White Cell Activation Research Articles

Utility of Differential White Cell Count and Cell Population Data for Ruling Out COVID-19 Infection in Patients With Community-Acquired Pneumonia

IntroductionThe main aim of this study was to assess the utility of differential white cell count and cell population data (CPD) for the detection of COVID-19 in patients admitted for community-acquired pneumonia (CAP) of different etiologies. MethodsThis was a multicenter, observational, prospective study of adults aged ≥18 years admitted to three teaching hospitals in Spain from November 2019 to November 2021 with a diagnosis of CAP. At baseline, a Sysmex XN-20 analyzer was used to obtain detailed information related to the activation status and functional activity of white cells. ResultsThe sample was split into derivation and validation cohorts of 1065 and 717 patients, respectively. In the derivation cohort, COVID-19 was confirmed in 791 patients and ruled out in 274 patients, with mean ages of 62.13 (14.37) and 65.42 (16.62) years, respectively (p<0.001). There were significant differences in all CPD parameters except MO-Y. The multivariate prediction model showed that lower NE-X, NE-WY, LY-Z, LY-WY, MO-WX, MO-WY, and MO-Z values and neutrophil-to-lymphocyte ratio were related to COVID-19 etiology with an AUC of 0.819 (0.790, 0.846). No significant differences were found comparing this model to another including biomarkers (p=0.18). ConclusionsAbnormalities in white blood cell morphology based on a few cell population data values as well as NLR were able to accurately identify COVID-19 etiology. Moreover, systemic inflammation biomarkers currently used were unable to improve the predictive ability. We conclude that new peripheral blood biomarkers can help determine the etiology of CAP fast and inexpensively.

The Multifaceted Benefits of Exercise in Prevention of COVID-19

The Multifaceted Benefits of Exercise in Prevention of COVID-19

Feasibility of quantifying change in immune white cells in abdominal adipose tissue in response to an immune modulator in clinical obesity.

BackgroundObesity is often associated with inflammation in adipose tissue (AT) with release of mediators of atherogenesis. We postulated that it would be feasible to collect sufficient abdominal AT to quantify changes in a broad array of adaptive and innate mononuclear white cells in obese non-diabetic adults in response to a dipeptidyl protease inhibitor (DPP4i), known to inhibit activation of immune white cells.MethodsAdults 18–55 years-of-age were screened for abdominal obesity and insulin resistance or impaired glucose tolerance but without known inflammatory conditions. Twenty-one eligible participants consented for study and were randomized 3:1 to receive sitagliptin (DPP4i) at 100mg or matching placebo daily for 28 days. Abdominal AT collected by percutaneous biopsy and peripheral blood mononuclear cell fractions were evaluated before and after treatment; plasma was stored for batch testing.ResultsHighly sensitive C-reactive protein, a global marker of inflammation, was not elevated in the study population. Innate lymphoid cells (ILC) type 3 (ILC-3) in abdominal AT decreased with active treatment compared with placebo (p = 0.04). Other immune white cells in AT and peripheral blood mononuclear cell (PBMC) fractions did not change with treatment compared to placebo (p>0.05); although ILC-2 declined in PBMCs (p = 0.007) in the sitagliptin treatment group. Two circulating biomarkers of atherogenesis, interferon-inducible protein-10 (IP-10) and sCD40L declined in plasma (p = 0.02 and p = 0.07, respectively) in the active treatment group, providing indirect validation of a net reduction in inflammation.ConclusionsIn this pilot study, two cell types of the innate lymphoid system, ILC-3 in AT and ILC-2 PBMCs declined during treatment and as did circulating biomarkers of atherogenesis. Changes in other immune cells were not demonstrable. The study showed that sufficient abdominal AT could be obtained to quantify white cells of both innate and adaptive immunity and to demonstrate changes during therapy with an immune inhibitor.Trial registrationClinicalTrials.gov identifier (NCT number): NCT02576

Studies of White Cell, Platelet, and Coagulation Activation with Plerixafor Administration in Patients with Sickle Cell Disease

