Sandra S. Kaplan

Inflammatory cytokine and nitric oxide responses in pediatric sepsis and organ failure

OBJECTIVE To examine the relationship of circulating proinflammatory and anti-inflammatory cytokine concentrations to nitric oxide and organ failure in pediatric sepsis. DESIGN Prospective study. SETTING Pediatric intensive care unit (ICU), childrens Hospital of Pittsburgh, University of Pittsburgh. PATIENTS Nineteen patients with a diagnosis of sepsis admitted to the pediatric ICU. Twelve uninfected critically iII patients served as controls. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Plasma interleukin (IL)-10, IL-6, and nitrite/nitrate concentrations were measured and compared with an index of organ failure daily for 3 days after presentation with the sepsis syndrome. Children with increased plasma IL-6 concentrations (n = 6) had increased plasma nitrite/nitrate concentrations (p < 0.01 on each day), increased organ failure scores (p < .05 on days 1 and 3), and the highest plasma IL-10 concentrations (p < .05 on days 1 and 3, p = .054 on day 2) when compared with children with sepsis and undetectable IL-6 concentrations. Children with sepsis and detectable IL-6 concentrations, and children with undetectable IL-6 concentrations, both had increased nitrite/nitrate concentrations (p < .005 on days 1 through 3) and increased IL-10 concentrations (p < .05 on days 1 and 2) compared with controls. Children with increased IL-6 concentrations had higher organ failure on each day (p < .01), and children with undetectable IL-6 concentrations had higher organ failure on days 1 and 2 only (p < .005) when compared with controls. Organ failure improved over time in the children with undetectable IL-6 concentrations (p < .005). CONCLUSIONS Increased plasma nitrite/nitrates and increased organ failure scores occurred in the children with sepsis who had an exaggerated proinflammatory state, despite a pronounced anti-inflammatory response. When the anti-inflammatory response predominated and the proinflammatory state was dampened, organ failure status improved.

Plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis

OBJECTIVE To determine whether plasma nitrite and nitrate concentrations are associated with the development of sepsis-induced multiple organ failure. DESIGN Prospective study. SETTING University childrens hospital. PATIENTS Fifty-three consecutive children meeting criteria for sepsis and not receiving exogenous sources of nitric oxide. INTERVENTIONS Plasma nitrite and nitrate concentrations were measured, and the number of organs failing was scored using an organ failure index on the first 3 days of sepsis. MEASUREMENTS AND MAIN RESULTS Children with three or more organs failing on day 3 of sepsis had higher plasma nitrite and nitrate concentrations than children who had resolution of failure of three or more organs by day 3 of sepsis (days 2 and 3) and children who never had three organs failing in the first 3 days of sepsis (days 1, 2, and 3). Children who developed sequential pulmonary/hepatic/renal organ failure had significantly higher plasma nitrite and nitrate concentrations (days 1, 2, and 3). Nonsurvivors had significantly higher plasma nitrite and nitrate concentrations (days 2 and 3) than survivors. Plasma nitrite and nitrate concentrations on day 1 predicted the development of persistent failure of three of more organs and sequential multiple organ failure but not mortality. CONCLUSION Increased plasma nitrite and nitrate concentrations are associated with the development of multiple organ failure in pediatric sepsis.

sFas and sFas ligand and pediatric sepsis-induced multiple organ failure syndrome.

