Effect of anterior quadratus lumborum block with ropivacaine on the immune response after laparoscopic surgery in colon cancer: a substudy of a randomized clinical trial.

BACKGROUND: Surgery induces a temporal change in the immune system, which might be modified by regional anesthesia. Applying a bilateral preoperative anterior quadratus lumborum block has proven to be a safe and effective technique in pain management after abdominal and retroperitoneal surgery, but the effect on the immune response is not thoroughly investigated. METHODS: This study is a substudy of a randomized, controlled, double-blinded trial of patients undergoing laparoscopic hemicolectomy due to colon cancer. Twenty-two patients were randomized to undergo either a bilateral anterior quadratus lumborum nerve block with a total of 60 mL ropivacaine 0.375% or placebo with corresponding isotonic saline injections. The main objective of this exploratory substudy was to investigate the systemic immune response in the first postoperative day by examining changes in blood transcript levels (n=750) and stimulated secretion of cytokines (n=17) on ex vivo activation with microbial ligands and anti-CD3/CD28. RESULTS: Using unsupervised data analysis tools, we observed no effect of the bilateral anterior quadratus lumborum nerve block on gene expression in immune cells (permutational multivariate analysis of variance using distance matrices: F=0.52, p=0.96), abundances of major immune cell populations (Wilcoxon rank-sum test: p>0.05), and stimulated cytokine secretion (Wilcoxon rank-sum test: p>0.05). CONCLUSIONS: Our study provides evidence that administration of bilateral anterior quadratus lumborum nerve block as a part of a multimodal analgesic regimen in an enhanced recovery after surgery for laparoscopic hemicolectomy in this cohort does not alter the systemic immune response.Trial registration number NCT03570541.

Assessment of source material for human intestinal organoid culture for research and clinical use.

OBJECTIVE: Human intestinal organoids (hIOs) have potential as a model for investigating intestinal diseases. The hIO system faces logistic challenges including limited access to biopsies or low expression of epithelial cell types. Previous research identified the feasibility of tissue from the transverse (TC) or sigmoid colon (SC), or from cryopreserved biopsies from regions of the gastrointestinal tract. We aimed to create a protocol for robust hIO generation that could be implemented across multiple centres, allowing for development of a consistent biobank of hIOs from diverse patients. RESULTS: TC and SC hIOs were expanded from fresh or frozen biopsies with standard or refined media. The expression of epithelial cells was evaluated via PCR. Growth of TC and SC hIO from healthy donors was reproducible from freshly acquired and frozen biopsies. A refined media including insulin-like growth factor (IGF)-1 and fibroblast growth factor (FGF)-2 enabled the expression of epithelial cells, including higher expression of goblet cells and enterocytes compared to standard organoid media. We identified a consistent time point where hIOs generated from frozen biopsies reflect similar hIO composition from freshly acquired samples. Feasibility of hIOs as a tool for research and clinical use, including the use of frozen biopsies, was demonstrated.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

TNFα-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression.

Maintenance of regulatory T cells CD4+CD25highFOXP3+ (Treg) stability is vital for proper Treg function and controlling the immune equilibrium. Treg cells are heterogeneous and can reveal plasticity, exemplified by their potential to express IL-17A. TNFα-TNFR2 signaling controls IL-17A expression in conventional T cells via the anti-inflammatory ubiquitin-editing and kinase activity regulating enzyme TNFAIP3/A20 (tumor necrosis factor-alpha-induced protein 3). To obtain a molecular understanding of TNFα signaling on IL-17 expression in the human effector (effTreg, CD25highCD45RA-) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve (naïveTreg, CD25highCD45RA+) and effTreg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; effTreg (TNFαlow/IL-17Ahigh) and naïveTreg (TNFαhigh/IL-17Alow). In effTreg, TNFα-TNFR2 signaling prevented IL-17A expression, whereas inhibition of TNFα signaling by clinically applied anti-TNF antibodies led to increased IL-17A expression. Inhibition of TNFα signaling led to reduced TNFAIP3 expression, which, by using siRNA inhibition of TNFAIP3, appeared causally linked to increased IL-17A expression in effTreg. Kinome activity screening of CD3/CD28-activated effTreg revealed that anti-TNF-mediated neutralization led to increased kinase activity. STRING association analysis revealed that the TNF suppression effTreg kinase activity network was strongly associated with kinases involved in TCR, JAK, MAPK, and PKC pathway signaling. Small-molecule-based inhibition of TCR and JAK pathways prevented the IL-17 expression in effTreg. Together, these findings stress the importance of TNF-TNFR2 in regulating the kinase architecture of antigen-activated effTreg and controlling IL-17 expression of the human Treg. These findings might be relevant for optimizing anti-TNF-based therapy and may aid in preventing Treg plasticity in case of Treg-based cell therapy.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2'-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

An Autocrine TNFα-Tumor Necrosis Factor Receptor 2 Loop Promotes Epigenetic Effects Inducing Human Treg Stability In Vitro.

