The cellular adhesion receptor αvβ6-integrin is highly expressed in many cancers, e.g., pancreatic, lung, head-and-neck, cervical, bladder, and esophageal carcinoma. Multimerization of αvβ6-integrin-specific RGD peptides increases the target affinity and retention but affects biodistribution and pharmacokinetics. Amide formation of the terminal carboxylic acid moieties of the square-symmetrical bifunctional chelator DOTPI with 3-azidopropylamine yields derivatives with 4, 3, and 2 terminal azides and zero, 1, and 2 remaining carboxylic acids, respectively, whereby formation of the 2-cis-isomer is preferred according to NMR investigation of the Eu(III)-complexes. Cu(II)-catalyzed alkyne-azide cycloaddition (CuAAC) of the alkyne-functionalized αvβ6-integrin binding peptide cyclo[YRGDLAYp(NMe)K(pent-4-ynoic amide)] (Tyr2) yields the respective di-, tri-, and tetrameric conjugates for Lu-177-labeling. In mice bearing αvβ6-integrin-expressing xenografts of H2009 (human lung adenocarcinoma) cells, the Lu-177-labeled trimer's tumor-to-blood ratio of 112 exceeds that of the tetramer (10.4) and the dimer (54). Co-infusion of gelofusine (succinylated gelatin) reduces the renal uptake of the trimer by 89%, resulting in a 10-fold better tumor-to-kidney ratio, while no improvement of that ratio is observed with arginine/lysine, para-aminohippuric acid (PAH), and hydroxyethyl starch (HES) coinfusions. Since the Lu-177-labeled Tyr2-trimer outperforms the dimer and the tetramer, such trimers are considered the best lead structures for the ongoing development of αvβ6-integrin targeted anticancer theranostics.
- MeSH
- antigeny nádorové * metabolismus MeSH
- chelátory * chemie MeSH
- click chemie MeSH
- integriny * metabolismus MeSH
- lidé MeSH
- lutecium * chemie MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie MeSH
- oligopeptidy * chemie farmakokinetika MeSH
- radiofarmaka farmakokinetika chemie terapeutické užití MeSH
- radionuklidy * chemie MeSH
- teranostická nanomedicína metody MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.
- MeSH
- buněčná adheze * účinky léků MeSH
- buněčná smrt účinky léků MeSH
- buňky Hep G2 MeSH
- integrin alfa2beta1 metabolismus MeSH
- integriny metabolismus genetika MeSH
- kolagen typu I * metabolismus genetika MeSH
- lidé MeSH
- nealkoholová steatóza jater metabolismus patologie MeSH
- palmitany toxicita farmakologie MeSH
- proliferace buněk * účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- viabilita buněk * účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Východiska: Triple-negativní karcinomy prsu (TNBC) jsou heterogenní skupinou nádorů s převážně agresivním chováním a špatnou prognózou. V souvislosti s jejich agresivním chováním a chemorezistencí vůči léčbě se do popředí dostal koncept epitelo-mezenchymové tranzice (EMT). Proteiny CD9 a CD29 jsou spojeny s EMT a mohou hrát roli v progresi TNBC. Naším cílem bylo prozkoumat asociaci těchto markerů s metastázami do lymfatických uzlin, gradingem tumoru, proliferační aktivitou a přežitím pacientů. Pacienti a metody: Náš soubor tvořilo 66 pacientek s TNBC bez neoadjuvantní terapie ve věku 26–81 let. Patologické stadium nádoru se pohybovalo od pT1b do pT3 a histologický stupeň od II do III podle systému Bloom-Richardson. Imunohistochemické hodnocení exprese CD9, CD29, E-cadherinu, vimentinu, androgenového receptoru a Ki-67 bylo provedeno semikvantitativně pomocí H-skóre. Exprese proteinů byla statisticky hodnocena ve vztahu ke klinicko-patologickým parametrům a přežití pacientů. Výsledky: Pozorovali jsme nižší expresi CD9 v metastázách lymfatických uzlin ve srovnání s primárním nádorem (p = 0,021). Exprese CD29 v primárním nádoru byla signifikantně nižší u pacientů s metastázami v lymfatických uzlinách ve srovnání s pacienty bez diseminace (p = 0,03). Ani exprese CD9 ani CD29 proteinu nebyla spojena s přežitím specifickým pro karcinom prsu (BCSS). Nižší exprese E-cadherinu na periferii primárního tumoru byla spojena s horším BCSS (p = 0,038). Pro grading ani přítomnost metastáz v lymfatických uzlinách nebyl nalezen signifikantní vztah s BCSS. Nižší exprese E-cadherinu na periferii byla také spojena s vyšší hladinou Ki67 (Rs −0,26) a vimentinu (Rs −0,33). Závěr: Snížená exprese proteinů CD9 a CD29 byla spojena s růstem metastáz v lymfatických uzlinách, avšak jejich souvislost s přežitím nebyla prokázána. Nižší exprese E-cadherinu na periferii primárního nádoru byla spojena s vysokou proliferací a špatným nádorově specifickým přežitím.
