Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for the generation of neutralizing antibodies.

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.

Bactericidal monoclonal antibodies specific to the lipopolysaccharide O antigen from multidrug-resistant Escherichia coli clone ST131-O25b:H4 elicit protection in mice.

The Escherichia coli sequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to live E. coli cells and the in vitro and in vivo efficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused by E. coli ST131-O25b:H4.

Influence of microvascular endothelial cells on transcriptional regulation of proximal tubular epithelial cells.

In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, close-proximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1 beta), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-alpha IV) and cytokines/growth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms.

Gene expression and biomarkers in renal transplant ischemia reperfusion injury.

The incidence of postischemic acute renal allograft failure (ARF) occurs in roughly 25% of cadaveric donor kidney recipients. This high rate remained virtually unchanged over the last decades despite modification in recipient management and modern immunosuppressive strategies. It has recently been shown that among other reasons, the systemic inflammation in the brain death cadaveric organ donor contributes to subsequent ARF in the recipient. This review focuses on the consequences of ischemia and reperfusion on the cellular level and offers potential solutions for the reduction of ARF. Genome-wide gene expression analysis together with sophisticated biostatistical analysis made it possible to identify several candidate gene products and proteins that may act as specific and sensitive biomarker for renal inflammation and ischemia. These markers may be very helpful in the clinical management of patients with a high a priori risk of subsequent ARF such as recipients of marginal donor kidneys. Ongoing clinical trials will evaluate whether immunosuppression of the cadaveric organ donor before organ harvest will have the potential to reduce inflammation in the transplant kidney and subsequently lead to a reduction in the rate of ARF.