Project description:The spread of antimicrobial resistance (AMR), coupled with the decline in antibiotic development, has become a major public health concern. Recent studies estimate that around 700,000 people die each year from infections caused by multidrug-resistant (MDR) bacteria. This led the WHO to publish the ESKAPEE list of high priority pathogens for AMR, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli. Among these, Gram-negative bacteria (K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp., and E. coli) are particularly overrepresented. This is mainly due to their high propensity to develop multiple resistance mechanisms, in addition to their intrinsic resistance to many antimicrobials, which is due to their membrane composition and the expression of broad-spectrum efflux pumps. One strategy to combat such AMR is the use of drug enhancers that are able to restore the antibacterial activity of poorly active antibiotics. In this context, we demonstrated that the polyamino-isoprenyl enhancer, NV716, efficiently potentiates the antibacterial activity of two families of multi-target Ser/Cys-based enzyme inhibitors, namely the oxadiazolone derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC), against Enterobacter cloacae, while remaining inactive against other Gram-negative bacteria. We confirmed that NV716 potentiates some OX & CyC compounds by permeabilizing the outer membrane and thus by increasing the inhibitor accumulation as shown by fluorescence confocal microscopy. By using bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) approach coupled to proteomic analysis, we also identified the target proteins of the best OX & CyC inhibitors from E. cloacae lysate, thereby confirming their multi-target nature. Interestingly, 6 of the latter proteins were also captured via CC-ABPP in P. aeruginosa lysate, and are highly conserved in all Gram-negative bacteria. These results provide proof of concept that both OX & CyC, if successfully potentiated, could be used against a wide range of ESKAPEE Gram-negative bacteria.

Project description:The presence of Donor-Specific anti-HLA Antibodies (DSA) is associated with an increased risk of both acute and chronic antibody-mediated rejection (AMR) in kidney allografts. AMR has remained challenging in kidney transplantation and is the major cause of late allograft loss. However, not all patients with DSA develop AMR, leading to the question of whether this represents accommodation, if other protective mechanisms exist or if this is actually a state of pre-rejection. Clinical and histological features, and gene expression profiles of kidney biopsy and blood samples of donor-specific antibody (DSA)+ patients without rejection were compared to antibody-mediated rejection (AMR) patients to elucidate the mechanisms involved in prevention of AMR. Of the 71 DSA+ patients, 46 had diagnosis of AMR and 25 did not show rejection. 50 DSA- patients without rejection were used as control. A subgroup of patients with available biopsy (n=61) and blood samples (n=54) were analyzed by microarrays. Both, DSA+/AMR+ and DSA+/AMR- biopsies showed increased expression of gene transcripts associated with cytotoxic T, natural killer cells, macrophages, interferon-gamma and rejection compared to DSA- biopsies. Regulatory T cell transcripts were up-regulated in DSA+/AMR+ and B cell transcripts in DSA+/AMR- biopsies. Whole blood gene expression analysis showed increased immune activity in only DSA+/AMR+ patients. There were no differentially expressed tolerant genes studied (n=14) in the blood or biopsy specimens of DSA+/AMR- patients. During a median 36 months follow-up, 4 DSA+/AMR- patients developed AMR, 12 continued to have DSAs but 9 lost DSAs. Gene expression profiles did not predict the development of AMR or persistence of DSAs. These results indicate increased immune activity in DSA+/AMR- biopsies despite lack of histologic findings of rejection. All clinically indicated kidney transplant biopsies performed at our institution after January 2009 were reviewed and 263 patients with anti-HLA antibody testing at the time of biopsy were identified. There were 71 DSA+ and 192 DSA- patients (Figure 1). Of the 71 DSA+ patients, 46 had biopsy diagnosis of acute AMR (n=9) or chronic AMR (n=37), and 25 had normal histopathology or minimal non-specific interstitial fibrosis/tubular atrophy (IFTA). Of the 192 DSA- patients, 50 patients with normal histology and/or mild non-specific IFTA were used as a control group. Clinical and histopathological findings of these 3 groups (DSA+/AMR+, DSA+/AMR- and DSA-) were analyzed. A subgroup of patients who were enrolled in the Institutional Review Board-approved âImmune Monitoring Studyâ who had clinically indicated biopsy (n=61) and whole blood samples (n=54) stored were used for genomic analysis. Twenty-eight biopsy and blood samples from DSA+/AMR+ patients, 13 biopsy and 14 blood samples from DSA+/AMR- patients, and 20 biopsy and 12 blood samples from DSA- patients, were available for microarray analysis.

Project description:Objective. Meningococcal sepsis remains an important cause of childhood morbidity and mortality. Largely due to logistic complexities of research in young children with acute life-threatening disease, very little is known regarding differential expression kinetics and molecular regulation of immune response genes in leukocyte subsets. Materials and methods. In this prospective case-control study, six children with meningococcal sepsis were included. Blood was drawn at four time points (t=0, t=8, t=24 and t=72 h after admission to the paediatric intensive care unit). Blood was also collected from matched controls. Detailed immunophenotyping of leukocytes was performed; RNA isolated from whole blood, lymphocytes, monocytes, and granulocytes was used to perform Affymetrix micro-array gene expression analysis. Results and conclusion. There were no differences in total leukocyte count between patients and controls. In contrast to previous in vitro studies we observed an unexpected decrease in NK cell numbers, as well as downregulation of NK cell specific and cytotoxic T-cell related gene expression in patients with meningococcal septic shock. By contrast, expression of genes, involved in innate immunity and several other pathways, differed between the different leukocyte subpopulations in a dynamic fashion. Compared to previously reported gene expression profiles, it was possible to define a meningococcal sepsis specific expression profile. Experiment Overall Design: In this prospective case-control study, six children with meningococcal sepsis were included. Blood was drawn at four time points (t=0, t=8, t=24 and t=72 h after admission to the paediatric intensive care unit). Blood was also collected from matched controls. Detailed immunophenotyping of leukocytes was performed; RNA isolated from whole blood, lymphocytes, monocytes, and granulocytes was used to perform Affymetrix micro-array gene expression analysis. Experiment Overall Design: No replicates were performed. Experiment Overall Design: Table 4. Results of between-subject analyses. Experiment Overall Design: Source Time (h) ca co/ca at t=T Experiment Overall Design: Blood t0: ca:2 3, co: a b c; t8: 2; t24 :ca:1 2 3; t72: ca:1 2. Experiment Overall Design: Lym t0: ca:2 3, co:a b c d;t8: ca:1 3 4;t24: ca:1 2 3 4 6; t72: ca: 1 2. Experiment Overall Design: Mono t0:ca: 1 3 4 6, co: a b d; t8: ca:1 2 3; t24:ca: 1 2 3; t72: ca:1. Experiment Overall Design: Numbers represent the patient identification numbers. Experiment Overall Design: Characters represent the individual controls.

			Action	DRS
	20250722_KC89_1A.raw	Raw
	20250722_KC89_1B.raw	Raw
	20250722_KC89_2A.raw	Raw
	20250722_KC89_2B.raw	Raw
	20250722_KC89_3A.raw	Raw

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Lyophilization prior to homogenisation and extraction increases membrane protein detection in Gram-negative bacterial proteomic analyses

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