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Pollution of the environment by crude oil and oil products (represented by various types of compounds, mainly aliphatic, mono- and polyaromatic hydrocarbons) poses a global problem. The strain Pseudomonas veronii 7–41 can grow on medium-chain n-alkanes (C8–C12) and polycyclic aromatic hydrocarbons such as naphthalene. We performed a genetic analysis and physiological/biochemical characterization of strain 7–41 cultivated in a mineral medium with decane, naphthalene or a mixture of the hydrocarbons. The genes responsible for the degradation of alkanes and PAHs are on the IncP-7 conjugative plasmid and are organized into the alk and nah operons typical of pseudomonads. A natural plasmid carrying functional operons for the degradation of two different classes of hydrocarbons was first described. In monosubstrate systems, 28.4% and 68.8% of decane and naphthalene, respectively, were biodegraded by the late stationary growth phase. In a bisubstrate system, these parameters were 25.4% and 20.8% by the end of the exponential growth phase. Then the biodegradation stopped, and the bacterial culture started dying due to the accumulation of salicylate (naphthalene-degradation metabolite), which is toxic in high concentrations. The activity of the salicylate oxidation enzymes was below the detection limit. These results indicate that the presence of decane and a high concentration of salicylate lead to impairment of hydrocarbon degradation by the strain.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pmr in mice. Complex therapy with polymyxin B (1 mg/kg) and 4-hexylresorcinol (30 mg/kg) led to cure in 80%; in 20% of these mice, no bacterial cells were found. After treatment with polymyxin B alone, only 50% animals survived and all of them contained bacterial cells. Comparative analysis of the results of monotherapy and combined treatment indicates that 4-hexylresorcinol not only increases the efficiency of antibiotic, but also minimizes persistence of the infection agent and therefore, the risk of development of antibiotic resistance.

Here, we review the data on mechanisms inhibiting cytotoxic effect of anthrax toxin on the immune system cells. Various disease forms, immunopathogenesis and contemporary methods for anthrax treatment are discussed. In addition, an anthrax toxin was outlined, whereas structural and functional organization of the protective antigen, lethal and edema factors was detailed. A mechanism for association of a protective antigen and lethal factor, protective antigen and edema factor leading to formation of a lethal toxin and edema toxin, respectively, was described. Participation of protective antigen domains in the process of interaction with surface receptors of imunocompetent cells as well as features of binding a protective antigen with lethal factor and edema factor are discussed. A mechanism of endosomal toxin complex internalization and subsequent transfer of effector molecules to the cytosol are described. Effects of the lethal factor and the edema factor on components of eukaryotic cells as well as cytotoxicity mechanisms are analyzed. The approaches to block anthrax toxin action at various stages of toxicoemia have been analyzed based on previously uncovered sequential signs of cytotoxic activity for Bacillus anthracis toxins. Currently available chimeric and humanized monoclonal antibodies are capable of neutralizing B. anthracis toxins at diverse assembly stages, particularly considering the drugs inhibiting: inter-receptor interaction between protective antigen with eukaryotic cells; furin-like enzymes activating prepore assembly; protective antigen oligomerization; binding of the lethal factor or edema factor to the protective antigen; translocation of the lethal factor or the edema factor into cell cytosol; transport of protective antigen with lethal factor or edema factor from endosomes; enzymatic activity of lethal factor or edematous factor. The anti-toxin agents approved for anthrax prevention and treatment in Russia and worldwide are discussed. The limitations of anti-toxin agents and perspectives for their improvement are also described including inhibition of lethal factor activity, interference with integration of toxin components, blockade of interactions between toxic complexes and immune cell receptors.

