The effect of physical activity on cytokine levels in adults living with type 1 diabetes-a preliminary study.

The aim of this study is to investigate whether physical activity and the level of body fat are factors reducing the level of pro-inflammatory cytokines in people with T1DM. Twenty-five men (27.8 ± 9.4 years old; 178.9 ± 6.9 cm; 80.6 ± 12 kg) and 18 women (28.1 ± 12.5 years old; 162.4 ± 5.5; 63.1 ± 9.9 kg) were divided into four groups based on body fat percentage and level of physical activity (AN-active people with normal body fat; IAN-inactive people with normal body fat; AO-active people with excessive body fat, IAO-inactive people with excessive body fat). The level of cytokines in the blood serum was assessed. The level of IL-8 was higher (measurable) in inactive men, regardless of adiposity degree and in women, only in the inactive group with normal body fat. IL-6 was found only in active men with excessive adiposity. In conclusion, the findings from this study allow to indicate that moderate level of physical activity may contribute to a reduction in the development of systemic low-grade inflammation in patients with T1DM, and thus, may reduce the risk of CVD.

Evolutionarily conserved mechanisms regulating stress-induced neutrophil redistribution in fish.

Introduction: Stress may pose a serious challenge to immune homeostasis. Stress however also may prepare the immune system for challenges such as wounding or infection, which are likely to happen during a fight or flight stress response. Methods: In common carp (Cyprinus carpio L.) we studied the stress-induced redistribution of neutrophils into circulation, and the expression of genes encoding CXC chemokines known to be involved in the regulation of neutrophil retention (CXCL12) and redistribution (CXCL8), and their receptors (CXCR4 and CXCR1-2, respectively) in blood leukocytes and in the fish hematopoietic organ - the head kidney. The potential involvement of CXC receptors and stress hormone receptors in stress-induced neutrophil redistribution was determined by an in vivo study with selective CXCR inhibitors and antagonists of the receptors involved in stress regulation: glucocorticoid/mineralocorticoid receptors (GRs/MRs), adrenergic receptors (ADRs) and the melanocortin 2 receptor (MC2R). Results: The stress-induced increase of blood neutrophils was accompanied by a neutrophil decrease in the hematopoietic organs. This increase was cortisol-induced and GR-dependent. Moreover, stress upregulated the expression of genes encoding CXCL12 and CXCL8 chemokines, their receptors, and the receptor for granulocytes colony-stimulation factor (GCSFR) and matrix metalloproteinase 9 (MMP9). Blocking of the CXCR4 and CXCR1 and 2 receptors with selective inhibitors inhibited the stress-induced neutrophil redistribution and affected the expression of genes encoding CXC chemokines and CXCRs as well as GCSFR and MMP9. Discussion: Our data demonstrate that acute stress leads to the mobilization of the immune system, characterized by neutrophilia. CXC chemokines and CXC receptors are involved in this stress-induced redistribution of neutrophils from the hematopoietic tissue into the peripheral blood. This phenomenon is directly regulated by interactions between cortisol and the GR/MR. Considering the pivotal importance of neutrophilic granulocytes in the first line of defense, this knowledge is important for aquaculture, but will also contribute to the mechanisms involved in the stress-induced perturbation in neutrophil redistribution as often observed in clinical practice.

Cross-reactivity of T cell-specific antibodies in the bank vole (Myodes glareolus).

