Ghrelin and obestatin in thyroid gland - immunohistochemical expression in nodular goiter, papillary and medullary cancer.

INTRODUCTION: Previous studies analyzing ghrelin and obestatin expression in thyroid gland tissue are not unanimous and are mostly related to ghrelin. The role of ghrelin and obestatin in the thyroid gland appears very interesting due to their probable involvement in cell proliferation. Furthermore, since the thyroid gland is associated with the maintenance of energy balance, the relationship between ghrelin, obestatin and thyroid function is worthy of consideration. The aim of the study was to assess ghrelin and obestatin immunocytochemical expression in nodular goiter (NG), papillary cancer (PTC) and medullary cancer (MTC). MATERIAL AND METHODS: Analyzed samples included 9 cases of NG, 8 cases of PTC and 11 cases of MTC. The analysis of ghrelin and obestatin expression was performed by use of the immunohistochemical (IHC) EnVision system and evaluated with filter HSV software (quantitative morphometric analysis). RESULTS: Quantitative ghrelin expression in MTC cells was higher than in NG (p = 0.013) and correlated negatively with the size of the tumor (r= -0.829, p < 0.05). We did not observe any differences in ghrelin expression neither between MTC and PTC nor between NG and PTC. Obestatin immunoexpression pattern in all analyzed specimens was irregular and poorly accented. The strongest immunoreactivity for obestatin was demonstrated in NG. In MTC obestatin expression was significantly weaker than in NG and PTC (p < 0.05 in both cases). In NG the intensity of obestatin immunostaining was significantly higher than that of ghrelin (p = 0.03). Conversely, ghrelin expression in MTC was definitely more evident than obestatin immunoreactivity (p < 0.01). There was no statistically significant difference between ghrelin and obestatin expression in PTC. No correlations were detected between reciprocal tissue expressions of ghrelin and obestatin in the analyzed specimens of NG, PTC or MTC. CONCLUSIONS: The differences between ghrelin expression in NG and MTC suggest that ghrelin may be involved in thyroid cell proliferation. The differences between ghrelin and obestatin immunoreactivity in benign and malignant thyroid tumors could support the theory of alternative transcription of the preproghrelin gene and independent production of ghrelin and obestatin.

Tissue expression of S100 proteins in gallbladder mucosa of the patients with calculous cholecystitis.

Proteins of S100 group, produced by phagocytes represent endogenous activators of innate immune responses. Role of these proteins in the etiopathogenesis of cholelithiasis remains unknown. The studies aimed at the morphometric evaluation of S100A8 and S100A9 protein expression in gallbladder mucosa in patients with acute and chronic calculous cholecystitis (n = 71). The presence of proteins was detected by immunohistochemistry while quantitative analysis employed the spatial visualization technique. We found the immunopositive expression of the two studied S100 proteins in neutrophils and monocytes/macrophages of the gallbladder's wall and a higher expression in acute cholecystitis. Quantitative study revealed higher immunoexpression of S100A9 over S100A8 in both studied groups of patients. Moreover, a reciprocal linear relationship between the expression of the studied proteins and a positive correlation between expression of either S100A8 or S100A9 and inflammatory activity (grading) in the gallbladder wall were found. The expression of S100A8 protein in the chronic cholecystitis group and in older patients correlated with leukocytosis, which suggests the role of S100A8 particularly at the chronic stage of cholecystitis. The obtained results indicated close relationship between S100A8 and S100A9 proteins in their proinflammatory functions. The increased expression of only one of them can be recognized as a useful index of local inflammatory activity in calculous cholecystitis.

Cytokeratin 8 and 18 tissue expression in gallbladder mucosa of patients with cholelithiasis.

