ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes.

Little is known about the mechanisms involved in the regulation of nociceptin and its receptor (nociceptin opioid peptide receptor, NOP) in response to inflammation and pain in humans. In this study, specific signaling pathways contributing to the regulation of nociceptin and NOP in human peripheral blood leukocytes were investigated. After approval by the ethics committee, peripheral blood obtained from healthy donors was cultured with or without phorbol-12-myristate-13-acetate (PMA). Prepronociceptin (ppNOC) and NOP mRNA were analyzed by real-time quantitative polymerase chain reaction, and nociceptin concentrations in culture supernatants by fluorescent enzyme immunoassay. Nociceptin and NOP protein levels in blood leukocyte subsets were determined using flow cytometry. To examine the contribution of signaling pathways to ppNOC and NOP regulation, blood was pre-treated with kinase inhibitors specific for ERK, JNK, p38, and NFκB pathways prior to culturing with or without PMA. PMA dose-dependently upregulated ppNOC mRNA but downregulated NOP mRNA in human peripheral blood leukocytes. PMA 10 ng/ml increased ppNOC after 6 h and suppressed NOP after 3 h compared to controls (both P <0.005). Nociceptin concentrations were increased in supernatants of PMA-induced blood samples after 24 h ( P <0.005), whereas expression of cell-membrane NOP was decreased by PMA in blood leukocyte subsets (all P <0.05). Blockade of ERK or p38 pathways partially prevented PMA effects on ppNOC and NOP mRNA (all P <0.05). The combination of ERK and p38 inhibitors completely reversed the effects of PMA ( P <0.05). ERK and p38 are two major signaling pathways regulating nociceptin and its receptor in human peripheral blood leukocytes under inflammatory conditions.

Neutrophil extracellular trap formation during surgical procedures: a pilot study.

Neutrophils can release neutrophil extracellular traps (NETs) containing DNA fibres and antimicrobial peptides to immobilize invading pathogens. NET formation (NETosis) plays a vital role in inflammation and immune responses. In this study we investigated the impact of surgical trauma on NETosis of neutrophils. Nine patients undergoing "Transcatheter/percutaneous aortic valve implantation" (TAVI/PAVI, mild surgical trauma), and ten undergoing "Aortocoronary bypass" (ACB, severe surgical trauma) were included in our pilot study. Peripheral blood was collected before, end of, and after surgery (24 h and 48 h). Neutrophilic granulocytes were isolated and stimulated in vitro with Phorbol-12-myristate-13-acetate (PMA). NETosis rate was examined by microscopy. In addition, HLA-DR surface expression on circulating monocytes was analysed by flow-cytometry as a prognostic marker of the immune status. Both surgical procedures led to significant down regulation of monocytic HLA-DR surface expression, albeit more pronounced in ACB patients, and there was a similar trend in NETosis regulation over the surgical 24H course. Upon PMA stimulation, no significant difference in NETosis was observed over time in TAVI/PAVI group; however, a decreasing NETosis trend with a significant drop upon ACB surgery was evident. The reduced PMA-induced NETosis in ACB group suggests that the inducibility of neutrophils to form NETs following severe surgical trauma may be compromised. Moreover, the decreased monocytic HLA-DR expression suggests a post-operative immunosuppressed status in all patients, with a bigger impact by ACB, which might be attributed to the extracorporeal circulation or tissue damage occurring during surgery.

Human ß-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Development and Evaluation of a Droplet Digital PCR Assay for 8p23 ß-Defensin Cluster Copy Number Determination.

INTRODUCTION: It has been proposed that the copy number (CN) variation (CNV) in ß-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available. OBJECTIVE: Droplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay. METHODS: The assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes. RESULTS: In a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy. CONCLUSIONS: ddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.

Technical Approach Determines Inflammatory Response after Surgical and Transcatheter Aortic Valve Replacement.

OBJECTIVE: To investigate the periprocedural inflammatory response in patients with isolated aortic valve stenosis undergoing surgical aortic valve replacement (SAVR) or transcatheter aortic valve implantation (TAVI) with different technical approaches. MATERIAL AND METHODS: Patients were prospectively allocated to one of the following treatments: SAVR using conventional extracorporeal circulation (CECC, n = 47) or minimized extracorporeal circulation (MECC, n = 15), or TAVI using either transapical (TA, n = 15) or transfemoral (TF, n = 24) access. Exclusion criteria included infection, pre-procedural immunosuppressive or antibiotic drug therapy and emergency indications. We investigated interleukin (IL)-6, IL-8, IL-10, human leukocyte antigen (HLA-DR), white blood cell count, high-sensitivity C-reactive protein (hs-CRP) and soluble L-selectin (sCD62L) levels before the procedure and at 4, 24, and 48 h after aortic valve replacement. Data are presented for group interaction (p-values for inter-group comparison) as determined by the Greenhouse-Geisser correction. RESULTS: SAVR on CECC was associated with the highest levels of IL-8 and hs-CRP (p<0.017, and 0.007, respectively). SAVR on MECC showed the highest descent in levels of HLA-DR and sCD62L (both p<0.001) in the perioperative period. TA-TAVI showed increased intraprocedural concentration and the highest peak of IL-6 (p = 0.017). Significantly smaller changes in the inflammatory markers were observed in TF-TAVI. CONCLUSION: Surgical and interventional approaches to aortic valve replacement result in inflammatory modulation which differs according to the invasiveness of the procedure. As expected, extracorporeal circulation is associated with the most marked pro-inflammatory activation, whereas TF-TAVI emerges as the approach with the most attenuated inflammatory response. Factors such as the pre-treatment patient condition and the extent of myocardial injury also significantly affect inflammatory biomarker patterns. Accordingly, TA-TAVI is to be classified not as an interventional but a true surgical procedure, with inflammatory biomarker profiles comparable to those found after SAVR. Our study could not establish an obvious link between the extent of the periprocedural inflammatory response and clinical outcome parameters.