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Biomedical subjects

J Grzybowski

Publications and source records attributed to J Grzybowski.

At least 19 recordsLinked to original sources
PubMed2005

In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients.

CONTEXT: Lamin A/C (LMNA) gene variations have been reported in more than one third of genotyped families with dilated cardiomyopathy (DCM). However, the relationship between LMNA mutation and the development of DCM is poorly understood. METHODS AND RESULTS: We found that end stage DCM patients carrying LMNA mutations displayed either dramatic ultrastructural changes of the cardiomyocyte nucleus (D192G) or nonspecific changes (R541S). Overexpression of the D192G lamin C dramatically increased the size of intranuclear speckles and reduced their number. This phenotype was only partially reversed by coexpression of the D192G and wild type lamin C. Moreover, the D192G mutation precludes insertion of lamin C into the nuclear envelope when co-transfected with the D192G lamin A. By contrast, the R541S phenotype was entirely reversed by coexpression of the R541S and wild type lamin C. As lamin speckle size is known to be correlated with regulation of transcription, we assessed the SUMO1 distribution pattern in the presence of mutated lamin C and showed that D192G lamin C expression totally disrupts the SUMO1 pattern. CONCLUSION: Our in vivo and in vitro results question the relationship of causality between LMNA mutations and the development of heart failure in some DCM patients and therefore, the reliability of genetic counselling. However, LMNA mutations producing speckles result not only in nuclear envelope structural damage, but may also lead to the dysregulation of cellular functions controlled by sumoylation, such as transcription, chromosome organisation, and nuclear trafficking.

Animals↗
PubMed2001

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Bacterial Proteins↗
PubMed2001

Photodynamic activity of the haematoporphyrin derivative with rutin and arginine substituents (HpD-Rut(2)-Arg(2)) against Staphylococcus aureus and Pseudomonas aeruginosa.

The impact of the new photosensitizer HpD-Rut(2)-Arg(2)on the growth of methicillin-resistant Staphylococcus aureus(MRSA), methicillin-susceptible Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa clinical strains isolated from infected burn wounds was examined. The susceptibility of the isolates to the photodynamic action of the sensitizer was evaluated by the determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the newly developed microdilution method. The results were compared with the previously investigated HpD-Arg(2). All clinical isolates examined proved to be susceptible to the photodynamic action of HpD-Rut(2)-Arg(2). The MIC of this newly synthetized photosensitizer ranged from 0.8 to 12.5 microg ml(-1) for MRSA, from 0.4 to 6.2 microg ml(-1) for MSSA and from 6.2 to 50 microg ml(-1) for P. aeruginosa. While MBC ranged from 1.6 to 12.5 microg ml(-1) for MRSA, 0.4 to 6.2 microg ml(-1) for MSSA and 6.2 to 100 microg ml(-1) for P. aeruginosa. This photosensitizer is more effective in its bactericidal photodynamic action than previously tested HpD-Arg(2).

Anti-Bacterial Agents↗
PubMed1999

New cytokine dressings. II. Stimulation of oxidative burst in leucocytes in vitro and reduction of viable bacteria within an infected wound.

Recently, we have developed the new dressings containing rhG-CSF or rhGM-CSF. In the present study we investigated either in vitro or in vivo biological activity of the dressings. Human whole blood samples were incubated with extracts from the collagen- or polyurethane-based dressings containing rhG-CSF or/and rhGM-CSF and phagocytic and oxidative metabolic activities were quantitated using Phagotest or Bursttest kits. The results indicate that both the number of phagocyting cells and the intensity of phagocytosis per cell, as well as the level of the oxidative burst in particular, were stimulated by one or both of the cytokines extracted. Next, the experimental skin wounds in mice were infected with 107 CFU of Pseudomonas aeruginosa strain ATCC 27853 and treated locally with the rhG-CSF-containing dressing. The analysis of the biopsies taken from the wounds indicated that in mice treated with the cytokine-containing dressing on the third day the log of CFU per biopsy was 5.0 vs. 6.2 in the control (P<0.001), and on the 8th day was lower than 4 vs. 5.4 in control (P<0.0001). Our findings clearly suggest that the newly designed dressings containing the incorporated CSFs can be used as effective topical cytokine-delivery system in the treatment of bacterial infections in wounds.