The Fas-Fas ligand system is important for apoptosis of activated immune cells. Perturbation of this system occurs in diseases with dysregulated inflammation. Increased soluble Fas (sFas) occurs in systemic inflammatory response syndrome (SIRS) and can block apoptosis. Increased shedding of FasL (sFasL) occurs in viral infection and hepatitis. Although dysregulated inflammation is associated with sepsis-induced multiple organ failure (MOF) in children, a role for Fas has not been established. We hypothesize that 1) sFas will be increased in children with severe and persistent sepsis-induced MOF and will correlate with inflammatory markers suggesting a role for sFas in inflammatory dysregulation in severe sepsis, and 2) sFasL will be increased when viral sepsis or sepsis-induced liver failure–associated MOF is present in children. Plasma sFas, sFasL, IL-6, IL-10, nitrite + nitrates, and organ failure scores were measured on d 1 and d 3 in 92 children with severe sepsis and 12 critically ill control children. sFas levels were increased in severe sepsis, continued to increase in persistent MOF and nonsurvivors, and were correlated with serum inflammatory markers (IL-6, IL-10, nitrite + nitrate levels). In contrast, sFasL was not increased in severe sepsis and did not correlate with inflammation. sFasL was, however, increased in liver failure–associated MOF and in nonsurvivors, and was associated with viral infection. At autopsy, hepatocyte destruction and lymphocyte infiltration were associated with increased sFas and sFasL levels. sFas may interfere with activated immune cell death and contribute to dysregulation of inflammation, worsening outcome from severe sepsis. sFasL may contribute to hepatic injury and the development of liver failure–associated MOF.

Biomaterial-neutrophil interactions: Dysregulation of oxidative functions of fresh neutrophils induced by prior neutrophil-biomaterial interaction

Biomaterial-associated infection results in increased morbidity and mortality, and may occur because of nonproductive premature activation of neutrophils resulting in impaired phagocyte function at the biomaterial surface in the event of bacterial challenge. To further explore the effects of this premature activation, we evaluated the supernatants of biomaterial associated neutrophils to determine whether soluble mediators were released, and the likely role of these mediators. We show that these supernatants contain a chemoattractant and thereby induce chemotaxis by fresh neutrophils. No evidence of enhanced oxidative free radical production by either unstimulated neutrophils or a primed response to other mediators occurs when neutrophils were incubated with these supernatants. We also examined the effect of adding fresh neutrophils to a biomaterial surface containing a previous inoculum of neutrophils, and observed that the fresh cells did not become stimulated to release reactive oxygen intermediates (ROI) and also exhibited impaired killing of staphylococci. These studies suggest that not only does the biomaterial surface activate the initial wave of neutrophils but that subsequent waves of neutrophils exhibit an impaired host-defense function. These results are consistent with the known impairment of host defense in the presence of biomaterials, and provide evidence for a long-term down-regulation of neutrophil function at biomaterial surfaces.

Platelets and ATP prime neutrophils for enhanced O2- generation at low concentrations but inhibit O2- generation at high concentration

Platelets are currently thought to play a role in tissue injury and inflammatory states both directly and indirectly through their action on neutrophils (PMNs). Both stimulation and inhibition of PMN superoxide anion (O2 ‐) production by platelets has been reported. To clarify these discrepant observations, we investigated the effects of wide ranges of platelet to PMN ratios as well as concentrations of ATP and ADP on human PMN O2 ‐ production. Platelets, at low platelet‐to‐PMN ratios (1:1 and 5:1), primed PMNs which were stimulated with either FMLP or PMA. However, at higher platelet‐to‐PMN ratios (25:1, 50:1, and 100:1), inhibition of O2 ‐ production was seen. ATP also had a biphasic effect on O2 ‐ production: low concentrations of ATP (1 x 10‐6 to 3.2 x 10‐4 M) increased O2 production and high concentrations of ATP (6.4 x 10‐4 M and above) inhibited O2 production. ADP, when added to stimulatory concentrations of ATP, also caused inhibition of O2 ‐ production. The incubation time for platelet‐neutrophil interactions in vitro was also crucial. Short incubation periods lead to priming, whereas longer periods (> 5 min) lead to inhibition. We believe that these studies help to resolve the controversy over the effects of platelets upon the production of O2 ‐ by human PMNs and lend further support to the notion that platelets may modulate injury resulting from neutrophil activation.

