Serum-induced macrophage activation is related to the severity of septic shock

Backgroud:  It seems that a balance between anti and pro-inflammatory responses must be kept to eliminate the pathogen without inducing inflammatory damage in the host. Thus we determined the relation between macrophage activation and the severity and clinical outcome in septic patients. Material and Methods:  This was a prospective study at a tertiary general intensive care unit. Thirty-three patients admitted with sepsis, severe sepsis or septic shock were included. As a control group, healthy volunteers were included matched to septic patients by age and sex. Peritoneal rat macrophages were cultured with 2% serum from healthy volunteers or from septic patients for determination of phagocytic potential or the capacity to produce cytokines. Results:  TNF and IL1 secretion by macrophages activated with serum from sepsis and severe sepsis patients was higher than with serum from healthy controls. In addition, proinflammatory cytokines released in vitro from macrophages, but not determined directly in the serum from patients, were lower in non-survivor septic patients when compared to survivors. In contrast, IL-10 secretion by macrophages activated with serum from septic patients was higher in nonsurvivors. In the septic shock group we observed a diminution in the phagocytic index compared to sepsis and severe sepsis groups, and the phagocytic index was higher in sepsis survivors. Conclusions:  Markers of antiinflammation are predominant in more severe types of sepsis suggesting that antiinflammation is related to mortality. Received 2 May 2008; returned for revision 23 June 2008; received from final revision 4 June 2008; accepted by C. Kasserra 17 June 2008     

Hydrogen sulphide is pro-inflammatory in haemorrhagic shock

Objective:  H₂S is pro-inflammatory in inflammatory models, thus we investigated whether H₂S plays a role in haemorrhagic shock (HS)-associated inflammation. Methods:  Male, Sprague-Dawley rats were given an inhibitor of H₂S biosynthesis, DL-propargylglycine (PAG, 50 mg/kg, i. v.) or saline (1 ml/kg) 30 min before blood withdrawal and subjected to HS (mean arterial pressure (MAP) of 40 mM Hg for 90 min) followed by reinfusion of shed blood. Animals were killed at 5, 90, 270 and 630 min after reinfusion. Results:  Pre-treatment of animals with PAG 1) increased the HR recovery rate (n = 6 – 12, P < 0.05); 2) attenuated the increase in plasma levels of TNF-α and IL-6 and reduced lung iNOS expression levels (n =5 P, < 0.05); and 3) attenuated the increase in plasma levels of ALT and reduced HS-induced increase in liver and lung myeloperoxidase (MPO) activity (n = 5, P < 0.05). Conclusions:  H₂S is pro-inflammatory in HS and inhibition of H₂S biosynthesis may reduce some HS-induced inflammatory responses and organ injury. Received 28 November 2007; returned for revision 25 February 2008; received from final revision 7 May 2008; accepted by G. Wallace 16 June 2008 The first two authors contributed equally to this work.     

Inhibition of neutrophil infiltration by a malleable protein matrix of lactic acid bacteria-fermented whey proteins in vivo

Objective:  A novel nutraceutical ingredient, the Malleable Protein Matrix (MPM), has previously demonstrated a significant anti-inflammatory effect in a systemic inflammatory disease model, comparable to conventional drugs. The objective of this study was to investigate the potential anti-inflammatory effects of MPM on neutrophil infiltration in vivo, phagocytosis activity as well as cytokine and chemokine production. Methods:  Groups of ten C57BL\6J mice received water or MPM per os for a period of 2 weeks prior to the creation of a murine air pouch. The subsequent neutrophil recruitment and activities were characterized following lipopolysaccharide injection. Results:  In the water control group, the number of recruited cells was 1.8X10⁷ cells/pouch, which was reduced to 9X10⁶ cells/pouch with oral MPM consumption, representing an inhibition of 50% of infiltrating leukocytes. A considerable reduction in the cytokine and chemokine production, mostly TNFα, IL-1β and IL-6 production in the MPM-treated group, suggested an inhibition of the mediators responsible for leukocyte extravasation. On the other hand, MPM consumption had no effect on neutrophil phagocytosis activity. Conclusion:  MPM administration demonstrates a significant reduction of neutrophil infiltration associated with an inhibition of cytokine and chemokine production. The air pouch model shares similarities with in vivo characteristics of rheumatoid arthritis and neutrophilic diseases, both of which would benefit from this 50% inhibition of neutrophil infiltration induced by MPM. Received 14 May 2007; returned for revision 7 October 2007; received from final revision 25 April 2008; accepted by S. Stimpson 27 June 2008     