Animals↗
PubMed1999

New cytokine dressings. I. Kinetics of the in vitro rhG-CSF, rhGM-CSF, and rhEGF release from the dressings.

Wound repair-stimulatory activities of various cytokines and growth factors depend on successful delivery of these factors to the injured sites. Here were present the design and preparation of the new collagen- and polyurethane-based dressings containing the recombinant human cytokines-rhG-CSF, rhGM-CSF or rhEGF. To test the efficacy of the retrieval of the incorporated cytokines, their controlled release from the dressings was carried out over three consecutive days using polyurethane sponge as a collector of the extracts. The maximum quantities of the released rhG-CSF, rhGM-CSF and rhEGF reached approximately 25, 50, and 10%, respectively, of the total cytokine contents of the dressings, as assessed by the specific ELISA tests. These data indicate that collagen- and polyurethane dressings containing rhGM-CSF and rhG/CSF may serve as effective tools for the topical delivery of cytokines to wounded tissues.

Bandages↗
PubMed1999

Examination of antibacterial activity of the photoactivated arginine haematoporphyrin derivative.

The antibacterial photodynamic effect of arginine haematoporphyrin derivative (HpD-Arg2) was investigated using two methods. The effect proved to be highly antibacterial when it was employed against P. aeruginosa and S. aureus cells floating in the nutrient broth. The reduction of approximately four logarithms of CFU ml-1 after light exposition, as compared to a dark control, was observed. In the second step of the study the photodynamic effect was employed against the same strains of bacteria after their colonization on isolated mouse muscles. In this case the antibacterial effect was observed as well. However, the reduction of CFU ml-1 achieved was much lower and reached one logarithm only. The optimal concentration of photoactivated HpD-Arg2 for S. aureus and P. aeruginosa was 25 microg ml-1 and 800 microg ml-1 for the floating bacteria, and 200 microg ml-1 and 1000 microg ml-1 for sessile bacteria, respectively. We have concluded from our study that the antibacterial effect investigated may offer, in the future, an alternative method for treatment of the tissues superficially infected with pathogens resistant to antibiotics. However, further studies in this field are necessary.

Animals↗
PubMed1999

Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia.

Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABL mRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4-9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 microM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 x 10(9)/l neutrophils was slow at 25-51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.

Antigens, CD34↗
PubMed1999

[Local application of G-CSF, GM-CSF and EGF in treatment of wounds].

G-CSF, GM-CSF and EGF play an important role in wound healing as components of the cytokine network which regulates cooperation of cells in the repair processes. The first two cytokines have been registered as hematopoietic drugs under the names Neupogen (Roche) and Leucomax (Sandoz). Both G-CSF and GM-CSF stimulate phagocytosis in maturated leucocytes and enhance proliferation of endothelial cells. G-CSF, GM-CSF and EGF which is known as mitogenic agent were applied as topical drugs for wound care; the application resulted in acceleration of wound healing. Preliminary suggestions as to the topical dosage of the cytokines in the treatment of wounds are put forward.

Administration, Topical↗
PubMed1999

Investigation of antistaphylococcal activity of collagen dressing containing bacitracin.

