Morphological changes in the enteric nervous system caused by carcinoma of the human large intestine.

The innervations of the large intestine is responsible for it peristalsis and contractibility. Investigations of the enteric nervous system in many colon diseases have revealed changes in this structure. No study has been carried out on morphological changes of the enteric nervous system in the human large intestine with carcinoma. The aim of this study was to investigate potential changes in the structure of the enteric neurons in patients with sigmoid and rectal cancer. Material for the study was obtained from patients undergoing operations due to carcinoma of the sigmoid colon and rectum. Microscopic observation of the cancerous tumor of the human large intestine revealed changes in the enteric nervous system innervating this part of the gastrointestinal tract. In the region of the enteric plexuses located close to the tumour, disruption of their correct placement and structure was observed. The changes also consisted of the disappearance of neurons and nerve fibers forming these plexuses. In the solid cancerous tumour, elements of the enteric nervous system were not present. Destruction of the enteric nervous system in the course of carcinoma of the large intestine may cause disruption of proper intestinal function and may be responsible for part of symptoms which the patients suffer

Somatostatin, substance P and calcitonin gene-related peptide-positive intramural nerve structures of the human large intestine affected by carcinoma.

The aim of this study was to investigate the arrangement and chemical coding of enteric nerve structures in the human large intestine affected by cancer. Tissue samples comprising all layers of the intestinal wall were collected during surgery form both morphologically unchanged and pathologically altered segments of the intestine (n=15), and fixed by immersion in buffered paraformaldehyde solution. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP). The microscopic observations revealed distinct morphological differences in the enteric nerve system structure between the region adjacent to the cancer invaded area and the intact part of the intestine. In general, infiltration of the cancer tissue resulted in the gradual (depending on the grade of invasion) first decomposition and reduction to final partial or complete destruction and absence of the neuronal elements. A comparative analysis of immunohistochemically labeled sections (from the unchanged and pathologically altered areas) revealed a statistically significant decrease in the number of CGRP-positive neurons and nerve fibres in both submucous and myenteric plexuses in the transitional zone between morphologically unchanged and cancer-invaded areas. In this zone, a decrease was also observed in the density of SP-positive nerve fibres in all intramural plexuses. Conversely, the investigations demonstrated statistically insignificant differences in number of SP- and SOM-positive neurons and a similar density of SOM-positive nerve fibres in the plexuses of the intact and pathologically changed areas. The differentiation between the potential adaptive changes in ENS or destruction of its elements by cancer invasion should be a subject of further investigations

Changes in vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide and neuropeptide Y-ergic structures of the enteric nervous system in the carcinoma of the human large intestine.

This investigation was aimed at immunohistochemical analysis of potential changes in the enteric nervous system caused by cancer of the large intestine. In this purpose, neurons and nerve fibers of intestinal plexuses containing neuropeptides: vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY), in pathologically changed part of the large intestine were microscpically observed and compared. Samples were taken from patients operated due to cancer of the sigmoid colon and rectum. The number of neurons and density of nerve fibres containing neuropeptides found in sections with cancer tissues were compared to those observed in sections from the uninvolved intestinal wall. Changes relating to reductions in the number of NPY-ergic neurons and density of nerve fibres in submucous and myenteric plexuses in the sections with cancer tissues (pathological sections) were statistically significant. A statistically similar presence of VIP-ergic and PACAP-ergic neurons in the submucosal and myenteric plexuses was observed in both the pathological and control sections. On the other hand, in the pathological sections, VIP-ergic nerve fibres in the myenteric plexuses and PACAP-ergic nerve fibres in the submucosal and myenteric plexuses were found to be less dense. Analysis revealed changes in pathologically affected part of the large intestine may caused disruption of proper intestinal function. Observed changes in the neural elements which are responsible for relaxation of the intestine may suggest dysfunction in the innervation of this part of the colon

Ultrastructural characteristics of myenteric plexus in patients with colorectal cancer

Introduction. It have been found previously that colorectal cancer (CRC) is accompanied by atrophy of myenteric plexuses (MPs) localized close to the tumor. The aim of the study was to compare ultrastructure of MPs localized in the unchanged part of the colon wall distant to CRC tumor with the ultrastructure of MPs in the vicinity of CRC tumor. Material and methods. The present study was conducted using post-operative material derived from 11 patients with CRC. Samples of colon wall were taken from the margin of cancer invasion and from a macroscopically unchanged segment of the large intestine, immediately fixed and processed according to the standard protocol for transmission electron microscopy studies. Results. In the MPs localized in the control part of colon wall the presence of numerous unmyelinated axons and cell bodies of neurons, interstitial cells of Cajal and enteroglial cells were observed. As compared to control samples, in the MPs located close to the tumor invasion, expansion of the extracellular matrix and myelin-like structures accompanying some nerve fibers were found. The appearance of mast and plasma cells was observed within MPs in the vicinity of CRC tumor. Sporadically, apoptotic cells were present inside the MPs. Conclusions. The presence of myelin-like structures and apoptotic cells within MPs located close to tumor invasion suggests that atrophy of MPs may be caused by factors released from CRC tumor