Suppression of Prostaglandin E2 by Malaria Parasite Products and Antipyretics Promotes Overproduction of Tumor Necrosis Factor–α: Association with the Pathogenesis of Childhood Malarial Anemia

Cytokines and effector molecules are important immunoregulatory molecules in human malaria. Tumor necrosis factor (TNF)-alpha limits malaria parasitemia but also promotes pathogenesis at high concentrations, whereas prostaglandin E2 (PGE2) inhibits TNF-alpha production and is reduced in childhood malaria, at least in part, through suppression of cyclooxygenase (COX)-2 following the ingestion of Plasmodium falciparum hemozoin (pfHz; malarial pigment) by peripheral blood mononuclear cells (PBMCs). Although molecular interactions between TNF-alpha and PGE2 are largely unexplored in human malaria, results presented here show that pfHz-induced suppression of PBMC COX-2 gene products induces overproduction of TNF-alpha. Moreover, addition of exogenous PGE2 to pfHz-treated PBMCs dose-dependently decreased TNF-alpha production, whereas experimental COX inhibitors and antipyretics used during human malaria generated increased TNF-alpha production. Healthy, malaria-exposed children had elevated levels of circulating bicyclo-PGE2/TNF-alpha, compared with children with malarial anemia (P<.01), with systemic bicyclo-PGE2 and TNF-alpha significantly associated with hemoglobin concentrations (r=0.745; P<.01). The results of the present study illustrate that pfHz-induced suppression of PGE2 promotes overproduction of TNF-alpha, which is associated with enhanced malarial anemia.

Increased plasma chemoattractant in Sweet's syndrome

The neutrophil function and plasma leukotactic activity of a patient with Sweets syndrome and cystonodular acne were evaluated during a 2 1/2-year period. These studies demonstrated that chemotaxis was frequently slightly increased, especially during an exacerbation of Sweets syndrome, but showed some decrease during isotretinoin therapy. Other functions, such as phagocytosis, metabolic activation, and bacterial killing, also were slightly increased. In addition, the patients serum contained a heat-stable, nonlipid chemoattractant that was present at all times except during a course of isotretinoin. Although his symptoms responded to aspirin, the plasma continued to show this chemoattractant. These findings are consistent with the hypothesis that excess chemoattractant in Sweets syndrome attracts neutrophils, which then mediate an inflammatory response. In addition, aspirin may be used to control Sweets syndrome symptoms, although it does not suppress the plasma chemoattractant.

Simultaneous occurrence of non‐Hodgkinapos;s lymphoma and acute myelomonocytic leukemia

The simultaneous occurrence of nodular poorly differentiated lymphocytic lymphoma (NLPD) and acute myelomonocytic leukemia (AMML) was confirmed by the histologic examination of lymph node section, peripheral blood, and bone marrow. This association in the absence of previous chemotherapy or radiotherapy has not been previously documented. There have been six previous case reports of patients with non‐Hodgkinapos;s lymphocytic lymphoma who, during treatment with alkylating agents or radiotherapy, developed acute myeloid leukemia (AML). These methods of therapy may have had a leukemogenic role in such patients. The possible relationship between these two diseases is discussed.

Clinical Predictors of Severe Malarial Anaemia in a Holoendemic Plasmodium falciparum Transmission Area

Severe malarial anaemia (SMA) is a common complication of Plasmodium falciparum infections, resulting in mortality rates that may exceed 30% in paediatric populations residing in holoendemic transmission areas. One strategy for reducing the morbidity and mortality associated with SMA is to identify clinical predictors that can be readily recognized by caregivers for prompt therapeutic interventions. To determine clinical predictors of SMA, Kenyan children (3–36 months, n = 671) presenting with acute illness at a rural hospital in Siaya District were recruited. Demographic, clinical, laboratory and haematological parameters were measured upon enrolment. As human immunodeficiency virus‐1 and bacteraemia promote reduced haemoglobin (Hb) concentrations, children with these infections were excluded from the analyses. Children with P. falciparum mono‐infections (n = 355) were stratified into three groups: uncomplicated malaria (Hb ≥ 110 g/l); non‐SMA (60 ≤ Hb < 109), and SMA (Hb < 60 g/l). SMA was characterized by a younger age, monocytosis, thrombocytopenia, reticulocytosis, reduced erythropoiesis, elevated pigment‐containing monocytes (PCM), respiratory distress, conjunctival and palmar pallor, splenomegaly, signs of malnutrition, and protracted fever and emesis. Logistic regression analysis demonstrated that age, reticulocyte count, presence of PCM and conjunctival and palmar pallor were significant predictors of SMA. Recognition of these clinical signs in children residing in resource‐poor settings may help to guide the identification and management of SMA.

Comparative Pharmacokinetics and Renal Effects of Cyclosporin A and Cyclosporin G in Renal Allograft Recipients

Cyclosporin G (CSG) has produced less nephrotoxicity than cyclosporin A (CSA) at equivalent doses in animal models. Conflicting results have been reported concerning differences in the pharmacokinetics of CSA and CSG in preclinical studies, and no data exist regarding the effect of steady‐state oral administration of CSG on renal function in transplant patients or CSG‐induced release of endothelin and nitric oxide (NO) in vivo. The objective of the study was to examine steady‐state pharmacokinetic profiles of adult renal allograft recipients receiving CSA and CSG in relation to concentrations of endothelin‐1 and NO2/NO3 in urine and plasma, creatinine clearance (Clcr), and urinary excretion of N‐acetyl‐β‐D‐glucosaminidase (NAG) 9 months after transplantation. Concentrations of CSA and CSG were measured in whole blood over a 12‐hour dose interval by both a monoclonal and polyclonal fluorescence polarization radioimmunoassay for CSA. A metabolite fraction was defined as the numerical difference between the levels obtained at each time point by both assays. Patient groups were defined as follows: group 1: initial CSA (n = 6); group 2: initial CSG (n = 7); group 3: five of the seven patients in group 2 taking CSG subsequently undergoing conversion to CSA; group 4: the same five patients in group 3 restudied 1 month after 1:1 dosage conversion to CSA; and group 5: CSA groups 1 and 4 combined (n = 11). In group 1, the metabolite fraction accounted for 32% to 54% of the total measurable drug concentration at each time point, whereas in group 2, the metabolite fraction accounted for at most 10% to 15% of the total drug levels measurable by polyclonal fluorescence polarization radioimmunoassay. Although there were no significant differences in any of the mean pharmacokinetic parameters between groups using monoclonal fluorescence polarization radioimmunoassay, the normalized area under the concentration—time curve (NAUC) value was less in four of five patients after conversion from CSG to CSA, with a more variable and delayed time to reach peak concentration (tmax) but equivalent apparent oral clearance (Clpo) values. Clcr was found to change significantly with time in groups 1 and 5 but not in group 2, with CSA producing a more profound and sustained decrease than CSG. Endothelin‐1 and NO2/NO3 levels in plasma and urine remained relatively constant after administration of both CSA and CSG, and there were no significant differences between groups 3 and 4 regarding mean endothelin‐1 and NO2/NO3 concentrations in plasma, urinary release of endothelin‐1 and NO2/NO3, and mean AUC of endothelin‐1 and AUC of NO2/NO3. However, monoclonal NAUC correlated significantly with total urinary endothelin‐1 within CSA groups 1 and 5 but not within CSG group 2. Metabolite NAUC correlated significantly with total urinary NAG within CSA group 1. Although limited by the small number of patients, this study suggests that 1) CSG may produce less of a reduction in Clcr over time after oral administration at steady state than does CSA, and 2) this beneficial effect of CSG may be in part due to decreased intrarenal release of endothelin‐1, as urinary excretion of endothelin‐1 seemed to correlate better with CSA than with CSG exposure.