Systemic Leukocyte Counts Research Articles

Shift Work Predicts Increases in Lipopolysaccharide-Binding Protein, Interleukin-10, and Leukocyte Counts in a Cross-Sectional Study of Healthy Volunteers Carrying Low-Grade Systemic Inflammation.

The disruption of inflammatory responses is a potential mechanism behind the harmful effects of shift work and is associated with increased risk of hypertension, stroke, obesity, diabetes, and cancer. These responses are linked to the proliferation of leukocytes in shift workers, suggesting a systemic signal as a potential mediator. The purpose of this study was to assess the relationship between systemic inflammation, leukocyte counts, and systemic endotoxemia in samples from a diverse cohort of day workers and shift workers. Participants (normothermic and normotensive) were healthy volunteers, non-smoking, and drug- and medication-free. The following outcomes were measured: C-reactive protein, TNF-α, IL-6, IL-1β, IL-10, leukocyte counts (monocytes, lymphocytes, and neutrophils), and lipopolysaccharide-binding protein (LBP). Risk factors that increase systemic inflammation, such as blood pressure, sleep loss, and cortisol, were also assessed. The results indicated that shift workers slept significantly less than day workers and had significantly increased concentrations of all of the cytokines measured as well as plasma cortisol. Regression models found that after controlling for covariates, shift-work exposure predicted the significant increase observed in IL-10, leukocyte counts, and LBP. Our results suggest that acute increases in low-grade systemic endotoxemia are unresolved during chronic shift-work exposure. This ongoing immune challenge may underlie the disrupted inflammatory responses characteristic of shift-work-related pathologies. Systemic endotoxemia may represent a novel target to investigate the early effects of exposure to shift-work schedules.

Prediction of in-hospital mortality of Clostriodiodes difficile infection using critical care database: a big data-driven, machine learning approach

Research objectivesClostriodiodes difficile infection (CDI) is a major cause of healthcare-associated diarrhoea with high mortality. There is a lack of validated predictors for severe outcomes in CDI. The aim of...

Leukocyte-Endothelium Interaction in the Sublingual Microcirculation of Coronary Artery Bypass Grafting Patients

Objective: The aim of this study was to apply an innovative methodology to incident dark-field (IDF) imaging in coronary artery bypass grafting (CABG) patients for the identification and quantification of rolling leukocytes along the sublingual microcirculatory endothelium. Methods: This study was a post hoc analysis of a prospective study that evaluated the perioperative course of the sublingual microcirculation in CABG patients. Video images were captured using IDF imaging following the induction of anesthesia (T₀) and cardiopulmonary bypass (CPB) (T₁) in 10 patients. Rolling leukocytes were identified and quantified using frame averaging, which is a technique that was developed for correctly identifying leukocytes. Results: The number of rolling leukocytes increased significantly from T₀ (7.5 [6.4–9.1] leukocytes/capillary-postcapillary venule/4 s) to T₁ (14.8 [13.2–15.5] leukocytes/capillary-postcapillary venule/4 s) (p < 0.0001). A significant increase in systemic leukocyte count was also detected from 7.4 ± 0.9 × 10⁹/L (preoperative) to 12.4 ± 4.4 × 10⁹/L (postoperative) (p < 0.01). Conclusion: The ability to directly visualize leukocyte-endothelium interaction using IDF imaging facilitates the diagnosis of a systemic inflammatory response after CPB via the identification of rolling leukocytes. Integration of the frame averaging algorithm into the software of handheld vital microscopes may enable the use of microcirculatory leukocyte count as a real-time parameter at the bedside.

Omega-9 Oleic Acid, the Main Compound of Olive Oil, Mitigates Inflammation during Experimental Sepsis.

