Iron accumulation has been implicated in degenerative retinal diseases. It can catalyze the production of damaging reactive oxygen species. Previous work has demonstrated iron accumulation in multiple retinal diseases, including age-related macular degeneration and diabetic retinopathy. In mice, systemic knockout of the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph), which oxidize iron, results in retinal iron accumulation and iron-induced degeneration. To determine the role of Heph in the retina, we generated a neural retina-specific Heph knockout on a background of systemic Cp knockout. This resulted in elevated neural retina iron. Conversely, retinal ganglion cells had elevated transferrin receptor and decreased ferritin, suggesting diminished iron levels. The retinal degeneration observed in systemic Cp-/-, Heph-/- mice did not occur. These findings indicate that Heph has a local role in regulating neural retina iron homeostasis, but also suggest that preserved Heph function in either the RPE or systemically mitigates the degeneration phenotype observed in the systemic Cp-/-, Heph-/- mice.

• MeSH
• ceruloplasmin genetika   metabolismus   MeSH
• homeostáza MeSH
• makulární degenerace * genetika   MeSH
• membránové proteiny * genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• retina metabolismus   MeSH
• železo 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

Iron-induced oxidative stress can cause or exacerbate retinal degenerative diseases. Retinal iron overload has been reported in several mouse disease models with systemic or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp and Heph can potentiate ferroportin (Fpn) mediated cellular iron export. Here, we used retina-specific Fpn KO mice to test the hypothesis that retinal iron overload in Cp/Heph DKO mice is caused by impaired iron export from neurons and glia. Surprisingly, there was no indication of retinal iron overload in retina-specific Fpn KO mice: the mRNA levels of transferrin receptor in the retina were not altered at 7-10-months age. Consistent with this, levels and localization of ferritin light chain were unchanged. To "stress the system", we injected iron intraperitoneally into Fpn KO mice with or without Cp KO. Only mice with both retina-specific Fpn KO and Cp KO had modestly elevated retinal iron levels. These results suggest that impaired iron export through Fpn is not sufficient to explain the retinal iron overload in Cp/Heph DKO mice. An increase in the levels of retinal ferrous iron caused by the absence of these ferroxidases, followed by uptake into cells by ferrous iron importers, is most likely necessary.

• MeSH
• ceruloplasmin genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• přetížení železem * MeSH
• proteiny přenášející kationty * genetika   MeSH
• retina metabolismus   MeSH
• železo 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

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.

• MeSH
• dědičné dystrofie rohovky diagnóza   genetika   metabolismus   MeSH
• dítě MeSH
• DNA genetika   MeSH
• dospělí MeSH
• exony MeSH
• heterozygot MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• sekvence nukleotidů MeSH
• sekvenční delece MeSH
• senioři MeSH
• transkripční faktor Zeb1 genetika   metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zinkové prsty MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.

• MeSH
• biokompatibilní potahované materiály MeSH
• biologické markery metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné kultury MeSH
• dárci tkání MeSH
• dospělí MeSH
• fibrin farmakologie   MeSH
• kultivační média MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae cytologie   metabolismus   MeSH
• počet buněk MeSH
• podkladové buňky MeSH
• proliferace buněk fyziologie   MeSH
• rohovkový epitel cytologie   metabolismus   MeSH
• senioři MeSH
• sérum * MeSH
• trombocyty * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

• MeSH
• biopsie MeSH
• imunohistochemie MeSH
• keratiny biosyntéza   genetika   MeSH
• kultivační média MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae metabolismus   ultrastruktura   MeSH
• nemoci rohovky genetika   patologie   chirurgie   MeSH
• regulace genové exprese * MeSH
• RNA genetika   MeSH
• rohovkový epitel metabolismus   ultrastruktura   MeSH
• senioři MeSH
• transmisní elektronová mikroskopie MeSH
• transplantace rohovky * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

