NLRP6 function in inflammatory monocytes reduces susceptibility to chemically induced intestinal injury.

NLRP6 is a member of the Nod-like receptor family, whose members are involved in the recognition of microbes and/or tissue injury. NLRP6 was previously demonstrated to regulate the production of interleukin (IL)-18 and is important for protecting mice against chemically induced intestinal injury and colitis-associated colon cancer. However, the cellular mechanisms by which NLRP6 reduces susceptibility to colonic inflammation remain unclear. Here, we determined that NLRP6 expression is specifically upregulated in Ly6Chi inflammatory monocytes that infiltrate into the colon during dextran sulfate sodium (DSS)-induced inflammation. Adoptive transfer of wild-type (WT) Ly6Chi inflammatory monocytes into Nlrp6-/- mice was sufficient to protect them from mortality, significantly reducing intestinal permeability and damage. NLRP6-deficient inflammatory monocytes were defective in tumor necrosis factor α (TNFα) production, which was important for reducing DSS-induced mortality and was dependent on autocrine IL-18 signaling by inflammatory monocytes. Our data reveal a previously unappreciated role for NLRP6 in inflammatory monocytes, which are recruited after DSS-induced intestinal injury to promote barrier function and limit bacteria-driven inflammation. This study highlights the importance of early cytokine responses, particularly NLRP6-dependent and IL-18-dependent TNFα production, in preventing chronic dysregulated inflammation.

Long-term, high-level hepatic secretion of acid α-glucosidase for Pompe disease achieved in non-human primates using helper-dependent adenovirus.

Pompe disease (glycogen storage disease type II (GSD-II)) is a myopathy caused by a genetic deficiency of acid α-glucosidase (GAA) leading to lysosomal glycogen accumulation causing muscle weakness, respiratory insufficiency and death. We previously demonstrated in GSD-II mice that a single injection of a helper-dependent adenovirus (HD-Ad) expressing GAA resulted in at least 300 days of liver secretion of GAA, correction of the glycogen storage in cardiac and skeletal muscles and improved muscle strength. Recent reports suggest that gene therapy modeling for lysososomal storage diseases in mice fails to predict outcomes in larger animal models. We therefore evaluated an HD-Ad expressing GAA in non-human primates. The baboons not only tolerated the procedure well, but the results also confirmed that a single dose of the HD-Ad allowed the livers of the treated animals to express and secrete large amounts of GAA for at least 6 months, at levels similar to those achieved in mice. Moreover, we detected liver-derived GAA in the heart, diaphragm and skeletal muscles of the treated animals for the duration of the study at levels that corrected glycogen accumulation in mice. This work validates our proof-of-concept studies in mice, and justifies future efforts using Ad-based vectors in Pompe disease patients.

[Wound bacterial flora by different methods of second-stage implant surgery].

Microbiological studies of wound around dental implants disclosed by different methods. Significant differences between the methods have not been identified that may be important in the choice of tactics of the operation.

[Impact of various second-stage implant surgery methods on adjacent gingival tissues microcirculation].

The paper presents the rezults of laser doppler flowmetry (LDF) studies of microcirculation in gingival tissues around dental implants disclosed by different methods. Microhemodynamics in gingival tissues when using a scalpel recovered after 14 days, carbon dioxide laser -- 7 days and erbium laser -- 3 days, which may be important in the choice of the operation tactics.

Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2.

The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

[Automated decision making support system for urologists on the prediction and the prevention of stone formation in urolithiasis].

This article describes a decision making support system for urologists on the prediction and management of preventive interventions for urolithiasis using fuzzy logic decision-making device. Dictionary of informative signs and alphabet of classes are formed. The formulas for calculating the membership functions according to the known features are developed; these formulas allow to calculate the certainty factors for pertaining of inspected object to the desired class by means of iterative rules of rule of logical inference. Based on comparison of the values obtained with the threshold certainty factors, dephasification of conclusion is produced. In accordance with the obtained decision rules, control algorithm for the prevention measures in urolithiasis is developed. To test the effectiveness of "operation" of the synthesized decision rules, the certainty factors were calculated or 200 patients with urolithiasis, which were divided into two groups according to the results of observation during the year depending on the presence of recurrence. The analysis of the intersection of histograms of distribution of coefficient values showed high diagnostic efficiency (0.94) of synthesized decision rules.

[Methods for the Crimean-Congo hemorrhage fever diagnosis].

Several distinct methods currently used for the Crimean-Congo Hemorrhage Fever diagnosis (CCHF) were suggested in this work. We demonstrated that the ELISA-based diagnostic kits, which are based on CCHFV recombinant antigens produced in E. coli cells, still possessed a few substantial shortcomings, which are yet to be addressed. In this work we presented the development of the unique CCHFV detection system fully based on reverse transcription--nested two-step polymerase chain reaction (RT-PCR). Our RT-PCR-based diagnostic kit for the CCHFV detection is now commercially available. We also developed a simple screening method for the samples, potentially containing CCHFV, which is based on restriction fragment length polymorphism (RFLP) amplicons analysis and allows for preliminary genotyping of the CCHFV isolates.

