Relative luciferase activity of the wild type promoter (WT) and a promoter construct bearing the -60 C/T mutation in homozygosis

<p><b>Copyright information:</b></p><p>Taken from "Genetic variants of chemokine receptor CCR7 in patients with systemic lupus erythematosus, Sjogren's syndrome and systemic sclerosis"</p><p>http://www.biomedcentral.com/1471-2156/8/33</p><p>BMC Genetics 2007;8():33-33.</p><p>Published online 22 Jun 2007</p><p>PMCID:PMC1913537.</p><p></p> (A): relative luciferase activity in HUT78 cells following transfection of the promoter constructs by electroporation. Data shown is representative of three independent experiments. (B): relative luciferase activity in primary T-cells that were transfected with the promoter constructs using Amaxa transfection technology. Two independent experiments (exp.) were performed. In each experiment primary T-cells from 13 donors were used. Shown are results from donor #2 and donor #9. No differences between WT and mutant promoter activity were observed in the remaining 11 donors. Error bars represent the SEM of triplicates. Statistic analysis was performed applying unpaired t-test. Values < 0.05 were considered significant (* = p < 0.05, ** = p < 0.01)

Image_2_Characterization of serum biomarkers and antibody responses against Prevotella spp. in preclinical and new-onset phase of rheumatic diseases.jpg

IntroductionThe characterization of the influence of the microbiota on the development and drug responses during rheumatic diseases has intensified in recent years. The role of specific bacteria during disease development has become a central research question. Notably, several lines of evidence point to distinct microbes, e.g., Prevotella copri (P. copri) being targeted by antibodies in clinical phases of rheumatic diseases.MethodsIn the present study, we compiled a broad collection of human serum samples from individuals at risk of developing RA, chronic RA patients as well as patients with new-onset of rheumatic diseases. We evaluated the presence of inflammatory biomarkers in our serum collection as well as serum antibody responses against novel, genetically distinct isolates of P. copri and several oral pathobionts.ResultsOur analysis revealed the presence of increased levels of inflammatory markers already in pre-clinical and new onset rheumatoid arthritis. However, antibody reactivity against the microbes did not differ between patient groups. Yet, we observed high variability between the different P. copri strains. We found total serum IgG levels to slightly correlate with IgG antibody responses against P. copri, but no relation between the latter and presence or prevalence of P. copri in the intestine.DiscussionIn conclusion, our work underlined the importance of strain-level characterization and its consideration during further investigations of host-microbiota interactions and the development of microbiome-based therapeutic approaches for treating rheumatic diseases.</p

Statiscal analysis of <i>LILRA3</i> deletion in B-NHL patients to controls.

<p>Statiscal analysis of <i>LILRA3</i> deletion in B-NHL patients to controls.</p

Association of the<i>LILRA3</i> Deletion with B-NHL and Functional Characterization of the Immunostimulatory Molecule

<div><p>LILRA3 is the sole soluble member of the LILR family. Previous studies from our group had shown that a 6.7 kb genetic deletion of <i>LILRA3</i> is associated with MS and Sjögren’s syndrome. An impairment of the immune response leads to a predisposition for B-NHL, so we wanted to study whether the deletion of <i>LILRA3</i> is also a risk factor for B-NHL, as well as the function of LILRA3. We discovered that the frequency of the homozygous <i>LILRA3</i> deletion was significantly higher in B-NHL (6%) than in blood donors (3%) (P = 0.03). We detected binding of fluorochrome-conjugated recombinant LILRA3 to monocytes and B-cells. Incubation of PBMCs with recombinant LILRA3 induced proliferation of CD8⁺ T-cells and NK cells, as determined by CFSE staining. Using a transwell system, we demonstrated that LILRA3-stimulated lymphocyte proliferation was mediated by monocytes and required both cell contact and soluble factors. Secretion of IL-6, IL-8, IL-1β and IL-10 in the cell supernatant was stimulated by LILRA3. We conclude that LILRA3 is an immunostimulatory molecule, whose deficiency is associated with higher frequency of B-NHL.</p></div

Binding of fluorochrome-conjugated LILRA3 to monocytes and B-cells.

<p>LILRA3 and BSA were conjugated with DL488 and used to stain PBMCs. (A) Representative histograms showing binding of LILRA3 or BSA, in the absence (no blocking) or presence (blocking) of excess unlabelled protein, to various PBMC cell subsets. (B) Triplicate analysis of the protein binding, expressed as the mean±SD of the MFIs, analysed using 2-way ANOVA with Bonferroni post test to compare every column. The experiment was duplicated in 2 further independent experiments which gave similar results. (***p<0.001, N.S.p>0.05).</p

LILRA3 mediated proliferation is dependent on monocytes.

<p>T-cell proliferation is dependent on cell-cell contact to monocytes whereas NK-cell proliferation is dependent on soluble factors. In a transwell chamber, using CFSE-stained PBMCs, whole PBMCs were filled in the upper chamber and PBMCs-MO in the lower chamber. In the absence or presence of 500 ng/mL LILRA3, irradiated allogeneic feeder PBMCs were added to either chambers. The proliferation of the various cell subsets was determined by flow cytometry after 7 days. (A) Representative histogram plots showing proliferation of CD4 T-cells (CD3⁺CD4⁺), CD8 Tcells (CD3⁺CD8⁺) and NK-cells (CD3⁻NKp46⁺) indicated by their CFSE fluorescence. The percentage of cells which had proliferated was obtained by gating to the left of the peak of unproliferated cells in unstimulated responders. The pictogram above the histograms shows whether the column of histograms belong to the upper chamber or lower chamber, and whether the allogeneic stimulation (+allo) was given to whole PBMCs or PBMCs-MO (B) Statistical analysis of results from 4 donors expressed as mean±SD, performed using 2-way ANOVA with Bonferroni post test. (**p<0.01, ***p<0.001, N.S.p>0.05).</p

Cell culture supernatants from previous CFSE proliferation experiments were harvested and used to determine concentrations of various cytokines via a multiplex bead array.

<p>Results expressed as mean±SD from 8 donors, statistically analysed for each cytokine using 1-way repeated measures ANOVA with Tukey post test. (*p<0.05, **p<0.01, ***p<0.001).</p

Induction of proliferation by LILRA3 in an MLR.

<p>Responder PBMCs were incubated in the absence or presence of suboptimal amounts of irradiated allogeneic feeder cells and varying concentrations of LILRA3, and the proliferation measured on the 7^th day with ³H-thymidine assay. Results expressed as mean±SD from 9 donors, statistically analysed using 1-way repeated measures ANOVA with Dunnett post test to compare to 0 ng/mL LILRA3 control. (*p<0.05, **p<0.01).</p

Patient characteristics.

<p>Patient characteristics.</p

Prevalence of autoimmune conditions among the B-NHL patients.

<p> A patient had both psoriasis and autoimmune hemolytic anemia.</p