Factors effecting the induction of rat forestomach hyperplasia induced by Swedish oral smokeless tobacco (snus).

Long term exposure to oral smokeless tobacco may induce lesions in the oral cavity characterized by a hyperplastic epithelium. The possible role of nicotine and the physical properties of oral tobacco for developing these lesions, as well as of dysplasia and neoplasia is unclear. Low nitrosamine Swedish snus as well as non-genotoxic butylated hydroxyanisole induces increased cellular proliferation in the rat forestomach epithelia. Using this model, we report here on the effects of nicotine, pH, and particle size. Snus with different properties had no impact on oxidative stress as determined by 8-oxo-7,8-dihydro-2'-deoxyguanosine, or on interleukin IL-1b. Whereas BHA boosted IL-6, probably due to the presence of nicotine. there was no significant enhancement of cell divisions with increasing particle size, although in individual samples the variations in proliferation rates increased greatly with increasing particle size. Conforming to human experience, the enhanced cell proliferation caused by snus was found to be completely reversible. A cacao bean extract had a protective action similar to that previously found for blueberries. The main cause of the observed tobacco induced cell proliferation could be mechanical irritation, possibly in combination with nicotine, whereas within the studied range, pH did not affect the rate of cell division.

ß-catenin and PPAR-γ levels in bone marrow of myeloproliferative neoplasm: an immunohistochemical and ultrastructural study.

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: ß-catenin, PPAR-γ and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of ß-catenin immunoreactive cells was significantly higher in PV then in ET (p < 0.01) or PMF group of patients (p < 0.01) and also in ET versus PMF group of patients (p < 0.01). Erythroid lineage showed absent ß-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-γ was localized mostly in megakaryocytes and the highest number of PPAR-γ immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed ß-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-γ immunopositive megakaryocytes characterized by abnormal maturation.

Inhibition by blueberries (bilberries) and extract from milk thistle of rat forestomach hyperplasia induced by oral smokeless tobacco (Swedish snus).

The aim of this study was to identify palatable additives which have a significant protective action against soft tissue changes in the oral cavity caused by Swedish smokeless tobacco ("snus"), and that satisfy existing legal requirements. Although the cancer risk from snus is extremely low, long term use may result in highly undesirable keratotic lesions and associated epithelial abnormalities in the oral cavity. The rat forestomach, which is vulnerable to the irritative action of non-genotoxic compounds like butylated hydroxyanisole, propionic acid as well as snus, was chosen as an experimental model. Studied toxicological endpoints included histopathology and cellular proliferation based on DNA incorporation of bromodeoxyuridine. After 6 weeks' exposure, blueberries (bilberries) and an extract from the common milk thistle were found to exert a highly significant inhibition of cell proliferation induced by snus in the rat forestomach epithelium, indicating a potential protection with respect soft tissue changes in the human oral cavity.

WITHDRAWN: BRCA1 protein expression and TOP2A gene aberration and protein expression in four molecular subtypes of breast cancer.

Ahead of Print article withdrawn by publisher.

Endocrine cells in human fetal corpus of stomach: appearance, distribution, and density.

BACKGROUND: Since reports on endocrine cells and their kinetics in the corpus of the human stomach are limited, the aim of this study was to examine the appearance, localization, density, and the relationship among the endocrine cell types in the corpus of the human stomach during prenatal and early postnatal development. METHODS: We examined chromogranin A, somatostatin, ghrelin, glucagon, and serotonin expression by immunohistochemistry in 2 embryos, 38 fetuses, and 3 infants in the corpus of human stomach. RESULTS: Chromogranin A secreting endocrine cells were identified in the corpus at week 10 of gestation. Somatostatin cells were present from the 10th week, ghrelin and serotonin cells from the 11th week, and glucagon cells from the 12th week of gestation. Endocrine cells were present individually or clustered within the glandular base and body during the first trimester, and were present separately within the basal and central parts of glands during the second and third trimesters. Somatostatin cells were the most common type of cells (~46 %) during the first trimester, while ghrelin cells were the most numerous during the second trimester (~34 %), and in infants (~28 %). The percentage of glucagon cells was significant only during the first trimester of pregnancy (5.5 %), and the percentage of serotonin cells was only significant just before birth (4.8 %). CONCLUSIONS: These results show, for the first time, that the largest number of endocrine cells are present in the corpus during the first trimester of prenatal development. Also, these results suggest that secretory products of endocrine cells play a role in the regulation of homeostasis, growth, and differentiation, and in human stomach function.

