Inflammatory Fluid Research Articles

Bactericidal properties of group IIa secreted phospholipase A(2) against Pseudomonas aeruginosa clinical isolates.

It has been shown that human group IIa secreted phospholipase A(2) (sPLA(2)), found at high levels in inflammatory fluids, displays direct bactericidal properties against Gram-positive bacteria, while activity against Gram-negative bacteria requires the complement system or additional co-factors produced by neutrophils. Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen, is the most common Gram-negative rod found in cystic fibrosis lung infections, where it is associated with an inflammatory environment. Because murine intestinal group II sPLA(2) produced by Paneth cells has been shown to be directly bactericidal against Gram-negative bacteria, IIa sPLA(2) activity against P. aeruginosa clinical isolates was evaluated and provides the first evidence that the enzyme can be fully bactericidal in a concentration- and time-dependent manner against Gram-negative rods. Furthermore, it was demonstrated that these bactericidal properties were unaffected by high protein and salt concentrations, as observed in cystic fibrosis secretions, and that bacterial killing paralleled phospholipid hydrolysis. Finally, no cytotoxicity was observed when IIa sPLA(2) was incubated with human pulmonary cells, highlighting its potential use to synergize bactericidal antibiotics by promoting sublethal alterations of the bacterial cell wall.

The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer’s patches

Salmonella enterica serovar Typhimurium is an important cause of enteric infections in farm animals and it is one of the most frequent food borne infections worldwide. Serovar Typhimurium lacking the sopB gene is attenuated for induction of host inflammatory response and fluid accumulation into the intestinal lumen, which correlates with clinical diarrhea. SopB is an inositol phosphate phosphatase, but its exact role in the pathogenesis of salmonellosis is still unclear. We employed the bovine ileal ligated loop model to compare the tissue distribution of a sopB mutant and its wild type parent serovar Typhimurium. Sections of the Peyer’s patches were histologically processed and immuno-stained for detection of serovar Typhimurium. In addition, samples were processed for transmission electron microscopy, and the profile of expression of host chemokine and cytokine responses was assessed. Ultrastructurally both strains had the same ability to invade intestinal epithelial cells. No differences were detected in the tissue distribution of the sopB mutant and the wild type organism and both strains elicited the same profile of chemokines and pro-inflammatory cytokines. In conclusion, our results indicate that the attenuation of the sopB mutant is associated with pathogenic mechanisms other than invasion and distribution in host intestinal tissues.

Identification of an autoantigen on the surface of apoptotic human T cells as a new protein interacting with inflammatory group IIA phospholipase A2

AbstractOne of the most studied secreted phospholipases A2 (sPLA2), the group IIA sPLA2, is found at high levels in inflammatory fluids of patients with autoimmune diseases. A characteristic of group IIA sPLA2 is its preference for negatively charged phospholipids, which become exposed on the extracellular leaflet of apoptotic cell membranes. We recently showed that low molecular weight heparan sulfate proteoglycans (HSPGs) and uncharacterized detergent-insoluble binding site(s) contribute to the enhanced binding of human group IIA PLA2 (hGIIA) to apoptotic human T cells. Using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry we now identify vimentin as the major HSPG-independent binding protein of hGIIA on apoptotic primary T lymphocytes. Vimentin is partially exposed on the surface of apoptotic T cells and binds hGIIA via its rod domain in a calcium-independent manner. Studies with hGIIA mutants showed that specific motifs in the interfacial binding surface are involved in the interaction with vimentin. The sPLA2 inhibitor LY311727, but not heparin, inhibited this interaction. In contrast, heparin but not LY311727 abrogated the binding of hGIIA to cellular HSPGs. Importantly, vimentin does not inhibit the catalytic activity of hGIIA. Altogether, the results show that vimentin, in conjunction with HSPGs, contributes to the enhanced binding of hGIIA to apoptotic T cells.

Synovite granulomateuse après injection de hylane GF-20. À propos de deux cas

Intra-articular hyaluronan injections are used to treat osteo-arthritis of the knee. Acute painful swelling with a joint effusion develops locally after the injection in about 10% of cases but resolves spontaneously. Crystals are identifiable in some patients, but the mechanism in crystal-negative cases remains unknown. We report knee arthritis with inflammatory joint fluid free of organisms and crystals in two patients after Hylan GF-20 treatment for femorotibial osteo-arthritis. Synovial membrane histology disclosed granulomatous synovitis with epithelioid histiocytic and multinucleate giant cells but no visible foreign bodies. These two cases suggest that crystal-negative arthritis after Hylan GF-20 injection may be ascribable to granulomatous synovitis of the foreign-body giant-cell type.

Granulomatous synovitis after intraarticular Hylan GF-20. A report of two cases

Intraarticular hyaluronan injections are used to treat osteoarthritis of the knee. Acute painful swelling with a joint effusion develops locally after the injection in about 10% of cases but resolves spontaneously. Crystals are identifiable in some patients, but the mechanism in crystal-negative cases remains unknown. We report knee arthritis with inflammatory joint fluid free of organisms and crystals in two patients after Hylan GF-20 treatment for femorotibial osteoarthritis. Synovial membrane histology disclosed granulomatous synovitis with epithelioid histiocytic and multinucleate giant cells but no visible foreign bodies. These two cases suggest that crystal-negative arthritis after Hylan GF-20 injection may be ascribable to granulomatous synovitis of the foreign-body giant-cell type.

Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids.

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

Ultrastructural evidence of early endothelial damage in coronary arteries of rat cardiac allografts

Background: Events that occur early after transplantation, particularly immune recognition of allo-endothelium, initiate transplant vascular disease (TVD). Previous work suggests an important compromise of endothelial integrity as the allo-immune milieu evolves, although mechanisms by which integrity is altered remain unclear. Increased vascular permeability caused by endothelial damage may allow inflammatory cells, lipoproteins, other proteins, and plasma fluid to enter the sub-endothelial space, thereby contributing to the initiation of atherosclerosis. In this study, we examined endothelial integrity in coronary arteries and the proximal aorta after cardiac transplantation in rats. Methods: We used Lewis-to-Lewis and Lewis-to-F344 rat heterotopic cardiac transplant models. We studied the effects of cyclosporine (5mg/kg/day) therapy compared with saline-treated controls. En face silver nitrate staining was performed to demonstrate endothelial cell borders and gaps. We used scanning electron microscopy to extend silver nitrate findings and to further define the presence and nature of endothelial disruptions. We used transmission electron microscopy to further characterize immune cell identity and interaction with endothelium. Results: Syngrafts and cyclosporine-treated allografts showed normal-looking endothelium similar to that observed in arteries from native hearts. However, saline-treated allografts displayed progressive endothelial destruction, including large intercellular gaps, missing cells, and areas of bare extracellular matrix. Exfoliated surfaces were covered by platelets at various stages of adhesion, activation, and spreading. Similarly, we observed numerous leukocytes as either adherent to the endothelial lining or transmigrating into the sub-endothelial space. Cessation of cyclosporine therapy was associated with the development of similar abnormalities. Conclusions: Our findings indicate that, especially when immunosuppression is insufficient, early endothelial damage may promote vascular permeability and thereby initiate TVD.

Romboencefalitis: una forma de infección por Listeria monocytogenes en el sistema nervioso central

Listeria monocytogenes infections can involve the central nervous system in the form of a rhomboencephalitis. Three possible cases of rhomboencephalitis by Listeria monocytogenes are reported (2 females, aged 44 and 49 and a man of 36 years old). The three cases were preceded by an unspecific prodrome of headache, vertigo and fever in absence of a meningeal syndrome. The neurological stage was defined by the unilateral involvement of cranial nerves and the cerebellum and a clear inflammatory cerebrospinal fluid (CSF) with the presence of polymorphonuclear leukocytes, and normal glucose and protein levels. A magnetic nuclear resonance (MRI) showed the appearance of characteristic images, present in the bulboprotuberancial region. These images are one of the most constant features of this disease, reported in the literature. The early diagnosis of rhomboencephalitis was based on the clinical picture, the study of the CSF and the MRI, allowing the use of antimicrobials, prior to microbiological identification. Therefore, the risk of brain stem and cardiac complications of the disease is reduced.

The CD56Bright Subset of NK Cells Is Greatly Expanded IN Inflammatory Synovial Fluid and Tissue

The CD56Bright Subset of NK Cells Is Greatly Expanded IN Inflammatory Synovial Fluid and Tissue

Fluid extravasation during cardiopulmonary bypass in piglets--effects of hypothermia and different cooling protocols.

Hypothermic cardiopulmonary bypass (CPB) is associated with capillary fluid leak and edema generation which may be secondary to hemodilution, inflammation and hypothermia. We evaluated how hypothermia and different cooling strategies influenced the fluid extravasation rate during CPB. Fourteen piglets were given 60 min normothermic CPB, followed by randomization to two groups: 1: rapid cooling (RC-group) ( approximately 15 min to 28 degrees C); 2: slow cooling (SC-group) ( approximately 60 min to 28 degrees C). Ringer's solution was used as CPB prime and for fluid supplementation. Fluid input/losses, plasma volume, colloid osmotic pressures (plasma, interstitial fluid), hematocrit, serum-proteins and total tissue water (TTW) were measured and fluid extravasation rates calculated. Start of normothermic CPB resulted in a 25% hemodilution. During the first 5-10 min the fluid level of the reservoir fell markedly due to an intravascular volume loss necessitating fluid supplementation. Thereafter a steady state was reached with a constant fluid need of 0.14 +/- 0.04 ml kg-1 min-1. After start of cooling the fluid needs increased in the following 30 min to 0.91 +/- 0.11 ml kg-1 min-1 in the RC group (P < 0.001) and 0.63 +/- 0.10 ml kg-1 min-1 in the SC-group (P < 0.001) with no statistical between-group differences. Fluid extravasation rates after start of hypothermic CPB increased from 0.20 +/- 0.08 ml kg-1 min-1 to 0.71 +/- 0.13 (P < 0.01) and 0.62 +/- 0.13 ml kg-1 min-1 (P < 0.05) in the RC- and SC-groups, respectively, without any changes in degree of hemodilution. TTW increased in most tissues, whereas the intravascular albumin and protein masses remained constant with no between group differences. Hypothermia increased fluid extravasation during CPB independent of cooling strategy. Intravascular albumin and protein masses remained constant. Since inflammatory fluid leakage usually results in protein rich exudates, our data with no net protein leakage may indicate that mechanisms other than inflammation could contribute to fluid extravasation during hypothermic CPB.

Flory syndrome or lymphomonocytic/low-neutrophil oligoarthritis: a diagnosis or a prognosis?

Objective. – To individualize a new clinical entity of chronic arthritis and/or a factor indicating good prognosis of chronic arthritis. Methods. – We retrospectively studied 12 cases of monoarthritis or oligoarthritis that met none of the criteria sets for known diseases, even after more than 10 years of follow-up. Results. – Features in these 12 patients included recurrent effusions of inflammatory joint fluid consistently showing a predominance of lymphocytes and monocytes, long periods of remission separating the flares, absence of clinical or radiological joint lesions despite prolonged follow-up (10–40 years), an excellent response to joint aspiration and intraarticular glucocorticoid injection, unresponsiveness to second-line drugs, and young age at onset. Males and females were equally affected. Absolute lymphocyte/monocyte counts in joint fluid were similar in the 12 study patients and in 59 patients with rheumatoid arthritis, whereas absolute and differential neutrophil counts were significantly lower in the study patients. Conclusion. – The above-described features and excellent functional outcome suggest that this clinical pattern may deserve to be viewed as a separate entity. We suggest the name “idiopathic lymphomonocytic arthritis” or, since a low-neutrophil count in joint fluid was also a conspicuous finding, “Flory syndrome”, Flory being the name of the first patient in our series.

The possibility of detecting subgingival calculus by laser-fluorescence in vitro.

The aim of the present study was to investigate laser-fluorescence effects on the root surface. The possibility of detecting subgingival calculus should be evaluated to control complete calculus removal after scaling and root planing. Twenty freshly extracted human teeth, partially covered with calculus on the root surface, were fixed on a translation stage. The specimens were irradiated by light from a laser source (diode laser, <1 mW) with a wavelength of 655 nm using the Diagnodent trade mark -system. To simulate the conditions in a periodontal pocket during the measurement procedure, the teeth were covered by a layer of physiological saline solution or blood in order to determine the influence of different inflammatory fluids. The root surface was scanned on a line of about 1 cm in steps of 0.5 mm. The start and end was marked by a notch. The results of the laser-fluorescence detection were compared to clinical and histological findings. Clinically apparent calculus on the root surface was always accompanied with a statistically significant increase of laser-fluorescence values ( p < 0.05). These values were not influenced by the different fluids ( p < 0.05). The laser-fluorescence values were reproducible ( p > 0.05). After scaling, when no debris remained at the root surface, laser-fluorescence values decreased significantly ( p < 0.05). The histological findings supported the clinical results. In conclusion, the present study indicates that laser-fluorescence values on the root surface are strongly correlated to the presence of calculus. This fluorescence effect can basically be used to develop a new detecting system for subgingival calculus in periodontal pockets.

Optimizing response of the neonate and infant to cardiopulmonary bypass

Abstract Despite the ability to surgically correct complex defects in neonates and infants, limitations in outcome are sometimes encountered in relation to exposure to cardiopulmonary bypass (CPB). The deleterious effects of CPB in neonates are often pronounced because of their immature tissue/organ function and the disparity between the CPB circuit size and the patient. A variety of techniques that have been developed to prevent or lessen tissue edema, including miniaturization of the circuit and oxygenator, prime additives (eg, albumin, steroids), biocompatible circuitry, and variations in perfusion strategies, are discussed as are post-CPB strategies such as modified ultrafiltration, which removes inflammatory mediator-rich fluid from the patient and bypass circuit. With optimization of the response of the infant to our systems used during repair of their cardiac lesion, we will see significant improvement in surgical outcomes. Copyright 2003 Elsevier, Inc. All rights reserved.

