Mojca Narat

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells†

ABSTRACT AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1−/− mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.

Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5+/-1.5%) and CEC-32 (RIF 7.0+/-0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R(low). Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2+/-0.3%) similar to that of R(low) (1.1+/-0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Nanoparticle interaction with the immune system.

Abstract When nanoparticles enter the body, their interactions with cells are almost unavoidable. Unintended nanoparticle interaction with immune cells may elicit a molecular response that can have toxic effects and lead to greater susceptibility to infectious diseases, autoimmune disorders, and cancer development. As evidenced by several studies, nanoparticle interactions with biological systems can stimulate inflammatory or allergic reactions and activate the complement system. Nanoparticles can also stimulate immune response by acting as adjuvants or as haptens. Immunosuppressive effects have also been reported. This article gives a brief review of in vitro and in vivo research evidencing stimulatory or suppressive effects of nanoparticles on the immune system of mammals. In order to ensure safe use of nanosized particles, future research should focus on how their physical and chemical properties influence their behaviour in the biological environment, as they not only greatly affect nanoparticle-immune system interactions but can also interfere with experimental assays Ko nanodelci vstopijo v organizem, pridejo v kontakt s celicami imunskega sistema. Nezaželene interakcije nanodelcev z imunskim sistemom lahko sprožijo molekularni odziv, ki lahko pripelje do toksičnih učinkov in povečane dovzetnosti organizma za okužbe, avtoimunska obolenja ter razvoj raka. Dosedanje raziskave so pokazale, da nanodelci lahko sprožijo vnetne in alergijske reakcije, lahko pa tudi aktivirajo sistem komplementa. Nanodelci lahko delujejo kot adjuvansi ali kot hapteni. Obstajajo pa tudi poročila, ki kažejo na sposobnost nanodelcev, da zavrejo imunski odziv. V članku bomo povzeli ugotovitve dosedanjih raziskav in vitro ter in vivo, ki so bile narejene na področju proučevanja vplivov nanodelcev na stimulacijo ali supresijo imunskega sistema sesalcev. Za zagotovitev varne uporabe nanodelcev moramo razumeti kako fizikalno-kemijske lastnosti nanodelcev vplivajo na njihovo obnašanje v biološkem okolju. Lastnosti nanodelcev moramo upoštevati tudi ob izvajanju poskusov, da se izognemo lažnim rezultatom zaradi potencialne interference nanodelcev z dejavniki v eksperimentalnem okolju. Čeprav je bilo do sedaj narejenih že več nanotoksikoloških raziskav, je vpliv nanodelcev na imunski sistem še vedno slabo razumljen. Sposobnost nanodelcev za modulacijo imunskega odziva narekuje potrebo po nadaljnjih raziskavah interakcij nanodelcev z imunskim sistemom.

A survey of avian Mycoplasma species for neuraminidase enzymatic activity

Among 23 currently recognized avian Mycoplasma (AM) species only Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae cause disease and loss of production in chickens and/or turkeys. Because neuraminidases are considered virulence factors in many pathogenic microorganisms the aim of our study was to determine which AM species possess neuraminidase enzymatic activity (NEAC). Small samples of AM cells were assayed for NEAC using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-alpha-d-N-acetylneuraminic acid. In the case of positive NEAC reaction the substrate gave the insoluble indigoblue product what enabled simple test and easy estimation of NEAC. M. gallisepticum and M. synoviae which share sequences of the gene encoding neuraminidase (sialidase NanH) exhibited considerable levels of NEAC. However, NEAC levels differed among their strains, as well as among cultures of different strains. Only certain cultures of the type strain of M. meleagridis showed NEAC, whereas among six serovars of M. iowae only serovar I (type strain 695) showed NEAC. Weak NEAC was detectable in M. anseris, M. cloacale and M. pullorum, whereas the type strain of M. corogypsi (BV1) showed strong NEAC. Our study provides novel informations about NEAC in AM species and suggests that higher invasiveness and possibly, the pathological processes might be associated with their NEAC.

Human intestinal mucosa-associated Lactobacillus and Bifidobacterium strains with probiotic properties modulate IL-10, IL-6 and IL-12 gene expression in THP-1 cells.

Lactobacilli and bifidobacteria are considered one of the permanent genera of the physiological human intestinal microbiota and represent an enormous pool of potential probiotic candidates. Approximately 450 isolates of presumptive Lactobacillus or Bifidobacterium strains were obtained from bioptic samples of colonic and ileal mucosa from 15 adolescents aged 12 to 18 years. On the basis of randomly amplified polymorphic DNA (RAPD)-PCR analysis, 20 strains were selected for further taxonomic classification and characterisation, as well as assessment of probiotic properties and safety. Importantly, selected strains showed the capability of colonising different parts of the intestine. The most frequently isolated species was Lactobacillus paracasei followed by Lactobacillus fermentum. The majority of isolates were susceptible to antimicrobials of human and veterinary importance, however, tetracycline and/or erythromycin resistance was observed in Lactobacillus plantarum and L. fermentum strains. Thirteen strains were able to ferment more than 19 different carbon sources and three out of five tested strains exerted antagonistic activity against several different indicator strains. Two Lactobacillus isolates (L. paracasei L350 and L. fermentum L930 bb) and one Bifidobacterium isolate (Bifidobacterium animalis subsp. animalis IM386) fulfilled in vitro selection criteria for probiotic strains and exhibited strong downregulation of pro-inflammatory cytokines IL-6 and IL-12 and upregulation of anti-inflammatory IL-10. The selected strains represent suitable candidates for further studies regarding their positive influence on host health and could play an important role in ameliorating the symptoms of inflammatory bowel diseases.

Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses

Avian-specific toll like receptor 15 (TLR15) is functionally equivalent to a group of TLR2 family proteins that the mammalian innate immune system utilizes to recognize a broad spectrum of microbe-associated molecular patterns, including bacterial lipoproteins. In this study we examined the role of chicken TLR2 family members in the innate immune response to the avian pathogenic bacterium, Mycoplasma synoviae. We found that Mycoplasma synoviae, and specifically the N-terminal diacylated lipopeptide (MDLP) representing the amino-terminal portion of its mature haemagglutinin protein, significantly induces the expression of TLR15, but not TLR1 and TLR2 in chicken macrophages and chondrocytes. TLR15 activation is specific and depends on diacylation of the lipopeptide. Activation of TLR15 after stimulation with Mycoplasma synoviae and MDLP triggers an increase in the expression of transcription factor nuclear factor kappa B and nitric oxide production. Moreover, transfection of avian macrophage cells with small interfering RNA reduces the expression of TLR15 after stimulation with MDLP. This leads to decreased activation of the innate immune response, as measured by nitric oxide production. Additionally, pretreatment of cells with neutralizing anti-TLR15 antibody results in a notable attenuation of MDLP-driven release of nitric oxide. This positive correlation may constitute a mechanism for stimulating the innate immune response against avian mycoplasmas in chicken cells via TLR15.

Adaptation of adhesion test using Caco-2 cells for anaerobic bacteriumPseudobutyrivibrio xylanivorans, a probiotic candidate

Pseudobutyrivibrio xylanivorans strain Mz5T, an anerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach,i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability ofP. xylanivorans Mz5T. The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.

The humoral and cellular immune response to a lipid attenuated pore-forming toxin from the sea anemone Actinia equina L.

The immunogenicity of a pore-forming polypeptide, equinatoxin II, from the sea anemone Actinia equina was studied after attenuation of the toxins lethal and cytolytic activity by autologous polar lipids. In BALB/c mice, the lipid-inactivated toxin was used to raise specific antibodies and cellular immunity, resulting in in vivo protection. In vitro, haemolytic activity could be diminished by both normal and immune serum, the latter being more efficient. Purified specific IgG1 and IgG2 did not or only poorly neutralized the haemolytic activity, therefore implying the marked role of serum lipoproteins in the toxin attenuation. In response at the cellular level, equinatoxin II activated specific splenocytes. Increased concanavalin A stimulation of specific splenocytes was observed in the absence of antigen.