The nerve-dependency of Merkel cell proliferation in cultured human fetal glabrous skin.

Merkel cells are thought to function as slowly adapting mechanoreceptors and are known as targets for sensory nerves. However, the nerve-dependency of Merkel cells remains controversial. In this respect, some investigators have found interregional differences between hairy and glabrous skin and others have shown intraregional differences within denervated rat touch domes. Differences between species have also been reported. This study was performed to determine whether Merkel cells proliferate in vitro in the absence of the systemic factors, blood vessels and the intact nerves in human skin. Suspension organ culture was performed using fetal digits to investigate their in vitro proliferation. Merkel cells and cutaneous nerves were identified using antibodies to cytokeratin 20 and protein gene product 9.5 (PGP 9.5), respectively. Fetal digits of 56-82 day gestational age were cultured in serum free medium in a high O2 (45%) environment. Tissues were harvested before starting culture (D0) and 1, 4, 7, 14, 28 d after culture. Merkel cells were observed in the volar pads and dorsal nail matrices at D0. After 28 d of suspension organ culture, digits looked healthy structurally and the number of Merkel cells had increased. However, PGP 9.5-immunoreactive nerves were markedly diminished after 1 day of culture and almost disappeared after 4 days. Merkel cell proliferation in vitro suggested that Merkel cell development is probably nerve-independent in human fetal glabrous skin.     

Postnatal differentiation of Merkel cells in the rat palatine mucosa, with special reference to the timing of peripheral nerve development and the potency of cell mitosis

The origin and mechanism of the differentiation and proliferation of Merkel cells are enigmatic. A preliminary study in our laboratory showed that Merkel cells in the rat palatine mucosa emerge after birth. This is in contrast to the case of similar cells in the skin that differentiate during the embryonic period prior to the establishment of peripheral nerve innervation. We studied immunohistochemically the developmental timings of Merkel cells and peripheral nerves in the rat palatine mucosa using antibodies to cytokeratins 18 and 20, PGP 9.5, and CGRP using developing palates of prenatal and postnatal rats. We also studied the potency of mitosis in Merkel cells by immunohistochemistry using antibodies for a cell proliferation marker Ki67 and cyclin D-kinase inhibitors p16, p21 and p27. It was shown that Merkel cells in the rat palatine mucosa differentiate postnatally, after the development of peripheral nerve fiber terminals was almost established. The emergence and increase in number of Merkel cells progressed in an anterior-to-posterior wave. Newly appearing Merkel cells were usually negative for anti-cytokeratin 20 antibody but gained affinity for the antibody with progress of maturation. All Merkel cells in the palatine mucosa were negative for anti-Ki67 antibody but positive for anti-p27 antibody. These results indicate that Merkel cells in the rat palatine mucosa are not responsible for the development of peripheral nerve fiber terminals and that these cells differentiate in situ from intraepithelial stem cells. Accepted: 14 June 2000     

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.     

Characterization of the peri-implant epithelium in hamster palatine mucosa: behavior of Merkel cells and nerve endings

The purpose of this study was to investigate the relationship between Merkel cells and nerve elements during tissue regeneration after receiving dental implants. Golden hamsters were divided into 3 groups and titanium alloy implants were fixed in their left-side maxilla through the third palatine rug. Animals were sacrificed at 1, 2, 3, 4, 5, 6, and 7 days after the implantation and tissues were characterized at the immunohistochemical and morphological levels. CK 20 and PGP 9.5 antibodies which react with Merkel cells and nerve fibers were used. Immunohistochemically, no CK 20-positive Merkel cells were seen in the peri-implant epithelium throughout the 7 days. However, starting at day 4, PGP 9.5-positive nerve fibers appeared in the connective tissue, and by day 7, nerve fibers had invaded the more superficial layer of the peri-implant epithelium compared to the mucosa removal control group. At the electron microscopic level, the intercellular spaces of the regenerating epithelium in the mucosa removal control group were small. In contrast, intercellular spaces of the peri-implant epithelium tended to be wide and regenerating nerve fibers invaded those intercellular spaces. In both the mucosa removal control group and the implantation group, the basal lamina and connective tissues regenerated completely. However, clear Merkel cells containing neurosecretory granules were not observed. Taken together, our results indicate that Merkel cells in the hamster palatine mucosa do not regenerate in the peri-implant epithelium. However, regenerative nerve fibers seem to play essential roles as part of the defense and sensory systems around the peri-implant epithelium to compensate for the weakened defense mechanism.     

A comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and S-100 protein in neuroblastoma, Ewing's sarcoma and other round cell tumours in children

A comparative study of immunohistochemical staining for neuron-specific enolase (NSE), protein-gene product 9.5 (PGP 9.5) and S-100 was made in 71 undifferentated round cell tumours from 65 children using formalin-fixed tissues and a standard alkaline phosphatase-anti-alkaline phosphatase method. All of 29 neuroblastomas marked for NSE and 27 for PGP 9.5; staining was diffuse and usually strong in all tumour elements, irrespective of the degree of differentiation. Patterns of staining remained consistent in primary, recurrent and metastatic tumours and were not modified by previous chemotherapy. S-100 staining was weak and confined to cell processes and schwannian elements in less than half of the tumours studied. Two primitive neuroectodermal tumours both stained strongly for NSE and PGP 9.5. Staining for NSE was observed in single maturing cells in 3/12 rhabdomyosarcomas and in tubular elements in 2/4 Wilms' tumours; primitive rhabdomyoblasts and undifferentiated renal blastema were negative; seven lymphomas were negative. Six of 17 skeletal Ewing's sarcomas showed light to moderate cytoplasmic staining for NSE and PGP 9.5. The site, histology and clinical course of these marker-positive Ewing's sarcomas showed no distinctive features. Staining for PGP 9.5 is a useful additional marker for neural differentiation in round cell tumours.     

Apoptosis of Merkel cells in neurotrophin-3 null mice

Postnatal mice lacking neurotrophin-3 (NT3) are deficient in Merkel cells of touch domes and whisker follicles. We examined the mechanism of Merkel cell loss by immunocytochemistry and electron microscopy. Merkel cell of whisker follicles of NT3 null newborns exhibited decreased immunoreactivity for cytokeratin 8 and contained apoptotic bodies that were positive for cleaved caspase-3, a marker of active apoptosis. By electron microscopy, the Merkel cells displayed aggregation of chromatin along the nuclear membrane, with the marginated chromatin forming caps at the periphery of the nucleus. Ribosomes aggregated in the cytoplasm, while dense core granules characteristic of Merkel cells were still discernible. Finally, the Merkel cells and their nuclei fragmented into apoptotic bodies. None of the apoptotic Merkel cells were contacted by nerve fibers, and their desmosomal contacts with surrounding keratinocytes disappeared. After postnatal day 6 apoptotic Merkel cells were no longer observed, and the number of surviving Merkel cells was severely reduced. They were flat and contained few osmiophilic granules. We conclude that perinatal apoptosis is responsible for the loss of Merkel cells lacking innervation in NT3 null mice.     

Richner-Hanhart's Syndrome: New Ultrastructural Observations on Skin Lesions of Two Cases

New ultrastructural observations are described in skin lesions of two brothers with Richner-Hanhart's syndrome (RHS). Physical examination of the two patients showed painful skin lesions of palms and soles combined with denderitic corneal ulceration and mental retardation. The diagnosis of RHS was confirmed biochemically with high tyrosine levels in both blood and urine. Examination by transmission electron microscopy revealed several abnormal ultrastructural changes in the epidermal cells. The horny cells contained heterogeneously, electron-dense cytoplasm with many lipid droplets. The granular cell cytoplasm contained abundant tonofibrils and keratohyaline granules. The spinous cell cytoplasm was vacuolated due to the presence of minute tyrosine crystals, which are known to have a lytic effect. The surrounding keratinocytes contained multilobed nuclei. The basal epidermal cells appeared normal except for Merkel cells, which were severely damaged by vacuolatio, also due to the presence of tyrosine crystals. This study showed that high tyrosine levels can induce several ultrastructural pathological changes in the epidermal cells, including the skin chemoreceptor Merkel cells.     

Immunohistochemical analysis of equine pulmonary granular cell tumours

Histopathological and immunohistochemical examinations were made on four female horses aged 9-12 years with pulmonary granular cell tumours (GCTs). The tumours, which were multiple, of varying size, firm and off-white in colour, surrounded the bronchi and bronchioles. Metastatic lesions were not detected. The tumour cells had abundant eosinophilic cytoplasm filled with prominent coarse eosinophilic granules. Immunohistochemically, these tumour cells reacted uniformly with vimentin and S100 antibodies. Most were immunolabelled by antibodies against glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and protein gene product 9.5 (PGP9.5), and a few cells were positive with Leu7 antibody. However, the tumour cells did not react with antibodies against neurofilament protein (NF), cytokeratin (CK), chromogranin, alpha1 antichymotrypsin (AACT), myoglobin, desmin, alpha-actin or alpha-smooth muscle actin (alpha-SMA). These immunohistochemical properties of tumour cells support the hypothesis that equine pulmonary GCTs are derived from Schwann cells of the peripheral nervous system in peribronchial and peribronchiolar tissues. GFAP, MBP, Leu7 and PGP9.5 antibodies should help to distinguish equine granular cell tumours from other tumours. Copyright Harcourt Publishers Ltd.     

Loss And Survival of Spiral Ganglion Neurons in the Guinea Pig After Intracochlear Perfusion with Aminoglycosides

Loss of cochlear hair cells results in a loss of ganglion cells and further neurodegenerative changes throughout the auditory pathway. Understanding more about the early stages of ganglion cell loss in vivo may lead to ways of ameliorating or preventing the loss of these neurons. To examine these stages, the effects of intracochlear perfusion with aminoglycoside antibiotics on the organ of Corti and spiral ganglion cells were evaluated in young adult guinea pigs at survival periods ranging from 1 hour to 12 weeks, using immunocytochemical and ultrastructural techniques. At 1 hour survival a base-to-apex gradient of damage was indicated in the cochlea by the appearance of severely damaged hair cells and injured ganglion cells in the basal coil while in the apical coil, hair cells were damaged but intact and ganglion cells appeared normal. By 4 hours the appearance of severely disrupted hair cells and damaged ganglion cells had extended throughout the cochlea. The ultrastructural appearance of many injured ganglion cells demonstrated features characteristic of cell death including condensed cytoplasm, non-marginal clumping of nuclear chromatin, and wrinkled nuclear membrane. Despite the loss of many ganglion cells, a population of these cells remained at 12 weeks survival. These contained large amounts of rough endoplasmic reticulum, were unmyelinated apart from the central process and were surrounded by satellite cells. These features are typical of ganglion cells during development, before the onset of hearing. Immunolabelling of cochlear whole mounts after hair cell destruction with protein gene product 9.5 (PGP 9.5) revealed the presence of neural elements in the organ of Corti at up to 12 weeks survival. These may associated with the remaining ganglion cells. In these surviving ganglion cells, the intense labelling with PGP 9.5 together with the increase in rough endoplasmic reticulum, indicates the presence of active protein synthesis which may be connected with their survival.     

蛋白基因产物9.5与宫颈癌临床病理表现的关系及其作用机制的实验研究

目的:研究蛋白基因产物9.5(PGP9.5)在宫颈癌病理标本中的表达与宫颈癌临床病理特征及预后的关系,并初步探讨利用PGP9.5-siRNA靶向治疗宫颈癌的潜在价值。方法:回顾性分析2008年1月~2015年6月在天津市第五中心医院妇科及天津市中心妇产科医院接受手术治疗并经病理确诊的180例宫颈癌患者临床资料。所有患者宫颈癌病理标本制作病理切片并进行SP法免疫组化染色。以PGP9.5染色后评分为依据将患者分为PGP9.5高表达组和PGP9.5低表达组。观察2组患者年龄、HPV感染、病理分级、肿瘤直径、淋巴结转移、浸润深度、累及脉管和临床分期等临床病理学参数的关系。采用Kaplan-Meier法和log-rank检验进行生存分析。采用PGP9.5-siRNA、NC-siRNA、PGP9.5真核表达质粒和对照空质粒按照脂质体载体试剂说明书转染Si Ha细胞,设立si-PGP9.5组、NC组、PGP9.5组和vector组。同时设立Si Ha细胞空白对照(control)组。分别采用Western blot实验、平板克隆形成实验和Transwell实验分析5组细胞PGP9.5蛋白的表达水平、克隆形成能力和侵袭能力。结果:2组患者病理分级、肿瘤直径、淋巴结转移、浸润深度、累及脉管和临床分期等临床病理参数与PGP9.5表达水平有关。PGP9.5高表达组患者3、5年总体生存率明显低于PGP9.5低表达组患者。与control组相比,si-PGP9.5组SiHa细胞中PGP9.5蛋白的表达量明显降低,克隆形成数量明显减少,过膜细胞数量明显减少;PGP9.5组Si Ha细胞中PGP9.5蛋白的表达量明显升高,克隆形成数量明显增多,过膜细胞数量明显增加。结论:PGP9.5蛋白高表达预示宫颈癌患者预后不良,可能成为预测宫颈癌患者预后的良好指标,对其表达进行抑制可能实现对宫颈癌的有效基因治疗。     