In the present paper the anti-bacterial effect of bacitracin released from the collagen-based dressing and penetrating to the Staphylococcus aureus-colonised polyurethane sponge was assessed. The bacteria-colonised sponge, regarded as the "in vitro model of an infected wound", was used as a modification of the previously utilised by us analogous experimental systems. In addition, kinetics of the penetration of bacitracin from the collagen dressing to the sponge was estimated. The results indicate that in spite of the fact that the amount of bacitracin in the polyurethane sponge exceeded the minimal bactericidal concentration (MBC), not all bacteria from the S. aureus species colonising the sponge could be killed. Moreover, it appeared that in order to efficiently eradicate the sponge-adherent staphylococci the concentration of bacitracin 50-fold higher than the MBC value must be used.

Bacitracin↗
PubMed1999

Antimicrobial properties of copper-coated electroconductive polyester fibers.

Three synthetic copper-coated EURO-static fibers (PET--polyester, PA--polyamide, and PAC--polyacrylamide) manufactured by EUROPA Corporation S.C., Poland, were tested as potential antimicrobial agents. The inhibitory properties of the fibers were examined using different microorganisms as follows: i. Staphylococcus aureus ATCC 25293, and Pseudomonas aeruginosa ATCC 27853 reference strains, ii. 8 strains of S. aureus (4 MRSA and 4 MSSA) and 5 strains of P. aeruginosa isolated from infected wounds, and iii. fungal pathogen Scopulariopsis sp. isolated from onychomycosis case. The results of experiments have evidenced that polyester (PET) copper-coated EURO-static fibers inhibit the growth of all the strains used.

Antisepsis↗
PubMed1998

Adhesion of Pseudomonas aeruginosa to collagen biomaterials: effect of amikacin and ciprofloxacin on the colonization and survival of the adherent organisms.

The adherence of P. aeruginosa to collagen membrane, sponge, and to a new anti-infective COLL dressing and the susceptibility of the organisms attached to the biomaterials to amikacin were investigated in vitro. After 17 h of attachment, the bacteria demonstrated an increased resistance to amikacin compared with their free-floating counterparts. Amikacin, even at a concentration exceeding 150 times the minimal bactericidal concentration (MBC) for the strain tested, did not eradicate the attached bacteria from the surface of collagen membrane. However, when the drug at a high concentration (over 16 times the minimal inhibitory concentration, MIC) was present in the incubation medium before it had been inoculated with P. aeruginosa, a reduction of 2 log10 units in the organisms adherent to the surface of collagen membrane was observed. We conclude that slow release of the antibiotic from the COLL dressing could control the bacterial colonization on the surface. In fact, the released amikacin at the final concentration of 32 times the MBC reduced the number of adherent bacteria by 6 log10 units. In contrast, ciprofloxacin at the same final bactericidal concentration completely eradicated the bacteria from the surface of COLL dressing. However, as ciprofloxacin is not recommended for use as a topical antimicrobial agent, a further search is needed to find an agent with a similar anticolonization activity.

Amikacin↗
PubMed1998

Improving the intracellular delivery and molecular efficacy of antisense oligonucleotides in chronic myeloid leukemia cells: a comparison of streptolysin-O permeabilization, electroporation, and lipophilic conjugation.