The Mediterranean diet, rich in olive oil, is beneficial, reducing the risk of cardiovascular diseases and cancer. Olive oil is mostly composed of the monounsaturated fatty acid omega-9. We showed omega-9 protects septic mice modulating lipid metabolism. Sepsis is initiated by the host response to infection with organ damage, increased plasma free fatty acids, high levels of cortisol, massive cytokine production, leukocyte activation, and endothelial dysfunction. We aimed to analyze the effect of omega-9 supplementation on corticosteroid unbalance, inflammation, bacterial elimination, and peroxisome proliferator-activated receptor (PPAR) gamma expression, an omega-9 receptor and inflammatory modulator. We treated mice for 14 days with omega-9 and induced sepsis by cecal ligation and puncture (CLP). We measured systemic corticosterone levels, cytokine production, leukocyte and bacterial counts in the peritoneum, and the expression of PPAR gamma in both liver and adipose tissues during experimental sepsis. We further studied omega-9 effects on leukocyte rolling in mouse cremaster muscle-inflamed postcapillary venules and in the cerebral microcirculation of septic mice. Here, we demonstrate that omega-9 treatment is associated with increased levels of the anti-inflammatory cytokine IL-10 and decreased levels of the proinflammatory cytokines TNF-α and IL-1β in peritoneal lavage fluid of mice with sepsis. Omega-9 treatment also decreased systemic corticosterone levels. Neutrophil migration from circulation to the peritoneal cavity and leukocyte rolling on the endothelium were decreased by omega-9 treatment. Omega-9 also decreased bacterial load in the peritoneal lavage and restored liver and adipose tissue PPAR gamma expression in septic animals. Our data suggest a beneficial anti-inflammatory role of omega-9 in sepsis, mitigating leukocyte rolling and leukocyte influx, balancing cytokine production, and controlling bacterial growth possibly through a PPAR gamma expression-dependent mechanism. The significant reduction of inflammation detected after omega-9 enteral injection can further contribute to the already known beneficial properties facilitated by unsaturated fatty acid-enriched diets.

C-Reactive Protein and Resistin Detect Bacterial Infections in Liver Cirrhosis

Introduction: Bacterial infection usually has poor outcome, but often full of diagnostic difficulties in cirrhotic patients. The role of clinical parameters such as systemic inflammatory response syndrome, leukocyte count, neutrophil count, and other markers remains unclear in liver cirrhosis patients. The aim of this study was to evaluate the usefulness of inflammatory markers and determined which markers were best for the diagnosis of infection in decompensated cirrhotic patients. Methods: This was a diagnostic study consisted of 80 cirrhotic patients admitted to Sanglah general hospital, Denpasar from August 2014 until July 2015. Prospective study was designed. Markers of infection consist of leukocyte count, neutrophil count, neutrophil to lymphocyte ratio (NLR), C-reactive protein (CRP) and resistin were measured. Accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were determined. Results: Bacterial infections were noted in twenty patients (25%) and spontaneous bacterial peritonitis (SBP) was the most common infections occurred. NLR, CRP, and resistin were higher in bacterial infections group (p < 0.05). Multiple logistic regression analyses showed that CRP and resistin were predictive factor for occurrence of bacterial infections (p < 0.05). For the diagnosis of infection, baseline CRP - using a 11.65 mg/L cut-off value - and resistin - using a 13 ng/mL cut-off value - generated area under the receiver operating characteristic (ROC) curve of 0.796 and 0.787, respectively. The sensitivity, specificity, PPV, and NPV for CRP were 90%, 73%, 52.9%, and 95.7%, respectively. For resistin, the sensitivity, specificity, PPV, and NPV were 90%, 59%, 41.9%, and 94.6% respectively. Conclusion: In the present study, CRP level of 11.65 mg/L or resistin 13 ng/mL seem adequate in rule out infections. Pieri et al in 2014 found out that CRP is still reliable in prediction of bacterial infection in cirrhosis. Johansson et al stated that resistin increase in noncirrhotic patients with severe sepsis. Currently there are no data on resistin levels in cirrhotic patients with bacterial infections. This study showed that resistin may have role in bacterial infections. In conclusion, resistin and CRP showed good accuracy as screening tests in diagnosing bacterial infection in cirrhotic patients. In the future, these modalities may aid clinician in predicting which patient more likely to have infections.Figure: Distribution of the serum concentration of resistin (A), C-reactive protein (CRP) (B), and neutrophil-lymphocyte ratio (NLR) (C) in liver cirrhosis patients with or without infections.Figure: ROC curves of CRP and resistin for the diagnosis of infection at admission.Table: Table. Diagnostic accuracy of C-reactive protein and resistin in diagnosing infection in liver cirrhosis.

Alcohol Intoxication Reduces Systemic Interleukin-6 Levels and Leukocyte Counts After Severe TBI Compared With Not Intoxicated TBI Patients.