Motilin-immunoreactive cells in the duodenum, pyloric stomach and pancreas of caimans (Caiman latirostris and Caiman crocodilus, alligatorinae): a further comparison using region-specific motilin antisera

Motilin-immunoreactive cells in the duodenum, pyloric stomach and pancreas of Caiman latirostris and Caiman crocodilus were investigated using region specific antisera for porcine and canine motilin molecules. Motilin-immunoreactive cells were found in the duodenum, pyloric stomach and pancreas of both caiman species. These cells were primarily open-type endocrine ones in the epithelium of the duodenum and pyloric stomach. Motilin-immunoreactive cells were observed in both the exocrine and endocrine portions of the pancreas, and frequently exhibited one or more cytoplasmic processes of variable length. Since motilin-immunoreactive cells do not cross-react with serotonin or any of the other pancreatic and gut hormones, they are considered to be cell type independent from any of the other known pancreatic or gut endocrine cells. The molecular similarity between caiman motilin and porcine and canine motilins and the heterogeneity of the motilin molecule in the caiman digestive system is discussed.     

Confocal imaging of HuC/D RNA-binding proteins in adult rat primary sensory neurons.

HuC/D RNA-binding proteins are antigens that are specifically expressed in nerve cells. In the present study, immunocytochemistry and laser confocal microscopy were used to study the cellular distribution of HuC/D RNA-binding proteins in adult rat primary sensory neurons. Colocalization with the protein gene product 9.5 (PGP 9.5) was used to identify sensory neurons. Confocal laser imaging showed that HuC/D antigens are expressed in the cytoplasm of all rat primary sensory neurons. Most neurons also showed anti-HuC/D immuno-positivity in the nucleus. The demonstration of the ubiquitous expression of HuC/D antigens in primary sensory neurons gives us a possible explanation for the severe pathological lesions observed in dorsal root ganglia in association with the paraneoplastic Hu Syndrome.     

Arrangement and innervation of the iliocostalis and longissimus muscles of the brown caiman (Caiman crocodilus fuscus: Alligatoridae,crocodilia)

The axial musculature of the brown caiman was investigated in detail with particular attention to the nerve supply, using a binocular stereomicroscope. Due to the prominent development of the longissimus (Lo) and the iliocostalis (IC) muscles of the caiman, the pattern of distribution of the spinal nerves in the body wall was unique; there also was less differentiation of the external intercostalis. There were four primary divisions of the spinal nerves in the thoracic region of the caiman, from ventral to dorsal: the intercostal nerve, the IC nerve, the Lo nerve, and the dorsal main trunk. Thus, the classic concept of the organization of the spinal nerves may not be suitable for the caiman. These findings suggest that evolutionary changes in the dorsolateral axial musculature have brought about the rearrangement of the organization of the spinal nerves. In addition, each clearly segmented myotome of the Lo and IC was innervated by more than two segments of the spinal nerves (pluriseg-mental innervation). The manner of formation of the myotome and its innervation is discussed from the viewpoint of comparative and developmental anatomy.     

Enkephalin-like immunoreactivity within the telencephalon of the reptile Caiman crocodilus

Immunohistochemical methods were used to characterize the distribution of staining for leucine enkephalin-like and methionine enkephalin-like immunoreactivities in the telencephalon of Caiman crocodilus. Very similar distributions of both leucine enkephalin-like and methionine enkephalin-like immunoreactivity were observed. The greatest accumulations of enkephalin-like immunoreactive material were observed within the ventrolateral area of the telencephalon, a region considered comparable to the mammalian corpus striatum and avian paleostriatal complex (i.e. basal ganglia) on the basis of embryological, anatomical and histochemical criteria. Within the ventrolateral area, many small immunoreactive neuron cell bodies were observed, particularly within the rostromedial small-celled component of the ventrolateral telencephalic area. A rich plexus of fibers displaying enkephalin-like immunoreactivity invests the entire ventrolateral area including the large-celled subdivision. A system of thick, coarse, radially-directed immunoreactive fibers running between medial and dorsal portions of the ventrolateral area and more ventral portions was observed in this study. Other structures in the caiman telencephalon, containing large numbers of neural elements displaying enkephalin-like immunoreactivity, were the ventral paleostriatum (a region considered comparable to the ventral pallidum of mammals), the lateral septal nucleus and the nucleus accumbens. The corticoid areas contained far fewer elements displaying enkephalin-like immunoreactivity, although immunoreactive fibers and cell bodies were observed within the medial, dorsal and lateral corticoid areas, particularly at caudal levels. The dorsal ventricular ridge contains the lowest number of immunoreactive cells and fibers of any structure within the caiman telencephalon although occasional neurons displaying enkephalin-like immunoreactivity were encountered in the dorsal ventricular ridge. The results are compared to the distribution of enkephalin within the cerebral hemispheres of mammals, birds and other reptiles.     

