A New "Non-Traditional" Antibacterial Drug Fluorothiazinone-Clinical Research in Patients with Complicated Urinary Tract Infections.

In order to combat resistance, it is necessary to develop antimicrobial agents that act differently from conventional antibiotics. Fluorothiazinone, 300 mg tablet (The Gamaleya National Research Center), is an original antibacterial drug based on a new small molecule T3SS and flagellum inhibitor. A total of 357 patients with complicated urinary tract infections (UTIs) were divided into two groups and given Fluorothiazinone 1200 mg/day or a placebo for 7 days to evaluate the efficacy and safety of the drug. Additionally, all patients were given Cefepime 2000 mg/day. Fluorothiazinone with Cefepime showed superiority over placebo/Cefepime based on the assessment of the proportion of patients with an overall outcome in the form of a cure after 21 days post-therapy (primary outcome), overall outcome in cure rates, clinical cure rates, and microbiological efficacy at the end of therapy and after 21 days post-therapy (secondary outcomes). In patients who received Fluorothiazinone, the rate of infection recurrences 53 and 83 days after the end of the therapy was lower by 18.9%, compared with patients who received placebo. Fluorothiazinone demonstrated a favorable safety profile with no serious unexpected adverse events reported. The results showed superiority of the therapy with Fluorothiazinone in combination with Cefepime compared with placebo/Cefepime in patients with cUTIs.

LTßR-RelB signaling in intestinal epithelial cells protects from chemotherapy-induced mucosal damage.

The intricate immune mechanisms governing mucosal healing following intestinal damage induced by cytotoxic drugs remain poorly understood. The goal of this study was to investigate the role of lymphotoxin beta receptor (LTßR) signaling in chemotherapy-induced intestinal damage. LTßR deficient mice exhibited heightened body weight loss, exacerbated intestinal pathology, increased proinflammatory cytokine expression, reduced IL-22 expression, and proliferation of intestinal epithelial cells following methotrexate (MTX) treatment. Furthermore, LTßR-/-IL-22-/- mice succumbed to MTX treatment, suggesting that LTßR- and IL-22- dependent pathways jointly promote mucosal repair. Although both LTßR ligands LIGHT and LTß were upregulated in the intestine early after MTX treatment, LIGHT-/- mice, but not LTß-/- mice, displayed exacerbated disease. Further, we revealed the critical role of T cells in mucosal repair as T cell-deficient mice failed to upregulate intestinal LIGHT expression and exhibited increased body weight loss and intestinal pathology. Analysis of mice with conditional inactivation of LTßR revealed that LTßR signaling in intestinal epithelial cells, but not in Lgr5+ intestinal stem cells, macrophages or dendritic cells was critical for mucosal repair. Furthermore, inactivation of the non-canonical NF-kB pathway member RelB in intestinal epithelial cells promoted MTX-induced disease. Based on these results, we propose a model wherein LIGHT produced by T cells activates LTßR-RelB signaling in intestinal epithelial cells to facilitate mucosal repair following chemotherapy treatment.

Anti-Idiotypic Nanobodies Mimicking an Epitope of the Needle Protein of the Chlamydial Type III Secretion System for Targeted Immune Stimulation.

The development of new approaches and drugs for effective control of the chronic and complicated forms of urogenital chlamydia caused by Chlamydia trachomatis, which is suspected to be one of the main causes of infertility in both women and men, is an urgent task. We used the technology of single-domain antibody (nanobody) generation both for the production of targeting anti-chlamydia molecules and for the subsequent acquisition of anti-idiotypic nanobodies (ai-Nbs) mimicking the structure of a given epitope of the pathogen (the epitope of the Chlamydial Type III Secretion System Needle Protein). In a mouse model, we have shown that the obtained ai-Nbs are able to induce a narrowly specific humoral immune response in the host, leading to the generation of intrinsic anti-Chlamydia antibodies, potentially therapeutic, specifically recognizing a given antigenic epitope of Chlamydia. The immune sera derived from mice immunized with ai-Nbs are able to suppress chlamydial infection in vitro. We hypothesize that the proposed method of the creation and use of ai-Nbs, which mimic and present to the host immune system exactly the desired region of the antigen, create a fundamentally new universal approach to generating molecular structures as a part of specific vaccine for the targeted induction of immune response, especially useful in cases where it is difficult to prepare an antigen preserving the desired epitope in its native conformation.