Background: Increased baseline white blood cell count (WBC) has been associated with vaso-occlusive complications in sickle cell disease (SCD), raising concern for the use of mobilizing agents that increase WBC count for hematopoietic progenitor cell (HPC) collection for gene therapy. Indeed, G-CSF, which predominantly increases neutrophil counts, when administered to SCD patients can precipitate life-threatening vaso-occlusion. Such vaso-occlusion, however, does not always correlate with degree of white blood cell count elevation (Fitzhugh C et al, Cytotherapy 2009) and G-CSF is associated with increases in neutrophil activation and adhesion. This raises the question whether WBC might be a surrogate for other proposed contributors to vaso-occlusion, such as activation of white cells, platelets, and coagulation. We examined this issue in the context of a clinical study of HPC mobilization with a single dose of plerixafor alone in clinically stable SCD patients.Materials and Methods: 14 patients (13 SS, 1 SB0, median age 31 with range 21-46) have completed the study so far (6 patients at 80 ug/kg, 3 patients at 160 ug/kg, and 5 patients at 240 ug/kg). 10 patients were on hydroxyurea (HU) at a median dose of 25 mg/kg (range 16-28 mg/kg) with a mean HbF of 17% versus 7% for the non-HU treated patients. Only one patient (non-HU treated) had received recent red cell transfusion (for leg ulcers) and had HbA (54%) present. Whole blood was collected at means of 4 hr pre-dose and 12 hr post-dose for activation marker studies by flow cytometry (cell studies) or ELISA (coagulation studies) as listed in Table 1.Results: Increases in absolute total white cell, neutrophil, monocyte, and lymphocyte concentrations in whole blood with plerixafor treatment were all highly significant (Table 2, p < 0.002), without significant differences between HU- and non-HU treated patients or between dose levels. Twelve of 14 patients, however, did not experience any significant adverse events from plerixafor administration. Absolute neutrophil counts (ANC) were strongly correlated with absolute concentrations (but not percentages) of activated β2 integrin neutrophils (r= 1.0) and activated Mac-1 neutrophils (r= 0.8) but no other activation markers. We observed significant plerixafor-associated increases in activation markers (Figure 1) only for ANC subsets positive for activated β2 integrin (1.7± 0.44 fold, mean ± SD, p=0.002) and activated Mac-1 (1.7 ± 1.2 fold, p=0.05), which the correlations with ANC partly explain. The activated Mac-1 significance, however, disappeared with Bonferroni correction. There were no significant differences in activation markers between dose levels or between HU- and non-HU treated patients. There were moderate correlations between activated β2 integrin or activated Mac-1 and absolute reticulocyte count (r= 0.6 for both) and HbF (r= -0.5 for both) but not between these activation markers and LDH. Two patients had pain crises starting at 48 hr (80 ug/kg dose) and 81 hr (240 ug/kg dose) post-plerixafor administration; their pre and post-dose (12 hr) white counts are shown in Table 3. The fold-increase in activation markers for these 2 patients is summarized in Figure 2 (for each activation marker, color-coded arrows point to the two patients)Conclusion: As with G-CSF, elevation of WBC or their subsets with plerixafor is not necessarily associated with vaso-occlusion, as manifested by pain crises. In our study, however, ANC elevation correlated with absolute concentrations of neutrophils positive for activated β2 integrin and activated Mac-1, which were the only activation markers significantly increased with plerixafor. These activation markers in turn correlated with reticulocyte count and HbF, which influence SCD vaso-occlusive severity. Vaso-occlusion as manifested by pain crises, however, was not consistently associated with the highest fold increases in ANC or activation markers. Therefore it remains unclear which bioassays correlate with vaso-occlusion associated with plerixafor mobilization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

White cell and marrow imaging: subtracting uncertainty.

A subtraction method for assessments of dual-isotope indium-111 (In)-labelled white cell and Tc-nanocoll was proposed, and following a pilot study was introduced in routine service to improve the confidence of reporters. This technical note describes a simple technique to produce subtraction images from routinely acquired images to investigate and exclude post joint replacement infection. Labelled leucocytes (with In) show areas of increased white cell accumulation, whereas Tc-nanocoll images show bone marrow distribution. Images resulting from the subtraction protocol highlight any areas of white cell activity which is not concurrent with the marrow distribution indicative of infection. Fusion images produced from these images sets also assists in localization of white cell accumulation(s). Key to the success of this technique is the use of simultaneous acquisition of both white cell and marrow images; thus removing any issues with patient movement and/or repositioning. Reporting clinicians convey a clear preference for the inclusion of the subtracted and fused images at clinical review.

Platelet activation and clotting cascade activation by dialyzers designed for high volume online hemodiafiltration.