The effect of alcohol consumption on inflammatory state and outcome in brain-injured patients remains controversial. We analyzed the influence of positive blood alcohol concentration (BAC) on inflammatory changes, inhospital complications, and mortality in traumatic brain injury (TBI) patients. Patients with an Injury Severity Score (ISS) at least 16 and Abbreviated Injury Scale of head (AIS-head) at least 3 were included upon arrival in the emergency room and grouped according to positive BAC (>0.5‰, BAC) vs. less than 0.5‰ alcohol (no BAC). Injury severity, vital signs, complications, mortality, and systemic interleukin (IL)-6 levels were prospectively determined, and BAC was quantified. According to ISS, AIS-head, age, and sex, we performed matched-pair analysis. A total of 101 TBI patients were included. Of them 74 patients were dedicated to no BAC group and 27 to BAC group. ISS was significantly higher in the no BAC group. Positive BAC group required significantly less packed red blood cells and fresh frozen plasma (P < 0.05). Shorter ICU stays were found in BAC-positive patients. Inhospital complications, including single/multiple organ failure, systemic inflammatory response syndrome, sepsis, pneumonia, and acute respiratory distress syndrome, showed no significant differences. Systemic IL-6 levels and leukocyte counts (IL-6: 65.0 ± 8.0 vs. 151.8 ± 22.3; leukocytes: 10.2 ± 0.9 vs. 13.2 ± 0.8, both P < 0.05) were significantly lower in BAC-positive patients. Matched-pair analysis was performed with 27 pairs. No significant differences in transfusions were monitored after matching. However, lowered systemic IL-6 levels and leukocyte counts in the BAC group were also detected after matching, indicating that this effect is ISS-independent. This study shows that positive BAC in TBI patients is associated with lower systemic IL-6 levels and leukocyte numbers, indicating that positive BAC may have immunosuppressive effects in this cohort of patients compared with TBI patients who were not alcohol intoxicated.

CORRELATION OF C-REACTIVE PROTEIN AND COPD SEVERITY

Chronic obstructive pulmonary disease (COPD) is a progressive pulmonary disease characterized by systemic inflammation. The aim of this study was to correlate the parameters of systemic inflammation, C-reactive protein (CPR) and total leukocyte count, with clinical indicators of the disease. Our study included 157 COPD patients, both outpatients and those hospitalized at the Knez Selo Department of Pulmonology of the Niš Clinical Centre during a six-month period, while in the phase of disease exacerbation. The symptoms of COPD in each patient were estimated by the COPD Assessment Test (CAT) and modified Medical Research Council (mMRC) dyspnea scale. The parameters of pulmonary function (FEV1 and FVC), acid-base status, body mass index, history of exacerbation and comorbidities were also evaluated. The level of CRP, but not leukocytes, showed significant correlation with the severity of clinical presentation according to GOLD classification. The higher the CRP concentration, the higher was the disease severity determined according to GOLD classification (p < 0.001). There was no statistically significant difference in CRP level and leukocyte count according to comorbidities (p = 0.29). The level of CRP was higher in patients with a high CAT score and mMRC scale (p < 0.001). The same trend was observed for leukocyte count when compared with CAT results, but not when correlated to mMRC scale. The level of CRP during COPD exacerbation can be an independent predictor of the disease severity and paraclinical findings.

Derivation and validation of a simple clinical bedside score (ATLAS) for Clostridium difficile infection which predicts response to therapy

BackgroundClostridium difficile infection (CDI) continues to be a frequent and potentially severe infection. There is currently no validated clinical tool for use at the time of CDI diagnosis to categorize patients in order to predict response to therapy.MethodsSix clinical and laboratory variables, measured at the time of CDI diagnosis, were combined in order to assess their correlation with treatment response in a large CDI clinical trial database (derivation cohort). The final categorization scheme was chosen in order to maximize the number of categories (discrimination) while maintaining a high correlation with clinical cure assessed two days after the end of therapy. Validation of the derived scoring scheme was done on a second large CDI clinical trial database (validation cohort). A third comparison was done on the two pooled databases (pooled cohort).ResultsIn the derivation cohort, the best discrimination and correlation with cure was seen with a five-component ATLAS score (age, treatment with systemic antibiotics, leukocyte count, albumin and serum creatinine as a measure of renal function), which divided CDI patients into 11 groups (scores of 0 to 10 inclusive) and was highly correlated with treatment outcome (R2=0.95; P<0.001). This scheme showed excellent prediction of cure in the validation cohort (overall Kappa=95.2%; P<0.0001), as well as in the pooled cohort, regardless of treatment (fidaxomicin or vancomycin).ConclusionsA combination of five simple and commonly available clinical and laboratory variables measured at the time of CDI diagnosis, combined into a scoring system (ATLAS), are able to accurately predict treatment response to CDI therapy. The ATLAS scoring system may be useful in stratifying CDI patients so that appropriate therapies can be chosen to maximize cure rates, as well as for categorization of patients in CDI therapeutic studies in order allow comparisons of patient groups.

