Lipopolysaccharide Antigens Research Articles

COVID-19 Impairs Immune Response to Candida albicans.

Infection with SARS-CoV-2 can lead to Coronavirus disease-2019 (COVID-19) and result in severe acute respiratory distress syndrome (ARDS). Recent reports indicate an increased rate of fungal coinfections during COVID-19. With incomplete understanding of the pathogenesis and without any causative therapy available, secondary infections may be detrimental to the prognosis. We monitored 11 COVID-19 patients with ARDS for their immune phenotype, plasma cytokines, and clinical parameters on the day of ICU admission and on day 4 and day 7 of their ICU stay. Whole blood stimulation assays with lipopolysaccharide (LPS), heat-killed Listeria monocytogenes (HKLM), Aspergillus fumigatus, and Candida albicans were used to mimic secondary infections, and changes in immune phenotype and cytokine release were assessed. COVID-19 patients displayed an immune phenotype characterized by increased HLA-DR+CD38+ and PD-1+ CD4+ and CD8+ T cells, and elevated CD8+CD244+ lymphocytes, compared to healthy controls. Monocyte activation markers and cytokines IL-6, IL-8, TNF, IL-10, and sIL2Rα were elevated, corresponding to monocyte activation syndrome, while IL-1β levels were low. LPS, HKLM and Aspergillus fumigatus antigen stimulation provoked an immune response that did not differ between COVID-19 patients and healthy controls, while COVID-19 patients showed an attenuated monocyte CD80 upregulation and abrogated release of IL-6, TNF, IL-1α, and IL-1β toward Candida albicans. This study adds further detail to the characterization of the immune response in critically ill COVID-19 patients and hints at an increased susceptibility for Candida albicans infection.

Combined Bacterial Antigen Lipopolysaccharide and Lipoteichoic Acid Increase Cal 27 Oral Cancer Cell Proliferation

Oral biofilms harbour gram-negative bacterial antigen lipopolysaccharide (LPS) involved in oral cancer progression and gram-positive bacterial surface-associated adhesive, lipoteichoic acid (LTA). Thus, we hypothesised that LPS and LTA together would increase the proliferation of cancer cells compared to stimulation by LPS alone. Oral cancer cell lines SCC4, SCC9, SCC25, Cal 27 and the normal oral cell line, OKF6, were studied. The bacterial antigen stimulation indices were determined using the MT Glo assay. Cell proliferation after bacterial antigen stimulation was validated by clonogenic assays. Phosphokinase array, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blot were employed to study proliferative and apoptotic pathways in bacterial antigen-stimulated cells. Bacterial antigens significantly stimulated Cal 27 (p ≤ 0.001) alone. SCC4 and SCC9 showed negligible stimulation with either antigen, while SCC25 results were comparable to OKF6. The combined antigen stimulation of Cal 27 led to a decrease in phosphorylated p53 and β-catenin and higher PI3K compared to LPS only stimulated cells (p ≤ 0.001). Combined bacterial antigen stimulation results in increased proliferation of Cal 27 cells due to lowering of tumor suppressor proteins and increased tumor proliferation-related proteins.

Autoimune component in the development of celebrovascular insufficiency in children with bronchial asthma

Bronchial asthma (BA) remains one of the most serious diseases of our time. A number of studies have shown that this disease in a number of cases, especially with the threat of termination of pregnancy, acute and chronic diseases of mothers, accompanied by fetal hypoxia, originates in the ante- and postnatal period. In general, taking into account the peculiarities of the cellular tissue organization of the brain and cerebrovascular blood supply, prolonged hypoxic lesions increase the activity of the hypothalamic-diencephalic structures and the cerebral cortex. The existing respiratory disorders lead to hemodynamic and metabolic disorders of cerebral structures, emotional sphere and autonomic regulation. However, this issue in childhood requires further study. The aim is to study autoimmune processes in the pathogenesis of cerebrovascular insufficiency in children with BA. Materials and methods. We examined 121 patients with asthma aged 5 to 15 years in the period of exacerbation. To study the role of the autoimmune component in the development of cerebrovascular insufficiency and its relationship with the autoimmune process in the bronchopulmonary system in AD in children, we used a method for the quantitative determination of autoantibodies to lipopolysaccharide antigens (LA) of cerebral vessels and topographic structures of the brain, as well as to homologous LA bronchi and lung tissue. The results of the studies have shown that the first signs of cerebral hemodynamic disturbance are recorded already in patients with mild disease and are aggravated depending on the severity of BA. Conclusions. The most characteristic changes are an increase in the tone of small and medium vessels and impaired cerebral venous circulation. It was also found that the levels of autoantibodies to lipopolysaccharide antigens of cerebral vessels and cellular tissue structures of the brain correlate with an increase in the level of autoantibodies to lipopolysaccharide antigens of the trachea, bronchi and lung tissue and reflect the severity of AD in children.

