Plasma-derived extracellular vesicle surface markers CD45, CD326 and CD56 correlate with the stage of osteoarthritis: a primary study of a novel and promising diagnostic tool of the disease.

Recently, there is a growing interest in the research based on extracellular vesicles (EVs) which represent paracrine factors secreted by almost all cell types. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and compositions. EVs play an important role in intercellular communication, mechanism and tissue repair. Moreover, EVs could help not only in the treatment of diseases but also in their diagnostics. This work focused on the evaluation of the potential of EVs being used as biomarkers for the diagnosis of osteoarthritis (OA) based on a comparison of the composition of EVs separated from platelet-poor plasma (PPP) of healthy donors and OA patients at different stages of OA. OA is established as a complex syndrome with extensive impact on multiple tissues within the synovial joint. It is a chronic disease of musculoskeletal system that mainly affects the elderly. Depending on the use of the Kellgren-Lawrence classification system, there are four grades of OA which have a negative impact on patients' quality of life. It is very difficult to detect OA in its early stages, so it is necessary to find a new diagnostic method for its timely detection. PPP samples were prepared from whole blood. PPP-EVs were separated from 3 groups of donors-healthy control, early stage OA, end-stage OA, and their content was compared and correlated. EVs from PPP were separated by size exclusion chromatography and characterized in terms of their size, yield and purity by NTA, western blotting, ELISA and flow cytometry. Detection of surface markers expression in EVs was performed using MACSPlex approach. Inflammatory and growth factors in EVs were analysed using MAGPix technology. Our study confirmed significant differences between EVs surface markers of patients and healthy controls correlating with the age of donor (CD63, CD31 and ROR1) and stage of OA (CD45, CD326 and CD56), respectively. Circulating EVs have been under extensive investigation for their capability to predict OA pathology diagnosis as potential targets for biomarker discovery. Taken together, obtained results indicated that PPP-EVs surface markers could be used as potential biomarkers in the early diagnosis of OA.

The Role of Mesenchymal Stromal Cells and Their Products in the Treatment of Injured Spinal Cords.

Spinal cord injury (SCI) is a destructive condition that results in lasting neurological damage resulting in disruption of the connection between the central nervous system and the rest of the body. Currently, there are several approaches in the treatment of a damaged spinal cord; however, none of the methods allow the patient to return to the original full-featured state of life before the injury. Cell transplantation therapies show great potential in the treatment of damaged spinal cords. The most examined type of cells used in SCI research are mesenchymal stromal cells (MSCs). These cells are at the center of interest of scientists because of their unique properties. MSCs regenerate the injured tissue in two ways: (i) they are able to differentiate into some types of cells and so can replace the cells of injured tissue and (ii) they regenerate tissue through their powerful known paracrine effect. This review presents information about SCI and the treatments usually used, aiming at cell therapy using MSCs and their products, among which active biomolecules and extracellular vesicles predominate.

Transcriptomic Analysis of Macrophage Polarization Protocols: Vitamin D3 or IL-4 and IL-13 Do Not Polarize THP-1 Monocytes into Reliable M2 Macrophages.

Two main types of macrophages (Mφ) include inflammatory (M1) and anti-inflammatory (M2) macrophages. These cells can be obtained in vitro by polarization of monocytic cell lines using various stimuli. Since there is currently no consensus on the best method for the acquisition of reliable M1 and M2 macrophages from the THP-1 cell line, we decided to compare three different polarization protocols at the transcriptomic level. Whole transcriptomes of Mφ polarized according to the chosen protocols were analyzed using RNA-seq. Differential expression of genes and functional enrichment for gene ontology terms were assessed. Compared with other protocols, M1 macrophages polarized using PMA (61.3 ng/mL) and IFN-γ along with LPS had the highest expression of M1-associated regulatory genes and genes for M1 cytokines and chemokines. According to the GO enrichment analysis, genes involved in defensive and inflammatory processes were differentially expressed in these Mφ. However, all three chosen protocols which use Vit D3, IL-13/IL-4, and IL-4, respectively, failed to promote the polarization of macrophages with a reliable M2 phenotype. Therefore, optimization or development of a new M2 polarization protocol is needed to achieve macrophages with a reliable anti-inflammatory phenotype.

