Preload-independent mechanisms contribute to increased stroke volume following large volume saline infusion in normal volunteers: a prospective interventional study

Introduction

Resuscitation with saline is a standard initial response to hypotension or shock of almost any cause. Saline resuscitation is thought to generate an increase in cardiac output through a preload-dependent (increased end-diastolic volume) augmentation of stroke volume. We sought to confirm this to be the mechanism by which high-volume saline administration (comparable to that used in resuscitation of shock) results in improved cardiac output in normal healthy volunteers.

Methods

Using a standardized protocol, 24 healthy male (group 1) and 12 healthy mixed sex (group 2) volunteers were infused with 3 l normal (0.9%) saline over 3 hours in a prospective interventional study. Individuals were studied at baseline and following volume infusion using volumetric echocardiography (group 1) or a combination of pulmonary artery catheterization and radionuclide cineangiography (group 2).

Results

Saline infusion resulted in minor effects on heart rate and arterial pressures. Stroke volume index increased significantly (by approximately 15–25%; P < 0.0001). Biventricular end-diastolic volumes were only inconsistently increased, whereas end-systolic volumes decreased almost uniformly. Decreased end-systolic volume contributed as much as 40–90% to the stroke volume index response. Indices of ventricular contractility including ejection fraction, ventricular stroke work and peak systolic pressure/end-systolic volume index ratio all increased significantly (minimum P < 0.01).

Conclusion

The increase in stroke volume associated with high-volume saline infusion into normal individuals is not only mediated by an increase in end-diastolic volume, as standard teaching suggests, but also involves a consistent and substantial decrease in end-systolic volumes and increases in basic indices of cardiac contractility. This phenomenon may be consistent with either an increase in biventricular contractility or a decrease in afterload.     

Generation of rabbit antibodies against death ligands by cDNA immunization

Specificity problems, especially in immunoblot analysis, have been shown for several commercial antibodies raised against the death ligand Fas ligand (FasL) using conventional protein and/or peptide immunizations. In this work, we have optimized the development of rabbit antisera and isolated pAb against the death ligands FasL, Apo2 ligand/TRAIL and Apo3 ligand/TWEAK by cDNA intramuscular immunization. This alternative approach has generated specific pAb in all three cases, which are useful for immunoblot purposes. The present data suggest that for the production of antibodies against certain glycosylated membrane proteins, cDNA immunization could be the method of choice.     

Behavior of Eosinophil Counts in Recovered and Deceased COVID-19 Patients over the Course of the Disease

Knowledge about the immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, particularly regarding the function of eosinophils, has been steadily emerging recently. There exists controversy regarding the implications of eosinophils in the coronavirus disease 2019 (COVID-19)’s pathology. We report a retrospective cohort study including the comparison of leukocyte counts in COVID-19 patients, considering the outcomes of recovery (n = 59) and death (n = 60). Among the different types of leukocytes, the eosinophil counts were those that showed the greatest difference between recovered and deceased patients. Eosinopenia (eosinophil count < 0.01 × 10⁹/L) was more frequently observed in deceased than recovered patients (p = 0.0012). The eosinophil counts more rapidly increased and showed a greater proportion over the course of the disease in the recovered than deceased patients. Furthermore, the estimated survival rate was greater in patients without eosinopenia than in patients with eosinopenia (p = 0.0070) during hospitalization. Importantly, recovered but not deceased patients showed high negative correlations of the eosinophils with the neutrophil-to-lymphocyte ratio (NLR) and neutrophil counts at Day 9 of the onset of clinical symptoms (p ≤ 0.0220). Our analysis suggests that eosinopenia may be associated with unfavorable disease outcomes and that the eosinophils have a beneficial function in COVID-19 patients, probably contributing by controlling the exacerbated inflammation induced by neutrophils.     