The bank vole is a common Cricetidae rodent that is a reservoir of several zoonotic pathogens and an emerging model in eco-immunology. Here, we add to a developing immunological toolkit for this species by testing the cross-reactivity of commercially available monoclonal antibodies (mAbs) to the bank vole lymphocyte differentiation molecules and a transcription factor. We show that a combination of mAbs against CD4, CD3, and Foxp3 allows flow cytometric distinction of the main subsets of T cells: putative helper CD4+, cytotoxic CD8+ (as CD3+CD4-) and regulatory CD4+Foxp3+. We also provide a comparative analysis of amino acid sequences of CD4, CD8αß, CD3εγδ and Foxp3 molecules for a number of commonly studied Cricetidae rodents and discuss mAb cross-reactivity patterns reported so far in this rodent family. We found that in case of mAbs targeting the extracellular portions of commonly used T cell markers, sequence similarity is a poor prognostic of cross-reactivity. Use of more conserved, intracellular molecules or molecule fragments is a more reliable approach in non-model species, but the necessity of cell fixation limit its application in, e.g. functional studies.

Evolutionary trade-offs constraining the MHC gene expansion: beyond simple TCR depletion model.

The immune system is as much shaped by the pressure of pathogens as it is by evolutionary trade-offs that constrain its structure and function. A perfect example comes from the major histocompatibility complex (MHC), molecules that initiate adaptive immune response by presentation of foreign antigens to T cells. The remarkable, population-level polymorphism of MHC genes is assumed to result mainly from a co-evolutionary arms race between hosts and pathogens, while the limited, within-individual number of functional MHC loci is thought to be the consequence of an evolutionary trade-off between enhanced pathogen recognition and excessive T cell depletion during negative selection in the thymus. Certain mathematical models and infection studies suggest that an intermediate individual MHC diversity would thus be optimal. A recent, more direct test of this hypothesis has shown that the effects of MHC diversity on T-cell receptor (TCR) repertoires may differ between MHC classes, supporting the depletion model only for MHC class I. Here, we used the bank vole (Myodes=Cletronomys glareolus), a rodent species with variable numbers of expressed MHC genes, to test how an individual MHC diversity influences the proportions and TCR repertoires of responding T cell subsets. We found a non-linear relationship between MHC diversity and T cell proportions (with intermediate MHC numbers coinciding with the largest T cell proportions), perhaps reflecting an optimality effect of balanced positive and negative thymic selection. The association was strongest for the relationship between MHC class I and splenic CD8+ T cells. The CD8+ TCR richness alone was unaffected by MHC class I diversity, suggesting that MHC class I expansion may be limited by decreasing T cell counts, rather than by direct depletion of TCR richness. In contrast, CD4+ TCR richness was positively correlated with MHC class II diversity, arguing against a universal TCR depletion. It also suggests that different evolutionary forces or trade-offs may limit the within-individual expansion of the MHC class II loci.

The effect of selected immunostimulants on hemocytes of the false black widow Steatoda grossa (Theridiidae) spiders under chronic exposition to cadmium.

The aim of this study was to analyze whether, and to what extent, long-term exposure to cadmium, administered in sublethal concentrations by the oral route, caused changes in the immune potential of hemocytes in adult female Steatoda grossa spiders. We used artificial and natural immunostimulants, namely phorbol 12-myristate 13-acetate (PMA) and bacterial cell suspension based on Gram-positive (G+, Staphylococcus aureus) and Gram-negative (G-, Pseudomonas fluorescens) bacteria, to compare the status of hemocytes in nonstimulated individuals and those subjected to immunostimulation. After cadmium exposure, the percentage of small nongranular hemocytes in response to G+ cell suspension and PMA mitogen was decreased. Furthermore, in the cadmium-intoxicated spiders the percentage of plasmatocytes after immunostimulation remained lower compared to the complementary control group. Exposure to cadmium also induced several degenerative changes, including typical apoptotic and necrotic changes, in the analyzed types of cells. Immunostimulation by PMA mitogen and G+ bacterial suspension resulted in an increase in the number of cisterns in the rough endoplasmic reticulum of granulocytes, in both the control group and cadmium-treated individuals. These changes were accompanied with a low level of metallothioneins in hemolymph. Chronic cadmium exposure may significantly weaken the immune defense system of spiders during infections.

Effect of Staphylococcus aureus infection on the heat stress protein 70 (HSP70) level in chicken embryo tissues.

