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Background Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. Apoptosis is a key mechanism involved in all the stages of the disease: vascular damage, immune dysfunction, and fibrosis. Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death. Dysregulation of caspases underlies human diseases including cancer and inflammatory disorders, and major efforts to design better therapies for these diseases seek to understand how these enzymes work and how they can be controlled. Objectives This pilot study aimed to explore the expression of caspase-3 in stomach tissue in SSc patients and possible correlation with disease activity and severity. Methods A total of thirteen stomach tissue biopsies (11 SSc and 2 controls) were collected. The same investigator, blind to clinical features, performed upper gastrointestinal endoscopy. The sections of stomach tissue were separately incubated for 45 min with rabbit anti-human/mouse active caspase-3 primary antibody. The intensity of apoptosis according to the caspase 3 activity was semiquantitatively selected into four categories: mild, moderate and abundant. We evaluated the disease activity and severity using clinical and laboratory parameters according to a modified Medsger severity scale and activity score. The disease activity was assessed according to Valentini's Scleroderma Disease Activity Index. In all the patients skin involvement was assessed by the modified Rodnan skin score (mRSS). The study was approved by the University Hospital Split Ethics Committee. Results Eleven SSc female patients [mean age 54.3 (13.6) years, median disease duration 13 years with minimum–maximum range 1–35 years] were enrolled in this study after they gave written informed consent. All patients fulfilled the ACR criteria for the diagnosis of SSc. Five SSc patients had moderate/abundant and six had mild apoptosis on the basis of the caspase 3 activity. In SSc patients, the number of apoptotic cells is high, and their distribution is observed in several layers of the stomach wall, including blood vessels (Figure 1). The skin involvement and disease duration were highly different between SSc patients regarding to the apoptosis level (P

U miševa s isključenim St8sia1 genom (St8sia1-KO), kojima nedostaje enzim GD3 sintaza, nakupljaju se gangliozidi a-serije, a nedostaju gangliozidi b- i c-serije. Iako se sastav gangliozida značajno mijenja, ukupna koncentracija gangliozida u KO miševa ostaje ista kao u miševa divljeg tipa (WT). Gangliozidi u staničnoj membrani nisu uniformno raspoređeni, nego su grupirani u lipidnim splavima, kao i transmembranski proteini koneksini (Cx), koji su ključni za međustaničnu komunikaciju u mnogim organima, a u nekoliko regulatornih mehanizama u bubrezima igraju funkcionalnu ulogu. S obzirom na to da promijenjeni sastav gangliozida utječe na distribuciju proteina u lipidnim splavima različitih staničnih membrana i tako vjerojatno utječe na distribuciju, transport i funkciju različitih izoformi Cx, cilj ovog istraživanja bio je utvrditi učinke promijenjenog sastava gangliozida na izražaj Cx37, Cx40, Cx43, Cx45 i Panx1 u različitim regijama bubrega St8sia1-KO miševa. Pokusi su provedeni na dvanaest mužjaka miševa u dobi od 6 mjeseci: četiri miša divljeg tipa (C57BL/6-tip, WT) i osam St8sia1-KO miševa. Uzorci mišjih bubrega su obrađeni za imunohistokemiju i potom analizirani. Izražaj Cx37, Cx40, Cx43, Cx45 i Panx1 ispitivan je imunofluorescentnim mikroskopom (BX61, Olympus, Tokio, Japan), dok je kvantitativna procjena imunoreaktivnosti provedena pomoću ImageJ softvera(National Institutes of Health, Bethesda, MD, SAD). Kako bi lokalizirali izražaj koneksina i Panx1 u različitim dijelovima bubrega miša, uradili smo dvostruko imunohistokemijsko bojenje s različitim biljezima tubularnih segmenata. Izražaj koneksina i Panx1 je određen u različitim regijama bubrega: kora (CTX.), vanjski sloj vanjske medule (O.S.), unutarnji sloj vanjske medule (IN.S.) i unutarnja medula (IN.MED. ). Cx37, Cx40, Cx45 i Panx1 značajno su niže izraženi u različitim dijelovima bubrega St8sia1-KO miševa u usporedbi s WT. Svi ovi ranije navedeni koneksini smanjenog su izražaja najkonzistentnije u O.S. u St8sia1-KO miševa, pa možemo zaključiti da su proksimalni tubuli i debeli uzlazni krakovi Henleove petlje dijelovi nefrona koji su najosjetljiviji na promjene u sastavu gangliozida. U CTX. regiji je zabilježen smanjen izražaj Cx37, Cx45 i Panx1, dok su u IN.S. regiji Cx37 i Panx1 bili smanjeno izraženi. Rezultati ovog istraživanja sugeriraju da nedostatak GD3 sintaze u St8sia1-KO miševa dovodi do poremećaja izražaja Cx u bubrezima, što je vjerojatno povezano s promjenom sastava gangliozida. Uočeni poremećaj izražaja Cx u bubrezima St8sia1-KO miševa daje važan uvid u promjene na staničnoj razini koje se mogu vidjeti u hiperglikemijskim stanjima, a time i vrijedne informacije za buduća istraživanja. Izmijenjen sastav gangliozida u bubrezima St8sia1-KO miševa može