Characterization of Non-canonical Way in Inflammasome Monocytes of Patients With Severe Sepsis

Study Overview
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Brief Summary
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Activation of caspase-4 and human caspase-5 (orthologs of caspase-11 in mice) in innate immune cells.

Detailed Description
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Lipopolysaccharide (LPS) of the wall of Gram-negative bacteria is one of pathogen associated molecular patterns (PAMPs), which are recognized by cells of the innate immune system via Toll-like receptor 4 (TLR4) . The LPS / TLR4 interaction induces the secretion of a variety of proinflammatory cytokines. Among them, interleukin-1β (IL-1β) is a major cytokine, and alterations to its secretion has been associated with various diseases, such as periodic syndrome associated cryopyrin (CAPS), gout, rheumatoid arthritis or multiple sclerosis. The release of IL-1β is controlled by a molecular platform protein, known as the inflammasome name. The canonical protein (that is to say conserved in evolution) of the inflammasome NLRP3 are NLRC4 and that engages the ASC adapter protein to activate caspase-1, which promotes the cleavage of interleukin IL-1β. Gram-negative bacteria such as Escherichia coli cause non-canonical activation of caspase-1, caspase-11In involving more NLRP3 and AUC. In mouse, caspase-11 is essential to the immune response to gram-negative bacteria, but bacterial PAMPs that are responsible for triggering of the non-canonical inflammasome remain to be identified. It has recently been shown that caspase-11 is involved in the death of the mice subjected to septic shock. The mouse model requires caspase-11-dependent mechanism, caspase-independent -1 since the CASP1 - / - mouses are also susceptible to LPS as wild type mice. A key question to be answered is whether LPS triggers similar events in human cells. If this is the case, this knowledge may be useful in drug development for treatment of sepsis.

Official Title
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Characterization of Non-canonical Way in Inflammasome Monocytes of Patients With Severe Sepsis

Conditions
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Severe Sepsis

Intervention / Treatment
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* Biological: blood samples


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: patients with sepsis severe (bacterial infection associated with deterioration of at least one vital function) defined according to the criteria consensus conference patients whose diagnosis is made within 24 hours of the opening hours of Pasteur Research Laboratory patients with arterial catheter (not to impose an additional puncture to the patient) will be recruited in the intensive care unit of the Hospital St. Joseph. Arterial blood (20 ml) will be charged within 24 hours of the onset of severe sepsis criteria, during working hours of the laboratory of the Pasteur Institute, using vacuum tubes under heparin (Vacutainer), sent by a courier within 2 hours following the removal (to ensure that the cells are sufficiently fresh during processing in the laboratory) Exclusion Criteria: none
Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| patients with severe sepsis<br>blood samples from patients with severe sepsis | Biological: blood samples<br>* Isolation and stimulation of monocytes, preparation of cell lysates, measurement of cytokine production, Western blot<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Characterize the expression of caspase-4 and -5 in by measuring the production of cytokines: IL-1β concentrations and IL-1α will determined by ELISA in the cell supernatant will be collected from monocytes stimulated or not |  | Two hours after blood collection : isolation and stimulation of monocytes than preparation of cell lysates | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Correlate the activation of caspases-4 and -5 with IL-1β production with (canonical inflammasome) and IL-1α (Non-canonical inflammasome) measured by Western blot |  | Two hours after blood collection : isolation and stimulation of monocytes than preparation of cell lysates | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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lipopolysaccharide, sepsis, caspase-4 and -5, monocytes, IL-1β and IL-1α