Border patrol gone awry: Lung NKT cell activation by Francisella tularensis exacerbates tularemia-like disease

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice

Construction of the Reagent Panel “GenPest-subspecies/Altai-RGF”

The aim of the study was to develop a test system that allows for detecting plague pathogen DNA in clinical and biological samples, environmental objects with the simultaneous determination of its appurtenance to the main and non-main subspecies, differentiation of the altai biovar central asiatica subspecies separately. Materials and methods. Primer sets for specific genetic markers have been selected using the VectorNTI 10 software, optimal conditions for PCR were determined for RotorGene devices. To assess the specificity and sensitivity of the developed set of reagents, 44 strains of microorganisms were used, of which 19 were Yersinia pestis strains and 25 strains of heterologous microorganisms. The diagnostic sensitivity of “GenPest-subspecies/Altai-RGF” is 98.6 % with a confidence level of probability of 91 %. The diagnostic specificity of “GenPest-subspecies/Altai-RGF” is ≥ 99 % with a confidence level of 91 %. Results and discussion. A medical product “A set of reagents for the detection and differentiation of plague pathogen strains of the main and non-main subspecies (altai biovar, subspecies central asiatica exclusively) by the polymerase chain reaction with hybridization-fluorescent registration of results in real-time mode (GenPest-subspecies/Altai-RGF)” has been developed. The set of reagents passed the state registration in accordance with the established procedure. The use of the developed set of reagents is relevant for the Gorno-Altai high-mountain plague focus of the Russian Federation and the adjacent part of Mongolia

Improvement of Approaches to the Verification of the Vaccine Strain <i>Francisella tularensis</i> 15 NIIEG during Long-Term Storage

The aim of the study was to improve the methods for verifying the vaccine strain Francisella tularensis 15 NIIEG during long-term storage under current conditions.Materials and methods. The paper summarizes the results of studying the phenotypic and genetic properties of lyophilized cultures of the vaccine strain F. tularensis 15 NIIEG (1953, 1966, 1969, 1987, 1990, 2003, 2012 and 2013) stored at SCEMAP for a period of one to 60 years.Results and discussion. Previous studies have revealed that freeze-dried cultures of F. tularensis 15 NIIEG generally had the characteristics of the vaccine strain, with the exception of deviations from the regulatory requirements for residual virulence and specific safety. The stability of preservation of deletions in the pilA and pilE genes (the region of differentiation RD19) and the genes encoding lpp lipoprotein (RD18) in the vaccine strain, which was stored for various periods of time in a lyophilized state, has been confirmed. The vaccine-strain-specific mutation C178404T (by the genome of F. tularensis LVS strain, GenBank NCBI no. CP009694) has been identified, and an approach to determine it has been developed. The data obtained are promising as regards using the above deletions in the RD18/RD19 regions in combination with the C178404T mutation to assess the authenticity of the vaccine strain using molecular genetic methods. Thus, the conducted retrospective analysis of the data on the cultures of tularemia microbe vaccine strain from the 1940s to 2013 and the gathered experimental data, made it possible to supplement the uniform requirements for the manufacture, study, maintenance, storage and movement of F. tularensis 15 NIIEG vaccine strain with new evidence. Based on the results obtained, the authors have drawn a draft methodological recommendations of the federal level “Vaccinal strain Francisella tularensis 15 NIIEG: order of handling”

Гомеостаз собак при их кормлении сухим кормом промышленного производства и кормом домашнего приготовления

The work aimed to test a newly developed new formulation of domestic dry food for dogs. Some homeostasis indicators in dogs transferred from natural feeding to complete dry food were assessed. The studies were conducted on two groups of dogs kept by their owners in apartment conditions and on personal plots. Dogs of the 1st group (experimental) were transferred from feeding homemade food to industrially produced complete dry food. The diet of dogs in group 2 (control) did not change and still consisted of meat products, cereals and vegetables.Biomaterial for research (blood, faeces) was taken at the beginning of the experiment and after two months of controlled feeding. The bioelemental and biochemical composition of blood, haematological parameters and faecal microbiology were studied. The dogs' and general clinical conditions were assessed, including determination of live weight at the beginning and end of the two-month study. The results of the elemental analysis showed that two months after the transfer of dogs of the 1st group from the "natural" type of feeding to dry food in the blood of the animals, the concentration of arsenic, lead, strontium, chromium, iodine, selenium and zinc decreased, but the levels of lithium increased nickel and molybdenum. Of the biochemical and haematological parameters, AST, ALT, β-lipoproteins, amylase, lipase, total protein and haemoglobin increased statistically significantly, but alkaline phosphatase level decreased. In addition, switching dogs to dry food contributed to a decrease in 1 g of faeces in the concentration of E. coli with regular enzymatic activity, lactose-negative E. coli, microflora of the genus Proteus and yeast-like fungi. During the experimental period, animals of the 2nd (control) group showed an increase in the amount of microflora of the genus Protea and lactose-negative E. coli. In the intestines of dogs of both groups during the study period, an increase in coccal flora and a decrease in the number of E. coli hemolytic and E. aerogenes were observed.Цель работы состояла в испытании вновь разработанной новой рецептуры отечественного сухого корма для собак. Проведена  оценка некоторых показателей гомеостаза собак, переведенных с натурального типа кормления на полнорационный сухой корм. Исследования проводили на двух группах собак, содержавшихся их владельцами в квартирных условиях и на приусадебных участках. Собаки 1-й группы (опытной) были при этом переведены с кормления кормом домашнего приготовления на полнорационный сухой корм промышленного производства. Рацион собак 2-й группы (контрольной)  не менялся и по-прежнему состоял из мясопродуктов, каш и овощей. Биоматериал  для исследований (кровь, фекалии) брали в начале опыта и спустя два месяца контролируемого кормления. Изучали биоэлементный и биохимический состав крови, гематологические показатели и микробиологию кала. Оценивали кондиции собак, их общее клиническое состояние, включая определение живой массы в начале и по окончании двухмесячного исследования. Полученные результаты элементного анализа показали, что спустя два месяца после перевода собак 1-й группы с «натурального» типа кормления на сухой корм в крови животных снизилась концентрация мышьяка, свинца, стронция, хрома, йода, селена и цинка, но повысились уровни лития, никеля и молибдена. Из биохимических и гематологических показателей статистически значимо выросли AST, ALT, β-липопротеиды, амилаза, липаза, общий белок и гемоглобин, но снизился уровень щелочной фосфатазы. Кроме того, перевод собак на сухой корм способствовал снижению в 1 г фекалий концентрации E.coli с нормальной ферментативной активностью, лактозонегативной E.coli, микрофлоры рода протея и дрожжеподобных грибов. У животных 2-й (контрольной) группы за период опыта отмечен рост количества микрофлоры рода протея и лактозонегативной E.coli. В кишечнике собак обеих групп в исследуемый период наблюдалось увеличение кокковой флоры и снижение количества E.coli hemolytic и E.aerogenes.