The significance of the distribution of the indicators of humoral and cell immunity and their role in the pathogenesis of bronchial asthma in children with the position of system analysis

Bronchial asthma is one of the common diseases in children of different ages. In recent years, around the world, including in Ukraine, there is a trend towards its sustainable growth. To date, there are no methods of systemic immunodiagnostics that would allow with high diagnostic accuracy to identify clinical forms and severity of asthma, which would allow more fully reveal the pathogenetic mechanisms and individualize approaches to the treatment of asthma in children. The aim of this work was to study the hierarchy of immunological parameters in the pathogenetic matrix, which will determine the features of clinical forms and severity of asthma in children on the basis of systematic analysis. A comprehensive clinical and immunological examination of 176 children with asthma aged 6 to 15 years. To detect the autoimmune component used lipopolysaccharide antigens obtained from homologous cell-tissue structures of the trachea, bronchi and lung tissue from sectional samples of the bronchopulmonary system from accidentally killed children with group I (0) blood 2–4 hours after death. The level of autoantibodies to lipopolysaccharide antigens of the bronchopulmonary system was determined by quantifying the autoantibody index – Qφ. As a result of the study for the first time to improve the diagnosis and differentiation of clinical forms and severity of asthma from the standpoint of system analysis was developed immunodiagnostic complex, which took into account the degree of deviation from the norm values (Student's t-test, t = 1.96) and their distribution in pathogenetic matrix. This approach to ranking the positions of immunological parameters allowed to determine the features of humoral and cellular immunity, the process of apoptosis of cell-tissue structures of the bronchopulmonary system and the autoimmune component in the pathogenesis of asthma in children, which opens approaches to individualization of pathogenetic therapy.

The emerging world of microbiome in autoimmune disorders: Opportunities and challenges

Trillions of commensal bacteria colonizing humans (microbiome) have emerged as essential player(s) in human health. The alteration of the same has been linked with diseases including autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Gut bacteria are separated from the host through a physical barrier such as skin or gut epithelial lining. However, the perturbation in the healthy bacterial community (gut dysbiosis) can compromise gut barrier integrity, resulting in translocation of bacterial contents across the epithelial barrier (leaky gut). Bacterial contents such as lipopolysaccharide and bacterial antigens can induce a systemic inflammatory environment through activation and induction of immune cells. The biggest question in the field is whether inflammation causes gut dysbiosis or dysbiosis leads to disease induction or propagation, i.e., it is inside out or outside in or both. In this review, we first discuss the microbiome profiling studies in various autoimmune disorders, followed by a discussion of potential mechanisms through which microbiome is involved in the pathobiology of diseases. A better understanding of the role of the microbiome in health and disease will help us harness the power of commensal bacteria for the development of novel therapeutic agents to treat autoimmune disorders.

Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain’s chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to β-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study

Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 μg (cohort 1) and 10 μg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 μg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 μg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 μg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. The European Union Seventh Framework Programme.

Immunological effects of two new nanovaccines against Brucella based on OPS and LPS antigens conjugated with PLGA nanoparticles

