Pharmaceutical grade synthetic peptide Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu ameliorates DSS-induced murine colitis by reducing the number and pro-inflammatory activity of colon tissue-infiltrating Ly6G+ granulocytes and Ly6C+ monocytes.

A pharmaceutical grade synthetic tetradecapeptide Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu (GEPON) that mimics the ezrin protein hinge region was studied in dextran sodium sulphate-induced murine experimental colitis (DSS colitis). We report that GEPON intraperitoneal injections significantly attenuated DSS-induced pathological manifestations in the large intestine, bloody diarrhoea, and body weight loss in C57BL/6 mice. GEPON markedly inhibited the transcription rate of pro-inflammatory Il1b, Il6, and Nos2 genes in the colon tissue, in contrast with those encoding anti-inflammatory factors, such as Tgfb1, I10, and Arg1, whose transcription rate did not change significantly. Using flow cytometry, we found that GEPON treatment significantly reduced the accumulation of Ly6G+ granulocytes and Ly6C+ monocytes in the colon infiltrate of DSS colitis mice. Analysis of the mRNA level in myeloid cells sorted from the colon tissue revealed that GEPON had decreased the expression of pro-inflammatory genes in both colon-infiltrating Ly6G+ granulocytes and Ly6C+ monocytes, but not in Ly6C-CD64+ macrophages of DSS-treated mice. The direct anti-inflammatory impact of GEPON was shown in an in vitro culture of Ly6C+ monocytes, as evidenced by an inhibition of IL-1 beta and IL-6 mRNA expression. Taken together, our results demonstrated that GEPON had a pronounced therapeutic effect on ulcerative colitis in a laboratory mice model and provided evidence of its curative efficacy via inhibition of colon tissue inflammation by decreasing Ly6G+ granulocyte and Ly6C+ monocyte infiltration and by reducing their pro-inflammatory activities.

The TLR4 Agonist Immunomax Affects the Phenotype of Mouse Lung Macrophages during Respiratory Syncytial Virus Infection.

In the study, the effect of the TLR4 agonist Immunomax was investigated in vitro and in vivo. In particular, Immunomax was shown to polarize mouse bone marrow macrophages from the M0 and M2 states into the M1 state (ARG1 and iNOS mRNA expression levels were used to identify the mouse M1 and M2 phenotypes). Next, we investigated the prophylactic antiviral effect of Immunomax in both a model of mouse respiratory syncytial virus (RSV) infection and a model of RSV-induced bronchial asthma (BA) exacerbation. In the experiment with RSV-induced BA exacerbation, Immunomax-treated mice were characterized by a significant decrease of the viral load in lung homogenates, an increased amount of M1 macrophages in the lung, a tendency toward Th2-dependent ovalbumin-specific IgG1 antibodies decrease in blood serum, a significant increase in RSV-activated CD4+ T cells secreting IFNγ (Th1 cells), and a simultaneous significant decrease in the amount of CD4+ cells secreting IL-4 (Th2 cells) in the mouse spleen, which were detected by ELISPOT 1.5 months after experiment. These findings suggest that treatment with the TLR4 agonist Immunomax polarizes the immune response towards antiviral Th1 and may be used for short-term antiviral prophylaxis to prevent acute respiratory viral infections in asthmatics.

[Possible use of the growing liver biological set for hepatic recovery after toxic damage (an experimental study)]. / Vozmozhnosti biologicheskoi kombinatsii, poluchennoi iz rastushchei pecheni, dlia ee vosstanovleniia pri toksicheskom povrezhdenii (éksperimental'noe issledovanie).