The hybrid gene BCR-ABL that typifies chronic myeloid leukemia (CML) represents an attractive target for therapy with antisense oligodeoxyribonucleotides (ODN). A central obstacle in the therapeutic application of ODN is their poor cellular uptake. Adding various lipophilic conjugates to the ODN backbone has been reported to improve uptake, and electroporation of target cells has also been shown to enhance intracellular ODN delivery. We have shown that (1) BCR-ABL-directed ODN will specifically decrease the level of BCR-ABL mRNA, provided that cells are first permeabilized with Streptolysin-O (SL-O), and (2) chimeric methylphosphonodiester:phosphodiester ODN directed against 9 bases either side of the BCR-ABL junction are more efficient ODN effectors than structures composed solely of phosphodiester or phosphorothioate linkages. In this study, we compared the efficacy of lipophilic conjugation, SL-O permeabilization and electroporation on the intracellular delivery and molecular effect of BCR-ABL-directed ODN. b2a2- and b3a2-directed chimeric ODN were synthesized either unmodified or with one of the following groups at the 5' end: cholesterol, vitamin E, polyethylene glycol of average molecular weight 2,000 or 5,000, N-octyl-oligo-oxyethylene, or dodecanol. ODN associated with Lipofectin was also studied. Comparison was made in untreated, electroporated, and SL-O permeabilized KYO1 cells. Uptake was examined by fluorescence microscopy and flow cytometry, using ODN structures that were 3' labeled with fluorescein. The effect on target BCR-ABL mRNA expression was analyzed by Northern blotting. Several conjugated structures associated avidly with the cell membrane without achieving significant intracellular uptake or molecular effect. Similarly, ODN:Lipofectin complexes moderately increased cell association, without enhancing intracellular levels of ODN or inducing detectable molecular effect. In SL-O permeabilized or electroporated cells, uptake was approximately 1 to 2 logs greater than in untreated cells, and rapid nuclear localization was seen, especially with unmodified chimeric ODN. In SL-O permeabilized cells treated with ODN directed to the b2a2 and b3a2 junctions respectively, b2a2 BCR-ABL mRNA levels at 4 hours were reduced to 2. 6% +/- 2.1% and 38.4% +/- 1.3% of control values. In cells permeabilized by electroporation, BCR-ABL mRNA levels were decreased to 4.0% +/- 1.4% of control levels by b2a2 directed ODN, although very little nontargeted suppression was seen with b3a2-targeted ODN (93.4% +/- 4.2% of control). Greater cell to cell variation in ODN uptake was seen for SL-O permeabilized cells when compared with electroporated cells, suggesting that, after SL-O permeabilization, relatively unpermeabilized and overpermeabilized populations may coexist. No structure had any effect on the level of irrelevant (p53, MYC, and GADPH) mRNA levels. We conclude that the conjugation of chimeric ODN with one of the above-mentioned lipophilic groups or the complexing of ODN with Liopfectin does not improve either intracellular delivery of ODN or the molecular effect. In contrast, both electroporation and SL-O permeabilization (1) considerably enhanced uptake of chimeric ODN (even for structures without a conjugate group) and (2) achieved significant suppression of target mRNA levels.

Bacterial Proteins↗
PubMed1998

Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells.

It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c- myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 microM but were inactive at 0.2 microM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.

Bacterial Proteins↗
PubMed1998

The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses.

During the course of a study aimed at improving antisense oligodeoxynucleotide-mediated ex vivo bone marrow purging of patients suffering from chronic myeloid leukemia (CML), the properties of a number of antisense structures intended to reduce the expression of c-myc, mutant p53, and bcr-abl mRNAs and proteins were examined. The majority of the antisense oligodeoxynucleotides were designed to be capable of directing ribonuclease H (RNase H) cleavage of their target mRNAs. Streptolysin O (SLO) reversible permeabilization was used to deliver the oligodeoxynucleotides into the CML line KYO-1. We found that the efficiency and specificity of antisense oligonucleotide-induced reductions of target protein expression depended on target protein half-life, the oligonucleotide structure, and the specific sequence within the target mRNA. Transient reductions of c-myc mRNA and protein were achieved with a chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to the initiation codon, but cell proliferation was unaffected. In contrast, a chimeric oligodeoxynucleotide of similar structure targeted to an alternative site in the coding region of c-myc mRNA reduced target mRNA and protein levels for over 24 hours and halted cell proliferation. Chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to a point mutation in KYO-1 p53 mRNA efficiently reduced target mRNA expression, but only small, transient reductions in p53 protein expression were observed. However, a chimeric methylphosphonate-phosphorothioate oligodeoxynucleotide targeted to the same site reduced p53 protein to 30% of control levels over a 48-hour period. BCR-ABL protein expression was unaffected by chimeric oligodeoxynucleotides targeted to the breakpoint in bcr-abl mRNA, even when mRNA levels at early times were substantially reduced.