biti dobar model za proučavanje posljedičnih promjena izražaja Cx u dijabetičkim uvjetima., In St8sia1 gene knockout mice (St8sia1-KO) lacking the GD3 synthase enzyme, a-series gangliosides accumulate, and b- and c-series gangliosides are deficient. Total concentration of gangiosides in the KO mice remains the same as in wild-type (WT) mice, while the ganglioside composition markedly changes. In the plasma membrane gangliosides are clustered in lipid rafts, same as connexins (Cx), that are transmembrane proteins critical for intercellular communication in many organs, and play a functional role in several regulatory mechanisms in kidney. Considering that altered ganglioside composition affects the distribution of proteins in lipid rafts of different cell membranes and in this way probably influences the distribution, transport, and function of different Cx isoforms, the aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1-KO mice. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1-KO mice. Mice kidney samples were processed for immunohistochemistry and analyzed. The expression of Cx37, Cx40, Cx43, Cx45 and Panx1 was examined by immunofluorescence microscopy (BX61, Olympus, Tokio, Japan), while quantitative evaluation of immunoreactivity was performed using ImageJ Software (National Institutes of Health, Bethesda, MD, SAD). To localize expression of connexins and Panx1 in different parts of the mice kidney, we performed double fluorescent staining with different tubular segment markers. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). Cx37, Cx40, Cx45, and Panx1 are significantly lower expressed in different parts of the kidneys of St8sia1-KO mice compared with WT. All of the above mentioned markers are decresased the most consistent in the O.S. in St8sia1-KO mice, so we can conclude that proximal tubules and thick ascending limbs are parts of the nephron most sensitive to changes in ganglioside composition. In the CTX. region reduced expression for Cx37, Cx45 and Panx1 was observed, while in the IN.S. region Cx37 and Panx1 were lower expressed. The results of the present study suggest that deficiency of GD3 synthase in St8sia1-KO mice leads to disturbance in renal Cx expression, which is probably related to alteration of ganglioside composition. The observed disruption of Cx expression in the kidney of St8sia1-KO mice gives important insight in changes on cellular level that can be seen in hyperglycemic conditions, and thus valuable informations for future studies. The disruption of ganglioside composition in the kidney of St8sia1-KO mice may be a good model for the alteration of ganglioside composition and properties of lipid rafts under diabetic conditions.

Čimbenici stanične proliferacije i diferencijacije, te pojavnost primarnih cilija u zamecima sjekutića/očnjaka u čovjeka tijekom razvojnih stadija pupoljka, kape i zvona, istražena je na serijskim rezovima zubnih tkiva 10 normalnih ljudskih fetusa (starosti 7-21 tjedana) primjenom imunohistokemijske metode i metode dvostruke imunofluorescencije za biljege Ki-67, α-tubulin, γ-tubulin, Oct-4, CK8 i vimentin. Udjeli stanica pozitivnih na Ki-67 analizirani su Kruskal-Wallis i Dunn-ovim post hoc testom. Tijekom promatranog razdoblja, udio stanica pozitivnih na Ki-67 je u vanjskom caklinskom epitelu pao sa 48.86% na 24.52%, dok se u unutarnjem caklinskom epitelu povećao sa 56.11% na 60.06%, da bi potom pao na 46.26%. U dentalnoj papili je bilo 46.26% stanica pozitivnih na Ki-67, a u stadiju zvona samo 22.08%. Opadanje udjela proliferirajućih stanica se vidjelo u zvjezdastom retikulumu (sa 46.26% na 15.49%). Jaki citoplazmatski izražaj Oct-4 opisan je u caklinskom organu i to osobito u diferencirajućim preameloblastima. Pomak od jezgrenog prema citoplazmatskom izražaju Oct-4 uočen je tijekom napredovanja razvoja i u stanicama dentalne papile. Primarne cilije (α-tubulin) nalazile su se na površini većine stanica zubnog zametka. Proliferirajuće stanice (bez cilija) je obilježavala prisutnost dvaju centriola (γ-tubulin), dok su kod nekih diferencirajućih stanica primarne cilije povezane s jednim od centriola. CK8 je bio izražen uglavnom u epitelnim dijelovima zubne osnove, dok je vimentin bio izraćen i u epitelnim, i u mezenhimskim tkivima zubnog zametka. Izražaj CK8 se pojačavao u preameloblastima s napredovanjem razvoja. Koekspresija dvaju biljega (CK8 i vimentin) primijećena je u caklinskom organu, a pojačavala se u preameloblastima i preodontoblastima tijekom njihove diferencijacije. Stanična proliferacija omogućava rast caklinskog organa, a njezino smanjenje u pojedinim tkivima upućuje na ispunjenje njihove razvojne funkcije i postupni nestanak. Smanjenje proliferacije se poklapa s početkom diferencijacije preameloblasta i preodontoblasta. Staničnu diferencijaciju prati pojačani izražaj Oct-4 i prisutnost primarnih cilija. Obrasci izražaja CK8 i vimentina upućuju na njihovu ulogu u proliferaciji, migraciji stanica te diferencijaciji preameloblasta i preodontoblasta.

Paneksini su glikoproteini koji formiraju transmembranske kanale velikog promjera putem kojih se otpušta ATP u izvanstanični milje. Na taj način sudjeluju u purinergičkoj parakrinoj komunikaciji između stanica te imaju važnu ulogu u različitim fiziološkim i patološkim procesima. Dijabetička nefropatija najčešći je uzročnik kroničnog bubrežnog zatajenja a njena patofiziologija i dalje nije potpuno jasna. Sve veći broj novijih studija ukazuje na ulogu izravne komunikacije između stanica i parakrinog ATP-a u patofiziologiji DN. U ovoj studiji istražena je glomerularna i tubularna ekspresija Panx1 u bolesnika sa šećernom bolešću tipa 2 i kontrolne skupine te korelacija sa serumskim kreatininom kao pokazateljem bubrežne funkcije. Nađena je značajna pozitivna korelacija između ekspresije Panx1 u glomerulima i kreatinina u serumu, koja je bila izraženija u podskupini bolesnika s dijabetesom, a nije nađena izolirano u skupini bez dijabetesa. Naši podaci sugeriraju da je glomerularna ekspresija Panx1 potencijalni pokazatelj pogoršanja bubrežne funkcije u bolesnika s dijabetesom tipa 2. S obzirom da do sada nema objavljenih podataka o ulozi Panx1 u razvoju bubrega, također je istražena ekspresija Panx1 u različitim fazama embrionalnog i ranog fetalnog razvoja. U 6. tjednu embrionalnog razvoja pronađena je ekspresija Panx1 u mezonefričkim glomerulima i mezonefričkim tubulima. Na prijelazu iz 6. u 7. tjedan embrionalnog razvoja, Panx1 imunoreaktivnost je nađena u mezonefričkim tubulima i mezonefričkom kanalu, kao i u metanefričkom pupoljku uretera i čašicama. U 7. tjednu embrionalnog razvoja, snažna Panx1 ekspresija primijećena je u mokraćovodnom pupoljku u metanefrosu, dok nije pronađena Panx1 imunoreaktivnost u metanefričkoj čašici. U 8. tjednu embrionalnog razvoja, ekspresija Panx1 također je pronađena u mokraćovodnom pupoljku u metanefrosu kao i u bubrežnom mjehuriću i nefronu u obliku zareza, te u epitelnim stanicama Bowmanove kapsule. Ekspresija Panx1 pronađena je u ranoj fazi u paramezonefričkom kanalu i u mezonefričkom kanalu, te se smanjivala prema 8. tjednu. Tijekom 6-10. tjedna kolokalizacija Panx1 s aSMA pronađena je u krvnim žilama u razvoju. U postnatalnom bubregu, jaka imunoreaktivnost Panx1 bila je prisutna u stanicama medularnog i kortikalnog sabirnog kanalića, stanicama koje proizvode renin i proksimalnim tubulima. Vrlo slaba Panx1 imunoreaktivnost pronađena je u određenim distalnim stanicama tubula i u silaznim tankim krakovima Henleove petlje. Imunoreaktivnost Panx1 također je pronađena u podocitima (obilježenih nefrinom). Panx1 nije bio kolokaliziran s aSMA imunoreaktivnošću u žilama postnatalnog ljudskog bubrega, ali je bio prisutan u endotelu. Prostorno-vremenska ekspresija Panx1 tijekom ranih faza razvoja ljudskog bubrega podržava njegovu moguću ulogu u procesima stanične diferencijacije, migracije i pozicioniranja u razvoju ljudskog bubrega., Pannexins are glycoproteins that form large diameter transmembrane channels through which ATP is released into the extracellular environment. In this way, they are involved in purinergic paracrine communication between cells and play an important role in various physiological and pathological processes. Diabetic nephropathy is the most common cause of chronic renal failure, and its pathophysiology is still not fully understood. A growing number of recent studies point to the role of direct communication between cell and paracrine ATP in the pathophysiology of DN. In this study, glomerular and tubular expression of Panx1 in patients with type 2 diabetes and control groups and its correlation with serum creatinine as an indicator of renal function were investigated. A significant positive correlation was found between glomerular Panx1 expression and serum creatinine, which was pronounced in the subgroup of patients with diabetes and was not found in isolation in the group without diabetes. Our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes. In the absence of published data on the role of Panx1 in kidney development, the expression of Panx1 at different stages of embryonic and early fetal development was also investigated. At week 6 of embryonic development, Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from the 6th to the 7th week of embryonic development, Panx1 immunoreactivity was found in the mesonephric tubules and the mesonephric duct, as well as in the metanephric ureteric bud and ampullae. At week 7 of embryonic development, strong Panx1 expression was observed in the ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. At week 8 of embryonic development, Panx1 expression was also detected in the ureteric bud in the metanephros as well as in the renal vesicle and comma-shaped nephron and in the epithelial cells of Bowman's capsule. Expression of Panx1 was found in the paramesonephric duct and mesonephric duct in the early phase and it diminished toward the 8th week. In the 6th to 10th week, colocalization of Panx1 with aSMA was detected in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and in the descending thin limbs of the loop of Henle. Panx1 immunoreactivity was also found in podocytes (labelled with nephrin). Panx1 was not colocalized with aSMA immunoreactivity in postnatal human kidney vessels but was present in the endothelium. The spatiotemporal expression of Panx1 at early stages of human kidney development argues for its possible role in the processes of cell differentiation, migration, and positioning in human kidney development.

