Subpopulation Heterogeneity of NK Cells during the Genetic Modification for Subsequent Use in Targeted Therapy.

Obtaining genetically engineered NK cells is a developing area of immunotherapy. In this work, we analyzed the subset heterogeneity of NK cells subjected to retroviral transduction, taking into account the content of adaptive NK cell progenitors. It was shown that subsets KIR2DL2/DL3+, as well as CD57-KIR2DL2/DL3+NKG2C+, can be modified with greater efficiency than the corresponding subsets that do not carry the KIR2DL2/DL3 and NKG2C markers. After genetic modification, the CD57-KIR2DL2/DL3+NKG2C+ cells began to express CD57 de novo, acquiring the adaptive NK cell phenotype.

A Novel Approach to Anticancer Therapy: Molecular Modules Based on the Barnase:Barstar Pair for Targeted Delivery of HSP70 to Tumor Cells.

One important distinction between many tumor cell types and normal cells consists in the translocation of a number of intracellular proteins, in particular the 70 kDa heat shock protein (HSP70), to the surface of the plasma membrane. It has been demonstrated that such surface localization of HSP70 on tumor cells is recognized by cytotoxic effectors of the immune system, which increases their cytolytic activity. The mechanisms behind this interaction are not fully clear; however, the phenomenon of surface localization of HSP70 on cancer cells can be used to develop new approaches to antitumor immunotherapy. At the same time, it is known that the presence of HSP70 on a cell's surface is not a universal feature of cancer cells. Many types of tumor tissues do not express membrane-associated HSP70, which limits the clinical potential of these approaches. In this context, targeted delivery of exogenous HSP70 to the surface of cancer cells with the aim of attracting and activating the cytotoxic effectors of the immune system can be considered a promising means of antitumor immunotherapy. Molecular constructs containing recombinant mini-antibodies specific to tumor-associated antigens (in particular, antibodies specific to HER2/neu-antigen and other markers highly expressed on the surface of a wide range of cancer cells) can be used to target the delivery of HSP70 to tumor tissues. In order to assess the feasibility and effectiveness of this approach, recombinant constructs containing a mini-antibody specific to the HER2/ neu-antigen in the first module and HSP70 molecule or a fragment of this protein in the second module were developed in this study. Strong selective interaction between the modules was ensured by a cohesive unit formed by the barnase:barstar pair, a heterodimer characterized by an unusually high constant of association. During testing of the developed constructs in in vitro models the constructs exhibited targeted binding to tumor cells expressing the HER2/neu antigen and the agents had a significant stimulating effect on the cytotoxic activity of NK cells against the respective cancer cells.

[Analysis of toxicity and biocompatibility of chitosan derivatives with different physico-chemical properties].

A comparative study of the toxicity and hemocompatibility of chitosan and its derivatives with different acetylation degrees, molecular masses, charges, and hydrophobicity has been performed. It has been shown that only positively charged chitosan derivatives activate platelets and suppress cell proliferation, regardless of the acetylation degree, molecular mass, and hydrophobicity. Chitosan quaternization decreases toxicity at a low degree of substitution and abruptly increases it at a high one. Negatively charged chitosan derivatives were nontoxic and compatible with blood components. It was concluded that the toxicity of chitosan and its derivatives is defined by their charge and solubility at a neutral pH.

Purification of high-molecular-weight subfraction from porcine skin inhibiting proliferation of A431 human carcinoma epidermoid cells.

Subfraction with a molecular weight >250 kDa isolated from porcine skin and inhibiting the proliferation of A431 human carcinoma epidermoid cells was purified by DEAE 32 anion exchange chromatography with NaCl concentration step-gradient. The effects of the initial subfraction and fractions obtained by separation in DEAE 32 on the proliferation of A431 human carcinoma epidermoid cells were studied in vitro in two tests (MTT and fluorescent test). The more sensitive fluorescent test showed the highest inhibitory activity of fraction No. 2 released from the column at 0.15 M NaCl. One major protein component and a series of minor protein components were detected in this fraction by vertical PAAG-SDS electrophoresis.

[Autocrine factor participation in defence of cytotoxic CTLL-2 cells from oxidative stress].

The role of autocrine factors (AF) secreted by cytotoxic IL-2-dependent CTLL-2 cells along with pyruvate in cell defense from oxidative stress was investigated. The addition of conditioned medium (CM) containing pyruvate and AF into CTLL-2 cell cultures increased significantly cell survival under oxidative stress condition. The kinetics of hydrogen peroxide removal from cell cultures under oxidative stress in the case of CM addition has been obtained. The removal of H2O2 mostly by means of its reaction with pyruvate that is contained in CM has been shown at the beginning of oxidative stress (up to 15 min). Pyruvate content in CM was determined as 138 +/- 7 microM. Cell filtration on column with Bio-Gel P-10 was used for removal pyruvate from CM. Three fractions of CM (A, B and C) were obtained as a result of gel filtration. Pyruvate was not detected in any fraction. The fraction A was eluted from column as the first one and contained the largest molecules. Cell survival test showed the fraction B to have the highest ability to protect CTLL-2 cells under oxidative stress. The fraction A supported cell survival to a less degree and fraction C was shown to have no protective ability. The addition of the fraction B to the cell cultures resulted in preservation of significantly higher intracellular ATP level in the cells under oxidative stress than in the control ones. Moreover, AF of the fraction B was shown to react directly with hydrogen peroxide and inactivate it in the absence of cells. AF of the fraction A did not have such properties.

[Influence of autocrine factor deficit in culture on survival and energy metabolism of CTLL-2 cells under oxidative stress].

A lot of data has shown recently that survival of mammalian cells is under a control of growth factors and autocrine survival factors (AF). We studied the influence of AF deficit on survival, intracellular ATP content, and transmembrane potential of mitochondria of IL-2-dependent CTLL-2 cells under oxidative stress. CTLL-2 cells cultivated under deficit of AF have been shown to be more susceptible to oxidative injury in comparison with the cells cultivated without deficit of AF (control); they died at smaller concentrations of H2O2 than control cells did. The ATP content in CTLL-2 cells was decreased under AF deficit conditions even without any stress and treatment of the cells by hydrogen peroxide resulted in additional large decrease of it. ATP depression was accompanied by disruption of cell membrane (blebbing) and drop of mitochondrial potential. Cell death under oxidative stress in the presence of AF deficit has been shown to proceed by both apoptosis and necrosis.