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  • Author: Andrzej Krzysztof Siwicki x
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Open access

Joanna Małaczewska and Andrzej Krzysztof Siwicki

Abstract

The purpose of the study was to determine the cytotoxicity of commercial silver, gold, and copper nanocolloids towards two established cell lines (NIH/3T3 and GMK) and primary chick embryo cell culture (CECC), using routine colorimetric assays: MTT, NRU, and LDH, which enable a preliminary evaluation of the mechanism of cytotoxic effect of the tested substances. The MTT assay evaluates the activity of mitochondria, NRU assay reveals the damage to lysosomes, while LDH assay shows injuries to the cytoplasmic membrane. The NRU assay proved to be non-applicable to the tested nanocolloids, most probably due to the interaction of nanoparticles with neutral red dye, which affected the colorimetric reaction. The MTT assay was more sensitive than LDH because the intercellular effect of a substance occurs before permanent damage to the cytoplasmic membrane. Silver nanocolloid was distinguished by the highest cytotoxicity, irrespective of the applied cell model, although the other two metals showed some cytotoxic effects as well, with gold nanocolloid being more toxic than copper one. Although the primary chick embryo cell culture, as a model reflecting more faithfully the conditions in a living organism than continuous cell lines, was undistinguished by elevated tolerance to the most toxic silver nanocolloid, it showed the tendency to recovery from the growth suppression with longer exposure after the application of less toxic gold and copper nanocolloids.

Open access

Sylwia Tobólska, Sylwia Terpiłowska, Jerzy Jaroszewski and Andrzej Krzysztof Siwicki

Abstract

Introduction

Inosine pranobex (Isoprinosine) stimulates cell-mediated immune responses to viral infections in humans and might have also therapeutic use in animals. The aim of this study was to compare three in vitro cytotoxicity assays on mouse embryo fibroblasts and liver cancer cells and determine their ability to detect early cytotoxic effects for inosine pranobex.

Material and Methods

BALB/3T3 clone A31and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 μg/mL. Cell viability was determined with the MTT reduction, the LHD release, and the NRU tests.

Results

A decrease in the cell viability was observed after incubating the BALB/3T3 clone A31and HepG2 cells with inosine pranobex.

Conclusions

Based on the cytotoxicity endpoints measured in these investigations in BALB/3T3 clone A31cells, it can be concluded that the cell membrane may be the first part of the cell to be affected by inosine pranobex. The disintegration of lysosomes and mitochondria follows mitochondria damage. In HepG2 cells likewise, the cell membrane may be the first part of the cell to be affected by inosine pranobex. Also in liver cancer cells, the disintegration of mitochondria (assessed with the MTT reduction assay) and next of lysosomes (assessed with the NRU assay) follows mitochondria damage.

Open access

Barbara Kazuń, Krzysztof Kazuń, Joanna Żylińska and Andrzej K. Siwicki

Abstract

The presence of lactic acid bacteria (LAB) favors the stabilization of intestinal flora, facilitates digestion, improves the assimilability of fodder, and has an immunomodulatory effect on the immune system. According to current research, the application of LAB following antibiotic treatment prevents the development of opportunistic bacteria inhabiting the digestive tract. In the study the potential probiotic properties of Lactobacillus plantarum strains, which can be administered as an alternative to antibiotic treatment in aquaculture, were investigated under in vitro conditions. The strains of L. plantarum were characterized for important properties such as the ability to grow in the presence of 10% fish bile, a tolerance of low pH, and antagonism to pathogens dangerous for fish such as Aeromonas salmonicida and Pseudomonas fluorescens; therefore, they meeting the criteria for strains with probiotic properties. In view of currently increasing resistance to antibiotics and a decrease of their efficiency, probiotic bacteria can serve to support immunity to infections in the future.

Open access

Sylwia Tobólska, Sylwia Terpiłowska, Jerzy Jaroszewski and Andrzej Krzysztof Siwicki

Abstract

Introduction

Inosine pranobex (Methisoprinol, ISO, Isoprinosine) is an immuno-modulatory antiviral drug that has been licensed since 1971 in several countries worldwide. In humans, the drug is approved for the treatment of viral infections, and it might also have therapeutic use in animals. The aims of the presented work were to investigate the genotoxicity of inosine pranobex on BALB/3T3 clone A1 and HepG2 cell lines and to elucidate its mutagenicity using the Ames test.

Material and Methods

The BALB/3T3 clone A1 and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 μg/mL. The genotoxicity was determined by comet and micronucleus assays, and the mutagenicity was determined by Ames assay.

Results

Inosine pranobex did not induce a significant dose-related increase in the number of comets or micronuclei in BALB/3T3 clone A1 and HepG2 cells. Moreover, based on the results of the Ames test, it was concluded that inosine pranobex is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Conclusion

Based on the results of a comet assay, micronucleus assay, and Ames test, it was concluded that inosine pranobex is neither genotoxic nor mutagenic.

