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Background: Preload responsive postoperative patients with signs of inadequate organ perfusion are commonly assumed to be hypovolemic and therefore treated with fluids to increase preload. However, preload is influenced not only by blood volume, but also by venous vascular tone and the contribution of these factors to preload responsiveness in this setting is unknown. Based on this, the objective of this study was to investigate blood volume status in preload-responsive postoperative patients., Methods: Data from a clinical trial including postoperative patients after major abdominal surgery were analyzed. Patients with signs of inadequate organ perfusion and with data from a passive leg raising test (PLR) were included. An increase in pulse pressure by ≥ 9% was used to identify patients likely to be preload responsive. Blood volume was calculated from plasma volume measured using radiolabelled albumin and hematocrit. Patients with a blood volume of at least 10% above or below estimated normal volume were considered hyper- and hypovolemic, respectively., Results: A total of 63 patients were included in the study. Median (IQR) blood volume in the total was 57 (50-65) ml/kg, and change in pulse pressure after PLR was 14 (7-24)%. A total of 43 patients were preload responsive. Of these patients, 44% were hypovolemic, 28% euvolemic and 28% hypervolemic., Conclusions: A large fraction of postoperative patients with signs of hypoperfusion that are likely to be preload responsive, are hypervolemic. In these patients, treatments other than fluid administration may be a more rational approach to increase cardiac output. Trial registration EudraCT 2013-004446-42., (© 2023. The Author(s).)

Macrophages are ubiquitously distributed throughout the various tissues of the body and perform many functions including the orchestration of inflammatory responses against pathogens by classically activated M1 macrophages and the regulation of wound healing and tissue remodeling by anti-inflammatory, alternatively activated M2 macrophages. The responsibility for these pleiotropic functions lies in the expression of a myriad of surface receptors unique to given subsets of macrophages. Much of what we know about the function of human macrophage subsets has been gleaned by studying in vitro generated macrophages matured in the presence of GM-CSF or M-CSF and polarized with different cytokines. Oftentimes, culture conditions, such as the type of serum used, the duration of the culture, and the use of polarizing cytokines, vary between studies making direct comparisons difficult. Sample preparation and processing (e.g., Ficoll ® enrichment of leukocytes from whole blood) can also influence gene expression on human monocytes. Furthermore, overlap in surface marker expression can make it difficult to distinguish between different macrophage subsets.We directly compared the expression of over 20 different surface markers on M1 and M2a macrophages cultured in either serum-free media or in the presence of fetal bovine serum or human AB serum and found that the presence or type of serum used affected the expression of several markers such as CD200R1 and CD32. Moreover, we compared the expression of these surface markers on polarized and unpolarized macrophages and determined that polarization was critical to the expression of several of these markers including CD38 and SLAM F7. Differences in sample processing can alter the expression of surface markers, such as ACE-2, on monocytes. We observe that ACE-2 expression is higher on human whole blood CD14 + monocytes versus Ficoll ® -enriched CD14 + monocytes derived from PBMCs (peripheral blood mononuclear cells), where expression can be reduced by up to 50%. These results indicate that differences in serum, culture media, and sample processing can alter gene expression in both human macrophages and monocytes. Importantly, the results of these studies significantly expand our knowledge of the phenotypic differences between human M1 and M2a macrophages and demonstrate the importance of culture conditions in generating these phenotypes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Optimal infusion rate of colloids in patients with suspected hypovolemia is unknown, and the primary objective of the present study was to test if plasma volume expansion by 5% albumin is greater if fluid is administered slowly rather than rapidly., Methods: Patients with signs of hypoperfusion after major abdominal surgery were randomized to intravenous infusion of 5% albumin at a dose of 10 ml/kg (ideal body weight) either rapidly (30 min) or slowly (180 min). Plasma volume was measured using radiolabeled albumin at baseline, at 30 min, and at 180 min after the start of infusion. Primary outcome was change in plasma volume from the start of infusion to 180 min after the start of infusion. Secondary outcomes included the change in the area under the plasma volume curve and transcapillary escape rate (TER) for albumin from 180 to 240 min after the start of albumin infusion., Results: A total of 33 and 31 patients were included in the analysis in the slow and rapid groups, respectively. The change in plasma volume from the start of infusion to 180 min did not differ between the slow and rapid infusion groups (7.4 ± 2.6 vs. 6.5 ± 4.1 ml/kg; absolute difference, 0.9 ml/kg [95%CI, - 0.8 to 2.6], P = 0.301). Change in the area under the plasma volume curve was smaller in the slow than in the rapid infusion group and was 866 ± 341 and 1226 ± 419 min ml/kg, respectively, P < 0.001. TER for albumin did not differ and was 5.3 ± 3.1%/h and 5.4 ± 3%/h in the slow and in the rapid infusion groups, respectively, P = 0.931., Conclusions: This study does not support our hypothesis that a slow infusion of colloid results in a greater plasma volume expansion than a rapid infusion. Instead, our result of a smaller change in the area under the plasma volume curve indicates that a slow infusion results in a less efficient plasma volume expansion, but further studies are required to confirm this finding. A rapid infusion has no effect on vascular leak as measured after completion of the infusion., Trial Registration: EudraCT2013-004446-42 registered December 23, 2014.

Neutrophils are crucial mediators of host defense that are recruited to the central nervous system (CNS) in large numbers during acute bacterial meningitis caused by Streptococcus pneumoniae. Neutrophils release neutrophil extracellular traps (NETs) during infections to trap and kill bacteria. Intact NETs are fibrous structures composed of decondensed DNA and neutrophil-derived antimicrobial proteins. Here we show NETs in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis, and their absence in other forms of meningitis with neutrophil influx into the CSF caused by viruses, Borrelia and subarachnoid hemorrhage. In a rat model of meningitis, a clinical strain of pneumococci induced NET formation in the CSF. Disrupting NETs using DNase I significantly reduces bacterial load, demonstrating that NETs contribute to pneumococcal meningitis pathogenesis in vivo. We conclude that NETs in the CNS reduce bacterial clearance and degrading NETs using DNase I may have significant therapeutic implications.

Flow cytometry is a powerful technique that allows simultaneous detection of multiple markers on a specific cell population. This method is virtually unlimited as long as the specimen of interest can be put into a single-cell suspension for staining and subsequent analysis by the flow cytometer. Most investigators using this methodology are doing so because their cell population is rare in frequency and requires multiple markers to characterize their population of interest; thus standard methods such as Western blot and IHC are unsuitable due to limitations in cell number and the number of markers available. Most investigators using this method are using 6-14 parameters to study their cell populations of interest: however, using a large number of fluorochrome-labeled antibodies is hampered by the fact that suboptimal fluorochromes must be used, and that high and low cell density markers must be chosen with care. This is further complicated when the cell markers of interest are cytokines, transcription factors, surface markers, and/or phosphorylated proteins, each potentially requiring a specialized buffer system for optimal detection of the antibody of interest. This chapter focuses on optimizing flow cytometry staining methods for simultaneous detection of surface markers, transcription factors, secreted cytokines, and phosphorylated antibodies in a single stain on CD4+ human Th2 cells.

Background: Administration of fluids to restore normovolaemia is one of the most common therapeutic interventions performed peri-operatively and in the critically ill, but no study has evaluated the importance of infusion rate for the plasma volume-expanding effect of a resuscitation fluid. The present study is designed to test the hypothesis that a slow infusion of resuscitation fluid results in better plasma volume expansion than a rapid infusion., Methods/design: The study is a single-centre, assessor-blinded, parallel-group, randomised prospective study. Patients over 40 years of age admitted to the post-operative care unit after a Whipple procedure or major gynaecological surgery and presenting with signs of hypovolaemia are eligible for inclusion. Patients are randomised in a 1:1 fashion with no stratification to either rapid (30 minutes) or slow (180 minutes) infusion of 5% albumin at a dose of 10 ml/kg ideal body weight. Plasma volume is measured using 125 I human serum albumin at baseline (prior to albumin infusion) as well as at 30 minutes and 180 minutes after infusion start. The primary endpoint is change in plasma volume from baseline to 180 minutes after the start of 5% albumin infusion. Secondary endpoints include the integral of plasma volume over time from baseline to 180 minutes after the start of the infusion and transcapillary escape rate of albumin (%/h) from 180 minutes to 240 minutes after the start of albumin infusion. In addition, diuresis, change in central venous oxygen saturation, lactate and blood pressure will be evaluated. A total of 70 patients will be included in the study, and the study has 80% power to detect a difference of 4 ml/kg in plasma volume expansion between the two groups., Discussion: The present study is the first clinical investigation of the importance of infusion rate for the plasma volume-expanding effect of a resuscitation fluid., Trial Registration: EudraCT identifier: 2013-004446-42 . Registration date: 20 December 2013. ClinicalTrials.gov identifier: NCT02728921 . Registration date: 31 March 2016.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays. In our study, we observed a general correlation in donors' ranking between ELISPOT and flow cytometry; ELISA values did not correlate with either ELISPOT or flow cytometry. However, a detailed donor-to-donor comparison between ELISPOT and flow cytometry revealed significant discrepancies: donors who have similar numbers of IFNγ-positive cells measured by flow cytometry show 2-3-fold differences in the number of spot-forming cells (SFCs) measured by ELISPOT; and donors who have the same number of SFCs measured by ELISPOT show 30% differences in the number of IFNγ-positive cells measured by flow cytometry. Significant discrepancies between donors were also found when comparing ELISA and ELISPOT techniques: donors who secreted the same amount of IFNγ measured by ELISA show six-fold differences in the number of SFCs measured by ELISPOT; and donors who have 5-7-times less secreted IFNγ measured by ELISA show a two-fold increase in the number of SFCs measured by ELISPOT compared to donors who show a more profound secretion of IFNγ measured by ELISA. The results of our study suggest that there can be a lack of correlation between IFNγ values measured by ELISPOT, ELISA and flow cytometry. The higher number of cytokine-positive cells determined by flow cytometry is not necessarily indicative of a higher number of cytokine-secreting cells when they are analyzed by either ELISPOT or ELISA. Our ELISPOT vs. ELISA comparison demonstrates that the higher number of SFCs observed in ELISPOT does not guarantee that these cells secrete larger amounts of cytokines compared to donors with lower SFC numbers. In addition, our data indicate that ELISPOT, ELISA and flow cytometry should be performed as complementary, rather than stand-alone assays: running these assays in parallel on samples from the same donors may help to better understand the mechanisms underlying the physiology of cytokine-secreting cells.

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a-Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca(2+) sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were approximately 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased alpha(1)-adrenergic contraction. Following inhibition of PKC, alpha(1)-adrenergic contraction was normalized. PDBu-induced Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca(2+) sensitivity.

Ca(2+) sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca(2+) sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving approximately 50% of maximal active force were compared in small intestinal preparations: 1). Ca(2+)-activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2). Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5'-3-O-(thio)triphosphate); and 3). PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca(2+) sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca(2+) sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca(2+) sensitization in smooth muscle.

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.

Agonist-induced activation of smooth muscle involves a rise in intracellular Ca(2+) concentration and sensitization of myosin light chain phosphorylation to Ca(2+). Sr(2+) can enter through Ca(2+) channels, be sequestered and released from sarcoplasmic reticulum, and replace Ca(2+) in activation of myosin light chain phosphorylation. Sr(2+) cannot replace Ca(2+) in facilitation of agonist-activated Ca(2+)-dependent nonselective cation channels. It is not known whether Sr(2+) can replace Ca(2+) in small G protein-mediated sensitization of phosphorylation. To explore mechanisms involved in alpha-receptor-activated contractions in smooth muscle, effects of replacing Ca(2+) with Sr(2+) were examined in rat portal vein. Norepinephrine (NE) at >3.0 x 10(-7) M in the presence of Ca(2+) resulted in a strong sustained contraction, whereas this sustained component was absent in the presence of Sr(2+); only the amplitude of phasic contractions increased. Pretreatment with low (approximately 0.05 mM) free Ca(2+) followed by 2.5 mM Sr(2+) resulted in a sustained component of the NE response. In beta-escin-permeabilized preparations, phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization to Sr(2+). In conclusion, a Ca(2+)-regulated membrane/channel process is required for the sustained component of NE responses in rat portal vein. Sensitization alone is not responsible for the sustained phase of the NE contraction.

Flow cytometry was used to study signaling events in individual CD4 T cells after antigen recognition in the body. Phosphorylation of c-jun and p38 mitogen-activated protein kinase was detected within minutes in all antigen-specific CD4 T cells in secondary lymphoid tissues after injection of peptide antigen into the bloodstream. The remarkable rapidity of this response correlated with the finding that most naive T cells are in constant contact with dendritic antigen-presenting cells. Contrary to predictions from in vitro experiments, antigen-induced c-jun and p38 mitogen-activated protein kinase phosphorylation did not depend on CD28 signals and was insensitive to inhibition by cyclosporin A. Our results highlight the efficiency of the in vivo immune response and underscore the need to verify which signaling pathways identified in vitro actually operate under physiological conditions.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.

Fetal thymic organ culture of TCR transgenic (Tg) tissue has been used to study issues of timing and specificity in T cell development. Because most TCR Tgs express a rearranged alphabeta TCR on the cell surface at an earlier stage in development than normal mice, there is a possibility that the conclusions of studies using TCR Tg cultures may not apply to normal development. In particular, in our studies of peptide-induced development of CD8 T cells, it is possible that the peptide acts on the immature double-negative cell, driving development of CD8 T cells without passing through a double-positive stage. This issue was examined by asking whether MHC class I restriction was required and by analyzing CD8beta levels and endogenous TCR alpha chain rearrangements. We found that if nonstimulatory peptides were used in fetal thymic organ culture, CD8 T cells developed via the conventional pathway, transiting through a double-positive stage. However, we could not rule out that cells selected in the presence of stimulatory peptides (agonists) did not develop directly from double-negative precursors.