Thrombocytopenia in the experimental leptospirosis of guinea pig is not related to disseminated intravascular coagulation

BACKGROUND: Thrombocytopenia is commonly observed in severe leptospirosis. However, previous studies on coagulation alterations during leptospirosis resulted in inconsistent conclusions. Some findings showed that the prominent levels of thrombocytopenia observed in severe leptospirosis did not reflect the occurrence of disseminated intravascular coagulation (DIC) syndrome, while the others reached the conclusion that the hemorrhages observed in leptospirosis were due to DIC. The aim of this study is to elucidate whether DIC is an important feature of leptospirosis. METHODS: The leptospirosis model of guinea pig was established by intraperitoneal inoculation of Leptospira interrogans strain Lai. Hematoxylin and eosin (HE) staining, electron microscopy and immunohistochemistry staining were used to detect the pathologic changes. Platelet thrombus or fibrin thrombus was detected by HE, Martius Scarlet Blue (MSB) staining and electron microscopy. Hemostatic molecular markers such as 11-dehydrogenate thromboxane B2 (11-DH-TXB2), thrombomodulin (TM), thrombin-antithrombin III complex (TAT), D-Dimer and fibrin (ogen) degradation products (FDPs) in the plasma were examined by quantitative enzyme-linked immunosorbent assay (ELISA) to evaluate the hematological coagulative alterations in leptospirosis models. RESULTS: Pulmonary hemorrhage appeared in the model guinea pig 24 hours after leptospires intraperitoneal inoculation, progressing to a peak at 96 hours after the infection. Leptospires were detected 24 hours post-inoculation in the liver, 48 hours in the lung and 72 hours in the kidney by immunohistochemistry staining. Spiral form of the bacteria was initially observed in the liver, lung and kidney suggestive of intact leptospires, granular form of leptospires was seen as the severity increased. Platelet aggregation in hepatic sinusoid as well as phagocytosis of erythrocytes and platelets by Kupffer cells were both observed. Neither platelet thrombus nor fibrin thrombus was found in the liver, lung or kidney via morphological observation. Thrombocytopenia was observed in all infected guinea pigs of our experimental leptospirosis study. Analysis of hematologic molecular markers showed that 11-DH-TXB2 and TM in the plasma were elevated significantly. TAT that reflects the thrombin activation had a trend of decline after infection. Although D-dimer and FDPs increased statistically, the increasing may not bear clinical significance. CONCLUSION: Pathologic and hematological studies for experimental leptospirosis of guinea pig indicated that the thrombocytopenia found in guinea pigs did not correlate with the occurrence of DIC. The platelet aggregation and Kupffer cells phagocytosis might be the potential causes of thrombocytopenia in severe leptospirosis

Alterations in the human lung proteome with lipopolysaccharide

<p>Abstract</p> <p>Background</p> <p>Recombinant human activated protein C (rhAPC) is associated with improved survival in high-risk patients with severe sepsis; however, the effects of both lipopolysaccharide (LPS) and rhAPC on the bronchoalveolar lavage fluid (BALF) proteome are unknown.</p> <p>Methods</p> <p>Using differential in gel electrophoresis (DIGE) we identified changes in the BALF proteome from 10 healthy volunteers given intrapulmonary LPS in one lobe and saline in another lobe. Subjects were randomized to pretreatment with saline or rhAPC.</p> <p>Results</p> <p>An average of 255 protein spots were detected in each proteome. We found 31 spots corresponding to 8 proteins that displayed abundance increased or decreased at least 2-fold after LPS. Proteins that decreased after LPS included surfactant protein A, immunoglobulin J chain, fibrinogen-γ, α₁-antitrypsin, immunoglobulin, and α₂-HS-glycoprotein. Haptoglobin increased after LPS-treatment. Treatment with rhAPC was associated with a larger relative decrease in immunoglobulin J chain, fibrinogen-γ, α₁-antitrypsin, and α₂-HS-glycoprotein.</p> <p>Conclusion</p> <p>Intrapulmonary LPS was associated with specific protein changes suggesting that the lung response to LPS is more than just a loss of integrity in the alveolar epithelial barrier; however, pretreatment with rhAPC resulted in minor changes in relative BALF protein abundance consistent with its lack of affect in ALI and milder forms of sepsis.</p

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system

Systemic versus localized coagulation activation contributing to organ failure in critically ill patients

In the pathogenesis of sepsis, inflammation and coagulation play a pivotal role. Increasing evidence points to an extensive cross-talk between these two systems, whereby inflammation not only leads to activation of coagulation but coagulation also considerably affects inflammatory activity. The intricate relationship between inflammation and coagulation may not only be relevant for vascular atherothrombotic disease in general but has in certain clinical settings considerable consequences, for example in the pathogenesis of microvascular failure and subsequent multiple organ failure, as a result of severe infection and the associated systemic inflammatory response. Molecular pathways that contribute to inflammation-induced activation of coagulation have been precisely identified. Pro-inflammatory cytokines and other mediators are capable of activating the coagulation system and downregulating important physiological anticoagulant pathways. Activation of the coagulation system and ensuing thrombin generation is dependent on an interleukin-6-induced expression of tissue factor on activated mononuclear cells and endothelial cells and is insufficiently counteracted by physiological anticoagulant mechanisms and endogenous fibrinolysis. Interestingly, apart from the overall systemic responses, a differential local response in various vascular beds related to specific organs may occur

Tissue Factor–Dependent Chemokine Production Aggravates Experimental Colitis

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction–dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage

Correlation of protease-activated receptor-2 expression and synovitis in rheumatoid and osteoarthritis

Protease-activated receptor-2 (PAR-2) is known to be pro-inflammatory and increasing evidence points to an inflammatory component in osteoarthritis. This investigation examined the relationship between synovitis and PAR-2 expression, histological and immunohistochemical analysis being performed on synovial samples obtained from OA and RA patients, along with non-arthritic samples obtained by post mortem (PM). Samples were also analysed for PAR-4 expression, this receptor also having putative pro-inflammatory roles. Analysis involved comparison of inflammatory indices (synovial thickness and monocyte infiltration) with expression of PAR-2 and PAR-4. Synovial explants were also analysed for TNFα generation in the presence of a PAR-2 antagonist (ENMD-1068) or vehicle. OA synovia showed heterogeneity of inflammatory indicators, some samples overlapping with those from the RA cohort whilst others appeared similar to the PM cohort. PAR-2 expression, both in the lining layer and the interstitium, correlated strongly and significantly with synovial thickness (r = 0.91) and monocyte infiltration (r = 0.83), respectively (P &#60; 0.001 in both cases), and this remains significant on individual cohort analysis. PAR-2 was co-localised to CD3 and CD68 cells in RA and OA synovium as well as fibroblasts derived from these synovia. PAR-4 was also expressed, but the relationship with inflammatory indicators was substantially weaker. Inflammatory indicators in OA synovia showed considerable variability, but correlated strongly with PAR-2 expression, suggesting PAR-2 upregulation in synovitis. Heterogeneity of inflammatory indicators was paralleled by wide variation in TNFα generation between samples. Secretion of this cytokine was dose-dependently inhibited by ENMD-1068, providing evidence of a functional role for PAR-2 in promoting synovitis

FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4.

Toll-like receptor 4 (Tlr4) has a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a Tlr4-induced gene. Here we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide. FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of Tlr4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling