Sanja Vignjević

Chronic psychological stress activates BMP4-dependent extramedullary erythropoiesis

Psychological stress affects different physiological processes including haematopoiesis. However, erythropoietic effects of chronic psychological stress remain largely unknown. The adult spleen contains a distinct microenvironment favourable for rapid expansion of erythroid progenitors in response to stressful stimuli, and emerging evidence suggests that inappropriate activation of stress erythropoiesis may predispose to leukaemic transformation. We used a mouse model to study the influence of chronic psychological stress on erythropoiesis in the spleen and to investigate potential mediators of observed effects. Adult mice were subjected to 2 hrs daily restraint stress for 7 or 14 consecutive days. Our results showed that chronic exposure to restraint stress decreased the concentration of haemoglobin in the blood, elevated circulating levels of erythropoietin and corticosterone, and resulted in markedly increased number of erythroid progenitors and precursors in the spleen. Western blot analysis revealed significantly decreased expression of both erythropoietin receptor and glucocorticoid receptor in the spleen of restrained mice. Furthermore, chronic stress enhanced the expression of stem cell factor receptor in the red pulp. Moreover, chronically stressed animals exhibited significantly increased expression of bone morphogenetic protein 4 (BMP4) in the red pulp as well as substantially enhanced mRNA expression levels of its receptors in the spleen. These findings demonstrate for the first time that chronic psychological stress activates BMP4‐dependent extramedullary erythropoiesis and leads to the prolonged activation of stress erythropoiesis pathways. Prolonged activation of these pathways along with an excessive production of immature erythroid cells may predispose chronically stressed subjects to a higher risk of leukaemic transformation.

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 β-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 β-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.

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.

Ethanol and nitric oxide modulate expression of glucocorticoid receptor in the rat adrenal cortex

BACKGROUND This study was performed to investigate expression and distribution of glucocorticoid receptor (GR) in the rat adrenal cortex, acute effect of ethanol on its expression and possible role of endogenous nitric oxide (NO) in this phenomenon. METHODS Adult female Wistar rats showing diestrus day 1 were treated with: a) ethanol (2 or 4 g/kg body weight (b.w.), ip), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), well-known competitive inhibitor of all isoforms of NO synthase (NOS), (30 mg/kg b.w., sc) followed by ethanol (4 g/kg, ip) 3 h later and c) L-NAME (30 mg/kg b.w., sc) followed by saline (ip) 3 h later. Untreated rats were used as controls. Adrenocortical expression of GR was estimated by immunohistochemistry. RESULTS Strong nuclear GR staining was observed throughout the cortex of control rats. Acute ethanol treatment significantly decreased the expression of GR in the zona fasciculata and zona reticularis. Blockade of NO formation had no influence on this effect of ethanol, whereas L-NAME itself induced significant decline in GR immunoreactivity. CONCLUSIONS Obtained findings are the first to demonstrate localization and distribution of the GR throughout the rat adrenal cortex and to suggest that ethanol as well as endogenous NO may modulate adrenocortical expression of this steroid receptor.

Ghrelin receptors in human gastrointestinal tract during prenatal and early postnatal development.

The aim of our study was to investigate the appearance, density and distribution of ghrelin cells and GHS-R1a and GHS-R1b in the human stomach and duodenum during prenatal and early postnatal development. We examined chromogranin-A and ghrelin cells in duodenum, and GHS-R1a and GHS-R1b expression in stomach and duodenum by immunohistochemistry in embryos, fetuses, and infants. Chromogranin-A and ghrelin cells were identified in the duodenum at weeks 10 and 11 of gestation. Ghrelin cells were detected individually or clustered within the base of duodenal crypts and villi during the first trimester, while they were presented separately within the basal and apical parts of crypts and villi during the second and third trimesters. Ghrelin cells were the most numerous during the first (∼11%) and third (∼10%) trimesters of gestation development. GHS-R1a and GHS-R1b were detected at 11 and 16 weeks of gestation, showed the highest level of expression in Brunners gland and in lower parts of duodenal crypts and villi during the second trimester in antrum, and during the third trimester in corpus and duodenum. Our findings demonstrated for the first time abundant duodenal expression of ghrelin cells and ghrelin receptors during human prenatal development indicating a role of ghrelin in the regulation of growth and differentiation of human gastrointestinal tract.

The acute effect of ethanol on adrenal cortex in female rats--possible role of nitric oxide.

AIMS The present study was designed to investigate a possible role of endogenous nitric oxide (NO) in the adrenal response to an acute alcohol administration in female rats. To this end, N(ω)-nitro-L-arginine-methyl ester (L-NAME), a competitive inhibitor of all isoforms of NO synthase, was used. METHODS Adult female Wistar rats showing diestrus Day 1 were treated with: (a) ethanol (2 or 4 g/kg, intraperitoneally); (b) L-NAME (30 or 50 mg/kg, subcutaneously) followed by either ethanol or saline 3 h later. Untreated and saline-injected rats were used as controls. The animals were killed 30 min after last injection. Adrenal cortex was analyzed morphometrically, and plasma levels of adrenocorticotropic hormone (ACTH) and serum concentrations of corticosterone were determined. RESULTS Acute ethanol treatment enhanced the levels of ACTH and corticosterone in a dose-dependent manner. Stereological analysis revealed that acute alcohol administration induced a significant increase in absolute volume of the cortex and the zona fasciculata (ZF). In addition, ethanol at a dose of 4 g/kg increased volume density and length of the capillaries in the ZF. However, other stereological parameters were unaffected by alcohol exposure. Pretreatment with both doses of L-NAME had no effect on ethanol-induced changes. CONCLUSION Obtained findings indicate that acute ethanol treatment stimulates the activity of the adrenal cortex and that this effect is not mediated by endogenous NO in female rats under these experimental conditions.

Ethiopathogenesis, diagnosis and therapy of acquired megacolon in dogs

Megacolon refers to an abnormal dilatation of the colon. This condition occurs in both humans and animals. Although it seems to be more common in cats, megacolon may also occur in dogs. However, data regarding the etiopathogenesis, clinical course and outcome of canine megacolon are scarce. The aim of this study is to present the experience of our team in diagnosis and therapy of canine acquired megacolon, with particular reference to etiopathogenetic aspects. The prospective study included 28 dogs affected with megacolon, aged 5-9 years. The 26 animals underwent a surgical procedure (colonotomy followed by manual extraction of faeces), and were followed up for a period of 28 days. On the basis of anamnestic data, clinical and radiographic findings, 7 dogs (25%) were presented with idiophatic acquired megacolon, while 75% of cases had secundary acquired megacolon of different etiology (including pelvic canal stenosis, lumbar and sacral spinal injuries or back leg fractures, in 46% od cases; keeping the animals in the backyard and irresponsibility of their owners, in 11%; non-adequate nutrition, in 11%; and decreased physical activity and keeping animals in small flats, in 7%). During early postoperative period, the medical treatment and dietary regimen enabled defecation in 65% of cases. The remaining 35% of cases were treated with Cisapride in order to establish spontaneous defecation. All dogs recovered completely during the 28- days follow-up period. According to the results of interviews with dog owners, all animals were in good condition six months after the surgical procedure.

Idiopathic and secondary acquired megacolon in dogs is associated with diminished vasoactive intestinal polypeptide innervation of the affected colon

It is well established that megacolon in carnivores, including both cats and dogs, is a common finding. Megacolon occurs more often in the cat that the dog. Based on current data idiopathic megacolon is a common cause of constipation in cats (62% of constipated cats are affected by diopathic megacolon). There is no evidence of idiopathic megacolon in dogs and publications about this disease in this species is very scarce. We investigated the enteric nervous system in the dilated portion (DP) of the colon in dogs with idiopathic aquired (n=7) or secondary aquired megacolon (n=21) and compared the results with a normal colon in control dogs (n=3). Colonic sections of surgical specimens were investigated by conventional and immunohistochemical methods, including pan-neuronal markers (NSE, synaptophisin, and neurofilament) and VIP, as well as S-100 protein for detection of ganglionic glial cells. Compared to controls, the two megacolon groups showed no changes of density of enteric neurons in both submucosal and myenteric nervous plexuses in DP of the colon and of enteric glial cells. However, compared to controls and dogs with secondary megacolon, there was a significant decrease in the density of NFP-ir nerve fibers in the longitudinal muscle layer in dogs with idiopathic acquired megacolon. In addition, dogs with idiopathic megacolon display decreased VIP-ir in the myenteric plexus and lamina propria mucosae, and absence of VIP-ir neurons in the submucosal plexus of DP of the colon. Similar alterations, although of lesser severity, may be found in dogs with secondary aquired megacolon. We consider that both idiopathic and secondary aquired megacolon might occur on the basis of a dysplastic changes of VIP-ir enteric neurons.

[Significance of pathohistological findings and the expression of Bcl-2 in diagnosis and treatment of oral planocellular carcinoma].

BACKGROUND/AIM Numerous studies were aimed to detect and characterize various tumor markers in patients with oral planocellular carcinoma in order to reduce moratlity and mobidity rates of these patients, as well as to establish the correlation between the expression of specific tumor marker and prognostic outcome. The aim of this study was to determine patohistological characteristics of tumor and peritumor tissue in patients with oral planocellular carcinoma, with special regard to the expression of Bcl-2, as well as to point out the significance of clinicomorphological correlations for clinical use. METHODS Sixty-two patients with oral planocellular carcinoma, stage II and III, were examined. The patients were surgically treated for this condition at the Clinic for Maxillofacial Surgery, Military Medical Academy, Belgrade. Surgical specimens were obtained from both tumor and peritumoral tissues. Patohistologic degree of tumor differentiation and the immunohistochemical expression of Bcl-2 were determinated for each specimens. RESULTS Twenty-four (39%) patients had tumor dimension T1, while six (9%) and thirty-two (52%) patients had tumor dimension T2 and T3, respectively. Patohistologic analysis of peritumor connective, fat, muscle and bone tissue samples confirmed the presence of tumor infiltration. The expression of Bcl-2 in peritumor tissue samples correlated significantly with tumors histologic grade (rho = 0.468; p < 0.001), nuclear grade (rho = 0.430; p < 0.001) and nucleocytoplasmic ratio (rho = 0.410; p = 0.001). CONCLUSION This results suggest that the expression of Bcl-2 in combination with patohistologic findings could have a prognostic value in patients with oral planocellular carcinoma.

Blockade of nitric oxide synthesis stimulates the activity of adrenal cortex

Objective. Although it is known that nitric oxide modulates the activity of hypothalamic-pituitary-adrenal axis, its functional significance has not been elucidated. Additionally, there is no information on the effect of nitric oxide on cortical expression of glucocorticoid receptor. The purpose of this study was to investigate the influence of endogenous nitric oxide on structure and function of rat adrenal cortex and adrenocortical expression of glucocorticoid receptor. Methods. Adult female Wistar rats showing diestrus day 1 were treated with Nω-nitro-L-arginine-methyl ester (LNAME), an inhibitor of all three isoforms of nitric oxide synthase, at a dose of 30 mg/kg, subcutaneously. The concentrations of adrenocorticotropic hormone (ACTH) and corticosterone were determined by radioimmunoassay. Stereological and immunohistochemical analyses were performed on paraffin sections. Results. Blockade of nitric oxide production significantly increased blood levels of ACTH and corticosterone. Stereological analysis showed that treatment with L-NAME significantly decreased numerical density of the cells in all cortical zones. Consistent with the decreased numerical density, L-NAME significantly increased the volume of cells in cortical zones. Inhibition of nitric oxide synthesis decreased expression of glucocorticoid receptor in zona fasciculata and zona reticularis. Conclusion. Obtained results indicate that endogenous nitric oxide inhibits activity of adrenal cortex and modulates expression of glucocorticoid receptor.