Hemodialysis patients are pro-thrombotic. Higher volume online postdilutional hemodiafiltration (OL-HDF), with increasing hematocrit increases the risk of clotting in the extracorporeal circuit (ECC). We wished to determine whether OL-HDF increased platelet activation and ECC clotting. Coagulation parameters, platelet, white cell, and endothelial activation markers were measured at the start and end of dialysis sessions in 10 patients and also pre- and post-dialyzer after 15 minutes using two different dialyzers designed for high volume OL-HDF; cellulose triacetate (TAGP) and polysulphone (PS), and polyvinylpyrrolidone (PVP). Patients were anticoagulated with a heparin bolus. At the start of OL-HDF, D dimers, thrombin antithrombin complexes (TATs), and soluble adhesions molecules (sICAM-1 and sVCAM-1) were increased. Post-treatment soluble P selectin (PS/PVP 26.7 ± 7.1 versus 36.6 ± 9.9; TAGP 28.7 ± 7.2 versus 43.5 ± 8.4 ng/ml, P < 0.001), and soluble CD40 ligand (PS/PVP 297 ± 228 versus 552 ± 272, TAGP 245 ± 187 versus 390 ± 205 ng/ml, P < 0.05) increased. Post-dialyzer concentrations increased versus pre-dialyzer for tissue factor (PS/PVP 117 ± 12 versus 136 ± 16, TAGP 100 ± 25 versus 128 ± 40 ng/ml, P < 0.05), factor VIIIc (PS/PVP 174 ± 54 versus 237 ± 83, TAGP 163 ± 60 versus 247 ± 102 IU/ml, P < 0.01), sVCAM-1 (PS/PVP 782 ± 64 versus 918 ± 140, TAGP 722 ± 121 versus 889 ± 168 ng/ml, P < 0.01), and D-dimers (PS/PVP 292 ± 132 versus 355 ± 167, TAGP 300 ± 129 versus 391 ± 171 ng/ml, P < 0.001). There was no macroscopic thrombus noted in the ECC, and no increase in microparticles, platelet factor-4, or TATs. Despite being pro-thrombotic, with activation of platelets, and lymphocytes during passage through ECC, no macroscopic clotting, or increased TATs were noted during OL-HDF, and no major differences between cellulosic and polysulphone dialyzers.

Gram Negative Bacterial Inflammation Ameliorated by the Plasma Protein Beta 2-Glycoprotein I.

Lipopolysaccharide (LPS) is a major component of the outer wall of gram negative bacteria. In high doses LPS contributes to the inflammation in gram negative sepsis, and in low doses contributes to the low grade inflammation characteristic of the metabolic syndrome. We wanted to assess the role of beta2-glycoprotein I (β2GPI) a highly conserved plasma protein and its different biochemical forms in a mouse model of LPS systemic inflammation. Normal and β2GPI deficient mice were administered LPS through their veins and assessed for a range of inflammation markers in their blood and liver. Different biochemical forms of β2GPI were measured in normal mice given either saline or LPS. We show that β2GPI has a significant role in inhibiting LPS induced inflammation. In this study we provide some evidence that β2GPI serves a protective role in a mouse model of LPS inflammation. This resolves the controversy of previous studies which used LPS and β2GPI in test tube based models of LPS induced activation of white cells. We also highlight the potential relevance of a newly discovered biochemical form of β2GPI in LPS mediated inflammation and we speculate that this form has a protective role against LPS induced pathology.

New light on white matter damage of the premature brain: a neonatologist's point of view

Periventricular leucomalacia (PVL) is traditionally considered a multifactorial lesion related to three main mechanisms: ischemia, inflammation and excitotoxicity. For years it was believed that hypoperfusion, associated with the peculiar vascular anatomy of the premature brain (border zones), was the conditio sine qua non in the pathogenesis of PVL. More recently this theory has been questioned. Many studies have stressed the importance of the association between inflammation/infection and white matter injury and have supported the multi hit hypothesis according to which several (genetic, hormonal, immune and nutritional) factors may team up in a multi-hit fashion. The emerging concept is that the fetal white cell activation together with the interaction between the innate and adaptive immune system play a main role in white matter damage. Currently there are increasing evidence that PVL is a disease of connectivity. In this article we review the news in the basics of pathogenesis, the incidence, the definition and the diagnosis of PVL. Furthermore, recent follow-up studies and neuroprotective therapies are mentioned. Proceedings of the International Course on Perinatal Pathology (part of the 10 th International Workshop on Neonatology · October 22 nd -25 th , 2014) · Cagliari (Italy) · October 25 th , 2014 · The role of the clinical pathological dialogue in problem solving Guest Editors: Gavino Faa, Vassilios Fanos, Peter Van Eyken

Effects of long-term cryopreservation on peripheral blood progenitor cells

The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34(+) cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.

