Apoptoza u bubrežnom tkivu štakora uzrokovana okratoksinom A

The aim of our study was to find whether ochratoxin A (OTA) induces the apoptosis and/or necrosis of kidney tissue in rats. In the first experiment, the highest number of apoptotic cells was found in rats sacrificed one day after OTA administration (1.00 mg/kg b.w., i.p.). The number of apoptotic cells reduced gradually and they were not seen nine days after OTA administration. A possible dose-dependence of histological changes was checked in kidney tissue of rats given 0.25, 0.50 or 1.00 mg of OTA/kg b.w., i.p. three times a week for four weeks. The number of apoptotic cells showed a clear dose-dependence, but necrosis was absent even at the highest doses. The time-dependent appearance of lesions related to OTA administration was checked by administering 0.50 mg OTA/kg body weight to rats, and sacrificing them one day after 1, 3, 6, and 9 doses/administrations, or 6 and 21 day after 12 doses/administrations. Long-term administration is associated with continued and increased apoptosis without necrosis, suggestive of OTA’s role in the pathogenesis of progressive renal atrophy.Okratoksin A (OTA) nefrotoksični je mikotoksin za koji se pretpostavlja da je uzročnik endemske nefropatije i tumora urotela koji se s većom učestalošću javljaju u području endemske nefropatije. Endemska nefropatija je smrtonosna bolest koja zahvaća oba bubrega, a primarno dolazi do oštećenja proksimalnih tubula bubrega. Za sada uzrok bolesti nije poznat. Međutim, niske koncentracije OTA čest su nalaz u krvi ljudi i iz drugih područja. Cilj našeg istraživanja bio je utvrditi da li OTA u bubregu pokusnih životinja uzrokuje apoptozu ili nekrozu. U prvom pokusu štakori su bili tretirani jednokratnom dozom OTA (1,00 mg/kg tj. t., ip.) te su žrtvovani u razdoblju od jedan do 9 dana. Najveći broj apoptotskih stanica nađen je u životinja koje su žrtvovane jedan dan nakon tretmana, njihov se broj postepeno smanjivao te u životinja koje su žrtvovane 9 dana nakon tretmana nisu nađene apoptotske stanice. Ovisnost nastanka histoloških promjena u bubregu štakora o dozi ispitana je u supkroničnom pokusu primjenom triju doza OTA (0,25, 0,50 i 1,00 mg/kg tj. t., ip.) tijekom 4 tjedna (3 puta na tjedan). Koncentracija OTA i nastanak apoptotskih stanica bili su ovisni o primijenjenoj dozi, a u histološkim preparatima nije nađena nekroza. Ovisnost nastanka apoptotskih stanica u bubregu o duljini tretmana s OTA ispitana je na štakorima koji su tretirani višekratno s OTA (0,50 mg/kg tj. t., ip.) i žrtvovani jedan dan nakon prvog, trećeg, šestog, devetog i dvanaestog tretmana, odnosno 6. i 21. dan nakon dvanaestog tretmana. Ovim je pokusom dokazano da je nastanak apoptotskih stanica u bubregu štakora povezan s duljinom tretmana s OTA

Retrospektivna procjena učinka nintedaniba u mišjem modelu plućne fibroze - usporedba učinkovitosti s kliničkim ishodom idiopatske plućne fibroze