The risk of catching brucellosis and the absence of effective human vaccines has led to the use of new strategies to produce new vaccines against infections caused by this bacterium. In this study, the effect of two new designed nanovaccines on the production of immunoglobulins and their proper functions was investigated. Firstly, the production of immunoglobulins in the serum of the mouse model after three vaccination periods with lipopolysaccharide and oligopolysaccharide antigens in conjugation with PLGA nanoparticles were studied. The mice were divided into 6 groups and each group received LPS-PLGA, OPS-PLGA, LPS, OPS, PLGA and PBS separately. Immunoglobulin titers were measured every two weeks after each vaccination period. Two weeks after the last vaccination, opsonophagocytosis test were performed to evaluate the efficacy and function of produced immunoglobulins. In order to investigate the protective effect of nanovaccines in vivo, the mice were challenged with Brucella abortus bacteria. Immune responses have increased on immunoglobulin production after each vaccination period. The results of the opsonophagocytosis assay showed that produced immunoglobulins have the superior performance to antibodies produced against the pure antigens. By performing the challenge assay, it was found that the immune system of mice receiving new nanovaccines were able to remove more bacteria from the body of mice. In general, it can be concluded that both new prepared nanovaccines can be considered to improve the immunization process in animals and humans against brucellosis although LPS-PLGA conjugate creates more immunogenicity compared to OPS-PLGA nanovaccine.

The Diversity of Lipopolysaccharide (O) and Capsular Polysaccharide (K) Antigens of Invasive Klebsiella pneumoniae in a Multi-Country Collection.

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (>500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645 K. pneumoniae isolates were collected from the blood of patients in 13 countries during 2005–2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and wzi capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of K. pneumoniae strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were gmlABC-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and wzi capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent K. pneumoniae invasive infection.

Anti-Vibrio cholerae responses in the human periphery and intestine following a live-attenuated vaccine

Abstract Vibrio cholerae is a prototypical mucosal pathogen that causes cholera, a severe diarrheal disease. Cholera affects an estimated 3 to 5 million people annually, resulting in over 100,000 deaths. Infection leads to serotype-specific immunity that can last for up to ten years. Immunity is likely mediated by intestinal antibodies, which primarily target the immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). However, how immunity is generated and maintained is not well understood. Here we have used the live-attenuated cholera vaccine, Vaxchora, to characterize the primary immune response in both the periphery and the human duodenum. Previously, this vaccine showed 80% protective efficacy 90 days after vaccination of naïve volunteers, and induced early antibody responses in levels similar to infection. We developed new technology to identify and characterize V. cholerae-specific plasmablasts and plasma cells by flow cytometry, and found that antigen-specific plasmablasts in peripheral blood quickly expand and upregulate mucosal trafficking markers CCR9 and CCR10. These B cells either develop locally or home to the small intestine, where anti-LPS and anti-CT antibody-secreting cells are present by day 28. Surprisingly, our preliminary data shows that vaccinees may exhibit robust anti-CT responses in the duodenum even when peripheral anti-CT antibody secreting cells are not detected, which provides novel insight into the generation of sustained protection. These data will help us understand not only protective immunity to V. cholerae, but also how the human mucosal plasma cell compartment is developed and maintained.

Factors Required for Adhesion of Salmonella enterica Serovar Typhimurium to Corn Salad (Valerianella locusta).

Salmonella enterica is a foodborne pathogen often leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, frequency of outbreaks linked to the consumption of fresh or minimally processed food of nonanimal origin is increasing. New infection routes of S. enterica by vegetables, fruits, nuts, and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g., salads, that are mainly consumed in a minimally processed form. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species and other vegetables. In this study, we systematically analyzed the contributions of the complete adhesiome, of lipopolysaccharide (LPS), and of flagellum-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to Valerianella locusta (corn salad). We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad, with particular regard to the expression of all known adhesive structures, using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagellum-mediated motility of STM in adhesion to corn salad leaves.IMPORTANCE Transmission of gastrointestinal pathogens by contaminated fresh produce is of increasing relevance to human health. However, the mechanisms of contamination of, persistence on, and transmission by fresh produce are poorly understood. We investigated the contributions of the various adhesive structures of STM to the initial event in transmission, i.e., binding to the plant surface. A reductionist system was used that allowed experimentally controlled surface expression of individual adhesive structures and analyses of the contribution to binding to leave surfaces of corn salad under laboratory conditions. The model system allowed the determination of the relative contributions of fimbrial and nonfimbrial adhesins, the type 3 secretion systems, the O antigen of lipopolysaccharide, the flagella, and chemotaxis of STM to binding to corn salad leaves. Based on these data, future work could reveal the mechanism of binding and the relevance of interaction under agricultural conditions.

Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth.

Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

Correlation between composition and activity of gold nanoparticle conjugates with streptococcal protein G

The composition of the conjugates of gold nanoparticles with streptococcal protein G was studied using fluorescence spectroscopy. The method for determining the composition is based on measuring the intrinsic fluorescence of tryptophan as part of the protein. The equilibrium constants of protein binding by the gold surface were determined using the Sketchard method. An increase in the dissociation constant of the protein–nanoparticle complex for increasing the amount of bound protein was demonstrated, and a relationship was established between the stability of the conjugates, their antigen-binding activity, and the dissociation constant. The effectiveness of the conjugates of different compositions in immunochromatographic assay of specific antibodies against the lipopolysaccharide antigen of Brucella abortus was compared. The binding ability of the conjugates increased along with the amount of protein G to ~200 molecules per nanoparticle. A further increase in the amount of adsorbed protein led to a deterioration in the functional activity of the conjugates.

Revelation of Immune Memory at the First Stage of Antigen-Specific Cell Response after Second Introduction of the Live Plague Vaccine

Background. The study of immune memory is necessary to evaluate the effectiveness of immunization against infection, including plague and to make a choice of vaccination scheme.Goals. The goal is to study the possible role of immune memory in the early stage of the antigen-specific response – the formation of cells with receptors for capsular (F1) and lipopolysaccharide (LPS) antigens of plague live EV vaccine.Methodology. Volunteers vaccinated with live plague vaccine EV for the first time (6 persons – group 1) and again (6 persons – group 2) were examined. In the mononuclear fraction of the blood of volunteers the cells binding antigens F1 and LPS Y. pestis (CBA) were determined.Results. In the volunteers group 2, the content of CBA at 2 days after vaccination was higher than in group 1. Between the 5th day and the end of the CBA detection, their content in group 2 decreased, and in group 1, it increased, but remained significantly less than in group 2 two days after immunization.Сonclusions. It is shown that the previous vaccination accelerates the first stage of the antigen-specific human response to second vaccination against plague. This reflects the role of immune memory in the formation of this stage of the immune response at vaccination against plague.

Evaluation of the Immunomodulatory Effects of Thymus vulgaris Ethanolic Leaf Extract Combination with Partially Purified Lipopolysaccharide from Proteus mirabilis in Mice

The current study was carried out to study a high injection dose of the ethanolic extract thymus vulgaris leaf (500 ug /Kg) against the immune response combination with partially purified extracted Lipopolysaccharide ( LPS) from Proteus mirablis.Study groups were included four groups; Group I :treated with normal saline. Group II : treated with LPS antigen, Group III: injected subcutaneously ((500 ug /Kg) from ethanolic extract thymus vulgaris, group IV : injected subcutaneously (500 ug /Kg) from ethanolic extract thymus vulgaris leaf and LPS antigen, the immunological assays were measured through the phagocytic activity as (non specific immunity) after day 8 by using the phagocytic activity index.After day I4 the lymphocyte proliferations was estimated by MTT index. For delayed-type hypersensitivity reaction,the result was measured at 24, 48 and 72 hours after LPS antigens injection.While for Humoral immune response, after day 21 and day 28 the antibody production was estimated by indirect immunoflourescent and by Gel electrophoreses.The results were showed no significant difference in the NBT index between Groups but noticed Group III had a value lower than Group II, While the MTT results were revealed, Group IV had the highest value. In the other side of the study the DTH results showed Group IV had the highest value after 48 hr with significant differences (P≤0.05), in addition, the humoral immune response results were consisted gel electrophoresis and indirect immunoflourescent results showed after day 21 and day 28 Group IV had the highest value. The results was showed the ethanolic extract thymus in a high concentration combination with LPS from P. mirabilis had effects on the immune response particularly Humoral immune response and Cellular immune response but still act as anti inflammatory role as revealed in many previous studies.