Dialyzers Designed to Increase Internal Filtration Do Not Result in Significantly Increased Platelet Activation and Thrombin Generation

Introduction: To increase middle molecule clearances, high-flux dialyzers with increased internal filtration have been developed. However, dialyzer design and structure may affect thrombin generation and platelet activation, thereby risking increased clotting and reduced dialyzer clearances. Methods: Coagulation parameters, platelet, white cell and endothelial activation markers were measured prior to and following dialysis sessions in 12 patients using two different dialyzers designed for increased internal filtration. Results: Prior to dialysis, patients had evidence of activation of coagulation with increased factor VIII:C, thrombin-antithrombin complexes and prothrombin fragment 1+2, increased platelet activation, with raised platelet factor 4, β-thromboglobulin levels and increased fibrinolysis (raised D-dimers). Dialysis was associated with the release of soluble platelet integrin, sP selectin, increased endothelial activation with increased levels of von Willebrand factor (vWF) antigen (vWF:Ag) and vWF propeptide (vWF:pp) and sE selectin. There was no difference in tinzaparin levels at the end of the dialysis session using either dialyzer, as shown by anti-Xa activity – 0.145 ± 0.027 versus 0.11 ± 0.017 IU/ml, respectively. Conclusion: Haemodialysis patients have an inflammatory phenoytype, characterized by increased activation of coagulation, platelets and also fibrinolysis. However, dialyzers designed to increase internal filtration did not significantly increase platelet activation or thrombin generation.

Effect of maternal fish oil and seaweed extract supplementation on colostrum and milk composition, humoral immune response, and performance of suckled piglets1

An experiment with a 2 x 2 factorial arrangement of treatments (n = 10 sows/treatment) was conducted to investigate the effect of maternal dietary supplementation with seaweed extract (SWE: 0 vs. 10.0 g/d) and fish oil (FO) inclusion (0 vs. 100 g/d) from d 109 of gestation until weaning (d 26) on sow colostrum and milk composition, humoral immune response on d 5 and 12 of lactation, and suckling piglet performance. Furthermore, the influence of dietary treatment on the phagocytic activity of whole blood white cells at weaning was examined. The SWE (10 g) contained laminarin (1 g), fucoidan (0.8 g), and ash (8.2 g) and was extracted from a Laminaria spp. The FO contained approximately 40% eicosapentaenoic acid and 25% docosahexaenoic acid. The SWE-supplemented sows had greater colostrum IgG (P < 0.01) and milk protein (P < 0.05) concentrations on d 12 of lactation compared with non-SWE-supplemented sows. Piglets suckling SWE-supplemented sows had greater serum IgG (P < 0.01) and IgA (P < 0.05) concentrations on d 5 and IgG concentrations on d 12 (P < 0.05) of lactation compared with those suckling non SWE-supplemented sows. In contrast, FO supplementation exerted a suppressive effect on piglet serum IgA concentrations on d 5 of lactation (P < 0.05) compared with non-FO-supplemented diets. Dietary FO supplementation enhanced the n-3 PUFA proportion of sow milk (P < 0.001) and piglet serum at weaning (P < 0.001). Piglets suckling SWE-supplemented sows had a greater percentage of Escherichia coli phagocytizing leukocytes (P < 0.05) and a reduced percentage of E. coli phagocytizing lymphocytes (P < 0.01) compared with non-SWE-supplemented sows. Piglets suckling FO-supplemented sows had a greater percentage of leukocytes (P < 0.05) and lymphocytes (P < 0.05) phagocytizing E. coli compared with non-FO-supplemented sows. However, total leukocyte, lymphocyte, monocyte, and neutrophil numbers were not influenced by sow dietary treatment. Average piglet weaning weight and ADG between birth and weaning were not influenced by sow dietary treatment. In conclusion, the current study demonstrates that SWE supplementation from d 109 of gestation until weaning enhanced colostral IgG concentrations and circulatory IgG concentrations in suckled piglets on d 5 and 12 of lactation. Furthermore, the percentage of leukocytes and lymphocytes phagocytizing E. coli at weaning increased in piglets suckling FO-supplemented sows, indicating an enhancement of immune function against presenting pathogens. However, the combination of SWE and FO bestowed no positive effect on immune responses investigated in the current study.

A Pilot Study of Tapered Oral Dexamethasone for the Acute Chest Syndrome of Sickle Cell Disease.