Open access

Sylwia Terpiłowska, Dorota Siwicka-Gieroba and Andrzej Krzysztof Siwicki

Abstract

Introduction

Nickel and iron are very commonly occurring metals. Nickel is used in industry, but nowadays it is also used in medical biomaterials. Iron is an element necessary for cell metabolism and is used in diet supplements and biomaterials, whence it may be released along with nickel.

Material and Methods

BALB/3T3 and HepG2 cells were incubated with iron chloride or nickel chloride at concentrations ranging from 100 to 1,400 μM. The following mixtures were used: iron chloride 200 μM plus nickel chloride 1,000 μM, or iron chloride 1,000 μM plus nickel chloride 200 μM. The cell viability was determined with MTT, LHD, and NRU tests.

Results

A decrease in cell viability was observed after incubating the BALB/3T3 and HepG2 cells with iron chloride or nickel chloride. A synergistic effect was observed after iron chloride 1,000 μM plus nickel chloride 200 μM treatment in all assays. Moreover, the same effect was observed in the pair iron chloride 200 μM plus nickel chloride 1,000 μM in the LDH and NRU assays.

Conclusions

Iron (III) and nickel (II) decrease cell viability. Iron chloride at a concentration of 200 μM protects mitochondria from nickel chloride toxicity.

Open access

Sylwia Terpiłowska, Dorota Siwicka-Gieroba and Andrzej Krzysztof Siwicki

Abstract

Introduction

Iron and molybdenum are essential trace elements for cell metabolism. They are involved in maintaining proper functions of enzymes, cell proliferation, and metabolism of DNA.

Material and Methods

BALB/3T3 and HepG2 cells were incubated with iron chloride or molybdenum trioxide at concentrations from 100 to 1,400 μM. The cells were also incubated in mixtures of iron chloride at 200 μM plus molybdenum trioxide at 1,000 μM or iron chloride at 1,000 μM plus molybdenum trioxide at 200 μM. Cell viability was determined with MTT reduction, LHD release, and NRU tests.

Results

A decrease in cell viability was observed after incubating both cell lines with iron chloride or molybdenum trioxide. In cells incubated with mixtures of these trace elements, a decrease in cell viability was observed, assessed by all the used assays.

Conclusion

Iron (III) and molybdenum (III) decrease cell viability in normal and cancer cells. A synergistic effect of the mixture of these elements was observed.

Open access

Barbara Kazuń, Joanna Małaczewska, Krzysztof Kazuń, Joanna Żylińska-Urban and Andrzej K. Siwicki

Abstract

Introduction

Immune-potentiating functions of Lactobacillus plantarum strains in the common carp were evaluated.

Material and Methods

Fourteen days of feeding fish dry diet supplemented with the bacteria provided parameters of nonspecific humoral immunity (lysozyme, ceruloplasmin, γ-globulin, total protein levels, and serum bactericidal activity) and cellular immunity (pinocytosis, respiratory burst activity, and potential killing activity of organ phagocytes), as well as the proliferative response of organ lymphocytes stimulated with mitogens. The resistance of fish to infection with Aeromonas hydrophila was also determined.

Results

Dietary supplementation with L. plantarum had a substantial influence on the activity of organ phagocytes, especially the potential killing activity of head kidney cells. A significant increase in the proliferative activity of LPS-stimulated B lymphocytes and in the levels of γ-globulins and total protein was observed. The supplemented diet conveyed higher resistance than the control diet as the cumulative fish mortalities after infection with A. hydrophila were 65% and 85%, respectively.

Conclusion

The results indicate that dietary supplementation with L. plantarum stimulates the antibacterial resistance of common carp and may reinforce defence against bacterial infections, but further studies need to be conducted.

Open access

Joanna Małaczewska, Edyta Kaczorek-Łukowska, Monika Szymańska-Czerwińska, Wojciech Rękawek, Roman Wójcik, Krzysztof Niemczuk and Andrzej Krzysztof Siwicki

Abstract

Introduction

Coxiella (C.) burnetii, the aetiological agent of Q fever, is able to modulate the macrophage/T-lymphocyte axis in an infected organism and impair synthesis of monokines and lymphokines.

Material and Methods

The purpose of this research was to determine the levels of the cytokines that play a key role in the response to C. burnetii antigens (IL-1β, IL-2, IL-6, IL-10, IFN-γ and TNF-α) in the serum of animals originating from an infected herd prior to vaccination (day 0) and at 1, 7, and 21 days afterwards.

Results

The vaccination of animals did not affect the production of IL-6, IL-1β, or IL-2. The serum levels of these cytokines were too low to measure in most of the samples. The initial levels of TNFα, IFNγ, and IL-10 were higher in seropositive than in seronegative animals, although significant differences between seropositive shedders and seropositive nonshedders appeared only in the levels of IFNγ and IL-10. Additionally, the course of the post-vaccination response concerning these two cytokines was different among seronegative nonshedders, seropositive nonshedders, and seropositive shedders.

Conclusion

It seems that analysis of the IFNγ and IL-10 concentrations in animal blood serum may have some practical value in an assessment of the health status of seropositive animals and post-vaccination response.