The review presents a general description of therapeutic monoclonal antibodies, cell lines used to obtain them, characterizes the reasons for the immunogenicity of recombinant antibodies, and approaches used to eliminate the side effects of therapeutic monoclonal antibodies. The focus is on resolving the immunogenicity problems of fully human therapeutic monoclonal antibodies. The most attention are concentrated on the data of antibody-producing cell genomic editing to increase the yield of the product, the stability of expression of the recombinant protein and reduce its immunogenicity. Modern methods of site-directed modification (zinc finger method, TALEN and CRISPR/CAS9) for editing the genome of the CHO cell line are analyzed. The strategies of genomic editing choice carrying out taking into account the advances of omix technologies are discussed. Approaches to increase the life span of producer cells are considered, including an increase in the expression of anti-apoptotic signals and the deletion of proapoptotic genes, an increase in the duration of the cell cycle of cells in the G0/G1 phase. The approaches used to regulate the posttranslational modification of monoclonal antibodies are considered. Significant part of the review are devoted to the discussion of the spesificity and differences of glycosylation, galactosylation and sialization of monoclonal antibodies in different expression systems and the associated different degree of immunogenicity of monoclonal antibodies. The main approaches to the regulation of the synthesis of monoclonal antibodies at the stage of translation using non-coding RNA are considered.

Currently, live anthrax vaccine has been used for vaccine prophylaxis in Russia and neighbor countries for seve ral decades, but precise mechanism of post-vaccination protection mechanism remains unclear. Here, we provide data on examining serum antibody level against protective antigen (PA) and lethal factor (LF) in repeatedly vaccinated volun teers at early stage (5–8 days) and 1 month after the performing pre-scheduled annual revaccination. Amount of peripheral blood antigen-specific memory T cells after previous vaccinations was analyzed. It was showed that frequency of CD3+CD45RO+CD62L– memory effector T cells was increased in the majority of volunteers on day 5-8 day after performing pre-scheduled annual revaccination that peaked at day 7 by elevating it by 2-fold compared with the control group. Percentage of anthrax-specific central memory T cells did not increase at early stage after vaccination, whereas amount of activated CD3+CD45RO+CD62L+HLA-DR+ subset within this memory T cell population was increased. Likewise, percentage of activated CD3+CD45RO+CD62L–HLA-DR+ effector memory T cell subset was also increased. Moreover, serum anti-PA IgG were detected on day 5–8 day after pre-scheduled annual revaccination in half of volunteers, whereas anti-LF IgG were found only in a single volunteer. Rapidly elevated amount of serum anthrax-specific IgG antibodies evidences about sustained memory B cell response in peripheral blood samples in volunteers after pre-scheduled annual revaccination. However, percentage of CD19+CD27+ memory B cells was not significantly elevated at early stage after revaccination that tended to increase. Both helper and cytotoxic T cell subsets were activated on day 5–8 after revaccination revealed by upregulated expression of CD69 and/or CD25 markers, with the latter predominantly found on helper T cells, thereby accounting for their high proliferative activity, whereas the former — on cytotoxic T cell subsets. Detection of anti-PA IgG antibodies correlates with protection against anthrax, which was confirmed in animal models. Unfortunately, the level of serum anti-PA IgG antibodies rapidly declines after vaccination. Ability of memory B cells to rapidly trigger production of anthrax-specific antibodies in response to revaccination suggests that anti-anthrax immunity may be evaluated by measuring frequency of peripheral blood anthrax-specific memory B and T cells.

Neutralization of the lethal toxin of Bacillus anthracis is an important topic of both fundamental medicine and practical health care, regarding the fight against highly dangerous infections. We have generated a neutralizing monoclonal antibody 1E10 against the lethal toxin of Bacillus anthracis and described the stages of receptor interaction between the protective antigen (PA) and the surface of eukaryotic cells, the formation of PA oligomers, assembly of the lethal toxin (LT), and its translocation by endocytosis into the eukaryotic cell, followed by the formation of a true pore and the release of LT into the cell cytosol. The antibody was shown to act selectively at the stage of interaction between Bacillus anthracis and the eukaryotic cell, and the mechanism of toxin-neutralizing activity of the 1E10 antibody was revealed. The interaction between the 1E10 monoclonal antibody and PA was found to lead to inhibition of the enzymatic activity of the lethal factor (LF), most likely due to a disruption of true pore formation by PA, which blocks the release of LF into the cytosol.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pm

