Cellular and molecular mechanisms underlying erythropoietic response to chronic stress

Stres je sastavni deo svakodnevnog života. Psihološki stresori pokreću kompleksan odgovor organizma na stres koji utiče na različite fiziološke procese uključujući hematopoezu. Poznato je da se u bazalnim uslovima proces eritrocitopoeze odvija prevashodno u kostnoj srži, dok se u stanjima povećanih potreba organizma za eritrocitopoezom ovaj proces, nazvan stres eritrocitopoeza (SE), aktivira i u slezini. Prema najnovijim saznanjima, neadekvatna regulacija SE može rezultirati prekomernom ekspanzijom i malignom transformacijom nezrelih ćelija crvene krvne loze. Do sada je proces SE ispitivan prevashodno na mišjim modelima anemije dok su literaturni podaci o efektima hroničnog psihološkog stresa na proces eritrocitopoeze veoma oskudni. Cilj ove studije je bio: 1) da se ispita uticaj hroničnog psihološkog stresa na proces eritrocitopoeze u kostnoj srži i slezini, i 2) da se istraže ćelijski i molekularni mehanizmi regulacije ispitivanog procesa. U tu svrhu su korišćeni odrasli mužjaci miševa visokorodnih sojeva CBA i C57/BL6, kao i MIF knockout miševi koji su bili podvrgnuti restraint stresu 7 ili 14 dana uzastopno, u trajanju od 2 sata dnevno. Za detekciju progenitorskih ćelija eritrocitne loze primenjen je metod kultivacije hematopoetskih ćelija na podlozi od metilceluloze obogaćene odgovarajućim citokinima. Metodama protočne citofluorometrije i imunohistohemije izvršena je detekcija i kvantifikacija eritroblasta u ispitivanim uzorcima kostne srži i slezine. Ekspresija ciljnih proteina je analizirana primenom Western blot-a i imunohistohemije dok je relativna ekspresija ispitivanih gena određena metodom Real-Тime PCR. Naši rezultati su pokazali da hronični psihološki stres redukuje koncentraciju gvožđa i hemoglobina u krvi, i da dovodi do stimulacije eritrocitopoeze u kostnoj srži i slezini miša. Inhibicija sinteze azot monoksida u potpunosti je sprečila stimulišući efekat hroničnog stresa na progenitorske ćelije crvene krvne loze u kostnoj srži, ukazujući prvi put da ovaj signalni molekul ima značajnu ulogu u ekspanziji eritroidnih progenitora tokom hroničnog psihološkog stresa. Pored povećanog broja progenitorskih ćelija, u slezini hronično stresiranih životinja smo detektovali i znatno veći procenat eritroblasta što ukazuje da hronični psihološki stres ima izraženiji efekat na ekstramedularnu eritrocitopoezu...Stress has become an important aspect of daily life. Exposure to a psychological stressor initiates an integrated response that affects different physiological processes including hematopoiesis. The majority of steady-state erythropoiesis occurs in the bone marrow. However, recent studies have shown that the adult spleen serves as the main site of red blood cell production under different stress conditions, collectively referred to as stress erythropoiesis (SE). Furthermore, emerging evidence suggests that inappropriate activation of SE may predispose to leukemic transformation. Much of what we know about SE comes from the analysis of murine models of anemia, but the erythropoietic effects of chronic psychological stress remain largely unknown. The aim of the current study was to examine the influence of chronic psychological stress on erythropoiesis in adult bone marrow and spleen, as well as to investigate cellular and molecular mechanisms underlying the observed effects. For this purpose, adult male CBA and C57BL/6 mice were subjected to 2 hrs. daily restraint stress for 7 or 14 consecutive days. In addition, we used macrophage migration inhibitory factor (MIF)-knockout mice on a C57BL/6 background to examine whether MIF is involved in the control of stress-induced erythropoiesis. The number of erythroid progenitors was determined using colony assays, whereas CD71/Ter119 profiles of bone marrow and splenic cells were analyzed by flow cytometry. The expression of target proteins were assessed by Western blot and immunohistochemistry, while gene expression analysis was performed using real-time PCR. Our results showed that chronic exposure to restraint stress decreased the concentration of iron and hemoglobin in the blood, and resulted in markedly increased number of erythroid progenitors in murine bone marrow and spleen. Blockade of nitric oxide synthesis completely abolished the stimulatory effect of chronic restraint stress on erytrhoid progenitors in the bone marrow, suggesting a role of nitric oxide in stress-induced erythroid cell growth. Although repeated restraint stress initiated an erythroid stress response in both bone marrow and spleen, the effects on growth and maturation of erythroid cells were more prominent in the spleen, showing that chronic psychological stress stimulates mainly extramedullary erythropoiesis..

Ćelijski i molekularni mehanizmi regulacije eritrocitopoeze u uslovima hroničnog stresa

Stress has become an important aspect of daily life. Exposure to a psychological stressor initiates an integrated response that affects different physiological processes including hematopoiesis. The majority of steady-state erythropoiesis occurs in the bone marrow. However, recent studies have shown that the adult spleen serves as the main site of red blood cell production under different stress conditions, collectively referred to as stress erythropoiesis (SE). Furthermore, emerging evidence suggests that inappropriate activation of SE may predispose to leukemic transformation. Much of what we know about SE comes from the analysis of murine models of anemia, but the erythropoietic effects of chronic psychological stress remain largely unknown. The aim of the current study was to examine the influence of chronic psychological stress on erythropoiesis in adult bone marrow and spleen, as well as to investigate cellular and molecular mechanisms underlying the observed effects. For this purpose, adult male CBA and C57BL/6 mice were subjected to 2 hrs. daily restraint stress for 7 or 14 consecutive days. In addition, we used macrophage migration inhibitory factor (MIF)-knockout mice on a C57BL/6 background to examine whether MIF is involved in the control of stress-induced erythropoiesis. The number of erythroid progenitors was determined using colony assays, whereas CD71/Ter119 profiles of bone marrow and splenic cells were analyzed by flow cytometry. The expression of target proteins were assessed by Western blot and immunohistochemistry, while gene expression analysis was performed using real-time PCR. Our results showed that chronic exposure to restraint stress decreased the concentration of iron and hemoglobin in the blood, and resulted in markedly increased number of erythroid progenitors in murine bone marrow and spleen. Blockade of nitric oxide synthesis completely abolished the stimulatory effect of chronic restraint stress on erytrhoid progenitors in the bone marrow, suggesting a role of nitric oxide in stress-induced erythroid cell growth. Although repeated restraint stress initiated an erythroid stress response in both bone marrow and spleen, the effects on growth and maturation of erythroid cells were more prominent in the spleen, showing that chronic psychological stress stimulates mainly extramedullary erythropoiesis...Stres je sastavni deo svakodnevnog života. Psihološki stresori pokreću kompleksan odgovor organizma na stres koji utiče na različite fiziološke procese uključujući hematopoezu. Poznato je da se u bazalnim uslovima proces eritrocitopoeze odvija prevashodno u kostnoj srži, dok se u stanjima povećanih potreba organizma za eritrocitopoezom ovaj proces, nazvan stres eritrocitopoeza (SE), aktivira i u slezini. Prema najnovijim saznanjima, neadekvatna regulacija SE može rezultirati prekomernom ekspanzijom i malignom transformacijom nezrelih ćelija crvene krvne loze. Do sada je proces SE ispitivan prevashodno na mišjim modelima anemije dok su literaturni podaci o efektima hroničnog psihološkog stresa na proces eritrocitopoeze veoma oskudni. Cilj ove studije je bio: 1) da se ispita uticaj hroničnog psihološkog stresa na proces eritrocitopoeze u kostnoj srži i slezini, i 2) da se istraže ćelijski i molekularni mehanizmi regulacije ispitivanog procesa. U tu svrhu su korišćeni odrasli mužjaci miševa visokorodnih sojeva CBA i C57/BL6, kao i MIF knockout miševi koji su bili podvrgnuti restraint stresu 7 ili 14 dana uzastopno, u trajanju od 2 sata dnevno. Za detekciju progenitorskih ćelija eritrocitne loze primenjen je metod kultivacije hematopoetskih ćelija na podlozi od metilceluloze obogaćene odgovarajućim citokinima. Metodama protočne citofluorometrije i imunohistohemije izvršena je detekcija i kvantifikacija eritroblasta u ispitivanim uzorcima kostne srži i slezine. Ekspresija ciljnih proteina je analizirana primenom Western blot-a i imunohistohemije dok je relativna ekspresija ispitivanih gena određena metodom Real-Тime PCR. Naši rezultati su pokazali da hronični psihološki stres redukuje koncentraciju gvožđa i hemoglobina u krvi, i da dovodi do stimulacije eritrocitopoeze u kostnoj srži i slezini miša. Inhibicija sinteze azot monoksida u potpunosti je sprečila stimulišući efekat hroničnog stresa na progenitorske ćelije crvene krvne loze u kostnoj srži, ukazujući prvi put da ovaj signalni molekul ima značajnu ulogu u ekspanziji eritroidnih progenitora tokom hroničnog psihološkog stresa. Pored povećanog broja progenitorskih ćelija, u slezini hronično stresiranih životinja smo detektovali i znatno veći procenat eritroblasta što ukazuje da hronični psihološki stres ima izraženiji efekat na ekstramedularnu eritrocitopoezu..

Etiopatogeneza, dijagnoza i terapija stečenog megakolona pasa

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% of 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.Megakolon označava abnormalnu dilataciju kolona. Javlja se i kod ljudi i kod životinja. Iako je oboljenje češće kod mačaka, javlja se i kod pasa. Međutim, podaci o etiopatogenezi, kliničkom toku i ishodu bolesti megakolona kod pasa su oskudni. Stoga je cilj ove studije da se iznesu sopstvena iskustva o dijagnostici i terapiji stečenog megakolona kod pasa, sa posebnim naglaskom na etiopatogenetski aspekt oboljenja. Studija je bila prospektivnog karaktera i obuhvatila je 28 pasa sa megakolonom, starosti 5-9 godina. Dvadeset šest životinja podvrgnuto je hirurškoj intervenciji (kolonotomija sa manuelnom ekstrakcijom fecesa) i praćeno postoperativno u toku 28 dana. Na osnovu anamnestičkih podataka dobijenih od vlasnika pasa, kliničkog i radiografskog nalaza, 7 pasa (25%) imalo je idiopatski stečeni megakolon, dok je kod 21 psa (75%) bolest okarakterisana kao sekundarni stečeni megakolon uzrokovan različitim etiološkim faktorima (uključujući stenozu pelvičnog kanala, oštećenje lumbalne i sakralne kičme ili frakturu zadnjih ekstremiteta, kod 46% pasa; čuvanje životinja u dvorištu i nedovoljni nadzor od strane vlasnika, kod 11%; neadekvatna ishrana, kod 11%; smanjenja fizičke aktivnosti i čuvanje životinja u skučenom prostoru, kod 7%). U ranom postoperativnom periodu, terapija i režim ishrane doveli su do uspostavljanja defekacije kod 65% pasa. Kod preostalih 35% slučajeva spontana defekacija je uspostavljena primenom terapije Cisapridom. Sve životinje su se u toku 28-dnevnog nadzora kompletno oporavile, a odlično opšte stanje zabeleženo je, na osnovu intervjua sa vlasnicima i 6 meseci nakon hirurške intervencije

Putative Role of Neutrophil Extracellular Trap Formation in Chronic Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are hematologic malignancies characterized by gene mutations that promote myeloproliferation and resistance to apoptosis via constitutively active signaling pathways, with Janus kinase 2-signal transducers and the activators of transcription (JAK-STAT) axis as a core part. Chronic inflammation has been described as a pivot for the development and advancement of MPNs from early stage cancer to pronounced bone marrow fibrosis, but there are still unresolved questions regarding this issue. The MPN neutrophils are characterized by upregulation of JAK target genes, they are in a state of activation and with deregulated apoptotic machinery. Deregulated neutrophil apoptotic cell death supports inflammation and steers them towards secondary necrosis or neutrophil extracellular trap (NET) formation, a trigger of inflammation both ways. NETs in proinflammatory bone marrow microenvironment induce hematopoietic precursor proliferation, which has an impact on hematopoietic disorders. In MPNs, neutrophils are primed for NET formation, and even though it seems obvious for NETs to intervene in the disease progression by supporting inflammation, no reliable data are available. We discuss in this review the potential pathophysiological relevance of NET formation in MPNs, with the intention of contributing to a better understanding of how neutrophils and neutrophil clonality can orchestrate the evolution of a pathological microenvironment in MPNs

Interplay between stress and cancer—A focus on inflammation

Stress is an integral part of life. While acute responses to stress are generally regarded as beneficial in dealing with immediate threats, chronic exposure to threatening stimuli exerts deleterious effects and can be either a contributing or an aggravating factor for many chronic diseases including cancer. Chronic psychological stress has been identified as a significant factor contributing to the development and progression of cancer, but the mechanisms that link chronic stress to cancer remain incompletely understood. Psychological stressors initiate multiple physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic nervous system, and the subsequent changes in immune function. Chronic stress exposure disrupts the homeostatic communication between the neuroendocrine and immune systems, shifting immune signaling toward a proinflammatory state. Stress-induced chronic low-grade inflammation and a decline in immune surveillance are both implicated in cancer development and progression. Conversely, tumor-induced inflammatory cytokines, apart from driving a tumor-supportive inflammatory microenvironment, can also exert their biological actions distantly via circulation and therefore adversely affect the stress response. In this minireview, we summarize the current findings on the relationship between stress and cancer, focusing on the role of inflammation in stress-induced neuroendocrine-immune crosstalk. We also discuss the underlying mechanisms and their potential for cancer treatment and prevention

Adipogenesis in Different Body Depots and Tumor Development

Adipose tissue (AT) forms depots at different anatomical locations throughout the body, being in subcutaneous and visceral regions, as well as the bone marrow. These ATs differ in the adipocyte functional profile, their insulin sensitivity, adipokines' production, lipolysis, and response to pathologic conditions. Despite the recent advances in lineage tracing, which have demonstrated that individual adipose depots are composed of adipocytes derived from distinct progenitor populations, the cellular and molecular dissection of the adipose clonogenic stem cell niche is still a great challenge. Additional complexity in AT regulation is associated with tumor-induced changes that affect adipocyte phenotype. As an integrative unit of cell differentiation, AT microenvironment regulates various phenotype outcomes of differentiating adipogenic lineages, which consequently may contribute to the neoplastic phenotype manifestations. Particularly interesting is the capacity of AT to impose and support the aberrant potency of stem cells that accompanies tumor development. In this review, we summarize the current findings on the communication between adipocytes and their progenitors with tumor cells, pointing out to the co-existence of healthy and neoplastic stem cell niches developed during tumor evolution. We also discuss tumor-induced adaptations in mature adipocytes and the involvement of alternative differentiation programs

Neuronal Nitric Oxide Synthase Mediates the Effect of Ethanol on IgA

Aims: We showed previously that the acute effect of ethanol on intestinal immunoglobulin A (IgA) expression might be mediated by endogenous nitric oxide (NO). To extend these findings, this study was designed to investigate a possible role of neuronal NO synthase (nNOS) in the observed phenomenon, using 7-nitroindazole (7-NI), a selective inhibitor of its activity. Methods: Adult male Wistar rats were treated with: (a) ethanol (4 g/kg, intraperitoneally, i.p.), (b) 7-NI (25 mg/kg, i.p.) followed by ethanol (4 g/kg, i.p.) 30 min later and (c) 7-NI (25 mg/kg, i.p.) followed by saline 30 min later. Untreated rats were used as controls. The concentrations of serum and intestinal IgA were measured by enzyme-linked immunosorbent assay, while the expression of nNOS was determined using western blot and immunohistochemistry. Results: Acute ethanol treatment significantly increased the concentration of IgA in the ileal extracts, whereas it decreased its serum level. Inhibition of nNOS activity by 7-NI abolished this action of alcohol on IgA. Additionally, western blot analysis revealed that the acute alcohol administration induced an increase in the expression of intestinal nNOS. Furthermore, nNOS-immunoreactive cells, observed within the lamina propria of small intestine, were numerous in ethanol-treated rats. Conclusion: Taken together, these results extended our previous findings suggesting that nNOS mediates the acute effect of ethanol on IgA and supported an immunomodulatory role of this enzyme isoform

Blokada sinteze azot-monoksida stimuliše aktivnost korteksa nadbubrežne žlezde

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.Cilj. Iako je poznato da azot-monoksid moduliše aktivnost hipotalamo-hipofizno-nadbubrežne osovine, funkcionalni značaj tog delovanja nije rasvetljen. Pored toga, dejstvo azot-monoksida na kortikalnu ekspresiju glukokortikoidnog receptora nije istraženo. Cilj ovog rada bio je da se ispita uticaj endogenog azot-monoksida na strukturu i funkciju kore nadbubrežne žlezde pacova i adrenokortikalnu ekspresiju glukokortikoidnog receptora. Metode. Odrasle ženke pacova Wistar soja u metestrusnoj fazi ciklusa tretirane su Nω-nitro-L-arginin metil esterom (L-NAME), inhibitorom sve tri izoforme azotmonoksid sintaze, u dozi 30 mg/kg, subkutano. Koncentracije adrenokortikotropnog hormona (ACTH) i kortikosterona u krvi određene su radioimunološkom metodom. Na parafinskim presecima nadbubrežne žlezde izvršena je stereološka i imunohistohemijska analiza korteksa. Rezultati. Blokada sinteze azot-monoksida značajno povećava koncentracije ACTH i kortikosterona u krvi. Stereološka analiza je pokazala da tretman sa L-NAME značajno smanjuje numeričku gustinu ćelija u svim kortikalnim zonama. U skladu sa smanjenom numeričkom gustinom, L-NAME značajno povećava volumen ćelija u sve tri zone korteksa. Inhibicija sinteze azot-monoksida smanjuje ekspresiju glukokortikoidnog receptora u zoni fascikulati i zoni retikularis. Zaključak. Dobijeni rezultati ukazuju na mogućnost da endogeni azot-monoksid inhibiše aktivnost kore nadbubrežne žlezde i da moduliše ekspresiju glukokortikoidnog receptora

Idiopatski i sekundarni stečeni megakolon kod pasa udruženi su sa smanjenom vip-inervacijom u oštećenom kolonu

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.Poznato je da se magakolon javlja kod mesojeda, uključujući mačke i pse, pri čemu je ovo oboljenje daleko učestalije kod mačaka. Na osnovu dosadašnjih saznanja, idiopatski megakolon je čest uzročnik konstipacije kod mačaka i 62% mačaka sa konstipacijom ima idiopatski megakolon. Istovremeno, podaci o psima sa idiopatskim megakolonom veoma su oskudni. U ovom radu je proučavan enterični nervni sistem u dilatiranom delu kolona kod 7 pasa sa idiopatskim megakolonom i 21 psa sa sekundarnim stečenim megakolonom, a rezultati su upoređeni sa normalnim kolonom kod 3 kontrolne zdrave životinje. Tkivni preseci kolona bojeni su klasičnim histološkim i imunohistohemijskim metodama, pri čemu su primenjeni pan-neuronski markeri (NSE, sinaptofizin i neurofilament) i VIP, kao i S-100 protein za detekciju glijalnih ćelija u enteričnim ganglijama. Nisu otkrivene razlike u gustini enteričnih neurona u submukoznom i mijenteričnom pleksusu kod životinja sa megakolonom, kao ni razlike u gustini glijalnih ćelija enteričnih ganglija, u odnosu na kontrolnu grupu životinja. Međutim, u odnosu na kontrolnu grupu, kod životinja sa idiopatskim megakolonom dokazana je smanjena VIP-imunoreaktivnost (ir) u mienteričnom pleksusu i krznu mukoze, kao i kompletno odsustvo VIP-ir neurona u submukoznom pleksusu dilatiranog dela kolona. Slične promene, ali u manjem stepenu, postojale su kod pasa sa sekundarnim stečenim megakolonom. Može da se zaključi da u patogenezi idiopatskog i sekundarnog stečenog megakolona značajnu ulogu imaju displastične promene u VIP-ergičkim neuronima enteričkog nervnog sistema

Ghrelin endocrine cells in the human stomach during prenatal and early postnatal development

The aim of this study was to investigate the appearance, localization and density of ghrelin cells in the human stomach during prenatal development. For this purpose the antrum and corpus of embryos, fetuses and infants are stained immunohistochemically by the streptavidin-biotin technique. The presence of P/D1 cells at 11 weeks of fetal development, their highest density during the first detection and higher density in the corpus than in antrum, and their localization in the glandular base of stomach gland, all suggest that ghrelin plays a major role in the early stages of the developing stomach