Safety of Intra-Articular Use of Atelocollagen for Enhanced Tissue Repair

Collagen is an important biomaterial in intra-articular tissue engineering, but there are unanswered questions about its safety. We hypothesize that the addition of type-I-collagen for primary repair of the Anterior Cruciate Ligament (ACL) might result in a local and systemic reaction in a porcine model after 15 weeks as demonstrated by joint effusion, synovial thickening, elevated intraarticular and systemic leukocyte counts. Further, this reaction might be aggravated by the addition of a platelet concentrate. Eighteen porcine ACLs were transected and repaired with either sutures (n=6), a collagen sponge (n=6), or a collagen-platelet-composite (CPC; n=6). Twelve intact contralateral knees served as controls (n=12). No significant synovial thickening or joint effusion was seen in the collagen-treated knees. Synovial fluid leukocyte counts showed no significant differences between surgically treated and intact knees, and no differences were seen in leukocyte counts of the peripheral blood. The addition of a platelet concentrate to the knee joint resulted in lower serum levels of IL-1β, but serum levels of TNF-α were not significantly different between groups. In conclusion, the presence of collagen, with or without added platelets, did not increase the local or systemic inflammatory reactions following surgery, suggesting that Type I collagen is safe to use in the knee joint.

Epidermal phospholipase Cδ1 regulates granulocyte counts and systemic interleukin-17 levels in mice

Phospholipase C is a key enzyme in phosphoinositide turnover. Although its functions have been extensively studied at the cellular level, many questions remain concerning its functions at the organ and individual animal levels. Here we demonstrate that mice lacking phospholipase Cδ1 develop granulocytosis associated with elevated serum levels of the granulopoietic cytokine interleukin-17. Re-introduction of phospholipase Cδ1 into keratinocytes of phospholipase Cδ1-deficient mice reverses this phenotype, whereas conditional ablation of phospholipase Cδ1 in keratinocytes recreates it. Interleukin-17 and its key upstream regulator interleukin-23 are also upregulated in epidermis. Loss of phospholipase Cδ1 from keratinocytes causes features of interleukin-17-associated inflammatory skin diseases. Phospholipase Cδ1 protein is downregulated in the epidermis of human psoriatic skin and in a mouse model of psoriasis. These results demonstrate that phosphoinositide turnover in keratinocytes regulates not only local inflammatory responses but also serum cytokine levels and systemic leukocyte counts, and affects distant haematopoietic organs.

Circadian Expression of Endothelial Selectins, Regulated by the Sympathetic Nervous System, Controls Peripheral Leukocyte Homeostasis

