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

Unprecedented Epimerization of an Azithromycin Analogue: Synthesis, Structure and Biological Activity of 2′-Dehydroxy-5″-Epi-Azithromycin

Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton–McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities

Macrolactonolides: a novel class of anti-inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described

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