Pulmonary function tests (PFTs) routinely implemented in clinics are the first step in the diagnosis of idiopathic pulmonary fibrosis. Evaluation of PFTs in the mouse model of pulmonary fibrosis accompanied by histological readouts may improve the clinical predictability of new therapeutic candidates. Forced vital capacity (FVC) is considered the most predictive of restrictive pulmonary disorders. This study aimed to test the improvement of PFT in mice lung fibrosis induced by treatment with an approved substance nintedanib, considered the gold standard. The hypothesis that treatment in animal models will demonstrate similar effects as in humans in the most relevant clinical outcomes was tested. Two experimental designs were enrolled in this study, a preventive regimen, with treatment initiation from the day of the challenge; and a therapeutic regimen, starting on day 7 postchallenge when fibrotic changes are present in the lungs. Experiments were terminated at two different time points, at 14 and 21 days postchallenge. C57BL/6 mice were administered with bleomycin (BLM) intranasally and treated with nintedanib from day 0 to day 14 or from day 7 until day 21. Fourteen or 21 days after the BLM challenge, PFTs were assessed using the in vivo invasive lung function measurement system Buxco® Pulmonary Function Test (PFT) (DSI™, New Brighton, USA). Histological evaluation was performed as a modified Ashcroft score. The bleomycin challenge induced a significant decrease of FVC in both experiments. However, nintedanib treatment given in both regimens significantly improved lung functionality. These findings were confirmed with histological analysis of the Ashcroft scoring system, modified by Matsuse. In conclusion, a good correlation between functional test parameters and the clinical effect of nintedanib was shown in both experiments: the preventive regimen was sampled 14 days post-challenge and the therapeutic regimen 21 days post-challenge. Based on these findings, the implementation of PFTs could be a good platform to increase the translational value of the model and potential new treatments.Testovi funkcije pluća prvi su klinički postupak u dijagnostici respiratornih bolesti kao što je idiopatska plućna fibroza (IPF). Mišji modeli plućne fibroze su vrlo važni u razvoju novih terapija. Uvođenje testova u životinjski model, zajedno s histološkom procjenom omogućit će bolji probir novih molekula i njihovo daljnje kliničko istraživanje. Cilj je ovog istraživanja bio odrediti precizne vrijednosti parametara plućnih funkcija korištenjem standardne humane terapije za IPF, nintedaniba na mišjim modelima u svrhu postavljanja platforme za učinkovitiji razvoj novih potencijalnih terapija. Testiranje je vršeno pod pretpostavkom da će odabrani tretman u mišjem modelu dati sličan efekt kao i kod ljudi u odabranoj dijagnostičkoj metodi. Testirana su dva eksperimentalna protokola: preventivnouvođenje terapije prije razvoja fibrotičnih promjena i terapijski protokol aplikacije terapije u vrijeme nastanka plućne fibroze. C57Bl/6 miševima je bleomicin apliciran intranazalno prvog dana studije, a terapija nintedanibom primijenjena je od prvog dana do 14. u preventivnom te od sedmog dana do 21. u terapijskom protokolu. Četrnaestog ili Retrospektivna procjena učinka nintedaniba u mišjem modelu plućne fibroze - usporedba učinkovitosti s kliničkim ishodom idiopatske plućne fibroze 21. dana nakon početka pokusa nakon bleomicinske primjene, testovi funkcije pluća su provedeni koristeći Buxco® Pulmonary Function Test (PFT) (DSI™, New Brighton, SAD) u in vivo invazivni sustav. Histološka procjena je provedena koristeći modificirani Ashcroft sustav ocjenjivanja količine patoloških promjena u plućima. Primjena bleomicina u plućima miševa je utjecala na značajno smanjenje parametra forsiranog vitalnog kapaciteta (FVC) u oba ispitana protokola, dok je terapija nintedanibom značajno poboljšala nastale promjene. Ovi rezultati su potvrđeni i ocjenom histoloških promjena u tkivu pluća. Testovi funkcionalnosti pluća u mišjem modelu značajno koreliraju s kliničkim efektom istraživane terapije u obje studije. U preventivnom protokolu istraženom 14. dana te u terapijskom 21. dana eksperimenta. Na temelju ovih saznanja, možemo zaključiti kako uvođenje ovog testa, kao relevantne kliničke dijagnostike, možemo poboljšati translacijsku vrijednost životinjskih modela u razvoju nove terapije

Comparison of systemic inflammatory and hematology parameters in normal C57BI/6 and genetically diabetic db/db mice during local wound repair

Uvod: Upala je početni odgovor domaćina na ozljedu. Ona nije ograničena samo na mjesto rane, nego izaziva sustavne promjene uključujući raznovrsne fiziološke i biokemijske promjene koje se skupno nazivaju odgovorom akutne faze. Ove se promjene nastavljaju tijekom rješavanja upale i procesa cijeljenja rane. U ovom ispitivanju smo usporedili serumski amiloid A protein (SAA), hematološke parametre (ukupna bijela krvna slika, postotak neutrofila i lim-focita) te koncentracije interferona-gama (IFN-γ) u serumu tijekom cijeljenja neokludirane, ekscizijske kožne rane u punoj debljini kod genetski dijabetičnih db/db miševa i nedijabetičnih C57Bl/6 miševa iz istoga legla. Materijal i metode: Područje rane izazvane „punch" biopsijom (promjera 8 mm) kod svakog je miša analizirano planimetrijski uz računalnu potporu. Trećeg, 6., 9. i 13. dana od ranjavanja SAA i IFN-g mjereni su u plazmi testovima ELISA, a hematološki parametri u punoj krvi na automatskom hematološkom analizatoru Sysmex SF 3000. Rezultati: Šestog i devetog dana jasno je zabilježeno kašnjenje u zatvaranju rane kod db/db miševa u usporedbi sa zdravim miševima. Ukupna bijela krvna slika bila je značajno viša u db/db miševa 9. i 13. dana. Kroz čitavo razdoblje obnove rane, diferencijalni broj neutrofila bio je viši, a broj limfocita niži kod db/db miševa u usporedbi s C57Bl/6 miševima. Vršne koncentracije SAA zabilježene su 3. dana kod C57Bl/6 miševa i db/db miševa (368,7 mg/L odnosno 173,5 mg/L), s težnjom prema nižim vrijednostima kod db/db miševa. Razine IFN-γ bile su značajno više (P < 0,05) 9. i 13. dana kod db/db miševa (75,3 pg/mLodnosno 89,9 pg/mL) u usporedbi s razinama kod C57Bl/6 miševa (66,6 pg/mL odnosno 57,2 pg/mL). Zaljučak: Lokalni proces tkivne regeneracije kod miševa nakon lokalne kožne ozljede uzrokuje sustavne promjene u perifernoj krvi. Niti određivanje koncentracije SAA niti IFN-γ nije se moglo rabiti za motrenje dinamike cijeljenja rane u ovim vremenskim točkama.Introduction: Inflammation is the initial host response to injury. It is not only localized to the wound site but also causes systemic changes, including a variety of physiological and biochemical changes collectively called the acute phase response. These changes continue during the resolution of inflammation and the wound healing process. In this study we compared serum amyloid A protein (SAA), hematological parameters (total white blood cell count, neutrophil and lymphocyte percentage) and interferon-gamma (IFN-γ) concentrations in serum during healing of non-occluded, excisional, full-thickness dermal wounds in genetically diabetic db/db mice and non-diabetic C57Bl/6 littermates. Materials and Methods: Area of a punch biopsy (8 mm in diameter) wound in each mouse was analyzed by computer-assisted planimetry. On days 3,6, 9 and 13 after wounding, SAA and IFN-γ were measured in plasma by ELISA assays and hematological parameters in whole blood by SysmexSF 3000 automatic hematology analyzer. Results: A delay in the closure of wounds in db/db in comparison to normal mice was clearly seen on days 6 and 9. Total white blood cell count was significantly higher on days 9 and 13 in db/db mice. Differential neutrophil counts were higher and lymphocyte counts lower in db/db mice in comparison to C57BL/6 mice throughout the wound repair period. Peak SAA concentrations were seen on day 3 in C57Bl/6 and db/db mice (368.7 mg/L and 173.5 mg/L, respectively), but tended to be lower in db/db mice. IFN-γ levels were significantly higher (P < 0.05) on days 9 and 13 in db/db (75.3 pg/mL and 89.9 pg/mL, respectively) in comparison to those in C57Bl/6 mice (66.6 pg/mL and 57.2 pg/mL, respectively). Conclusion. The local tissue regeneration process in mice after local skin injury causes systemic changes in peripheral blood. Determination of neither SAA nor IFN-γ concentrations could be used to monitor wound healing dynamics at these time points