We have recently shown that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner, controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature 2008; 452:442). In addition to the oscillations in circulating HSC numbers, we have also observed circadian fluctuations in the numbers of blood leukocytes in mice maintained on a standard 12h light - 12h dark cycle. Systemic leukocyte counts (4129 ± 521/μl (12 pm) vs 2000 ± 233/μl (8 pm); p=0.003) and their subsets, neutrophils (2762 ± 118 vs 732 ± 45/μl; p=0.02) and lymphocytes (5997 ± 151 vs 1867 ± 661/μl; p=0.03), were significantly reduced at night compared to the morning. We hypothesized that peripheral leukocyte counts are regulated by their interactions with endothelial cells in the BM microcirculation. To test this hypothesis we analyzed the constitutive interactions of leukocytes with BM microvessels using intravital microscopy to evaluate whether the trafficking of leukocytes in the BM microvasculature is also subjected to circadian regulation. We found that the absolute number of rolling leukocytes (18.0 ± 0.2 (12 pm) vs 31.1 ± 3.5 (8 pm) x103/mm2;p=0.004) and the rolling flux fraction (RFF, i.e. rolling/systemic leukocyte counts)(6.5 ± 0.6 vs 20.7 ± 3.1 %; p=0.0001) were increased three-fold at night, inversely correlating with the number of circulating leukocytes. Adoptive transfer experiments of fluorescently labeled BM leukocytes also revealed a two-fold increase in the number of recruited adherent leukocytes at night compared to when cells were injected in the morning (0.97 ± 0.17 (12 pm) vs 2.54 ± 0.53 (8 pm)/vessel area (μm2);p=0.007). Flow cytometry analyses revealed that the majority of these recruited cells were Mac-1+/Gr-1+ myeloid cells. This suggests that the capacity of the BM to recruit leukocytes fluctuates over the course of a day. Moreover, adoptively transferred cells formed clusters at specific sites in the BM at night. To investigate the mechanisms, we first subjected mice lacking both endothelial selectins (P-and E-selectins) to the same experimental protocols. Double deficient mice did not exhibit circadian variations in PB (9725 ± 1185/μl (12 pm) vs 8271 ± 1394/μl (8 pm); p=0.50) or BM (RFF 0.28 ± 0.05 vs 0.34 ± 0.05 %; p=0.48), and did not show clusters of adoptively transferred cells in the BM. To investigate the role of the SNS, we sympathectomized mice with 6-hydroxydopamine. Chemical sympathectomy significantly reduced the circadian differences of leukocyte rolling on BM endothelium (RFF 7.8 ± 1.1% (12 pm) vs 14.9 ± 2.8% (8 pm); p=0.01) and leukocyte numbers in PB (2750 ± 322 vs 2225 ± 363/μl; p=0.32). In addition, the number of adherent adoptively transferred cells in the BM did not show significant fluctuation (1.66 ± 0.26 vs 1.54 ± 0.20/vessel area(μm2);p=0.72). These results suggest that the process of homing/cluster formation is dependent on constitutive, oscillatory expression of P- and/or E-selectins and regulated by the SNS.

Combined Therapeutic Hypothermia and Barbiturate Coma Reduces Interleukin-6 in the Cerebrospinal Fluid After Aneurysmal Subarachnoid Hemorrhage

Inflammatory response with cytokine release is reported to correlate with clinical outcome after aneurysmal subarachnoid hemorrhage (SAH). In selected cases, hypothermia and barbiturate coma are applied as means for neuroprotection after severe SAH. Hypothermia and high-dose barbiturate are reported to attenuate the inflammatory response. In this pilot study, we assessed the effect of the combined therapy on the inflammatory response. In 15 patients with SAH, daily cerebrospinal fluid (CSF) and plasma samples were collected. Interleukin (IL)-6, tumor necrosis factor alpha (TNF-alpha), IL-1beta, systemic leukocyte, and leukocyte counts in the CSF were quantified. Group 1 represented 7 cases treated with combined therapeutic hypothermia (33 degrees C) and barbiturate coma. Group 2 represented 8 cases without combined therapy. Compared with the systemic levels, all cases showed higher cytokine levels in the CSF. Mean IL-6 level in the CSF was significantly lower in group 1 (P<0.001). The ratio between IL-6 levels in the CSF and plasma, as a parameter for intrathecal synthesis, was significantly lower in group 1 (P=0.014). Mean CSF and systemic levels of TNF-alpha of group 1 were significantly higher compared with group 2 (P=0.009 and P<0.001). The mean systemic IL-1beta level was significantly lower in group 1 (P<0.001), as well as the leukocyte counts, both, systemic and in the CSF (P<0.001 and P=0.032). The present data show a most pronounced decrease of IL-6 levels in the CSF, beside decrease in systemic IL-1beta levels, systemic leukocyte counts, and CSF leukocyte counts in group 1, which would be expected to reflect an attenuation of inflammatory response. The impact and role of TNF-alpha remains unclear.

