INTRODUCTION:Sepsis and septic shock are associated with microcirculatory dysfunction, significantly impacting patient outcomes. This study aimed to evaluate the effects of a 20 % albumin bolus on microcirculation compared to crystalloid resuscitation in fluid-responsive patients (ClinicalTrials.govID:NCT05357339). METHODS:We conducted a single-centre randomised controlled trial, enrolling 103 patients (Albumin n = 52, Control n = 51). Fluid responsiveness was assessed, and fluid was administered in boluses of 100 ml to clinical effect. Microcirculation was measured using the Side stream Dark Field camera and AVA 4.3 software. Baseline characteristics, macrohaemodynamics, and microcirculation parameters were recorded. Three patients were excluded from analysis. RESULTS:The final cohort comprised 100 patients, 35 (35 %) females with a mean age of 58 years (range: 18-86). The mean APACHE score was 28 (range: 7-45), and the mean SOFA score was 9.4 (range: 1-17). No significant differences in APACHE (26.24 vs. 29.4, p = 0.069) or SOFA (9.08 vs. 9.78, p = 0.32) scores were found for albumin and control group respectively. The albumin group had worse microcirculation at baseline but demonstrated significant improvements in microvascular density and activity at 15 min and 60 min (p < 0.005), while the control group exhibited no significant changes. Additionally, both groups were fluid responsive, with a mean pulse pressure variability of 17 % at admission. There were no significant differences in overall fluid balances, vasopressor days, length of ICU stay, or mortality between groups. CONCLUSION:This study demonstrates that a 20 % albumin bolus significantly enhances microcirculation in fluid-responsive patients with septic shock. These findings underscore the potential benefits of targeted microcirculation therapy in critically ill patients.

IntroductionSepsis is responsible for 1 in 5 deaths globally and the majority of those who survive have lasting health issues. A hallmark of sepsis is a deregulated inflammatory response to infection, with leukocytes playing a critical role.MethodsThis study utilised a targeted single-cell multi-omics approach to characterise peripheral blood mononuclear cell (PBMC) populations and their transcriptomic profiles in an Irish cohort of people with (i) sepsis and (ii) bacteraemia without sepsis (defined as clinically significant positive blood culture without sepsis as assessed by the Clinical Microbiology service).ResultsVariable leukocyte distributions were identified, with decreased cytotoxic lymphocytes, including CD8+ T cells, natural killer cells, CD56+ T cells, γδ T cells, mucosal-associated invariant T cells, and increased T helper (Th) cell subsets within sepsis samples. Additionally, PBMCs from sepsis samples displayed an impaired expression profile in effector T cells, resulting in widespread suppression of PBMC cytotoxic activity.DiscussionThese results identify potential mechanisms underlying the functional impairment witnessed in sepsis. Such mechanisms may inform future diagnostic and treatment strategies.

Background/Objectives: In septic shock, microcirculatory dysfunction contributes to organ failure and mortality. While sidestream dark-field (SDF) imaging is the reference method for assessing microvascular perfusion, its complexity limits routine use. This study evaluates near-infrared spectroscopy (NIRS) with vascular occlusion testing (VOT) as a potential bedside tool for monitoring microcirculatory changes following fluid resuscitation. Methods: Sixty-three fluid-responsive patients with sepsis were randomized to receive either 20% albumin or crystalloid. NIRS-VOT and sublingual SDF measurements were obtained at baseline and 60 min post-resuscitation. The reoxygenation slope (ReOx) derived from NIRS was calculated and compared with clinical severity scores and SDF-derived microcirculatory parameters. Results: ReOx significantly increased from baseline to 60 min in the albumin group (p = 0.025), but not in the crystalloid group. However, between-group differences at 60 min were not statistically significant. ReOx at 60 min was inversely correlated with APACHE II score (ρ = −0.325) and lactate (ρ = −0.277) and showed a weak inverse trend with norepinephrine dose. AUROC for ICU survival based on ReOx was 0.616. NIRS ReOx showed weak correlations with SDF parameters, including the number of crossings (p = 0.03) and the consensus proportion of perfused vessels (CPPV; p = 0.004). Conclusions: NIRS-VOT detected microcirculatory trends after albumin administration but showed limited agreement with SDF imaging. These findings suggest that NIRS and SDF assess different physiological domains. Further studies are warranted to define the clinical utility of NIRS as a microcirculation monitoring tool (Clinicaltrials.gov: NCT05357339).

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy controls (control group) in the setting of acute medical wards and intensive care units in a 1000-bed university hospital. A total of 42 patients with sepsis, 30 patients with infection, and 30 healthy controls were studied. The differentiation states of circulating mucosal associated invariant T (MAIT) cells and Natural Killer T (NKT) cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA−, CD197+), effector memory (CD45RA−, CD197−), or terminally differentiated (CD45RA+, CD197−). The differentiation states of circulating gamma-delta T lymphocytes were characterised as naive (CD45RA+, CD27+), central memory (CD45RA−, CD27+), effector memory (CD45RA−, CD27−), or terminally differentiated (CD45RA+, CD27−). The expression of IL-12 and IL-23 receptors, the transcription factors T-Bet and RORγt, and interferon-γ and IL-17a were analysed. Results: MAIT cell counts were lower in the septic group (p = 0.002) and the infection group (p < 0.001) than in the control group. The MAIT cell T-Bet expression in the infection group was greater than in the septic group (p = 0.012). The MAIT RORγt expression in the septic group was lower than in the control group (p = 0.003). The NK cell counts differed in the three groups (p < 0.001), with lower Natural Killer (NK) cell counts in the septic group (p < 0.001) and in the infection group (p = 0.001) than in the control group. The NK cell counts increased in the septic group in the 3 weeks following the onset of sepsis (p = 0.028). In lymphocyte stimulation experiments, fewer NK cells expressed T-Bet in the septic group than in the infection group (p = 0.002), and fewer NK cells expressed IFN-γ in the septic group than in the control group (p = 0.002). The NKT cell counts were lower in the septic group than both the control group (p = 0.05) and the infection group (p = 0.04). Fewer NKT cells expressed T-Bet in the septic group than in the infection group (p = 0.004). Fewer NKT cells expressed RORγt in the septic group than in the control group (p = 0.003). Fewer NKT cells expressed IFN-γ in the septic group than in both the control group (p = 0.002) and the infection group (p = 0.036). Conclusion: The clinical presentation of infection and or sepsis in patients is linked with a mosaic of changes in the innate lymphocyte Th1 and Th17 phenotypes. The manipulation of the innate lymphocyte phenotype offers a potential avenue for immune modulation in patients with sepsis.

Antibody panels for flow cytometry were prepared with specific antibodies for identification of lymphocyte populations, and antibodies specific to IL-23 receptor andIL-12 receptorβeta-2 subunit. Single stains were used for compensation controls and fluorescence minus one (FMO) controls were used to help set gates for flow cytometry.

Stimulation of PBMCs to study TH1 and TH17 Innate Lymphocytes. PBMCs were incubated in medium alone, or stimulated for 5 hours with 50 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin. Wells intended for intracellular staining for IL-17A and IFN-γ were stimulated in the presence of brefeldin A to prevent cytokine release from the cells. Antibody panels with 0.2% saponin were used for intracellular staining of IL17A and IFNγ. Antibody panels with Permeabilization Buffer (Miltenyi FoxP3 Staining Buffer Set) were made for intracellular staining of RORγ(t) and T-Bet.

How to prepare PBMCs for Stimulation experiment

Antibody panels for flow cytometry were prepared with specific antibodies for identification of lymphocyte populations, and antibodies specific to IL-23 receptor andIL-12 receptorβeta-2 subunit. Single stains were used for compensation controls and fluorescence minus one (FMO) controls were used to help set gates for flow cytometry.

OBJECTIVE:The role of Th1 and Th17 lymphocyte responses in human infection and sepsis of elderly patients has yet to be clarified. DESIGN:A prospective observational study of patients with sepsis, infection only and healthy controls. SETTING:The acute medical wards and intensive care units in a 1000 bed university hospital. PATIENTS:32 patients with sepsis, 20 patients with infection, and 20 healthy controls. Patients and controls were older than 65 years of age. Patients with recognised underlying immune compromise were excluded. METHODS:Phenotype, differentiation status and cytokine production by T lymphocytes were determined by flow cytometry. MEASUREMENTS:The differentiation states of circulating CD3+, CD4+, and CD8+ T cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentated (CD45RA+, CD197-). Expression of IL-12 and IL-23 receptors, and the transcription factors T-bet and RORγt, was analysed in circulating T lymphocytes. Expression of interferon- γ and IL-17A were analysed following stimulation in vitro. RESULTS:CD4+ T cells from patients with infection predominantly expressed effector-memory or terminally differentiated phenotypes but CD4+ T cells from patients with severe sepsis predominantly expressed naive phenotypes (p<0.0001). CD4+ T cells expressing IL-23 receptor were lower in patients with sepsis compared to patients with infection alone (p = 0.007). RORγt expression by CD4+ T cells was less frequent in patients with sepsis (p<0.001), whereas T-bet expressing CD8+ T cells that do not express RORγt was lower in the sepsis patients. HLA-DR expression by monocytes was lower in patients with sepsis. In septic patients fewer monocytes expressed IL-23. CONCLUSION:Persistent failure of T cell activation was observed in patients with sepsis. Sepsis was associated with attenuated CD8+Th1 and CD4+Th17 based lymphocyte response.

Stimulation of PBMCs to study TH1 and TH17 Lymphocytes. PBMCs were incubated in medium alone, or stimulated for 5 hours with functional mAbs specific for CD3 and CD28, 0.05 μg/ml lipopolysaccharide (LPS), or 50 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin. Wells intended for intracellular staining for IL-23, IL-12, IL-17A, and IFN-γ were stimulated in the presence of brefeldin A to prevent cytokine release from the cells. Antibody panels with 0.2% saponin were used for intracellular staining of IL12, IL23, IL17A and IFNγ. Antibody panels with Permeabilization Buffer (Miltenyi FoxP3 Staining Buffer Set) were made for intracellular staining of RORγ(t) and T-Bet.

Recent advances in sepsis therapy exclusively involve improvements in supportive care, while sepsis mortality rates remain disturbingly high at 30%. These persistently high sepsis mortality rates arise from the absence of sepsis specific therapies. However with improvements in supportive care, patients with septic shock commonly partially recover from the infection that precipitated their initial illness, yet they frequently succumb to subsequent health care associated infections. Remarkably today the pathophysiology of sepsis in humans, a common disease in western society, remains largely a conundrum. Conventionally sepsis was regarded as primarily a disorder of inflammation. More recently the importance of immune compromise in the pathophysiology of sepsis and health care associated infection has now become more widely accepted. Accordingly a review of the human evidence for this novel sepsis paradigm is timely. Septic patients appear to exhibit a complex and long-lasting immune deficiency state, involving lymphocytes of both the innate and adaptive immune responses that have been linked with mortality and the occurrence of health care associated infection. Such is the pervasive nature of immune compromise in sepsis that ultimately immune modulation will play a crucial role in sepsis therapies of the future.