Background: Triple-negative breast carcinomas (TNBC) are a heterogeneous group of tumors with mostly aggressive behaviour and poor prognosis. In association with their aggressive behavior and chemoresistance to treatment, the concept of epithelial-mesenchymal transition (EMT) has come to the fore. CD9 and CD29 proteins are associated with EMT and may play a role in TNBC progression. Our aim was to investigate association of these markers with the lymph node metastasis, tumor grade, proliferative activity, and patient survival. Patients and methods: Our cohort consisted of 66 TNBC patients without neoadjuvant therapy, aged 26–81 years. The pathological tumor stages ranged from pT1b to pT3 and histological grades ranged from II to III, according to the Bloom-Richardson system. Immunohistochemical evaluation of CD9, CD29, E-cadherin, vimentin, androgen receptor and Ki-67 expression was performed semiquantitatively using the H-score. Expression of the proteins was statistically evaluated in relation to the clinicopathological parameters and survival of the patients. Results: We observed lower expression of CD9 in lymph node metastases compared to the primary tumor (P = 0.021). The CD29 expression in primary tumor was significantly lower in patients with lymph node metastases compared to patients without cancer dissemination (P = 0.03). Neither CD9 nor CD29 protein expression was associated with breast cancer-specific survival (BCSS). Lower expression of E-cadherin at the periphery of the primary tumor was associated with worse BCSS (P = 0.038). Neither grade nor the presence of lymph node metastases reached significant association with the BCSS. Lower expression of E-cadherin at the periphery was also associated with higher Ki67 (Rs −0.26) and vimentin (Rs −0.33). Conclusion: Decreased protein expression of CD9 and CD29 were associated with lymph node metastasis growth, however, their association with survival was not proved. Lower expression of E-cadherin at the periphery of the primary tumor was associated with high proliferation and poor breast cancer-specific survival.
- MeSH
- antigeny CD29 analýza MeSH
- antigeny CD9 analýza MeSH
- epitelo-mezenchymální tranzice imunologie MeSH
- imunohistochemie * metody MeSH
- kadheriny analýza MeSH
- kohortové studie MeSH
- lidé MeSH
- lymfatické metastázy diagnostické zobrazování imunologie MeSH
- prognóza MeSH
- triple-negativní karcinom prsu * diagnostické zobrazování imunologie sekundární MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs.
The fungal pathogen Candida albicans is linked to chronic brain diseases such as Alzheimer's disease (AD), but the molecular basis of brain anti-Candida immunity remains unknown. We show that C. albicans enters the mouse brain from the blood and induces two neuroimmune sensing mechanisms involving secreted aspartic proteinases (Saps) and candidalysin. Saps disrupt tight junction proteins of the blood-brain barrier (BBB) to permit fungal brain invasion. Saps also hydrolyze amyloid precursor protein (APP) into amyloid β (Aβ)-like peptides that bind to Toll-like receptor 4 (TLR4) and promote fungal killing in vitro while candidalysin engages the integrin CD11b (Mac-1) on microglia. Recognition of Aβ-like peptides and candidalysin promotes fungal clearance from the brain, and disruption of candidalysin recognition through CD11b markedly prolongs C. albicans cerebral mycosis. Thus, C. albicans is cleared from the brain through innate immune mechanisms involving Saps, Aβ, candidalysin, and CD11b.
- MeSH
- Alzheimerova nemoc metabolismus mikrobiologie MeSH
- amyloidní beta-protein metabolismus MeSH
- antigeny CD11b * metabolismus MeSH
- Candida albicans metabolismus MeSH
- fungální proteiny metabolismus MeSH
- mikroglie * metabolismus mikrobiologie MeSH
- mykózy * genetika metabolismus MeSH
- myši MeSH
- toll-like receptor 4 * metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- MeSH
- humanizované monoklonální protilátky aplikace a dávkování farmakologie klasifikace terapeutické užití MeSH
- injekce subkutánní metody MeSH
- integrin alfa4 antagonisté a inhibitory MeSH
- lidé středního věku MeSH
- lidé MeSH
- natalizumab * aplikace a dávkování farmakologie terapeutické užití MeSH
- nukleární magnetická rezonance biomolekulární klasifikace metody MeSH
- roztroušená skleróza diagnóza farmakoterapie MeSH
- virus JC imunologie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.
- MeSH
- axony * fyziologie MeSH
- integriny metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mícha metabolismus MeSH
- nervové receptory fyziologie MeSH
- poranění míchy * terapie metabolismus MeSH
- potkani Sprague-Dawley MeSH
- regenerace nervu fyziologie MeSH
- spinální ganglia metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm β1 integrin. Males and females with β1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm β1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin β1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.
- MeSH
- antigeny CD29 * genetika metabolismus MeSH
- fertilizace MeSH
- integriny metabolismus MeSH
- interakce spermie a vajíčka * MeSH
- myši MeSH
- oocyty metabolismus MeSH
- sperma metabolismus MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.
- MeSH
- antigeny CD18 MeSH
- bakteriální adheze MeSH
- bakteriální adheziny metabolismus MeSH
- Bordetella pertussis * metabolismus MeSH
- faktory virulence rodu Bordetella MeSH
- glykosaminoglykany MeSH
- hemaglutininy metabolismus MeSH
- heparin MeSH
- integriny MeSH
- lidé MeSH
- makrofágový antigen 1 MeSH
- pertuse * MeSH
- proteasy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
During pancreas development endocrine cells leave the ductal epithelium to form the islets of Langerhans, but the morphogenetic mechanisms are incompletely understood. Here, we identify the Ca2+-independent atypical Synaptotagmin-13 (Syt13) as a key regulator of endocrine cell egression and islet formation. We detect specific upregulation of the Syt13 gene and encoded protein in endocrine precursors and the respective lineage during islet formation. The Syt13 protein is localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells, marking a previously unidentified apical-basal to front-rear repolarization during endocrine precursor cell egression. Knockout of Syt13 impairs endocrine cell egression and skews the α-to-β-cell ratio. Mechanistically, Syt13 is a vesicle trafficking protein, transported via the microtubule cytoskeleton, and interacts with phosphatidylinositol phospholipids for polarized localization. By internalizing a subset of plasma membrane proteins at the front domain, including α6β4 integrins, Syt13 modulates cell-matrix adhesion and allows efficient endocrine cell egression. Altogether, these findings uncover an unexpected role for Syt13 as a morphogenetic driver of endocrinogenesis and islet formation.
