP 9.5 can remove ubiquitin from proteins destined for proteasomal degradation, rather the substrate(s) of the enzyme appear to be one or more as yet unidentified short ubiquitin C-terminal extensions. Other suggested functions of the protein are plausible but largely unconfirmed. Isolated loss of UCHL1/PGP 9.5 function seen in the gracile axonal dystrophy (GAD) mouse due to a deletion in its gene results in a failure of axonal transport and a “dying-back” axonopathy beginning distally in long axons. The evidence that mutations in the UCHL1/PGP 9.5 gene lead to either significant susceptibility to or protection from Parkinson's disease (or other human neurodegenerative disorders) is weak. Antibodies to the protein have found remarkably widespread application in the detection of fine nerves in peripheral tissues of many vertebrate species.     

Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation

We have characterized the distribution of neural tissue and its primary target tissue, airway smooth muscle (ASM), in an in vitro mouse model of early lung development comprising left lung lobes at embryonic Day 12, cultured for 2 or 5 d. Neural tissue was detected with antibodies to protein gene product 9.5 (PGP 9.5), synapsin, and p75NTR (the low-affinity neurotrophin receptor), and smooth muscle with an antibody to alpha-actin. Imaging by confocal microscopy revealed few PGP 9.5-positive neurons at the start of culture; after 2 d clusters of neurons and nerve fibers had appeared along the lobar bronchus and after 5 d along the secondary and tertiary branches. Neural tissue did not just follow the smooth muscle-covered tubules, as seen in vivo, but also grew outside the lobes onto a wide layer of alpha-actin-positive cells, suggesting that smooth muscle may express a trophic factor that attracts nerves. Explants cultured with glial-derived neurotrophic factor (GDNF) exhibited a striking increase in the amount of p75NTR- and PGP 9.5-positive tissue outside the lobes, whereas GDNF-impregnated beads attracted neuronal precursors and influenced the direction of neurite extension. We show that the mouse lung explant is suitable for investigating trophic signals involved in pulmonary innervation and that GDNF may have a role in the early innervation of the developing airways.     

Pulmonary neuroendocrine cell hyperplasia: identification, diagnostic criteria and incidence in untreated ageing rats of different strains

Pulmonary Neuroendocrine Cells (PNEC) are found as clusters called neuroepithelial bodies (NEB) or as single cells scattered in the respiratory epithelium. Pulmonary neuroendocrine cell hyperplasia is recorded in humans and experimentally manipulated rodents. The objectives of this work were to identify the optimal immunohistochemical markers for PNEC in the rat for use on paraffin-embedded, formalin-fixed material and to provide the first comparative incidence of PNEC hyperplasia in untreated 2-year-old rats of different strains. Calcitonin-gene related peptide (CGRP) and protein G product 9.5 (PGP9.5) antibodies identified PNEC consistently and selectively. In contrast, PNEC did not express chromogranin-A or S-100. PNEC hyperplasia was defined as foci of PNEC with greater than 40 nuclei, excluding overlying respiratory epithelium and submucosal PNEC. PNEC hyperplasia was observed at low incidence (0-7%) in untreated 2-year-old Sprague-Dawley, Han Wistar and Wistar rats but not Fischer 344 rats. This is the first report of spontaneous PNEC hyperplasia in rats. The cause of this hyperplasia is unknown, but experimental models that induce PNEC hyperplasia by causing bronchiolar cell injury are discussed. PNEC neoplasia in the rat is unreported in the literature and was not observed in animals examined in this study.     

Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.     

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12–13) — a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14–16) — thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17–18) — thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0–3] many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5–10) — a small number of PGP 9.5 IR cells in the taste buds appeared, and their number increased gradually. Stage VI (PI4-adult) — the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.     