The purpose of this work was to obtain genus-specific monoclonal antibodies against the

Ever decreasing efficiency of antibiotic treatment due to growing antibiotic resistance of pathogenic bacteria is a critical issue in clinical practice. The two generally accepted major approaches to this problem are the search for new antibiotics and the development of antibiotic adjuvants to enhance the antimicrobial activity of known compounds. It was therefore the aim of the present study to test whether alkylresorcinols, a class of phenolic lipids, can be used as adjuvants to potentiate the effect of various classes of antibiotics. Alkylresorcinols were combined with 12 clinically used antibiotics. Growth-inhibiting activity against a broad range of pro- and eukaryotic microorganisms was determined. Test organisms did comprise 10 bacterial and 2 fungal collection strains, including E. coli and S. aureus, and clinical isolates of K. pneumoniae. The highest adjuvant activity was observed in the case of 4-hexylresorcinol (4-HR), a natural compound found in plants with antimicrobial activity. 50% of the minimal inhibitory concentration (MIC) of 4-HR caused an up to 50-fold decrease in the MIC of antibiotics of various classes. Application of 4-HR as an adjuvant revealed its efficiency against germination of bacterial dormant forms (spores) and prevented formation of antibiotic-tolerant persister cells. Using an in vivo mouse model of K. pneumoniae-induced sepsis, we could demonstrate that the combination of 4-HR and polymyxin was highly effective. 75% of animals were free of infection after treatment as compared to none of the animals receiving the antibiotic alone. We conclude that alkylresorcinols such as 4-HR can be used as an adjuvant to increase the efficiency of several known antibiotics. We suggest that by this approach the risk for development of genetically determined antibiotic resistance can be minimized due to the multimodal mode of action of 4-HR.

Nowadays healthcare-associated infections caused Pseudomonas aeruginosa remain actual problem due to P. aeruginosa resistance to a wide range of antimicrobials and its ability to form biofilms that are 1000 times more resistant to antibiotics than free-living (plankton) cultures. P. aeruginosa biofilms forming is regulated by Quorum Sensing (QS) communication system which is controlled by inhibitors (Quorum sensing inhibitors, QSIs). An essential role in stabilization of biofilms belongs to extracellular DNA (eDNA) as a structural polyanionic polymer whereas its genetic function is not applicable. In addition, internal signal molecules c-di-GMP, cascade Gac/Rsm also participate in formation of P. aeruginosa biofilms. A review provides a detailed description of the biofilm molecular regulation by means of QS inhibitors and QS modulators of signaling molecules QS.

An efficient method of highly sensitive, specific determination of botulinic type A neurotoxin (which allows the detection of a toxin in a concentration from 1 pg/mL (10 fg per one PCR reaction) was created based on the technology of immuno-PCR. Solid phase consisting of paramagnetic organo–silicious particles carrying a detection complex of the toxin heavy chain with a pair of polyclonal antibodies to it was the most appropriate in the optimal scheme of the analysis. One of the antibodies bound to DNA (which is a matrix for PCR amplification) by means of biotin–neutravidin interaction. Matrix DNA was a molecular “net” generated by biotinylated DNA molecules with tetravalent neutravidin. The binding signal was detected by real time PCR (by the TaqMan method). An immuno-PCR method for determination of type A botulotoxin was developed.

