Human gallbladder carcinoma: Role of neurotrophins, MIB-1, CD34 and CA15-3.

Gallbladder carcinoma is the most common biliary tract tumor and the fifth most common gastrointestinal tract cancer .The prognosis of gallbladder carcinoma is poor and less than 5% of the patients are still alive five years postoperatively. Gallbladder specimens were obtained during surgical operations performed in eleven patients for resection of a gallbladder carcinoma, and during five autopsies (control cases selected among patients who died from for other causes, excluding those suffering from biliary or hepatic diseases). Immunohistochemical characterization and distribution of neurotrophins, with their respective receptors, were analyzed. The actual role played by these neurotrophic factors in the general regulation, vascular permeability, algic responsiveness, release of locally active substances and potential tumorigenesis in the gallbladder and biliary ducts compartment remains controversial. Our study revealed an increased immunohistochemical expression of NGF and TrKA in the epithelium and in the epithelial glands of the gallbladder carcinoma together with an evident immunoreactivity for BDNF in the same neoplastic areas. An evident immunoreactivity for NGF, TrKA and BDNF was observed in control specimens of gallbladder obtained during autopsies, whereas a weak or quite absent immunoreactivity was observed in the same specimens for NT4, TrKC and p75. On the contrary an appreciable immunoreactivity for p75 was observed in the specimens harvested from patients with gallbladder carcinoma. We also investigated the expression of some known tumor markers such as MIB-1 (anti Ki-67), CD34 and CA15-3, to identify a possible correlation between the expression of these molecular factors and the prognosis of gallbladder carcinoma. They resulted highly expressed in the stroma (CD34 and CA 15-3) and in the epithelium/epithelial glands (MIB-1) of the neoplastic areas and appeared to be almost absent in the control cases, suggesting that these markers, taken together, could be adopted as a panel of prognostic factors in the evaluation of the gallbladder carcinoma.

Dopamine nerve fibres and related receptors in bronchus-associated lymphoid tissue (BALT).

Age-related changes of the dopamine nerve fibres of bronchus associated lymphoid tissue (BALT) were investigated in male Wistar rats of 3 months (young), and 24 months (old/aged). Dopamine histofluorescence techniques have been used, associated with image analysis for the detection of dopamine nerve fibres. In young rats, white, fluorescent nerve fibres supply BALT. This tissue is innervated by a delicate network of nerve fibres rich in varicosities. In old rats these fluorescent nerve fibres are strongly reduced. Moreover, dopamine D1a and D1b receptors were stained using fluorescent monoclonal antibodies. The BALT of young rats possesses a higher number of D1a and D1b receptors, while, in the old rats, these receptors are strongly reduced. The possible significance of reduced dopamine neurotransmission in BALT of aged rats is discussed.

Diabetic rats treated by low molecular weight heparin OP 2123/parnaparin: morphological changes in the kidney and heart.

AIM: Early morphological alterations in the rat kidney and heart due to experimentally induced diabetes are described in order to evaluate the possible therapeutic role of low molecular weight heparin (LMWH; OP 2123/parnaparin). METHODS: Our findings concern the alterations observed in the rat kidney and heart because these are the organs (together with the retina) mainly involved in the early morphological angiopathic modifications associated with diabetic damage of organs and tissues. In diabetic animals treated with LMWH, the Periodic Acid-Schiff (PAS) reaction showed a slight decrease when compared with the diabetic control group. Photographs were submitted to the quantitative analysis of images using a Quantimet 500 Image Analyzer (Leica) equipped with specific software. The following parameters were measured: (1) total area occupied by alkaline phosphatase (AP)-positive capillaries; (2) number and diameter of AP-positive capillaries; (3) distribution and total area occupied by PAS-positive structures (related to the intensity of the reaction resulting from the different amount of mucopolysaccharides). RESULTS: LMWH treatment is efficient in preventing these modifications, above all in the kidney. The histological study of the heart and kidney shows no significant, relevant alterations. However, the histological study of the mucopolysaccharides in diabetic animals highlighted a tendency for the heart to accumulate these substances. LMWH treatment only modestly reduced this accumulation. CONCLUSIONS: Previous evidence demonstrating a beneficial effect of therapy based on heparan sulphate proteoglycans and/or other heparin-like substances in insulin-dependent diabetes mellitus seems to be confirmed by our experimental results in different organs of adult rats. In fact, parnaparin treatment is effective (in our experience) for ameliorating the morphological pattern observed early in some diabetic tissues of rats and, above all, in the kidney.