The lack of acceptable pharmacological approaches for restoration of the injured liver is associated with complex of mechanisms involved in hepatic regeneration and with difficulty of the target selection. The aim of this research was to study the hepatoprotective function of the extract from both the growing and regenerating liver containing a natural set of factors crucial for the hepatic restoration. Extracts from both regenerating liver of rats after 70% hepatic resection and the growing liver of neonatal pigs were obtained using own original technique. The set of resultant extracts was named as the hepatic regeneration set (HRS). HRS fractionation was carried out using the Toyopearl HW-50S sorbent. The efficiency of HRS and its fractions was estimated using a model of the mouse liver thioacetamide injury and monitoring hepatic enzyme activity in blood serum. The activities of AST and ALT in intact animals were 50 U/l and 80 U/l, respectively; after thioacetamide administration they increased to 2059±212 U/l and 4280±440 E/l, respectively (p<0.05). Treatment of injured animals with HRS from the rat regenerating liver resulted in a significant decrease of transaminase activities to 924±148 U/l (AST; p<0.05) and 1633±308 U/l (ALT; p<0.05). A similar effect was observed after treatment with HRS from the neonatal pig liver: the AST decreased to 937±138 U/l (p<0.05), while ALT activity decreased to 1710±237 U/l (p<0.05). HRs fractionation resulted in identification two active fractions characterized by much higher (8-29) hepatotropic effect that that of the whole extract. These fractions contained peptide/protein components with the range of molecular mass of 3-60 kDa (fraction 1) and 3-25 kDa (fraction 2a). Fraction 1 also contained some polynucleotides in fraction 1. Subsequent studies of these fractions exceeding the hepatotropic effect of original HRS is clearly needed to identify their individual components by immunochromatography methods, ELISA, MRM mass spectrometry and quantitative PCR.

Regulation of the target protein (transgene) expression in the adenovirus vector using agonists of toll-like receptors.

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.

[Comparative analysis of the immune response to DNA constructions encoding hepatitis C virus nonstructural proteins].

A promising approach to construction of antiviral vaccines consists in activation of cellular immunity with the DNA vaccines. The goal of this work was to evaluate the efficacy of genetic immunization of mice with DNA pcNS3-NS5B encoding five hepatitis C virus (HCV) nonstructural proteins: NS3, NS4A, NS4B, NS5A, and NS5B in comparison with plasmids containing genes of same individual nonstructural proteins. The DNA constructions were injected intramuscularly in DBA mice three times. The humoral immune response was assessed with ELISA; cellular immune response--in blast transformation reaction, by quantitation of CD4+ and CD8+ T cell proliferation using flow cytofluorometry, by intracellular synthesis and secretion of IFN-gamma and IL-2 in ELISpot and ELISA. It was found that the functionally active T cell response was achieved to antigens presenting NS3, NS4, NS5A, and NS5B epitopes of different HCV genotypes in response to pcNS3-NS5B plasmid and was stronger than that to plasmids carrying individual genes. A high proliferation rate of CD4+ T cells, secretion of IL-2 and IFN-gamma, induction of anti-NS3 and anti-NS5B IgG2a were demonstrated. These findings indicate that DNA construction pcNS3-NS5B is one of promising candidates for anti-HCV vaccine developing.

[DNA immunization with a plasmid containing gene of hepatitis C virus protein 5A (NS5A) induces the effective cellular immune response].

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti-NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against abroad spectrum of NSSA epitopes as assessed by T-cell proliferation andsecretion of antiviral cytokines IFN-gamma and IL-2. In in vitro T-cell stimulation experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.

[Postinfection irritable bowel syndrome].

AIM: To characterize pathogenesis, clinicolaboratory criteria and treatment of postinfection irritable bowel syndrome (PICS). MATERIAL AND METHODS: The examination including histological study of the small and large intestine mucosa, polymerase chain reaction (PCR), coagglutination reaction using shigella, salmonella, yersinia, campilobacter jejuni diagnosticums, indirect hemagglutination reaction for identification of antibodies to these agents in the blood serum was conducted in 750 patients with PICS. Fecal seeding on selective media was made as well as the respiratory test for bacterial growth in the small intestine. Immune status was studied with laser cytometry, chemiluminescence, immunodiffusion, immunofluorescence, flow laser cytofluorometry. Personality profile was assessed by MMPI. RESULTS: PICS was diagnosed in 599 (79.9%) of 750 patients. Most of them had diarrhea, abnormal fecal microflora, antigens of acute intestinal infection agents in circulating immune complexes of the serum and coprofiltrates. Immune system was characterized by low phagocytic activity, attenuation of cell and humoral immunity. Etiotropic and pathogenetic treatment including intestinal antiseptics, probiotics and immunomodulators produced persistent remission during a year in 79.3% PICS patients. CONCLUSION: PICS is described which differs from ICS by registration of markers of acute intestinal infections in biological media, bacterial overgrowth in the small intestine and dysbiosis in the large intestine, immunodeficiency. A positive response was observed to treatment with intestinal antiseptic and enterosorbent drugs, probiotics and immunomodulators.

