Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

The versatile role of the contact system in cardiovascular disease, inflammation, sepsis and cancer.

The human contact system consists of plasma proteins, which - after contact to foreign surfaces - are bound to them, thereby activating the zymogens of the system into enzymes. This activation mechanism gave the system its name - contact system. It is considered as a procoagulant and proinflammatory response mechanism, as activation finally leads to the generation of fibrin and bradykinin. To date, no physiological processes have been described that are mediated by contact activation. However, contact system factors play a pathophysiological role in numerous diseases, such as cardiovascular diseases, arthritis, colitis, sepsis, and cancer. Contact system factors are therefore an interesting target for new therapeutic options in different clinical conditions.

Kininogen supports inflammation and bacterial spreading during Streptococccus Pyogenes Sepsis.

BACKGROUND: High-molecular-weight kininogen is a cofactor of the human contact system, an inflammatory response mechanism that is activated during sepsis. It has been shown that high-molecular-weight kininogen contributes to endotoxemia, but is not critical for local host defense during pneumonia by Gram-negative bacteria. However, some important pathogens, such as Streptococcus pyogenes, can cleave kininogen by contact system activation. Whether kininogen causally affects antibacterial host defense in S. pyogenes infection, remains unknown. METHODS: Kininogen concentration was determined in course plasma samples from septic patients. mRNA expression and degradation of kininogen was determined in liver or plasma of septic mice. Kininogen was depleted in mice by treatment with selective kininogen directed antisense oligonucleotides (ASOs) or a scrambled control ASO for 3 weeks prior to infection. 24 h after infection, infection parameters were determined. FINDINGS: Data from human and mice samples indicate that kininogen is a positive acute phase protein. Lower kininogen concentration in plasma correlate with a higher APACHE II score in septic patients. We show that ASO-mediated depletion of kininogen in mice indeed restrains streptococcal spreading, reduces levels of proinflammatory cytokines such as IL-1ß and IFNγ, but increased intravascular tissue factor and fibrin deposition in kidneys of septic animals. INTERPRETATION: Mechanistically, kininogen depletion results in reduced plasma kallikrein levels and, during sepsis, in increased intravascular tissue factor that may reinforce immunothrombosis, and thus reduce streptococcal spreading. These novel findings point to an anticoagulant and profibrinolytic role of kininogens during streptococcal sepsis. FUNDING: Full details are provided in the Acknowledgements section.

The synthetic LPS binding peptide 19-2.5 interferes with clotting and prevents degradation of high molecular weight kininogen in plasma.

Sepsis and septic shock are life-threatening conditions and remain an important medical problem, emphasizing the need to identify novel therapeutic approaches. Coagulation dysfunction, hypotension, disturbed microcirculation and multiorgan failure occur frequently. These severe conditions result from an overwhelming inflammatory response, induced by pathogen and damage associated molecular patterns (PAMPs and DAMPs) released into the bloodstream. In the present study, we demonstrated that the synthetic Lipopolysaccharid (LPS)-binding peptide 19-2.5 interferes with the activation of the coagulation and contact system. Moreover, binding of LPS to high molecular weight kininogen (HK), one of the major LPS carrier in blood, could be prevented by the peptide. Thus, peptide 19-2.5 might represent a promising target in the treatment of endotoxemia and sepsis, not only by its anti-inflammatory potential, but also by the anticoagulant effect, together with its ability to prevent degradation of HK.

Streptococcus gallolyticus abrogates anti-carcinogenic properties of tannic acid on low-passage colorectal carcinomas.

The tannase-producing Gram-positive bacterial species Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathogen of the human gut and strongly associated with colorectal cancer (CRC). A unique feature of Sgg is its ability to degrade tannic acids (TA). TA constitute an important part of the human diet with known anti-tumorigenic properties. Here, we examined whether Sgg is able to protect tumor cells from the toxic effect of TA and thus drive tumorigenesis indirectly. Human CRC cell lines (n = 8) were treated with increasing concentrations of TA. We confirmed the cytotoxic activity of TA in a dose-dependent manner. In virtually all cell lines, viability decreased significantly (>60% inhibition). Moreover, pyrogallol, the degradation product of TA, had no effect on the tested cell lines. This suggests a specific effect of TA. Cytotoxicity was due to necrosis and induction of senescence in residual cells. Finally, when TA was degraded by Sgg, the cytotoxic effect could be abolished. Tumor cells even responded with boosted cell proliferation, highlighting the impact of Sgg on CRC progression. We here provide another piece of evidence for the active interplay between Sgg and cancer preventive components. These data will help to move forward in designing concepts for therapeutic and eventually also prophylactic approaches to combat gastrointestinal malignancies.

Prothrombotic and Proinflammatory Activities of the ß-Hemolytic Group B Streptococcal Pigment.

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1ß, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.

Adipose-derived mesenchymal stem cells release microvesicles with procoagulant activity.

Extracellular vesicles are produced by a number of different cell types, among them mesenchymal stromal/stem cells (MSC) of different sources. It has been shown that extracellular vesicles of MSC exert similar therapeutic effects as the cells themselves. Here, we isolated and characterized extracellular vesicles produced by adipose-derived MSC (adMSC) in vitro upon stimulation with the proinflammatory substances lipopolysaccharide (LPS) and tumor necrosis factor (TNF). We found that the number of vesicles produced by adMSC does not change upon stimulation of the cells with LPS and TNF. Furthermore, adMSC-derived extracellular vesicles exert procoagulant activity independent of previous stimulation with LPS or TNF. We found evidence that the vesicles induce coagulation via both the intrinsic and the extrinsic pathway of coagulation.

Deletion of the L-Lactate Dehydrogenase Gene ldh in Streptococcus pyogenes Leads to a Loss of SpeB Activity and a Hypovirulent Phenotype.

Streptococcus pyogenes uses lactic acid fermentation for the generation of ATP. Here, we analyzed the impact of a deletion of the L-lactate dehydrogenase gene ldh on the virulence of S. pyogenes M49. While the ldh deletion does not cause a general growth deficiency in laboratory media, the growth in human blood and plasma is significantly hampered. The ldh deletion strain is furthermore less virulent in a Galleria mellonella infection model. We show that the ldh deletion leads to a decrease in the activity of the cysteine protease SpeB, an important secreted virulence factor of S. pyogenes. The reduced SpeB activity is caused by a hampered autocatalytic activation of the SpeB zymogen into the mature SpeB. The missing SpeB activity furthermore leads to increased plasmin activation and a reduced activation of the contact system on the surface of S. pyogenes. All these effects can be reversed when ldh is reintroduced into the mutant via a plasmid. The results demonstrate a previously unappreciated role for LDH in modulation of SpeB maturation.

The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.