Dab1 protein (Disabled-1) je unutarstanični adaptor reelin signalnog puta koji sudjeluje u prenatalnoj migraciji neurona, neurotransmisiji, formiranju pamćenja i sinaptičkoj plastičnosti. Yotari je autosomno recesivna mutanta mišjeg Dab1 gena koju fenotipski karakterizira nestabilan hod, tremor i prerana smrt. Dosadašnja istraživanja uglavnom su bila vezana za neurološke nepravilnosti uzrokovane mutacijom Dab1, a uloga reelin signalnog puta u tkivima i organima izvan živčanog sustava nije ispitivana. Hepatociti, najbrojnije stanice jetre komuniciraju putem pukotinastih veza (GJ, engl. gap junction) sastavljenih od koneksina. U ovom radu istražen je poremećaj u staničnoj komunikaciji u yotari miševima analizom izražaja koneksina (Cxs) Cx26, Cx32, Cx37, Cx40, Cx43 i Cx45 tijekom razvoja jetre embrionalnih dana E13.5 i E15.5. Korištena je imunofluorescentna metoda, analiza urađena na svjetlosnom i fluorescencijskom mikroskopu, a rezultat kvantificiran kao površina vidnog polja prekrivena pozitivnim signalom. Podaci su izraženi kao srednja vrijednost±SD (standardna devijacija) korištenjem jednosmjernog ANOVA testa. Svi ispitani koneksini bili su značajno manje izraženi u yotari miševa u usporedbi s kontrolnom wt skupinom u E13.5. Izražaj koneksina u E15.5 je sličan, s izuzetkom Cx37 koji u wt miša pokazuje neznatan izražaj. Stvaranje kanala u patološkim uvjetima i sklonost apoptozi istraženi su analizom izražaja paneksina 1 (Panx1), inducirajućeg čimbenika apoptoze (AIF) i kaspaze 3 tijekom navedenih embrionalnih razdoblja. Porast izražaja Panx1 u E15.5 u yotari miševa, te veći izražaj AIF u oba razdoblja daje pretpostavku da su yotari skloniji apoptozi. Naši rezultati ističu važnost međustanične komunikacije (GJIC) tijekom razvoja jetre i njihovu moguću ulogu potencijalnih dijagnostičkih biljega u bolestima jetre., Dab1 protein (Disabled-1) is an intracellular reelin signaling pathway adapter involved in prenatal neuronal migration, neurotransmission, memory formation, and synaptic plasticity. Yotari is an autosomal recessive mutant of the mouse Dab1 gene, phenotypically characterized by unstable gait, tremor, and premature death. Previous research has been mainly related to neurological abnormalities caused by the Dab1 mutation, and the role of the reelin signaling pathway in tissues and organs outside the nervous system has not been investigated. Hepatocytes, the most numerous liver cells, communicate through gap junctions (GJs) composed of connexins. In this study, a disorder in cellular communication in yotari mice was investigated by analyzing the expression of connexins (Cxs) Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 during embryonic day E13.5 and E15.5. Immunofluorescent method was used, analysis was performed on a light and fluorescence microscope, and the result was quantified as the surface of the field of view covered with a positive signal. Data were expressed as mean ± SD (standard deviation) using a one-way ANOVA test. All tested connexins were significantly less pronounced in the yotari mice compared to the control wt mice in E13.5. The expression of analysed connexins in E15.5 is similar, with the exception of Cx37 which in wt mouse shows very slight expression. Channel formation under pathological conditions and propensity to apoptosis were investigated by analysis of the expression of Panexin 1 (Panx1), AIF and caspase 3 during these embryonic periods. The increase in Panx1 expression in E15.5 in the yotari mice, and the higher expression of AIF in both periods possibly indicate that yotari are more prone to apoptosis. Our results highlight the importance of intercellular communication (GJIC) during liver development and their possible role as potential diagnostic markers in liver disease.

Objectives: The expression and localization of Greb1l in the nephrons of yotari (Dab1-/-) and wildtype (Dab1+/+) mice was analyzed to further develop its suggested importance in mammal kidneys overall, but also its significance particularly in yotari mice nephrons. Materials and methods: yotari and wildtype mice were sacrificed on the 13.5th embryonal day, and 4th and 14th postnatal day. Paraffin embedded kidney tissue sections were analyzed by immunofluorescence using Greb1l antibodies. Kidney structures were then examined by fluorescence microscope. The percentages of positive cells between each group were compared and analyzed by a Kruskal-Wallis-test, followed by a t-test. Results: In 14D kidneys of both genotypes, DCT was the structure with the highest percentage of Greb1l immunoreactive cells, particularly in the yotari group (p, Cilj: Analizirali smo izražaj i lokalizaciju Greb1l u nefronima yotari miševa (Dab1-/-) i divljeg tipa (Dab1+/+) kako bi se dalje utvrdio njegov predloženi značaj u bubrezima sisavaca općenito, kao i njegov značaj posebno u bubrezima yotari miševa. Materijali i metode: yotari miševi i miševi divljeg tipa žrtvovani su 13.5-og embrionalnog dana, te četvrtog i četrnaestog postnatalnog dana. Parafinski rezovi bubrežnog tkiva analizirani su imunofluorescencijom pomoću Greb1l protutijela. Bubrežne strukture ispitivane su fluorescencijskim mikroskopom. Postotak pozitivnih stanica između svake skupine uspoređen je i analiziran pomoću Kruskal-Wallis testa, a zatim t-testa. Rezultati: U 14 dana starom bubregu u oba genotipa, DCT je bio struktura s najvećim postotkom Greb1l imunoreaktivnih stanica, posebno u yotari grupi (p