Trophic venous ulcers and their relationship with the immune status.

This paper describes the results of an original study into the systemic and local immunity in 25 patients with trophic venous ulcers. The authors discovered a number of significant parameters demonstrating the influence of the immune status on the formation of venous ulcers and tempo of their healing. They revealed previously unknown relationships between the immune status, clinical manifestations arid natural history of venous ulcers. Presented herein are the first results of the local use of the immunomodulator gepon and evidence for its efficacy in the treatment of chronic trophic venous ulcers.

[Immune status of patients with cardiac arrhythmias: idiopathic and in primary heart disease].

AIM: A detailed description of immune status abnormalities of adult patients with heart arrhythmia either idiopathic or in combination with primary heart disease such as chronic myocarditis and dilated cardiomyopathy (DCMP). MATERIAL AND METHODS: Eighty two consecutive patients aged 16-57 years admitted to the L.A. Myasnikov Institute of Clinical Cardiology (Moscow) for heart arrhythmia were studied. Among them 35 patients had idiopathic heart arrhythmia (IHA, group 1) with no evidence of any primary heart disease, while other 47 patients (group 2) had heart arrhythmia combined with primary heart disease (chronic myocarditis or DCMP). In group 1 ventricular arrhythmia was recorded in 27 patients (12 cases with ventricular tachyarrhrythmia ?VTA, 15 cases with ventricular extrasystolia- VE). Supraventricular heart arrhythmia was found in 6 patients (3 cases of constantly recurring supraventriccular tachycardia, 2 cases of paroxysmal and 1 with constant atrial fibrillation). The intermittent atrioventricular block of the second-third degree was recorded in 2 patients. The patients of group 2 were divided into subgroups 2a, 2b and 2c. In subgroup 2a (patients with DCMP without signs of heart failure) ventricular arrhythmia was found in 7 patients (VT ? 5, VE ? 2). Supraventricular arrhythmia was recorded in 7 patients 5 of which had constantly recurring supraventricular tachycardia, 1 ? paroxysmal and 1 constant atrial fibrillation. In subgroup 2b (DCMP patients with obvious signs of heart failure) ventricular arrhythmia was recorded in 12 patients, among them 6 had VT and 6 ? VE, 2 ? constant atrial fibrillation). In subgroup 2c (patients with chronic myocarditis) ventricular arrhythmia was in 7 patients (VT ? 5, VE ? 2), constant atrial fibrillation ? in 2, heart conduction abnormalities ? in 3 patients, atrioventricular block of the first or second degree ? in 2, sick sinus syndrome ? in 1. To verify the diagnosis, all the patients have undergone physical examination, blood cell counts and biochemical tests, urine clinical analysis, ECG and ultrasound heart examination as well as 24h ECG monitoring. On demand, bicycle exercise test or treadmill test, coronaroangiography, endomyocardial biopsy and invasive electrophysiological examination were made. RESULTS: Immune status abnormalities found in patients with heart arrhythmia both idiopathic and combined with primary heart diseases such as chronic myocarditis and DCMO correspond to immune defense response during chronic infection. Activation of different anti-infection defense mechanisms was recorded in patients with idiopathic heart rhythm and conductivity abnormalities. Immune deficiency was found in arrhythmia and conductivity abnormalities combined with primary heart diseases (chronic myocarditis or DCMP). A positive correlation exists between the degree of immune defense failure and reduction of myocardial contractility. CONCLUSION: There exists a characteristic pattern of immune status abnormalities in patients with arrhythmia, both idiopathic or combined with primary heart disease (myocarditis, DCMP). The abnormalities depend on severity of arrhythmia, intensity of inflammatory processes in the myocardium and on the degree of left ventricular contractility dysfunction in patients with primary heart diseases.

[Epitope mapping of antigenic determinants of hepatitis C virus proteins by phage display].