Base Sequence↗
PubMed1997

A new anti-infective collagen dressing containing antibiotics.

A new antibacterial dressing for infected wounds was prepared. The dressing was composed of a collagen membrane and collagen sponge; both biomaterials possess good tissue biocompatibility. An active antibacterial layer of limited hydrophobicity was placed between the membrane and the sponge and into the upper part of the sponge. The dressing contained gentamycin or amikacin at concentrations of 0.3 microgram/cm2 (loading level of the drug utilized during preparation of the dressing). Either the antibiotic or its concentration easily can be changed in the dressing by the manufacturer. The dressing was stable for several months. The antibiotic was released slowly from the dressing in in vitro experiments for 3 days. Antibacterial activity of the dressing was tested using a mouse wound model experimentally infected with Pseudomonas aeruginosa. Both dressings, containing either amikacin or gentamycin, reduced the number of living bacterial cells in the infected tissue almost to zero during the course of observation. The new dressing may be effective in the treatment of infected wounds in patients.

Amikacin↗
PubMed1997

Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients.

Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have clinical potential for purging harvests from chronic myeloid leukaemia (CML) patients, prior to autografting. Whilst ODN are highly effective in cell-free systems, their uptake into intact cells is very poor. We have previously reported that reversible permeabilisation of CML cell lines with Streptolysin-O (SL-O) can dramatically increase intracytoplasmic and nuclear ODN uptake. In this study, we examined whether SL-O permeabilisation could be used to enhance ODN uptake into bone marrow (BM) and peripheral blood stem cell (PBSC) harvests from CML patients, without undue toxicity. All 19 harvests studied were from patients in stable chronic phase of CML. Samples studied were either fresh BM harvests following leucoconcentration, fresh PBSC collections, or from previously cryopreserved harvests. Cells were permeabilised by SL-O to load them with fluorescein-labelled ODN. The proportion of permeabilised and viable cells was assessed by fluorescein uptake and propidium iodide exclusion, respectively, by flow cytometry. The effect of SL-O on ODN uptake and cell toxicity was unpredictable on simple mononuclear fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-positive cells, and this was achieved without either direct toxicity or inhibition of CFU-GM growth. The SL-O concentration required for optimal permeabilisation varied considerably from case to case, in line with previous data on cell lines. PBSC harvests positively selected for CD34-positive cells tended to achieve superior permeabilisation to CD34 positively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harvests which are first selected for CD34-positive cells.

Adult↗
PubMed1997

Release of antibiotics from collagen dressing.

Our new collagen dressing has been developed recently. Three types (A, B, and C) of the dressing were prepared in this study. Each type contained bacitracin, neomycin or colistin. The antibiotic was input into: i. collagen sponge (CS)--type A, ii. layer of limited hydrophobicity (LLH)--type B, and iii. into both CS and LLH layers--type C. The final concentration of the antibiotic that resulted from the loading level was 2 mg/cm2 for the dressings of type A and B and 4 mg/cm2 for the dressing of type C. The antibiotics were then extracted from the pieces of dressings for two days through dialysis membrane. Susceptibility of 54 bacterial strains (S. aureus, P. aeruginosa, and Acinetobacter) isolated from burn wounds were tested to the three antibiotics used for preparation of the dressings. The results of the study evidenced that efficiency of released of antibiotics into the extracts depended on the kind of antibiotic and on the type of dressing. The concentration of the antibiotics proved to be much higher than MIC90 values of the bacterial isolates tested in respect to their susceptibility. The dressing containing mixture of the three antibiotics in two layers--CS and LLH is now considered as potentially effective for care of infected wounds. It may be useful for the treatment of infected wounds or for profilaxis of contaminated wounds, ensuring: i. sufficient antimicrobial activity in wound, and ii. optimal wound environment for the presence of collagenic biomaterial on the damaged tissue.

Acinetobacter↗