Cytokeratins (CKs) 8 and 18 are normally expressed in simple epithelia. This unique pair of CKs is believed to be involved in hepatic diseases and many human cancers. Little is known about the role of tissue expression of both CKs in patients with cholelithiasis (CH). The aim of the study was to analyse tissue expression of CK8 and 18 in the specimens of gallbladder mucosa in 35 young (up to 25 years of age) and 20 older patients (approximately 50 years of age) with CH. An immunocytochemical ABC method and the spatial visualization technique were conducted. Our study demonstrated significantly lower amounts of both CKs in young patients, as compared to older patients. The higher cellular expression of CK8 in older patients was linked to acute clinical course vs. chronic ones. Tissue expression of neither CK correlated with inflammatory activity (grading) of the gallbladder mucosa. A positive correlation between reciprocal expressions of the two CKs may confirm a cytoprotective role of the two proteins in both groups of patients with symptomatic CH. Significantly higher expression of CK18 than that of CK8 in younger patients suggests a different role of CK8 and 18 in lithogenesis in this group.

Assessment of S100 protein expression in the epididymis of juvenile and adult European bison.

In our study, we decided to compare S100 protein expression in the material obtained from the epididymes of 5- and 12-month-old calves, and adult European bison, and to detect any differences in S100 expression according to the animal age and size of the organ examined. We used the epididymes obtained from 6 adult European bison aged 6-12 years, from 6 at the age of 12 months and 6 calves aged 5 months. Immunocytochemical reactions were performed using the avidin-biotinylated-peroxidase (ABC) technique according to HSU. Specific polyclonal rabbit antiserum against bovine S100 protein (Bio Genex Laboratories) at a dilution at 1:400 was applied. We found the expression of S100 protein in endothelial cells of arteries, veins and lymphatic vessels in all the study animals. At the same time, we found no differences in the expression of S100 protein in vascular endothelial cells. Our observations seem to indicate that S100 expression in endothelial cells of European bison epididymis is not correlated with age or maturity of the organ tested. We found S100 protein in smooth muscle cells of arteries and veins in all European bison specimens examined. Interestingly in the current study, in young 5-month-old sexually immature European bison specimens we observed weaker expression of S100 protein in smooth muscle cells of small vessels as compared to the same cell type both in large vessels in these animals and in small vessels in adult specimens.

The effect of extracellular substance components on the proliferation and expression of hormones in cultured cells of medullary thyroid carcinomas.

The effects were examined of selected extracellular medium (ECM) components on the proliferation of medullary thyroid carcinoma cells and on the production of calcitonin and CGRP. Human TT cells and rat rMTC cells were cultured for 24, 48 and 72 hours on glass coated with type I collagen, fibronectin or poly-D-lysine. More pronounced proliferation was demonstrated by TT cells grown on poly-Dlysine or collagen in comparison with the control and less pronounced proliferation was typical of cells grown on fibronectin. On the other hand, rMTC cells were more markedly manifest on any ECM substrates than that on glass. Alterations in the proliferation were paralleled by changes in the expression of CT and CGRP.

Expression of cytokines (TNF-alpha, IL-1alpha, and IL-2) in chronic hepatitis C: comparative hybridocytochemical and immunocytochemical study in children and adult patients.

Hepatitis C virus (HCV) is one of the principal causes of hepatitis, which in more than 80% of cases leads to chronic lesions in the liver and involvement of extrahepatic organs. It remains unknown why the infection so frequently turns chronic, independently of patient age. Using immunocytochemistry (IHC) and in situ hybridization (ISH) (both linked to the ImmunoMax technique) we examined cell sources of TNF-alpha, IL-1alpha, and IL-2 in control and HCV-infected children and adults. We demonstrated augmented expression of all the cytokines in HCV-infected patients compared to controls. No differences were detected in amounts of studied transcripts or cytokine proteins between biopsies taken from HCV-infected children and adults. Expression of TNF-alpha was localized mainly in liver sinusoidal cells (macrophages, endothelial cells). A high proportion of hepatocytes demonstrated expression of TNF-alpha, IL-1alpha, and IL-2. In both groups of patients, higher amounts of cytokine proteins than studied transcripts were demonstrated. The augmented expression of TNF-alpha, IL-1alpha, and IL-2 in liver with a similar proportion of involved cells (mainly hepatocytes) in children and in adults points to participation of the cytokines in the pathogenesis of chronic hepatitis C. The expression is insufficient to terminate the infection and may be linked with the comparably frequent chronic transformation of HCV infection noted in children and adults.

The effect of calcitriol and its analogues on proliferation and hormone expression in cultured cells of thyroid medullary carcinomas.

The study aimed at evaluating the effects of calcitriol and of its analogues on the proliferation of TT and rMTC cells (human and rat line tumour cells originating from thyroid medullary carcinoma) and at examining the effects of the substances on the secretion of the principal hormones of the cells, calcitonin (CT) and calcitonin gene-related peptide (CGRP). Cells of thyroid medullary carcinoma (human TT cells and rat rMTC cells) were cultured for 5 days in the absence or in the presence of calcitriol and of its two analogues (PRI-1906 and PRI-2191) in concentrations of 10(-9) to 10(-6) M. Calcitriol and the applied analogues weakly inhibited proliferation of thyroid medullary carcinoma in in vitro conditions. The evident effect of analogues on hormone secretion points to their effect on the process of CT gene expression.

The expression of selected neuroendocrine markers and of anti-neoplastic cytokines (IL-2 and IL-12) in lung cancers.

We have continued our studies by detecting three markers of neuroendocrine tumours of the lungs, including chromogranin A, NSE and synaptophysin, to confirm the neuroendocrine origin of lung tumours and by examining the content of two anti-neoplastic cytokines, IL-2 and IL-12 in the tumours. The studies were performed on paraffin sections of lung carcinoids (n = 13) and small cell lung carcinomas (SCLC) (n = 15). Pronounced expression of all 3 markers of neuroendocrine tumours was detected in most of the pulmonary carcinoids and in 5/15 of SCLC. Co-expression of the two cytokines (IL-2 and IL-12) in tumour cells was detected in 12/13 patients with lung carcinoid and expression of at least one cytokine in 12/15 patients with SCLC. Significantly lower numbers of cells immunoreactive to both cytokines were detected in SCLC as compared to lung carcinoids. The studies have confirmed the literature data on the lowered secretion of IL-2 in SCLC and extend the data by supplying information on the expression of IL-12. The lowered expression of the two cytokines at the time of diagnosis may represent a prognostic factor for survival in SCLC.

Development of innervation in primary incisors in the foetal period.

Sections from the frontal part of the mandible of 43 human foetuses from 9 to 39 weeks of prenatal age, which contained two, three and sometimes four lower incisors were immunohistochemically examined using protein gene product and neuron specific enolase (NSE) antibodies in order to establish the time of appearance of nerve fibres in the developing tooth germ and to define their topography. Nerve fibres were first detected in the dental follicle in the 11th week of intrauterine life. Their presence in the dental papilla was confirmed in the 18th week when the first layers of dentine and enamel were deposited. In the 24th week of intrauterine life, the nerve fibres first reached the subodontoblastic region. In the subsequent weeks, an increase in the number of nerve fibres accompanying blood vessels in the central portion of the dental papilla resulted in the formation of neuro-vascular bundles. Moreover, the progressive deposition of enamel and dentine was accompanied by branching of papillary nerves, which thereby formed a fan-pattern. In the foetal period, no evidence was found for the formation of a subodontoblastic plexus. However, we did observe single nerve fibres in close proximity to the odontoblast layer at the end of intrauterine life. Nerve fibres were not detected in either predentine or dentine throughout foetal life.

Immunocytochemical analysis of the tissue location of cytokines (IL-2 and IL-12) in neuroendocrine lung cancer.