Autoregulative function in the brain in an endotoxic rat shock model

Objective and design:  Autoregulative function in the brain gets relevant in hypodynamic conditions of a sepsis syndrome. We investigated the temporal pattern and dose dependent effects of LPS-induced shock on autoregulative function in rats. Material and subjects:  Chloralose-anesthetized and mechanically ventilated male CD-rats (n = 30). Treatment:  Animals were subjected to vehicle, 1 or 5 mg/kg b.w. lipopolysaccharide (LPS) from E. coli given intravenously. Methods:  Autoregulative function was tested repeatedly with a carotid compression technique assessing the transient hyperemic response ratio (THRR) in the cortex with laser Doppler flowmetry up to 270 min. THRR data from exsanguination experiments served as controls. Results:  Despite lower blood pressure levels in the high dose group (control: 114 ± 7 mmHg; 1 mg/kg LPS group: 82 ± 16 mmHg; 5 mg/kg LPS group: 62 ± 16 mmHg; p < 0.05) progressive cerebral hyperemia occurred similarly in both groups. Compared to exsanguinations experiments autoregulative compensation for lower blood pressure levels was lacking in the high LPS dose group at the end of experiments. Conclusions:  Cerebral autoregulation was affected by LPS-induced shock supporting the notion of vasoregulative failure in endotoxic shock Received 22 October 2007; returned for revision 20 February 2008; received from final revision 23 May 2008; accepted by K. Visvanathan 8 July 2008     

Treatment with bindarit, an inhibitor of MCP-1 synthesis, protects mice against trinitrobenzene sulfonic acid-induced colitis

OBJECTIVE: Chemokines play a fundamental role in trafficking and activation of leukocytes in colonic inflammation. We investigated the ability of bindarit, an inhibitor of monocyte chemoattractant protein-1 (MCP-1/CCL2) synthesis, to inhibit chemokine production by human intestinal epithelial cells (HT-29) and its effect in trinitro-benzene sulfonic acid (TNBS)-induced colitis in mice.MATERIALS AND METHODS: HT-29 cells were incubated with bindarit in the presence of TNF-alpha/IFN-gamma and 24 h later supernatants were collected for MCP-1, IL-8 and RANTES measurement. A 1 mg enema of TNBS was given to BALB/c mice, and bindarit (100 mg/kg) was orally administered twice daily starting from two days before colitis induction. Weight loss, histology, and MCP-1 level and myeloperoxidase (MPO) activity in colon extracts were assessed.RESULTS: In HT-29 cells, bindarit concentration-dependently and selectively inhibited MCP-1 secretion (as well as mRNA expression) primed by TNF-alpha/IFN-gamma. Moreover treatment with bindarit reduced clinical and histopathological severity of TNBS-induced colitis. These effects were associated with significant inhibition of MCP-1 and MPO in colon extracts.CONCLUSIONS: Bindarit exhibits a potent bioactivity in reducing leukocyte infiltration, down-regulating MCP-1 synthesis, and preventing the development of severe colitis in a mice model of TNBS-induced colitis. These observations suggest a potential use of MCP-1 synthesis blockers in intestinal inflammation in humans.     

