Interlobular Connective Tissue Research Articles

The value of optical coherence tomography and morphometry in evaluating the peripancreatic adipose tissue in infected pancreatic necrosis

To investigate the morphometric and optical coherence tomography (OCT) characteristics of the microstructure of retroperitoneal peripancreatic adipose tissue during passive drainage and active perfusion-aspiration drainage for infected pancreatic necrosis. The authors analyzed 74 samples of peripancreatic adipose tissue obtained from 37 patients with passive (Group 1) and active perfusion-aspiration drainage (Group 2) of a focus of peripancreatitis. The tissues were examined with OCT, then fixed in a formalin solution and underwent histological examination, including morphometric one. The examined groups showed qualitative and quantitative differences in the tissue microstructure. In Group 1 samples, the proportion (median (upper quartile; lower quartile)) of interlobular connective tissue was 5.6 (3.9; 6.1)%; and that of adipocytes was 44.6 (41.2; 51.6)%. Necrotic tissue occupied 46.2 (35.6; 56.1)% of the area of specimens. The OCT images of necrotic tissue displayed a preponderance of unstructured regions with a high signal level while the parenchymal areas showed a cellular structured pattern. In Group 2 samples, the proportion of interlobular connective tissue was 16.2 (11.4; 19.7)%, and that of adipocytes was 68.5 (59.7; 71.2)%. At the same time, Group 2 exhibited necrosis - 14.4 (11.5; 19.2%) that was 3.2 times less than in Group 1. The OCT images of Group 2 samples showed a preponderance of cellular structured regions characteristic of the parenchyma, with pronounced stromal elements, which corresponded to the histological pattern. Active perfusion-aspiration drainage of peripancreatic adipose tissue versus passive drainage is associated with a smaller proportion of necrotic tissue (14.4% versus 46.2%; p≤0.05) and a larger proportion of a stromal vascular component (16.2% vs. 5.6%; p≤0.05). OCT could qualitatively distinguish viable fat tissue parenchyma from necrotic areas, without specially preparing the samples, which was confirmed by histomorphometric results.

MORPHOLOGICAL CHANGES PANCREAS AFTER OF MELATONIN'S EFFECT IN DIFFERENT SEASONS.

Morphological changes of the pancreas of young rats after exposure of exogenous melatonin in the spring and autumn periods was investigated. Exogenous melatonin (Unipharm Inc., USA) was administered to experimental group of animals daily'at a dose 5 mg/kg. The duration of the experiment was 28 days. It was revealed that the exocrine part of the.pancreas responds differently to the effects of melatonin at different times of the year. Thus, after administration of melatonin in the spring increase of the size of acinus, the height of the epithelium (by 7 %), area exocrinocytes (by 58 %), of their nucleus (by 20 %) and cytoplasm (69 %), the amount of nucleoli in cells (18 %), reduction the amount of connective tissue elements. Melatonin introduction in the autumn decrease in the size of acinus, height and area of exocrinocytes, growth the number of exocrinocytes in the acinus, nucleoli and width layers interlobular connective tissue in the gland. This may indicate that melatonin increases in the spring of the synthetic activity of the exocrine pancreas, whereas in the autumn (for the majority of the morphometric parameters) - somewhat reduces its functional state. The administration of melatonin in the spring (mostly) and in the autumn periods increased the functional activity of the endocrine pancreas. This is indicated by growth in the size of Langerhans islets, increasing the number and density of the (autumn) endocrinocytes.

Pancreatic response of spontaneously hypertensive rats to the exogenous administration of melatonin in the spring and autumn