Objectives: The expression and localization of Creld2 in the nephrons of yotari (Dab1-/-) and wildtype (Dab1+/+) mice was analyzed to further develop its suggested importance in mammal kidneys overall, and its significance particularly in yotari mice nephrons. Materials and methods: Yotari and wildtype mice were sacrificed on the 13.5th embryonal day, and the 4th and 14th postnatal day. Paraffin embedded kidney tissue sections were analyzed by immunofluorescence using Creld2 antibodies. Kidney structures were then examined by fluorescence microscope. The percentages of positive cells in the different nephron structures were compared between yotari and wildtype mice and analyzed by a Kruskal-Wallis test, followed by a t-test. Results: The PCT of postnatal 14D wildtype nephrons showed a strong Creld2 expression signal in the cell cytoplasm and was the structure with the highest percentage of immunoreactive cells (95%) with significantly higher expressions compared to the yotari mice group (P, Cilj: Analizirali smo izražaj i lokalizaciju Creld2 u nefronima yotari miševa (Dab1-/-) i divljeg tipa (Dab1+/+) kako bi se dalje utvrdio njegov predloženi značaj u bubrezima sisavaca općenito, kao i njegov značaj posebno u bubrezima yotari miševa. Materijali i metode: Yotari miševi i miševi divljeg tipa žrtvovani su 13.5-og embrionalnog dana, te četvrtog i četrnaestog postnatalnog dana. Parafinski rezovi bubrežnog tkiva analizirani su imunofluorescencijom pomoću Creld2 protutijela. Bubrežne strukture ispitivane su fluorescencijskim mikroskopom. Postotak pozitivnih stanica između svake skupine uspoređen je i analiziran pomoću Kruskal-Wallis testa i potom korištenjem t-testa. Rezultati: PCT 14 postnatalnih dana starog nefrona divljeg tipa pokazao je jaku ekspresiju Creld2 signala u staničnoj citoplazmi i bila je struktura sa najvećim postotkom imunoreaktivnih stanica (95%) sa signifikantno višom ekspresijom u usporedbi s grupom yotari miševa (P

U istraživanju „Izražaj apoptotskih i proliferacijskih čimbenika u želučanoj sluznici oboljelih od sistemske skleroze“ histološki su analizirane razlike u proliferaciji, apoptozi i profibrotičkim čimbenicima u uzorcima želučane sluznice oboljelih od sistemske skleroze (SSc) i kontrolne skupine. U istraživanju je sudjelovao 15 bolesnika sa SSc i 5 kontrolnih ispitanika bez SSc. Bolesnici su sistematizirani s obzirom na oblik i težinu kliničke slike sistemske skleroze. Pet bolesnika je klasificirano u skupinu s ograničenom kožnom SSc (lcSSc) i blagom bolešću, pet s umjerenom difuznom i pet s teškom difuznom kožnom SSc (dcSSc). Uzorci tkiva su imunohistokemijski obojeni protutijelom Ki-67 (proliferacija), kaspazom-3 (apoptoza), sindekanom-1 (epitelni biljeg), CTGF (profibrotički biljeg) i α-SMA (biljeg miofibroblasta). Istovremena prisutnost (kolokalizacija) Ki-67 pozitivnih stanica s biljegom za miofibroblaste (α-SMA) određena je upotrebom dvostruke imunofluorescentne metode. Postotak reaktivnih stanica analiziran je primjenom Kruskal-Wallis testa i Dunn-ovog post hoc testa, a P, In the study "Expression of apoptotic and proliferative factors in the gastric mucosa of patients with systemic sclerosis" differences in proliferation, apoptosis and profibrotic factors were histologically analyzed in samples of gastric mucosa in patients with systemic sclerosis (SSc) and control group. The study included 15 patients with SSc and 5 control subjects without SSc. Patients were systematized with respect to the type and severity of systemic sclerosis. Five patients were classified into the group with limited cutaneous SSc (lcSSc) and mild disease, five with moderate diffuse, and five with severe diffuse cutaneous SSc (dcSSc). Tissue samples were immunohistochemically stained with Ki-67 antibody (proliferation), caspase-3 (apoptosis), syndecan-1 (epithelial marker), CTGF (profibrotic marker), and α-SMA (myofibroblast marker). The simultaneous presence (colocalization) of Ki-67 positive cells with α-SMA was determined using double immunofluorescence method. The percentage of reactive cells was analyzed by a Kruskal-Wallis test and the Dunn post hoc test. P

Cilj istraživanja Cilj istraživanja bio je ispitati moguću povezanost između izražaja hijaluronske kiseline (HA) i njenog receptora CD44 s prisustvom upalnog infiltrata u uzorcima tkiva gingive pacijenata oboljelih od uznapredovalog generaliziranog parodontitisa. Materijali i metode U svrhu istraživanja metodom imunofluorescencije (IF) obrađeno je 20 uzoraka gingive ispitanika sa zdravim parodontom (kontrole, n = 20) i 20 uzoraka gingive ispitanika s uznapredovalim generaliziranim parodontitisom stadija 3 i 4 (ispitna skupina, n = 20). Za IF bojenje korištena su primarna protutijela na HA i CD44 te na opći biljeg upalnih stanica CD45 za prikazivanje upalnog infiltrata. Preparati su fotografirani tehnikom panoramskog snimanja. Obrada panoramskih snimki provedena je u programu Adobe Photoshop® CC (2019). Izračun izražajnih domena i prostornih gradijenata IF signala napravljen je u programu ImageJ (NIH, Public Domain). Za statističku analizu podataka korišteni su Microsoft Office Excel 2016 (Microsoft Corp, Redmond, WA, USA) i GraphPad v8 (GraphPad Software, La Jolla, CA, USA). Rezultati U uzorcima gingive obje skupine, HA i CD44 izražavaju različite populacije stanica uključujući i upalne stanice. Nije nađena razlika u ukupnom izražaju HA i CD44 u zdravoj i bolesnoj gingivi. Međutim, raspodjela signala na HA i CD44 prema tkivnim odjeljcima (epitel, subepitelna stroma) u bolesnoj gingivi je bila različita što se pokazalo statistički značajnim povećanjem stromalnog izražaja oba ispitivana čimbenika u gingivi ispitanika s uznapredovalim generaliziranim parodontitisom. Ni HA ni CD44 prostorno ne koreliraju s prisustvom upalnog infiltrata što se može objasniti njihovim ubikvitarnim izražajem u gingivi u usporedbi sa znatno ograničenijim