Adrenomedullin and endothelin-1 are related to inflammation in chronic heart failure

Background:  Adrenomedullin (ADM) and endothelin-1 (ET-1) are novel promising peptide biomarkers in chronic heart failure (CHF). According to recent studies among their pleiotropic effect they play roles in the regulation of inflammation. The aim of the study was to measure the above mentioned two vasoactive peptides in parallel in a well characterized population of patients with CHF, and study their associations with inflammatory markers. Materials and methods:  A total of 186 patients (138 male, 48 female) with <45% left ventricular ejection fraction (LVEF), and without acute inflammatory disease, were enrolled. Plasma midregional-proADM (MR-proADM) and C-terminal-proET-1 (CT-proET-1) were determined by a novel sandwich immunoluminomertic assay. Results:  Increased MR-proADM and CT-proET-1 plasma levels were measured in patients with severe CHF (NYHA III-IV) as compared to the group of NYHA I-II (p<0.0001). MR-proADM and CT-proET-1 levels showed significant negative correlation with serum albumin and prealbumin levels (p≤0.0001), while positive correlations were found with levels of CRP, TNF-alpha, soluble TNF receptors and IL-6 (p≤0.0001). In multiple linear regression models after adjustments for several potential confounders (disease severity [LV-EF, NYHA classes, NT-proBNP], ion and water homeostasis [sodium and presence of peripheral oedema], renal function [serum creatine]) the relationship between ADM and albumin, CRP, soluble TNF receptors and between ET-1 and CRP, TNF receptors and IL-6 remained significant. Conclusions:  Vasoregulation and inflammation may be connected in heart failure patients independently of the disease severity. The observed link may contribute to the understanding of the complex pathomechanism in CHF. Received 14 August 2008; returned for revision 31 October 2008; received from final revision 9 November 2008; accepted by C. Kasserra 18 November 2008     

Leukocyte depletion during cardiac surgery with extracorporeal circulation in high risk patients

Background:  Cardiopulmonary bypass is associated with systemic inflammation that may contribute to increased perioperative mortality. Depletion of circulating leukocytes may reduce the inflammatory response. We studied the effect of a leukocyte depleting filter on leukocyte activation during cardiopulmonary bypass in high risk patients. Methods:  Fifty patients undergoing coronary artery bypass grafting with a preoperative high risk were randomly placed in an arterial line leukocyte filter group (n = 25) with a leukocyte depleting filter. Blood sampling took place from the arterial line to analyze polymorphnuclear elastase and myeloperoxidase at six time points, including: A) before the induction of anesthesia, B) before the induction of the cardiopulmonary bypass C) 1 min after the release of the aorta clamp, D) the end of the operation, E) 1 h postoperative, and F) 24 h postoperative. Results:  Levels of polymorphonuclear elastase, (PMNE), and myeloperoxidase (MPO) were found to be higher after the release of the aortic cross clamp in the leukocyte filter group; these levels remained elevated until 24 hours after surgery and were high in comparison to preoperative baseline levels. The differences in PMNE between both groups at time points C and D (p < 0.005) and E (p < 0.05) were statistically significant. The serum levels of the S-100B and neuron specific enolase (NSE) were found to be elevated between time points C and E in both groups without statistical significance. The in-hospital mortality was 16% (4 patients) in leukocyte filter group and 4% in control group (1 patient). Conclusions:  Interestingly, the activation of neutrophils was more pronounced in the LF group. The use of a leucocyte depleting filter was not advantageous for this patient cohort for clinical or biomedical endpoints. Received 17 February 2008; returned for revision 21 May 2008; received from final revision 6 August 2008; accepted by M. Katori 8 August 2008     

Temporal expression of cyclooxygenase-2 in peritoneal macrophages of rats and effects of panax notoginseng saponins

Objective:  To explore the temporal expression pattern of cyclooxygenase (COX)-2 and effects of panax notogensing saponins (PNS) in peritoneal macrophages of rats. Materials and methods:  Phagocytosis function of peritoneal macrophages was measured by chicken red blood cell phagocytosis assay in vitro. Expression of COX-2 mRNA and protein, PGE₂ and PGD₂ production were determined with real-time PCR, Western blotting and radioimmunoassay, respectively. Results:  Phagocytosis function of macrophages increased significantly after stimulation and reached peak during 2–3h. Expression of COX-2 mRNA and its protein increased markedly after stimulation and reached the first peak at 2 h and 3h, respectively; and then decreased to reach a minimum at 24h. The second peak appeared at 36h. PNS (50, 100, 200 mg/kg) increased the phagocytosis function obviously at 2h, decreased the expression level of COX-2 and PGE₂ production at 2 h and elevated COX-2 expression and PGD₂ production at 36 h, respectively. Conclusion:  COX-2 expression in peritoneal macrophages has a double-hump feature after stimulation. PNS enhanced phagocytosis, inhibiting COX-2 expression at an early stage and elevating it at a later stage. Received 29 February 2008; returned for revision 27 March 2008; received from final revision 14 May 2008; accepted by G. Wallace 9 June 2008 The first two authors contributed equally to this work.     

Immunological system status and the appearance of respiratory system disturbances in thymectomized patients

INTRODUCTION: Adult-onset thymoma may be responsible for several diseases, such as pure red cell aplasia, myasthenia gravis, and immunodeficiency (Good's syndrome). Thymectomy does not always improve the patient's condition, and may even produce additional symptoms. Its pathogenesis is still not entirely understood, but autoimmunological processes and bone marrow defect are the most frequently suggested. MATERIALS AND METHODS: Eleven patients (mean age: 56.2+/-15.5 years) were analyzed 6 months to 10 years after thymectomy due to thymoma as were 25 healthy persons serving as controls. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry techniques were used to evaluate the immunological status of the subjects. RESULTS: Good's syndrome was diagnosed in one patient, 4 subjects suffered from myasthenia gravis, and recurrent infections of upper and lower respiratory tract appeared in 9 patients. The immunological analyses (ELISA and flow cytometry) revealed a significantly lower IgG level (p<0.05), percentage of peripheral blood B lymphocytes (p<0.0005), and CD4:CD8 ratio (p<0.05) in thymectomized patients compared with the healthy controls. The percentages of CD4+ and CD8+ T lymphocytes expressing CD28 antigen were significantly lower in thymectomized patients than in healthy subjects (p<0.005 and p<0.01, respectively). The percentage of naive T helper lymphocytes was significantly lower in the patients than in the control group (p<0.05). CONCLUSIONS: Immunodeficiency and recurrent infections may be the first symptoms of immunological disturbances after thymectomy in adults. It is suggested that regular medical monitoring of these patients is important in preventing further complications, which may result in irreversible lung tissue destruction.     

Cytokine profile in collagen-induced arthritis: Differences between syngeneic and allogeneic pregnancy

OBJECTIVE: To identify the differences in cytokine profile between allogeneic and syngeneic pregnancy in mice with collagen-induced arthritis (CIA). METHODS: Mice (strain B10.RIII) were injected with bovine collagen. Females were mated with males of the same strain (syngeneic pregnancy) or with males of strain B10. Q (allogeneic pregnancy). Concentrations of cytokines were measured during pregnancy and after delivery, and the onset and evolution of arthritis was followed in all female animals throughout the study period. RESULTS: In female mice that developed CIA, cytokine concentrations were lower in allogeneic pregnancies than syngeneic pregnancies. When paired cytokine concentrations were compared in each animal during and after pregnancy, MCP-1 was lower during gestation than after delivery in both groups of pregnant mice, IL-6 was lower during gestation than after delivery only in allogeneic pregnancies, and IL-10 was lower during gestation than after delivery in allogeneic pregnancies, whereas in syngeneic pregnancies IL-10 was higher during gestation than after delivery. CONCLUSIONS: Allogeneic pregnancy was associated with less arthritis because of lower concentrations of proinflammatory cytokines (IL-6 and others), not because of an increase in the concentration of antiinflammatory cytokines (IL-10).     

