N. I. Chalisova

A Method of Creating a Cell Monolayer Based on Organotypic Culture for Screening of Physiologically Active Substances

We developed a method of culturing and phenotyping of a monolayer of cells of the retinal tissue, thymus and spleen on the basis of organotypic culture. All characteristic types of neurons and fi broblasts were found in their microenvironment in the retinal cell monolayer. Lymphocytes, macrophages, and fi broblasts were verifi ed in the monolayer of thymus and spleen cells. Histological staining, immunocytochemistry, and electron microscopy demonstrated the possibility of assessing the differentiation degree and functional activity of the cell monolayer. The developed technique preserves cell-cell interactions and a variety of cell types characteristic of the examined organ in the monolayer. This opens up new prospects for its application in basic research and in screening of different physiologically active substances.

The dynamics of stimulating and inhibiting influence on organoid cultures of nervous and lymphoid tissues.

Organoid cultures of tissue explants are successfully used to evaluate rapidly and quantitatively the specifically the type of the effect of a substance to be tested. A representative system of this kind is an organoid culture of cerebrospinal ganglia isolated from 10to 11-dayold chicken embryos [8, 9, 14], in which axon growth is indicative of the effect of stimulating cytokines. Explants of other tissues are also used as organoid cultures to evaluate the role of proliferation, migration, adhesion, and changes of cell types, which produce a combined effect resulting in an increase or decrease of the explant growth zone. Repair processes are known to be regulated in opposite directions: cell proliferation can be either stimulated or inhibited via apoptosis [14]. In an organoid culture, the dose dependence of the effects of cytokines usually fit a dome-shaped curve [1, 7, 11, 13]. In other words, the stimulating effects of these agents are observed at specific concentrations, whereas at both lower and higher concentrations, the response remains at the control level. There is evidence that similar nonlinear dose-dependent relationships are characteristic of the effects of some neuropeptides on learning [2, 3]. The question arises as to what mechanisms underlie stimulating and inhibiting effects observed in organoid tissue cultures. In this work, we attempted to determine the stimulating and inhibiting dose-dependent effects of various biologically active substances on organoid cultures. In particular, we studied the effect of axon-stimulating components secreted by the medicinal leech salivary gland (protease, destabilase, and egline C (an inhibitor of serine proteases) [10, 15]) on the organoid culture of cerebrospinal ganglia from chicken embryos, as well as the effect of the mitogen concanavalin A on a rat spleen organoid culture. The effects of these substances were also studied in the presence of neurotrophin, a nerve growth factor (NGF). It was demonstrated that, at concentrations exceeding the optimal ones, cytokines inhibited cell proliferation when applied alone. In combination with NGF, cytokines had a similar effect. Thus, overstimulation of the organoid tissue cultures “switched off” the proliferative processes, which reflects the cell system adaptability.

Studies of cytokines in nerve tissue cultures.

The effects of cortexin, epithalamin, and synthetic peptides on the growth of processes in sensory neurons and on the development of fragments of cortical and subcortical brain structures were studied in organotypic cultures from 10-11-day chick embryos. Cortexin (20 and 100 ng/ml), epithalamin (20 and 200 ng/ml), polypeptides M and P (2 and 20 ng/ml) had neurite-stimulating actions, evident on day 3 of dorsal root ganglion culture. Addition of cortexin (100 ng/ml) or polypeptide M (20 ng/ml) to the culture medium of cerebral cortex explants stimulated explant development. Addition of cortexin at the same concentration to explants of subcortical formations suppressed their development. Epithalamin (200 ng/ml) or polypeptide M (100 ng/ml) stimulated the development of explants from subcortical formations, the existence of the neurite-stimulating effect effects of these cytokines provided the basis for identifying the mechanism of action of brain peptides.

A regulatory effect of amino acids in organotypic cultures of lymphoid tissues with various degrees of immunological maturity.

Repair processes in tissues are oppositely directed: they either stimulate or depress cell proliferation [4–7]. In our recent work [3], we demonstrated opposite directions of the effects of synthetic dipeptides of the thymus, L-Glu-L-Trp (timogen) and L-Lys-L-Glu (vilon), depending on the age and immunological status of animals. However, it can be assumed that the amino acids contained in cytokines (as structural elements of small peptides or substrate of protein synthesis) may exhibit certain regulatory properties with respect to target tissues per se . This aspect of the amino acid effect is poorly understood thus far. There are only a few data (e.g., the data that glutamate, which is also a neurotransmitter, may induce apoptosis [1, 2]). The most adequate method for a rapid quantitative evaluation of the direction of the effects of biologically active compounds (or their combinations) is organotypic culturing of tissue fragments.

Modulating and Protective Effects of Thymic Peptides in Lymphoid Tissue Culture

The modern concept of an integrated neuroimmunoendocrine system places a strong emphasis on regulatory peptides that participate in intercellular and intracellular transduction of signals. In recent years, special focus has been on cytomedins, complex polypeptides isolated from various organs and tissues [1–3]. On the basis of cytomedins, new-generation pharmaceuticals have been developed. For example, thymalin has been isolated from the bovine thymus, and two so-called cytogens, immunomodulating peptides LGluL -Trp (thymogen) and L -LysL -Glu (vilon), have been synthesized [1, 2]. Thymalin has been registered by the Russian Federation State Pharmacopoeia and is widely applied for treatment of immune disorders [3]. A recent work [4] described multidirectional effects of individual thymic peptides administered in both isolated form and in the background of the mitogenic effect of concanavalin A (Con A). At the same time, interactions of thymic peptides with cytokines, e.g., neurotrophic factors, remain unclear. It is known that nerve growth factor (NGF) has a mitogenic effect on mononuclear spleen cells [5]. It has also been shown that NGF interacts with immune cells and plays a role in regulation of some immune reactions [5, 6]. In the presence of NGF, DNA synthesis in a rat mononuclear cell culture is elevated. However, the mechanisms of NGF interactions with immune cells yet remain unclear. There is ground to believe that NGF molecules act differently on different components of the neuroendocrineimmune system and may depend on immunomodulating peptides.

The neurite-stimulating effect of peptides from brain in dorsal root ganglion neuron organotypic culture

The effect of natural brain peptides (cortexin and epithalamin) as well as synthetic tetrapeptides (brain peptide and epiphysis peptide) were investigated in organotypic cultures of dorsal root ganglion (DRG) neurons and explants of brain tissue from 10-11 day old chick embryos. Cortexin (2-100 ng/ml), epithalamin (200 ng/ml), brain peptide (2 ng/ml) and epiphysis peptide (20 ng/ml) showed a neurite-stimulating effect in DRG cultures as compared to the control explants. Cortexin at 100 ng/ml and brain peptide at 20 ng/ml showed a stimulating effect in cortical cultures, and epithalamin at 200 ng/ml and epiphysis peptide at 100 ng/ml showed a stimulating effect in cultures of subcortical structures as compared to the control explants.

Effect of tripeptide Lys-Glu-Asp on physiological activity of neuroimmunoendocrine system cells.

Tripeptide Lys-Glu-Asp stimulates proliferation and inhibits apoptosis in organotypic cultures of neuroimmunoendocrine system cells. Lys-Glu-Asp accelerates cell renewal processes (decrease of apoptosis marker p53 and increase of proliferation marker Ki-67) in the pineal gland; this effect is more pronounced in cultures derived from old animals than in young cultures. The tripeptide induces the expression of low-differentiated lymphocyte marker CD5 and macrophage marker CD68, but in “old” cultures this effect is less pronounced than in “young” ones. Thus, in tissue culture Lys-Glu-Asp primarily affects the nervous and endocrine tissues during aging and produces a less pronounced effect on the nervous tissue. Physiological activity of the tripeptide consists in modulation of associative learning of honey bee in the model of short-term and the long-term memory.

Modulating effect of microflora metabolites of the human and animals on lymphoid tissue culture

395 Today, human and animal microflora is regarded as an extracorporal organ involved in nearly all physio logical processes taking place in the microorganism [11, 15]. It is necessary to reveal the regulatory rela tionships coordinating the interaction between the microflora and the microorganism during their devel opment and life activity. It is known that bacteria secrete diverse metabolic products into the environ ment, including carboxylic acids, amino acids, alco hols, vitamins, and some other compounds. The majority of these compounds, synthesized at quanti ties significantly exceeding own needs of bacteria, were regarded earlier as by products because of their imbalanced metabolism. The authors of recent works [1, 2, 6] showed that these metabolites are required for initiation and termination of the growth of the bacteria that secrete them, which means that these compounds are factors of antagonistic activity. Ovalbumin induced immunity stimulation in animals that received a complex of metabolites produced by probi otic bacterial strains potentiated splenocyte prolifera tion, increased the number of antibody producing cells in the spleen, and stimulated interleukin 2 (IL 2) production [3, 5]; i.e., a stimulatory effect on the immune system was observed. It can be assumed that the metabolites involved in the regulation of the com position and activity of microbiocenosis may also have a direct regulatory effect on the host’s organism, first getting into the bloodstream and then into tissues. The most adequate and convenient method for primary screening assessment of the effect of relevant biologi cally active compounds is organotypic culturing of tis sues and analysis of explant growth zone [4, 8, 9, 12]. The advantage of organotypic cultures is that they retain the hierarchy of cell populations and the struc tural characteristics of tissue [7, 13].

Stimulation of cell proliferation and apoptosis in the presence of amino acids in organotypic culture of tissues of different degree of maturity

7 Data confirming the concept that an organism possesses sufficiently independent regulatory systems— peptide and amino acid—have been accumulated to date [1, 11, 15]. For example, the studies of the parameters of specific and nonspecific resistance showed that lysine, arginine, glutamic and aspartic acids, and tryptophane exhibit different immunity and phagocytosisstimulating and detoxicating properties. When injected subcutaneously to mice, lysine and arginine only stimulated phagocytosis but did not protect from toxic substances; lysine did not change the immune response, whereas arginine suppressed it [1]. Using PC3 and DU145 cell lines of androgen-independent prostate cancer, it was shown that methionine, tyrosine, or phenylalanine deprivation has an inhibitory effect, with the cell cycle being arrested at stage G0/G1. Methionine deprivation enhanced apoptosis in PC3 cells; tyrosine and phenylalanine deprivation, in DU145 cells [14]. Methionine, tyrosine, and phenylalanine deprivation inhibited the invasion of both cell lines; glutamine deprivation, only DU145 cells. Therefore, invasion inhibition does not depend on apoptosis induction [12, 13]. Our recent studies demonstrated modulating (stimulatory and inhibitory) effects of different amino acids, which depended of the type of tissue and age of animals [3, 4, 6]. The most adequate and convenient method for a rapid quantitative estimation of the direction of the effect of biologically active compounds is organotypic culturing of tissue fragments and analysis of the growth zone of explants. This is due to the fact that changes in the number of cells may serve as a criterion of primary integrated estimation of biological activity of substances, and a change in the number of cells itself may be the result of stimulation or inhibition of proliferation. Inhibition of proliferation due to apoptosis is studied by the methods of molecular biology, which allow detection of expression of proapoptotic proteins [12–14].

The regulatory effects of coded amino acids on basic cellular processes in young and old animals

Aspects of the regulatory effects of coded amino acids on basic cellular processes, viz., proliferation and apoptosis, are discussed. These effects occur due to the regulation of specific genes by amino acids at the transcriptional and translational levels, which leads to the initiation of regulatory cascades of many of the main cellular processes. Investigations in organotypic cultures of tissues of different geneses have demonstrated that different amino acids stimulate cellular proliferation or apoptosis. The group of low-molecular weight hydrophilic amino acids with charged chains influences cell proliferation in tissues of mesodermal genesis. Another group of high-molecular weight hydrophobic amino acids acts on tissues of ectodermal genesis. Thus, the coded amino acids are not only the structural elements of proteins, but can also actively participate in the regulation of specific genes that control the cellular cycle. The number of active amino acids is decreased by 2.7 times in explants from old animals as compared to young ones, reflecting the disturbance in amino-acid transport and gene expression by aging.