Remodeling of O Antigen in Mucoid Pseudomonas aeruginosa via Transcriptional Repression of wzz2.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people with cystic fibrosis (CF). Chronic P. aeruginosa isolates generally do not express O antigen and often have a mucoid phenotype, which is characterized by the overproduction of the exopolysaccharide alginate. Therefore, O antigen expression and the mucoid phenotype may be coordinately regulated upon chronic adaption to the CF lung. Here we demonstrate that PDO300, a mucoid strain derived from the nonmucoid laboratory isolate PAO1, does not produce very long O antigen due to decreased expression of Wzz2, the very long O antigen chain length control protein, and that mucoid clinical isolates express reduced levels of Wzz2 compared to nonmucoid isolates. Further, we show that forcing the expression of very long O antigen by PDO300, by providing wzz2 in trans, does not alter alginate production, suggesting that sugar precursors are not limited between the two biosynthesis pathways. Moreover, we confirm that AmrZ, a transcription factor highly expressed in mucoid strains, is a negative regulator of wzz2 promoter activity and very long O antigen expression. These experiments identify the first transcriptional regulator of O antigen chain length in P. aeruginosa and support a model where transition to a chronic mucoid phenotype is correlated with downregulation of very long O antigen through decreased Wzz2 production.IMPORTANCE Detection of mucoid Pseudomonas aeruginosa, characterized by the overproduction of alginate, is correlated with the establishment of a chronic pulmonary infection and disease progression in people with cystic fibrosis (CF). In addition to the overproduction of alginate, loss of O antigen lipopolysaccharide production is also selected for in chronic infection isolates. In this study, we have identified the regulatory network that inversely regulates O antigen and alginate production. Understanding the regulation of these chronic phenotypes will elucidate mechanisms that are important for the establishment of a long-term P. aeruginosa lung infection and ultimately provide an opportunity for intervention. Preventing P. aeruginosa from chronically adapting to the CF lung environment could provide a better outcome for people who are infected.

Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice

BackgroundRodentibacter (R.) pneumotropicus colonizes the respiratory and urogenital tracts of laboratory mice with a reported moderate serological prevalence from 4 to 13%. Thus, regular tests to identify this pathogen in mice are recommended for animal facilities. However, a recent study indicated that current serological assays are partly insensitive, as C57BL/6 and BALB/c mice infected with R. pneumotropicus were incorrectly screened as seronegative.ResultsHere, we report a systematic analysis of protein and lipopolysaccharides antigens by immunoblot and ELISA that allowed establishing a sensitive test system able to differentiate between R. pneumotropicus and the closely related species R. heylii. Furthermore, the main immunogen, designated as ‘characteristic antigen for Rodentibacter of laboratory origin 1’ (CARLO-1), was identified by two-dimensional gel electrophoresis followed by immunoblot and tandem mass spectrometry in a preparation of outer membrane proteins. An indirect ELISA relying on the recombinantly expressed protein provided high sensitivity, specificity, and selectivity. The corresponding carlo1 gene was highly conserved (> 97%) among 21 isolates of R. pneumotropicus and R. heylii.ConclusionThe newly identified protein CARLO-1 is well suited for the sensitive and specific serological detection of Rodentibacter infections in mice. Indirect differentiation of R. pneumotropicus and R. heylii infections may be possible using an ELISA based on a whole-cell antigen preparation. All four established ELISA systems using a whole-cell preparation, lipopolysaccharides, outer-membrane proteins and protein CARLO-1 as antigen, respectively, outperformed a commercial ELISA in terms of sensitivity.

Autoimmune reactions in the bronchial asthma in children induced heterophilic antigenes of microorganism of bronchopulmants

Introduction. At the present stage of development of the problem in the etiopathogenesis of bronchial asthma (BA) in children, regardless of form, one of the leading places belongs to the microbial factor. Aim. The aim of the work was to study the development of autoimmune reactions to the cellular tissue structures of the trachea, bronchi and lung tissue, stimulated by heterophilic antigens of the microbiota of the bronchopulmonary system of children with BA. Materials and methods. A total of 97 children with BA aged 7 to 15 years were examined. The diagnosis of the disease was established according to GINA (2017) and the order of the Ministry of Health of Ukraine dated 08.10.2013 No. 868. Heterophilic antigens of bronchopulmonary structures in microbiota were determined using hyperimmune organ-specific rabbit sera to antigens of the trachea, bronchi and lung tissue. Lipopolysaccharide antigens from homologous cell-tissue structures of the trachea, pulmonary bronchi were determined, water-salt antigens from the structures of the trachea, bronchi, and lung tissue were obtained from accidentally dead children with I (0) blood group. The level of autoantibodies to antigens of the bronchopulmonary system with the quantitative calculation of the indicator Qφ was determined in the nephelometric reaction. Results. In the work it was shown experimentally that microorganisms, isolated from sputum of children, patients with asthma in the period of exacerbation, varying their antigenic potential, are able to include in their structure heterophilic antigens of cell-tissue structures of the bronchopulmonary system. Microorganisms including in their structure heterophilic antigens of the trachea, bronchi and lung tissue not only determine the induction of the pathological process in the bronchopulmonary system, but also translate it into an autoimmune basis, exacerbating the severity of the course of the disease. Conclusions. The study showed that the proposed methods are important for clarifying the etiopathogenesis of BA in children and disclosing the mechanism for switching the pathological process in the bronchopulmonary system to an autoimmune basis and can be used to develop new approaches for the etiopathogenetic treatment of the disease.