Ekstracelularni matriks kod primarnih glomerulonefritisa

Aim: The extracellular matrix is a complex superstructure surrounding cells composed of various proteins. In the kidney, ECM proteins are part of basement membranes, glomerular mesangium, interstitium and vessel walls. Their role is not only mechanical support of the cells, but also modification of cell phenotype and mediation of cellular response. The composition of glomerular and interstitial extracellular matrix (ECM) in glomerulonephritis (GN) is changing. Chronic renal insufficiency develops only in those cases of glomerulonephritis in which the lumina of postglomerular capillaries in the renal cortex are narrowed due to expansion of extracellular matrix (ECM).The aim of this study was to elucidate the distribution of extracellular matrix proteins in various forms of primary GN and to compare the composition of interstitial ECM proteins with clinical data in various forms of primary glomerulonephritis (GN). Methods: We examined renal biopsies from 55 GN patients, while normal renal tissue was obtained from nephrectomy due to small renal tumour. Specimens fixed in B5 and/or Dubosque fixative were analysed by immunohistochemistry (APPAP-method) using a monoclonal antibodies against tenascin (TN), fibronectin (FN), collagen I, III and IV (COI, COIII, COIV) (DAKO). The same fixatives were used for controle renal tissue. Clinical data were: proteinuria and creatinine level, obtained by standard laboratory methods, immunotherapy and ACE-genotype. Results: In normal renal tissue glomerular mesangium is TN, FN and weakly COI positive. Glomerular and tubular basal membrane stained with COIV antibody. FN is found around TBM. There was a weak COI expression in interstitium. Intensive TN staining was found in medulla interstitium only. The wall of larger blood vessels was TN, FN and COI positive. Irrespectably of histological form of GN foci of glomerular sclerosis were TN, FN, COI, COIII and COIV positive. In focal glomerulosclerosis mesangium, apart from sclerotic foci, was FN negative. There was simetimes de novo COIII and COIV staining of mesangium in GN with mesangial proliferation. Fibrous cresscents showed TN, FN, COI, COIII and COIV staining. Thickened Bowman’s capsule expressed COI, COIV and TN. At sites of interstitial inflammation there was a de novo TN expression. At sites of interstitial fibrosis FN and COIV were invariably present. TN was found at sites of interstitial fibrosis in 20 of 33 cases with interstitial fibrosis, mainly in biopsies with concomitant interstitial inflammation. COIII staining was found at sites of fibrosis only in 5 biopsies. COI was expressed more intesively in interstitium when inflammation or fibrosis were present. Fifty patients had proteinuria at time of biopsy and 33 of them continued to have one after follow up period. Patients with persistent proteinuria had more often interstitial fibrosis with elevated content of COI and COIV. Presence of fibrin/fibrinogen deposits in interstium of patients with nephrotic proteinuria is negative prognostic factor (p=0,035). Those patients did not respond well to therapy. There were 12 patients with elevated creatinine at biopsy with similar composition of ECM as patients with proteinuria. Majority of patients with interstitial fibrosis had ACE-genotyp II although this genotype wasn’t the most common in the cochort. Conclusions: Our study showed that persistant proteinuria could be linked to interstitial fibrosis and fibrin deposits

Claudins: Beyond Tight Junctions in Human IBD and Murine Models

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well ; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations ; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study