In this study we tried to develop a new approach to suppress inflammation and neovascularization in the alkali-injured rabbit cornea. For this reason Cyclosporine A (CsA)-loaded electrospun nanofibers were transferred onto the ocular surface injured with alkali (0.25 N NaOH). Damaged corneas were divided into the following groups: untreated, treated with CsA eye drops, treated with nanofibers drug-free and treated with CsA-loaded nanofibers. Healthy rabbit corneas served as controls. Drug-free nanofibers and CsA-loaded nanofibers were transferred onto the damaged corneal surface immediately after the injury and sutured to conjunctiva. On day five after the injury the nanofibers were removed. The animals from all groups were sacrificed on day twelve after the injury. The extent of the inflammatory reaction and corneal healing were examined macroscopically, immunohistochemically and biochemically. The central corneal thickness was measured using an ultrasonic pachymeter. When compared with untreated injured corneas, injured corneas treated with drug-free nanofibers or injured corneas treated with CsA eye drops, the number of CD3-positive cells (T lymphocytes) and the production of pro-inflammatory cytokines were strongly reduced in corneas treated with CsA-loaded nanofibers, which was associated with the significantly decreased expression of matrix metalloproteinase 9, inducible nitric oxide synthase, vascular endothelial growth factor and active caspase-3. CsA-loaded nanofibers effectively suppressed corneal inflammation and corneal neovascularization. Central corneal thickness restored to levels before injury only in corneas treated with CsA-loaded nanofibers. Corneal transparency was highly restored in these corneas. It is suggested that the beneficial effect of CsA-loaded nanofibers was associated with the continuous release of CsA from nanofibers and continuous affection of damaged cornea by CsA. The suture of nanofibers to conjunctiva and the closed eyes contributed to beneficial corneal healing. This is in contrast to CsA eye drops, which are quickly washed from the ocular surface and the contact of CsA with the damaged cornea was limited. In conclusion, the approach with CsA-loaded nanofibers could represent an effective alternative mode of therapy for corneal chemical burns.

• MeSH
• alkálie škodlivé účinky   MeSH
• chemické popálení farmakoterapie   MeSH
• cyklosporin aplikace a dávkování   terapeutické užití   MeSH
• imunohistochemie MeSH
• imunosupresiva terapeutické užití   MeSH
• kaspasa 3 metabolismus   MeSH
• králíci MeSH
• modely nemocí na zvířatech MeSH
• nanovlákna MeSH
• neovaskularizace rohovky patologie   MeSH
• popálení oka farmakoterapie   metabolismus   patologie   MeSH
• poranění rohovky chemicky indukované   metabolismus   patologie   MeSH
• T-lymfocyty patologie   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The purpose of this study was to investigate whether rabbit bone marrow-derived mesenchymal stem cells (MSCs) effectively decrease alkali-induced oxidative stress in the rabbit cornea. The alkali (0.15 N NaOH) was applied on the corneas of the right eyes and then rinsed with tap water. In the first group of rabbits the injured corneas remained untreated. In the second group MSCs were applied on the injured corneal surface immediately after the injury and eyelids sutured for two days. Then the sutures were removed. In the third group nanofiber scaffolds seeded with MSCs (and in the fourth group nanofibers alone) were transferred onto the corneas immediately after the injury and the eyelids sutured. Two days later the eyelid sutures were removed together with the nanofiber scaffolds. The rabbits were sacrificed on days four, ten or fifteen after the injury, and the corneas were examined immunohistochemically, morphologically, for the central corneal thickness (taken as an index of corneal hydration) using an ultrasonic pachymeter and by real-time PCR. Results show that in untreated injured corneas the expression of malondialdehyde (MDA) and nitrotyrosine (NT) (important markers of lipid peroxidation and oxidative stress) appeared in the epithelium. The antioxidant aldehyde dehydrogenase 3A1 (ALDH3A1) decreased in the corneal epithelium, particularly in superficial parts, where apoptotic cell death (detected by active caspase-3) was high. (In control corneal epithelium MDA and NT are absent and ALDH3A1 highly present in all layers of the epithelium. Cell apoptosis are sporadic). In injured untreated cornea further corneal disturbances developed: The expressions of matrix metalloproteinase 9 (MMP9) and proinflammatory cytokines, were high. At the end of experiment (on day 15) the injured untreated corneas were vascularized and numerous inflammatory cells were present in the corneal stroma. Vascular endothelial growth factor (VEGF) expression and number of macrophages were high. The results obtained in injured corneas covered with nanofiber scaffolds alone (without MSCs) or in injured corneas treated with MSCs only (transferred without scaffolds) did not significantly differ from the results found in untreated injured corneas. In contrast, in the injured corneas treated with MSCs on nanofiber scaffolds, ALDH3A1 expression remained high in the epithelium (as in the control cornea) and positive expression of the other immunohistochemical markers employed was very low (MMP9) or absent (NT, MDA, proinflammatory cytokines), also similarly as in the control cornea. Corneal neovascularization and the infiltration of the corneal stroma with inflammatory cells were significantly suppressed in the injured corneas treated with MSCs compared to the untreated injured ones. The increased central corneal thickness together with corneal opalescency appearing after alkali injury returned to normal levels over the course of ten days only in the injured corneas treated with MSCs on nanofiber scaffolds. The expression of genes for the proinflammatory cytokines corresponded with their immunohistochemical expression. In conclusion, MSCs on nanofiber scaffolds protected the formation of toxic peroxynitrite (detected by NT residues), lowered apoptotic cell death and decreased matrix metalloproteinase and pro-inflammatory cytokine production. This resulted in reduced corneal inflammation as well as neovascularization and significantly accelerated corneal healing.