A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro, in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro. Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα-TNFR2 underlying the maintenance of Treg stability in vitro. Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα-TNFR2 loop plays an important role in endorsing Treg stability.

TNF-α-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-α blockade-driven IL-17A expression.

BACKGROUND: Anti-TNF inhibitors successfully improve the quality of life of patients with inflammatory disease. Unfortunately, not all patients respond to anti-TNF therapy, and some patients show paradoxical immune side effects, which are poorly understood. Surprisingly, anti-TNF agents were shown to promote IL-17A production with as yet unknown clinical implications. OBJECTIVE: We sought to investigate the molecular mechanism underlying anti-TNF-driven IL-17A expression and the clinical implications of this phenomenon. METHODS: Fluorescence-activated cell sorting, RNA sequencing, quantitative real-time PCR, Western blotting, small interfering RNA interference, and kinase inhibitors were used to study the molecular mechanisms in isolated human CD4+ T cells from healthy donors. The clinical implication was studied in blood samples of patients with inflammatory bowel disease (IBD) receiving anti-TNF therapy. RESULTS: Here we show that anti-TNF treatment results in inhibition of the anti-inflammatory molecule TNF-α-induced protein 3 (TNFAIP3)/A20 in memory CD4+ T cells. We found an inverse relationship between TNFAIP3/A20 expression levels and IL-17A production. Inhibition of TNFAIP3/A20 promotes kinase activity of p38 mitogen-activated protein kinase and protein kinase C, which drives IL-17A expression. Regulation of TNFAIP3/A20 expression and cognate IL-17A production in T cells are specifically mediated through TNF receptor 2 signaling. Ex vivo, in patients with IBD treated with anti-TNF, we found further evidence for an inverse relationship between TNFAIP3/A20 expression levels and IL-17A-producing T cells. CONCLUSION: Anti-TNF treatment interferes in the TNFAIP3/A20-mediated anti-inflammatory feedback loop in CD4+ T cells and promotes kinase activity. This puts TNFAIP3/A20, combined with IL-17A expression, on the map as a potential tool for predicting therapy responsiveness or side effects of anti-TNF therapy. Moreover, it provides novel targets related to TNFAIP3/A20 activity for superior therapeutic regimens in patients with IBD.

Differential Effects of Environmental and Genetic Factors on T and B Cell Immune Traits.

Effective immunity requires a complex network of cellular and humoral components that interact with each other and are influenced by different environmental and host factors. We used a systems biology approach to comprehensively assess the impact of environmental and genetic factors on immune cell populations in peripheral blood, including associations with immunoglobulin concentrations, from ∼500 healthy volunteers from the Human Functional Genomics Project. Genetic heritability estimation showed that variations in T cell numbers are more strongly driven by genetic factors, while B cell counts are more environmentally influenced. Quantitative trait loci (QTL) mapping identified eight independent genomic loci associated with leukocyte count variation, including four associations with T and B cell subtypes. The QTLs identified were enriched among genome-wide association study (GWAS) SNPs reported to increase susceptibility to immune-mediated diseases. Our systems approach provides insights into cellular and humoral immune trait variability in humans.

Etanercept biosimilars.

Etanercept was the first tumour necrosis factor alpha antagonist approved in the USA for the treatment of rheumatoid arthritis, in 1998, and then for other diseases. With the etanercept patent set to expire in the EU in 2015, a number of etanercept copies have reached the production phase and are undergoing clinical trials, with the promise of being cheaper alternatives to the reference product. In a global scenario that is favourable to the entry of biosimilars, this article discusses the stage of development, manufacture, clinical trials and the regulatory process involved in the approval of etanercept biosimilars, compiling the literature data. Reducing treatment cost is the principal attraction for biosimilars to emerge in the global market. It is essential for the doctors' decision on the prescription of these medications, as well as for payers, to have clearly defined studies of clinical equivalence, quality, and safety in order to better evaluate the various copies of etanercept. The authors discuss the need to harmonize different national regulations and the introduction of effective pharmacosurveillance systems for prompt recognition of adverse effects in copies of biopharmaceuticals that differ from those found in the reference products.