Effect of photobiomodulation therapy on the morphology, intracellular calcium concentration, free radical generation, apoptosis and necrosis of human mesenchymal stem cells-an in vitro study.

The aim of the study was to investigate the impact of multiwave locked system (MLS M1) emitting synchronized laser radiation at 2 wavelength simultaneous (λ = 808 nm, λ = 905 nm) on the mesenchymal stem cells (MSCs). Human MSCs were exposed to MLS M1 system laser radiation with the power density 195-318 mW/cm2 and doses of energy 3-20 J, in continuous wave emission (CW) or pulsed emission (PE). After irradiation exposure in doses of energy 3 J, 10 J (CW, ƒ = 1000 Hz), and 20 J (ƒ = 2000 Hz), increased proliferation of MSCs was observed. Significant reduction of Fluo-4 Direct™ Ca2+ indicator fluorescence over controls after CW and PE with 3 J, 10 J, and 20 J was noticed. A decrease in fluorescence intensity after the application of radiation with a frequency of 2000 Hz in doses of 3 J, 10 J, and 20 J was observed. In contrary, an increase in DCF fluorescence intensity after irradiation with laser radiation of 3 J, 10 J, and 20 J (CW, ƒ = 1000 Hz and ƒ = 2000 Hz) was also shown. Laser irradiation at a dose of 20 J, emitted at 1000 Hz and 2000 Hz, and 3 J emitted at a frequency of 2000 Hz caused a statistically significant loss of MSC viability. The applied photobiomodulation therapy induced a strong pro-apoptotic effect dependent on the laser irradiation exposure time, while the application of a sufficiently high-energy dose and frequency with a sufficiently long exposure time significantly increased intracellular calcium ion concentration and free radical production by MSCs.

There and back again: a dendrimer's tale.

The development of molecular nanostructures with well-defined particle size and shape is of eminent interest in biomedicine. Among many studied nanostructures, dendrimers represent the group of those most thoroughly characterized ones. Due to their unique structure and properties, dendrimers are very attractive for medical and pharmaceutical applications. Owing to the controllable cavities inside the dendrimer, guest molecules may be encapsulated, and highly reactive terminal groups are susceptible to further modifications, e.g., to facilitate target delivery. To understand the potential of these nanoparticles and to predict and avoid any adverse cellular reactions, it is necessary to know the mechanisms responsible for an efficient dendrimer uptake and the destination of their intracellular journey. In this article, we summarize the results of studies describing the dendrimer uptake, traffic, and efflux mechanisms depending on features of specific nanoparticles and cell types. We also present mechanisms of dendrimers responsible for toxicity and alteration in signal transduction pathways at the cellular level.

Neurotoxicity of poly(propylene imine) glycodendrimers.

Published results of studies on poly(propylene imine) (PPI) dendrimers indicate their potential use in the treatment of brain cancer or neurodegenerative diseases due to their ability to cross the blood-brain barrier. However, depending on dose, neurotoxicity may occur. Here, we discuss the impact of maltotriose modified PPI dendrimers on rat's nervous system. Wistar rats were treated intravenously for 14 consecutive days with densely (dense-shell; DS) and partly (open-shell; OS) modified PPI dendrimers at doses established as safe in the previous experiment following a single DS or OS administration. The examination included an estimation of the motility and the clinical symptoms of the respiratory, nervous, and cardiovascular systems. Both DS and OS glycodendrimers (GDs) induced adverse effects at the doses tested. Multiple administrations of PPI-OS had a detrimental influence on rats' survival. These findings suggest that the dendrimers adversely influence the nervous system and their toxic effects accumulate over time. In PPI-DS treated animals, the harmful effects were less severe but still present. However, with each treatment day, the clinical symptoms in both groups were less severe as if the animals developed tolerance to GDs. We hypothesize that the neurotoxicity of tested dendrimers is related to nanoparticles-induced autophagy.

Maltotriose-modified poly(propylene imine) Glycodendrimers as a potential novel platform in the treatment of chronic lymphocytic Leukemia. A proof-of-concept pilot study in the animal model of CLL.

