Antibodies to the N-Terminal Domain of Angiotensin-Converting Enzyme (ACE2) That Block Its Interaction with SARS-CoV-2 S Protein.

SARS-CoV-2 is a new coronavirus that is the cause of COVID-19 pandemic. To enter the cell, the virus interacts via its surface S protein with angiotensin-converting enzyme 2 (ACE2), the main entry receptor on the cell membrane. Most of protective antibodies, including those induced by vaccinations, target the S protein, preventing its interaction with the ACE2 receptor. We have evaluated an alternative strategy for blocking the S-ACE2 interaction using new antipeptide antibodies to the N-terminus of the ACE2 molecule. These antibodies allow detection of human ACE2 in vitro and ex vivo.

[Proinflammatory Cytokines and Skin Wound Healing in Mice].

Skin wound healing is subject to an intricate regulation, involves many cell populations and molecular mediators, and is one of the key mechanisms that ensures the barrier functions of the skin and the maintenance of body homeostasis. The efficiency of this process is largely determined by the balance of proinflammatory and proregenerative signals, which are mediated by cytokines. The review summarizes the latest data on the role of proinflammatory cytokines, mainly tumor necrosis factor (TNF), interleukin 6 (IL-6), interleukin 1 (IL-1), and interferons (IFNs), in skin wound healing, including those obtained with the use of genome editing techniques and methods of reverse genetics to establish relevant animal models. The roles that proinflammatory cytokines play at various stages of skin regeneration are discussed for both normal state and systemic pathologies, such as diabetes. Promising approaches to treating poorly healing wounds are summarized.

[The Role of Microglia in the Homeostasis of the Central Nervous System and Neuroinflammation].

Recently, much attention has been drawn to unraveling the mechanisms of neurodegenerative and neuroinflammatory disease pathogenesis. A special role in the development of neuropathologies is assigned to the interaction of the nervous and the immune systems. Microglia are the cells of the immune system that function as resident macrophages of the central nervous system (CNS) and are involved in the development of CNS, as well as in homeostatic interactions. Impaired microglia can contribute to neuroinflammation and neurodegeneration. With the help of genome editing technologies, the main paradigms in the development and functions of microglia have been addressed. At the same time, an understanding of the mechanisms of regulation of microglia in normal and pathological conditions is necessary to create an effective therapy aimed at treating various neurological diseases. This review focuses on recent findings on the origin of microglia, its regulatory role in the central nervous system, as well as its contribution to the development of neuroinflammation.

[Humanized Mouse Models as a Tool to Study Proinflammatory Cytokine Overexpression].

Dysregulated proinflammatory cytokine expression may result in the development of severe pathologies, such as rheumatoid arthritis, psoriasis, and neurodegenerative diseases. Transgenic mice and, in particular, those with controllable systemic overexpression of proinflammatory cytokines have recently become an essential instrument to study the molecular mechanisms underlying disease development. Importantly, many of the models are humanized by introducing a human cytokine gene, while leaving or removing the respective endogenous mouse gene. Humanized mice are especially valuable for biomedical research as they provide a relevant model to develop therapies based on blocking the pathogenic activity of a cytokine or to establish the functional significance of genome polymorphisms. The review discusses the available humanized mouse models with overexpression of key proinflammatory cytokines (TNF, IL-ip, and IL-6) and inflammatory cytokines with more specific functions (IL-8, IL-17, and IL-32) and their significance for basic and clinical research.

Cytokines as Mediators of Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Cytokines play a pivotal role in maintaining homeostasis of the immune system and in regulation of the immune response. Cytokine dysregulation is often associated with development of various pathological conditions, including autoimmunity. Recent studies have provided insights into the cytokine signaling pathways that are involved not only in pathogenesis of autoimmune neuroinflammatory disorders, such as multiple sclerosis, but also in neurodegenerative states, for example, Alzheimer's disease. Understanding the exact molecular mechanisms of disease pathogenesis and evaluation of relevant experimental animal models are necessary for development of effective therapeutic approaches.

[Proinflammatory and Immunoregulatory Functions of Interleukin 6 as Identified by Reverse Genetics].

Reverse genetics approach, involving genome editing, makes it possible not only to establish the nonredundant and unique functions of genes and their products, but also to construct animal models for biomedical research. Interleukin 6 (IL-6) is an important immunoregulatory and proinflammatory cytokine that differs from many related proteins in having a rather complicated signal transduction scheme. Apart from the multiple functions of IL-6, the most relevant biological problem of recent years was establishing what cells produce IL-6, in what form IL-6 is produced, what cells are recipients of the IL-6 signal, and what are the downstream events and physiological consequences of the IL-6 signaling cascade. Because IL-6 is involved in the pathogenesis of many diseases and is a drug target, understanding the mechanisms of its normal and pathogenic effects is important for the clinics. The review summarizes the recent data available in the field.

Neonatal Lethality and Inflammatory Phenotype of the New Transgenic Mice with Overexpression of Human Interleukin-6 in Myeloid Cells.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

Myeloid-Derived Suppressor Cells and Proinflammatory Cytokines as Targets for Cancer Therapy.

Myeloid-derived suppressor cells represent a heterogeneous population of immature myeloid cells. Under normal conditions, these cells differentiate into macrophages, dendritic cells, and granulocytes. However, in pathological states such as inflammation, infection, or tumor growth, there is an arrest of their differentiation that results in the accumulation of immature myeloid cells in the organism. In addition, these cells acquire a suppressor phenotype, expressing anti-inflammatory cytokines and reactive oxygen and nitrogen species, and suppress T-cell immune response. Myeloid-derived suppressor cells (MDSC) contribute to cancerogenesis by forming a favorable microenvironment for tumor growth. Proinflammatory cytokines, secreted by tumor cells and the tumor microenvironment, induce angiogenesis and metastasis and promote tumor growth. They also provide signals necessary for survival, accumulation, and function of MDSC. Understanding the mechanisms of myeloid suppressor cell development and the use of proinflammatory cytokine inhibitors may prove beneficial for tumor therapy.

Mechanisms of Changes in Immune Response during Bacterial Coinfections of the Respiratory Tract.

Acute diseases of the respiratory tract are often caused by viral pathogens and accompanying secondary bacterial infections. It is known that the development of such bacterial complications is caused mainly by a decreased infiltration with immune system cells and by suppressed inflammation in the lungs. There are significant advances in understanding the mechanisms of secondary infections, although many details remain unclear. This review summarizes current knowledge of the molecular and cellular changes in the host organism that can influence the course of bacterial coinfections in the respiratory tract.

Effects of Fibroin Microcarriers on Inflammation and Regeneration of Deep Skin Wounds in Mice.

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1ß, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.

Microbiota Induces Expression of Tumor Necrosis Factor in Postnatal Mouse Skin.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates many important processes in the body. TNF production in a physiological state supports the structure of lymphoid organs and determines the development of lymphoid cells in hematopoiesis. However, chronic TNF overexpression leads to the development of various autoimmune disorders. Sites of TNF production in the naïve state remain unclear due to the lack of in vivo models. In the present study, we used TNF-2A-Kat reporter mice to monitor the expression of TNF in different tissues. Comparative analysis of tissue fluorescence in TNF-2A-Kat reporter mice and wild type mice revealed constitutive expression of TNF in the skin of naïve adult mice. In the skin of TNF-2A-Kat reporter mouse embryos, no statistically significant differences in the expression of TNF compared to wild type animals were observed. Furthermore, we established that local depletion of microflora with topical antibiotics leads to a reduction in the fluorescence signal. Thus, we assume that the skin microflora is responsible for the expression of TNF in the skin of mice.

New Silk Fibroin-Based Bioresorbable Microcarriers.

We fabricated bioresorbable microcarriers from water solution of Bombyx mori silk fi broin. The microcarriers are 3D structures with intricate surface and pores allowing penetration of culture medium, gas exchange, and cell adhesion. Fibroin molecules form hydrophobic structures and normally have a negative charge, which stimulates migration, but inhibits cell adhesion and makes it less effective. In order to improve adhesion efficiency and velocity, gelatin (hydrophilic biopolymer with integrin-recognizing RGD sequence) was added to the microcarrier composition. The resultant bioresorbable microcarriers support adhesion and proliferation of 3T3 murine fibroblasts.

Modeling of viral-bacterial coinfections at the molecular level using agonists of innate immunity receptors.

The combined effect of innate immunity receptors in viral-bacterial coinfections was studied in vitro using the primary culture of murine macrophages activated by different combinations of ligands of innate immunity receptors belonging to the family of Toll-like receptors. The activation of macrophages first with a viral ligand and then with a bacterial one significantly decreased the expression of proinflammatory cytokine genes. Such attenuation of immune responses may occur during the development of bacterial complications in viral infections.

[Interleukin-6 From molecular mechanisms of signal transduction to physiological properties and therapeutic targeting].

Interleukin-6 (IL-6)--one of the most important pro-inflammatory cytokines that has a broad spectrum of immunoregulatory properties. Molecular mechanisms of signal transduction of IL-6 and its receptor, which were previously established, have recently been supplemented with a concept of trans-signaling. Selective inhibition of this signaling cascade would allow to modulate the pathological effects of IL-6. Methods of reverse genetics have helped to establish the physiological functions of IL-6 in normal state and in various diseases, including neoplasias. Therapeutic inhibitors of IL-6 or its receptor are already used for the treatment of several autoimmune diseases, however, systemic inhibition inevitably also neutralizes the protective functions of this cytokine. It is expected that in the future systemic therapy will be replaced by more specific and effective approaches that take into account the peculiarities of molecular signaling pathways in target cells and differences in the function of IL-6, depending on the cell source.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

Modern anti-cytokine therapy of autoimmune diseases.

The emergence of genetically engineered biological agents opened new prospects in the treatment of autoimmune and inflammatory diseases. Cytokines responsible for regulation of a wide range of processes during development of the normal immune response are among the most successful therapeutic targets. Studies carried out in recent decades and accompanied by rapid development of biotechnology have promoted establishing in detail the role and place of cytokines in autoimmune and inflammatory pathologies. Nevertheless, mechanisms that underlie anti-cytokine therapy are still not fully understood. This review examines the role of such cytokines as TNF, IL-1, and IL-6 in the development of inflammatory processes and the action mechanisms of their inhibitors.

Formation of compact aggregates of B-lymphocytes in lung tissue during mycobacterial infection in mice depends on TNF production by these cells and is not an element of the host's immunological protection.

Tumor necrosis factor (TNF) plays a pivotal role in the early control of Mycobacterium tuberculosis and M. avium infections by a host. It was previously shown that both phagocyte-derived and T-cell-derived TNF productions are critical for protective immunity against M. tuberculosis, but the role of TNF produced by B-cells remained unclear. By comparing mice with B-cell-specific TNF deletion to littermate control mice, here we show that TNF production by B-lymphocytes is essential for the formation of infection-specific aggregates of B-cells in the lung. It is likely that these compact foci represent a pathogenic feature of inflammatory response rather than an element of protective immunity, since the capacity to form aggregates has no influence on the severity of M. tuberculosis- and M. avium-triggered diseases.

Experimental models of arthritis in which pathogenesis is dependent on TNF expression.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by joint damage as well as systemic manifestations. The exact cause of RA is not known. Both genetic and environmental factors are believed to contribute to the development of this disease. Increased expression of tumor necrosis factor (TNF) has been implicated in the pathogenesis of RA. Currently, the use of anti-TNF drugs is one of the most effective strategies for the treatment of RA, although therapeutic response is not observed in all patients. Furthermore, due to non-redundant protective functions of TNF, systemic anti-TNF therapy is often associated with unwanted side effects such as increased frequency of infectious diseases. Development of experimental models of arthritis in mice is necessary for studies on the mechanisms of pathogenesis of this disease and can be useful for comparative evaluation of various anti-TNF drugs. Here we provide an overview of the field and present our own data with two experimental models of autoimmune arthritis - collagen-induced arthritis and antibody-induced arthritis in C57Bl/6 and BALB/c mice, as well as in tnf-humanized mice generated on C57Bl/6 background. We show that TNF-deficient mice are resistant to the development of collagen-induced arthritis, and the use of anti-TNF therapy significantly reduces the disease symptoms. We also generated and evaluated a fluorescent detector of TNF overexpression in vivo. Overall, we have developed an experimental platform for studying the mechanisms of action of existing and newly developed anti-TNF drugs for the treatment of rheumatoid arthritis.

Mouse lymphomyeloid cells can function with significantly decreased expression levels of cytochrome C.

Cytochrome c is an indispensable electron carrier in the mitochondrial respiratory chain and also an important mediator of the internal pathway triggering apoptosis. Mice with a complete deficiency of the Cycs gene encoding the somatic cytochrome c die during the embryogenesis. Using the technology of LoxP-cre-dependent tissue-specific recombination, we obtained some mouse strains with significantly reduced expression of cytochrome c in certain cell types ("conditional genetic knockdown"). This knockdown was achieved by abrogation of the normal splicing of the Cycs locus pre-mRNA due to an additional acceptor site inside the stop-cassette neo(r). Previously, we observed embryonic lethality in homozygous mice with the same knockdown of cytochrome c in all cells of the organism. In the present work we studied two novel mouse strains with conditional knockdown of the Cycs gene in T lymphocytes and macrophages. Somewhat surprisingly, the mice of these two strains under normal conditions were not phenotypically different from the wild-type mice, either on the whole organism level or on the level of activity of individual target cells. Thus, the amount of cytochrome c in lymphomyeloid cells does not affect their development and normal functioning.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10-C12) than those in lipid A from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.