Genome-wide identification of trihelix transcription factors in the apple genome in silico.

Trihelix transcription factors are involved in the growth and development of plants, as well as various stress responses. In the study presented, we identified 37 trihelix family genes in the apple genome (MdTH). The trihelix genes were located on 13 chromosomes. Phylogenetic analysis of these MdTH and the trihelix genes of other species divided them into six subfamilies: GT-1, GT-2, SH4, SIP1, GTγ, and GTδ. The genes of different groups significantly diverged in their gene structure and conserved functional domains. Cis-element analysis showed that promoter sequences of MdTH genes contained light response elements, phytohormone response elements, and stress-related cis-elements. The expression pattern analysis results demonstrated that MdTH were regulated by drought, salinity, as well as high and low temperatures. MdTH4 and MdTH24 were highly regulated by soil salinity, MdTH4-by drought. MdTH30 showed high expression under low temperature; MdTH8, MdTH20, and MdTH36-under high temperature.

Transcriptional control of ILC identity.

Innate lymphoid cells (ILCs) are heterogeneous innate immune cells which participate in host defense, mucosal repair and immunopathology by producing effector cytokines similarly to their adaptive immune cell counterparts. The development of ILC1, 2, and 3 subsets is controlled by core transcription factors: T-bet, GATA3, and RORγt, respectively. ILCs can undergo plasticity and transdifferentiate to other ILC subsets in response to invading pathogens and changes in local tissue environment. Accumulating evidence suggests that the plasticity and the maintenance of ILC identity is controlled by a balance between these and additional transcription factors such as STATs, Batf, Ikaros, Runx3, c-Maf, Bcl11b, and Zbtb46, activated in response to lineage-guiding cytokines. However, how interplay between these transcription factors leads to ILC plasticity and the maintenance of ILC identity remains hypothetical. In this review, we discuss recent advances in understanding transcriptional regulation of ILCs in homeostatic and inflammatory conditions.

Innate Lymphoid Cell Plasticity in Mucosal Infections.

Mucosal tissue homeostasis is a dynamic process that involves multiple mechanisms including regulation of innate lymphoid cells (ILCs). ILCs are mostly tissue-resident cells which are critical for tissue homeostasis and immune response against pathogens. ILCs can sense environmental changes and rapidly respond by producing effector cytokines to limit pathogen spread and initiate tissue recovery. However, dysregulation of ILCs can also lead to immunopathology. Accumulating evidence suggests that ILCs are dynamic population that can change their phenotype and functions under rapidly changing tissue microenvironment. However, the significance of ILC plasticity in response to pathogens remains poorly understood. Therefore, in this review, we discuss recent advances in understanding the mechanisms regulating ILC plasticity in response to intestinal, respiratory and genital tract pathogens. Key transcription factors and lineage-guiding cytokines regulate this plasticity. Additionally, we discuss the emerging data on the role of tissue microenvironment, gut microbiota, and hypoxia in ILC plasticity in response to mucosal pathogens. The identification of new pathways and molecular mechanisms that control functions and plasticity of ILCs could uncover more specific and effective therapeutic targets for infectious and autoimmune diseases where ILCs become dysregulated.

Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice.

The high prevalence of multidrug-resistant Acinetobacter baumannii has emerged as a serious problem in the treatment of nosocomial infections in the past three decades. Recently, we developed a new small-molecule inhibitor belonging to a class of 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT, previously called CL-55). FT effectively suppressed the T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella sp. without affecting bacterial growth in vitro. In this study, we describe that prophylactic use of FT for 4 days prior to challenge with resistant clinical isolates of A. baumannii (ABT-897-17 and 52TS19) suppressed septic infection in mice, resulting in improved survival, limited bacteraemia and decreased bacterial load in the organs of the mice. We show that FT had an inhibitory effect on A. baumannii biofilm formation in vitro and, to a greater extent, on biofilm maturation. In addition, FT inhibited Acinetobacter isolate-induced death of HeLa cells, which morphologically manifested as apoptosis. The mechanism of FT action on A. baumannii is currently being studied. FT may be a promising candidate for the development of a broad-spectrum anti-virulence drug to use in the prevention of nosocomial infections.

Innate Lymphoid Cells in Response to Intracellular Pathogens: Protection Versus Immunopathology.

Innate lymphoid cells (ILCs) are a heterogeneous group of cytokine-producing lymphocytes which are predominantly located at mucosal barrier surfaces, such as skin, lungs, and gastrointestinal tract. ILCs contribute to tissue homeostasis, regulate microbiota-derived signals, and protect against mucosal pathogens. ILCs are classified into five major groups by their developmental origin and distinct cytokine production. A recently emerged intriguing feature of ILCs is their ability to alter their phenotype and function in response to changing local environmental cues such as pathogen invasion. Once the pathogen crosses host barriers, ILCs quickly activate cytokine production to limit the spread of the pathogen. However, the dysregulated ILC responses can lead to tissue inflammation and damage. Furthermore, the interplay between ILCs and other immune cell types shapes the outcome of the immune response. Recent studies highlighted the important role of ILCs for host defense against intracellular pathogens. Here, we review recent advances in understanding the mechanisms controlling protective and pathogenic ILC responses to intracellular pathogens. This knowledge can help develop new ILC-targeted strategies to control infectious diseases and immunopathology.

Redefining the Role of Lymphotoxin Beta Receptor in the Maintenance of Lymphoid Organs and Immune Cell Homeostasis in Adulthood.

Lymphotoxin beta receptor (LTßR) is a promising therapeutic target in autoimmune and infectious diseases as well as cancer. Mice with genetic inactivation of LTßR display multiple defects in development and organization of lymphoid organs, mucosal immune responses, IgA production and an autoimmune phenotype. As these defects are imprinted in embryogenesis and neonate stages, the impact of LTßR signaling in adulthood remains unclear. Here, to overcome developmental defects, we generated mice with inducible ubiquitous genetic inactivation of LTßR in adult mice (iLTßRΔ/Δ mice) and redefined the role of LTßR signaling in organization of lymphoid organs, immune response to mucosal bacterial pathogen, IgA production and autoimmunity. In spleen, postnatal LTßR signaling is required for development of B cell follicles, follicular dendritic cells (FDCs), recruitment of neutrophils and maintenance of the marginal zone. Lymph nodes of iLTßRΔ/Δ mice were reduced in size, lacked FDCs, and had disorganized subcapsular sinus macrophages. Peyer`s patches were smaller in size and numbers, and displayed reduced FDCs. The number of isolated lymphoid follicles in small intestine and colon were also reduced. In contrast to LTßR-/- mice, iLTßRΔ/Δ mice displayed normal thymus structure and did not develop signs of systemic inflammation and autoimmunity. Further, our results suggest that LTßR signaling in adulthood is required for homeostasis of neutrophils, NK, and iNKT cells, but is dispensable for the maintenance of polyclonal IgA production. However, iLTßRΔ/Δ mice exhibited an increased sensitivity to C. rodentium infection and failed to develop pathogen-specific IgA responses. Collectively, our study uncovers new insights of LTßR signaling in adulthood for the maintenance of lymphoid organs, neutrophils, NK and iNKT cells, and IgA production in response to mucosal bacterial pathogen.

Clinical Evaluation of Different Treatment Strategies for Motor Recovery in Poststroke Rehabilitation during the First 90 Days.

BACKGROUND: Motor recovery after stroke is based on neuronal plasticity and the structural reorganization of the brain. Questions are debated about the proper moment to start rehabilitation in the acute period of stroke, the significance of rehabilitation interventions during the so-called "plastic window", and the advantages of modern and traditional programs. The aims of this study were to evaluate the role of different rehabilitation strategies and their combinations for motor recovery and the impact on functional disability by way of neurological and functional outcomes 3 months after ischemic stroke. METHODS: We used three rehabilitation approaches: early rehabilitation from the first day of stroke (Phase I), traditional exercise programs (Phase II), and an author's new method of biofeedback rehabilitation using motion sensors and augmented reality (AR) rehabilitation (Phase III). Clinical and functional outcomes were measured on the 90th day after stroke. We developed algorithms for quantifying the quality of movements during the execution of tasks in the motor domains of the AR rehabilitation program. RESULTS: Phase I of rehabilitation led to an improvement in functional independence, and the recovery of motor functions of the extremities with an absence of mortality and clinical deterioration. AR rehabilitation led to significant improvement both with respect to clinical and functional scores on scales and to variables reflecting the quality of movements. Patients who were actively treated during Phases II and III achieved the same final level of motor recovery and functional outcomes as that of participants who had only received AR rehabilitation during Phase III. Patients who underwent outpatient observation after Phase I showed a deficit of spontaneous motor recovery on the 90th day after stroke. CONCLUSIONS: Early rehabilitation was successful but was not enough; rehabilitation programs should be carried out throughout the entire "sensitive period" of poststroke plasticity. The newly developed AR biofeedback motion training is effective and safe as a separate rehabilitation method in the early recovery period of moderately severe, hemiparalytic, and ischemic stroke. These two rehabilitation approaches must be applied together or after each other, not instead of each other, as shown in clinical practice.

Campylobacter infection promotes IFNγ-dependent intestinal pathology via ILC3 to ILC1 conversion.

Innate lymphoid cells (ILCs) are a heterogeneous family of immune regulators that protect against mucosal pathogens but can also promote intestinal pathology. Although the plasticity between ILCs populations has been described, the role of mucosal pathogens in inducing ILC conversion leading to intestinal pathology remains unclear. Here we demonstrate that IFNγ-producing ILCs are responsible for promoting intestinal pathology in a mouse model of enterocolitis caused by Campylobacter jejuni, a common human enteric pathogen. Phenotypic analysis revealed a distinct population of IFNγ-producing NK1.1-T-bet+ILCs that accumulated in the intestine of C. jejuni-infected mice. Adoptive transfer experiments demonstrated their capacity to promote intestinal pathology. Inactivation of T-bet in NKp46+ ILCs ameliorated disease. Transcriptome analysis and cell-fate mapping experiments revealed that IFNγ-producing NK1.1-ILCs correspond to ILC1 profile and develop from RORγt+ progenitors. Collectively, we identified a distinct population of NK1.1-ex-ILC3s that promotes intestinal pathology through IFNγ production in response to C. jejuni infection.

Serum BDNF's Role as a Biomarker for Motor Training in the Context of AR-Based Rehabilitation after Ischemic Stroke.

BACKGROUND: brain-derived neurotrophic factor (BDNF) may play a role during neurorehabilitation following ischemic stroke. This study aimed to elucidate the possible role of BDNF during early recovery from ischemic stroke assisted by motor training. METHODS: fifty patients were included after acute recovery from ischemic stroke: 21 first received classical rehabilitation followed by 'motor rehabilitation using motion sensors and augmented reality' (AR-rehabilitation), 14 only received AR-rehabilitation, and 15 were only observed. Serum BDNF levels were measured on the first day of stroke, on the 14th day, before AR-based rehabilitation (median, 45th day), and after the AR-based rehabilitation (median, 82nd day). Motor impairment was quantified clinically using the Fugl-Meyer scale (FMA); functional disability and activities of daily living (ADL) were measured using the Modified Rankin Scale (mRS). For comparison, serum BDNF was measured in 50 healthy individuals. RESULTS: BDNF levels were found to significantly increase during the phase with AR-based rehabilitation. The pattern of the sequentially measured BDNF levels was similar in the treated patients. Untreated patients had significantly lower BDNF levels at the endpoint. CONCLUSIONS: the fluctuations of BDNF levels are not consistently related to motor improvement but seem to react to active treatment. Without active rehabilitation treatment, BDNF tends to decrease.

IL-23 Contributes to Campylobacter jejuni-Induced Intestinal Pathology via Promoting IL-17 and IFNγ Responses by Innate Lymphoid Cells.

Human pathogen Campylobacter jejuni is a significant risk factor for the development of long-term intestinal dysfunction although the cellular and molecular mechanisms remain scantily defined. IL-23 is an emerging therapeutic target for the treatment of inflammatory intestinal diseases, however its role in C. jejuni-driven intestinal pathology is not fully understood. IL-10 deficient mice represent a robust model to study the pathogenesis of C. jejuni infection because C. jejuni infection of mice lacking IL-10 results in symptoms and pathology that resemble human campylobacteriosis. To determine the role of IL-23 in C. jejuni-driven intestinal inflammation, we studied the disease pathogenesis in IL-23-/- mice with inhibited IL-10Rα signaling. These mice exhibited reduced intestinal pathology independent from bacterial clearance. Further, levels of IFNγ, IL-17, IL-22, TNF, and IL-6 were reduced and associated with reduced accumulation of neutrophils, monocytes and macrophages in the colon. Flow cytometry analysis revealed reduced production of IL-17 and IFNγ by group 1 and 3 innate lymphoid cells. Thus, our data suggest that IL-23 contributes to intestinal inflammation in C. jejuni infected mice by promoting IL-17 and IFNγ production by innate lymphoid cells.

Lymphotoxin in physiology of lymphoid tissues - Implication for antiviral defense.

Lymphotoxin (LT) is a member of the tumor necrosis factor (TNF) superfamily of cytokines which serves multiple functions, including the control of lymphoid organ development and maintenance, as well as regulation of inflammation and autoimmunity. Although the role of LT in organogenesis and maintenance of lymphoid organs is well established, the contribution of LT pathway to homeostasis of lymphoid organs during the immune response to pathogens is less understood. In this review, we highlight recent advances on the role of LT pathway in antiviral immune responses. We discuss the role of LT signaling in lymphoid organ integrity, type I IFN production and regulation of protection and immunopathology during viral infections. We further discuss the potential of therapeutic targeting LT pathway for controlling immunopathology and antiviral protection.

Perivascular Fibroblasts of the Developing Spleen Act as LTα1ß2-Dependent Precursors of Both T and B Zone Organizer Cells.

T and B cell compartmentalization is a hallmark of secondary lymphoid organs and is maintained by chemokine-expressing stromal cells. How this stromal cell network initially develops and differentiates into two distinct subsets is poorly known, especially for the splenic white pulp (WP). Here, we show that perivascular fibroblast precursors are triggered by LTα1ß2 signals to expand, express CCL19/21, and then differentiate into two functionally distinct fibroblast subsets responsible for B and T cell clustering and WP compartmentalization. Failure to express or sense CCL19 leads to impaired T zone development, while lack of B cells or LTα1ß2 leads to an earlier and stronger impairment in WP development. We therefore propose that WP development proceeds in multiple steps, with LTα1ß2+ B cells acting as major inducer cells driving the expansion and gradual differentiation of perivascular fibroblasts into T and B zone organizer cells.

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection.

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine.

Europium-Doped Cerium Oxide Nanoparticles Limit Reactive Oxygen Species Formation and Ameliorate Intestinal Ischemia-Reperfusion Injury.

Accumulating evidence suggests that ischemia-reperfusion-induced injury is associated with the formation of reactive oxygen species (ROS). This study demonstrates the therapeutic effectiveness of novel europium-doped cerium oxide nanoparticles (Eu-doped Ceria NPs) as ROS scavengers in a mouse model of intestinal ischemia-reperfusion-induced injury. An increased production of superoxide radicals is detected in the intestine throughout the ischemia stage and again after initiating reperfusion. These changes in superoxide radical formation are associated with the induction of inflammatory cytokines in the intestine. This study further shows that Eu-Ceria NPs exhibit superoxide scavenging activity in vitro. Importantly, administration of Eu-Ceria NPs into the intestinal lumen during the onset of ischemia effectively blocks superoxide accumulation, reduces the expression of IL-1b, and ameliorates the intestinal pathology. These results suggest that early increased production of ROS during the ischemia-reperfusion promotes intestinal pathology and that mucosal delivery of Eu-Ceria NPs may be a potential therapeutic approach to block ROS accumulation and ameliorate the severity of intestinal disease.

Longitudinal conductivity of LaF3/SrF2 multilayer heterostructures.

LaF3/SrF2 multilayer heterostructures with thicknesses of individual layers in the range 5-100 nm have been grown on MgO(100) substrates using molecular beam epitaxy. The longitudinal conductivity of the films has been measured using impedance spectroscopy in the frequency range 10-1-106 Hz and a temperature range 300-570 K. The ionic DC conductivities have been determined from Nyquist impedance diagrams and activation energies from the Arrhenius-Frenkel equation. An increase of the DC conductivity has been observed to accompany decreased layer thickness for various thicknesses as small as 25 nm. The greatest conductivity has been shown for a multilayer heterostructure having thicknesses of 25 nm per layer. The structure has a conductivity two orders of magnitude greater than pure LaF3 bulk material. The increasing conductivity can be understood as a redistribution of charge carriers through the interface due to differing chemical potentials of the materials, by strong lattice-constant mismatch, and/or by formation of a solid La1-xSrxF3-x solution at the interface during the growth process.

Murine Model of Intestinal Ischemia-reperfusion Injury.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies. Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.