Catecholaminergic nerve fibers in bronchus-associated lymphoid tissue: age-related changes.

Age-related changes of the catecholaminergic nerve fibers of the trachea, bronchial smooth muscle, lung capillaries and bronchus-associated lymphoid tissue (BALT) were studied in male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old/aged). Catecholamine histo- and immuno-fluorescence techniques were used, associated with image analysis and high pressure liquid chromatography with electrochemical detection of nor-epinephrine (nor-adrenaline). In young rats, blue-green fluorescent nerve fibers supply the trachea-bronchial smooth muscle and tracheal and bronchial glands. These structures are innervated by a delicate network of nerve fibers, being rich in varicosities. Pulmonary capillaries are sparsely innervated. The highest nor-epinephrine concentration was found in the trachea and bronchi, followed by BALT. The density and the pattern of noradrenergic nerve fibers of the trachea-bronchial tree or of the pulmonary vessels were similar in young and adult rats. In aged rats, a loss of noradrenergic nerve fibers, involving primarily the supply to the smooth muscle of the trachea-bronchial tree, was observed. Fluorescence microscopic techniques demonstrated a higher sensitivity than nor-epinephrine assay in detecting changes of the sympathetic nerve supply of the trachea-bronchial tree, pulmonary vessels and BALT. The possible significance of reduced noradrenergic nerve supply of the trachea-bronchial-pulmonary tree in aged rats is discussed.

Dopaminergic receptors in rat dura mater: pharmacological characteristics.

1. The location and distribution of dopaminergic receptors in rat dura mater was studied by examining several dural zones (vascular, perivascular, intervascular) in different cranial and spinal regions. 2. The pharmacological characteristics and anatomical distribution of dopamine D1- and D2-like receptors sites were investigated using combined pharmacological techniques and immunofluorescent microscopy. 3. Samples of rat dura mater were obtained from 10 adult Wistar rats. On frozen slices, dopaminergic D1 and D2 receptors were stained immunohistochemically using monoclonal antibodies. 4. Inhibition studies were performed using fluorescent and non-fluorescent agonists or antagonists to define the pharmacological specificity of the immunostaining. 5. The greater sensitivity to displacement by amisulpride, bromocryptine, domperidone, haloperidol, raclopride and l-sulpiride than to displacement by N-propyl-nor-apomorphine, quinpirole and clozapine suggests that the immunofluorescent sites observed in these experiments are likely to belong to the dopamine D2 receptor subtype. 6. Our observations provide evidence of the presence of D1 and D2 receptors in the wall of meningeal vessels. The dopaminergic receptors are located in the adventitia, media and intima of dural arteries. Furthermore, the density of receptors is higher in close proximity to arteries and decreases passing from the vascular to the perivascular and intervascular zones. 7. In the rat dura mater, dopamine regulates the meningeal blood vessels and, through this action, dopamine and its receptors can play an important role in the pathogenesis of cephalalgia.

Cholinergic innervation of BALT (bronchus associated lymphoid tissue) in rat.

The presence and distribution of acetylcholinesterase (AChE) and cholineacetyl transferase activities (Chat) were examined in the bronchus-associated lymphoid tissue (BALT) of juvenile, adult and old rats. Histoenzymatic and immunochemical methods were used in association with quantitative analysis of images and statistical analysis of the data. Our results showed that both AChE and Chat activities were primarily confined to the BALT lymphoid cells. Only a low level of activity was observed in the sub-pleural parenchyma of the lung and in the wall of the bronchus. Moreover, both AChE and Chat activities in the BALT are specifically located in the lymphoid cells. Histoenzymatic staining and corresponding values of quantitative analysis of images confirmed morphological and immunochemical results. Finally, the intensity of histoenzymatic staining for AChE and of immunochemical staining for Chat in BALT of rats strongly decreases with age. On the basis of our results we hypothesize that both AChE and Chat activities may play an important role in BALT and both these enzymes undergo specific age-related changes.