The aim of the study was to investigate and compare the effect of exogenous melatonin administration on the morphofunctional state of the exocrine and endocrine parts of the pancreases of spontaneously-hypertensive rats (SHR line) in spring and autumn. The research was conducted on 48 male SHR line rats, age 4 months. The rats of the experimental group received melatonin (Unipharm Inc.,USA) every day (at 10 am) orally at a dose of 5 mg/kg of body weight. The duration of the experiment was 28 days. Morphofunctional activity of the exocrine and endocrine parts of the pancreas was assessed using morphological and morphometric methods. The histological preparations were made by the standard methods. The slides were photographed using a digital camera on a Nikon microscope. The morphometric analysis was performed on digital images using the computer program Image J. The morphological signs of the exocrine pancreas activity were greater in the control rats in spring compared to autumn. Conversely, the activity of the endocrine part of the pancreas in control animals was higher in autumn, than in spring. Exocrine pancreas activity increases after administration of melatonin regardless of time of the year. This was evidenced by the increase in size and height of the epithelial acini, and the number of exocrinocytes placed in them. The width of the interlobular and interacinus connective tissue layers in the pancreas decreased in spring (mostly) and in autumn. The activity of the endocrine part of the pancreas increased after administration of melatonin in spring. Proof of this was an increase in the number and size of islets of Langerhans and the number of endocrinocytes they contained, compared with control values. The number and size of islets of Langerhans, the number and density of the endocrinocytes they contained decreased in the rats which received melatonin in autumn. This indicates a reduction of the endocrine pancreas activity. These results may have implications for therapeutic dosing of melatonin in patients with hypertension in different seasons of the year.

Prenatal and neonatal development of mandibular salivary gland of Indian buffalo

ABSTRACTThe primordial anlage of mandibular salivary gland was seen at 2 cm CVRL (37th day) whereas primary ducts were first observed at 11.5 cm CVRL (80th day), in the form of cords. However, the capsule formation around the gland was initiated at 21.1 cm CVRL (121st day). The lobulation was appeared at 15.3 cm CVRL (97th day). The duct system was completed at 21.1 cm CVRL (121st day). At 18.6 cm CVRL (112nd day), the terminal tubules (TT) attained the structure of the acini. The myoepithelial cells were first appeared as flattened basal cells initially around the developing acinar cells at 28.5 cm CVRL (138th day). The typical compound tubulo-acinar nature of the gland was first observed at 21.2 cm CVRL (122nd day). The parenchyma showed predominantly mucous type of cells from 28.3 cm CVRL (137th day) onwards, while initial appearance of serous demilunes was also observed at this stage. In neonatal age groups, the mandibular gland was of compound tubulo-acinar nature with a well-defined capsule. Localization of acidic as well as neutral mucopolysaccharides was observed in mucous cells and goblet cells in prenatal and neonatal age groups. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue, however, phospholipids were observed in the cell membrane of secretory cells and ducts.

Macroscopic Anatomy of the Saimaa Ringed Seal (Phoca hispida saimensis) Lower Respiratory Tract.

We studied the macroscopic anatomy of the lower respiratory tract of the endangered Saimaa ringed seal (Phoca hispida saimensis). Examination of one adult and one young individual found dead showed that trachea had 85 and 86 complete cartilage rings. The adjacent cartilages exhibited very few random anastomoses. There was variation in the confirmation of the trachea between the cranial and caudal part of the trachea. The right lung was divided by partly incomplete inter-lobar fissures into cranial, middle, caudal, and accessory lobes. The left lung consisted of cranial, middle, and caudal lobes. The lungs were characterized by a high amount of interlobular connective tissue. Silicone casts were prepared of the two specimens to visualize the tracheobronchial branching which was similar to that of marine ringed seals but in the Saimaa ringed seal the right middle lobar bronchus originated at the same level as the accessory lobar bronchus.

Development of the Liver in Alpaca (Vicugna pacos): A Microscopic and Macroscopic Description.

South American camelids have several biological, morphological and behavioural adaptations that allow them to live in geographical areas dominated by high altitudes. The liver has hematopoietic functions during the prenatal life, which could be modified in response to the unfavorable habitat. However, there are no previous data on the prenatal development of the liver in these species. In the present work, a study on the macroscopic and microscopic morphology of the liver of the alpaca during ontogeny was performed. Forty-one animals ranging in age from 20 days of embryonic development to adults were studied. Macroscopic and microscopic observations were performed on samples subjected to different techniques. Less than 7-g specimens were studied with stereoscopic magnifying glass. The general characteristics of the prenatal liver are similar to those of other mammals, and the structures related to hematopoietic function follow an ontogenic pattern similar to that of previously studied precocial species. However, there are differences in morphology when compared to descriptions for the Old World camelids, including the absence of relation between the caudate lobe and the right kidney and the lack of interlobular connective tissue.