Human blood group antigen H is not the specific marker for type I cells in the taste buds

We examined the localization of human blood antigen H (AbH) and its correlation with other cell type markers in the taste buds of circumvallate papillae of the adult rat. Immunoreactivity for AbH was localized in the membrane of two cell populations in the taste buds: in spindle-shaped cells extending from base to the apical portion of the taste buds as well as in round-shaped cells at the basal portion of the taste buds. Quantitative analysis revealed that approximately 47.8%, 24.4%, and 14.6% of cells within the taste buds displayed AbH-, alpha-gustducin- or protein gene product 9.5 (PGP 9.5)-immunoreactivity, respectively. Approximately 16.3% and 6.6% of AbH-immunoreactive taste bud cells displayed alpha-gustducin- or PGP 9.5-immunoreactivity, respectively. Although previous studies proposed that AbH immunoreactivity was specific for type I cells (dark cells or supporting cells), the present results indicate that AbH immunoreactivity is also present in some type II cells (alpha-gustducin immunoreactive cells) and type III cells (PGP 9.5-immunoreactive cells).     

Evaluation of PGP9.5 in the diagnosis of Hirschsprung's disease.

The ability of an acetylcholinesterase-stained frozen section to detect an increase in large cholinergic nerve fibres within the muscularis mucosae and extending into the lamina propria was a significant step forward in the diagnosis of Hirschsprung's disease (HD). However, such frozen section diagnosis is not always possible. The purpose of this study was to assess the ability of PGP9.5 to detect this pattern of mucosal nerve fibre staining immunohistochemically. Sixty-four specimens were included in the study. Twenty-six of these had been diagnosed as HD by conventional means. All cases were stained immunohistochemically with PGP9.5, S100, and anti-neurofilaments (NF). Twenty-four cases of HD were also stained with neurone-specific enolase (NSE). PGP9.5 reliably stained fibres in the mucosal and submucosal plexuses, and ganglion cells, when the latter were present. This positive staining of ganglion cells was more intense than that seen with NSE, and the positive fibre staining was more intense than that seen with NF. Increased lamina propria fibres were detected with PGP9.5 in only 37 per cent of HD cases compared with S100 positive staining in 60 per cent of cases. However, when S100 staining was assessed alone, it gave a higher false-negative rate in diagnosing HD than PGP9.5 used alone. Therefore we would recommend the use of PGP9.5 and S100 together for the immunohistochemical diagnosis of HD in formalin-fixed biopsies.     

Merkel cell tumor of the skin: an immunohistochemical study

Skin biopsy specimens from 12 elderly patients with Merkel cell tumors were investigated. Conventional light microscopy and immunohistochemical techniques were used. All of the tumors had similar morphologic features. Immunoreactivity for neuronspecific enolase, gastrin, calcitonin, and epithelial membrane-like antigen was demonstrated, and both neurofilaments and keratin filaments were observed. The immunohistochemical findings supported a Merkel cell origin for these Merkel cell tumors. The co-expression of neuroendocrine and epithelial markers in Merkel cell carcinomas is suggestive of neuroendocrine differentiation in a neoplasm of epithelial origin. Merkel cell carcinomas share many characteristics with neuroendocrine tumors of the bronchopulmonary and gastrointestinal tracts. All of these neoplasms may originate from cells of similar types that are present in several organs.     

Neuron-specific enolase in the Merkel cells of mammalian skin. The use of specific antibody as a simple and reliable histologic marker

Merkel cells are specialized skin receptor cells, characterized by their particular location in the epidermis and close association with nerve terminals. Although they can be distinguished ultrastructurally by their small, electron-dense secretory granules, there is no specific and reliable method for identifying them by light microscopy. Using antibodies to neuron-specific enolase (NSE), the authors have shown sparsely distributed groups of specifically immunostained cells and associated nerve terminals in the nose skin of cats and rats. These cells were easily distinguished from other epithelial cell types, including melanocytes and Langerhans cells and had all the morphologic features of Merkel cells and their so-called neurite complexes, including the characteristic cytoplasmic secretory granules (60 nm in diameter). NSE immunostaining is a simple and reliable method for the specific light-microscopic staining of Merkel cells and provides further evidence for NSE as a marker for the diffuse neuroendocrine system.     

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.