Cancer nanotherapeutics have shown promise in resolving some of the limitations of conventional drug delivery systems such as nonspecific biodistribution and targeting, lack of water solubility, and low therapeutic indices, Among the various nanoparticles that are available, dendrimers, highly branched macromolecules with a specific size and shape, are one of the most promising ones. In this preliminary study, we tested the anti-tumor activity of maltotriose-modified fourth-generation poly(propylene imine) glycodendrimers (PPI-G4-M3) in vivo in the subcutaneous MEC-1 xenograft model of human chronic lymphocytic leukemia (CLL) in NOD scid gamma mice. Fludarabine was used for model validation and as a positive treatment control. The anti-tumor response was calculated as tumor volume, tumor control ratio, and tumor growth inhibition. The study showed that PPI-G4-M3 inhibited subcutaneous tumor growth more efficiently than fludarabine. The anti-tumor response was dose-dependent. Cationic PPI-G4-M3 showed the highest anti-tumor activity but also higher toxicity than the neutral dendrimers and fludarabine. These first promising results warrant further studies in the optimization of dendrimers charge, dose, route and schedule of administration to combat CLL.

Anti-Tumour Activity of Glycodendrimer Nanoparticles in a Subcutaneous MEC-1 Xenograft Model of Human Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic Lymphocytic Leukaemia (CLL) is an indolent disorder, which mainly affects older adults. Since the advent of chemoimmunotherapy, great progress has been made in its treatment. However, some patients develop a more aggressive form of the disease and are included in the group of high-risk CLL patients with a dismal prognosis and a need for new therapies. OBJECTIVE: Maltotriose-modified poly(propylene imine) dendrimers were presented as potential agents in targeted therapy for CLL in the murine xenograft model. METHODS: Tumour, brain and internal organs resected from NOD scid gamma mice were subjected to gross and histopathological evaluation. RESULTS: The results of ex vivo tissue examination indicated that open-shell glycodendrimers prevented/inhibited the spread of CLL into the brain and internal organs and its transformation into a more aggressive form. CONCLUSION: The results of the study have a potentially important impact on the design of future personalized therapies as well as clinical trials.

Effect of Photobiomodulation Therapy on the Increase of Viability and Proliferation of Human Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: We have investigated how low intensity laser irradiation emitted by a multiwave-locked system (MLS M1) affects the viability and proliferation of human bone marrow mesenchymal stem cells (MSCs) depending on the parameters of the irradiation. STUDY DESIGN/MATERIALS AND METHODS: Cells isolated surgically from the femoral bone during surgery were identified by flow cytometry and cell differentiation assays. For irradiation, two wavelengths (808 and 905 nm) with the following parameters were used: power density 195, 230, and 318 mW/cm 2 , doses of energy 3, 10, and 20 J (energy density 0.93-6.27 J/cm 2 ), and in continuous (CW) or pulsed emission (PE) (frequencies 1,000 and 2,000 Hz). RESULTS: There were statistically significant increases of cell viability and proliferation after irradiation at 3 J (CW; 1,000 Hz), 10 J (1,000 Hz), and 20 J (2,000 Hz). CONCLUSIONS: Irradiation with the MLS M1 system can be used in vitro to modulate MSCs in preparation for therapeutic applications. This will assist in designing further studies to optimize the radiation parameters and elucidate the molecular mechanisms of action of the radiation. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Affecting NF-κB cell signaling pathway in chronic lymphocytic leukemia by dendrimers-based nanoparticles.

The complex genetic diversity of chronic lymphocytic leukemia (CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven, maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) modulate BCR, TRAIL and WNT signaling pathway gene expression in CLL cells and strongly influence their survival by inducing apoptosis and inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60 K microarray, as it is one of the major factors in the pathogenesis of B-cell neoplasms. The findings were compared with those obtained with Fludarabine (FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sized macromolecules for closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties.

Blockage of Wnt/ß-Catenin Signaling by Nanoparticles Reduces Survival and Proliferation of CLL Cells In Vitro-Preliminary Study.

The Wnt/ß-catenin signaling pathway is shown to play a significant role in the control of the survival, proliferation, and differentiation of hematopoietic cells. Studies have confirmed that aberrant activation of canonical Wnt signaling occurs in various forms of leukemia, and is crucial for chronic lymphocytic leukemia (CLL) pathogenesis. The aim of the study is to evaluate the influence of maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) on Wnt/ß-catenin pathway gene expression in CLL (MEC-1) cells and to compare these findings with those obtained with fludarabine (FA). Microarray data analysis reveals seven of 19 Wnt/ß-catenin pathway genes whose expression changes significantly during dendrimer and FA treatment: WNT10A, WNT6, and CDH1 among others. PPI-G4-M3 is already known to influence MEC-1 cell apoptosis and proliferation. The obtained results suggest that the reduction in cell survival under the influence of glycodendrimers and FA may be due to loss of Wnt signaling.