Immunotherapy of type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease in which the insulin-producing beta cells are destroyed. Diabetic patients manage their hyperglycemia by daily insulin injections. However, insulin therapy is by no means a cure. Accordingly, a significant effort has been ongoing to develop immunotherapies that effectively prevent and/or treat T1D in the clinic. This review focuses on antigen- and antibody-based immunotherapies and discusses the respective strengths and weaknesses of these approaches.     

The effect of adjunctive chlorhexidine mouthrinse on clinical parameters and gingival crevicular fluid cytokine levels in untreated plaque-associated gingivitis

Objective and design:  To examine the effectiveness of chlorhexidine mouthrinse (CHX) in addition to daily plaque control on gingival inflammation. Methods:  Fifty gingivitis patients were randomized to CHX or placebo groups. In addition to proper plaque control, CHX group rinsed with CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. Gingival crevicular fluid (GCF) samples were collected and clinical parameters including plaque index (PI), papillary bleeding index (PBI), calculus index and probing depth (PD) were recorded at baseline and repeated at 4 week. GCF IL-1α, IL-1β, IL-1Ra, and IL-8 levels were determined by ELISA. Results:  Whole mouth clinical parameters were significantly improved in both groups at 4 weeks. CHX group showed greater reduction in the mean PI scores than placebo at 4 weeks (p < 0.05). GCF IL-8 levels of anterior sites significantly reduced in CHX and placebo group at 4 weeks (p < 0.05). GCF IL-1α, IL-1β, IL-1Ra levels remained unchanged at 4 weeks in both groups. GCF cytokine levels of CHX group were similar to those of placebo at 4 weeks. Conclusions:  Within the limitations of this study, CHX mouthrinse as adjuncts to daily plaque control could be useful in management of plaque-associated gingivitis, although ineffective on GCF cytokine levels. Received 13 June 2008; returned for revision 9 July 2008; received from final revision 19 July 2008; accepted by C. Kasserra 19 August 2008     

Acute oxygen-ozone administration to rats protects the heart from ischemia reperfusion infarct

OBJECTIVE AND DESIGN: We tested here the effects of acute administration of an oxygen/ozone (O3) mixture on the myocardial tissue damage following an ischemic event.MATERIAL OR SUBJECTS: The study was done in Sprague-Dawley rats subjected to acute myocardial ischemia/reperfusion (I/R).TREATMENT: 100; 150; and 300 microg/kg oxygen/O3 mixture were insufflated intraperitoneally 1 h prior to I/R.METHODS: Myocardial infarct size measurement and immunhistochemistry or ELISA for nitrotyrosine, CD68, CD8,CD4 and caspase-3 were done.RESULTS: I/R produced a marked damage in the rat left ventricle with an infarct size as percentage of the area at risk (IS/ AR) of approximately 45 +/- 4% . Rats insufflated with a oxygen/O3 mixture showed a significant 2-h cardio-protection (e. g. infarct size over area at risk for the dose of 300 microg/kg was approximately 30 +/- 3%,) as compared with control rats (P <0.01). This effect was paralleled by a decrease in tissue levels of immunostaining for biomarkers of nitrosative stress (nitrotyrosine), inflammation (CD68) and immunity response (CD8 and CD4) between heart tissues from infarcted rats and infarcted O3 treated rats.CONCLUSIONS: These data indicate that the tissue and biochemical damages associated with myocardial ischemia/reperfusion can be counteracted by an acute O3 pretreatment.     

Bacterial DNA Induces the <Emphasis Type="Italic">Complement</Emphasis> System Activation in Serum and Ascitic Fluid from Patients with Advanced Cirrhosis