Optimizing response of the neonate and infant to cardiopulmonary bypass.

Despite the ability to surgically correct complex defects in neonates and infants, limitations in outcome are sometimes encountered in relation to exposure to cardiopulmonary bypass (CPB). The deleterious effects of CPB in neonates are often pronounced because of their immature tissue/organ function and the disparity between the CPB circuit size and the patient. A variety of techniques that have been developed to prevent or lessen tissue edema, including miniaturization of the circuit and oxygenator, prime additives (eg, albumin, steroids), biocompatible circuitry, and variations in perfusion strategies, are discussed as are post-CPB strategies such as modified ultrafiltration, which removes inflammatory mediator-rich fluid from the patient and bypass circuit. With optimization of the response of the infant to our systems used during repair of their cardiac lesion, we will see significant improvement in surgical outcomes.

Mactinin: a modulator of the monocyte response to inflammation.

During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. alpha-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of alpha-actinin, is generated by the degradation of extracellular alpha-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5-10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.

Surface fissures in articular cartilage: effect of pathological changes in synovial fluid

Objective. A unified mathematical model of two different modes of inception of fissures at the surface of articular cartilage in healthy and pathological joints. Design. The superficial tangential zone of articular cartilage is modeled as a three-phase medium consisting of collagen fibers, matrix, and of infiltrated thin constituent of synovial fluid. Background. The author’s general mesomechanical concept is applied to the analysis of deterioration of articular cartilage. Methods. Theoretical analysis based on the results of the author’s preceding paper. Results. The presented analysis shows that superficial fissures in articular cartilage can also be caused by pathological thinning of synovial fluid. Conclusions. Whereas in healthy joints the probable cause of creation of fissures at the surface of cartilage was shown to be fast impact loading, in joints with inflammatory synovial fluid the fissures can be caused by plain walking. Relevance Appearance of surface fissures in articular cartilage is a serious, still not fully clarified problem that deserves attention.

The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

Urokinase is required for the formation of mactinin, an α-actinin fragment that promotes monocyte/macrophage maturation

We have previously shown that lysates from HL-60 myeloid leukemia cells or from peripheral blood monocytes are able to degrade α-actinin to form a 31-kDa amino-terminal fragment with monocyte/macrophage maturation promoting activity. In contrast, intact α-actinin, which is a 100-kDa actin-binding protein, has no differentiating activity. The aim of this study was to investigate the enzyme responsible for the degradation of α-actinin to form this fragment, named mactinin. The ability of cell lysates to degrade [ 125I]α-actinin in the presence of various enzyme inhibitors, including inhibitors of metalloproteinases, cysteine proteinases, and serine proteases, was measured. Phenylmethylsulfonyl fluoride (PMSF) was the only inhibitor able to prevent formation of mactinin by cell lysate degradation of α-actinin, suggesting that a serine protease is responsible for the digestion. Of the various serine proteases tested (thrombin, plasmin, and urokinase), only urokinase was able to produce a 31-kDa band. The urokinase-generated 31-kDa band promoted maturation in HL-60 cells. Amiloride, a specific inhibitor of urokinase, inhibited production of the 31-kDa α-actinin fragment by HL-60 cell lysates. For in vivo tests, inflammatory fluid (from bronchoalvelolar lavage) was collected from uPA (urokinase) knockout mice and their wild-type counterparts after intratracheal challenge with Pneumocystis carinii. Although most (6 of 8) wild-type mice had mactinin in their inflammatory fluid samples, none (0 of 8) of the uPA knockout mice had mactinin present ( P<0.01). These results demonstrate that urokinase is necessary and sufficient for the formation of the monocyte/macrophage maturation promoting fragment, mactinin, in vitro and in vivo. These findings support the role of urokinase in the regulation of monocyte/macrophage functions, such as that occurring in inflammatory reactions.

Penetration of telithromycin (HMR 3647), a new ketolide antimicrobial, into inflammatory blister fluid following oral administration.

The penetration of telithromycin (HMR 3647), a novel ketolide antimicrobial, has been assessed in an open, single-dose study in eight healthy male subjects. Following a single, oral, 600 mg dose the mean ratio of the concentration of telithromycin in blister fluid over plasma was 1.38. Significant blister fluid concentrations were maintained up to 24 h post-dose. These results indicate that telithromycin penetrates well into inflammatory extracellular fluid, achieving high and sustained concentrations in this medium.

Myeloperoxidase produces nitrating oxidants in vivo.

Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.