Decreased expression of p73 in colorectal cancer

Introduction. The colorectal cancer (CRC) is one of the most frequent cancer in Poland and worldwide. This disease is characterized by distinct genetic alterations. p73 belongs to the p53 gene family; however, its role in the pathogenesis of CRC has not been completely understood. p73 gene encodes several mRNA variants and protein isoforms with its longest and fully functional p73a (mRNA) and TAp73a (protein) isoform. The aim of the study was to investigate p73 gene expression at the mRNA (p73a) and protein (TAp73a) levels in CRC. Material and methods. Small sections of the crc tumor tissue and macroscopically unchanged colon mucosa and submucosa from the dissection margin were collected from 23 patients diagnosed with CRC. p73 mRNA levels were measured by Real-time PCR (QPCR) method and the expression level of TAp73a protein was assessed by Western blotting (WB) and immunohistochemical (IHC) staining. Results. We found a 37% decrease in the level of p73a mRNA in neoplastically changed (tumor) compared with unchanged normal colon tissue from the surgical margin (p = 0.041). No correlations were found between mRNA levels in cancer tissue and clinical-pathological parameters. The semi-quantification of TAp73a protein revealed lower and higher TAp73a protein contents in 11/23 and 12/23 of tumor samples, respectively, when compared with the median value of TAp73a protein in normal colon tissue (p = 0.61). The level of TAp73a protein level was 5 times lower in poorly differentiated cancer cells (G3) in comparison to moderately differentiated ones (G2; p = 0.02). No statistically significant correlations were observed between the level of the TAp73a protein and clinical-pathological patients’ characteristics. The IHC analysis of TAp73a protein presence in CRC samples showed decreased immunoreactivity when compared with matched sections of the unchanged colon wall in 4/9 patients, similar intensity of the IHC reaction in 4/9 patients and increased immunoreactivity in 1/9 patients. The TAp73a protein was localized mainly in the cytoplasm of the cancer cells. No statistically significant correlations between IHC results and clinical-pathological features of the patients were found. Conclusions. The obtained results suggest that the p73 gene may play a role as a tumor suppressor in the CRC progression

Myenteric plexuses atrophy in the vicinity of colorectal cancer tissue is not caused by apoptosis or necrosis

Introduction. The previously performed studies showed that the presence of colorectal cancer (CRC) tumor is associated with the atrophy of myenteric plexuses in the vicinity of cancer invasion; however, the possible mechanisms of this phenomenon are not known. The aim of the present study was to determine whether the atrophic changes of the enteric nervous system (ENS) within an intestine wall of the CRC patients were caused by apoptosis or necrosis and whether they were associated with changes in the number of galanin-immunore­active (GAL-Ir) neurons. Material and methods. Samples of the large intestine wall located close to the CRC invasion and control, distally-located part of the colon, were collected from 9 CRC patients. The size of ENS plexuses and the number of neurons were compared. Triple immunofluorescent staining was used to visualize the co-expression of caspase 3 (CASP3) or caspase 8 (CASP8) with GAL and protein gene-product 9.5 (PGP 9.5, panneuronal marker) in the submucosal and myenteric ENS plexuses. The cells expressing myeloperoxidase (MPO, marker of neutrophils) and CD68 (marker of macrophages) were detected by immunohistochemistry around/in myenteric plexuses (MPs) and in the muscularis externa of the colon wall in the vicinity of tumor invasion. Results. Myenteric plexuses in the vicinity of the CRC tissue were significantly smaller and had lower number of neurons per plexus than distantly located plexuses. The number of CASP8- and CASP3-Ir neurons in the ENS plexuses was similar in the colon wall both close to and distally from tumor invasion. The number of CASP8-Ir neurons within MPs located close to CRC invasion was higher than of CASP3-Ir neurons. The percentage of neurons co-expressing CASP8 and GAL in myenteric plexuses close and distantly from tumor was three-fold lower than of those co-expressing CASP3 and GAL. The mean number of neutrophils and macrophages inside and around myenteric plexuses located close to tumor invasion was higher or similar, respectively, as compared with adjacent muscularis externa. Conclusions. The atrophy of myenteric plexuses in the vicinity of CRC invasion is not caused by apoptosis or necrosis. The differences in the proportions of neurons expressing galanin and the studied caspases suggest as yet unknown role of this neuropeptide in the mechanisms of neuron’s atrophy in MPs located close to CRC tumor

Alterations in porcine intrahepatic sympathetic nerves after bisphenol A administration