Similar developmental patterns of ghrelin- and glucagon-expressing cells in the human pancreas.

The pancreas appears to be a major source of ghrelin during fetal development, but the ontogeny of ghrelin cells in the human pancreas and their developmental relationship with α- and ß-cells remain largely unknown. In the present study, we examined the dynamics of ghrelin cell growth, colocalization of ghrelin with major pancreatic hormones and defined the similarities and differences among developmental patterns of ghrelin-, glucagon- and insulin-expressing cells in the human pancreas. To this end, paraffin-embedded pancreatic tissue sections from human embryos and fetuses were assessed by immunohistochemistry. Ghrelin-positive cells were first detected in the pancreas of 11-week-old fetuses. With advancing gestational age, both ghrelin- and glucagon-expressing cells were increasingly observed at the periphery of the developing islets, whereas insulin-containing cells were typically found in the islet core. Double immunohistochemistry showed that ghrelin-expressing cells were clearly separate from insulin-, somatostatin- and pancreatic polypeptide-containing cells. In contrast, cells coexpressing ghrelin and glucagon were sporadically detected during both the early and late fetal periods. Furthermore, morphometric analysis revealed a similar trend in the volume density of ghrelin- and glucagon-positive cells, and a contrasting pattern in ß-cell density at specific time points during the development of the human pancreas. This study demonstrates that the developmental pattern of ghrelin cells, although clearly distinct, is quite similar to that of glucagon-expressing cells. The obtained findings indicate a close lineage relationship between these cell populations, a functional relationship between their secretory products and an auto/paracrine mode of ghrelin-glucagon interaction in pancreatic development.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization.

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

The context of tetanus toxoid application influences the outcome of antigen-specific and self-directed humoral immune response.

Results are presented concerning our attempts to create a suitable model system for studying the connection between microbial antigen (micAg), autoimmunity and autoimmune disease on the basis of hyper-immunization and application of micAg in different contexts. Our research was focused on tetanus toxoid (TTd) as a model micAg. Non-pretreated and complete Freund's adjuvant pretreated BALB/c mice were immunized with high doses of TTd mixed with glycerol or aluminum hydroxide as adjuvants. The main aims of the experiments were to evaluate the properties of induced humoral immune responses, evaluate the pathological potential of induced immune responses and determine possible correlations between the properties of a humoral immune response and its pathological potential. The production of TTd-specific and self-reactive beta(2)-glycoprotein I (beta(2)-GP I)-specific antibodies (Abs) was detected in all groups but with specific, context-related properties. Analysis of pregnancy-related pathology (anti-beta(2)-GP I Abs-associated) showed differences in the pathological potential of the induced immune response. It was demonstrated that severity of pathology is positively correlated to the abundance of IgG that recognizes beta(2)-GP I adsorbed onto phosphatidylserine, and to IgG affinity. Furthermore, it was demonstrated that molecular mimicry, which results in generation of anti-beta(2)-GP I Abs upon TTd immunization, is necessary but not sufficient for the development of pregnancy-related pathology.

Murine monoclonal antibody 26 raised against tetanus toxoid cross-reacts with beta2-glycoprotein I: its characteristics and role in molecular mimicry.