English

The histology and ultrastructure of thyroid gland of White Fulani (Zebu) cattle were examined during the postnatal developmental period to show if variations in morphology exist with age and in relation to tropical climate. Eight prepubertal and ten pubertal cattle of abattoir origin were used to carry out this study. Histologically, the thyroid consisted of well-developed capsule, interlobular connective tissue, follicular and parafollicular cells from prepubertal to pubertal age of 5 to 7 years. The follicular cells were cuboidal in prepubertal period with some variations, became predominantly columnar in pubertal age of 2 to 4 years and highly flattened cells were common in the older pubertal animals of  5 to 7 years. The colloid was observed in the lumen at all age and was PAS-positive with abundant peripheral vacuoles in the pubertal age of 2 to 4 years. Significant variations (pË‚0.05) in structure of the thyroids were observed at all age and the follicular size and number of follicles increased as the cattle matured. Follicular height was reduced significantly in the older pubertal cattle of 5 to 7 years. Ultrastructurally, the follicular cells showed highly dilated cisternae of rough endoplasmic reticulum which decreased with age. Flattened follicular cells seen in the older pubertal cattle contained few cytoplasmic organelles and microvilli. Apically placed pseudopods and blebs were commonly observed in the pubertal age of 2 to 4 years. Lysosomal bodies increased with age of development. The parafollicular cells were encountered frequently in the prepubertal age than in the older cattle. They consisted of numerous dense secretory granules which increased in number with age. The present results suggest an optimum thyroid function during the pubertal. Our findings would be useful in interpreting changes in thyroid morphology during experimental and pathological conditions.   Key words:  Cattle, postnatal thyroid, histology, ultrastructure.

Distribution of Phosphatases, Oxidoreductases and Esterases in Sublingual Salivary Gland of Buffalo During Neonatal Development

The present study was aimed to elucidate the distribution of various enzymes in sublingual salivary gland of neonatal buffalo (n = 10), ranging from 1 to 3 months age, and correlate these enzyme systems with the secretory activity of gland. At 1 month age, a uniform moderate to strong positive diffused granular alkaline phosphatase activity was observed in serous acini and ductal epithelium. A moderate positive glucose-6-phosphatase and succinic dehydrogenase activity was seen close to the basement membrane. At 2 months age, glutamic dehydrogenase showed moderate activity in basal portions of serous acinar cells, while succinic dehydrogenase activity was strong, granular and evenly distributed in basement membrane of serous cells. Weak activity of monoamine oxidase and moderate non-specific esterase activity was seen. At 3 months age, alkaline phosphatase showed strong positive perinuclear activity in acinar cells, while glucose-6-phosphatase showed a moderate to strong activity in acinar cells and in ductal epithelium. Moderate to strong positive non-specific esterase activity was localized in nerve fibres of intralobular and interlobular connective tissues. Glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide diaphorase showed strong positive granular activity in basement membrane of serous acinar cells and ducts. The high basal activities of oxidoreductases were because of basal mitochondria and were associated with high energy production to meet the energy requirement for active and rapid transport of ions and water.

Mobilization of lymphatic endothelial progenitor cells and lymphatic neovascularization in primary Sjögren’s syndrome

Primary Sjogren’s syndrome (pSS) is a systemic autoimmune disorder charac- terized by chronic lymphocytic infiltration of exocrine glands leading to progressive functional impairment. Lympangiogenesis is a common finding in chronic inflamma- tory diseases; however, its role in pSS remains to be elucidated. Inflammation induces the production of growth factors for lymphatic vessels, such as vascular endothelial growth factor-C (VEGF-C). Recent growing evidence indicates that bone marrowderived lymphatic endothelial progenitor cells (LEPCs) may differentiate into lymphatic endothelial cells via VEGF-C/VEGFR-3 signaling contributing to lymphangiogenesis. This study was designed to investigate the circulating levels of LEPCs and the occurrence of lymphangiogenesis in pSS. Pheripheral blood mononuclear cells were collected from 10 female pSS patients and 11 healthy females. LEPCs, defined as CD34+CD133+VEGFR-3+ cells, were identified by FACS using CD34-FITC, CD133-APC and VEGFR-3-PE antibodies. Results were expressed as percentage of CD133+VEGFR-3+ cells among CD34+ cells. Labial minor salivary gland (MSG) biop- sies were obtained from 12 female pSS patients and 16 sicca non-pSS control females. MSGs were evaluated by haematoxylin-eosin and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germi- nal center-like structures. Lymphatic vessels were identified by immunohistochemistry for podoplanin (D2-40), a mucin-type transmembrane protein expressed by lymphatic endothelial cells but not by blood vessels. VEGF-C/VEGFR-3 expression in MSGs was investigated by immunofluorescence. An average ten-fold increase in circulating levels of LEPCs was found in pSS (35.2±2.7%) compared with controls (3.4±0.8%) (p=0.0003). In control MSGs, lymphatic vessels were only detected around excretory ducts in the interlobular connective tissue. In pSS MSGs, the number of lymphatic vessels was increased around interlobular excretory ducts and a newly formed lymphatic capil lary network was found within inflammatory foci. A strong expression of VEGF-C was detected in ductal cells, vessels and inflammatory cells in pSS MSGs. VEGFR-3 expres- sion was observed in a subset of vessels and infiltrating mononuclear cells. Our find ings suggest that LEPC mobilization and MSG lymphatic vessel reorganization may take center stage in the chronic inflammatory process of pSS.

Influence of mother HIV infection on pancreas development of the fetus.

Background. Current stage of HIV infection is characterized by a significant increase in the incidence of infection among women of reproductive age and pregnant women. Consequently it will lead to the complex of problems related to the health of children born to them. Objective. To identify the impact of HIV infection of the mother on the formation of the pancreas of the fetus. Methods. Pancreases were measured and weighed. Histological sections were stained with hematoxylin and eosin, Van Gison and Mallory methods. Immunohistochemical examination was performed using the indirect method of Coons in modifications of M.Brosman. α- and β-cells were detected using monoclonal antibodies to glucagon and insulin. Immunohistochemical study was carried out in the luminescent microscope «Axioskor 40" using the software Biostat.exe. All digital data is processed by methods of mathematical statistics using variations, alternative and correlation analysis. Results. Analyzing the morphometric parameters of the pancreas of stillbirths from HIV-infected mothers, comparing them with the control group we found a massive expansion of the stroma on the background of decreasing both quantitative and qualitative composition of the islets of Langerhans, as well as a violation of the ratio of populations α- and β-cells in the direction of decreasing β-endocrine cells (1: 1.47). The latter ratio is known to play a leading role in the development of the endocrine pancreas dysfunction. Decrease in the density of Langerhans islets due to the growth of the stroma by the newly formed interlobular and intralobular connective tissue, as indicated by stromal-parenchymal ratio of the organ, as well as reducing of the diameter of the island and the number of endocrine cells in it. β-cells of the islets of Langerhans of children of M group had evidences of functional inhibition manifesting in reducing of the cellular diameter and increase of nuclear/cytoplasmic ratio, decrease in the intensity of ß-cells luminescence. Conclusion. Immunohistochemical study of pancreatic stillbirths from HIV-infected mothers showed a decrease in the intensity of luminescence of ß-cells in sections treated with mAb to insulin, which was the strong evidence of their functional inhibition.

Histological Evaluation of Selected Organs of the Eurasian Beavers (Castor fiber) Inhabiting Poland.

There is a general scarcity of data on the histological structure of major organs in the Eurasian beaver (Castor fiber). This study presents the histological characteristics of beaver organs such as the liver, spleen, cardiac muscle, lungs and kidneys. Tissue samples were collected from 21 beavers and analysed. Selected samples of tail tissue were additionally examined. Tissue samples were placed in neutral buffered formalin and embedded in paraffin. 4-μm-thick sections were stained with haematoxylin and eosin, and other staining techniques were also used. Scant amounts of inter-lobular connective tissue were found in the liver. Ion or copper deposition was not observed, but scattered cytoplasmic glycogen deposits were present in hepatocytes. Our results suggest that beavers have defensive rather than storage spleens. Interestingly, the presence of melanin in splenic red pulp was noted. The histological structure of the examined organs closely resembled that of other rodent species. According to our knowledge, this is the first study evaluating the histological structure of beaver organs. Nevertheless, precise characterization of the evaluated organs requires further work with the involvement of accurate and reliable techniques, such as molecular biology or electron microscopy methods.

GROSS ANATOMY OF THE RESPIRATORY SYSTEM OF MIZO LOCAL PIG (ZO VAWK)

The present investigation provides a baseline data on gross anatomy of respiratory system of Mizo local pig (Zo vawk). The values of the biometrical measurements were less corresponding to the smaller shape, size and lesser body weight of Mizo local pigs. They have relatively longer nasal cavities corresponding to the long facial part of the head. The laryngeal prominence is found absent on the ventral surface of thyroid cartilage. The first tracheal ring is slightly wider and larger and no fusion between tracheal rings. The right extra pulmonary bronchus is shorter and wider than left extra pulmonary bronchus. The surface lobulations are clearly visible on the lung corresponding to the thick interlobar and interlobular connective tissue.

Structural and functional effects of early exposure to 4-nonylphenol on gonadal development of Nile tilapia (Oreochromis niloticus): b-histological alterations in testes.

The present study investigates the effects of different doses (0, 40, 60, 100 μg/L) of 4-nonylphenol (NP) on testis histology and sperms motility of mature Oreochromis niloticus. A tendency for a dose-dependent reduction in the gonado-somatic index (GSI) in the NP-exposed groups was observed. Histological examination revealed that high doses of NP (60 and 100 μg/L) cause hyperplasia of interlobular connective tissue components coincide with decrease in the size of seminiferous lobules and amount of lumen spermatozoa in comparison with the control and low-dose (40 μg/L)-exposed groups. In addition, within the seminiferous lobules, rupture of spermatogenic cysts, vacuolation and necrosis of primary spermatocytes due to damage of sertoli cells were clearly observed and lead to decrease in advanced spermatogenic cells. The highest dose caused significant decrease in the GSI associated with appearance of testis-ova (intersex) state. The percentage of abnormal sperms increased with increasing the dose of NP reaching to 96 % for those subjected to 100 μg NP/L, while it was only 11 % for the control group specimens. The study indicates that NP has estrogenic potency induced marked alteration in the sexual development including inhibition in testicular growth and deformation in the sperms.

PATHOHISTOLOGICAL CHANGES IN INBRED WHITE RATS’ THYMUS AND SPLEEN DURING ACUTE STRESS

Aim. To characterize in experiment pathological changes in the thymus and spleen of white rats in acute stress. Materials and methods. The experiment included 40 animals. Two experimental groups, 20 animals each, was formed: group I - intact animals living in standard vivarium conditions; group II - acute cold stress modeled in the following way: temperature +4 °C, exposition - 1,5 hours. On the day 10 th of the experiment the animals were withdrawn from the experiment and autopsy material was taken for subsequent histological study (spleen, thymus, Peyer’s plaques). Results. In the group with cold stress, vascular disorders (connective tissue edema and vascular plethora) and inversion of layers were observed in thymic tissue by the day 10 th of the experiment. In the cerebral and cortical layers, the areas of lymphocyte death, stroma collagenization were visualized and a large number of thymic bodies were formed. In thymic tissue, a great number of mast cells in the interlobular connective tissue, connective tissue of septa inside the lobules near the blood vessels as well as in the thymic parenchyma - in the lobular cortical substance and in the areas of lymphoid cell death - were detected. In the experimental group, marked signs of accidental thymic involution against the background of hemodynamic disorders as well as reaction of reticuloepithelial elements and mast cells were revealed. In the spleen of animals of this group there was decrease in white pulp volume - 22,98±2,45%; p <0,05 as compared to the control group. Conclusion. While comparing the data of histological study of animal organs the following patterns were found in both experimental and control groups: in animals exposed to acute stress changes typical for acute stress including reduction of thymic and splenic lymphoid tissue were revealed.

Histological and Histochemical Studies on the Paraorbital Gland in Broilers

The objective of this investigation was to study histological and histochemical structure of the paraorbital gland in broilers. Samples were obtained from 10 male and 10 female adult healthy Ross broilers. Tissue sections were stained with hematoxylin eosin, Verhoeff’s, Masson’s trichrome, alcian blue (pH 2.5), periodic acid-Schiff and Gomori’s method for reticulum. Results revealed the paraorbital gland of Ross broilers was covered by a thin capsule, which contributed trabeculae that divide the organ into lobes and lobules. The capsule consisted of parasympathetic ganglia, nerve bundles, adipose tissue, elastic, collagen and reticular fibers. The glandular epithelium was multilobular and compound tubuloalveolar. Plasma cells were present in interlobular connective tissues. The paraorbital duct was lined by columnar epithelial cells of varying height. The goblet cells were not found in paraorbital duct. Histochemical stainings revealed that the secretion of paraorbital gland was mucous and all epithelial cells of both corpus glandulae and ducts contained both neutral and acidic mucins. The histological and histochemical properties of the gland showed no gender differences. It is concluded that the general histological and histochemical structure of the paraorbital gland in broilers was similar to that of native chickens and domestic geese, but there were also some differences.

Fetal Parotid Gland Structural Remodeling In Case Of Intrauterine Growth Retardation

The important biological role of saliva in maintaining of the homeostasis of the oral cavity environment, preventing infection and tooth decay is widely accepted. Salivary glands insufficiency may violate the balance between health and disease. Clinical data indicated that the destruction, agenesis and aplasia of salivary gland commonly followed with hypo salivation, low salivary flow, which resulted in severe caries and periodontal disease. Salivary gland dysfunction also frequently found in preterm and low birth weight newborns. But the information about structural background of abnormal salivation in early childhood is still limited. A presence of any correlation between salivary glands’ structural development and intrauterine restrictions of fetal growth (IUGR) is unclear. The aim of present study was to determine morphological and morphometric peculiarities of human parotid gland in case of IUGR at late gestation. Material and methods Parotid glands of twenty human fetuses 20-22 weeks of gestation with diagnosed IUGR from late abortions material were compared with ten fetal glands in cases of induced abortions due to psychological reasons (control group). Tissue samples were immersion-fixed in 10% buffered formalin solution, embedded in paraffin wax. Histological slides were stained routinely with hematoxylin &amp; eosin, with Van Gieson's Stain. Microscopical examination was performed on magnification x 40 and x 100. Stereometric study by point count method at magnification x 40 allowed finding out volume fractions (VF) of gland’s parenchyma and stroma. VF of lobule’s components (gland’s wall, gland’s lumen, duct’s wall, duct’s lumen, vessels, intralobular connective tissue) were registered at magnification x100. Morphometry of the secretory portion of the parotid gland was conducted on the Zeiss microscope with the help of the AxioVision Rel.4.8 program. The mature (differentiated) end pieces were measured, including their area (in mkm2), width, height, perimeter (all in mkm). Similar measurements were done in the foci of immature secretory ends of a gland. Additionally the mean height of the epithelial cells layer within the mature secretory end pieces was measured. The differences were analyzed by methods of mathematical statistics using the software Microsoft Excel; data was compared with control measurements by Student’s t-test.Results The results of present research have shown the delayed differentiation of fetal parotid gland’s parenchymal components in case of IUGR. The parenchymal VF did not reach control values. Furthermore, VF of lobule’s components was also decreased. Ducts lumens appeared to be significantly narrower than at physiological gestation. Interlobular and intralobular connective tissue stroma, in contrast to the controls, occupied vast areas, and their volume fraction was increased. Deficit of the parenchymal components of the gland was enhanced by slower maturation of glands. In cases with IUGR, differentiated glands occupied smaller area, with reduced width, height and perimeter. Epithelium lining the differentiated glands is characterized by significantly lower height compared to the control group. Delayed differentiation resulted in higher proportion of immature glands. Their area, width, height and perimeter increased. IUGR was also accompanied with a variety of pathological changes. Conclusion Present evidences suggest that IUGR leads to impaired growth and maturation of the parotid gland. Structural immaturity and lack of differentiated parenchymal elements of the organ may form the basis of its secretory function’s lesion. The finding tends to support the hypothesis that the mechanism behind the increased risk of dental pathology in preterm, low birth weight and retarded children is centred at structural and functional immaturity of salivary gland.

Vaccination with epigenetically treated mesothelioma cells induces immunisation and blocks tumour growth

Malignant mesothelioma (MM) is an aggressive tumour associated with poor outcome in patients. Current treatments for MM are of limited efficacy. Our recent findings suggest that epigenetic drugs may induce both cytotoxicity and an immune response against MM cells. Thus, we used a mouse model of MM (AK7) to analyse how epigenetic drugs could modulate MM development in vivo. The treatment of tumour-bearing mice with an epigenetic drug already tested in clinical MM treatments (SAHA/Vorinostat) reduced the tumour mass and induced a moderate lymphocytic infiltration. However, the treatment did not stop tumour development. In order to show the potential effect of this epigenetic drug on tumour immunogenicity, in addition to cell cytotoxicity, we immunised mice either with AK7 cells pre-treated with SAHA, or with one of two cytotoxic drugs (curcumin or selenite), prior to transplantation of live AK7 cells. A specific immune response was observed only in mice immunised with AK7 cells pre-treated with the epigenetic drug (SAHA) and the tumour growth was arrested. An increase in the proportion of CD3+ CD8+ lymphocytes occurred in the peritoneal cavity. We also observed large conglomerates of immune cells in the omentum with clusters of CD8+ T cells, together with lymphocytes directed against residual AK7 cells in the interlobular connective tissue of the pancreas. Our data demonstrate that epigenetic drugs, such as SAHA, can stimulate tumour immunogenicity and improve the recognition of aggressive MM cells by the immune system in vivo.

Immunohistochemical Study of the Lymphatic Vessels in Major Salivary Glands of the Rat

This study was designed to examine whether lymphatic vessels are present in the lobules of major salivary glands in the rat. Immunostaining with an antibody against podoplanin, a lymphatic endothelial cell marker, was performed on sections of the submandibular, sublingual and parotid glands. Light microscopy demonstrated podoplanin-positive lymphatic vessels around the interlobular ducts and the interlobular arteries and veins in the interlobular connective tissue in all of the major salivary glands. No podoplanin-positive lymphatic vessels were found in the lobules. Electron microscopy also demonstrated lymphatic endothelial cells showing podoplanin expression only in the interlobular connective tissue. These findings suggest that the lymphatic system of the rat major salivary glands originates in the interlobular connective tissue, and not in the lobules.

Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues

The function and development of the rat mammary gland is dependent on the oestrus cycle. Normalization of gene expression in mammary gland samples assessed by quantitative RT-PCR therefore requires housekeeping genes (HKGs) which are stably expressed during the oestrus cycle. mRNA expression of 10 HKGs was measured in the rat mammary gland at different phases of the oestrus cycle. In addition, mRNA expression of the HKGs was measured in a panel of other rat tissues comprising laser microdissected mammary gland alveolar lobules and interlobular connective tissue and macrodissected mammary gland, liver, skeletal muscle, colon and ovary samples. Expression and ranking of HKGs varied between tissues and oestrus cycle phases and several HKGs were necessary for normalization between samples. In the mammary gland samples, three HKGs (Sdha, Tbp, and Atp5b) were identified as the optimal combination of stably expressed genes across oestrus cycle phases. For normalization between samples from the entire panel of rat tissues, eight HKGs (Rps18, Eef1a1, B2m, Actb, Tbp, Hprt, Pgk1, and Sdha) were identified as the optimal combination. These HKGs are of general relevance for studies comparing gene expression between different rat tissues.

Cyclic Morphological Changes in the Beagle Mammary Gland

Interpretation of chemical-induced effects on the female beagle mammary gland can be difficult owing to the wide variation of normal glandular morphology. In this retrospective study, morphological features of the gland in four (proestrus, estrus, diestrus, and anestrus) phases of the cycle are described. The gland was quiescent (inactive) in proestrus and estrus. In diestrus, with the rise of progesterone, four (I-IV) distinct morphological changes were evident. In phase I, there was exuberant stromal and ductal proliferation. In phase II, there was early lobular development with branching ducts and alveolar proliferation. In phase III, there was an abundance of glandular tissue with large lobules containing secretory material, whereas phase IV had features of early regression, increased interlobular connective tissue, and eosinophilic secretions in distended ducts and acini. In early anestrus, ducts were distended, with eosinophilic secretions with alveolar regression, whereas regression was complete in late anestrus. Glandular morphology was slightly variable in the mammary chain within the same dog. Progesterone receptor expression was prominent in estrus and early diestrus, and peak estrogen receptor expression was noted in diestrus II. Expression of proliferation marker ki-67 was highest in diestrus I, followed by diestrus II. There was excellent concordance between the estrous stage and the glandular morphology.