Abstract 1515Poster Board I-538 BACKGROUNDAcute chest syndrome (ACS) is a common cause of morbidity and mortality for individuals with sickle cell disease (SCD). The treatment of ACS is mainly supportive. Prior studies have shown that intravenous pulse-dose corticosteroids, such as dexamethasone, can decrease the duration and morbidity of ACS. However, such treatment may also precipitate ‘rebound‘ episodes of vaso-occlusive pain in some individuals that might negate the overall benefit of corticosteroids. Tapered corticosteroid therapy might decrease the rebound effect while maintaining therapeutic benefit. Therefore, we designed a prospective study to begin to test this hypothesis and to assess biomarkers that might clarify the therapeutic and toxic mechanisms of corticosteroids in SCD, predict outcome, and guide therapy. METHODSWe conducted a multi-center, placebo-controlled pilot study to test the feasibility and safety of high-dose oral dexamethasone followed by a taper for ACS. Children and adults with SCD and ACS of any severity were randomized to dexamethasone (0.3 mg/kg q12h x 2, 0.3 mg/kg q24h x 2, 0.2 mg/kg q24h x 2, 0.1 mg/kg q24h x2, then stop) or placebo to start within 24 hours of diagnosis. All subjects received standard, protocol-directed supportive care for ACS. We defined the duration of ACS using a novel, objective tool that assessed rate and effort of breathing, oxygen saturation in room air, thoracic pain, and use of supplemental oxygen and ventilatory support. The primary outcomes were the duration of ACS and the duration of hospitalization for ACS. We also measured a panel of biomarkers before, during and after therapy. Inflammatory biomarkers included high sensitivity C-reactive protein and secretory phospholipase A2 (sPLA2). White cell and endothelial activation markers included sVCAM-1, sICAM-1, vWF:Ag and vWF:RCoF, sE-selectin, sP-selectin, sL-selectin, nitric oxide metabolites (NO), and whole blood tissue factor. We used generalized linear mixed models controlling for age to test for differences between treatment groups. RESULTSWe enrolled 12 subjects (9 children, 3 adults; mean age 17.3 years, range 5 - 45) with homozygous sickle cell anemia at 4 centers and randomized 11 (1 drop-out) to either dexamethasone (N=5) or placebo (N=6). The objective ACS assessment tool was completed on all subjects without difficulty. In this pilot study, dexamethasone reduced the duration of hospitalization (41.5 vs 62.3 hrs; P=0.024), but not the duration of ACS (log of duration 2.4 vs 3.5 hrs; P=0.127), supplemental oxygen (17.5 vs 41.2 hrs; P=0.876), hypoxemia (13.8 vs 34.3 hrs; P=0.770), or total opioid usage in morphine equivalents (54.4 vs 68.8 mg; P=0.885). There were no statistically significant differences in adverse events between arms. However, 3 patients treated with dexamethasone had a painful event in the 2 weeks after hospital discharge (1 required re-hospitalization), compared to 1 patient in the placebo group (0 re-hospitalizations). No marked leukocytosis occurred in either treatment group, but the leukocyte count at 1-week follow-up had decreased less from baseline in the dexamethasone group compared to placebo (-17.7 vs -37.6%; P=0.028). The baseline sPLA2 concentration was 3 100 ng/mL in 4/5 and 4/6 patients in the dexamethasone and placebo arms. The white cell activation marker, sL-selectin was significantly decreased at 1-week follow-up in the dexamethasone group compared to placebo (573.8 ng/mL vs 742.8; change from baseline -121.2 vs 57.2; P<0.001). Patients who had rebound pain (N=4) had a significantly higher sL-selectin concentration after 24 hours of study drug (746.0 ng/mL vs 568.7, P=0.042). NO metabolites were significantly decreased after 24 and 48 hours of dexamethasone group compared to placebo (6.5 nMol/mL vs 9.1; change from baseline -2.7 vs 4.4; P=0.041). CONCLUSIONSA multi-center, randomized trial of tapered oral dexamethasone is feasible. Our novel ACS assessment tool is also feasible and useful for objective clinical ratings of ACS. Dexamethasone shortened the duration of hospitalization for ACS in this pilot study. Rebound vaso-occlusive pain occurred in both treatment groups. The WBC activation marker sL-selectin appears to be a promising biomarker of ACS therapy and rebound vaso-occlusion. Our results justify a definitive study of tapered oral dexamethasone for ACS that incorporates objective assessment tools and biomarkers. DisclosuresOff Label Use: Dexamethasone is a corticosteroid agent. We test its use for acute chest syndrome in this study.

