Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study.

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127-0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage-antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363 .

Leaving out control groups: an internal contrast analysis of gene expression profiles in atrial fibrillation patients--a systems biology approach to clinical categorization.

Atrial fibrillation (AF) is a frequent chronic dysrythmia with an incidence that increases with age (>40). Because of its medical and socio-economic impacts it is expected to become an increasing burden on most health care systems. AF is a multi-factorial disease for which the identification of subtypes is warranted. Novel approaches based on the broad concepts of systems biology may overcome the blurred notion of normal and pathological phenotype, which is inherent to high throughput molecular arrays analysis. Here we apply an internal contrast algorithm on AF patient data with an analytical focus on potential entry pathways into the disease. We used a RMA (Robust Multichip Average) normalized Affymetrix micro-array data set from 10 AF patients (geo_accession #GSE2240). Four series of probes were selected based on physiopathogenic links with AF entryways: apoptosis (remodeling), MAP kinase (cell remodeling), OXPHOS (ability to sustain hemodynamic workload) and glycolysis (ischemia). Annotated probe lists were polled with Bioconductor packages in R (version 2.7.1). Genetic profile contrasts were analysed with hierarchical clustering and principal component analysis. The analysis revealed distinct patient groups for all probe sets. A substantial part (54% till 67%) of the variance is explained in the first 2 principal components. Genes in PC1/2 with high discriminatory value were selected and analyzed in detail. We aim for reliable molecular stratification of AF. We show that stratification is possible based on physiologically relevant gene sets. Genes with high contrast value are likely to give pathophysiological insight into permanent AF subtypes.

Hunting interactomes of a membrane protein: obtaining the largest set of voltage-dependent anion channel-interacting protein epitopes.

The identification of epitopes involved in protein-protein interactions is essential for understanding protein structure and function. Large scale efforts, although identifying the interactions, did not always yield these epitopes, could not confirm most of the known interactions, and seemed particularly unsuccessful for native intrinsic membrane proteins. We have developed a fluidics-based approach (non-steady-state kinetics) to obtain the broadest set of the epitopes interacting with a given target and applied it to a phage display methodology optimized for membrane proteins. Phages expressing a liver cDNA library were screened against a membrane protein (voltage-dependent anion channel) reconstituted into liposomes and captured on a chip surface. The controlled fluidics was obtained by a surface plasmon resonance (SPR) device that combined the advantages of working with minute reaction volumes and non-equilibrium conditions. We demonstrated selective enrichment of binders and could even select for different binding affinities by fractionation of the selected outputs at various elution times. With voltage-dependent anion channel as bait (a mitochondrial channel critical for cellular metabolism and apoptosis) we found at least 40% of its already reported ligands and independently confirmed 55 novel functional interactions, some of which fully blocked the channel. This highly efficient approach is generally applicable for any protein and could be automated and scaled up even without the use of a SPR device. The epitopes directly identified by this method are useful not only for unraveling interactomes but also for drug design and therapeutics.

Direct measurement of VDAC-actin interaction by surface plasmon resonance.

VDAC--a mitochondrial channel involved in the control of aerobic metabolism and apoptosis--interacts in vitro and in vivo with a wide repertoire of proteins including cytoskeletal elements. A functional interaction between actin and Neurospora crassa VDAC was reported, excluding other VDAC isoforms. From a recent genome-wide screen of the VDAC interactome, we found that human actin is a putative ligand of yeast VDAC. Since such interaction may have broader implications for various mitochondrial processes, we probed it with Surface Plasmon Resonance (SPR) technology using purified yeast VDAC (YVDAC) and rabbit muscle G-actin (RGA). We show that RGA binds to immobilized YVDAC in a reversible and dose-dependent manner with saturating kinetics and an apparent K(D) of 50 microg/ml (1.2 microM actin). BSA does not bind VDAC regardless of the concentrations. Alternatively, VDAC binds similarly to immobilized RGA but without saturating kinetics. VDAC being known to interact with itself, this latter interaction was directly measured to interpret the RGA signals. VDAC could bind to VDAC without saturating kinetics as expected if higher order binding occurred, and could account for maximally 66% of the non-saturating behavior of VDAC binding onto RGA. Hence, actin-VDAC interactions are not a species-specific oddity and may be a more general phenomenon, the role of which ought to be further investigated.

In vitro interactions between the two mitochondrial membrane proteins VDAC and cytochrome c oxidase.

VDAC, a mitochondrial outer membrane channel, is involved in the control of aerobic metabolism and in apoptotic processes via numerous protein-protein interactions. To unveil those interactions, we screened a human liver cDNA library with the phage display methodology optimized to target VDAC reconstituted into a membrane environment. One positively selected clone yielded a sequence matching a part of the subunit I of human cytochrome c oxidase (COX), a mitochondrial inner membrane enzyme. Such putative interaction was never reported before. This interaction proved to be functional as evidenced by the effect of the human and yeast isoforms of VDAC on the oxidation of cytochrome c by the pure holoenzyme and by the effect of the COX epitope on VDAC permeability. Our results providing four independently obtained evidences of VDAC-COX interaction in vitro, would support a novel and potentially important level of mitochondrial regulation given the respective locations and functions of both proteins.