Category A select agents continue to be major threat to human population both as naturally occurring diseases and as potential weapon of bioterrorists. Anthrax and botulism are probably the most threatening agents as both have virtually uncontrolled natural reservoirs from which they can be isolated and propagated. Available specific antitoxin therapy of both diseases is outdated; its efficiency is questionable as well as safety of reactogenic or human-derived components used in treatment. Highly sensitive toxin detection techniques are still not as widespread as it needed for timely alerting medical services. There is urgent need of pre-exposure prophylaxis and postexposure specific antitoxin therapy for anthrax and botulism. Analysis of modern studies in the field suggests oligoclonal antibodies acting against receptor-binding toxin subunits and nucleic acid aptamers as allosteric inhibitors of metlloproteolytic toxin components as the most promising candidates for development of efficient antitoxin therapy

Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported worldwide and represent a serious problem for public healthcare. In the Russian Federation there is little information on epidemiology and etiology of STEC-infections as well as on molecular-genetic peculiarities of STEC pathogens. Objective : Our aim was to describe a food-borne outbreak as hemorrhagic colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute gastroenteritis in children in St.-Petersburg in 2013. Methods : Epidemiological, microbiological, molecular-genetic and bioinformati с methods were applied. Results : Objects to study were clinical specimens, milk and food samples, as well as STEC strains isolated during the outbreak. The outbreak of food-borne infection was found to be caused by STEC-contaminated raw milk as confirmed by epidemiological analysis, detection of STEC DNA and isolation of relevant pathogens in milk and sick children fecal specimens. The whole-genome sequencing revealed two groups of pathogens, E. coli O157:H7 and E. coli О 101:H33 among collected strains. Group I strains were attributed to the previously known sequence type ST24, while group II strains belonged to the previously non-described sequence type ST145. In strain genomes of both groups there were identified nucleotide sequences of VT2-like prophage carrying stx2 с gene, plasmid enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. coli O101:H33 strains. The latter previously was identified only in enteropathogenic E. coli (EPEC) strains. Conclusion : The additional knowledge of epidemiology and biology of STEC pathogens would assist clinicians and epidemiologists in diagnosing, treating and preventing hemorrhagic colitis.

An enzyme immunoassay-based test system for Y. pestis V antigen detection was developed. The specificity and sensitivity of this system met the requirements for medical immunobiological preparations for the identification of causative agents of highly fatal diseases. The sensitivity of the test system was assessed, and its high specificity was also demonstrated: the test system did not detect bacterial cells of closely related (four Y. pseudotuberculosis strains) and heterologous microorganism strains. The test system developed was able to detect the V antigen at concentrations as low as 2.0 ng/mL in cells of nine experimental Y. pestis cultures. The obtained preparation can be recommended for use in laboratory diagnostics of plaque.

A model of a controlled conversion of polysaccharide Vi-antigen of S. typhi into zwitterionic antigen is proposed. The immunological properties of modifications of this antigen conjugated to a protein support were studied.

Monoclonal antibodies (MCAs) to the E. coli O157: H7 O-antigen characterized by a high level of activity and specificity have been obtained. We investigated their biochemical properties and diagnostic importance. Based on these results, we constructed a latex assay using monoclonal antibodies for the identification of enterohemorrhagic E. coli belonging to the O157 serogroup. This assay was tested on pure cultures of 31 strains of closely related and other microorganisms. The designed assay allows one to detect microbial cells of E. coli O157:H7 at concentrations of 2.5 × 105 cell/ml and higher.

Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means of environmental monitoring. The properties of aptamers permit them to be regarded as fully synthetic analogs of antibodies. Moreover, certain properties of aptamers render them superior to antibodies in terms of the development of new diagnostic and monitoring systems that combine high sensitivity and specificity with high reproducibility and inexpensive manufacture. In particular, aptamer-based technologies for detection of biomolecules and whole microorganisms can be employed in solving the problems of highly sensitive express diagnostics of bacterial pathogens. The present review summarizes the current state of the techniques developed for aptamer-based detection of bacteria and their components and discusses the potential of their practical application.

