Peripheral Blood Polymorphonuclear Leukocytes Research Articles

Decreased polymorphonuclear leucocyte chemotactic response to leukotriene B4 in cystic fibrosis.

Evidence that leukotriene B4 (LTB4) is a significant inflammatory mediator in chronic pseudomonal respiratory disease was sought in adolescents and young adults with cystic fibrosis. Specific chemotaxis of peripheral blood polymorphonuclear leucocytes (PMN) was used as an indirect measure of remote in vivo exposure to LTB4. PMN from 17 patients showed a significant decrease in chemotaxis to 10(-7)-10(-9) M LTB4, but normal responses to 10(-8) M n-formyl-methionyl-leucyl-phenylalanine and 4 mg/ml casein, when compared with 17 healthy age- and sex-matched controls. This result is consistent with chronic production of LTB4, and specific deactivation of circulating PMN receptors for LTB4 in patients with cystic fibrosis. Pharmacologic inhibition of LTB4 production in vivo may help elucidate its role in the pathogenesis of lung damage in cystic fibrosis.

In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS). Peripheral blood PMNs obtained from 12 healthy Beagles. The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry. The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA. Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA-induced generation of reactive oxygen species.

Activation of human leukocytes on tantalum trabecular metal in comparison to commonly used orthopedic metal implant materials

We analyzed leukocyte functions and cytokine response of human leukocytes toward porous tantalum foam biomaterial (Trabecular Metaltrade mark, TM) in comparison to equally sized solid orthopedic metal implant materials (pure titanium, titanium alloy, stainless steel, pure tantalum, and tantalum coated stainless steel). Isolated peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) were cocultured with equally sized metallic test discs for 24 h. Supernatants were analyzed for cytokine content by enzyme-linked immunosorbent assay. Compared to the other used test materials there was a significant increase in the release of IL (interleukin)-1ra and IL-8 from PMN, and of IL-1ra, IL-6, and TNF-alpha from PBMC in response to the TM material. The cytokine release correlated with surface roughness of the materials. In contrast, the release of IL-2 was not induced showing that mainly myeloid leukocytes were activated. In addition, supernatants of these leukocyte/material interaction (conditioned media, CM) were subjected to whole blood cell function assays (phagocytosis, chemotaxis, bacterial killing). There was a significant increase in the phagocytotic capacity of leukocytes in the presence of TM-conditioned media. The chemotactic response of leukocytes toward TM-conditioned media was significantly higher compared to CM obtained from other test materials. Furthermore, the bactericidal capacity of whole blood was enhanced in the presence of TM-conditioned media. These results indicate that leukocyte activation at the surface of TM material induces a microenvironment, which may enhance local host defense mechanisms.

Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: A study on human leukocytes

Aim of the study Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of “Marcela”. Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). Materials and methods Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. Results In PBMCs, Achyrocline satureioides infusion in the 0.06–0.24 μg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-γ and interleukin (IL)-4. Lower concentrations of the infusion (0.006–0.03 μg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-γ and IL-4 and decreased the ratio IFN-γ/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations ≥0.06 μg/ml QE. On the contrary, in the 0.0012–0.03 μg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. Conclusions The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.

Association of reduced heme oxygenase-1 with excessive Toll-like receptor 4 expression in peripheral blood mononuclear cells in Behçet's disease

IntroductionToll-like receptors (TLRs) mediate signaling that triggers activation of the innate immune system, whereas heme oxygenase (HO)-1 (an inducible heme-degrading enzyme that is induced by various stresses) suppresses inflammatory responses. We investigated the interaction between TLR and HO-1 in an inflammatory disorder, namely Behçet's disease.MethodsThirty-three patients with Behçet's disease and 30 healthy control individuals were included in the study. Expression levels of HO-1, TLR2 and TLR4 mRNA were semiquantitatively analyzed using a real-time PCR technique, and HO-1 protein level was determined by immunoblotting in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes. In some experiments, cells were stimulated with lipopolysaccharide or heat shock protein-60; these proteins are known to be ligands for TLR2 and 4.ResultsLevels of expression of HO-1 mRNA were significantly reduced in PBMCs from patients with active Behçet's disease, whereas those of TLR4, but not TLR2, were increased in PBMCs, regardless of disease activity. Moreover, HO-1 expression in PBMCs from patients with Behçet's disease was repressed in the presence of either lipopolysaccharide or heat shock protein-60.ConclusionThe results suggest that upregulated TLR4 is associated with HO-1 reduction in PBMCs from patients with Behçet's disease, leading to augmented inflammatory responses.

Bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin-stimulated polymorphonuclear leukocytes

It has been known that administration of antibiotics may lead to excessive release of bacterial endotoxins and complicate clinical course of patients with Gram-negative infections. This concern may also apply to phages. Endotoxin may in turn activate neutrophils to produce reactive oxygen species (ROS) that are believed to play an important role in the pathogenesis of multiple organ dysfunction in the course of sepsis.We showed that a purified T4 phage preparation with low-endotoxin content could significantly diminish the luminol-dependent chemiluminescence (CL) of peripheral blood polymorphonuclear leukocytes (PMNs) both stimulated by lipopolysaccharides (LPSs) isolated from different Escherichia coli strains. This effect was also observed for live bacteria used for PMNs stimulation and was independent of bacterial susceptibility for T4-mediated lysis. Our data suggest, that phage-mediated inhibition of LPS- or bacteria-stimulated ROS production by PMNs may be attributed not only to phage–PMNs interactions, but also to phage–LPS interactions and bacterial lysis (in case of homologous phage). Interestingly, the T4 preparation did not influence ROS formation by PMNs stimulated with PMA. This suggests that the observed phenomena are also dependent upon the nature of activator.Bacteriophage-mediated inhibition of ROS formation by cells exposed to endotoxin provides new evidence for possible interactions between phages and mammalian cells. It helps in understanding the role of phages in our environment and may also be of important clinical significance.

Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.

Nuclear factor-kappaB expression in peripheral blood polymorphonuclear leukocytes of patients with acute pancreatitis and the preventive effectiveness of pyrrolidine dithiocarbamate on NF-kappaB in vitro

To investigate the expression of NF-kappaB in peripheral blood polymorphonuclear leukocyte (PMN) of acute pancreatitis (AP) and to assess the preventive effectiveness of pyrrolidine dithiocarbamate (PDTC) on NF-kappaB in vitro. Nineteen patients and 16 healthy individuals as control were enrolled in this study. The expression of NF-kappaB in PMNs was determined by gel electrophoretic mobility shift assay (EMSA). Routine clinical examination results and computed tomography findings of AP were recorded in all patients. The PMNs from the patients with AP showed higher levels of NF-kappaB activities than those from control subjects (P < 0.01), severe acute pancreatitis (SAP) group showed much higher than mild acute pancreatitis (MAP) group (P < 0.05). In vitro, PDTC could reduce the NF-kappaB activity in PMNs of patients with AP, and its effectiveness at 2 mmol/L was stronger than at 1 mmol/L (P < 0.05). The PMNs from control subjects pretreated with 2 mmol/L PDTC before stimulation with the plasma from patients with SAP showed lower levels of NF-kappaB activities than did those untreated (P < 0.05). The NF-kappaB activation in peripheral blood PMNs participate in the course of acute pancreatitis and can be inhibited by PDTC in vitro.

