Malgorzata Krzyzowska

Loss of IL-7Ralpha is associated with CD4 T-cell depletion, high interleukin-7 levels and CD28 down-regulation in HIV infected patients

Objective:Elevated levels of interleukin (IL)-7 are present in the blood of HIV-positive patients and it is known that IL-7 receptor (IL-7R)α expression decreases on T cells during HIV infection. The subset(s) of T cells with low IL-7Rα and the consequence of low IL-7Rα expression for T-cell survival are poorly characterized. Design:The frequency of IL-7Rα-negative T cells in HIV-positive patients was studied in relation to CD4 T-cell counts, IL-7 concentration and survival in culture. We analysed IL-7Rα expression in different T-cell populations and in relation to Bcl-2 expression. Methods:Specimens from 38 HIV-1 patients and 17 controls were examined. IL-7Rα and Bcl-2 expression in different T-cell populations was studied by flow cytometry. The influence of IL-7Rα expression on T-cell survival was studied by culturing T cells in the presence of IL-7. Results:Down-regulation of IL-7Rα on T cells correlated with depletion of CD4 T cells (P < 0.001) and also with increased concentration of serum IL-7 (P < 0.05). The decreased IL-7Rα expression was associated with low Bcl-2 expression and with the reduced survival capacity of T cells in the presence of IL-7 in vitro. Particularly, T cells with memory phenotype showed a decreased IL-7Rα expression in association with CD28 down-regulation. Conclusions:The positive effects of IL-7 on survival and homeostatic proliferation of T cells might be severely impaired in HIV-infected individuals due to IL-7Rα down-regulation. Differentiation towards a CD28-negative memory phenotype in response to chronic activation may lead to an overall decrease of IL-7 mediated survival within the peripheral T-cell pool.

Tannic acid modified silver nanoparticles show antiviral activity in herpes simplex virus type 2 infection.

The interaction between silver nanoparticles and herpesviruses is attracting great interest due to their antiviral activity and possibility to use as microbicides for oral and anogenital herpes. In this work, we demonstrate that tannic acid modified silver nanoparticles sized 13 nm, 33 nm and 46 nm are capable of reducing HSV-2 infectivity both in vitro and in vivo. The antiviral activity of tannic acid modified silver nanoparticles was size-related, required direct interaction and blocked virus attachment, penetration and further spread. All tested tannic acid modified silver nanoparticles reduced both infection and inflammatory reaction in the mouse model of HSV-2 infection when used at infection or for a post-infection treatment. Smaller-sized nanoparticles induced production of cytokines and chemokines important for anti-viral response. The corresponding control buffers with tannic acid showed inferior antiviral effects in vitro and were ineffective in blocking in vivo infection. Our results show that tannic acid modified silver nanoparticles are good candidates for microbicides used in treatment of herpesvirus infections.

Assessment of in vitro cellular responses of monocytes and keratinocytes to tannic acid modified silver nanoparticles

Hydrolyzable tannins are known to exhibit diverse biological effects, which can be used in combination with silver nanoparticles (AgNPs). In this study, we tested toxic and inflammatory properties of tannic-acid modified 13, 33, 46 nm and unmodified 10-65 nm AgNPs using murine 291.03C keratinocyte and RAW 264.7 monocyte cell lines. Both cell lines exposed for 24h to 1-10 μg/ml of 13 nm, 33 nm, 46 nm and unmodified AgNPs showed dose-dependent toxicity and decreased cell proliferation. Only small-sized AgNPs induced production of ROS by monocytes, but not keratinocytes. Monocytes internalized large aggregates of 33, 46 nm and 10-65 nm AgNPs in cytoplasmic vacuoles, whereas keratinocytes accumulated less particles. AgNPs of 13 nm were localized ubiquitously within both cell types. The tested AgNPs strongly down-regulated production of tumor necrosis factor-α (TNF-α) by monocytes, whereas keratinocytes exposed to AgNPs showed an opposite effect. Unmodified but not tannic acid-modified AgNPs increased production of the pro-inflammatory MCP-1 by monocytes and keratinocytes. In summary, low inflammatory potential and lack of ROS production by tannic-acid modified AgNPs sized above 30 nm suggests that tannic acid modification of large silver nanoparticles may help to increase AgNPs biosafety.

Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways

Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways.

The role of Chromium III in the organism and its possible use in diabetes and obesity treatment.

INTRODUCTION Diabetes and obesity are diseases characterized by their increasing incidence every year. When comparing with healthy subjects, the serum levels of chromium (Cr) are lowered in these two diseases. Several studies conducted in laboratory animals with experimentally- induced diabetes demonstrated that supplementation with chromium ions (III) decreased glucose concentration in the blood, reduced the probability of atherosclerosis and heart attack, lowered the levels of cholesterol and low density lipoprotein (LDL). The Importance of chromium is actually challenged due to lack of clear manifestations of Cr deficiency in humans and animals. OBJECTIVE The aim of this review was to present current knowledge about Cr its role in the organism and possible mechanisms of its action also in metabolic disorders such as diabetes or obesity. STATE OF KNOWLEDGE In the last decade, Cr was established to be rather a beneficial than essential trace element in mammals, and has gained popularity as a nutritional supplement and a component of many multivitamin/mineral formulations, fortified food and energy drinks. Cr supplements are widespread for diabetes and obesity treatment, despite conflicting reports on its efficacy. It was suggested that Cr shows a beneficial influence upon glucose and lipid disturbances. CONCLUSIONS The recent clinical trials provided evidence both in favor and against the importance of Cr in healthy and ill organisms. Unfortunately, also the molecular mechanism by which chromium affects glucose and lipid metabolism is still unclear. Beneficial effects of diet supplementation with different sources of Cr³⁺ can be potentially explained by rather pharmacological than nutritional effects.

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies.

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.

Long‐lasting immunity by early infection of maternal‐antibody‐protected infants

Newborn higher vertebrates are largely immuno‐incompetent and generally survive infections – including poxviruses – by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re‐emerging pathogens.

HSV-2 Regulates Monocyte Inflammatory Response via the Fas/FasL Pathway

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Faslpr/J (Fas−/−) and C3-Faslgld/J (FasL−/−) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.

Molecular cloning and characterisation of in vitro immune response against astacin-like metalloprotease Ace-MTP-2 from Ancylostoma ceylanicum.

Ancylostoma ceylanicum belongs to the group of parasites commonly known as hookworms, blood-sucking nematodes which infect around 576 million people and hundreds of millions of animals. The interactions between these parasites and host immune systems are complicated and yet to be determined. Hookworm infections are usually long lasting and recurrent, due in part to their ability to synthesize macromolecules capable of modulating the host immune response. The interaction of parasite proteins with host immune systems has been proven, but so far there is no data describing the influence of astacin-like metalloproteases (expressed among different parasitic nematodes) on the human immune system. The cDNA encoding A. ceylanicum metalloprotease 2 (Ace-mtp-2) was cloned using RACE-PCR. Computational analysis was used to examine the immunogenicity and recombinant Ace-MTP-2 was used to investigate its influence on human THP-1 monocytes and macrophages. The Ace-mtp-2 gene encodes an astascin-like metalloprotease, with a theoretical molecular mass of 26.7 kDa. The protease has a putative signal peptide, 11 potential phosphorylation sites, and two disulfide bridges revealed by computational analysis. Maximal expression of Ace-mtp-2 by A. ceylanicum occurs in the adult stage of the parasite, and Western blot indicates the secretory nature of the protease. This suggests the protease is working at the host-parasite interface and would likely be exposed to the hosts immune response. Recombinant protein were expressed in Escherichia coli and Pichia pastoris. Recombinant Ace-MTP-2 amplified the in vitro release of TNFα and induced release of IFNγ by lipopolysaccharide activated THP-1 macrophages. The presence of Ace-MTP-2 in secretory products of the adult parasite and the induction of IFNγ release may suggest an important role for Ace-MTP-2 in host-parasite interactions since IFNγ is suggested to be responsible for the protective immune response against adult hookworms.

The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles

AbstractWe describe herein the significance of a sodium citrate and tannic acid mixture in the synthesis of spherical silver nanoparticles (AgNPs). Monodisperse AgNPs were synthesized via reduction of silver nitrate using a mixture of two chemical agents: sodium citrate and tannic acid. The shape, size and size distribution of silver particles were determined by UV–Vis spectroscopy, dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). Special attention is given to understanding and experimentally confirming the exact role of the reagents (sodium citrate and tannic acid present in the reaction mixture) in AgNP synthesis. The oxidation and reduction potentials of silver, tannic acid and sodium citrate in their mixtures were determined using cyclic voltammetry. Possible structures of tannic acid and its adducts with citric acid were investigated in aqueous solution by performing computer simulations in conjunction with the semi-empirical PM7 method. The lowest energy structures found from the preliminary conformational search are shown, and the strength of the interaction between the two molecules was calculated. The compounds present on the surface of the AgNPs were identified using FT-IR spectroscopy, and the results are compared with the IR spectrum of tannic acid theoretically calculated using PM6 and PM7 methods. The obtained results clearly indicate that the combined use of sodium citrate and tannic acid produces monodisperse spherical AgNPs, as it allows control of the nucleation, growth and stabilization of the synthesis process. Graphical abstractᅟ