Category A select agents continue to be major threat to human population both as naturally occurring diseases and as potential weapon of bioterrorists. Anthrax and botulism are probably the most threatening agents as both have virtually uncontrolled natural reservoirs from which they can be isolated and propagated. Available specific antitoxin therapy of both diseases is outdated; its efficiency is questionable as well as safety of reactogenic or human-derived components used in treatment. Highly sensitive toxin detection techniques are still not as widespread as it needed for timely alerting medical services. There is urgent need of pre-exposure prophylaxis and postexposure specific antitoxin therapy for anthrax and botulism. Analysis of modern studies in the field suggests oligoclonal antibodies acting against receptor-binding toxin subunits and nucleic acid aptamers as allosteric inhibitors of metlloproteolytic toxin components as the most promising candidates for development of efficient antitoxin therapy.

Magnetic immunosorbents (MIS) have been designed using antianthrax monoclonal antibodies immobilized on aluminum silicate—iron oxide matrix activated by sodium perchlorate. MIS allows us to concentrate the analyzed microorganism and to decontaminate culture from concomitant microflora. Diagnostic test systems constructed on the basis of MIS were tested on pure Bacillus anthracis cultures and in the model experiments. The results testify to the high specificity and sensitivity (102–103 spore/ml) of the designed test systems.

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.

Isoniazid resistance in Mycobacterium tuberculosis (MBT) is associated with point mutations in codon 315 of the katG gene. Two PCR technique were developed for detection of point mutations in codon 315. Most frequent point mutations (AGC → ACC and AGC → AGA) were identified in codon 315 by using two sets of primers, either of which included an additional competitive blocking primer with a 3′-terminal phosphate group in order to prevent nonspecific amplification. PCR with a set of two primers, one of which contained five locked nucleic acid monomers (LNA), permits one to detect any of six known mutations in codon 315 of katG and, thereby, discriminate between isoniazid-sensitive and resistant MBT isolates. The structure and purity of the 17-nt long LNA-containing oligonucleotides were characterized by MALDI-TOF mass spectrometry; and the 17 bp duplex formed by two LNA-containing complementary oligonucleotides was analyzed by thermal denaturation.

Lipid-1a-phosphatase LpxE cleaves the phosphate group in the first position of lipid A of lipopolysaccharide in Gram-negative bacteria, which reduces toxicity and reactogenicity of bacterial endotoxin while maintaining its immunogenicity. Treatment with these enzymes in preparations of recombinant proteins obtained in bacterial producers, for example, in E. coli, may find application in the purification techniques of various pharmaceutical substances, primarily various protein components of vaccines. This work is devoted to the creation of an efficient method for obtaining an F. tularensis LpxE soluble protein in a heterologous expression system based on E. coli. Optimization of the bacterial production of LpxE enzyme is achieved by creating a SUMO-LpxE chimeric protein in which the SUMO polypeptide acts as a folding catalyst and an affinity target carrier for purifying the expression product. An additional increase in the level of synthesis of LpxE and reduction of its toxicity for a bacterial strain is achieved against the background of superproduction of bacterial periplasmic chaperones encoded by an auxiliary low copy plasmid. Subsequent chromatographic purification of the target recombinant protein involves the use of affinity metal chelate chromatography and a SUMO protease cleavage step of the SUMO peptide carrying the His6 N-terminal peptide with the reduction of natural primary structure of the enzyme. The developed method makes it possible to obtain a highly purified active enzyme LpxE F. tularensis with a yield of 0.26 g/L of bacterial culture without fermentation.

A description is given of the pyrazinamide-resistant clinical strains of M. tuberculosis derived from the sputum of patients treated in tuberculosis clinics in Tula (June 2001–July 2002). It was demonstrated that 30.3% (n = 91) of strains were resistant to pyrazinamide. It was learned that these strains were resistant to other antituberculosis drugs in most cases. The method of PCR sequencing was used to find mutations in the pncA gene, which determines the resistance to pyrazinamide. 44 different types of mutations localized in 28 codons were detected. The predominance of mutations in codons 57 (13.2%), 63 (7.7%), 97 (7.7%), 12 (6.6%), and 103 (6.6%) and in the −11 (6.6%) promoter of pncA was observed in the pyrazinamide-resistant strains. Several new mutations that determine the resistance of clinical strains of M. tuberculosis to pyrazinamide were described. A strong correlation between the resistance of mycobacteria to pyrazinamide and activity of pyrazinamidase was observed.