Local and Systemic Effects of a Tissue Engineered Neobladder in a Canine Cystoplasty Model

Tissue engineered bladders are emerging as a potential treatment option in urological surgery. Although successful neobladders can be engineered with autologous cells on a biodegradable polymer scaffold, studies of the local and systemic effects on host tissue have not been extensively pursued. We examined such effects at predetermined time points after implantation of tissue engineered neobladders in a canine cystoplasty model. Eight dogs underwent trigone sparing cystectomies. Six dogs (experimental group) received bladder augmentation with tissue engineered constructs produced from autologous urothelial and smooth muscle cells on a prefabricated polyglycolic acid polymer scaffold. Two beagles (control group) received bladder augmentation with the polyglycolic acid scaffold alone. Serial urodynamic studies, cystograms, peripheral blood smears, urinalysis, serum chemistry, complete blood count and electrolytes were done at predetermined time points postoperatively. The bladder, and local and distant organs were retrieved 6 months after surgery for analysis. Capacity and compliance of the engineered bladders reached normal levels by 6 months. Engineered bladders showed tissue composition similar to that of normal bladders. Infiltration of inflammatory cells was minimal and subsided with time. An increase in the total systemic leukocyte count and in bacteriuria was evident initially at 1 week but they returned to normal levels by 1 month postoperatively. Other systemic parameters remained within normal levels at all time points. There was no evidence of abnormal findings in local or distant organs. Implantation of polymer molds seeded with autologous bladder cells did not show significant local or systemic toxicity in a canine model. This study suggests that such engineered neobladders are safe and effective for reconstructive surgery.

In vivo Visualization of Early Microcirculatory Changes following Ischemia/Reperfusion Injury in Human Kidney Transplantation

To determine whether microcirculatory changes following ischemia/reperfusion (I/R) may serve as predictors for subsequent graft dysfunction, we used noninvasive orthogonal polarization spectral (OPS) imaging to directly visualize and quantify cortical kidney microcirculation. In a total of 13 combined kidney/pancreas recipients, following reperfusion (5/30 min) microcirculatory parameters such as capillary diameter, functional capillary density (FCD) and red-blood-cell velocity (V_RBC) of the renal graft were analyzed. From these parameters, a heterogeneity index (HI) and volumetric capillary blood flow (vCBF) were calculated. In addition, the extent of graft injury was determined by daily analysis of serum creatinine, blood urea nitrogen, C-reactive protein and systemic leukocyte count for 7 days post-transplant. At early reperfusion, a heterogeneous perfusion pattern with oscillating flow and scattered microvascular thrombosis of peritubular capillaries, resembling a ‘no reflow’, was observed. FCD was constant throughout the entire reperfusion period, whereas HI, capillary diameters, V_RBC and vCBF increased. The latter showed a significant positive correlation with creatinine changes between days 1 and 3. So far our finding of a positive correlation of early microvascular changes (vCBF) and clinical parameters (creatinine) indicate a possible therapeutic implication of OPS imaging to predict early I/R-induced renal graft dysfunction.

N-3 Polyunsaturated fatty acid-enriched diet does not protect from liver injury but attenuates mortality rate in a rat model of systemic endotoxemia*

We investigated the potential of dietary fish oil containing n-3 polyunsaturated fatty acids to attenuate hepatic injury and mortality rate of rats in response to systemic endotoxemia. Prospective, randomized, controlled animal study. University laboratory. A total of 43 male Sprague Dawley rats. Rats were fed either fish oil supplement or regular standard lab chow. After 8 wks of feeding, each diet group was subjected to a single exposure of lipopolysaccharide (Escherichia coli, 10 mg/kg intravenously) or saline. Hepatic microvascular response and liver injury were assessed by in vivo analysis of Kupffer cell phagocytic activity, leukocyte-endothelial cell interaction, nutritive sinusoidal perfusion failure, and parenchymal cell apoptosis (intravital fluorescence epi-illumination technique) as well as bile flow, serum liver enzyme activities, and tissue histomorphology. In animals fed a standard diet, livers at 16 hrs after lipopolysaccharide-exposure exhibited depressed Kupffer cell phagocytic activity, enhanced hepatic microvascular leukocyte activation, leukocytic tissue infiltration, sinusoidal perfusion failure, and parenchymal cell apoptosis. Hepatic microvascular injury was further accompanied by reduced bile flow and enhanced liver enzyme release. The fish oil enriched diet did not significantly change the multiple features of endotoxemia-associated liver injury; however, it maintained arterial blood pressure, systemic leukocyte count, and acid base balance and showed a tendency toward improved survival on lipopolysaccharide exposure with a 16 hr-survival rate of 80% (p =.06 vs. survival rate of 40% in animals fed a regular diet). Moreover, slightly increased serum concentrations of interleukin-10 coincided with enhanced concentrations of interleukin-6 in fish oil fed endotoxemic animals. Healthy, non-lipopolysaccharide-exposed, fish oil fed animals did not differ from those fed with the regular diet, except for dampened Kupffer cell phagocytic activity. Fish oil feeding does not protect from local endotoxemia-induced hepatic microvascular dysfunction. However, dietary modulation of inflammatory mediator response by macrophages, constituting an appropriate immune response, could add to the survival advantage seen in fish oil-fed animals on exposure to lipopolysaccharide.

Especially Polymorphonuclear Leukocytes, but also Monomorphonuclear Leukocytes, Roll Spontaneously in Venules of Intact Rat Skin: Involvement of E-Selectin

White blood cells roll spontaneously in venules of intact, noninflamed rat skin. We investigated noninvasively in two experimental series which leukocyte subtypes participate in this phenomenon and the possible involvement of E-selectin. Male Lewis rats were anesthetized with sodium pentobarbital, and intravital video microscopy was performed on postcapillary venules in the nail-fold of a hind leg. In series 1 acridine yellow was infused for 15 min (50 mg per kg intravenously) to stain the leukocyte nuclei in situ. With the use of fluorescence microscopy rolling leukocytes could be classified unequivocally as polymorphonuclear (granulocytes) or monomorphonuclear (lymphocytes/monocytes) by the shape of their nucleus. Irrespective of vessel depth beneath the skin surface (25-45 microm), most identified rolling leukocytes were classified as granulocytes (72%-100%; median 89%). This percentage was independent of total rolling leukocyte flux, systemic leukocyte count, or their in vitro differentiation pattern. In series 2, rats were treated with either a synthetic, highly selective E-selectin blocking peptide or a control peptide (intravenously, 12 mg peptide per kg bolus, followed by 50 mg per kg per h). E-selectin blockade significantly reduced the leukocyte rolling level to about 50% of baseline (p <0.01), whereas the rolling velocity increased (p <0.01); the control peptide had no effect. In summary, most of the leukocytes rolling spontaneously in postcapillary venules of intact rat skin are granulocytes, despite the absence of an acute inflammatory reaction. One of the adhesion molecules involved in this phenomenon is E-selectin.

Linomide abolishes leukocyte adhesion and extravascular recruitment induced by tumor necrosis factor αin vivo

The immunomodulator Linomide (roquinimex) ameliorates the development of numerous inflammatory and immunological diseases, including sepsis, arthritis, and encephalomyelitis. However, the mechanism underlying this protective effect of Linomide remains unclear. In this study, we wanted to evaluate the effect of Linomide treatment on the different steps in the extravasation process of leukocytes stimulated by tumor necrosis factor alpha (TNF-alpha) in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle microcirculation. We found that pretreatment with Linomide dose-dependently (3-300 mg/kg) reduced TNF-alpha-induced leukocyte adhesion and tissue recruitment. Notably, at 300 mg/kg response to TNF-alpha was nearly abolished, i.e. leukocyte adhesion was decreased by 83% and recruitment by 86%. In fact, the anti-inflammatory effect of this dose of Linomide corresponded in magnitude to the potency of 10 mg/kg of dexamethasone. Moreover, administration of Linomide did not alter the systemic leukocyte counts. On the other hand, 1-10 mg/kg of dexamethasone decreased the circulating number of mononuclear leukocytes by 77%. Taken together, our novel findings demonstrate that Linomide is a potent inhibitor of leukocyte adhesion and recruitment in cytokine-activated tissues. These data may help explain the documented protection provided by Linomide in inflammatory diseases characterized by cytokine and leukocyte accumulation.

The effects of inhibiting leukocyte migration with fucoidin in a rat peritonitis model.