izražajem općeg biljega upalnih stanica CD45. Nađena je umjerena međusobna prostorna korelacija izražaja HA i CD44 u bolesnoj gingivi što upućuje na to da CD44 samo djelomično posreduje učinke i/ili sudjeluje u metabolizmu HA u gingivi ispitanika s uznapredovalim generaliziranim parodontitisom. Zaključak Navedeni rezultati ne isključuju ulogu HA i CD44 u regulaciji upalnog odgovora u uznapredovalom generaliziranom parodontitisu, ali potrebna su daljnja istraživanja kako bi se priroda te uloge rasvijetlila., Objectives The aim of the study was to investigate association between the expression of hyaluronic acid (HA) and its main receptor CD44 with the presence of inflammatory infilitrate in gingiva samples of patients suffering from advanced generalized periodontal disease. Material and Methods For this study, 20 samples of gingival tissue from healthy donors (controls, n = 20) and patients suffering from advanced generalized periodontitis stage 3 and 4 (test group, n = 20) were obtained. For the immunofluorescence (IF) staining, primary antibodies against HA, CD44 and general inflammatory cell marker CD45 were used. The microscopic slides were photographed using panoramic technique. Editing of panoramic images was performed in Adobe Photoshop® CC (2019). Quantification of IF signals' expression domains and spatial gradients was performed in ImageJ (NIH, Public Domain). For statistical analysis Microsoft Office Excel 2016 (Microsoft Corp, Redmond, WA, USA) and GraphPad v8 (GraphPad Software, La Jolla, CA, USA) software packages were used. Results In healthy and diseased gingiva, different types of cells (including the inflammatory cells) express HA and CD44. No difference in the total expression of HA and CD44 in healthy and diseased gingiva was found. However, the pattern of distribution of HA and CD44 signals was somewhat different between two groups of samples with regard to increased stromal expression of HA and CD44 in diseased gingiva compared to healthy gingiva. No spatial correlation between HA and CD44 expression with the presence of inflammatory infiltrate was found, which might be explained by the fact that HA and CD44 are ubiquitously expressed in gingival tissue. Moderate spatial correlation between HA and CD44 was found in diseased gingiva, implying its partial involvement in HA signaling and HA metabolism during the advanced generalized periodontitis. Conclusion HA and CD44 might play a role in regulation of inflammatory response in advanced generalized periodontitis. However, further studies are needed in order to properly characterize such role of both investigated factors.

Uvod: Karcinom bubrežnih stanica (RCC) najčešći je karcinom bubrega s kontinuiranim porastom incidencije i smrtnosti u svijetu. Stoga su napori brojnih istraživačkih skupina usmjereni na otkrivanje značajnih dijagnostičkih, prognostičkih i terapijskih biljega koji bi poboljšali kvalitetu zdravstvene usluge za pacijente oboljele od karcinoma bubrežnih stanica. Ciljevi: Ciljevi našeg istraživanja bili su utvrđivanje izražaja VEGF, FGFR1 i FGFR2 biljega u različitim stadijima karcinoma bubrežnih stanica u odnosu na izražaj u normalnom tkivu te utvrđivanje kolokalizacije VEGF-a s FGFR1 i FGFR2 u karcinomu bubrega s obzirom na patohistološki stupanj tumora. Materijali i metode: Istraživanje smo proveli na uzorcima tumorskog tkiva i tkiva zdravog ruba bubrega, uzetim od ispitanika kojima je dijagnosticiran karcinom bubrega niskog (5 uzoraka) i visokog (5 uzoraka) stupnja. Provedeno je dvostruko imunofluorescencijsko bojenje tkivnih rezova karcinoma bubrega i kontrola na kombinaciju VEGF-a s FGFR1 i FGFR2. Postotak pozitivnih stanica izračunali smo na deset reprezentativnih polja iz svakog reza i izrazili kao srednju vrijednost ± SD, a u odnosu između skupina (niski i visoki stadij te kontrolna skupina). Za analizu podataka koristili smo Kruskal-Wallis i Dunn posthoc test. Rezultati: Rezultati našeg istraživanja pokazali su pozitivan imunohistokemijski izražaj VEGF, FGFR1 i FGFR2 u tkivu karcinoma bubrega niskog i visokog stupnja, kao i u normalnom bubrežnom tkivu. Ustanovljena je povremena kolokalizacija FGFR1 i VEGF u normalnom bubrežnom tkivu te kolokalizacija FGFR1 i FGFR2 s VEGF kod karcinoma bubrega niskog i visokog stupnja. U normalnom bubrežnom tkivu, FGFR1, FGFR2 i VEGF pokazali su blagu reaktivnost. U tkivu karcinoma bubrega niskog stupnja FGFR1 je pokazao umjerenu reaktivnost, dok su FGFR2 i VEGF pokazali blagu reaktivnost. U tkivu karcinoma bubrega visokog stupnja, svi biljezi su pokazali umjerenu reaktivnost. Utvrđena je kolokalizacija VEGF - a s FGFR1 i FGFR2 u karcinomu bubrega niskog i visokog stupnja. Zaključak: Temeljem rezultata utvrđen je izražaj i kolokalizacija VEGF, FGFR1 i FGFR2 biljega u različitim stadijima karcinoma bubrežnih stanica i u normalnom bubrežnom tkivu. Navedeni rezultati doprinose boljem razumijevanju uloge ovih čimbenika u različitim patohistološkim stadijima karcinoma bubrega., Introduction: Renal cell carcinoma (RCC) is the most common type of kidney cancer with a continuous increase in incidence and mortality in the world. Therefore, the efforts of numerous research groups are focused on the detection of significant diagnostic, prognostic and therapeutic markers that would improve the quality of health care for patients with renal cell carcinoma. Objectives: The aims of our study were to determine the expression of VEGF, FGFR1 and FGFR2 markers in different stages of renal cell carcinoma in regard to the expression in normal tissue and to determine the colocalization of VEGF with FGFR1 and FGFR2 in renal cell carcinoma considering the pathohistological tumor