[The production of high-yielding strain of rubella virus from wild type virus extracted from a patient in the Western Siberia in 2006].

The study was targeted to investigate the propagation of rubella virus in the cell cultures of various origins and with different cultivation methods. The high-yielding strain of rubella virus was produced. The "spinner-culture" cultivation method was applied and the strain's RNA was detected in 10-8 dilution in real time mode. This strain is supposed to be used in preparation of the standard antigen to implement in the development of immune enzyme test system targeted to the rubella virus specific antibodies.

[Differentiation of genetic variants of Crimean-Congo hemorrhagic fever virus].

The paper describes a simple, rapid screening of samples potentially containing Crimean-Congo hemorrhagic fever (CCHF) virus strains, by applying the restriction analysis of amplicones, for the differentiation of CCHF virus genotypes that are characteristic of Europe from virus biovariants uncharacteristic of this area, this technique requiring no sequence at the first stage. For this screening, the authors propose to use the PCR fragment of CCHF L segment that comprises a variable region, as well as Alul and Haelll restriction endonucleases. The screening scheme proposed for samples potentially containing CCHF virus may aid investigators to monitor in order to detect uncharacteristic genotypic virus variants in the Russian Federation and other European countries.

[Entification of the Rubella virus genotype 1H in Western Siberia].

Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes all three Rubella structural proteins (C, E2, and E1), was implemented. This analysis provides characterization of this strain and classifies it as 1H genotype, thereby correcting previous classification of this strain based upon shorter nucleotide sequence, only encoding E1 protein. Therefore, this study identified the genotype of the Rubella virus not previously detected in Western Siberia (and even entire Russian Federation), which highlights the importance of more extensive characterization of genetic variability of the Rubella virus, especially with regard to potential influence of vaccination on the Rubella virus mutagenesis.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses.

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing.

[Automatic decision support system in prognostication, diagnosis, treatment and prophylaxis of chronic prostatitis].

A great variety of subjective and objective disorders related with chronic prostatitis make difficulties in its diagnosis. The proposed principle of chronic prostatitis classification identifies 33 clinically significant variants of diagnosis by combination of classification grades. The system of fuzzy decisive rules for prediction and differential diagnosis of chronic prostatitis supports decision on the level 0.9 and higher. Use of energetic characteristics of diagnostically significant biologically active points raises quality of prognosis of the onset and exacerbation of chronic prostatitis and helps to select optimal schemes of acpuncture. Application of decision support systems for urological decision on prognosis, diagnosis, prevention and treatment of chronic prostatitis improves quality of medical aid and adequate management of patients with chronic prostatitis.

CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies.

Human complement receptors 1 and 2 are well described as important regulators of innate and adaptive immune responses, having pivotal roles in regulating complement activation (CR1) and B-cell maturation/survival. In contrast, the role of the murine homologs of CR1 and CR2 (mCR1/2) have been primarily defined as modulating activation of the adaptive immune system, with very little evidence available about the role of mCR1/2 in regulating the innate immune responses to pathogens. In this paper, we confirm that mCR1/2 plays an important role in regulating both the innate and adaptive immune responses noted after Adenovirus (Ad)-mediated gene transfer. Our results uncovered a novel role of mCR1/2 in downregulating several complement-dependent innate immune responses. We also unveiled the mechanism underlying the complement-dependent induction of neutralizing antibodies to Ad capsids as a CR1/2-dependent phenomenon that correlates with B-cell activation. These results confirm that Ad interactions with the complement system are pivotal in understanding how to maximize the safety or potency of Ad-mediated gene transfer for both gene therapy and vaccine applications.

Complex interactions with several arms of the complement system dictate innate and humoral immunity to adenoviral vectors.

The complement system is known to play critical roles in pathogen identification, initiation of innate immune responses and facilitation of adaptive immune responses. Several studies have suggested that recombinant adenoviruses (rAds) interact with proteins of the complement system within minutes of administration. In this study, we assessed the roles of the alternative (Factor B), classical (C1q and C4) and common (C3) arms of the complement system in the innate and humoral response to systemic rAd administration using mice genetically deficient for each of these functions. Although most plasma cytokines and chemokines induced by Ads appeared to be elicited in a C3-dependent manner, we found that rAd-induced thrombocytopenia was dependent on Factor B and C3, implicating the alternative pathway as responsible for this response. Alteration of the complement-dependent transcriptome response after rAd-induced liver gene expression was also found to be Factor B- and C3-dependent. Ad interactions with the classical and alternative arms of the complement system can also be redundant, as many complement-dependent, Ad-induced innate immune responses appeared to be primarily C3-dependent. We also identified a C3 dependence of Ad-mediated induction of the nuclear factor-kappaB (NF-kappaB) activation pathway. Finally, we confirmed that humoral immune responses to the vector capsid, and the transgene it encodes, are also complement-dependent.