Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

Exome sequencing identifies ACSF3 as a cause of combined malonic and methylmalonic aciduria

We used exome sequencing to identify the genetic basis of combined malonic and methylmalonic aciduria (CMAMMA). We sequenced the exome of an individual with CMAMMA and followed up with sequencing of eight additional affected individuals (cases). This included one individual who was identified and diagnosed by searching an exome database. We identify mutations in ACSF3, encoding a putative methylmalonyl-CoA and malonyl-CoA synthetase as a cause of CMAMMA. We also examined a canine model of CMAMMA, which showed pathogenic mutations in a predicted ACSF3 ortholog. ACSF3 mutant alleles occur with a minor allele frequency of 0.0058 in ～1,000 control individuals, predicting a CMAMMA population incidence of ～1:30,000. ACSF3 deficiency is the first human disorder identified as caused by mutations in a gene encoding a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins, and may emerge as one of the more common human metabolic disorders.     

Molecular cloning and analysis of the protein modules of aggrecans

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.     

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.