Acute Rejection and Cardiac Allograft Vascular Disease Is Reduced by Suppression of Subclinical Cytomegalovirus Infection

Anticytomegalovirus (CMV) prophylaxis prevents the acute disease but its impact on subclinical infection and allograft outcome is unknown. We sought to determine whether CMV prophylaxis administered for three months after heart transplant would improve patient outcomes. This prospective cohort study of 66 heart transplant recipients compared aggressive CMV prophylaxis (n = 21, CMV hyperimmune globulin [CMVIG] plus four weeks of intravenous ganciclovir followed by two months of valganciclovir); with standard prophylaxis (n = 45, intravenous ganciclovir for four weeks). Prophylaxis was based on pretransplant donor (D) and recipient (R) CMV serology: R-/D+ received aggressive prophylaxis; R+ received standard prophylaxis. Outcome measures were: CMV infection assessed by DNA-polymerase chain reaction on peripheral blood polymorphonuclear leukocytes, acute rejection, and cardiac allograft vascular disease (CAV) assessed by intravascular ultrasound. All patients completed one year of follow-up. RESULTS.: CMV infection was subclinical in all but four patients (two in each group). Aggressively treated patients had a lower incidence of CMV infection (73 +/- 10% vs. 94 +/- 4%; P = 0.038), and an independent reduced relative risk for acute rejection graded > or =3A (relative risk [95% CI] = 0.55 [0.26-0.96]; P = 0.03), as compared with the standard prophylaxis group. Aggressively prophylaxed patients also showed a slower progression of CAV, in terms of coronary artery lumen loss (lumen volume change=-21 +/- 13% vs. -10+/-14%; P = 0.05); and vessel shrinkage (vessel volume change = -15 +/- 11% vs. -3 +/- 18%; P = 0.03). Prolonged (val)ganciclovir plus CMVIG reduces viral levels, acute rejection, and allograft vascular disease, suggesting a role for chronic subclinical infection in the pathophysiology of the most common diseases affecting heart transplant recipients.

Activation state-dependent interaction between Galphai and p67phox.

The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Galphai subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Galphai. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Galphai. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Galphai. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Galphai, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Galphai form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Galphai in the regulation or activation of NADPH oxidase.

Effect of cyclosporin A on human neutrophil and monocyte function.

The effect of various concentrations of cyclosporin A (CyA), ranging from below peak blood levels to 20 times higher than blood levels of human peripheral blood polymorphonuclear and mononuclear leukocytes, was examined. CyA was found to bind to neutrophils with Kd values in the range of 20-50 nM. CyA at clinically obtainable blood level concentrations had no effect on neutrophil and monocyte chemotaxis, neutrophil oxidative burst, monocyte phagocytosis, or neutrophil bactericidal activity. The data on the release of lactoferrin, a secondary granule substance, from activated neutrophils showed that the calcium ionophore A 23187-induced lactoferrin release was inhibited by treatment of cells with 4 microM CyA, whereas release of lactoferrin from zymosan- or phorbol myristate acetate-activated neutrophils was not affected by the same concentration of CyA. This effect could either be due to differences in the degree of cell membrane perturbation by the various activators or to calcium dependence of neutrophil activation. A third possibility may be that CyA acts at some subsequent steps in the release process of neutrophils. It is concluded that CyA does not interfere with important functions of human phagocytes, the cells that play a major role in the defence against invading microorganisms.

Regulation of leukocyte adhesion molecules by leukocyte/biomaterial‐conditioned media: A study with calcium‐phosphate‐coated and non‐coated NiTi‐shape memory alloys

AbstractTo evaluate the role of soluble mediators released after interaction of leukocytes with biomaterials on cells of the implant microenvironment, the surface expression of adhesion molecules (CD11b, CD62L, CD66b, ICAM‐1) on freshly isolated whole blood leukocytes (WBL) was analyzed by flow cytometry after incubation in leukocyte/nickel‐titanium shape‐memory alloy (NiTi‐SMA)‐conditioned media. NiTi‐SMA samples were either coated with calcium phosphate by dipping in oversaturated calcium phosphate solution (CaP‐coating) or were non‐coated. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) were isolated and seeded on both samples to generate respective conditioned media. In comparison with conditioned media obtained by non‐coated NiTi‐SMA, the expression of CD11b, CD66b, and ICAM‐1 on WBL was upregulated by conditioned media obtained from CaP‐coated samples. In contrast, the expression of CD62L on WBL was decreased after incubation in conditioned medium obtained from CaP‐coated NiTi‐samples compared to non‐coated NiTi. These data demonstrate the possible influence of released mediators elicited by leukocyte‐biomaterial interactions on cells of the implant microenvironment.