PPI-G4 Glycodendrimers Upregulate TRAIL-Induced Apoptosis in Chronic Lymphocytic Leukemia Cells.

Although chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western world, it remains incurable with conventional chemotherapeutic agents. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study examines the proapoptotic effects of poly(propylene imine) (PPI) glycodendrimers modified with the maltotriose residues (PPI-G4-OS-Mal-III and PPI-G4-DS-Mal-III) on the TNF family in CLL cells. The combination of an understanding of the signaling pathways associated with CLL and the development of a molecular profiling is a key issue for the design of personalized approaches to therapy. Gene expression is determined with two-color microarray 8 × 60K. The findings indicate that PPI-G4-OS/DS-Mal-III affect gene expression from the TRAIL apoptotic pathway and exert a strong effect on CLL cells comparable with fludarabine. Dendrimer-targeted technology may well prove to bridge the gap between the ineffective treatment of today and the effective personalized therapy of the future.

Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment. OBJECTIVE: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier. METHOD: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes. RESULTS: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells. CONCLUSION: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.

How to study dendrimers and dendriplexes III. Biodistribution, pharmacokinetics and toxicity in vivo.

This paper reviews the biodistribution, toxicity and pharmacokinetics of pure dendrimers and their complexes with nucleic acids (dendriplexes) in animals, including mice, rats, rabbits, and guinea pigs. Methods and results will both be discussed. The paradigm about dendrimers' toxicity based on in vitro studies should be revised; almost all dendrimers of low and middle generations are non-toxic in vivo, despite showing some cytotoxic effects in vitro. Only the high generations of unmodified cationic dendrimers in high doses have some toxicity in vivo. Modifications of dendrimers decrease their toxicity, even when this has already been acceptable with regard to unmodified dendrimers. Several undesirable effects following administration of unmodified cationic dendrimers diminish during prolonged dosing because of the development of counteracting mechanisms. Disturbances tend to return to normal levels during the recovery period after dendrimers have ceased to be administered to animals. Neutralization of the surface charge of dendrimers in their dendriplexes leads to less toxicity in vivo. Although dendrimers and dendriplexes accumulate temporarily in liver, pancreas, heart, and kidneys, they do not do permanent damage to them, i.e. the risk of irreversible damage or malfunction of these internal organs is slight. Chemical modifications of dendrimers determine the desired location of multifunctional dendrimer-based conjugates and dendriplexes in a targeted organ. Clearance of dendrimers also strongly depends on their chemical structure. When nucleic acids are complexed with dendrimers (forming so-called dendriplexes), they are more stable, having longer circulation times than free and PEI-complexed ones. Dendrimers are highly efficient in transfection and can be targeted to any organ, e.g. brain, lung and kidneys. The vast majority of dendrimers and dendriplexes are non-immunogenic. To sum up, these promising results from in vivo studies open up the possibility of dendrimers being applied as a new generation of nano-therapeutic agents in medicine, especially in human gene therapy.

Toxicity and proapoptotic activity of poly(propylene imine) glycodendrimers in vitro: considering their contrary potential as biocompatible entity and drug molecule in cancer.

Since the first dendrimers were synthesized, scientists around the world have studied their properties and potential applications. Cationic dendrimers are characterized by significant toxicity due to their interactions with cells components. The replacement of all cationic surface groups by neutral ones and therefore diminishing positive charge reduces the toxicity but may also lead to loss of dendrimers' desirable properties and restrict their biomedical applications. We have compared the cytotoxicity, as well as proapoptotic and antiproliferative activity of unmodified fourth generation PPI dendrimer (PPI-G4) and dendrimers modified with maltose (Mal) or maltotriose (Mal-III) - for full (dense shell - DS) or partial (open shell - OS) surface modifications. We have proved that among glycodendrimers, the OS-Mal PPI-G4 dendrimer is the most toxic, whereas DS-Mal-III molecule shows relatively weak or even no effect. We have also confirmed that OS dendrimers, both maltotriose and maltose modified, not only reduce cancer cells viability by inducing apoptosis but also inhibit their proliferation. The use of dendrimers as an active substance, which may be a drug per se is one of the most exciting and clinically important applications of cancer nanotechnology, therefore a partial modification of the surface appears to be a perfect solution for this purpose.