   Introduction. Bisphenol A (BPA) is used in the chemical industry for manufacturing plastics which are used as food packaging. Data indicate that BPA is released from such products and is widely present in the environment and the human body. So far, the EFSA and the US FDA have determined “safe” BPA oral exposure levels, and a large amount of data indicates that BPA is harmful even at low-doses. Our previously performed analyses concerning BPA exposure demonstrated the impact of this substance on parasympathetic and peptidergic nerve fibers present within the liver. Therefore, this study concerns BPA exposure and sympathetic intrahepatic in­nervation in reference to several neuropeptides which modulate neuronal responses: cocaine and amphetamine regulated transcript (CART), galanin (GAL), calcitonin gene-regulated peptide (CGRP) and substance P (SP). Materials and methods. Fifteen young swine at 8 weeks of age were used as experimental models of the juvenile human liver. The pigs were divided into 3 groups and received capsules orally with bisphenol at a dose of 0.05 mg/kg b.w./day; a dose of 0.5 mg/kg b.w./day and placebo capsules as a control. After 28 days of oral BPA intake, the animals were euthanized, perfused with 4% paraformaldehyde (PFA), and livers were collected and fixed in PFA. The cryostat sections were subjected to a routine double-labeling immunofluorescence technique. The primary antibodies were directed against dopamine beta hydroxylase (DbH), which is a marker for sympathetic nerves, and one of the investigated neuropeptides: CART, GAL, CGRP and SP, which co-localized the inves­tigated nerves. Immunoreactive nerves were counted in the liver and the percentage presence of each neuronal combination in particular samples of each experimental group were determined and analyzed statistically. Results. The BPA oral intake at low and ten times higher dosage caused an increase of the number of sympa­thetic nerve fibers within the porcine liver by 48.6% and 63.7%, respectively. Moreover, BPA exposure caused an increased presence of sympathetic nerve fibers in these two experimental groups, which were co-localized with CART and GAL up to 65.9%/173.2% and 147.4%/126.3%, respectively. At the lower BPA doses of 50 μg/kg b.w./day, the percentages of SP+/DbH+ and CGRP+/DbH+ nerve fibers were similar to the control. However at a ten times higher dose, BPA caused an increased number of SP+/ DbH+ and CGRP+/ DbH+ nerve fibers in the liver, up to 46.4% and 73.5% respectively. Conclusions. BPA caused an increase in the number of sympathetic nerve fibers as well as sympathetic nerve fibers which co-localized with neuropeptides in the porcine liver. The increase in CART and GAL were excep­tionally high even at low BPA doses. BPA food contamination may dysregulate liver sympathetic innervation, and thereby may change the oxygenated blood supply, alter metabolism and disrupt the activity of hepatic pa­renchymal cells

Plasticity of neuropeptidergic neoplasm cells in the primary and metastatic Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and highly aggressive cutaneous carcinoma with characteristics of neuroendocrine tumor. We performed immunohistochemical analysis to demonstrate the presence of various neuropeptides within cells of MCC resected from a 75-year old woman. The cells of primary tumor of cheek were compared with the cells of regional right submandibular metastatic tumor which was found eight months later. A double- staining IHC for the pan-neuronal marker, PGP 9.5, and selected neuropeptides in the tissue material obtained from both locations was performed. Single multipolar cells in the main mass of primary tumor stained positively for PGP 9.5 and such neuropeptides as GAL, VIP, PACAP, NPY and CGRP. Moreover, we demonstrated for the first time the presence of neuropeptides in metastatic MCC cells. In the metastatic tumor, cells showing the co-localization of PGP-9.5 and neuropeptides were more numerous, mostly of oval shape, and significantly smaller than in the primary tumor. Thus, the progression of MCC may be associated with the acquisition by its cells of new morphological and biological features

PLAGL1 protein is differentially expressed in the nephron segments and collecting ducts in human kidney

Introduction. PLAGL1 (pleiomorphic adenoma gene-like 1) is a C2H2-type zinc finger transcription factor associated with the regulation of cell growth and development. Although PLAGL1 expression in kidney was assessed by biochemical methods, the exact localization of the PLAGL1 protein in human kidney has not yet been described. Material and methods. Macroscopically unchanged specimens of kidney tissue were collected from 39 patients undergoing nephrectomy due to renal cell carcinoma. H & E staining of paraffin sections was used to assess histology of the kidney whereas immunohistochemistry was used to localize PLAGL1 protein in kidney compartments. In addition, database sequences search for putative PLAGL1 binding sites among the kidney-related genes was performed. Results. PLAGL1 staining intensity differed depending on the kidney compartment. Strong PLAGL1 immunoreactivity was found in thick ascending limbs of Henle’s loop, distal tubules and collecting ducts, whereas PLAGL1 expression in proximal tubules and renal corpuscles (including podocytes) was moderate and weak, respectively. By the in sillico screening of promoter sequences for PLAGL1 specific DNA-binding sites GGG­GCCCC we designated 43 candidate genes for PLAGL1-regulated genes. Analysis of their functional annotations identified three significantly over-represented gene sets: inositol phosphate metabolic processes (GO), endocrine and other factor-regulated calcium reabsorption (KEGG) and calcium signaling pathways (KEGG). Conclusion. Differences in the renal expression of PLAGL1 suggest that this protein may be involved in the regulation of several cellular pathways both as transcriptional factor and coactivator/corepressor of other tran­scription factors reflecting its role in the cell type-specific control of gene expression.