Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds.

Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.

Collagen-induced arthritis: severity and immune response attenuation using multivalent N-acetyl glucosamine.

Rheumatoid arthritis is an autoimmunity leading to considerable impairment of quality of life. N-acetyl glucosamine (GlcNAc) has been described previously as a potent modulator of experimental arthritis in animal models and is used for osteoarthritis treatment in humans, praised for its lack of adverse effects. In this study we present a comprehensive immunological analysis of multivalent GlcNAc-terminated glycoconjugate (GC) application in the treatment of collagen-induced arthritis (CIA) and its clinical outcome. We used immunohistochemistry and FACS to describe conditions on the inflammation site. Systemic and clinical effects were evaluated by FACS, cytotoxicity assay, ELISA, cytometric bead array (CBA), RT-PCR and clinical scoring. We found reduced inflammatory infiltration, NKG2D expression on NK and suppression of T, B and antigen-presenting cells (APC) in the synovia. On the systemic level, GCs prevented the activation of monocyte- and B cell-derived APCs, the rise of TNF-α and IFN-γ levels, and subsequent type II collagen (CII)-specific IgG2a formation. Moreover, we detected an increase of anti-inflammatory IL-4 mRNA in the spleen. Similar to the synovia, the GCs caused a significant reduction of NKG2D-expressing NK cells in the spleen without influencing their lytic function. GCs effectively postponed the onset of arthritic symptoms, reduced their severity and in 18% (GN8P) and 31% (GN4C) of the cases completely prevented their appearance. Our data prove that GlcNAc glycoconjugates prevent the inflammatory response, involving proinflammatory cytokine rise, APC activation and NKG2D expression, leading to the attenuation of clinical symptoms. These results support the glycobiological approach to the treatment of collagen-induced arthritis/rheumatoid arthritis (CIA/RA) as a way of bringing new prospects for more effective therapeutic interventions.

Antitumor activity of N-acetyl-D-glucosamine-substituted glycoconjugates and combined therapy with keyhole limpet hemocyanin in B16F10 mouse melanoma model.

N-Acetyl-D-glucosamine-substituted glycoconjugates (GCJs) with the polyamidoamine (GN8P) or calix[4]arene (GN4C) scaffold represent ligands for NKR-P1 molecule and induce NK cell-mediated cytotoxicity in vitro. The in vivo effect of these GCJs on mouse melanoma model was determined when administered either alone or in combination with non-specific immunostimulator keyhole limpet hemocyanin (KLH). All types of treatment significantly reduced the tumor growth on day 23, while GN4C as well as KLH were effective continuously (from day 14). The GN4C also induced the longest mean survival time (46.3 ± 11.1 d), followed by KLH+GN4C (36.4 ± 12.1), KLH (35.6 ± 6.5), KLH+GN8P (35.6 ± 6.7), and GN8P (32.4 ± 7.0), compared to controls (29.8 ± 3.6). The B16F10 specific cytotoxicity of peripheral blood cells was significantly elevated by both KLH and GN8P, whereas not by GN4C. KLH increased the effect of the GN4C, but did not influence that of GN8P. GN4C was proved to exert anticancer activity in mouse melanoma model. The combination of KLH with GCJs did not generate synergism.

CD161 receptor participates in both impairing NK cell cytotoxicity and the response to glycans and vimentin in patients with rheumatoid arthritis.

We investigated the role of natural killer (NK) cells and CD161, their primary C-type-lectin-like receptor in rheumatoid arthritis (RA). Samples were compared with healthy donors (HD), dermatomyositic (DM), polymyositic (PM), and osteoarthritic (OA) patients. RA, PM, and DM NK cell cytotoxicities significantly decreased relative to the HD and OA NK cells (p<0.0001). These results correlated with an increased expression of NK cell inhibitory receptor CD161, in active disease RA patients. We demonstrated that NK cells are able to respond to mutated citrullinated vimentin (MCV), an RA-specific autoantigen, leading to increases in both PAD4 enzyme and CD161 mRNA expression. MGAT5 glycosidase involvement was detected in GlcNAc metabolism within the synoviocytes of RA patients. Our findings reveal a functional relationship between CD161 expression and NK cell cytotoxicity as well as reactivity to glycans and MCV, thus providing new insight into the pathogenesis of RA and confirming the involvement of surface glycosylation.

NK cell-mediated cytotoxicity modulation by A(2) adenosine receptor agonist in different mammalian species.