The influence of maltotriose-modified poly(propylene imine) dendrimers on the chronic lymphocytic leukemia cells in vitro: dense shell G4 PPI.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Europe and North America. For many years scientists and doctors have been working on introducing the most effective therapy into CLL as prognosis of survival time and the course of the disease differ among patients, which might pose a problem in treating. Nanotechnology is providing new insights into diagnosis and, compared with conventional treatments, more efficient treatments, which might improve patients' comfort by decreasing side effects. Among the various nanoparticles that are available, dendrimers are one of the most promising. The aim of this study was a preliminary assessment of the clinical value of treating CLL patients with fourth generation poly(propylene imine) (PPI) dendrimers-either unmodified (PPI-G4) or approximately 90% maltotriose-modified (PPI-G4-DS-Mal-III). PPI-G4-DS-Mal-III dendrimers have, in contrast to the cationic PPI-G4, a neutral surface charge and are characterized by low cyto-, geno-, and hematotoxicity in vitro and in vivo. For the in vitro study we used blood mononuclear cells collected from both untreated CLL patients and from healthy donors. Apoptosis was measured by an annexin-V (Ann-V)/propidium iodide (IP) assay, and mitochondrial membrane potential was estimated with use of Mito Tracker Red CMXRos. Presented results confirm the influence of dendrimers PPI-G4 and PPI-G4-DS-Mal-III on apoptosis and CLL lymphocytes viability in in vitro cultures. Both tested dendrimers demonstrated higher cytotoxicity to CLL cells than to healthy donors cells, whereas unmodified dendrimers were more hematotoxic. The surface modification clearly makes glycodendrimers much more suitable for biomedical applications than unmodified PPI-G4; therefore further biological evaluations of these nanoparticles are conducted in our laboratories.

Modulation of biogenic amines content by poly(propylene imine) dendrimers in rats

Biogenic amines and polyamines participate in all vital organism functions, their levels being important function determinants. Studies were performed to check whether repeated administration of poly(propylene imine) (PPI) dendrimers, synthetic macromolecules with diaminobutane core, and peripheral primary amine groups, may influence the endogenous level of amines, as represented by the two of them: spermidine, a natural derivative of diaminobutane, and histamine. The experiment was carried out on Wistar rats. Fourth generation PPI dendrimer, as well as maltotriose-modified fourth generation PPI dendrimers with (a) cationic open sugar shell and (b) neutral dense sugar shell that possess a higher biocompatibility, was used. Applying the combination of column chromatography on Cellex P and spectrofluorimetric assays of o-phthaldialdehyde, the final amine condensation products were employed to analyze tissue spermidine and histamine outside the central nervous system. Furthermore, radioenzymatic assay was used to measure histamine levels in the brain. The obtained results indicate that in some tissues, the endogenous concentrations of histamine and spermidine may be affected by dendrimers depending on their dose and type of dendrimers (AU)

Influence of fourth generation poly(propyleneimine) dendrimers on blood cells.

Dendrimers provide many exciting opportunities for potential biomedical applications. However, owing to their positively charged surfaces, poly(propyleneimine) (PPI) dendrimers show toxic and haemolytic activities. One of the methods for masking the peripheral cationic groups is to modify them using carbohydrate residues. In this study, three types of the fourth generation PPI dendrimers-uncoated (PPI-g4), approximately 35% maltotriose (Mal-III)-coated (PPI-g4-OS), and approximately 90% Mal-III-coated (PPI-g4-DS) were investigated by assessing their effects on red blood cell (RBC) haemolysis in samples of pure RBCs, RBCs in the presence of human serum albumin (HSA) or human plasma, and RBCs in whole blood. Lymphocyte proliferation and platelet (PLT) aggregation were also studied in the presence of various concentrations of dendrimers. Although all dendrimers examined affected all the blood cells studied, the unmodified PPI-g4 had the most damaging effect. It caused high RBC haemolysis rates and PLT aggregation and greatly inhibited lymphocyte proliferation. These effects were caused by the cationic surface of this polymer. The modification of PPI-g4 with Mal-III reduced the effect of the dendrimer on all blood cells. The presence of HSA or plasma in the buffer containing the RBCs or RBC in whole blood significantly decreased the extent of dendrimer-driven haemolysis.

Genotoxicity of poly(propylene imine) dendrimers.

Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers.

Modulation of biogenic amines content by poly(propylene imine) dendrimers in rats.