We and other authors have recently shown that the pattern of the immune response to components of anthrax, the Bacillus anthracis lethal toxin, is complex. In addition to the neutralizing antibodies, the antitoxin antibody pool contains antibodies enhancing the toxin lethal action. We mapped the epitopes in the protective antigen that are responsible for the induction of both antibody types. In this study, we obtained new data on the cytotoxicity of the B. anthracis lethal toxin toward the J774 A.1 cell line in the presence of monoclonal antibodies to various domains of the protective antigen and the lethal factor. The role of the Fc fragment of immunoglobulins in enhancing the lethal toxin action was shown. These results may serve as a basis for the development of a new generation vaccine for anthrax.

A correlation was observed between the Beijing/W and LAM families of Mycobacterium tuberculosis and drug resistance, and the transmissiveness of multidrug-resistant strains belonging to these two families was also shown.

Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C → T) of Beijing and 100% (513A → C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored.

Anthrax and botulism are dangerous infectious diseases that can be fatal unless detected and treated quickly. Fatalities from these diseases are primarily due to endopeptidase toxins secreted by the pathogens. Rapid and sensitive detection of the presence of active toxins is the key element for protection from natural outbreaks of anthrax and botulism, as well as from the threat of bioterrorism. We describe an ultrasensitive polymerase chain reaction (PCR)-based assay for detecting proteolytic activity of anthrax and botulinum toxins using composite probes consisting of covalent peptide-DNA conjugate for the detection of anthrax, and noncovalent protein-aptamer assembly to assay botulinum toxin activity. Probes immobilized on the solid-phase support are cleaved by toxins to release DNA, which is detected by real-time PCR. Both assays can detect subpicogram quantities of active toxins isolated from composite matrices. Special procedures were developed to isolate intact toxins from the matrices under mild conditions. The assay is rapid, uses proven technologies, and can be modified to detect other proteolytic and biopolymer-degrading enzymes.

The genotypic characteristics and drug susceptibility profiles of clinical isolates of Mycobacterium tuberculosis recovered from prison hospital patients in the Tula region (central Russia) during 2001 and 2002 are reported. The emergence of multi-drug-resistant tuberculosis (TB) poses a major health risk to the population, with economic implications for TB control. Prisons serve as a continuous source of TB transmission. The results showed that members of the LAM and Beijing families are major contributors to the epidemiological picture of TB in the population studied. The two families of strains accounted for most of the drug-resistant TB in the population. The genotypic characteristics of the M. tuberculosis predominant LAM strain that was responsible for 31 % of TB cases in this setting are presented.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Phage display has proved itself as a powerful and convenient approach for rapid sampling of billions of protein-aceous structures against a ligand of interest. The most widespread system uses display of proteins and peptides as fusions to coat proteins III or VIII of the M13/fd phage. Efficient screening of phage display library requires high purity of the input phage particles. Double precipitation by polyeth-ylene glycol (PEG) with an average molecular weight of 8000 Da (PEG-8000) is a standard technique for purification of filamentous bacterio-phages (1,2). Certain contaminants could still remain in the PEG-purified phage preparation. Although efficient removal of contaminating RNases from bacteriophage preparation using treatment by ionic detergent followed by ultracentrifugation in CsCl gradient was reported (3), this method is costly, time-consuming, and is not applicable if the native state of the phage-displayed protein or peptide is affected by ionic detergents and high salt. Size-exclusion chromatography (SEC) has been used in preparation of pure eukaryotic viruses (4,5) and bacteriophage λ (6). We assumed that SEC can be the method of choice for efficient purification of filamentous phages under mild conditions. In this work we describe a rapid and simple technique for chromatographic purifi-cation of filamentous phage particles in Sephacryl™ S-500 resin (Amersham Biosciences, Piscataway, NJ, USA).The purity of phage preparations obtained after double PEG precipi-tation was assayed by electrophoresis in Tris-tricine (7). Phage particles displayed the single-chain variable fragment (scFv) of the MRL-4 anti-DNA monoclonal antibody (MAb) (8) fused to the C-terminal portion of the pIII minor phage coat protein (δpIII). Production of the scFv-δpIII fusion cloned into the pTScDisp phagemid (GenBank

We analyzed IS 6110 -associated polymorphisms in the phospholipase C genes of 107 isolates of Mycobacterium tuberculosis selected to be representative of isolates circulating in central Russia. We found that the majority of Latin American-Mediterranean family strains contained an insertion in a unique position in the plcA gene, suggesting a common ancestor. This insertion can serve as a specific genetic marker for this group, which we designate the LAM-RUS family.

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity.

We studied the effects of alpha1-acid glycoprotein on tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to alpha1-acid glycoprotein: first, stimulation of TNF-alpha and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-alpha/IL-10 ratio remained unchanged after addition of native alpha1-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of alpha1-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca(2+) and Mn(2+). Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Tuberculosis imposes a major burden of death and disease on the human population. Each year, tens of millions of people become infected with Mycobacterium tuberculosis , several millions develop clinical disease, and more than 2 million die of tuberculosis (1)(2). The successful treatment of tuberculosis depends heavily on timely diagnostics and selection of an adequate treatment strategy. Use of genotyping tools based on molecular techniques decreases the time necessary for the detection of drug resistance in M. tuberculosis from several weeks to a few days or even less, and a patient’s treatment regimen can be adjusted more rapidly to account for any detected drug resistance. Several mutations conferring resistance to the first-line drugs used for antituberculosis treatment have been described [for a review, see Ref (3)], making detection of these mutations by molecular biology techniques feasible. Rifampin and isoniazid belong to the first-line drugs used for treatment of tuberculosis (4). Resistance to rifampin, one of the most efficient antitubercular drugs, is conferred mainly by mutations in the rpoB gene. Ninety-six percent of rifampin-resistant cases are attributable to point mutations, insertions, or deletions located in an 81-nucleotide segment of the rpoB gene (5). Although resistance to isoniazid can be conferred by mutations in several different genes, mutations in katG represent the most frequent cause of the resistance (3)(6). Here we describe the allele-specific depletory PCR (ASD-PCR) assay, which was developed for the detection of mutations associated with M. tuberculosis resistance to first-line drugs, specifically isoniazid and rifampin. The accuracy and reproducibility of classic allele-specific PCR assays depend greatly on the careful selection of amplification conditions, primer design, and template concentration. Elongation through the mismatched duplex leads to accumulation of mutant template fully complementary to the initially mismatched primer, yielding the false-positive amplicon. Depending on the nature of …

Tuberculosis ward of a prison in Russia.Molecular characterization of drug-resistant isolates.Isolates were collected from all tuberculosis patients occurring in the prison over a 1-year period.Of 130 patients studied, 17 patients produced pan-susceptible isolates and 113 produced isolates resistant to at least one drug, including 85 multidrug-resistant isolates. Mutations at katG315 occurred in 98% of isoniazid-resistant isolates. Mutations in rpoB were found in 89% of rifampicin-resistant isolates. Mutations in pncA occurred in 13% of the 75 isolates tested. By spoligotyping, members of the Beijing (55 isolates) and LAM (31 isolates) families were identified. By IS6110 genotyping, two groups (34 and 55 isolates) of related isolates were found, including three clusters (10, 12, and 16 isolates) with identical patterns. In a study of samples collected 3 months apart from 28 patients, four patients produced isolates containing a mixture of strains and five patients produced specimens containing distinctly different isolates. Isolates of nine patients acquired additional drug resistance.Three families of strains accounted for much of the drug-resistant tuberculosis in this population. Multiple resistance, acquisition of resistance, and infection with two or more strains as well as reinfection were observed.