grade. Materials and methods: The study was performed on samples of tumor tissue and normal renal tissue, taken from respondents diagnosed with low (5 samples) and high grade (5 samples) renal cell carcinoma according to the WHO. Double imunofluorescence staining of renal cell carcinoma tissue sections was performed and controlled on combination of VEGF with FGFR1 and FGFR2. The percentage of positive cells was calculated on ten representative fields from each section and expressed as mean ± SD and in the relationship between the groups (low and high stage and control group). We used the Kruskal-Wallis and Dunn posthoc test for data analysis. Results: The results of our study showed a positive immunohistochemical expression of VEGF, FGFR1 and FGFR2 in low and high grade renal cell carcinoma tissue, as well as in normal renal tissue. Occasional colocalization of FGFR1 and VEGF in normal renal tissue and colocalization of FGFR1 and FGFR2 with VEGF in low and high grade renal carcinoma, was established. In normal renal tissue, FGFR1, FGFR2 and VEGF showed mild reactivity. In low grade renal cell carcinoma tissue, FGFR1 showed moderate reactivity, while FGFR2 and VEGF showed mild reactivity. In high grade renal cell carcinoma tissue, all markers showed moderate reactivity. The colocalization of VEGF with FGFR1 and FGFR2 in low and high grade renal carcinoma, was determined. Conclusion: Based on the results, the expression and colocalization of VEGF, FGFR1 and FGFR2 markers in different stages of renal cell carcinoma and in normal renal tissue, were determined. These results contribute to a better understanding of the role of these factors in different pathohistological stages of renal cell carcinoma.

Cilj: Snail je transkripcijski faktor koji sudjeluje u regulaciji epitelno-mezenhimalne tranzicije (EMT) suprimirajući ekspresiju ektodermalnih gena u mezodermu i potičući ekspresiju mezenhimalnih gena. EMT je ključni proces u embrionalnom razvoju, a postnatalno se povezuje s mnogim patološkim stanjima, kao što su karcinogeneza i fibroza. Ekspresija Dab1 gena je ključna za normalan neurološki razvoj, ali njegov utjecaj na razvoj bubrega je još uvijek nedovoljno istražen. Analizirajući izražaj Snail-a u bubregu, istraživali smo postoji li povezanost između mutacije na Dab1 genu i oštećenja bubrega. Materijali i metode: analizirali smo uzorke bubrega Dab1-/- miševa starih 11 i 14 dana i usporedili ih sa zdravom kontrolnom skupinom iste dobi. Na uzorke smo nanijeli primarno, a zatim i sekundarno protutijelo te smo promatrali izražaj Snail-a u proksimalnim tubulima, distalnim tubulima i glomerulima u kori bubrega. Također smo koristili i bojanje hemalaun-eozinom i DAPI bojanje. Rezultati: Najveći broj Snail pozitivnih stanica bio je 11. dan u distalnim tubulima Dab1 knock-out miša, a tada se vidi i ulazak Snail-a u jezgru glomerula. Najjači signal stanice proksimalnog tubula, distalnog tubula i glomerula u Dab1-/- miša pokazuju 11. dan, a 14. jačina signala u glomerulima se smanjuje, a u proksimalnom i distalnom tubulu ostaje ista. 14. postnatalni dan primjećeno je pojačano stvaranje vakuola u proksimalnim tubulima Dab1-/- miša. Kod kontrolne skupine, jačina bojanja Snail-a se povećava 14. postnatalni dan u proksimalnim i distalnim tubulima, dok se u glomerulima smanjuje u odnosu na 11. dan. Zaključak: Naši rezultati pokazuju veći porast Snail-a u bubregu knock out miša u odnosu na zdravu kontrolnu skupinu, što upućuje na to da Dab1 gen ima veliku ulogu u razvoju bubrega i njegova mutacija potencijalno uzrokuje oštećenja. Budući da do sada uloga Snail-a u bubrezima Dab1-/- miša još nije istražena, potrebno je provesti još istraživanja., Objective: Snail is a transcription factor that has an important role in the regulation of epithelial-mesenchymal transition (EMT) by suppressing the expression of ectodermal genes in the mesoderm and stimulating the expression of mesenchymal genes. EMT is a crucial process in embryonic development, postnatally associated with carcinogenesis and fibrosis. Dab1 expression is crucial for normal neurological development, but its impact on renal development is still insufficiently reaserched. Analyzing the expression of Snail in the kidney, we investigated whether there is a link between mutations in the Dab1 gene and kidney damage. Materials and methods: We analyzed kidney samples of Dab1-/- mice aged 11 and 14 days and compared them with healthy control groups. We applied primary antibody to the samples and secondary antibody one day later. We observed Snail expression in the proximal tubules, distal tubules, and glomeruli in the renal cortex. Hemalaun-eosin staining and DAPI staining were also performed. Results: The highest number of Snail positive cells was on day 11 in the distal tubules of the Dab1 knock-out mouse. The entry of Snail into the glomerular nucleus was also seen at day 11. The strongest signal of the cell of the proximal tubule, distal tubule and glomerulus Dab1-/- mouse samples show the 11th day. The 14th signal strength in the glomeruli decreases but in the proximal and distal tubule it remains the same. In the control group, the intensity of Snail staining increased on the 14th postnatal day in the proximal and distal tubules, while in the glomeruli it decreased. Conclusion: Our results show a greater increase of Snail in the kidney of the knock-out mouse compared to the healthy control group. These results indicate that the Dab1 gene has a great ability in kidney development and its mutation potentially causes damage to the kidneys. Since the role of Snail in the kidneys of Dab1-/- mice has not yet been investigated, more research is needed.