Cyclosporine, an Immunosuppressant, Attenuates Phorbol-Induced Lung Injury in Rats

Objective White cell activation in the lung plays a critical role to induce lung injury and lymphocytes in the thoracic duct system may also participate. We evaluated the effect of cyclosporine on phorbol myristate acetate (PMA)-induced lung injury. Materials and Methods We used an in situ isolated, blood perfused rat lung model to measure pulmonary arterial pressure (PAP) and lung weight gain (LWG; g) for 50 minutes after a bolus injection of PMA (0.05 μg/mL). Oxygen radical release was estimated by an LKB 1251 luminometer and by nitric oxide (NO) release as measured by an ENO-20 NO analyzer. Results In the group exposed to PMA alone, the mean PAP increased from 16.53 ± 1.28 to 43.33 ± 3.40 mm Hg ( P < .001), and lung weight increased by 4.35 ± 0.67 g during the 50-minute perfusion after PMA challenge ( P < .001). In vitro measurement showed that PMA induced a significant increase in oxygen radical release ( P < .001). PMA attenuated NO release ( P < .001) into the perfusion system. Pretreatment with cyclosporine (3 mg/kg) for 3 days prevented the increases in both PAP ( P < .01) and LWG ( P < .001). NO release was maintained in cyclosporine-pretreated rats. Cyclosporine also showed dose-dependent attenuation of oxygen radical release by PMA-activated white blood cells. Conclusion The mechanisms responsible for the protective effect of cyclosporine on the lung injury induced by phorbol may be related to an attenuation of oxygen radical production with maintenance of NO release.

Vascular disease and diabetes: is hypoglycaemia an aggravating factor?

Acute hypoglycaemia provokes profound physiological changes affecting the cardiovascular system and several haematological parameters, principally as a consequence of sympatho-adrenal activation and counter-regulatory hormonal secretion. Many of these responses have an important role in protecting the brain from neuroglycopenia, through altering regional blood flow and promoting metabolic changes that will restore blood glucose to normal. In healthy young adults the cardiovascular effects are transient and have no obvious detrimental consequences. However, some of the effected changes are potentially pathophysiological and in people with diabetes who have developed endothelial dysfunction, they may have an adverse impact on a vasculature that is already damaged. The acute haemodynamic and haematological changes may increase the risk of localized tissue ischaemia, and major vascular events can certainly be precipitated by acute hypoglycaemia. These include myocardial and cerebral ischaemia and occasionally infarction. Established diabetic retinopathy often deteriorates after strict glycaemic control is instituted, the latter being associated with a threefold increase in frequency of severe hypoglycaemia, and enhanced exposure to mild hypoglycaemia. The possible mechanisms underlying these hypoglycaemia-induced effects include haemorrheological changes, white cell activation, vasoconstriction, and the release of inflammatory mediators and cytokines. The concept that acute hypoglycaemia could aggravate vascular complications associated with diabetes is discussed in relation to evolving comprehension of the pathogenesis of atherosclerosis and blood vessel disease.

Livedo : de la physiopathologie au diagnostic

We propose a diagnostic approach when facing a livedo. First, the pathophysiology of the livedo is reviewed using key articles barely quoted in the literature. Then the topic is handled in two ways. Figures and tables allow a rapid reading convenient "at the patient's bedside". The subject is also reviewed thoroughly, and we emphasize the intricacy of the various pathophysiological mechanisms involved for each livedo's related disease. Diseases associated with livedo are then briefly described with emphasis on the key diagnostic features and prevalence. Usefulness of the main diagnostic procedures is discussed. A livedo is a usually purplish-blue erythema, reticulated (small and complete meshes) or racemosa (large broken circular segments) which is related to a slowdown of the blood flow in the dermic venules. These venules form adjacent circles communicating with each other, parallel to the skin surface. The blood flow slowdown may be due to a local vasoconstriction (vasomotor livedo) or to an arteriolar occlusion. Arteriolar occlusion may be related to blood abnormalities (thrombosis, high viscosity, embolus) or to increased parietal thickness (vasculitis, calcic deposition, intimal hyperplasia). It is not always possible to clinically distinguish a vasomotor livedo from those associated with diseases. Diagnostic procedures should be oriented by the medical history, the features of the livedo, and associated symptoms. Usefulness of the skin biopsy is usually limited to the situations where the livedo is infiltrated or necrotic. To raise controversy about their importance and implications, some pathophysiological data are presented: intimal pseudohyperplasia in Sneddon's syndrome and antiphospholipid syndrome, and white cell activation in essential thrombocythemia.

