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Background: Procalcitonin is an indicator of systemic inflammation associated with major surgery or sepsis. Procalcitonin exists in a full-length and truncated variant as a result of dipeptidylpeptidase-4 (DPP4)-cleavage. We recently identified differential biological activity of both variants. Here, we present an immunoassay-based method for the separate detection of procalcitonin variants and correlation to clinical data in patients with severe systemic inflammation., Methods: Rabbits were immunized with peptides of N-terminal sequences of both human procalcitonin variants and polyclonal antibodies purified from rabbit plasma. Antibodies were used for the detection of procalcitonin variants in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available monoclonal anti-procalcitonin antibody as capture. Serum was collected from 19 septic patients exhibiting hyperprocalcitonemia as part of a cross-sectional study; clinical data were analyzed and correlated with procalcitonin variant measurements. DPP4 activity was determined by a DPP4 activity assay., Results: Purified antibodies allowed for the separate detection of both procalcitonin variants in all patients. Levels of truncated procalcitonin (truncPCT) correlated with DPP4-activity (Pearson's R = 0.85, P < .001) and negatively correlated with patients' Sequential Organ Failure Score (SOFA) scores (Pearson's R = -0.56, P = .013). In contrast, the correlation between full-length procalcitonin (fullPCT) and SOFA scores was positive (Pearson's R = 0.56, P = .013). Separation of the patient collective into groups with higher amounts of fullPCT versus truncPCT revealed higher SOFA scores in patients with fullPCT > truncPCT (mean ± standard error of the mean; 11. 3 ± 0.8 vs 6. 1 ± 1.5, P = .003). Patients with fullPCT > truncPCT showed a tendency towards higher doses of vasopressor (0. 2 ± 0.1 vs 0. 1 ± 0.03 µg/kg/min norepinephrine within the first 24 hours after sepsis diagnosis, P = .062) and exhibited higher creatinine (2. 0 ± 0.2 vs 1. 4 ± 0.3mg/dL, P = .019) and leukocyte levels (31. 0 ± 5.4 vs 12. 8 ± 1.9cells/µL, P = .012). In addition, patients with fullPCT > truncPCT were more often subjected to treatment with hydrocortisone (49.0 vs 0%, P = .018)., Conclusions: Polyclonal antibodies generated using procalcitonin N-terminal variant peptides as immunogens are suitable for procalcitonin variant assessment. The separate detection of procalcitonin variants may offer additional diagnostic value and can be correlated with organ dysfunction and clinical outcomes in patients with systemic inflammation., Competing Interests: Conflicts of Interest, Funding: Please see DISCLOSURES at the end of this article., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Anesthesia Research Society.)

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34 + cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34 + cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAM low CD103 - XCR1 - CD172a + CD11b + cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4 -/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAM low cDC2 (cDC2B) development and survival in vitro., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4 + T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3 - IL-10 + Tr1 cells but not Foxp3 + Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thomann, Schneider, Cyran, Eckert, Kerstan and Lutz.)

Objective: We wanted to characterize patient accompaniment to medical encounters and to explore the rationale and influence of the companion on the primary care medical encounter., Study Design: This was a descriptive study., Population: Academic general internal medicine physicians, patients, and patient companions participated., Outcomes Measured: We measured the frequency of waiting and examination room companions, the reasons for accompaniment, the influence on the encounter, and the overall helpfulness of the companion as assessed by patients and companions. We also determined the physicianamprsquos assessment of the companionamprsquos influence, helpfulness, and behavior during the encounter., Results: Companions were in the examination room for 16% of visits; 93% were family members. The rationales for waiting and examination room companions were to help with transportation, provide emotional support, and provide company. Examination room companions helped communicate concerns to the physician, remember the physicianamprsquos advice, make decisions, and communicate their own concerns to the physician. Patients believed that examination room companions influenced 75% of medical encounters, mainly by improving communication between physician and patient. Physicians agreed that examination room companions favorably influenced physician and patient understanding (60% and 46% of encounters, respectively). Patients indicated that waiting and examination room companions were very helpful for 71% and 83% of visits, respectively., Conclusions: Companions frequently accompany patients to their primary care medical encounters. They are often family members, and they assume important roles in enhancing patient and physician understanding.

Objective: To test the hypothesis that there is substantial use of a practitioner of alternative/complementary medicine by patients traditionally considered to be underserved., Design: Cross-sectional, self-administered survey study., Settings: Three university hospital-affiliated general ambulatory clinics serving patients of different socioeconomic status and racial origin., Subjects: Five hundred and thirty-six (93% of those attending) consecutive clinic attendees., Outcome Measures: Past use and desired future use of one or more practitioners of five modalities of alternative/complementary medicine and willingness to pay for these modalities out-of-pocket., Results: Past usage and desired future usage of one or more practitioners of alternative/complementary medicine was comparable at the three clinic sites despite wide differences in socioeconomic status and willingness/ability to pay out-of-pocket for these services. Multivariable analyses revealed lower self-rated health status and female gender (both p < 0.006) but not income, race, age or education as independent, significant predictors of use of a practitioner of alternative/complementary medicine., Conclusion: Usage of alternative/complementary medicine is not confined to any well-circumscribed socioeconomic group and is common in patients often considered to be underserved. Self-assessed lower health status is significantly and independently associated with use of a practitioner of alternative/complementary care.