• MeSH
• alkálie toxicita   MeSH
• chemické popálení patologie   chirurgie   MeSH
• hojení ran MeSH
• králíci MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• modely nemocí na zvířatech MeSH
• nanovlákna terapeutické užití   MeSH
• oxidační stres * MeSH
• poranění rohovky MeSH
• rohovka patologie   chirurgie   MeSH
• tkáňové podpůrné struktury * MeSH
• transplantace mezenchymálních kmenových buněk metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/μg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/μg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

• MeSH
• dospělí MeSH
• fluorescenční protilátková technika nepřímá MeSH
• keratokonus enzymologie   patologie   chirurgie   MeSH
• konjunktiva enzymologie   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae enzymologie   MeSH
• lysyloxidasa metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• oxidoreduktasy aminokyselin metabolismus   MeSH
• rohovka enzymologie   MeSH
• rohovkové keratocyty enzymologie   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The goal of our study was to determine whether proteins typical of the human mesothelial cell phenotype, such as mesothelin, HBME-1 (Hector Battifora mesothelial cell-1) protein and calbindin 2, are expressed in the human cornea, especially in endothelial cells. Cryosections and endothelial and epithelial imprints of sixteen human cadaverous corneoscleral discs were used. The presence of proteins was examined using immunohistochemistry and Western blotting, while mRNA levels were determined by qRT-PCR. A strong signal for mesothelin was present in the corneal epithelium, while less intense staining was visible in the endothelium. Similarly, higher and lower mRNA levels were detected using qRT-PCR in the corneal epithelium and endothelium, respectively. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Marked positivity was present in the corneal stromal extracellular matrix, while no staining was present in the sclera. Calbindin 2 was detected using immunohistochemistry and Western blotting in the corneal epithelium, endothelium and stroma. qRT-PCR confirmed its expression in epithelial and endothelial cells. Three proteins expressed constitutively in mesothelial cells were detected in the human cornea. The possible function of mesothelin in cell-cell contact on the ocular surface is discussed. The presence of HBME-1 protein in the endothelial layer may indicate a still unknown function that could be shared with mesothelial cells of the pleura and peritoneum. The much more pronounced occurrence of calbindin 2 in the corneal epithelium compared to fewer positive endothelial cells explains the higher turnover of epithelial cells compared to the proliferatively inactive endothelium.

• MeSH
• fluorescenční protilátková technika nepřímá MeSH
• GPI-vázané proteiny MeSH
• imunoenzymatické techniky MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• oční proteiny metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• rohovkový endotel metabolismus   MeSH
• rohovkový epitel metabolismus   MeSH
• S100 kalcium vázající protein G genetika   metabolismus   MeSH
• stroma rohovky metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to compare the presence and activity of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9 and 13 in human melted and cadaverous corneas. Twelve melted corneal specimens from three patients with rheumatoid arthritis, one patient with ocular cicatricial pemphigoid and one patient with melting attributed to spastic entropion and ten control corneal buttons were used. The presence of MMPs was detected using indirect enzyme immunohistochemistry. The active forms of MMP-2 and -9 and MMP-3 and -7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP-1 and -3 were measured using activity assays. Increased immunostaining intensity for MMP-1 and -9 was seen in the corneal epithelium and the anterior stroma of all, and for MMP-2, -3, -7 and -8 of almost all, melted corneas compared to the negative or slightly positive staining of the controls. The posterior stroma showed the presence of MMP-1, -2, -3 and -9 in almost all and of MMP-7 and -8 in half of all melted specimens. A markedly higher level of active MMP-2 was detected in six and active MMP-9 in all of eleven pathologic specimens compared to control specimens, using gelatin zymography. The proenzymes of MMP-3 and -7 and the MMP-7 intermediate cleavage product were detected only in melted corneas using casein zymography. Significantly increased MMP-1 and -3 activity was also found in the melted corneas using activity assays. The markedly increased immunostaining for MMP-1, -2, -3, -7, -8 and -9 as well as the elevated levels of the active forms of MMP-1, -2, -3 and -9 in melted corneal specimens from patients with various diagnoses suggest that although different stimuli may trigger the pathways that lead to the destruction of the extracellular matrix, these enzymes could play a subsequent role in this process.

• MeSH
• benigní pemfigoid sliznice komplikace   MeSH
• dárci tkání MeSH
• dospělí MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• imunoenzymatické techniky MeSH
• keratoplastika perforující MeSH
• lidé středního věku MeSH
• lidé MeSH
• matrixové metaloproteinasy fyziologie   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nemoci rohovky enzymologie   chirurgie   MeSH
• recidiva MeSH
• revmatoidní artritida komplikace   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH