Metamizole (Dipyrone) as an Alternative Agent in Postoperative Analgesia in Patients with Contraindications for Nonsteroidal Anti-Inflammatory Drugs.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) play an important role in multimodal pain management. In patients with a contraindication for NSAIDs, pain management is challenging. A recent Dutch anesthesiology guideline propagates the use of metamizole (dipyrone) in these patients. Metamizole is a controversial drug, its use being previously discouraged because of the risk for agranulocytosis. We discuss whether metamizole could be an alternative to classical NSAIDs and opioids in postoperative pain management despite this drawback. METHOD: Literature review and pharmacovigilance research based on World Health Organization adverse effect registrations. RESULTS: Metamizole causes fewer gastric and duodenal ulcers than other nonselective NSAIDs, and the risk for bleeding is limited. It is unknown whether it is safer than a nonselective NSAID combined with a proton pump inhibitor. Although the drug appears to be safe for renal function in healthy volunteers, data in high-risk patients (eg, those with heart or renal failure) are lacking. The incidence of metamizole-induced agranulocytosis is controversial, but the risk is likely to be limited with short-term postoperative use in this selected group of patients. CONCLUSION: Although firm evidence is lacking, metamizole may be safer for the upper intestinal tract and kidneys than other NSAIDs, and could alternatively be used in patients with an increased risk for stomach or renal problems. Hereby, improved postoperative pain relief can potentially be achieved. The risk for metamizole-induced agranulocytosis is judged to be acceptable.

Low-affinity TCR engagement drives IL-2-dependent post-thymic maintenance of naive CD4+ T cells in aged humans.

Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA(+) CD25(dim) CD4(+) T cells can develop from conventional naive CD25(-) CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA(+) CD25(dim) CD4+ T cells expressed a broad TCRVß repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA(+) CD25(dim) CD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.

Human secondary lymphoid organs typically contain polyclonally-activated proliferating regulatory T cells.

Immunomodulating regulatory T-cell (Treg) therapy is a promising strategy in autoimmunity and transplantation. However, to achieve full clinical efficacy, better understanding of in vivo human Treg biology is warranted. Here, we demonstrate that in contrast to blood and bone marrow Tregs, which showed a resting phenotype, the majority of CD4(pos)CD25(pos)CD127(neg)FoxP3(pos) Tregs in secondary lymphoid organs were proliferating activated CD69(pos)CD45RA(neg) cells with a hyperdemethylated FOXP3 gene and a broad T-cell receptor-Vß repertoire, implying polyclonal activation. Activated CD69(pos) Tregs were distributed over both T-cell and B-cell areas, distant from Aire(pos) and CD11c(pos) cells. In contrast to the anergic peripheral blood Tregs, lymphoid organ Tregs had significant ex vivo proliferative capacity and produced cytokines like interleukin-2, while revealing similar suppressive potential. Also, next to Treg-expressing chemokine receptors important for a prolonged stay in lymphoid organs, a significant part of the cells expressed peripheral tissue-associated, functional homing markers. In conclusion, our data suggest that human secondary lymphoid organs aid in the maintenance and regulation of Treg function and homeostasis. This knowledge may be exploited for further optimization of Treg immunotherapy, for example, by ex vivo selection of Tregs with capacity to migrate to lymphoid organs providing an in vivo platform for further Treg expansion.

Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations.

BACKGROUND: Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. OBJECTIVE: We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. METHODS: A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. RESULTS: Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. CONCLUSION: We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases.

Immunotherapy with regulatory T cells in transplantation.

Regulatory T cell (Treg)-based immunotherapy is of great interest to induce tolerance in clinical transplantation settings. In fact, the first clinical trials of Treg infusion after stem cell transplantation have recently begun. However, many important issues regarding human Treg immunotherapy are still to be resolved. In this review, we provide a short update on Tregs and elaborate on various strategies for Treg-based immunotherapy. First, infusion of ex vivo-selected naturally occurring Tregs is addressed, with emphasis on Treg isolation, expansion, antigen specificity, homing and stability. Next, the potential of ex vivo-induced Treg transfusion strategies is discussed. Finally, therapies aimed at in vivo increase of Treg numbers or function are addressed. In addition, we summarize the current knowledge on effects of immunosuppressive drugs on Tregs. In the following years, we expect exciting new data regarding the clinical application of Treg immunotherapy in transplantation to be released.

Clinical grade Treg: GMP isolation, improvement of purity by CD127 Depletion, Treg expansion, and Treg cryopreservation.

BACKGROUND: Treg based immunotherapy is of great interest to facilitate tolerance in autoimmunity and transplantation. For clinical trials, it is essential to have a clinical grade Treg isolation protocol in accordance with Good Manufacturing Practice (GMP) guidelines. To obtain sufficient Treg for immunotherapy, subsequent ex vivo expansion might be needed. METHODOLOGY/PRINCIPAL FINDINGS: Treg were isolated from leukapheresis products by CliniMACS based GMP isolation strategies, using anti-CD25, anti-CD8 and anti-CD19 coated microbeads. CliniMACS isolation procedures led to 40-60% pure CD4(pos)CD25(high)FoxP3(pos) Treg populations that were anergic and had moderate suppressive activity. Such CliniMACS isolated Treg populations could be expanded with maintenance of suppressive function. Alloantigen stimulated expansion caused an enrichment of alloantigen-specific Treg. Depletion of unwanted CD19(pos) cells during CliniMACS Treg isolation proved necessary to prevent B-cell outgrowth during expansion. CD4(pos)CD127(pos) conventional T cells were the major contaminating cell type in CliniMACS isolated Treg populations. Depletion of CD127(pos) cells improved the purity of CD4(pos)CD25(high)FoxP3(pos) Treg in CliniMACS isolated cell populations to approximately 90%. Expanded CD127(neg) CliniMACS isolated Treg populations showed very potent suppressive capacity and high FoxP3 expression. Furthermore, our data show that cryopreservation of CliniMACS isolated Treg is feasible, but that activation after thawing is necessary to restore suppressive potential. CONCLUSIONS/SIGNIFICANCE: The feasibility of Treg based therapy is widely accepted, provided that tailor-made clinical grade procedures for isolation and ex vivo cell handling are available. We here provide further support for this approach by showing that a high Treg purity can be reached, and that isolated cells can be cryopreserved and expanded successfully.

Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(pos)CD25(high) T cells for immunotherapy.

BACKGROUND: Regulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(pos)CD25(high) Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent. CONCLUSIONS/SIGNIFICANCE: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.