Mesenchymal Stem Cells in the Treatment of Human Spinal Cord Injury: The Effect on Individual Values of pNF-H, GFAP, S100 Proteins and Selected Growth Factors, Cytokines and Chemokines.

At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers (S100, GFAP, and pNF-H) as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury.

The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis.

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1ß and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs' response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy.

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs' uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.

Effect of Intra-Articular Injection of Platelet-Rich Plasma on the Serum Levels of Osteoarthritic Biomarkers in Patients with Unilateral Knee Osteoarthritis.

BACKGROUND: The aim of this study is to determine the effect of three doses of intra-articular injection of platelet-rich plasma (PRP) into the osteoarthritic (OA) knee joint on the functional status and on the changes in the levels of specific OA biomarkers in blood serum. METHODS: Forty patients with unilateral primary knee osteoarthritis were enrolled in this single center, prospective clinical trial. For each patient, three intra-articular PRP injections were administered one week apart. Clinical and laboratory assessment was performed before the first PRP injection (baseline), and 3 months after the third PRP application (3-month follow up). Pain in the affected knee joint was assessed with the Visual Analog Scale for Pain (VAS). Change in clinical status was evaluated with the Western Ontario and McMaster Universities Arthritis Index Questionnaire (WOMAC). Concentrations of 19 biomarkers (EGF, Eotaxin, FGF-2, GRO, IL-10, IL-1RA, IL-8, IP-10, MCP-1, PDGF-AB/BB, RANTES, MMP-3, MMP-13, Collagen type 2, BMP-2, TIMP-1, TIMP-2, TGF beta 1, and COMP) in the serum of studied patients were quantified. RESULTS: At 3-month follow up, there was a significant decrease in the VAS score and significant improvement in the WOMAC score. There was a significant decrease in the levels of Eotaxin, MCP-1, MMP-1, IL-10, EGF, PDGF-AB/BB, TGF- ß1 compared to baseline levels. A significant increase in markers BMP-2, COMP, Collagen type 2 and GRO was found at the same time point. There was no significant change in the concentrations of other biomarkers (FGF-2, IL-1RA, IL-8, IL-10, MMP-3, RANTES, TIMP-1, TIMP-3). CONCLUSIONS: We found an increase in specific pro-anabolic and anti-inflammatory biomarkers with a concomitant decrease in pro-inflammatory biomarkers at 3 months after three intra-articular applications of PRP. Significant improvement in VAS and WOMAC scores was observed. Treatment with PRP may be an effective therapeutic option with anti-inflammatory and regenerative potential in patients with primary knee OA.

New therapeutic approaches of mesenchymal stem cells-derived exosomes.

Mesenchymal stem cells (MSCs) have been demonstrated to have a great potential in the treatment of several diseases due to their differentiation and immunomodulatory capabilities and their ability to be easily cultured and manipulated. Recent investigations revealed that their therapeutic effect is largely mediated by the secretion of paracrine factors including exosomes. Exosomes reflect biophysical features of MSCs and are considered more effective than MSCs themselves. Alternative approaches based on MSC-derived exosomes can offer appreciable promise in overcoming the limitations and practical challenges observed in cell-based therapy. Furthermore, MSC-derived exosomes may provide a potent therapeutic strategy for various diseases and are promising candidates for cell-based and cell-free regenerative medicine. This review briefly summarizes the development of MSCs as a treatment for human diseases as well as describes our current knowledge about exosomes: their biogenesis and molecular composition, and how they exert their effects on target cells. Particularly, the therapeutic potential of MSC-derived exosomes in experimental models and recent clinical trials to evaluate their safety and efficacy are summarized in this study. Overall, this paper provides a current overview of exosomes as a new cell-free therapeutic agent.

A Preliminary Study of Combined Detection of COMP, TIMP-1, and MMP-3 in Synovial Fluid: Potential Indicators of Osteoarthritis Progression.

OBJECTIVE: Osteoarthritis (OA) commonly affects weight-bearing joints and is characterized by articular cartilage breakdown combined with osteophyte formation at the joint margins and chronic nonspecific inflammation of synovium. Understanding the profile of inflammation in a patient population is an essential starting point to predict or prevent OA progression. The aim of this study was to identify the profile of selected biomolecules in synovial fluid (SF) and investigate the correlation according to gender, age, and severity of the disease within patients from among the general knee OA population. DESIGN: In our study SF samples were aspirated from the knees of 65 OA patients (46 patients with early knee OA and 19 patients with end-stage knee OA according to the Kellgren-Lawrence grading scale). The concentration of interleukins (IL-6, IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-13), MMPs inhibitors (TIMP-1, TIMP-2), cartilage oligomeric matrix protein (COMP), and adiponectin was analyzed using a multiplex ELISA-based approach. CONCLUSIONS: Our results indicate significant linear correlation of MMP-13 and COMP concentration with age (P < 0.05), but not with OA severity. In fact, 3 of the examined biomolecules, MMP-3 (P < 0.01), TIMP-1 (P < 0.01), and COMP (P < 0.05) significantly correlate with the grade of knee OA and might be associated with OA severity.

3D printed Polylactid Acid based porous scaffold for bone tissue engineering: an in vitro study.

PURPOSE: The objective of this study was to fabricate PLA-based porous scaffold by 3D printing technology and to evaluate their cytotoxicity and biocompatibility under in vitro conditions in respect to bone tissue engineering. MATERIAL AND METHODS: Pure PLA in filamentous form was processed via 3D printing technology of fused filament fabrication into porous scaffolds. The structure and porosity of scaffolds were measured by metrotomography. PLA scaffolds were pre-treated by human serum, foetal bovine serum and complete cell culture medium to enhance bio-attractivity of the scaffold's surface for the adherence of the cells. Cells were enzymatically isolated from the periosteum of the proximal tibia and then expanded in monolayer. Periosteum-derived osteoprogenitors (PDOs) were seeded on the pre-treated PLA scaffolds and subsequent cell proliferation was measured by commercially available cell proliferation assay. Adherence of PDOs on the PLA scaffold was confirmed by scanning electron microscopy (SEM). RESULTS: Prepared scaffolds had well-defined structure and were characterized by uniform distribution of pores. They were non-toxic and biocompatible with PDOs, however, PLA scaffold with the periosteum-derived progenitor cells was significantly better in the group of scaffolds pre-treated with normal human serum. CONCLUSIONS: The obtained PLA porous scaffolds favored attachment of periosteum derived progenitors and proliferation, furthermore, cells penetrated into the scaffold through the interstitial pores which was meaningful for cytocompatibility evaluation.

Influence of Kartogenin on Chondrogenic Differentiation of Human Bone Marrow-Derived MSCs in 2D Culture and in Co-Cultivation with OA Osteochondral Explant.

Articular cartilage has limited capacity for natural regeneration and repair. In the present study, we evaluated kartogenin (KGN), a bioactive small heterocyclic molecule, for its effect on in vitro proliferation and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) in monolayer culture and in co-culture models in vitro. OA osteochondral cylinders and hBMSCs were collected during total knee replacement. The effect of KGN on hBMSCs during 21 days of culture was monitored by real-time proliferation assay, immunofluorescence staining, histological assay, scanning electron microscopy (SEM) (imaging and multiplex enzyme-linked immunosorbent assay) ELISA assay. The rate of proliferation of hBMSCs was significantly increased by treatment with 10 µM KGN during nine days of culture. Histological and SEM analyses showed the ability of hBMSCs in the presence of KGN to colonize the surface of OA cartilage and to produce glycosaminoglycans and proteoglycans after 21 days of co-culture. KGN treated hBMSCs secreted higher concentrations of TIMPs and the secretion of pro-inflammatory molecules (MMP 13, TNF-α) were significantly suppressed in comparison with control without hBMSCs. Our preliminary results support the concept that 10 µM KGN enhances proliferation and chondrogenic differentiation of hBMSCs and suggest that KGN is a potential promoter for cell-based therapeutic application for cartilage regeneration.