Staphylococcus aureus bacteria are components of physiological biocenosis of skin or mucous membranes in some animals' genera but also they are dangerous opportunistic pathogens responsible for infections of various localization, course or manifestations. Proteins produced by these bacteria destroy tissues, leukocytes and cause haemolysis of erythrocytes. Host organisms respond by defence mechanisms. Production of heat stress proteins (HSPs) is one of defence responses of infected host organism. To evaluate infection and host defence mechanisms some animal models of experimental infection are reported. Use of chick embryo model allows demonstrating adequate differences in staphylococcal virulence depending on the strain genotype. The aim of the study was to examine the changes in heat shock protein HSP70 levels in chick embryo tissues after infection caused by S. aureus strains no. tu2, pa3, ch5, ch10, ch24, and ch25 isolated from chickens. The bacteria were injected directly into fluid of amnion cavity and incubated for 10 days. The mortality of particular chick embryos was reported and the tissues for further analysis were taken every day from day 13 to day 19. The levels of heat stress protein HSP70 were determined by dot-blot method. Results showed that the strains no. ch5, ch24, and ch25 were the most virulent. HSP70 levels increased in all groups of injected embryos at the same time the hatching process was started. The presented study showed that the infected chick embryos were characterized by higher HSP level from 12.3% up to 19.7% compared to the control group. The morphological analysis showed numerous erythrocytes with damaged cell membranes and morphological changes of erythrocytes. Changes in the level of HSP70 protein can be a useful indicator of infection caused by S. aureus bacteria. Additionally, chicken embryo is a helpful research model in studies of pathogenesis of diseases caused by bacteria.

Arginase Activity in Eisenia andrei Coelomocytes: Function in the Earthworm Innate Response.

Arginase is the manganese metalloenzyme catalyzing the conversion of l-arginine to l-ornithine and urea. In vertebrates, arginase is involved in the immune response, tissue regeneration, and wound healing and is an important marker of alternative anti-inflammatory polarization of macrophages. In invertebrates, data concerning the role of arginase in these processes are very limited. Therefore, in the present study, we focused on the changes in arginase activity in the coelomocytes of Eisenia andrei. We studied the effects of lipopolysaccharide (LPS), hydrogen peroxide (H2O2), heavy metals ions (e.g., Mn2+), parasite infection, wound healing, and short-term fasting (5 days) on arginase activity. For the first time in earthworms, we described arginase activity in the coelomocytes and found that it can be up-regulated upon in vitro stimulation with LPS and H2O2 and in the presence of Mn2+ ions. Moreover, arginase activity was also up-regulated in animals in vivo infected with nematodes or experiencing segment amputation, but not in fasting earthworms. Furthermore, we confirmed that the activity of coelomocyte arginase can be suppressed by l-norvaline. Our studies strongly suggest that similarly to the vertebrates, also in the earthworms, coelomocyte arginase is an important element of the immune response and wound healing processes.

Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs.

Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses. Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells. The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones. Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity. It was confirmed that coelomocytes-immunocompetent cells of the earthworm Eisenia andrei-are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton. Similar to vertebrate leukocytes (e.g., neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors. ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, e.g., heat shock proteins HSP27. The latter fact confirms that mechanisms of ET release are conserved in evolution. The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa.

Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

Metallothionein 2 and Heat Shock Protein 72 Protect Allolobophora chlorotica from Cadmium But Not Nickel or Copper Exposure: Body Malformation and Coelomocyte Functioning.

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements.

ß1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells.

Integrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of ß1,6-acetylglucosaminyltransferase V, we showed that ß1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvß3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvß3 caring overexpressed ß1,6-branched structures, important for FAK upregulation.

Antioxidative and immunological responses in the haemolymph of wolf spider Xerolycosa nemoralis (Lycosidae) exposed to starvation and dimethoate.

