Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Determination of Interleukin-6 and Tumor Necrosis Factor-alpha concentrations in Iranian-Khorasanian patients with preeclampsia

BACKGROUND: Our objective was to determine the role of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha), markers of immune activation and endothelial dysfunction, in patients with preeclampsia. METHODS: Twenty four women with preeclampsia and eighteen antepartum normotensive pregnant women were recruited as controls. Serum levels of IL-6 and TNF-alpha were measured by enzyme-linked immunosorbent assay. We used independent-samples t test to assess the differences in the concentration of cytokines in preeclamptic patients and control subjects. RESULTS: IL-6 levels [mean (S.D.)] were significantly higher in preeclamptic women [5.8 (4.85) pg/ml] compared to normal pregnant women [3.01 (2.45) pg/ml] (p = 0.02). There was no significant change in concentration of TNF-alpha in preeclamptic women [53.8 (30.0) pg/ml] compared to normal pregnant women [51.9 (33.8) pg/ml] (p > 0.1). CONCLUSION: The results of this study show that IL-6 as a pro-inflammatory cytokine is present in higher concentration in women with preeclampsia. The study was undertaken in women with established preeclampsia and it is not possible to determine whether the increased concentration of IL-6 is a cause or consequence of the disease. Furthermore, these findings suggest that serum TNF-alpha level is not associated with preeclampsia

Naturally Occurring Lipid A Mutants in Neisseria meningitidis from Patients with Invasive Meningococcal Disease Are Associated with Reduced Coagulopathy

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA).FCT fellowship: (SFRH/BPD/92860/2013)

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