Abstract 3715: Inflammatory and Thrombotic Changes in Bereavement: A Potential Mechanism of Increased Cardiovascular Risk

Background: Bereavement is associated with increased risk of cardiovascular disease (CVD) especially in early bereavement. The mechanism of increased risk has not been well studied. This prospective study evaluated inflammatory and thrombotic measures in bereaved spouses and parents, within 2 weeks of bereavement and again at 6 months. Methods: Fifty-one subjects (35F/17M; aged 65.2 ± 13.1 years (mean ± SD) were prospectively studied within 2 weeks of bereavement and at 6 months, and compared to 43 non-bereaved relatives of discharged patients; age 61.1 ± 11.1 years). All participants had haemostatic measures obtained between 8 –11 am and processed within one hour. White cell subpopulations and activation marker levels were obtained using flow cytometry. Group results were analysed using ANOVA controlling for age. Results: Acute bereavement versus controls: Compared to controls, acutely bereaved had a higher white cell count (WBC) 7.1 vs 6.2 10 9 /L (p&lt;0.001), higher neutrophil count 4.4 vs 3.5 10 9 /L (p=0.001) and a trend towards higher monocyte count 468 vs 525/uL (p=0.06). Acutely bereaved subjects also had a reduced T suppressor cell number (CD8 + ) (406 vs 496 MFI, p=0.02), increased activation marker CD26 on CD4 T cells (541 vs 496 MFI, p =0.02) and increased NK cells positive for CD57 (46.5% vs 39.8%, p=0.05). Acutely bereaved subjects had higher levels of vWF-Ag than controls 126% vs 116% (p=0.04), and higher cortisol levels (340 vs 277 nmol/L (p=0.007). Bereavement - acute versus 6 months: Compared to acute assessment results shown above, the bereaved at 6 months showed reduced WBC count (6.2 10 9 /L; p=0.003), neutrophil count (3.7 10 9 /L; p&lt;0.001), monocyte count (486, p=0.009) and CD57 + NK cells (p &lt;0.001). vWF-Ag was also lower (114%; p=0.02) as was factor VIII-ac (1.33 vs 1.17, p = 0.001). Fibrinogen and C-reactive protein did not change significantly from acute to 6 months. Conclusion: Acute bereavement is associated with increased inflammatory and prothrombotic changes. These findings provide insight into potential mechanisms for the increased CVD risk with bereavement and clues to potential preventive measures.

Cerebral Endothelial Nitric Oxide Synthase Expression is Reduced After Very Low Flow Bypass

In previous studies we have shown that delayed capillary reperfusion after low flow bypass predicts neurologic injury. In this acute study, we hypothesized that low flow reduces endothelial nitric oxide synthase (eNOS) expression, which may lead to more profound inflammatory response and delayed capillary perfusion. Twelve piglets (13.2 +/- 0.7 kg) had a cranial window placed over the parietal cerebral cortex for direct examination of the microcirculation using intravital fluorescence microscopy. Animals were cooled to 15 degrees C or 34 degrees C on cardiopulmonary bypass (pH stat, hematocrit 30%, pump flow 100 mL/kg/minute) followed by 2 hours of low flow (50 mL/kg/minute) or very low flow (10 mL/kg/minute). Rhodamine staining was used to observe adherent and rolling leukocytes in postcapillary venules. The eNOS protein expression was determined by Western immunoblotting. High temperature and low flow rate correlated with significantly reduced eNOS expression (p < 0.01). Univariate comparisons based on Student t tests indicated that eNOS protein levels were lower at 34 degrees C than at 15 degrees C (0.7 +/- 0.6 vs 1.7 +/- 0.5, p < 0.01) and at 10 mL/kg per minute compared with 50 mL/kg per minute (0.8 +/- 0.7 vs 1.6 +/- 0.5, p = 0.03). Moreover, two-way analysis of variance revealed that temperature (F = 21.6, p < 0.001) and flow rate (F = 13.8, p = 0.005) were independent multivariate predictors of eNOS expression. During low flow bypass, eNOS was inversely correlated with numbers of adherent (p = 0.002) and rolling (p = 0.006) leukocytes, following an exponential decay curve closely. eNOS expression is reduced after very low flow bypass, particularly at a higher bypass temperature. This is associated with delayed capillary reperfusion. Reduced eNOS is also associated with increased white cell activation which may lead to greater neurologic injury.

Fetal Hemoglobin Protects Against Activation of Hemostasis Endothelial and Circulating Blood Cells in Sickle Cell Anemia.