Adenosines, endogenous purine nucleosides, appear in the extracellular space under metabolically stressful conditions associated with ischemia, inflammation, and cell damage. Their activity on innate immunity is prevalently inhibitory and can develop both in infectious and neoplastic diseases. During cancer development, tumor cells that release high concentrations of adenosines can impair the function of tumor-infiltrating lymphocytes and assist tumor growth by neo-angiogenesis. We evaluated the influence of A(2) adenosine receptor (A(2)AR) agonist on cytotoxic-cell response comparing human with other mammalian species (rodents, pigs, goats), both in healthy and in cancer conditions. The A(2)AR agonist developed dose-dependent inhibition of the cytotoxic activity of immune effector cells in all studied species. However, variability of the response was observed in relation to the species and the target cells that were used. Altogether, our data indicate that the A(2)AR plays a central role in adenosine-mediated inhibition of immune response to tumors.

Immunological response in the mouse melanoma model after local hyperthermia.

Our study was aimed to characterize the phenotype and functional endpoints of local microwave hyperthermia (LHT, 42 degrees C) on tumor infiltrating and spleen leukocytes. The effectiveness of LHT applied into the tumor of B16F10 melanoma-bearing C57/BL6 mice was compared with anesthetized and non-treated animals. Subpopulations of leukocytes were analyzed using the flow cytometry, and the cytotoxic activity of splenocytes against syngeneic B16F10 melanoma and NK-sensitive YAC-1 tumor cell lines was evaluated in (51)Cr-release assay. Similarly, the in vitro modification of the heat treatment was performed using healthy and melanoma-bearing splenocytes. We found a 40 % increase of activated monocytes (CD11b+CD69+) infiltration into the tumor microenvironment. In the spleen of experimental animals, the numbers of cytotoxic T lymphocytes (CTLs-CD3+CD8+) and NK cell (CD49b+NK1.1+) raised by 22 % and 14 %, respectively, while the NK1.1+ monocytes decreases by 37 %. This was accompanied by an enhancement of cytotoxic effector function against B16F10 and YAC-1 targets in both in vivo and in vitro conditions. These results demonstrate that LHT induces better killing of syngeneic melanoma targets. Furthermore, LHT evokes the homing of activated monocytes into the tumor microenvironment and increases the counts of NK cells and CTL in the spleen.

Neurobiological aspects of depressive disorder and antidepressant treatment: role of glia.

Depression is a complex disorder related to chronic inflammatory processes, chronic stress changes and a hippocampal response. There is a increasing knowledge about the role of glial cells in nutrient supply to neurons, maintenance of synaptic contacts and tissue homeostasis within the CNS. Glial cells, viewed in the past as passive elements with a limited influence on neuronal function, are becoming recognized as active partners of neurons and are starting to be discussed as a possible therapeutic target. Their role in the pathogenesis of depressive disorders is also being reconsidered. Attention is devoted to studies of the different types of antidepressants and their effects on transmembrane signaling, including levels of alpha subunits of G proteins in C6 glioma cells in vitro as a model of postsynaptic changes in vivo. These models indicate similarities in antidepressant effects on G proteins of brain cells and effector cells of natural immunity, natural killers and granulocytes. Thus, an antidepressant response can exhibit certain common characteristics in functionally different systems which also participate in disease pathogenesis. There are, however, differences in the astrocyte G-protein responses to antidepressant treatment, indicating that antidepressants differ in their effect on glial signalization. Today mainstream approach to neurobiological basis of depressive disorders and other mood illnesses is linked to abnormalities in transmembrane signal transduction via G-protein coupled receptors. Intracellular signalization cascade modulation results in the activation of transcription factors with subsequent increased production of a wide array of products including growth factors and to changes in cellular activity and reactivity.

Neisseria meningitidis strains from patients with invasive meningococcal disease differ in stimulation of cytokine production.

Cytokine production was determined in vitro after stimulation with three different Neisseria meningitidis (NM) strains. Virulent NM B strain isolated from a patient with mild course of invasive meningococcal disease (IMD) elicited higher cytokine production than NM B and NM C hypervirulent strains isolated from patients with moderate and fatal course of IMD, respectively. Endotoxin concentration after in vitro stimulation correlated with cytokine production: the highest endotoxin levels were observed with virulent NM B strain. Serum cytokines and endotoxin levels showed an opposite trend. These results suggest that inflammatory response during IMD is predominantly influenced by host factors.

Anticoagulant drugs increase natural killer cell activity in lung cancer.

BACKGROUND: In preclinical studies in animal models and in initial clinical trials, anticoagulation drugs have been shown to be effective in the prevention and treatment of haematogenous metastasis, and in the prolongation of survival in animal models. However, only a few studies have been performed on the direct influence of anticoagulation drugs on the immune system. OBJECTIVE: The purpose of this study is to determine the effect of warfarin, unfractioned heparin, low molecular weight heparins (LMWHs), and acetylsalicylic acid anticoagulants on the functional activity of natural killer (NK) cells. PATIENTS AND METHOD: Cytotoxic activity in patients with early, operable stages of non-small-cell lung cancer was compared with healthy volunteers. Cytotoxic studies were also carried out in tumor-bearing animals. RESULTS: Lung-cancer patients were characterized by significantly lower NK cell cytotoxicity (7.07 +/- 3.15) than healthy donors (44.12 +/- 10.62, P < 0.001). NK cell activation was found in both in vitro experiments using peripheral blood mononuclear cells (PBMC) from healthy donors and ex vivo in lung carcinoma patients after treatment with unfractionated heparin and fraxiparine. Similarly, potentiation of NK cell activity in Lewis lung carcinoma-bearing mice was found after therapy with unfractionated heparin. NK cell activity is lower in lung cancer patients than in normal subjects. CONCLUSIONS: NK cell activation was increased by LMWHs. Other anticoagulants augment the effector function of NK cells in cancer patients and in an animal model of lung cancer. This is a novel effect of these compounds, which were thought previously to exert their effect only via their anticoagulant properties.

Mass spectrometry is a powerful tool for identification of proteins associated with lipid rafts of Jurkat T-cell line.

Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

Detection of 13q abnormalities in multiple myeloma using immunomagnetically selected plasma cells.

Accurate prognostic evaluation of patients with multiple myeloma (MM) is required for their stratification for more adequate therapy. Chromosomal G-banding and interphase fluorescence in situ hybridization (FISH) on cell-nonspecific samples and on myeloma cells selected by magnetic-activated cell separation (MACS) were used to study 13 samples from 12 multiple myeloma (MM) patients. Bone marrow (BM) samples were analysed using three approaches. Standard mitotic samples were prepared and analysed after G-banding. Interphase FISH was performed to detect the 13q14 deletion in unselected BM cells. In parallel, myeloma cells were selected from the BM using the CD138-specific antibody. The high-purity myeloma cell suspension was then analysed by interphase FISH for the 13q14 deletion. Magnetic separation yielded enriched myeloma cell suspensions with the mean viability of 98.0% (range: 97.0%-99.0%), and the purity of 97.6% (range: 87.2%-99.2%) as detected morphologically, and 85.2% (range: 44.8%-98.4%) as detected by immunophenotyping for CD138+ cells. Interphase FISH revealed the 13q14.3 deletion in 5 of 13 (38.5%) of cell-nonspecific samples and in 9 of 13 (69.2%) of enriched myeloma cell suspensions. In conclusion, interphase FISH on immunomagnetically selected MM cells increases the detection of the 13q14 deletion in BM samples from the patients with MM.

Effect of agroclavine on NK activity in vivo under normal and stress conditions in rats.

Agroclavine is a natural, clavine type of ergot alkaloid with D1 dopamine and a-adrenoceptor agonistic properties. We showed previously that in vitro agroclavine enhances natural killer (NK) cell activity, increases interleukin-2 and interferon-gamma production and prolongs the survival time of tumor-bearing mice. The aim of this study was 1) to test the effect of agroclavine on NK activity in vivo, and 2) to assess the potential toxicity of high doses of agroclavine on cardiac and liver functions using creatine kinase MB (CKMB) and alanine aminotransferase (ALT) as biochemical markers in normal and stressed animals. The effect of stress was studied because we examined promising anticancer properties of agroclavine and malignant diseases are supposed to be a potent stressful event for patients. In our experiments 3-month-old male rats of the Wistar-Kyoto strain were used. Agroclavine was injected intraperitoneally (0.5 mg/kg or 0.05 mg/kg) 30 min before stress (four hours' restraint and immersion in 23 degrees C water). The animals were killed 30 min after stress, blood was collected and the spleen was removed. Non-stressed animals treated with agroclavine were killed 5 h after the drug administration. The results confirmed our previous in vitro results and showed that also in vivo agroclavine increases NK cell activity under non-stress conditions. Agroclavine only slightly increased CKMB and had no influence on ALT in non-stressed animals. These promising results are limited by the fact that agroclavine (0.5 mg/kg) diminished NK cell activity and significantly increased ALT and CKMB under stress conditions.

Cancer immunomodulation by carbohydrate ligands for the lymphocyte receptor NKR-P1.

Natural killer (NK) and T cells express a superfamily of proteins with structural features of C-type lectins. Recombinantly prepared soluble form of rat NKR-P1 (CD161) recognized carbohydrate GalNac and GlcNac moieties. Ganglioside GM2 and heparin related-IS oligosaccharides representing the high affinity ligands for this receptor, increased the sensitivity of targets for killing by the rat effectors isolated from blood and spleen in vitro. Based on these results, we investigated in vivo the possible therapeutic effect of GM2 and IS carried by liposomes during induced rat colorectal carcinogenesis. The reduction of cancer incidence versus the controls (50% vs 88.88%), approached the 5-fluorouracil treatment (41.66%).

Association of human NK cell surface receptors NKR-P1 and CD94 with Src-family protein kinases.

Human natural killer (NK) cells express on their surface several members of the C-type lectin family such as NKR-P1, CD94, and NKG2 that are probably involved in recognition of target cells and delivery of signals modulating NK cell cytotoxicity. To elucidate the mechanisms involved in signaling via these receptors, we solubilized in vitro cultured human NK cells by a mild detergent, Brij-58, immunoprecipitated molecular complexes containing the NKR-P1 or CD94 molecules, respectively, by specific monoclonal antibodies, and performed in vitro kinase assays on the immunoprecipitates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and phospho-amino acid analysis revealed the presence of in vitro tyrosine phosphorylated proteins that were subsequently identified by re-precipitation (and/or by western blotting) as the respective C-type lectin molecules and Src family kinases Lck, Lyn, and Fyn. The NKR-P1 and the CD94-containing complexes were independent of each other and both very large, as judged by Sepharose 4B gel chromatography. Crosslinking of NKR-P1 on the cell surface induced transient in vivo tyrosine phosphorylation of cellular protein substrates. These results indicate involvement of the associated Src-family kinases in signaling via the NKR-P1 and CD94 receptors.

The effect of drugs on the mortality of mice inoculated with Friend leukaemia virus or toxoplasma gondii.

Infection and tumors provoke substantial changes accompanied with the disbalance of many neuroendocrine factors which in their summarizing effects influence the life span of animals. Our previous results showed enhanced mortality after one injection of morphine in association with Friend leukaemia virus infection. The aim of this study was to examine the effects of some other opioids (pethidine and pentazocine) and an acetylcholine esterase inhibitor neostigmine on the survival of animals under two conditions: (1) Friend leukaemia virus infection which mostly depressed immune functions, and (2) Toxoplasma gondii infection which in general enhanced the immune status. In contrast to our previous observation with morphine, the mortality induced by single doses of pethidine (150 mg/kg) or pentazocine (50-75 mg/kg) was unchanged during the Friend leukaemia virus infection. A single injection of neostigmine (0.42 or 0.56 mg/kg) was significantly more lethal in DBA-2 mice infected with Friend leukaemia virus. Neostigmine in doses of 0.33 and 0.4 mg/kg caused death in 46 % and 57 %, respectively, of animals infected with Toxoplasma gondii which was significantly higher in comparison with only 8 % and 12.5 % in control groups. Pethidine (150 mg/kg) killed 70 % of Toxoplasma gondii infected animals and even 90 % of non-infected mice. Thus, the Friend leukaemia virus and Toxoplasma gondii infections increased toxicity only of some drugs which may, at least partly, be associated with altered immune status during infection and involvement of the cholinergic system.

Neuroimmunomodulation of natural killer (NK) cells by ergot alkaloid derivatives.

Ergot alkaloids (EAs), products of Claviceps spp., are widely used in various fields of clinical medicine (neurology, psychiatry, endocrinology). In the present work we studied the neuroimmunomodulative effect of EAs on activation of NK cells and their signalling pathways. Furthermore, the killing capability of rat NK cells in vitro was examined in the presence of glycosidic derivatives of elymoclavine, agroclavine, and liposome-encapsulated EAs. The engagement of appropriate NK cell membrane receptors by EAs cause an indirect enhancement of adenylyl cyclase system through inhibition of G-protein al,2-subunit (up to 50 % of control values). All of the tested EAs enhanced the rat NK cell-mediated cytotoxic activity in vitro, particularly against target cells of astrocyte origin (C-6 glioma). The present results argue for a possible EA immunomodulatory role of cell-mediated immunity in tumour regression processes.

Ergot alkaloid glycosides with immunomodulatory activities.

New glycosides derived from ergot alkaloids elymoclavine and DH-lysergol were synthesized by chemoenzymatic methods. beta-Glucosides were obtained either by chemical method or by transglycosylation (glycosidase from Aspergillus oryzae), lactosides were prepared by further extension of carbohydrate chain using beta-1,4-galactosyltransferase (bovine milk) and alpha-5-N-acetylneuraminyl-(2-->6)-beta-D-galactopyranosyl-(l-->4)-2- acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->O)-elymoclavine was prepared using alpha-2,6-sialyltransferase (rat liver). Immunomodulatory activity of elymoclavine and 9,10-dihydrolysergol and their glycosylated derivatives on natural killer (NK) cell-mediated cytotoxicity of human resting and activated human peripheral blood mononuclear cells (PBMC) was investigated. Addition of ergot alkaloid glycosides to the mixtures of effector and target cells potentiated the PBMC cytotoxicity against both NK-sensitive and -resistant target cells. The glycoconjugates of elymoclavine enhanced cytotoxicity of PBMC against NK-resistant target cells. The glycoconjugates of DH-lysergol potentiated NK cytotoxicity of PBMC against NK-sensitive target cells.