Alpha‐1‐acid Glycoprotein Inhibits Phorbol Ester‐induced but not Fc‐Receptor‐induced Generation of Reactive Oxygen Species in Bovine Peripheral Blood Neutrophils

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein with anti-inflammatory and immunomodulating properties. AGP is described as a potent inhibitor of the production of reactive oxygen species (ROS) in human neutrophils. However, published reports about the mechanism of inhibition are conflicting. The influence of bovine AGP on the production of ROS by bovine peripheral blood polymorphonuclear leucocytes (PMN) was studied using a highly sensitive method approaching its inhibitory mechanism. ROS production in PMN was induced with phorbol 12-myristate 13-acetate (PMA) or opsonized Staphylococcus aureus bacteria. ROS generation was quantified and evaluated by flow cytometry. AGP efficiently suppressed PMA, but did not opsonize bacteria-induced ROS generation in vitro. The suppressive effect was concentration-dependent and adversely proportional to PMA concentration. The selective inhibitory potential of AGP in comparison with ovalbumin (OVA) and bovine serum albumin (BSA) showed that ROS inhibition was not a mere protein effect. ROS production was suppressed only if AGP and PMA were simultaneously present with PMN. Pre-incubation of PMN with AGP did not alter the PMN response to PMA. Moreover, AGP could not suppress ROS production after pre-stimulation of PMN with PMA. Human and bovine AGP did not differ in their inhibitory potential to the PMA-induced ROS production in bovine, human and equine PMN. The results show that AGP does not modulate bovine neutrophil functions directly, but acts as a scavenger of PMA.

Effects of resveratrol, piceatannol, tri-acetoxystilbene, and genistein on the inflammatory response of human peripheral blood leukocytes

Inflammatory processes are involved in the etiology of diseases. We analyzed the effect of resveratrol, piceatannol, synthetic tri-acetoxystilbene (TAS), and genistein (Bonistein(TM)) on the production of inflammatory mediators including prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha), interleukins, and chemokines, which participate in the progression of inflammation. In order to induce inflammatory responses, human peripheral blood mononuclear and/or polymorphonuclear leukocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN(gamma)) and the production of PGE2, interleukin-8 (IL-8), and TNF-alpha was determined. In response to the stimuli, genes were substantially activated within < 2 h (e. g., TNF-alpha, IL-1alpha), or at a later stage, (e. g., COX-2, IL-6, IL-8). Unlike genistein, resveratrol and related compounds dose-dependently reduced PGE2 production. Genistein, piceatannol, and TAS diminished secretion of TNF-alpha, and IL-8. TAS reduced mRNA levels of COX-2, TNF-alpha, IL-8, IL-6, and IL-1alpha, while resveratrol impaired early expression of IL-8 and TNF-alpha. Piceatannol out-performed resveratrol, yet without matching TAS. Genistein downregulated TNF-alpha and IL-8 expression. These substances altered the LPS/IFNgamma-induced gene expression in mononuclear cells rather than in polymorphonuclear leukocytes. Immunoblot analyses corroborated the distinct activity pattern of resveratrol and genistein. In conclusion, resveratrol and their derivatives attenuated the inflammatory response of PBLs at several levels, whereas genistein acts on cytokines and pro-inflammatory interleukins.

Comparative analysis of a double primer PCR assay with plasma, leukocytes and antigenemia for diagnosis of active human cytomegalovirus infection in bone marrow transplant patients

The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment.

Food protein-induced enterocolitis syndrome: Case presentations and management lessons

Enterocolitis induced in infants by cow's milk and/or soy protein has been recognized for decades. Symptoms typically begin in the first month of life in association with failure to thrive and may progress to acidemia and shock. Symptoms resolve after the causal protein is removed from the diet but recur with a characteristic symptom pattern on re-exposure. Approximately 2 hours after reintroduction of the protein, vomiting ensues, followed by an elevation of the peripheral blood polymorphonuclear leukocyte count, diarrhea, and possibly lethargy and hypotension. The disorder is generally not associated with detectable food-specific IgE antibody. There are increasing reports of additional causal foods, prolonged clinical courses, and onset outside of early infancy, leading to description of a food protein-induced enterocolitis syndrome. The disorder poses numerous diagnostic and therapeutic challenges. The purpose of this report is to delineate the characteristic clinical features and review the possible pathophysiologic basis to frame a rational strategy toward management.

A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis

Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions. We have isolated an exopolysaccharide-producing P. nigrescens, strain 22, from a chronic periodontitis lesion. The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate. The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis. To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established. The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes. Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 microg/mg), glucose (25.6 microg/mg), fructose (65.8 microg/mg), galactose (12.5 microg/mg), arabinose (6.2 microg/mg), xylose (3.2 microg/mg), rhamnose (6.1 microg/mg), and ribose (0.6 microg/mg). Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3). Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328. The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328. Exopolysaccharide alone failed to induce abscess formation in mice. Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors. The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated.

Inhibitory effect of furosemide on activation of human peripheral blood polymorphonuclear leukocytes stimulated with n-formyl-methionyl-leucyl-phenylalanine

The clinical efficacy of inhalatory furosemide (Fu) has been extensively studied in bronchial asthma patients but there are only a few studies addressing its action on cells participating in the underlying inflammatory process. Therefore, we investigated the effect of Fu on human peripheral blood polymorphonuclear leukocytes (PMNL) at concentrations that can be achieved in the bronchial lining fluid by inhalation, i.e. 10 −5, 10 −4 and 10 −3 M. The influence of Fu on the following PMNL parameters was investigated: intracellular calcium changes ([Ca 2+] i) as a part of signal transduction and luminol dependent chemiluminescence (LCL) as an indirect measure of NADPH-oxidase activation upon n-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation; chemotaxis to fMLP, phagocytosis and intracellular killing of Staphylococcus aureus. Incubation with Fu resulted in a concentration dependent reduction of Ca 2+ influx and Fu (10 −3 M) decreased the main Ca 2+ parameters to one half of the control values and to the level obtained in calcium-free buffer. In contrast, Fu had no effect if preincubated with the cells and then removed by washing. The LCL signal was reduced by Fu (10 −3 M) from 2000±870 to 550±440 arbitrary units [aU] ( p<0.05). In contrast to the [Ca 2+] i measurements, a slightly diminished LCL was also observed following preincubation with Fu and washing. No effect of Fu was found on phagocytosis and intracellular killing of St. aureus. Fu diminished chemotaxis to fMLP but at 10 −3 M it also displayed weak chemoattractant properties. The differential action of Fu on human PMNL may add to the understanding of its topical and restricted efficacy in bronchial asthma.

Clinical manifestations and molecular epidemiology of necrotizing pneumonia and empyema caused by Streptococcus pneumoniae in children in Taiwan.

Recently, there have been increasing numbers of pneumococcal pneumonia cases, with their associated complications. We conducted a retrospective review to increase the understanding of childhood pneumococcal pneumonia. Seventy-one patients with pneumococcal pneumonia were identified. Forty (56.3%) of them developed complicated pneumonia. Multivariate analysis showed that presence of immature polymorphonuclear leukocytes in peripheral blood (odds ratio [OR], 3.67; 95% confidence interval [CI], 1.08-12.63), high C-reactive protein levels (>12 mg/dL) (OR, 5.24; 95% CI, 1.10-24.93), and no underlying disease at presentation (OR, 5.48; 95% CI, 1.06-28.25) were independent predictors of the occurrence of necrosis or/and abscess. Fourteen isolates (35%), which were genotypically identical and had the same pulsed-field gel electrophoresis pattern (serogroup 14, with MICs of penicillin of 0.1-0.5 mu g/mL), were significantly associated with complicated pneumonia (P=.047). Whether the virulence of antibiotic-resistant pneumococci is evolving deserves further investigation.