Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.
Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics infl uence cellular metabolism during cancer progression. Here, we show that EZH2 and NRAS G12D mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-defi cient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2defi cient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRAS G12D to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-defi cient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-defi cient leukemiainitiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics.
Skoda et al. provide new insights into the collaboration between epigenetic regulator Ezh2 and a key hematopoietic tyrosine kinase in disease initiation and progression.
Familial erythrocytosis with elevated erythropoietin levels is frequently caused by mutations in genes that regulate oxygen-dependent transcription of the gene encoding erythropoietin ( EPO). We identified a mutation in EPO that cosegregated with disease with a logarithm of the odds (LOD) score of 3.3 in a family with autosomal dominant erythrocytosis. This mutation, a single-nucleotide deletion (c.32delG), introduces a frameshift in exon 2 that interrupts translation of the main EPO messenger RNA (mRNA) transcript but initiates excess production of erythropoietin from what is normally a noncoding EPO mRNA transcribed from an alternative promoter located in intron 1. (Funded by the Gebert Rüf Foundation and others.).
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. Escherichia coli is a member of the intestinal commensal flora. Certain variants (pathotypes) of the species, however, can cause either intestinal or extraintestinal infections, such as urinary tract infection, meningitis, or bacteremia (1). Extraintestinal pathogenic E. coli (ExPEC) strains harbor a large array of virulence traits that enable them to cause disease outside the intestinal tract. ExPEC strains have been evolving antibiotic resistance, often a combined resistance against most of the clinically relevant antibiotics, such as fluoroquinolones, aminoglycosides, and -lactam antibiotics. Typically, multidrug-resistant (MDR) strains are compromised in their fitness and virulence, which restricts their prevalence to a nosocomial setting and conversely limits their spread in the community. Some successful MDR clonal lineages do, however, retain high virulence potential (2, 3). The E. coli clonal lineage sequence type 131 (ST131)-O25b:H4, first described in 2008 (4, 5), has spread globally not only in hospitals (as do most other MDR clones) but also in the community (6-9). This clone is responsible for ϳ15% (up to 25% [10,11]) of all extraintestinal E. coli infections and represents the majority of fluoroquinolone-resistant isolates (12) and about half of the extended-spectrum -lactamase (ESBL)-producing isolates (13). The progressive acquisition of additional resistance phenotypes in ST131-O25b:H4 strains leaves very few effective antibiotics for treatment of patients infected by members of this lineage (14). Even more alarming is the recent appearance of carbapenem-resistant ST131 isolates (15-17). Recently, ST131-O25b:H4 strains were shown to predominate among carbapenem-resistant E. coli isolates (18). A major clinical concern is the lack of development of novel antibiotics against Gram-negative pathogens, again leaving very limited treatment options (19). The p...
Mesenchymal stromal cells (MSC) exert either tumor-stimulatory or tumor-inhibitory effect. The outcome of the tumor-MSC interaction is dictated by the tumor-specific activating signals. We analyzed the alterations in MSC phenotype in response to stimulation by tumor-secreted paracrine factors. Paracrine factors from human melanoma A375 and glioblastoma 8MGBA cells were used for prolonged culture of MSC to produce derived cells designated DIFF(A)-MSC or DIFF(G)-MSC, respectively. Derived cells were analyzed for the specific surface markers, the expression pattern of MSC markers and fibroblast-specific proteins. Changes in the cell phenotype were evaluated using scratch wound assay and tube formation in vitro; and xenotransplant growth in vivo. Our data show induced expression of vascular endothelial growth factor 2, CD146, fibroblast-specific protein, vimentin and endosialin in DIFF(A)-MSC cells. This indicates their differentiation towards the cells with features of tumor-associated fibroblasts upon stimulation with melanoma-secreted cytokines. Paracrine stimulation in DIFF(G)-MSC led to up-regulation of the genes involved in the MSC differentiation. MSC-specific surface marker characteristics were preserved in derived DIFF(A)-MSC and DIFF(G)-MSC cells. However, we observed increased proportion of CD146 and GD2 (neural ganglioside) positive cells and decreased expression of marker NG2 in the MSC exposed to tumor-conditioned medium. Melanoma-CM increased MSC migration, glioblastoma-CM compromised angiogenic capacity of MSC in vitro and the protumorigenic effect in vivo. Our data directly compare the pleiotropic effects mediated by the malignant cells on the MSC. Secreted paracrine factors from melanoma or glioblastoma differently changed molecular traits in MSC, which explains the dual role of MSC in tumor growth.
Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10–15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.
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