A Preliminary Study Comparing Microfracture and Local Adherent Transplantation of Autologous Adipose-Derived Stem Cells Followed by Intraarticular Injection of Platelet-Rich Plasma for the Treatment of Chondral Defects in Rabbits.

Objective This study aimed to compare microfracture and application of adipose-derived stem cells (ADSCs) by local adherent technique enhanced by platelet-rich plasma (PRP) to provide a new approach for the repair of cartilage defect. Design Full-thickness cylindrical defects were created in the medial femoral condyle in 9 New Zealand White rabbits (5 months old, 4.65 ± 0.20 kg). Two groups of rabbits ( n = 3) were either treated with ADSCs (Group 1) or the microfracture technique (Group 2) following intraarticular injection of PRP 3 times in weekly intervals. Rabbits in control group ( n = 3) remained untreated. The outcome was assessed macroscopically, histologically, and immunohistochemically. Results At the end of week 12, Group 1 showed better defect filling compared with Group 2. Specimens treated with the combination of ADSCs and PRP exhibited significant differences from the other groups in all criteria of International Cartilage Repair Society macroscopic scoring system. Conclusions Intraarticular injection of autologous PRP in combination with transplantation of autologous ADSCs by local adherent technique enhances the quality of cartilage defect repair with better results in comparison with microfracture surgery in a rabbit model.

Mesenchymal stem cells in the treatment of type 1 diabetes mellitus.

Diabetes mellitus type 1 is a form of diabetes mellitus that results from the autoimmune destruction of insulin-producing beta cells in the pancreas. The current gold standard therapy for pancreas transplantation has limitations because of the long list of waiting patients and the limited supply of donor pancreas. Mesenchymal stem cells (MSCs), a relatively new potential therapy in various fields, have already made their mark in the young field of regenerative medicine. Recent studies have shown that the implantation of MSCs decreases glucose levels through paracrine influences rather than through direct transdifferentiation into insulin-producing cells. Therefore, these cells may use pro-angiogenic and immunomodulatory effects to control diabetes following the cotransplantation with pancreatic islets. In this review, we present and discuss new approaches of using MSCs in the treatment of diabetes mellitus type 1.

Novel trisubstituted acridines as human telomeric quadruplex binding ligands.

A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV-vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the "braco"-like derivatives was to arrest the cells in the S- and G2M-phases of the cell cycle within the first 72h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.

Isolation, cultivation and characterisation of pigeon osteoblasts seeded on xenogeneic demineralised cancellous bone scaffold for bone grafting.

Avian osteoblasts have been isolated particularly from chicken embryo, but data about other functional tissue sources of adult avian osteoblast precursors are missing. The method of preparation of pigeon osteoblasts is described in this study. We demonstrate that pigeon cancellous bone derived osteoblasts have particular proliferative capacity in vitro in comparison to mammalian species and developed endogenous ALP. Calcium deposits formation in vitro was confirmed by alizarin red staining. Only a few studies have attempted to investigate bone grafting and treatment of bone loss in birds. Lack of autologous bone grafts in birds has prompted investigation into the use of avian xenografts for bone augmentation. Here we present a method of xenografting of ostrich demineralised cancellous bone scaffold seeded with allogeneic adult pigeon osteoblasts. Ostrich demineralised cancellous bone scaffold supported proliferation of pigeon osteoblasts during two weeks of co - cultivation in vitro. Scanning electron microscopy demonstrated homogeneous adult pigeon osteoblasts attachment and distribution on the surface of xenogeneic ostrich demineralised cancellous bone. Our preliminary in vitro results indicate that demineralised cancellous bone from ostrich tibia could provide an effective biological support for growth and proliferation of allogeneic osteoblasts derived from cancellous bone of pigeons.