Age-related changes of monoaminooxidases in rat cerebellar cortex.

Age-related changes of the monoaminoxidases, evaluated by enzymatic staining, quantitative analysis of images, biochemical assay and statistical analysis of data were studied in cerebellar cortex of young (3-month-old) and aged (26-month-old) male Sprague-Dawley rats. The enzymatic staining shows the presence of monoamino-oxidases within the molecular and granular layers as well as within the Purkinje neurons of the cerebellum of young and aged animals. In molecular layer, and in Purkinje neurons the levels of monoaminooxidases were strongly increased in old rats. The granular layer showed, on the contrary, an age-dependent loss of enzymatic staining. These morphological findings were confirmed by biochemical results. The possibility that age-related changes in monoaminooxidase levels may be due to impaired energy production mechanisms and/or represent the consequence of reduced energetic needs is discussed.

Effect of interferon on human thymus microenvironment.

Interferon, a thymic immunostimulator, was used with the aim of assessing the importance of adrenergic nerve fibers (ANF) and/or AChE-positive nerve fibers (AChENF) in the regulation of some immunological functions in humans. Thymic normal control fragments and/or thymic fragments of immunostimulated patients were removed during surgical biopsies. Thymic slices were stained with eosin-orange (for the recognition of microanatomical details of the microenvironment) and with Bodian's method for staining of nerve fibers. Histofluorescence microscopy was employed for staining ANF. AChENFs were detected by means of the direct-coloring thiocholine method. All images were submitted to quantitative morphometrical analysis and statistical comparisons of data. Moreover, the amount of proteins and noradrenaline was measured on thymic homogenates of the same patients. Treatment with interferon induces substantial changes in the thymic microenvironment, on ANF, on AChENFs and on the total amount of proteins and noradrenaline in thymic tissue homogenates. In conclusion immunostimulation with interferon induces substantial changes in the whole thymus and in its microenvironment, involving both sympathetic and parasympathetic nerve fibers.

Distribution of peptidergic nerve fibres in the guinea pig trabecular meshwork.

A quantitative analysis of peptidergic nerve fibres located in the trabecular meshwork of the guinea pig has been performed. Our results confirm that this structure contains VIP-, NPY- and substance P-like immunoreactivity as major neurotransmitters. These findings were obtained using immunohistochemical techniques. For this purpose serial sections of the eye were stained by immunohistochemistry for each of three neurotransmitters and stained sections were analysed by quantitative image analysis. Our findings demonstrate that SP-positive, NPY-positive and VIP-positive nerve fibres occupy 11.2, 4.9 and 2.4%, respectively, of the observed area (expressed as conventional units, C.U.) in the trabecular meshwork of the guinea pig eye. It is relevant to emphasize that the area containing these three types of peptidergic nerve fibres appears to be large (18.5 +/- 6.6 C.U.) in proportion to the total observed area. The innervation of the drainage angle of the guinea pig eye has been well described by many authors. This is the first study to report quantitative measurements of three types of peptidergic nerve fibres identified and measured in this area. The presence of these three neurotransmitters in the trabecular meshwork of guinea pig eye suggests their possible participation in aqueous humor regulation.

Nerve fibers--mast cells correlation in the rat parietal pleura.

Our results show that a morphological correlation exists between catecholaminergic nerve fibers and mast cells in the rat parietal pleura. Mast cells are found in proximity to catecholaminergic nerve fibers. Moreover we have demonstrated that mast cells and nerve fibers in the perivascular areas of rat parietal pleura show the same formaldehyde fluorescence. In rats previously sympathectomized with neurotoxin 6-OHDA the nerve fibers appear disarranged and poorly fluorescent and related mast cells show a low fluorescence. The importance of this correlation and the possible role of mast cells are discussed.