DIAGNOSTIC POTENTIAL OF THE ERYTHROCYTIC IMMUNOGLOBULIN DIAGNOSTICUM FOR INDICATION AND IDENTIFICATION OF THE CAUSATIVE AGENTS OF PARTICULARLY DANGEROUS (DEEP) MYCOSES

Objective of the study was to assess analytical and diagnostic sensitivity and specificity of the “Reagent kit. Erythrocytic coccidioidomycosal and histoplasmosal immunoglobulin dry diagnosticum”, designed for identification of causative agents of coccidioidomycosis and histoplasmosis in isolated cultures of micromycetes, as well as in clinical and biological samples using indirect hemagglutination test.Materials and methods. The investigation included 264 positive samples (216 samples of micromycete suspensions, 48 samples of biological and clinical material) containing pathogens of histoplasmosis and coccidioidomycosis concentrated up to 3,12·106 and 1,56·106 cells/ml, respectively, and 128 negative samples containing heterologous microorganisms in concentrations equal to 5·106 cells/ml. The study was carried out using biological samples that were artificially contaminated with stated pathogens of particularly dangerous mycoses and samples, obtained from bioassay animals with experimental infection.Results and conclusions. It is established that diagnostic sensitivity of the reagent kit is not less than 99,0 %. The diagnostic specificity is not less than 98,0 %. Reproducibility of the results in all cases was 100 %. The results obtained testify to the prospect of introduction of the developed kit into the health care practice

Comparison of chromosomal aberrations frequency and polymorphism of GSTs genes in workers occupationally exposed to cytostatics or anaesthetics

Authors compared the incidence of chromosomal aberrations (CAs) of workers occupationally exposed to cytostatics (group EXP1) or anaesthetics (group EXP2) in relationship to polymorphism of GSTM1, GSTP1 and GSTT1 genes. The cytogenetic analysis for chromosomal aberrations frequency and for polymorphisms of genes the PCR and PCR-RFLP method were used. Statistically higher frequency of total CAs was detected in both exposed groups: group EXP1 1.90±1.34%; Mann-Whitney U-test, p=0.001; group EXP2 2.53±1.46%, p=0.0008) as compared to control (1.26±0.93%). In group EXP2 was detected statistically higher frequency of aberrations CSA-type as compared to CTA-type. In xenobiotic metabolizing genes for GST higher frequency of total CAs and constituent types chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) of genes GSTM1 and GSTT1 with null genotype was detected. Statistically significant difference was detected only in CSA-type of aberrations in GSTT1 gene. In gene GSTP1 was not detected any difference in frequency of aberrations in presence of the variant allele. Presented results point out importance of individual susceptibility in evaluation of genotoxic agents of anaesthetics or cytostatics

CD1d^-/- mice exhibit a tempered inflammatory response to LVS.

<p>Cytokine levels were measured at 7d p.i. in lung homogenates <b>(A)</b> or serum <b>(B)</b> by CBA. Data are combined from at least 3 experiments with 10–20 mice/group. Bars are mean+SD. Comparisons in these experiments were made as indicated in Materials and Methods. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

CD1d^-/- mice have less severe neutrophilia after intranasal LVS infection.

<p><b>(A)</b> AST and ALT levels were measured in serum of infected mice on d7 p.i. Data are combined from 3 experiments (<i>n</i> = 10–12). <u>Mean+SD</u>. Shaded regions denote reference range. <b>(B)</b> CBC analysis of blood from LVS-infected mice performed on d7. Data are combined from 2 separate experiments (<i>n</i> = 15 mice/group). Bars are mean+SD. <b>(C)</b> Percent neutrophils in blood at the indicated time points p.i. Data are combined from 2 separate experiments. Bars are mean+SD of 5–15 mice/group/time point. <b>(D)</b> On d7 serum and lung G-CSF levels were measured by CBA. Data are combined from 2 experiments (<i>n</i> = 15 mice/group). Bars are mean+SD. Comparisons were made as indicated in Materials and Methods. *<i>p</i><0.05, ***<i>p</i><0.001.</p