The study aimed at immunocytochemical evaluation of the cellular expression of two cytokines, IL-2 and IL-12 in lung carcinoids (n = 10), following the earlier demonstration of two markers of endocrine tumours (chromogranin A and NSE--neuron-specific enolase). In the immunocytochemical studies the classical avidin-biotinylated peroxidase (ABC) technique was used. Results of the tests were semiquantitatively appraised employing the IRS scale. In 9/10 cases intense reaction (score: 6-12 points) was noted for both lung neuroendocrine markers. In all cases of carcinoma co-expression of IL-2 and IL-12 was demonstrated in cells of the tumours. The cytokines showed a cytoplasmic localisation of mean (score: 3-4 points) or high (score: 6-12 points) intensity of reaction. Our studies point to a possible role of the two cytokines in the proliferation of lung neuroendocrine carcinomas but more detailed analysis is required on a broader material.

Comparison of PTHrP expression in the epididymis of juvenile and adult European bisons.

The aim of the present study was to compare the expression of PTHrP in the epididymes of adult European bisons, and 12- and 5-month-old calves. The highest PTHrP expression was observed in adult animals in muscle cells and endothelium of large vessels, and in muscle cells of the epididymal duct. In one-year-old calves, the reaction was weaker than in adult bulls, being the weakest in 5-month-old calves. However, in small vessels of adult animals, in vascular cells and smooth muscle cells the reaction for PTHrP was considerably weak, being weaker in one-year-old calves, and negative in 5-month-old calves. A similar trace reaction was observed in muscle cells of the epididymal duct in 5- and 12-month-old calves. The present study has revealed that PTHrP expression in vascular and extravascular smooth muscle cells and endothelial cells in European bison is correlated with the animal age and size of the organ.

Immunolocalization of PTHrP in prepubertal and pubertal testis of European bison.

The present study deals with immunohistochemical localization of PTHrP in prepubertal and pubertal testis of European bison. PTHrP immunoreactivity was observed in germinal cells in the testis of both prepubertal and pubertal animals. In calves, PTHrP was found in germinal cells, in seminiferous tubules lacking the lumen. The reaction was strong and regularly distributed within the cytoplasm. In adult animals, the reaction showed differentiation in spermatogenic cells. Some cells were strongly and diffusely stained, others exhibited weaker reaction of granular pattern. Sertoli cells and Leydig cells were PTHrP-negative in calves and adult animals.

Detecting the replication of the hepatitis B virus using the ImmunoMax technique following treatment with interferon-alpha in children with chronic hepatitis.

BACKGROUND: Children with HBV in Poland are treated with preparations of interferon-alpha (IFN-alpha). The continuing lack of complete response to this type of anti-viral therapy remains to be explained. The application of cell biology techniques to identify the viral components in situ makes it possible to clarify the association between the distribution of the virus and morphological injury to the liver, the immune response of the host, and clinical symptoms in the natural course of infection. Our study was intended to evaluate HBV expression in liver biopsies taken an average of two years after completion of IFN-a therapy in 10 children with serological markers of persistent HBV infection. MATERIAL/METHODS: For the immunocytochemical detection of HBcAg and for the hybridocytochemical detection of HBV-DNA, the avidin-biotin-peroxidase (ABC) technique was employed, as well as classical in situ hybridization, both additionally amplified using the ImmunoMax technique. HBcAg and HBV-DNA levels were estimated using a semiquantitative technique. RESULTS: Our study demonstrated persistent active replication of HBV in the liver in all examined children. A mixed pattern of HBcAg localization prevailed (noted in cell nuclei, cytoplasm and cell membranes) with a somewhat lower proportion of involved cells and a more evident membrane localization of HBcAg, as compared to results obtained before treatment. HBV-DNA was observed in the cytoplasm of a fraction of hepatocytes similar to that noted before therapy. CONCLUSIONS: The ImmunoMax technique was found to be highly suitable for in situ monitoring of HBV replication after termination of IFN-a treatment. Children with focal distribution of HBcAg and HBV-DNA have the potential for earlier eradication of the virus from their livers.