Role of oxidative damage and IL-1{beta}-converting enzyme-like proteases in Fas-based cytotoxicity exerted by effector T cells

The Implication of oxidative damage and/or intact mitochondrialfunction in physiological Fas-based cytotoxicity has been testedusing the cytolytic hybridoma d11S and the CD8⁺ CTL clone KB5.C20,previously stimulated to express Fas ligand (FasL) on theirsurface, as effectors and U937 or U937-p^° cells (depletedof mitochondrial DNA) as targets. Immobilized anti-Fas mAb,which induced death of U937 cells, Inhibited the growth of U937-p°cells but without inducing cell death. By contrast, FasL-expressingeffectors readily killed both targets, with induction of DNAfragmentation, in 20 h assays. These results demonstrate thelack of involvement of mitochondrial-derived free radicals and/orIntact mitochondrial function in physiological Fas-based cytotoxicity.Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas)with a polyunsaturated fatty acid, which induces cell deaththrough the generation of lipid free radicals, resulted in thepotentiation of Fas-based cytotoxicity. This potentiating effect,but not Fas-based cytotoxicity Itself, was eliminated by thephysiological antioxidant vitamin E. On the other hand, theIL-1ß-converting enzyme (ICE)-like protease tetrapeptideinhibitor Ac-YVAD-cmk partially Inhibited Fas-based cytotoxicity,while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was amuch more effective inhibitor of Fas-Induced apoptosis. It wasconcluded that Fas-Induced cytotoxicity was clearly dependenton ICE-LIke protease activation, and especially on that of CPP32in Fas-sensitive cells, including mitochondrial DNA-depletedones.     

Seasonal changes in sperm chromatin condensation in ram (Ovis aries), Iberian red deer (Cervus elaphus hispanicus), and brown bear (Ursus arctos)

The effect of seasonality (temperate environment, Spain) on the chromatin status of ovine (Churra breed), Iberian red deer, and brown bear spermatozoa was studied. This work aims to improve genetic resource banks (GRBs) by enhancing existing knowledge of the effect of season on sperm quality. Samples were obtained by electroejaculation in Iberian red deer and brown bear and by artificial vagina in ram. We used the sperm chromatin structure assay (SCSA) to study the level of chromatin condensation of the spermatozoa in each studied period. These periods were: ram, breeding season (from September to January), nonbreeding season (from February to June), and summer (July and August); red deer, breeding season (September and October), postbreeding (November and December), and nonbreeding (the rest of the year); brown bear, prebreeding (March and April), breeding (May and June), postbreeding (July and August), and nonbreeding (September to February). Chromatin in ram was more decondensated in summer, and no differences were observed between the breeding and nonbreeding season. However, in red deer, spermatozoa obtained during the nonbreeding season showed more condensed chromatin than those obtained in the rut and postrut periods. Similarly, brown bear rendered sperm with loose chromatin in the prebreeding and breeding seasons. Less condensed chromatin in the breeding season may be related to faster epididymal transit due to enhanced spermatogenesis.     

Two signaling pathways can lead to Fas ligand expression in CD8+ cytotoxic T lymphocyte clones

As shown previously, a given cytotoxic T lymphocyte (CTL) clone (KB5.C20) could be induced to express the Fas ligand (FasL) by either T cell receptor (TCR) engagement or phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. In contrast, another CTL clone (BM3.3) has now been found to exert Fas-based cytotoxicity only after TCR engagement, but not after PMA/ionomycin stimulation. This suggested the existence of a PMA-insensitive, antigeninduced pathway leading to FasL expression. The inability of PMA to promote Fas-based cytotoxicity in BM3.3 cells was correlated with a defect in expression of the classical protein kinase C (PKC) isoforms α and βI. In KB5.C20 cells depleted of PMA-sensitive PKC isoforms and thus no longer responsive to PMA, Fas-based cytotoxicity could still be induced via the TCR/CD3 pathway. On the other hand, a requirement for phosphatidylinositol-3 kinase (PI3K) selectively in this TCR/CD3-induced pathway was demonstrated by specific inhibition with wortmannin. These results suggest that FasL expression when induced via the TCR/CD3 involves PI3K, and when induced by PMA/ionomycin requires the expression of PMA-sensitive PKC isoforms absent in clone BM3.3. Additional data suggest that in neither case was NF-χB activation implicated in FasL expression.