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The intestinal microbiota is increasingly recognized as a major modulator of health and disease (1). The dramatic explosion of genetic tools and metagenomic techniques in the last decade allowed to characterize the composition of the microbiome from several areas of the body. In addition, this knowledge increasingly enables scientists to link composition and changes with human health and more importantly with various disorders far beyond the gastrointestinal tract (2,3).

Several members of the neuropeptide family exert chemotactic actions on blood monocytes consistent with neurogenic inflammation. Furthermore, chromogranin A (CgA) containing Alzheimer plaques are characterized by extensive microglia activation and such activation induces neuronal damage. We therefore hypothesized that the catecholamine release inhibitory peptide catestatin (hCgA(352-372)) would induce directed monocyte migration. We demonstrate that catestatin dose-dependently stimulates chemotaxis of human peripheral blood monocytes, exhibiting its maximal effect at a concentration of 1 nM comparable to the established chemoattractant formylated peptide Met-Leu-Phe (fMLP). The naturally occurring catestatin variants differed in their chemotactic property insofar as that the Pro370Leu variant was even more potent than wild type, whereas the Gly364Ser variant was less effective. Specificity of this effect was shown by inhibition of catestatin-induced chemotaxis by a specific neutralizing antibody. In addition, catestatin mediated effect was blocked by dimethylsphingosine and treatment with endothelial differentiation gene (Edg)-1 and Edg-3 antisense RNA as well as by incubation with pertussis toxin and genistein indicating involvement of tyrosine kinase receptor-, G-protein- and sphingosine-1-phosphate signaling. Catestatin also stimulated Akt- and extracellular signal related kinase (ERK)-phosphorylation and catestatin-induced chemotaxis was blocked by blockers of phosphoinositide-3 (PI-3) kinase and nitric oxide as well as by inhibition of the mitogen-activated protein kinases (MAPK) system indicating involvement of these signal transduction pathways. In summary, our data indicate that catestatin induces monocyte chemotaxis by activation of a variety of signal transduction pathways suggesting a role of this peptide as an inflammatory cytokine.

Tissue concentrations of amphotericin B were determined in autopsy material of patients who had been treated with liposomal amphotericin B or amphotericin B colloidal dispersion (colloidal amphotericin B) for suspected or proven invasive fungal infection.Amphotericin B tissue levels were measured in liver, spleen, lung, kidney, and myocardial and brain tissue of 20 patients who had been treated with lipid-formulated amphotericin B, before they died from multi-organ failure. Seven patients had been treated with liposomal amphotericin B (AmBisome) and thirteen with colloidal amphotericin B (Amphocil). Tissue samples were obtained during routine autopsy, homogenized and extracted with methanol. Amphotericin B concentrations were measured using HPLC after purification by solid phase extraction.The highest amphotericin B levels were found in liver and spleen, followed by kidney, lung, myocardium and brain. In the lung higher amphotericin B concentrations were found after treatment with amphotericin B colloidal dispersion than after therapy with liposomal amphotericin B.The choice of lipid formulation may influence amphotericin B penetration into the lung.

Chronic inflammation is characterized by tissue infiltration with monocytes/macrophages, which possess broad proinflammatory, destructive, and remodeling capacities. Elevated levels of osteoprotegerin, an important regulator of differentiation and activation of osteoclasts that also affects different cells of the immune system, were found in the serum of patients with chronic inflammatory diseases. The study of whether osteoprotegerin affects monocyte locomotion in vitro and the possible mechanisms and pathways involved was investigated using Boyden microchemotaxis chambers and Western blot analyses. Osteoprotegerin significantly stimulated monocyte chemotaxis, whereas preincubation of monocytes with osteoprotegerin inhibited monocyte migration toward optimal concentrations of regulated upon activation normal T cell expressed and secreted, monocyte chemotactic protein -1, and procalcitonin. The effects of osteoprotegerin were abolished by pretreating cells with heparinase I and chondroitinase or antibodies against the ectodomain of syndecan-1. Osteoprotegerin signaling was shown to involve protein kinase C, phosphatidylinositol 3-kinase/Akt, and tyrosine kinase. Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.

Increasing evidence shows the importance of platelet-endothelial cell interactions in the progression of atherosclerosis. Platelets contribute to coronary events both as major components of thrombi and as a triggering factor in inflammation that leads to plaque vulnerability. Recent data suggest that statins, besides their lipid-lowering properties, exert pleiotropic effects that may be beneficial in atherosclerosis. Whether activated platelets influence cyclooxygenase-2 (COX-2) expression in human umbilical vein endothelial cells (HUVEC), the effect of atorvastatin, and possible mechanisms were investigated. COX-2 gene expression in HUVEC was studied using real-time polymerase chain reaction. CD40 ligand surface expression of platelets was tested by fluorescence-activated cell sorting analyses. Activated platelets significantly up-regulated COX-2 gene expression in HUVEC. Co-incubation of platelets with atorvastatin was shown to reverse this up-regulation via reduction of CD40 ligand surface expression on platelets. Data suggest that atorvastatin influences CD40-CD40-ligand-dependent platelet-endothelial interaction and that this influence affects platelet-induced COX-2 expression in HUVEC.

Activation of neuropeptide receptors on leukocytes induces chemotaxis. We determined in Boyden chambers with micropore filters, whether in human monocytes and lymphocytes this migratory response is heparan sulfate proteoglycan (HSPG) dependent. Chemotaxis toward calcitonin gene-related peptide, secretoneurin, vasoactive intestinal peptide (VIP), and substance P (SP) was abolished by removal of heparan sulfate side chains from cell surface proteoglycans or by addition of anti-syndecan-4 antibodies. Inhibition of neuropeptide-induced chemotaxis by dimethyl sphingosine (DMS), an inhibitor of sphingosine kinase, indicates transactivation of the sphingosine-1-phosphate chemotaxis pathway which was previously identified as being syndecan-4-related. Data suggest that HSPGs are involved in neuropeptide-induced chemotaxis of leukocytes.

Propanolol and metoprolol exert adrenoceptor-independent effects including scavenging of free radicals and inhibition of protein kinase C leading to inhibition of leukocyte migration and radical release as a consequence. Whether topically used metipranolol and timolol exert such effects is unknown. Neutrophil chemotaxis was tested using modified Boyden microchemotaxis chambers. Respiratory burst activity of neutrophils was detected fluorometrically. Radical scavenging properties were tested using 2′,7′-dichlorofluorescein diacetate. Metipranolol and timolol inhibited neutrophil chemotaxis at doses in the micromolar range, oxygen free radical production triggered with formyl-Met-Leu-Phe was inhibited at higher concentration. Protein kinase C involvement, suggested to trigger free radical production with phorbol myristate acetate, was antagonized. A direct radical scavenging effect of the β-blockers was also seen. Inhibition of neutrophil chemotaxis and free radical production is a novel mode of action of metipranolol and timolol that may be relevant for beneficial effects in the topical treatment of eye disease.

Heat waves result in excess deaths, excess emergency department visits, and intensive care unit admissions for heat stroke. We describe the clinical features and 3-month outcome of a patient with near-fatal heat stroke, admitted to our intensive care unit in July, 2001. After heavily working for hours at a construction site during a heat wave, the 28-year-old male presented with 41.4 degrees C body temperature and multiorgan failure, consisting of neurological impairment, rhabdomyolysis, acute renal failure, disseminated intravascular coagulation, and acute respiratory distress syndrome (ARDS). In the first week there was no evidence of infection. Treatment included cooling, aggressive volume resuscitation, administration of antithrombin-III concentrates and steroids. The patient survived and recovered normal neurological, renal, respiratory and haematological function, and no disability persisted. This case illustrates survival and complete recovery after multiorgan failure in heat stroke with vigorous intensive care. Treatment with antithrombin and steroids and may well have contributed to the favourable outcome. Correction of reduced antithrombin III levels to supranormal by therapeutic administration of antithrombin III concentrate in disseminated intravascular coagulation of heat stroke was not associated with any bleeding complications.