Peptidergic not monoaminergic fibers profusely innervate the young adult human testis

Numerous studies have reported that intratesticular nerves exert important regulatory effects on the functions of the male gonad; however, as yet little is known about their distribution in the young adult human testis. The purpose of this study was to explore whether peptidergic and adrenergic nerves occur in the male gonad of this age, and, if present, to depict their distribution further. Thirty testes were collected from 15 reproductively healthy donors aged 21–32 years. Antibodies against protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), C-terminal flanking peptide of NPY (CPON) and vasoactive intestinal peptide (VIP) were employed for immunohistochemical detection of intratesticular peptidergic nerves, and those against dopamine-beta-hydroxylase (DBH) and 5-hydroxytryptamine (5-HT) for monoaminergic ones. The testicular parenchyma exhibited a rich innervation by PGP 9.5-positive fibers, mainly associated with Leydig cell nests, blood vessels, and seminiferous tubules. Numerous NPY- and CPON-immunoreactive (IR) nerves also appeared in the gonads, but the vast majority were confined to blood vessels. A small number of VIP-IR fibers were detected in some arterioles. By contrast, however, no fibers displaying DBH or 5-HT immunoreactivity were observed within the testis. Additionally, expression of PGP-9.5, NPY, CPON, VIP, DBH and 5-HT was found in Leydig cells, PGP 9.5 in spermatogonia, and NPY and CPON in peritubular myoid cells. Our results suggest that the young adult human testis is devoid of monoaminergic nerves but profusely innervated by peptidergic fibers, which may serve as major neuronal regulators for testicular functions at this age.     

The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies

In order to assess the potential of protein gene product (PGP) 9.5 as a marker of the nervous and neuroendocrine systems, we examined its immunolocation in human, rat and guinea-pig tissues, using a rabbit polyclonal antiserum and two new mouse monoclonal antisera, I3C4 and 3IA3. Our results demonstrate immunoreactive PGP 9.5 in neurons and nerve fibres at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in part of the renal tubule, in spermatogonia and Leydig cells of the testis, and in ova and in some cells of the pregnant and non-pregnant corpus luteum. In routinely processed tissues, standard immunohistochemical techniques using the polyclonal antibody demonstrated peripheral nerve fibres of all sizes with striking clarity.     

Development of nerve fibres in the temporomandibular joint of the human fetus

The development of nerve fibres in the temporomandibular joint (TMJ) in relation to the development of bone, muscle and fibre components was investigated in human fetuses ranging from 9 weeks of gestation to birth. Immunohistochemistry for the glia-associated protein S-100 and for the neuro-specific marker protein gene product 9.5 (PGP 9.5) were used; specimens were compared to specimens of adult TMJ capsule and disc. At 9–10 weeks, a small number of neural elements are already present in the connective tissue around the joint and in the mesenchyme between the two articular blastemas from which the disc will differentiate. By 19 weeks many nerve fibres are clearly visible. Immunohistochemical results suggest diffuse disc innervation extending along the entire disc but not in the thin central area. More complex structures, i.e. encapsulated corpuscles, were also seen. The fetal disc appears highly innervated compared to adult tissue; already at this developmental stage morphology and distribution of nerves and corpuscles in the joint capsule are comparable to those in the adult joint. It may be concluded that the innervation of the TMJ is detectable from the end of the second month and that it develops fully between the third and the fifth month of gestation. Nerve endings in the disc are most numerous at 20 weeks, after which a progressive reduction, possibly secondary to the growth of articular tissues, is observed throughout the last trimester of fetal life and into adult life. The innervation of the lateral pterygoid muscle, on the contrary, is much less than that seen in adult muscles, even at full-term.     

The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.

In order to assess the potential of protein gene product (PGP) 9.5 as a marker of the nervous and neuroendocrine systems, we examined its immunolocation in human, rat and guinea-pig tissues, using a rabbit polyclonal antiserum and two new mouse monoclonal antisera, I3C4 and 3IA3. Our results demonstrate immunoreactive PGP 9.5 in neurons and nerve fibres at all levels of the central and peripheral nervous system, in many neuroendocrine cells, in part of the renal tubule, in spermatogonia and Leydig cells of the testis, and in ova and in some cells of the pregnant and non-pregnant corpus luteum. In routinely processed tissues, standard immunohistochemical techniques using the polyclonal antibody demonstrated peripheral nerve fibres of all sizes with striking clarity.     

