Immunoreactivity of the AAA plus chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals

Citation: Krajewska, J., Arent, Z., Wieckowski, D., Zolkiewski, M., & Kedzierska-Mieszkowska, S. (2016). Immunoreactivity of the AAA plus chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals. Bmc Microbiology, 16, 8. doi:10.1186/s12866-016-0774-8Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpB(Li)) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpB(Li) as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. Results: ClpB(Li) consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpB(Li) and E. coli ClpB is 52 %. The coding sequence of the clpB(Li) gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpB(Li) protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpB(Li) activates the host immune system, as evidenced by an increased level of antibodies against ClpB(Li) in the sera from infected animals, as compared to the control group. Additionally, ClpB(Li) was found in kidney tissues of Leptospira-infected hamsters. Conclusions: ClpB(Li) is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpB(Li) point to its potential value as a diagnostic antigen for the detection of leptospirosis

Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca(2+) Homeostasis via Cross-Linking RAP1GDS1

BACKGROUND: Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet. RESULTS: Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca(2+) uptake. Ca(2+)-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca(2+) release from the endoplasmic reticulum via both Ins3P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca(2+)uptake. CONCLUSIONS: Our data indicate that TG2 might act as a Ca(2+) sensor to amplify endoplasmic reticulum-derived Ca(2+) signals to enhance mitochondria Ca(2+) uptake. Since enhanced mitochondrial Ca(2+) levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca(2+) signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca(2+) homeostasis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Investigation of the structure of rhodium films electrodeposited on Au(111) single crystals.

The structure of rhodium films electrodeposited on (111) surfaces of gold was investigated using cyclic voltammetry, x-ray scattering measurements and scanning tunneling microscopy (STM). A better understanding of the rhodium deposition process and film structure was gained. After mechanical polishing the Au(111) crystal was electropolished and annealed in an hydrogen flame. These crystal preparation procedures yielded a gold single crystal with electrochemical characteristics comparable to previously published cyclic voltammagrams. The rhodium film was then deposited electrochemically from a 0.5 mM rhodium chloride and 0.5 M perchloric acid solution at 100 mV versus SCE at 70 C until the desired amount of charge had passed. The deposition of rhodium is a 3 electron process following this electrode reaction, [RhCl{sub 6}]{sup 3{minus}} (aq) + 3 e{sup {minus}} {r_arrow} Rh (s) + 6 Cl{sup {minus}} (aq). X-ray measurements were performed at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory beamlines X10a and X25 using focused, monochromatic radiation ({lambda} = 1.494 and 1.282 angstroms, respectively)