Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions.

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.

Influence of retinoic acid on human gingival epithelial barriers.

BACKGROUND AND OBJECTIVES: The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form a barrier and show various cellular contacts, including tight junctions (TJ). To analyse the barrier function in vitro the transepithelial electrical resistance (TER) is commonly used. Retinoic acid (RA) is an important signalling molecule in most tissues, including epithelial differentiation. RA signalling is mediated through three RA receptors. The aim of the study was to investigate the influence of RA on human gingival barriers in vitro. MATERIAL AND METHODS: Immortalized human gingival keratinocytes were seeded on culture plate inserts. The effect of RA with and without infection with Porphyromonas gingivalis W83 on the barrier was analysed by TER measurements. The expression of TJ proteins was investigated by western blot. RESULTS: During differentiation, mean TER increased from 16 (1 h), 43 (4 h) to 62 (6 h) Ohm × cm2 . Addition of 15 µm RA increased TER by +19 after 1 h, +25 after 4 h and +16 Ohm × cm2 after 6 h. The pan-RA receptor inhibitor BMS 493 resulted in TER values comparable to the control. The mean established TER of the control was approximately 110 Ohm × cm2 . Addition of 15 µm RA elevated TER to 127 Ohm × cm2 after 1 h, 150 Ohm × cm2 after 4 h and 189 Ohm × cm2 after 6 h (p ≤ 0.01). RA plus infection with P. gingivalis W83 further increased the TER increasing effect but could not prevent the destruction of TER induced by bacterial infection. The protein expression of the TJ proteins claudin 4 and occludin was enhanced while ZO-1 was downregulated after 1 h of RA incubation. CONCLUSION: RA provides barrier-positive elements to the gingival epithelial cell model that is accompanied by altered expression of TJ proteins.

Increased rate of prematurity associated with antenatal antiretroviral therapy in a German/Austrian cohort of HIV-1-infected women.

OBJECTIVE: The aim of the study was to assess the risk of adverse pregnancy outcomes after antenatal antiretroviral therapy in a well-defined prospective cohort of nontransmitting HIV-infected women. METHODS: Prospective monitoring of 183 mother-child pairs from 13 centres in Germany and Austria, delivering between 1995 and 2001, was carried out. Following German-Austrian guidelines recommending an elective Caesarean section (CS) at 36 weeks, prematurity was defined as <36 weeks' gestation for these analyses. RESULTS: Of 183 mother-child pairs, 42% were exposed to antenatal monotherapy and 17% to dual therapy. Of the 75 women exposed to highly active antiretroviral therapy (HAART), 21 (28%) received protease inhibitor (PI)-based HAART and the remaining 54 received nonnucleoside reverse transcriptase inhibitor-based HAART. In multivariable analysis (176 pregnancies), PI-based HAART exposure during pregnancy was associated with an increased risk of premature delivery [adjusted odds ratio 3.40; 95% confidence interval (CI) 1.13-10.2; P=0.029, compared with monotherapy]. Congenital abnormalities affected 3.3% infants. Perinatally, 18.9% of children (34 of 179) had respiratory problems requiring interventions, which were associated with prematurity but not with type of treatment exposure. From adjusted regression analysis, the mean birth weight z-score for children exposed to HAART with PI (+0.46; 95% CI 0.01-0.92; P=0.047) or dual therapy (+0.43; 95% CI 0.03-0.82; P=0.034) was slightly but significantly higher than that for those exposed to monotherapy; head circumference was appropriate for gestational age and there were no significant differences between treatment groups. CONCLUSIONS: Use of antenatal PI-based HAART initiated before or during pregnancy was associated with a significantly increased risk of premature delivery at <36 weeks' gestation. The overall crude prematurity rate was 34% (63 of 183; 95% CI 28-42).

Design and Performance of Laser-Pumped Cs-Magnetometers for the Planned UCN EDM Experiment at PSI.

We designed laser-pumped cesium vapor magnetometers in the M x configuration for the control and stabilization of magnetic field fluctuations and gradients in a new experiment searching for a permanent electric dipole moment of the neutron. The intrinsic sensitivity of the device was determined to be 30 fT in a measurement bandwidth of 1 Hz, limited by laser noise. In the shot noise limit the magnetometer can reach a sensitivity of 7 fT for 1 s integration time. Test measurements of the stability of a 2 µT magnetic field in a threefold magnetic shield have revealed fluctuations on the order of 200 fT to 300 fT with integration times in the range of 2 s to 100 s. Those fluctuations were traced back to the stability of the power supply used to generate the magnetic field. The laser-pumped magnetometer fulfills the requirements set by the planned neutron electric dipole moment experiment.

Identification of cytokeratins in bovine sperm outer dense fibre fractions.

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll(R) density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1-18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella.

Lanthionine macrocyclization by in situ activation of serine.

The present report details a straightforward, solid-phase approach to cyclolanthionine peptides. After stepwise assembly of the linear sequence and transformation of a single exposed serine to bromoalanine using P(Ph)3/CBr4, the detritilation of a cysteine side-chain sets the stage for a base-promoted macrocyclization. The entire procedure can be carried out in a solid-phase vessel using conventional 9-fluorenylmethyloxycarbonyl/tert-butyl-based chemistry and is amenable to automated format. The utility of this novel procedure is demonstrated by the synthesis of two previously reported lanthionine-containing cyclic peptides.