A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 µM BrdU and 10 µM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 µM BrdU+ and 77.6 ± 4.6% 5 µM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 µM BrdU+, and 76.9 ± 0.9% 10 µM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

Impact of different pancreatic microenvironments on improvement in hyperglycemia and insulin deficiency in diabetic rats after transplantation of allogeneic mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) in the pancreatic microenvironment can improve diabetes mellitus (DM). The aim of the present study was to determine whether different pancreatic microenvironments influence the improvement of hyperglycemia and insulin deficiency. METHODS: MSCs isolated from rat bone marrow were transplanted directly into different pancreatic microenvironments in male DM rats. DM was induced in the rats by streptozotocin injection. The rats were divided into 5 groups: normal control rats, DM control rats, and 3 experimental groups (DM rats plus MSCs injected into the head of the pancreas, the tail of the pancreas, or the whole pancreas). The body weight and blood glucose of the rats were monitored during the experiment after transplantation of the MSCs. Histopathologic and immunohistochemical analyses were used to detect the presence and number of islets and insulin production in the pancreatic tissue of the rats after MSC transplantation. RESULTS: At 28 days after MSC transplantation, we observed a statistically significant decrease in the blood glucose level and an increase in weight in DM rats compared with DM control rats (P < 0.0001 and P < 0.03, respectively). A comparison of each of the DM rat groups treated with MSCs showed no significant differences in the blood glucose levels or body weight. CONCLUSION: Our results suggest that transplantation of MSCs could improve DM in the pancreatic microenvironment in an animal model with streptozotocin-induced DM. The different pancreatic areas into which the MSCs were implanted had no significant influence on the improvement in hyperglycemia and insulin deficiency.

Treatment of knee joint osteoarthritis with autologous platelet-rich plasma in comparison with hyaluronic acid.

OBJECTIVE: This study aimed to find a simple, cost-effective, and time-efficient method for the preparation of platelet-rich plasma (PRP), so the acquired benefits will be readily available for multiple procedures in smaller outpatient clinics and to explore the safety and efficacy of the application of PRP in the treatment of degenerative lesions of articular cartilage of the knee. DESIGN: The study was designed as a prospective, cohort study with a control group. A total of 120 patients with Grade 1, 2, or 3 osteoarthritis according to the Kellgren and Lawrence grading scale were enrolled in the study. One group of patients was treated using three intra-articular applications of PRP, and the second group of patients was given three injections of hyaluronic acid. Outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index and the 11-point pain intensity Numeric Rating Scale. RESULTS: On average, a 4.5-fold increase in platelet concentration was obtained in the PRP group. No severe adverse events were observed. Statistically significantly better results in the Western Ontario and McMaster Universities Osteoarthritis Index and Numeric Rating Scale scores were recorded in a group of patients who received PRP injections after a 3- and 6-mo follow-up. CONCLUSIONS: Our preliminary findings support the application of autologous PRP as an effective and safe method in the treatment of the initial stages of knee osteoarthritis. Further studies are needed to confirm these results and to investigate the persistence of the beneficial effects observed.

Pathological findings in cornea tissue of patients with penetrating keratoplasty.

The aim of this clinical study was to examine corneas extracted from the patients during penetrating keratoplasty for the presence of immunoglobulins and inflammatory cells that can be warning for the graft failure. Individual clinical diagnoses were correlated to the presence of the inflammatory signs in corneal tissue. The signs of inflammation in corneal layers were detected especially in group of patients classified as viral keratitis but were also found in corneas of patients with degenerative diseases of the cornea. Depending on the number of keratoplasties no statistical difference in analysed parameters was found. Inflammatory process represented by slight positive presence cellular infiltration and/or immunoglobulins could be present in corneal tissue also in absence of acute manifestation. Histological and immunohistochemical evaluation of corneal tissue from patients undergoing penetrating keratoplasty could improve estimation of correct diagnosis. Subsequently prompt adequate therapy could improve worse prognosis of corneal graft with evident immune process.