Statins are thought to play a role in directly affecting immune and mesenchymal cells. Since cerivastatin's pleiotropic effects are poorly investigated, we were interested to find out whether this drug can modulate leukocyte and vessel wall cell functions. Leukocyte migration was tested in modified Boyden microchemotaxis chambers and oxygen radical production was measured fluorometrically. Transendothelial migration experiments were performed with human umbilical vein endothelial cells and neutrophils. Neutrophil, monocyte, and vascular smooth muscle cell caspase-3 activity and annexin-V binding were quantified by FIENA and FACS, respectively. Cerivastatin [10 pM to 100 μM] decreased leukocyte chemotaxis towards interleukin-8 or RANTES. Migration of cells was completely restored by addition of mevalonic acid. In neutrophils, cerivastatin [100 μM] reduced transendothelial migration, whereas treatment of endothelial cells failed to affect transmigration. Neutrophil respiratory burst activity was unaffected by cerivastatin. At concentrations of 10 nM or higher, cerivastatin increased the rate of apoptosis in phagocytes and smooth muscle cells. Results show that cerivastatin is able to inhibit leukocyte chemotaxis, and that cerivastatin induces neutrophil, monocyte, and smooth muscle cell apoptosis. The drug's impact on transendothelial migration is due to its effects on neutrophils. In addition to its lipid-lowering effects, pharmacological properties of cerivastatin may include modulatory actions in leukocytes and mesenchymal cells.

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Cathelicidins are mammalian proteins containing a C-terminal cationic antimicrobial domain. Porcine PR-39 cathelicidin affects leukocyte biology. Mechanisms of action may involve alteration of heparan sulfate proteoglycan-dependent functions in inflammatory cells. It was tested whether PR-39 affects human neutrophil migration and if such effects involve heparan sulphate proteoglycans. Neutrophils were from forearm venous blood of healthy donors. Migration was tested in modified Boyden chamber assays. Involvement of heparan sulfate proteoglycans was tested by their chemical modification and by the use of specific antibodies. PR-39 induced migration in neutrophils in a concentration dependent manner. Modification of heparan sulfate proteoglycans with sodium chlorate inhibited migration whereas chemotaxis toward the chemoattractant formyl-Met-Leu-Phe was not affected. Removal of heparan sulfates or chondroitin sulfates from the surface of neutrophils by heparinase or chondroitinase inhibited migration toward PR-39. In conclusion, antimicrobial PR-39 stimulates human neutrophil chemotaxis in a heparan sulfate proteoglycan-dependent manner. Involvment of syndecans is likely as both heparinase and chondroitinase were abrogating. Data suggest active participation of heparan sulfate proteoglycans of neutrophils in cathelicidin peptide-mediated regulation of the antimicrobial host defense.

After an ischemic event vascular growth factors are involved in regulating leukocyte infiltration in inflammatory processes. This study focused on effects of 2 other angiogenic growth factors, angiopoietin-1 and angiopoietin-2, on human neutrophils and on the involvement of the angiopoietin receptor Tie-2.Neutrophils were from venous blood of healthy donors and cell migration was studied by micropore filter assays. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA and fluorescence-activated cell-sorter scanner (FACS) analysis. Signaling mechanisms required for angiopoietin-dependent effects were tested functionally by using signaling enzyme blockers.The angiopoietins were chemotactic for neutrophils. They showed antagonistic effects on each other and both inhibited VEGF-directed migration of neutrophils. The effects of both angiopoietins were Tie-2 dependent. Tie-2 receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by Tie-2 receptor mRNA expression as demonstrated by reverse transcriptase PCR.Data suggest that a Tie-2 receptor is expressed by human neutrophils whose active site ligation with either angiopoietin-1 or angiopoietin-2 exerts migratory effects on the one hand and arrests VEGF-mediated chemotaxis on the other. These effects suggest a role of angiopoietins in modulating neutrophilic inflammation.

Accumulation of inflammatory mononuclear phagocytes in Alzheimer's senile plaques, a hallmark of the innate immune response to beta-amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimer's disease. Phagocytes migrate toward amyloid beta-protein involving formyl peptide receptor like-1-dependent signaling. Using human peripheral blood monocytes in Boyden chamber micropore filter assays, we show that the amyloid beta-protein- and amyloid beta-precursor protein-induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid beta-protein stimulated in monocytes the gene expression for sphingosine-1-phosphate receptors 2 and 5, but not 1, 3, and 4. FTY720 that acts as a sphingosine-1-phosphate receptor agonist after endogenous phosphorylation by sphingosine kinase, as well as various neuropeptides that are known to be monocyte chemoattractants, dose-dependently inhibited amyloid beta-protein-induced migration. These data demonstrate that the migratory effects of beta-amyloid in human monocytes involve spingosine-1-phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high beta-amyloid concentrations, interference with the amyloid beta-protein-dependent sphingosine-1-phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in beta-amyloid-related inflammatory diseases.

Pharmacokinetics of amphotericin B lipid complex (ABLC) was determined in two critically ill patients requiring continuous veno-venous haemofiltration (CVVH) because of acute renal failure. ABLC was administered at a mean daily dose of 4.94 mg/kg for suspected invasive mycosis. Mean C max was 0.56 μg/ml, the mean AUC 0−24 h was 7.46 mg h/l, V ss 9.13 l/kg, and t 1/2 was 13.21 h. The haemofilter clearance accounted about 20% of the total ABLC clearance. In one patient sampling was repeated after CVVH had been discontinued. The concentration–time profiles were very similar on and off haemofiltration. Data on our two patients suggest, that pharmacokinetics of ABLC is not significantly affected by CVVH and that ABLC can be administered at the standard doses during CVVH.

The neuropeptide substance P (SP) can modulate a number of immunological functions in vitro and in vivo. Here, we investigated if SP boosts migration and cytotoxicity of natural killer cells, thus providing a further link between "innate immunity" and neurogenic inflammatory processes like asthma bronchiale. We demonstrate a dose-dependent effect of SP on natural killer cell migration with a maximal response at 10(-8) M SP. SP was shown to stimulate unstimulated as well as interleukin-2 (IL-2)-activated natural killer cells. Stimulation of natural killer cell migration was neurokinin-1 receptor dependent. Furthermore, mRNA encoding the neurokinin-1 receptor was demonstrated as being present in natural killer cells using RT-PCR while mRNA of the neurokinin-2 receptor was not detectable. Additionally, SP seems to influence specific cytotoxicity against Raji and K567 effector cells by a receptor-independent mechanism. In conclusion, our data indicate that functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity.

Background: Eosinophil infiltration is a characteristic feature of allergic inflammation. Allergic responses are associated with local activation of the coagulation pathway and accumulation of fibrin. Objective: We tested whether protein C and activated protein C (APC), which are endogenous anti-inflammatory coagulation inhibitors, affect eosinophil function. Methods: Eosinophils were from venous blood of healthy donors. Cell migration and apoptosis were studied by using micropore filter assays and fluorometry, respectively. Receptor expression was investigated by means of RT-PCR and SDS-PAGE of immunoprecipitated protein. Results: Protein C and APC had no significant chemotactic effects on eosinophils. Eosinophils pretreated with protein C or APC showed significantly reduced migration toward chemoattractants. No effect of either protein C preparation was seen in eosinophil apoptosis assays. The inhibiting effect on migration was reversed by an antibody against the endothelial protein C receptor (EPCR). Synthesis of EPCR by eosinophils is suggested by demonstration of receptor mRNA expression and detection of metabolically labeled receptor protein. Conclusions: Data suggest that an EPCR is expressed by eosinophils whose activation with protein C or APC arrests directed migration. Protein C-affected eosinophil chemotaxis is a novel thrombin-independent component of the protein C pathway. (J Allergy Clin Immunol 2003;112:375-81.)

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase–polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti–endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation–blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C–based treatment of severe sepsis.