Study of individual hepatitis C (HCV) proteins could help to find a molecular structure and conformation, localization of antigenic and immunogenic determinants, to reveal of protective epitopes. It is necessary for practical medicine - development of diagnostic test-systems, vaccines and therapeutics. Linear and conformation dependent epitopes of HCV proteins was localized in this work and immunogenic properties of phage displayed peptides screened on monoclonal antibodies to HCV proteins have been investigated. Eleven epitopes of four HCV proteins have been studied. Three epitopes was found as linear, two epitopes were dependent on secondary structure of proteins and one epitope was dependent on tertiary structure of NS3 protein. Aminoacid sequences of other determinants have been determined and the distinct localization of these determinants will be continued after discovering of tertiary structure of HCV proteins. It was shown, that phage mimotope 3f4 is immunogenic and could induce specific hu- moral immune response to NS5A HCV protein. The data obtained could be useful for improving of HCV diagnostic test-systems, studying of amino acid substitutions and its influence on antigenic properties of the HCV proteins. The results could help to study an immune response in patients infected with different genotypes of HCV. Phage displayed peptides mimicking the antigenic epitopes of HCV proteins could be applied to development of HCV vaccine.

[Administration of the immunomodulator gepon after tonsillectomy].

The study included 37 patients with chronic tonsillitis. Before and after tonsillectomy all the patients' humoral and cellular immunities were studied. Local thermometry and physiological test "pharynx" were made. For optimization of early postoperative period, all the tonsillectomized patients were given a domestic immunomodulator with local anti-inflammatory activity gepon. The results of gepon therapy allowed conclusion that local use of immunomodulator gepon early after bilateral tonsillectomy is justified and can be recommended for wide clinical practice.

[Use of the Gepon immunomodulator for ulcerative colitis treatment].

The efficacy of the Gepon immunomodulator was studied in 36 patients with ulcerative colitis (UC) having distal lesions in the resistant form. Taking into account the fact that immunological abnormalities play the main role in UC pathogenesis, the drug with immunomodulatory action was used for overcoming resistance to basic anti-inflammatory drugs. The clinical and immunological reaction to the used drugs was found to be ambiguous. Most of the patients (83.3%) had clinical and endoscopic amelioration after taking Gepon and managed to overcome the resistance to basic anti-inflammatory drugs.

[The antiviral activity of peptide immunomodulator "Gepon" in experimental infections caused by herpes simplex viruses types 1 and 2]. / Protivovirusnaia aktivnost' peptidnogo immunomoduliatora "Gepon" pri éksperimental'nykh infektsiiakh, vyzvannykh virusami prostogo gerpesa tipov 1 i 2.

Experimental data are reported on the antiviral activity of peptide immune-modulator "Gepon" in infections caused by herpes simplex virus (HSV), types 1 and 2, in vitro and in vivo. The drug proved to be non-toxic and did not possess any viricidal action. Its antiviral effect was registered in experiments with a multiple-infection cells' culture. The maximal effect was a 100-fold reduction of the viral titer when it was used in a concentration of 6.25 mcg/I as a preventive measure 24 hours before triggering the infection. The mentioned drug's effect was reliably higher than its use within a treatment scheme (1 hour after the infection onset). "Gepon" possessed the reliable protecting qualities (36% of protection with a mean increase of the infected mouse life by 1.9 days) in experiments with intraperitoneally infected mice (10 LD50/mouse of HSV, type 2), when the drug was administered in doses of 0.1 and 1 mcg/mouse.

[The antiviral activity of the peptide immunomodulator "Gepon" in experimental models of street rabies virus]. / Protivovirusnaia aktivnost' peptidnogo immunomodulatora "Gepon" v éksperimentakh na modeli ulichnogo beshenstva.

Intracerebral injections of the Gepon drug were shown to be non-toxic when administered to outbred white mice, 0.01-0.1 micrograms. The drug protects 30-40% of mice (p < 95-99%) in contamination by street rables virus. The animals' protection depends on a dose and drug application scheme. The most pronounced protection was observed in its intramuscular administration, 0.1 microgram in the virus locus (infection opening). It should be necessarily pointed out that such pronounced protection (40%; p < 0.01) was noted within the therapeutic scheme of drug administration after virus infected the animals. A new method is described, which is related with evaluating the antiviral drugs' quality, i.e. with registering the dynamics of morbidity and death of infected animals.

[Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]. / Immunomoduliator "Gepon" podavliaet replikatsiiu virusa gepatita C v kul'ture kletok cheloveka in vitro.

