Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p < 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies

The complementarity-determining region sequences in IgY antivenom hypervariable regions

The data presented in this article are related to the research article entitled ''Development of IgY antibodies against anti-snake toxins endowed with highly lethal neutralizing activity'' (da Rocha et al., 2017) [1]. Complementarity-determining region (CDR) sequences are variable antibody (Ab) sequences that respond with specificity, duration and strength to identify and bind to antigen (Ag) epitopes. B lymphocytes isolated from hens immunized with Bitis arietans (Ba) and anti-Crotalus durissus terrificus (Cdt) venoms and expressing high specificity, affinity and toxicity neutralizing antibody titers were used as DNA sources. The VLF1, CDR1, CDR2, VLR1 and CDR3 sequences were validated by BLASTp, and values corresponding to IgY VL and VH anti-Ba or anti-Cdt venoms were identified, registered [Gallus gallus IgY Fv Light chain (GU815099)/Gallus gallus IgY Fv Heavy chain (GU815098)] and used for molecular modeling of IgY scFv anti-Ba. The resulting CDR1, CDR2 and CDR3 sequences were combined to construct the three - dimensional structure of the Ab paratope. Keywords: PCR, Sequencing, Modeling of biomolecule

New Insights into Immunopathology Associated to <i>Bothrops lanceolatus</i> Snake Envenomation: Focus on PLA₂ Toxin

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient’s prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals

Terminal Complement Complex (TCC) generation induced by the <i>Premolis semirufa</i>’<i>s</i> bristles extract.

<p>Samples of NHS (50 μL) were incubated with the bristles extract (175 μg/mL) or PBS, in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), at 37°C for 30 min, and SC5b-9 complex present in human serum samples was measured using the MicroVue SC5b-9 Plus EIA Kit. Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of SC5b-9 complex per mL of human serum (ng/mL) ± SD. (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the buffer and between the treatments.</p

Proteolytic action of Ps82 on purified human C components C3, C4 and C5.

<p>Samples of 0.1 μg of Ps82 were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) <b>[A]</b>, human C4 (3 μg) <b>[B]</b> and human C5 (3 μg) <b>[C]</b> at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1^st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2^nd lanes: incubation of purified components with Ps82; 3^rd lanes: incubation of the mixture with PMSF and 4^th lanes: incubation of the mixture with Phenanthroline. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.</p

Proteolytic action of the <i>Premolis semirufa’s</i> bristles extract on purified human C-components C3, C4 and C5.

<p>Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) <b>[A]</b>, human C4 (3 μg) <b>[B]</b> and human C5 (3 μg) <b>[C]</b> at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1^st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2^nd lanes: incubation of purified components with the extract; 3^rd lanes: incubation of the mixture with PMSF; 4^th lanes: incubation of the mixture with Phenanthroline and 5^th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.</p

Chromatography of the <i>Premolis semirufa’s</i> bristles extract and isolation of the protease responsible for complement activation.

<p><b>[A]</b> Chromatography of 1 mg of the extract on a FPLC-GP-250 Plus system using a molecular exclusion column (Superdex 200 10/300 GL), in 0.05 M ammonium bicarbonate buffer, pH 7.4. <b>[B]</b> SDS-PAGE of fractions screened by their ability to cleave the component C3. <b>[C]</b> SDS-PAGE (12%) of 10 μg of the extract and 0.25 μg of the fraction 10, under reducing conditions, followed by silver staining.</p

Action of Ps82 on the complement pathways.

<p>Samples (50 μL) of normal human serum (NHS) were pre-incubated with 0.25, 0.33 or 0.5 μg/mL of Ps82 or with 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract. The mixtures were pre-incubated for 30 min for the alternative pathway <b>[A]</b> or 1 h for the classical pathway <b>[C]</b> at 37°C, and then assayed for the residual complement activity in haemolytic tests. For lectin pathway <b>[B]</b>, samples of 50 μL of NHS were pre-incubated, in the presence or absence of 10 mM 1,10-Phenanthroline, with 0.5 μg/mL of Ps82 or 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract for 30 min at 37°C and then evaluated for the residual complement activity by ELISA. Alternatively, samples of NHS incubated with Ps82 or the <i>Premolis semirufa</i>’s bristles extract were evaluated in conditions for activation of the classical pathway, by the deposition of complement component C4b by ELISA <b>[D]</b>. Data are representative for three separate experiments, performed in duplicate, and the results are expressed as percentage of haemolysis (%) ± SD (for A and C) and percentage of C4b deposition (%) ± SD (for B and D) in relation to the control samples (NHS + buffer) and between the treatments. (*) <i>p</i> < 0.05 (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001. The symbol (#) indicates significant differences between the extract and Ps82.</p

Anaphylatoxins generation from purified human C5 induced by the <i>Premolis semirufa’s</i> bristles extract and Ps82.

<p>Samples of 3 μg of the extract or 0.1 μg of Ps82 were incubated, in the absence or presence of 10 mM PMSF, with human C5 (3 μg) for 30 min at 37°C. The generation of the anaphylatoxin C5a was measured using the "Human C5a ELISA Kit". Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of anaphylatoxin per mL (ng/mL) ± SD. (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the Buffer and the treatments.</p