Substantial heterogeneity of inflammatory cytokine production and its inhibition by a triple cocktail of toll-like receptor blockers in early sepsis.

Introduction: Early sepsis is a life-threatening immune dysregulation believed to feature a "cytokine storm" due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade. Methods: Whole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1α and IL-1ß were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies. Results: Only a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24 h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29-0.68; p<0.0001-0.04) and cytokine baseline concentrations (r=0.55; p<0.0001). Discussion: Upon meeting Sepsis-3 criteria for less than 24 h, a mere quarter of patient samples exhibits a strong inflammatory phenotype, as characterized by increased baseline inflammatory cytokine concentrations and a stark TLR-dependent increase upon further ex vivo incubation. Thus, early sepsis patient cohorts as defined by Sepsis-3 criteria are very heterogeneous in regard to inflammation. Accordingly, proper ex vivo assays may be useful in septic individuals before embarking on immunomodulatory treatments.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

COVID-19 in Elderly, Immunocompromised or Diabetic Patients-From Immune Monitoring to Clinical Management in the Hospital.

The novel, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic of acute respiratory illness worldwide and remains a huge threat to the healthcare system's capacity to respond to COVID-19. Elderly and immunocompromised patients are at increased risk for a severe course of COVID-19. These high-risk groups have been identified as developing diminished humoral and cellular immune responses. Notably, SARS-CoV-2 RNA remains detectable in nasopharyngeal swabs of these patients for a prolonged period of time. These factors complicate the clinical management of these vulnerable patient groups. To date, there are no well-defined guidelines for an appropriate duration of isolation for elderly and immunocompromised patients, especially in hospitals or nursing homes. The aim of the present study was to characterize at-risk patient cohorts capable of producing a replication-competent virus over an extended period after symptomatic COVID-19, and to investigate the humoral and cellular immune responses and infectivity to provide a better basis for future clinical management. In our cohort, the rate of positive viral cultures and the sensitivity of SARS-CoV-2 antigen tests correlated with higher viral loads. Elderly patients and patients with diabetes mellitus had adequate cellular and humoral immune responses to SARS-CoV-2 infection, while immunocompromised patients had reduced humoral and cellular immune responses. Our patient cohort was hospitalized for longer compared with previously published cohorts. Longer hospitalization was associated with a high number of nosocomial infections, representing a potential hazard for additional complications to patients. Most importantly, regardless of positive SARS-CoV-2 RNA detection, no virus was culturable beyond a cycle threshold (ct) value of 33 in the majority of samples. Our data clearly indicate that elderly and diabetic patients develop a robust immune response to SARS-CoV-2 and may be safely de-isolated at a ct value of more than 35.