Objectives: The expression and localization of Dab1 adaptor proteins and Notch2 receptors in the nephrons of yotari (Dab1 -/-), heterozygote (Dab1 +/-), and Wildtype (Dab1 +/+) mice was analyzed to further develop their suggested importance in mammal kidneys overall, but also their significance particularly in yotari mice nephrons. Materials and methods: yotari, heterozygote and wildtype mice were sacrificed on the 4th postnatal day. Paraffin embedded kidney tissue sections were analyzed by immunofluorescence using the antibodies, Notch2 and Dab1. Kidney structures were examined by fluorescence microscope. The percentage of positive cells between each group were compared and analyzed by a Kruskal-Wallis test. Results: In the DCT of all genotypes, a strong Dab1 expression signal was seen mostly to be localized in the cell membranes and was the structure with the highest percentage of immunoreactive cells for both Notch2 and Dab1. The highest percentage of positive cell expression for Notch2 stained kidneys was in the heterozygote genotype for all structures. In Dab1 immunofluorescence samples, the highest percentage of immunoreactive cells was observed in the DCT and glomeruli of heterozygotes and in the PCT of the wildtype genotype. Minimal positive cell expression in the yotari PCTs, and glomeruli of yotari and wildtype samples stained with Dab1 antibodies was found. There was minimal positive expression in the Notch2 stained wildtype glomeruli. Conclusions:The expression patterns of Dab1 and Notch2 in the nephron structures of the three genotypes of mice implicate not only the potential importance of Dab1 in DCT cellular signaling cascades for fluid and electrolyte homeostasis, but also suggests the possible signaling interaction of Dab1 and Notch2 in this structure. Overall, the yotari nephron showed significant DCT Dab1 expression, and variable Notch2 expression in all structures, with Notch2 expression in the glomeruli implicating glomerular injury as a possible cause of death in these knockout mice., Ciljevi: Analizirali smo izražaj i lokalizaciju Dab1 adapterske bjelaničevine i Notch2 receptora u nefronima miševa yotari (Dab1 - / -), heterozigota (Dab1 +/-) i divljeg tipa (Dab1 +/+) kako bi se dalje utvrdio njihov predloženi značaj u bubrezima sisavaca općenito, ali i njihov značaj posebno u nefrona yotari miševa. Materijali i metode: yotari, heterozigoti i divlji miševi žrtvovani su četvrtog postnatalnog dana. Parafinski rezovi bubrežnog tkiva analizirani su imunofluorescencijom pomoću protutijela, Notch2 i Dab1. Bubrežne strukture ispitivane su fluorescencijskim mikroskopom. Postotak pozitivnih stanica između svake skupine uspoređen je i analiziran Kruskal-Wallisovim testom. Rezultati: U DCT svih genotipova imali smo snažni izražaj Dab1 signala koji je uglavnom lokaliziran u staničnim membranama i bio je struktura s najvećim postotkom imunoreaktivnih stanica i za Notch2 i Dab1. Izražaj Notch2 imao je najveći postotak pozitivnih stanica bubrega u heterozogotnom genotipu za sve strukture. Imunofluorescencijski zražaj Dab1 imao je najveći postotak imunoreaktivnih stanica zabilježen u DCT i glomerulama heterozigota i u PCT miševa divljeg tipa. Minimalni pozitivn izražaj Dab1 nađen je u yotari miševa u PCT i glomerulima uzoracima divljeg tipa. Minimalan pozitivan izražaj Notch2 nađen je u glomerulima divljeg tipa. Zaključci: Izražaj Dab1 i Notch2 u nefronskim strukturama tri genotipa miševa implicira ne samo potencijalnu važnost Dab1 u DCT staničnim signalnim kaskadama za homeostazu tekućina i elektrolita, već također sugeriraju moguću signalnu interakciju Dab1 i Notch2 u ovim strukturama. Sveukupno, yotari nefron pokazao je značajan izražaj Dab1 u DCT i promjenjiv Notch2 izražaj u svim strukturama, gdje Notch2 izražaj u glomerulima implicira glomerularnu ozljedu kao mogući uzrok smrti kod ovih knockout miševa.