Phage typing, antibiotic resistance and genomic rep-PCR fingerprinting of clinical Salmonella isolates from the Caucasus region

Background: In reaction to the use of antibiotics in farming and veterinary practices, MDR phenotypes have emerged. As Salmonella enterica serovars are widely diverse and spread through the food chain, rapid increase in antibiotic resistance is also observed in clinical strains. Therefore, rapid identification and treatment solutions are direly needed to treat MDR infections. The use of lytic phage-based systems offers a triple benefit: rapid and cost effective typing using therapeutic agents, which can potentially lyse the examined MDR strains. Methods & Materials: In this study we have focused on isolates from patients with Salmonellosis, who have not yet received any antibiotic therapy. In total, 71 non-typhoid Salmonella enterica clinical isolates from the “Nork” Clinical Hospital of Infectious Diseases (Yerevan, Armenia) and 22 strains from the Referral Laboratory at the Republican Clinical Hospital of Infectious Diseases (RCHID, Tbilisi, Georgia) were selected. Serotyping by polyvalent antisera for flagellar (H) and lipopolysaccharide (O) antigens, and by biochemical analysis, were performed in the respective clinical settings. MALDI-TOF analysis was performed to confirm the identification at the genus level. Semi-automated rep-PCR fingerprinting profiles were determined using the DiversiLab® system and analyzed using Pearson's correlation analysis. The obtained clusters were compared with Salmonella group B, C, D, E, F, G, K, L, O, R reference libraries for serotype identification. Antibiotic resistance profiles were determined in accordance with the CLSI guidelines for standard disk diffusion assays. Phage typing was performed using the line spot-test method. Results: Species identification by phage typing confirmed the MALDI-TOF identifications. Forty-one percent of the tested clinical isolates were shown to be MDR, while none of the strains showed complete resistance to the tested phages. According to rep-PCR fingerprinting, 93 examined isolates clustered into 24 different cluster-serotypes. One isolate from RCHID showed 97.3% similarity with a Salmonella Dublin reference strain, one cluster harbored 14 strains with >95% similarity to a Salmonella typhimurium reference strain and another cluster consisted of 21 strains identified as Salmonella enteritidis. As expected, no correlation was observed between strain genotypes and phage types. Conclusion: A lytic phage-based characterization system can be relevant for epidemiological studies and rapid screening for therapeutic phage applications.

Effects of Lipopolysaccharides of Pseudomonas Aeruginosa and Aqueous Extract of Ginkgo Biloba, Ginkgoaceae, on Cellular Immune Response in Mice Balb/c

In this study, lipopolysaccharides (LPS) of Pseudomonas aeruginosa IAN5 isolate and aqueous extract of Ginkgo biloba leaves were used to investigate the cellular immunity in mice Balb/c (in vivo). Some parameters were used to achieve this study, are percentages of polymorphonuclear neutrophils (PMNs), phagocytosis coefficient of PMNs at different time periods 30, 60, 90 and 120 minutes, Formazan granules formation in them, and migration inhibition factor (MIF). The immunization of mice with the LPS antigen affected delay-type hypersensitivity and increased activity of PMNs in Candida sp. and reduced NBT, while inhibited migration of PMNs. The immunization did not affect the macrophages of PMNs. The injected treatment with LPS and Ginkgo biloba extract showed the best results significantly (p<0.05) compared to LPS and control treatments, whereas the Ginkgo biloba extract alone showed no significant differences (p<0.05).