Endocrine differentiation of extra-pulmonary small cell carcinoma demonstrated by immunohistochemistry using antibodies to PGP 9.5, neuron-specific enolase and the C-flanking peptide of human pro-bombesin

Several recent studies have confirmed the endocrine nature of small cell carcinoma of the lung. In extra-pulmonary sites, small cell 'undifferentiated' carcinomas have classical morphological features similar to their pulmonary counterpart. We therefore investigated, using immunocytochemistry, the possibility that the non-pulmonary neoplasms may also be endocrine in nature. Sections of 29 small cell carcinomas from oesophagus, stomach, larynx, colon and urinary bladder were immunostained using antisera to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), cytokeratin, leucocyte common antigen and peptides including bombesin, the C-flanking peptide of human probombesin, adrenocorticotrophic hormone, neurotensin, calcitonin and pancreatic polypeptide. All the tumours showed immunoreactivity for at least one of the two general endocrine markers PGP 9.5 and NSE. Twenty-three of the 29 cases were immunoreactive for PGP 9.5, 27 for NSE. All were positive for cytokeratin and negative for leucocyte common antigen. Of the regulatory peptides, immunoreactivity was obtained with antisera to bombesin (one case), the C-flanking peptide of human pro-bombesin (14 cases), adrenocorticotrophic hormone (one case) and calcitonin (three cases). No PGP 9.5-, NSE- or peptide-like immunoreactivity was detected in 25 control tumours from similar sites, including lymphomas and poorly differentiated tumours. These results suggest that non-pulmonary small cell carcinoma has an endocrine character.     

Capsaicin-induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats

To evaluate the potential for neuronal replacement following destruction of vagal afferent neurons, we examined nodose ganglia following i.p. capsaicin treatment of adult rats. Rats received capsaicin or vehicle followed by a regimen of 5'-bromo-2'-deoxyuridine injections (BrdU) to reveal DNA replication. Nodose ganglia were harvested at various times post-treatment and processed for 4',6-diamidino-2-phenylindole (DAPI) nuclear staining and immunofluorescence to estimate neuronal numbers and to determine vanilloid receptor, cleaved caspase 3, TUNEL, BrdU, the neuron-selective marker protein gene product (PGP) -9.5 and neurofilament-M-immunoreactivity. Twenty-four hours after capsaicin approximately 40% of nodose ganglion neurons expressed cleaved caspase 3-immunoreactivity and 16% revealed TUNEL staining, indicating that primary sensory neurons are killed by the capsaicin treatment of adult rats. The occurrence of neuronal death was confirmed by counts of DAPI-stained neuronal nuclei, which revealed >or=50% reduction of nodose neuron number by 30 days post-capsaicin. However, by 60 days post-capsaicin, the total numbers of neuronal nuclei in nodose ganglia from capsaicin-treated rats were not different from controls, suggesting that new neurons had been added to the nodose ganglia. Neuronal proliferation was confirmed by significant BrdU incorporation in nuclei of nodose ganglion cells immunoreactive for the neuron-specific antigen PGP-9.5 revealed 30 and 60 days post-capsaicin. Collectively, these observations suggest that in adult rats massive scale neurogenesis occurs in nodose ganglia following capsaicin-induced neuronal destruction. The adult nodose ganglion, therefore, provides a novel system for studying neural plasticity and adult neurogenesis after peripheral injury of primary sensory neurons.     

The three-dimensional distribution of nerves along the entire intrapulmonary airway tree of the adult rat and the anatomical relationship between nerves and neuroepithelial bodies

Using airway microdissection and three-dimensional confocal microscopy techniques in combination with the immunomarkers protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP), we defined the distribution of small afferent nerves fibers and all nerves throughout the intrapulmonary airways, along with the distribution of airway neuroendocrine cells and neuroepithelial bodies. We found (i) the presence of CGRP-and PGP 9.5-positive structures along the entire intrapulmonary airway tree of adult rats, (ii) decreasing nerve density from more proximal to more distal generations of conducting airways, (iii) the presence of nerve fibers in terminal bronchioles, (iv) the asymmetrical distribution of nerves within a single generation of intrapulmonary airway with regard to associated vessels, (v) the frequent interchange of single nerve fibers across epithelial and sub-epithelial compartments without termination, and (vi) a definably intimate relationship between afferent nerves and neuroepithelial bodies (NEBs) (i.e., 58% of NEBs studied were observed to have nerve fibers coursing through them, indicating direct connections). We conclude that the distribution of nervous elements (nerve fibers and neuroendocrine cells) within the intrapulmonary airways is highly heterogeneous, varying between airway levels and locally within a specific airway level.