Biogenic amines and polyamines participate in all vital organism functions, their levels being important function determinants. Studies were performed to check whether repeated administration of poly(propylene imine) (PPI) dendrimers, synthetic macromolecules with diaminobutane core, and peripheral primary amine groups, may influence the endogenous level of amines, as represented by the two of them: spermidine, a natural derivative of diaminobutane, and histamine. The experiment was carried out on Wistar rats. Fourth generation PPI dendrimer, as well as maltotriose-modified fourth generation PPI dendrimers with (a) cationic open sugar shell and (b) neutral dense sugar shell that possess a higher biocompatibility, was used. Applying the combination of column chromatography on Cellex P and spectrofluorimetric assays of o-phthaldialdehyde, the final amine condensation products were employed to analyze tissue spermidine and histamine outside the central nervous system. Furthermore, radioenzymatic assay was used to measure histamine levels in the brain. The obtained results indicate that in some tissues, the endogenous concentrations of histamine and spermidine may be affected by dendrimers depending on their dose and type of dendrimers.

Influence of dendrimers on red blood cells.

Dendrimers, highly branched macromolecules with a specific size and shape, provide many exciting opportunities for biomedical applications. However, most dendrimers demonstrate toxic and haemolytic activity because of their positively charged surface. Masking the peripheral cationic groups by coating them with biocompatible molecules is a method to reduce it. It was proven that modified dendrimers can even diminish haemolytic activity of encapsulated drugs. Experiments confirmed that anionic dendrimers are less haemotoxic than cationic ones. Due to the high affinity of dendrimers for serum proteins, presence of these components in an incubation buffer might also influence red blood cell (RBC)-dendrimer interactions and decrease the haemolysis level. Generally, haemotoxicity of dendrimers is concentration-, generation-, and time-dependent. Various changes in the RBCs' shape in response to interactions with dendrimers have been observed, from echinocytic transformations through cell aggregation to cluster formation, depending on the dendrimer's type and concentration. Understanding the physical and chemical origins of dendrimers' influences on RBCs might advance scientists' ability to construct dendrimers more suitable for medical applications.

In vivo toxicity of poly(propyleneimine) dendrimers.

Dendrimers are highly branched macromolecules with the potential to be used for biomedical applications. Several dendrimers are toxic owing to their positively charged surfaces. However, this toxicity can be reduced by coating these peripheral cationic groups with carbohydrate residues. In this study, the toxicity of three types of 4th generation poly (propyleneimine) dendrimers were investigated in vivo; uncoated (PPI-g4) dendrimers, and dendrimers in which 25% or 100% of surface amino groups were coated with maltotriose (PPI-g4-25%m or PPI-g4-100%m), were administered to Wistar rats. Body weight, food and water consumption, and urine excretion were monitored daily. Blood was collected to investigate biochemical and hematological parameters, and the general condition and behavior of the animals were analyzed. Unmodified PPI dendrimers caused changes in the behavior of rats, a decrease in food and water consumption, and lower body weight gain. In the case of PPI-g4 and PPI-g4-25%m dendrimers, disturbances in urine and hematological and biochemical profiles returned to normal during the recovery period. PPI-g4-100%m was harmless to rats. The PPI dendrimers demonstrated dose- and sugar-modification-degree dependent toxicity. A higher dose of uncoated PPI dendrimers caused toxicity, but surface modification almost completely abolished this toxic effect.

[Etiological factors of vascular grafts infections and their resistance to antibiotics]. / Czynniki etiologiczne zakazen przeszczepów naczyniowych i ocena ich lekoopornosci.

THE AIM: of our study was the identification of microorganisms causing vascular graft infections and the evaluation of their antimicrobial susceptibility. MATERIAL AND METHODS: 25 patients with infected vascular graft, took part in our research. In 19 patients late type of infection was recognized, in 6 the infection was qualified as early. Purulent discharge obtained from the fistula was inoculated on the bacteriological media. Antimicrobial susceptibility was assessed by disc-diffusion method. RESULTS: Staphylococcus aureus and Pseudomonas aeruginosa proved to be the most frequently isolated microorganisms. Mixed infection, caused by two distinct bacteria, occurred in 5 patients; in all cases one species belonged to Gram-positive, and the second one to Gram-negative bacteria. In 50% of patients with early type infection different species of Gram-negative rods were present, in 37,5% of them S. aureus and S. epidermidis were isolated. In late type infection Gram-negative rods were isolated from 54,5% of patients and Gram-positive bacteria from 31,5% of patients. The most frequently isolated species appeared to be Pseudomonas aeruginosa. The isolated species of bacteria varied depending on the degree of infection (according to Shilagy and Samson). CONCLUSIONS: A diversity of isolated species, the presence of mixed infections and resistance patterns typical for hospital flora among bacteria infecting vascular grafts cause that antibiotic therapy should always be based on the results of microbiological examination.