PROBLEM: Studies on experimental antiphospholipid syndrome (APS) models proved that molecular mimicry between plasma protein beta(2)-glycoprotein I (beta(2)GPI) and structure within micro-organisms or their products, might be a cause for experimental APS. Considering the heterogeneity of polyclonal antiphospholipid antibodies (aPLs), it is important to define the precise characteristics of pathogenic aPLs. To avoid the influence of polyclonality and to further analyse the connection between molecular mimicry and APS, we produced monoclonal antibodies (MAbs) against tetanus toxoid (TTd) and tested their reactivity against beta(2)GPI. METHOD OF STUDY: In this report, we analysed the characteristics of MAb26 raised against TTd and cross-reactive with beta(2)GPI: its binding properties in various in vitro immunoassays, its specific interactions with surface epitopes expressed on apoptotic cells and its role in vivo. RESULTS: We have demonstrated that MAb26: (i) binds beta(2)GPI being immobilized on an appropriate surface: irradiated polystyrene plates, non-irradiated plates pre-coated with anionic phospholipids and polyvinylidene fluoride membrane; (ii) binds specifically to apoptotic but not to viable cells and the binding is beta(2)GPI-dependent; and (iii) induces a pathologic pregnancy outcome when passively injected into BALB/c mice. CONCLUSION: This study concluded that certain subpopulations of antibodies raised against TTd and cross-reactive with beta(2)GPI, because of the molecular mimicry mechanism, could have pathologic potential.

Insulin-like growth factor-I in wound healing of rat skin.

Growth factors play an important role in orchestrating and enabling the cellular responses required for successful wound healing. In the present study, rat surgical incision was used to investigate insulin-like growth factor-I (IGF-I) expression in skin cells as well as its systemic and cutaneous tissue concentrations during acute phase of wound healing. Thirty two animals were sacrificed at days 2, 3, 5 and 9 after surgery. Eight animals were used as control. Tissue expression of IGF-I in both incisional and periincisional skin areas, as well as in skin of control unwounded animals was determined by immunohistochemistry. Serum and tissue concentrations of IGF-I were measured using RIA. Immunohistochemical analysis revealed enhanced IGF-I immunostaining in the incisional area at day 2 post-wounding. Presence of IGF-I immunoreactivity in the epidermis, as well as in dermal fibroblasts and monocytes within perivascular inflammatory infiltrate suggests its local synthesis. Although serum levels of IGF-I were not altered during wound healing, their tissue contents in the incisional area were significantly increased compared with periincisional area at days 2 and 3 after injury, as well as compared with skin content of unwounded control rats in all examined time points. Obtained results support a paracrine role of IGF-I during the acute phase of wound healing by primary intention in the rat.

G cells and gastrin in chronic alcohol-treated rats.

Numerous reports have described gastric mucosal injury in rats treated with high ethanol concentrations. However, to the best of our knowledge, ultrastructural characteristics of G cells and antral gastrin levels have not been previously reported, either in rats that chronically consumed alcohol or in human alcoholics. The goal of this study was to examine the effect of ethanol consumption (8.5 g/kg) over a 4-month period, under controlled nutritional conditions, on antral and plasma levels of gastrin, ultrastructure of G cells, morphometric characteristics of G cells by stereological methods, and analysis of endocrine cells in the gastric mucosa by immunohistochemistry. The chronic alcohol consumption resulted in a nonsignificant decrease in gastrin plasma levels and unchanged antral gastrin concentrations. A slightly damaged glandular portion of the gastric mucosa and dilatation of small blood vessels detected by histological analysis, suggests that ethanol has a toxic effect on the mucosal surface. Chronic alcohol treatment significantly decreased the number of antral G cells per unit area, and increased their cellular, nuclear, and cytoplasmatic profile areas. In addition, the volume density and diameter of G-cell granules, predominantly the pale and lucent types, were increased, indicating inhibition of gastrin release. Ethanol treatment also decreased the number of gastric somatostatin-, serotonin-, and histamine-immunoreactive cells, except the somatostatin cells in the pyloric mucosa, as well as both G: D: enterochromaffin cells (EC) cell ratios in the antrum and D: ECL cell ratios in the fundus. These results indicate that the change of morphometric parameters in G cells may be related to cellular dysfunction. Our findings also suggest that regulation of G-cell secretion was not mediated by locally produced somatostatin in ethanol-consuming rats, but may involve gastric luminal content and/or neurotransmitters of gastric nerve fibers.

Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by somatostatin-14.

The role of somatostatin on inhibition of both normal and tumor cell cycle, secretion of endocrine and exocrine cells, as well as induction apoptosis is well documented. However, its effect on T cell development and thymic structure is not fully clarified. In order to investigate the influence of somatostatin in vivo on the thymus structure and T cell development, the young adult Albino Oxford male rats were intracerebroventriculary treated with somatostatin-14. We examined the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method, and the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) alpha beta by flow cytometry. Additionally, we also determined the body and thymus weight of the rats, during the first three months of life, to define the time of SRIH-14 application. A decrease of relative thymus weight from the fourth weeks of postnatal life, and an unchanged relative thymus weight obtained in treated group indicates that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. The changes in the absolute number and numerical density of cortical thymocytes indicate that SRIH-14 alters the true lymphoid tissue. SRIH-14 changes relation between thymocyte subsets, increase number of CD4(-)CD8(-)TCR alpha beta(-) and CD4(-)CD8(+)TCR alpha beta(hi) thymocyte subsets as well as the CD4(-)CD8(-)TCR alpha beta(low/hi) thymocytes, while decrease number of CD4(+)CD8(+) TCR alpha beta(-/low/hi) thymocyte subsets. These results indicate that somatostatin-14 is not involved in the control of the physiologic involution of the thymus, although induces thymic weight loss through the reduction of true lymphoid tissue. In addition, changes in frequency of thymocyte subpopulations, especially immature cells, indicate that SRIH-14 modulates thymocytes development and maturation.

The effects of chronic stress on thymus innervation in the adult rat.

Various stressors induce changes in the immune system. However, it has not yet been analyzed how stressors affect thymus innervation. To examine whether chronic stress alters the morphology of the thymus by changing the nerve components of the thymus, adult male rats, 9-weeks old, were exposed to forced swimming during 21 successive days. The animals were sacrificed by decapitation after the last session and their thymuses were used for analysis of (i) the thymus compartments, (ii) distribution patterns of monoamine-containing nerve profiles and (iii) distribution patterns of acetylcholinesterase (AChE)-containing nerve profiles. Our results show that chronic stress in rats reduces the volume of both thymus cortex and medulla, numbers of thymocytes in the deep cortex and medulla and the density of fluorescent nerve profiles, whereas it increases density of fluorescent cells. The distribution patterns of nerve profiles containing monoamine and AChE were not affected. These changes indicate that chronic stress affects thymus development and T cell maturation by altering the sympathetic nerve component.

Exposure to forced swim stress alters morphofunctional characteristics of the rat thymus.

The aim of this study was to investigate whether chronic stress, induced by repeated daily swimming during 21 days, alters the morphofunctional parameters in the thymus of adult rats. Our results showed that chronic stress reduced thymus mass, total number of thymocytes, volume of the thymus compartments and numerical density of thymocytes within thymus inner cortex and medulla. However, the percentage of apoptotic cells and the level of corticosterone were significantly increased. The percentages of CD4-CD8-TCRalphabeta(low/high) and CD4-CD8+TCRalphabeta(-)thymocytes were significantly increased, while the percentage of the least mature CD4+CD8-SP TCRalphabeta(-) thymocytes was significantly decreased. These results show that recurred swimming procedure induces thymus hypotrophy and elevated percentage of DN TCRalphabeta(+) cells.

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28.

The aim of this study was to investigate the effects of centrally applied somatostatin-28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin-28 (experimental group) or saline (control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin-28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double-positive cells was found. The cellularity of double-positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin-28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin-28 evoked disturbance in transition of double-negative to double-positive thymocytes.