The aim of this study was to assess the intensity of enzymatic antioxidative parameters [catalase (CAT), glutathione peroxidase (GSTPx), glutathione reductase (GR), total antioxidant capacity (TAC)] and percentage of high granularity cells as well as low to medium granularity cells in haemolymph of wolf spiders Xerolycosa nemoralis exposed to starvation and dimethoate under laboratory conditions. Only in starved males, haemolymph included a lower percentage of high granularity cells, accompanied by high activity of CAT and GSTPx, than in the control. Exposure of males to dimethoate increased CAT activity, after single application, and significantly enhanced GR activity, after five-time application. In females, five-time contact with dimethoate elevated the percentage of high granularity cells. As in comparison to females, male X. nemoralis were more sensitive to the applied stressing factors, it may be concluded that in natural conditions both food deficiency and chemical stress may diminish the immune response of their organisms.

Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes.

Parameters such as total number of coelomocytes, riboflavin content in coelomocytes, expression of genes implied in metal homeostasis, and detoxification mechanisms can be used as biomarkers to assess the impact of metals on annelids. Defense biomarkers (detoxification gene expressions and coelomocyte parameters) were investigated in the ecotoxicologically important species Eisenia andrei following in vivo exposure to 5 different metals (zinc, copper, nickel, lead, and cadmium) at known concentrations. Coelomocyte numbers and riboflavin content were not affected by metallic exposure, but metal-specific gene expression variations were evidenced.

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei.

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.

Metal-specific effects on metallothionein gene induction and riboflavin content in coelomocytes of Allolobophora chlorotica.

Metal pollution affects earthworm coelomocytes, including their differential counts, riboflavin content and metallothioneins (MT) involved in metal homoeostasis and detoxification. The present work shows effects of Ni, Cu, Zn, Cd, and Pb at the same molarity (1mM) on coelomocytes of Allolobophora chlorotica after 2-day worm dermal exposure to metal chlorides. Numbers of coelomocytes/eleocytes extruded by electric shock and amounts of riboflavin in coelomocyte lysates were significantly decreased in Cu-exposed worms, less diminished in response to Ni, Zn, Cd, and unaffected by Pb. In sharp contrast, real-time PCR revealed a very strong (272 fold) MT-mRNA induction in response to Cd only. The induction was very low in response to Zn, Cu, Pb, and Ni ions (2.6, 2.1, 1.4, and 1.3-fold, respectively). In conclusion, decreased cell counts and riboflavin content are molecular biomarkers of Cu exposure while induction of MT-mRNA is a molecular biomarker of worm Cd exposure.

Flow cytometric quantification of proliferating coelomocytes non-invasively retrieved from the earthworm, Dendrobaena veneta.

Earthworms irritated naturally (e.g. by predators) or experimentally extrude coelomocyte-containing coelomic fluid through the dorsal pores of the body wall. In the present study, the earthworms, Dendrobaena veneta, experimentally depleted of free-floating coelomocytes by multiple electric shocks (1 min, 4.5 V) remained fully vital and coelomocyte depletion was followed by the extensive cell replenishment, which was more efficient in the case of amoebocytes than autofluorescent eleocytes/chloragocytes, quantified by flow cytometry. Immunohistochemical procedure with antibodies against human Ki-67 proliferation antigens revealed proliferating cells on cytospin preparations of coelomocytes extruded by electric shock. Quantification of proliferating cells in the suspension of extruded coelomocytes was performed by flow cytometry on FL-2 profiles of propidium iodide-stained samples; riboflavin-derived autofluorescence of eleocytes/chloragocytes was lost during detergent treatment. As expected, the percentage of coelomocytes proliferating in coelomic fluid was increased during restoration of coelomocyte number after experimental depletion. The method described here may be very useful for investigations of antigen-driven proliferation of earthworm coelomocytes.

Riboflavin content in autofluorescent earthworm coelomocytes is species-specific.

We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia jetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy.

Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions.

This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes.