Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Recombinant Human Erythropoietin with Additional Processable Protein Domains: Purification of Protein Synthesized in Escherichia coli Heterologous Expression System.

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

[EFFICACY OF CYCLOFERON LINIMENT IN THE COMBINED TREATMENT OF CHRONIC GINGIVITIS IN PATIENTS WITH CHRONIC INFECTIOUS DISEASES].

In order to study the clinical-pathogenetic efficacy of using cycloferon liniment in the combined therapy of patients with gingivitis on the background of chronic infectious diseases (HIV infection, hepatitis C, brucellosis), medical examination and treatment of 42 patients with this diagnosis has been carried out. It is established, that the use of cycloferon liniment in the combined therapy decreases the infection load in periodontal recess and manifestation of local inflammation, normalizes the immunity indices, and reduces the level of endogenous intoxication. All these factors provide acceleration of the recuperation processes and decrease the frequency of recidivating.

Effects of combined treatment with complex S. typhimurium antigens and factors stimulating osteogenesis (curettage, BMP-2) on multipotent bone marrow stromal cells and serum concentration of cytokines in CBA mice.

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P(+) colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P(+) colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was significantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1ß, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.

[Regulation of the binding of the BMP-2 growth factor with collagen by blood plasma fibronectin].

The release kinetics of recombinant human bone morphogenic factor 2 (rhBMP-2) from collageneous hydrogel in the presence of human blood plasma have been studied. The expulsion of rhBMP-2 from the collagen-BMP-2 complex by the competitive adhesion of collagen-binding proteins penetrating from plasma was firstly recognized. It was experimentally proven that that blood plasma fibronectin is the main collagen-binding protein, which is responsible for the controlled release of rhBMP-2. As a result, a new collageneous hydrogel with the incorporation of fibronectin was created which retained rhBMP-2 for a twice longer period as compared to the ordinary collageneous hydrogel. A distinctive feature of this new collagen-fibronectin matrix is the slow release of rhBMP-2 in the first three days which allows for the avoiding of adverse effects in clinics caused by the rapid release of large amounts of rhBMP-2 from collageneous hydrogel.

[Results and prospects of interferone inducers using in infectious diseases treatment].

There is represented the current classification of interferon inducers of various chemical groups related to antiviral agents, described the mechanisms of synthesis of different types of endogenous interferon in blood serum. The effectiveness of methylglucamine acridonacetates in integrated treatment of chronic hepatitis C, tuberculosis with HIV infection background, chronic brucellosis, arboviral diseases, including West Nile fever, as well as influenza and acute respiratory infections are shown. For successful treatment of acute and chronic diseases with endogenous interferon inducers should be applied as early as possible, at an average level of viremia , to enhance the effect of drugs with the directed etiotropic action and immunomodulators, which achieves the optimal pharmacotherapeutic effect.

[The use of cytoflavin for the treatment of chronic brucellosis].

Clinical and pathogenetic efficacy of cytoflavin as a component of combined therapy of chronic brucellosis was tested in 50 patients who repeated its beneficial effect on the quality of life. The mechanism of cytoflavin action consists of lowering severity of endogenous intoxication and systemic inflammation.

[Improvement of treatment of inflammatory diseases in oral cavity].

In order to determine the anti-pathogenic clinical efficacy of cycloferon liniment in the combined treatment of herpetic stomatitis and periodontitis, medical examination and treatment of these disorders have been carried out in a group of 80 patients. It is established that the use of cycloferon liniment in the combined treatment of herpetic stomatitis and periodontitis decreases the infectious load in parodontal recess, reduces the manifestations of local inflammation, normalizes the immunity indices, and decreases the level of endogenous intoxication, which ensures the acceleration of recuperation processes and lowers the frequency of recurrences.

[Efficacy of cycloferon in the treatment of brucellosis].

Therapeutic potential of cyclopheron was evaluated as exemplified by the treatment of patients with chronic brucellosis. Positive clinical dynamics during combined treatment including cyclopheron is apparent as reduced duration of intoxication and inflammation, improved quality of life, decreased frequency of exacerbation of the infectious process and development of intercurrent diseases. These effects are shown to be due to immunomodulating activity of cyclopheron and suppression of lipid peroxidation. Also, the drug increases activity of antioxidants, decreases the levels of proinflammatory (and to a lesser degree of anti-inflammatory) cytokines.

[Improvement of parodontitis therapy of patients with HIV-infection].

For the purpose to determine the clinic-pathogenetic efficacy of cycloferon liniment in the combined therapy of periodontitis of patients with subclinical stage of HIV-infection medical examination and treatment of 40 patients was carried out. It was established that use of liniment cycloferon in the combined treatment of patients with subclinical stage of HIV-infection allowed to accelerate process of normalization of lipid peroxidation parameters and antioxidant potential of blood, to decrease infection load (herpes symplex virus I, Candida albicans, Staphylococcus aureus) in parodontal recess and evidence of local inflammation with reduction of activity of the tumours necrosis factor and interleukin 1beta, what provided acceleration of recuperation processes, lowering the frequency of parodontitis relapses.

[Parodontitis immunotropic therapy in patients with chronic viral and bacterial infections].

80 patients with chronic hepatitis and brucellosis were examined in order to determine the efficacy of cycloferon liniment in parodontitis treatment. It was established that the use of local cycloferon form in comprehensive treatment of parodontitis helped to speed up recovery process and to reduce the frequency of disease relapses. Normalization of lipid peroxidation and antioxidant defense processes indices with reduction of infectious loading of parodontal pockets fluid and normalization of local cytokines' status was noted.

[Clinical laboratory approaches to parodontitis treatment optimization].

In order to determine cycloferon liniment clinical-pathogenetic efficacy in comprehensive parodontitis therapy examination and treatment of 80 patients was done. It was determined that the cycloferon liniment use in comprehensive treatment of patients with parodontitis let to reduce infectious load in parodontal pockets and local inflammation intensity, to normalize the secretory immunoglobulin level and immune status indices that provided speed up of healing process and reduction relapse frequency.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Clinical and laboratory indicators of systemic inflammation and endotoxicosis in the evaluation of the activity of an infectious process].

Patients with chronic brucellosis underwent a complex clinical and laboratory studies to determine the indicators reflecting the degree of systemic inflammation and endotoxicosis. On the basis of an analysis of the findings, an integral quantitative approach was applied to describing the associations between various pathophysiological processes and clinical manifestations; criteria for the activity of the infectious process were objectivized, and the form of chronic brucellosis was defined. The mathematical simulation method was shown to be an adequate tool to objectivized the differential diagnosis of the clinical forms of the disease.