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.

[Stimulating effect of polyion on the production of cytolytic T lymphocytes in a cell culture in vitro]. / Stimuliruiushchee vliianie poliona na produktsiiu tsitoliticheskikh T-limfotsitov v kul'ture kletok in vitro.

Introduction of polyion "vegetan" at the concentrations varying from 20 to 100 micrograms/ml into mixed lymphocyte culture (MLC) with insufficient antigenic stimulus, low immunogenic MLC, for the whole of the incubation period, leads to 30-70% increase in T-lymphocyte cytolytic activity. The effect of vegetan on the cytolytic activity of T-lymphocytes from normal MLC is less marked. In the in vivo model of primary synthesis of antibodies against heterologous erythrocyte antigens, vegetant causes 20-50-fold enhancement of a weak immune response compared to 1.5-2-fold when the latter approaches the highest levels. These findings indicate an inverse relationship between the immunostimulating activity of vegetan and the levels of the immune response, the latter being the target of activation. Vegetan was shown to induce more than 2-fold increase in the proliferation in both types of MLC as well as in mouse spleen lymphocyte monoculture. It is reasonable to propose that the preparation may stimulate the proliferation of both T-killers and other lymphocyte subpopulations.

[Increase in the permeability of the lymphocyte plasma membrane for uni- and bivalent cations and low-molecular metabolites following exposure to mitogenic polyanions]. / Povyshenie pronitsaemosti plazmaticheskoi membrany limfotsitov dlia odno- i dvukhvalentnykh kationov i nizkomolekuliarnykh metabolitov pod deistviem mitogennykh polianionov.

The paper is concerned with a study of the molecular mechanisms responsible for activation of B lymphocyte division by polyanions, polyacrylic acid (PAA) and dextran sulfate (DS). The mitogenic doses of PAA and DS were discovered to provoke an early increase in lymphocyte plasma membrane permeability. The cell membrane permeability for K+, Ca2+ and for labeled thymidine and uridine was measured in murine spleen cultures in vitro The K+ outflow from the cells was recorded according to variation in K+ concentration in the extracellular medium with the aid of a selective valinomycin electrode. The intensity of cell penetration by exogenous 45Ca, 3H-uridine or 3H-thymidine was determined by radioindicator analysis of the cytoplasma isotope pool. One to 2 min after adding the mitogenic doses of PAA or DS, the K+ outflow from lymphocytes markedly increased. It has been proved that this effect is not connected with inhibition of Na+, K+-ATPase of the plasma membrane. The increased membrane permeability for 45Ca and 3H-uridine was recorded 30-40 min after lymphocyte activation with the polyanion, that for 3H-thymidine was seen later (after 4-6 h). It is assumed that the differences between the time of recording high accumulation in the cytoplasm of 45Ca, 3H-uridine and 3H-thymidine and the time of recording high outflow of K+ from lymphocytes are determined by the differences in the sensitivity of the methods applied.

[Analysis of the cyclase enzyme activity of the cell membrane following lymphocyte stimulation with a mitogenic polyanion]. / Analiz aktivnosti tsiklaznykh fermentov kletochnoi membrany posle stimuliatsii limfotsitov mitogennym polianionom.

Stimulant action of the mitogenic polyanion, polyacrylic acid (PAA) was investigated in mouse lymphocyte culture in vitro. B cell division was induced by "impulsive" PAA treatment. Shortly after PAA treatment the activity of the membrane enzymes, adenylate and guanylate cyclases, was assayed according to the changes in the concentration of cAMP and cGMP. The effect of PAA on the time course of cAMP and cGMP in lymphocytes was compared to the effect of B cell mitogen of other chemical nature--bacterial lipopolysaccharide (LPS). PAA was demonstrated to produce no effect on the activity of membrane cyclase enzymes. On the contrary, following LPS addition guanylate cyclase in the lymphocyte membrane was activated within the first 5-10 minutes. Later on (after 2h) the cells activated with LPS showed an increase in adenylate cyclase activity. By the 12th-24th hour the concentration of cAMP in the LPS-stimulated cells reached 250% of the control level. The differences are discussed between the mitogenic polyanion (PAA) and the lipid-modifying mitogen (LPS) in the molecular mechanisms by which the lymphocyte responses are activated.