To determine whether fetal hemoglobin (HbF) protects against activation of hemostasis, endotheliium, and circulating blood elements, infants, children and young adults with homozygous SS disease (n=35) were studied. F-cell numbers, biomarkers of endothelial activation (including sVCAM-1, sE- and sP-selectin), white cell and platelet activation (sL-selectin and sP-selectin respectively) and markers of hemostatic activation, including D-Dimer and prothrombin fragment F1.2 were analyzed. Subjects with HbSS demonstrated significant increases (P<0.001) in all cell activation and hemostatic biomarkers. Analyses revealed a dominant, statistically significant correlation between F-cell and biomarkers of endothelial, white cell and hemostatic activation. F-cells correlated inversely with sVCAM-1 (R = −0.64, P<0.00005), sE-selectin (R = −0.51, P = 0.03) and sP-selectin (R = −0.41, P<0.02). Since HbF protects against HbS polymerization, our data suggests a role for the sickle erythrocyte in endothelial activation and is consistent with reported decreases in sVCAM-1 following hydroxyurea therapy or a chronic transfusion regimen. sP- and sE-selectin levels correlated with neutrophil counts (R = 0.63 and R = 0.42, P<0.00006 and P<0.02 respectively) while no relationship between platelets and sP-selectin was noted. An additional correlation between sP- and sE-selectin (R = 0.50, P<0.003) was also observed, reflecting enhanced neutrophil-endothelial interaction as part of an inflammatory and prothrombotic HbSS phenotype. Surprisingly, a positive correlation between sL-selectin and F-cells occurred (R = 0.4, P<0.02) with the young infant with HbSS demonstrating higher levels of this leucocyte activation marker than the older subject. A dysregulation of L-selectin shedding following neutrophil activation has been proposed to occur in sickle cell anemia. Our results suggest that HbF protects against this dysregulation. D-Dimer levels correlated negatively with F-cells (R = −0.76, P<0.000001), with a striking relationship between D-Dimer and markers of endothelial activation, including sVCAM-1 (R = 0.53, P<0.001), sE-selectin (R = 0.39, P<0.03) and sP-selectin (R = 0.41, P<0.02). Such findings are in keeping with D-Dimer's well-documented role as a biomarker in other inflammatory and ischemia-related disorders including coronary, cerebrovascular and peripheral arterial ischemic states. This report expands our knowledge regarding the protective effects of HbF against endothelial and hemostatic activation, and suggests a role for D-Dimer as a prothrombotic and proinflammatory marker in sickle cell anemia.

REPERFUSION INJURY IN THE HUMAN FOREARM IS MILD AND NOT ATTENUATED BY SHORT‐TERM ISCHAEMIC PRECONDITIONING

1. Ischaemia-reperfusion (IR) injury is an important contributor to tissue damage and has been shown to be attenuated by preconditioning (PC) in some animal models. A recent report has suggested that the forearm can be used for the study of this phenomenon in humans. We aimed to reproduce and further characterize this model. 2. Healthy young adult volunteers (mean (+/-SEM) age 32+/-6 years) were studied on two occasions. During one visit, IR alone was induced by 10 min of upper arm cuff occlusion, whereas on another occasion a PC stimulus (three 3 min cuff inflations) preceded IR. Endothelial function in the ischaemic arm was assessed by measuring arterial flow-mediated dilatation (FMD) and by calculation of forearm blood flow at baseline and 15 and 60 min after IR. Systemic venous blood was sampled from the non-ischaemic arm at baseline, after PC and at 2, 15 and 30 min after IR to assess neutrophil/leucocyte (CD11b) and platelet (bound glycoprotein IIb/IIIa and fibrinogen) activation, as well as numbers of platelet-leucocyte complexes, which were determined by flow cytometry. Because of a lack of measurable effects, the IR experiment was repeated with 20 min ischaemia in six subjects. 3. Five females and eight males completed the study. Flow-mediated dilatation was significantly impaired 30 min after IR (4.1 vs 6.2% at baseline; P<0.05);however, this was not significantly attenuated by ischaemic PC (FMD reduction at 30 min compared with baseline was 2.1+/-0.5% with IR alone and 2.6+/-1.4% with IR after PC; NS). No significant effect was seen on the number of platelet-leucocyte aggregates or on white cell or platelet activation after IR alone or after IR with PC (P>0.6 for all comparisons). Similar results were obtained in six subjects studied subjected to 20 min ischaemia. 4. In conclusion, in healthy young adults, brief periods of skeletal muscle ischaemia lead to arterial endothelial dysfunction, but no significant platelet or white cell activation. Preconditioning does not attenuate this effect on the endothelium. Further experiments with longer ischaemia times and varying PC stimuli may be necessary to produce measurable effects; however, this may prove difficult in conscious human subjects.

Higher bypass temperature correlates with increased white cell activation in the cerebral microcirculation*1

Higher bypass temperature correlates with increased white cell activation in the cerebral microcirculation*1