To study the effects of fucoidin on leukocyte rolling and emigration and bacterial colonization in a peritonitis sepsis model in rats. A controlled study in 64 male Wistar rats, anesthetized and rendered septic by cecal ligation and puncture (CLP). Immediately after CLP 32 animals received a continuous infusion of fucoidin and 32 a continuous infusion of Ringer's lactate. Systemic leukocyte counts were determined every 2 h after CLP. Surviving animals were anesthetized 24 h after CLP, and intravital measurements of leukocyte rolling in venules in the cremaster muscle were performed. The animals were then killed and their organs harvested for histological and microbiological examinations. The 24-h survival was comparable in the two groups. Fucoidin-treated animals had higher leukocyte counts in the systemic circulation and lower counts in the lungs, liver, abdominal cavity, and brain than control animals. The number of bacterial colony forming units in the abdominal cavity, lungs, liver, brain and blood did not differ in the two groups. Fucoidin treatment changed the type of bacteria predominantly found in the examined organs from Escherichia coli to Pseudomonas aeruginosa. In an intra-abdominal model of sepsis we found that treatment with fucoidin induces leukocytosis inhibits leukocyte rolling and reduces leukocyte emigration in the abdominal cavity, lungs, and liver. Reduction in the number of emigrating leukocytes was not associated with an increase in bacterial counts found in the examined organs.

The polysaccharide fucoidan inhibits microvascular thrombus formation independently from P- and L-selectin function in vivo.

Adhesion molecules of the selectin family (mainly P- and L-selectin) have been suggested to mediate interactions between platelets, leukocytes and endothelial cells in thrombus formation. The polysaccharide fucoidan has anticoagulative properties, but is also able to bind and block the function of the selectins. Here, we investigated in vivo (i) if fucoidan can prevent microvascular thrombus formation, and (ii) whether this is potentially mediated by the inhibition of P-and/or L-selectin. For this purpose, we used intravital microscopy in the mouse cremaster microcirculation in which thrombosis was induced photochemically by light exposure to individual arterioles and venules after intravenous (i.v.) injection of FITC-dextran. We found that intravenous administration of fucoidan significantly prolonged the time required for complete occlusion in arterioles and venules by almost seven- and nine-fold, respectively. In contrast, treatment with monoclonal antibodies against P- and L-selectin had no effect on the development of microvascular thrombosis. Fucoidan and also the anti-P-selectin antibody completely inhibited baseline venular leukocyte rolling in the cremaster muscle, indicating that these treatment regimes abolished P-selectin function. Importantly, fucoidan and the anti-P-selectin antibody had no effect on systemic platelet and leukocyte counts. On the other hand, we found that fucoidan treatment significantly altered coagulation parameters, including prothrombin time (Quick percentage), activated partial thromboplastin time (APTT) and thrombin clotting time (TCT), which may explain the potent in vivo anticoagulative effect of fucoidan observed here. Taken together, our novel findings suggest that fucoidan effectively prevents microvascular thrombus formation induced by endothelial damage in arterioles and venules in vivo. This protective effect of fucoidan is not attributable to inhibition of P- and L-selectin function but may instead be related to the anticoagulative capacity of fucoidan.

In vivo visualization of hemodialysis-induced alterations in leukocyte–endothelial interactions

The aim of this study was to develop a model for hemodialysis (HD) in small animals using conventional dialysis equipment that would allow the intravital microscopic observation of leukocyte-endothelial interactions in vivo. Cuprophan dialyzers were adapted to obtain a similar ratio of membrane area to blood volume as in clinical HD. A silicone ring was inserted into the dialyzer's inlet to limit the number of blood-perfused capillaries. Rabbits were dialyzed for one hour without a dialysate flow. Extracorporeal circulation with the cuprophan dialyzer resulted in a transient leukopenia and complement activation. At the nadir of leukopenia, leukocytes that rolled along the venular wall were scarcely observed, whereas rolling was abundant (54 +/- 9 per min) prior to extracorporeal circulation. The adhesion of leukocytes to the vascular endothelium was not induced. After 60 minutes, rolling of leukocytes was still reduced by 73 +/- 5.5%, despite the full recovery of circulating leukocyte counts. Extracorporeal circulation without a dialyzer also tended to reduce leukocyte rolling, although systemic leukocyte counts were not affected. The use of adapted conventional cuprophan hemodialyzers in rabbits yielded a transient leukopenia similar to that in clinical HD. Using intravital microscopy, we demonstrated impairment of leukocyte-endothelial interactions. In addition, our data indicate that tissues, in which leukocytes can roll and adhere, are not automatically sites of leukocyte sequestration during HD-induced leukopenia.