Translocation of intestinal bacteria to ascitic fluid is, probably, the first step in the development of spontaneous bacterial peritonitis in patients with cirrhosis. Proteins of the complement system are soluble mediators implicated in the host immune response to bacterial infections and its activation has been traditionally considered to be an endotoxin-induced phenomenon. The aim of this study was to compare the modulation of these proteins in response to the presence of bacterial DNA and/or endotoxin in patients with advanced cirrhosis and ascites in different clinical conditions. Groups I and II consisted of patients without/with bacterial DNA. Group III included patients with spontaneous bacterial peritonitis and Group IV with patients receiving norfloxacin as secondary long-term prophylaxis of spontaneous bacterial peritonitis. Serum and ascitic fluid levels of endotoxin and truncated residues of the complement system were measured by ELISA. The complement system is triggered in response to bacterial DNA, as evidenced by significantly increased levels of C3b, membrane attack complex, and C5a in patients from Groups II and III compared with patients without bacterial DNA (Group I) and those receiving norfloxacin (Group IV). Gram classification did not further differentiate the immune response between patients within groups II and III, even though endotoxin levels were, as expected, significantly higher in patients with bacterial DNA from gram-negative microorganisms. The complement protein activation observed in patients with bacterial DNA in blood and ascitic fluid is indistinguishable from that observed in patients with spontaneous bacterial peritonitis and may occur in an endotoxin-independent manner.     

Effects of Cognitive Adaptation on the Expectation-Burnout Relationship Among Nurses1

Burnout has traditionally been thought to result from unrealistically high expectations although research has provided only equivocal support. We explored the impact of cognitive adaptation disposition, including mastery, optimism, and self-esteem, on the expectations-burnout relationship. Nurses (N = 341) who were recruited at professional conferences completed questionnaires that assessed cognitive adaptation, burnout, and initial and current expectations of control. As expected, cognitive adaptation predicted fewer unmet expectations of control, which in turn predicted lower burnout. In addition, optimism and self-esteem predicted lower burnout regardless of perceived initial expectations, whereas mastery lessened the negative consequences of initially high expectations on burnout.     

Influence of mouse genetic background on wear particle-induced in vivo inflammatory osteolysis

OBJECTIVE AND DESIGN: Genetics may influence wear particle-induced inflammatory osteolysis after joint replacement. In the present work, mice with three different genetic backgrounds were used to test this hypothesis. TREATMENT: C57BL/6J, Balb/c and Kunming mouse were used. Each kind of mouse was divided into those receiving 30 mg UHMWPE particle implantation onto the calvariae and those receiving a sham operation. METHODS: Mice of each group were sacrificed one week after surgery. Calvariae were harvested for immunological assay of TNF-alpha and IL-1 beta secretion in supernatants of calvariae organ culture and histological analysis of calvarial sagittal suture osteolysis and osteoclastogenesis. RESULTS: Although UHMWPE particles induced obvious calvarial sagittal suture osteolysis and osteoclastogenesis in all strains as compared with their corresponding control mice, the most significant change was found in C57BL/6J mice, less severe in Balb/c mice and much less severe in Kunming mice. In agreement with pathological findings, UHMWPE particles induced the highest IL-1 beta secretion in C57BL/6J mice, compared with Balb/c and Kunming mice. However, no difference was observed concerning TNF-alpha secretion among these mice. CONCLUSION: Our data suggests that genetics had a significant influence on wear particle-induced inflammation, osteoclastogenesis and osteolysis. The influence of genetic background on implant life in patients with joint replacement warrants further investigation.     

In vitro effects of Helicobacter pylori-induced infection in gastric epithelial AGS cells on microglia-mediated toxicity in neuroblastoma SH-SY5Y cells

Objective:  To investigate the in vitro effects of H. pylori-conditioned medium (HCM) from gastric epithelial AGS cell cultures on microglia and neuronal cells. Material:  H. pylori, human gastric epithelial AGS cells, microglia-like BV-2 cells and human neuroblastoma SH-SY5Y cells. Treatment:  Treated AGS cells with H. pylori at ratios from 1:100 to 1:900 for 24 h. Cultured BV-2 cells and SH-SY5Y cells were treated with HCM from AGS cell cultures. Methods:  Cell viability was measured by a quantitative colorimetric assay with MTT. Nitric oxide (NO) was determined by using Griess reagent. IL-8 was measured by an enzyme-linked immunosorbent assay. Protein expressions were revealed by western blot analysis. Results:  H. pylori increased IL-8, NO, COX-2 and gp91^phox in AGS cell cultures. When BV-2 cells were cocultured with AGS cells, HCM increased COX-2, gp91^phox, iNOS and NO of BV-2 cells. HCM also enhanced the degradation of IκBα in BV-2 cells. HCM up-regulated expression of nNOS, COX-2, and gp91^phox of SH-SY5Y cells co-cultured with BV-2 cells. Particularly, the decrease of cell viability of SH-SY5Y induced by HCM was dependent on the presence of BV-2 cells. Conclusions:  H. pylori-induced infection induces microglia-mediated inflammation and neurotoxicity. The present results suggest that microglia play a critical role in HCM-induced toxicity of neuronal SH-SY5Y cells. Received 15 April 2008; returned for revision 10 May 2008; received from final revision 14 September 2008; accepted by M. Katori 18 September 2008     

Suramin inhibits macrophage activation by human group IIA phospholipase A2, but does not affect bactericidal activity of the enzyme

Objective:  Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A₂ (hsPLA₂GIIA). Materials and Methods:  The hsPLA₂GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA₂ GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. Results:  The hydrolytic activity of the hsPLA₂ GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA₂ GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA₂ GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 μM. Conclusions:  These results demonstrate that suramin selectively inhibits the activity of the hsPLA₂ GIIA against macrophages, whilst leaving the anti-bacterial function unchanged. Received 26 June 2008; returned for revision 8 August 2008; received from final revision 11 September 2008; accepted by J.Skotnicki 8 October 2008     

Abnormal cytokine production by bone marrow stromal cells of multiple myeloma patients in response to RPMI8226 myeloma cells

INTRODUCTION: Recent studies indicate that bone marrow stromal cells (BMSCs) derived from patients with multiple myeloma (MM) differ from those of healthy donors in their expression of extracellular matrix compounds and in cytokine production. It is not known whether these abnormalities are primary or are acquired by BMSCs on contact with MM cells. MATERIALS AND METHODS: Interleukin (IL)-6, IL-11, IL-10, and tumor necrosis factor (TNF)-alpha production by CD166+ mesenchymal BMSCs and the CD38+/CD138+ RPMI8226 myeloma cell line cultivated in vitro in monocultures or co-cultivated under cell-to-cell contact or non-contact conditions in the presence of a tissue culture insert were measured. Intracellular cytokines were measured by flow cytometry analysis as the percentage of cytokine-producing cells or by mean fluorescence intensity as the level of cytokine expression in cells. Additionally, ELISA was used to measure IL-6, soluble IL-6 receptor (sIL-6R), IL-11, IL-10, TNF-alpha, B-cell-activating factor of the TNF family (BAFF), hepatocyte growth factor (HGF), and osteopontin (OPN) production in the supernatants of the cultures and co-cultures. RESULTS: A higher ability of the BMSCs of MM patients than in controls was detected to produce IL-6, IL-10, TNF-alpha, OPN, and especially HGF and BAFF in response to the RPMI8226 cells. Moreover, the BMSCs of the MM patients significantly enhanced the production of sIL-6R by the RPMI8226 cells. DISCUSSION: Cytokines over-expressed by BMSCs of MM patients can function as growth factors for myeloma cells (IL-6, IL-10, HGF), migration stimulatory factors for tumor plasma cells (TNF-alpha, HGF), adhesion stimulatory factors (HGF, BAFF and OPN), stimulators of osteoclastogenesis (IL-6, TNF-alpha), and angiogenic factors (TNF-alpha). The results of this experiment strongly suggest that the BMSCs from MM patients differed in spontaneous and myeloma cell-induced production of cytokines, especially of HGF and BAFF, and these abnormalities were both primary and acquired by the BMSCs on contact with the MM cells. This in turn suggests the presence of an undefined, autocrine stimulation pathway resulting in a prolonged production of cytokines even in long-term cultures in vitro and in vivo. These abnormalities might provide optimal conditions for the proliferation and differentiation of residual tumor cells or their precursors in the affected bone marrow.