Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases

Analysis of Several PLA2 mRNA in Human Meningiomas

In view of the important oncogenic action of phospholipase A2(PLA2) we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC), one Ca2+-independent high molecular weight PLA2 (PLA2-VI) and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII) are expressed with PLA2-IVA, PLA2-IVB, PLA2-VI, and PLA2-XIIA as the major expressed forms. PLA2-IIE, PLA2-IIF, PLA2-IVD, and PLA2-XIIB are not detected. Plasma (PLA2-VIIA) and intracellular (PLA2-VIIB) platelet-activating factor acetylhydrolase transcripts are expressed in human meningiomas. However no difference was found for PLA2 transcript amounts in relation to the tumor grade, the subtype of meningiomas, the presence of inflammatory infiltrated cells, of an associated edema, mitosis, brain invasion, vascularisation or necrosis. In conclusion numerous genes encoding multiples forms of PLA2 are expressed in meningiomas where they might act on the phospholipid remodeling and on the local eicosanoid and/or cytokine networks

Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

Background : Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.[br/] Results: In this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence.[br/] Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.[br/] Conclusions : These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways

Isatuximab, carfilzomib, and dexamethasone in patients with relapsed multiple myeloma: updated results from IKEMA, a randomized Phase 3 study

Longer-term outcomes with the anti-CD38 antibody isatuximab in combination with carfilzomib-dexamethasone (Isa-Kd) were evaluated in the randomized Phase 3 trial IKEMA (NCT03275285), in a prespecified, follow-up analysis of progression-free survival (PFS, primary study endpoint), final complete response (CR) using Hydrashift Isa immunofixation assay, minimal residual disease (MRD) negativity, and safety. Enrolled patients had relapsed/refractory multiple myeloma (1-3 prior treatment lines). Isa 10 mg/kg was administered intravenously weekly in cycle 1 then biweekly. Efficacy analyses were performed in the intent-to-treat population (Isa-Kd: n = 179, Kd: n = 123) and safety evaluated in treated patients (Isa-Kd: n = 177, Kd: n = 122). Consistent with the primary interim analysis, the addition of Isa to Kd prolonged PFS (HR 0.58, 95.4% CI: 0.42-0.79; median PFS 35.7 [95% CI: 25.8-44.0] vs 19.2 [95% CI: 15.8-25.0] months). PFS benefit was observed with Isa-Kd across subgroups, including patients with poor prognosis. The stringent CR/CR rate was 44.1% vs 28.5% (odds-ratio: 2.09, 95% CI: 1.26-3.48), the MRD negativity rate 33.5% vs 15.4% (odds-ratio: 2.78, 95% CI: 1.55-4.99) and the MRD negativity CR rate 26.3% vs 12.2%, with Isa-Kd vs Kd. The safety profile of Isa-Kd was similar to that reported in the prior interim analysis. These findings further support Isa-Kd as a standard-of-care treatment for relapsed multiple myeloma patients.Clinical trial information: ClinicalTrials.gov, NCT03275285

Isatuximab plus carfilzomib and dexamethasone in patients with early <i>versus</i> late relapsed multiple myeloma: IKEMA subgroup analysis

Patients with multiple myeloma (MM) who experience early relapse within 12 months of therapy initiation are considered functional high-risk and represent an unmet need, needing better therapies to improve outcomes. The final IKEMA (clinicaltrials gov. identifier: NCT03275285) progression-free survival (PFS) analysis confirmed the significant PFS improvement reported at interim analysis with isatuximab (Isa) plus carfilzomib and dexamethasone (Kd; Isa-Kd) versus Kd in patients with relapsed MM (updated median PFS: 35.7 vs. 19.2 months; hazard ratio [HR] =0.58, 95% confidence interval [CI]: 0.42- 0.79). This IKEMA subgroup analysis examined efficacy and safety of Isa-Kd versus Kd in patients who experienced early (n=61 [Isa-Kd], n=46 [Kd]) vs. late relapse (n=104 [Isa-Kd], n=72 [Kd]). As expected, more aggressive features in baseline characteristics were observed in early relapse patients. Consistent with IKEMA overall population results, median PFS (early relapse: 24.7 vs. 17.2 months, HR=0.662, 95% CI: 0.407-1.077; late relapse: 42.7 vs. 21.9 months, HR=0.542, 95% CI: 0.355- 0.826), minimal residual disease negativity (MRD−) (early relapse: 24.6% vs. 15.2%; late relapse: 37.5% vs. 16.7%), and MRD− complete response (≥CR) rates (early relapse: 18.0% vs. 10.9%; late relapse: 30.8% vs. 13.9%) were higher with Isa-Kd versus Kd, respectively, in both early and late relapse patients. Grade ≥3, serious treatment-emergent adverse events, and death rates were higher in the late relapse Isa-Kd arm. However, the numbers of deaths were low and treatment exposure was significantly longer in Isa-Kd versus Kd late relapse patients. These results support the addition of Isa to Kd as standardof- care therapy for relapsed and/or refractory MM regardless of relapse timing

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy

CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival

Covichem: A biochemical severity risk score of COVID-19 upon hospital admission

Clinical and laboratory predictors of COVID-19 severity are now well described and combined to propose mortality or severity scores. However, they all necessitate saturable equipment such as scanners, or procedures difficult to implement such as blood gas measures. To provide an easy and fast COVID-19 severity risk score upon hospital admission, and keeping in mind the above limits, we sought for a scoring system needing limited invasive data such as a simple blood test and co-morbidity assessment by anamnesis. A retrospective study of 303 patients (203 from Bordeaux University hospital and an external independent cohort of 100 patients from Paris Pitié-Salpêtrière hospital) collected clinical and biochemical parameters at admission. Using stepwise model selection by Akaike Information Criterion (AIC), we built the severity score Covichem. Among 26 tested variables, 7: obesity, cardiovascular conditions, plasma sodium, albumin, ferritin, LDH and CK were the independent predictors of severity used in Covichem (accuracy 0.87, AUROC 0.91). Accuracy was 0.92 in the external validation cohort (89% sensitivity and 95% specificity). Covichem score could be useful as a rapid, costless and easy to implement severity assessment tool during acute COVID-19 pandemic waves