Use of Toll-like receptor assays for the detection of bacterial contaminations in icodextrin batches released for peritoneal dialysis.

Icodextrin is a starch derivative used for preparing solutions of peritoneal dialysis. Unfortunately, peptidoglycans (PGN) and lipopolysaccharides (LPS) have been reported to contaminate certain icodextrin batches and to contribute to the development of sterile peritonitis. The decision of selecting or rejecting icodextrin batches is however difficult, because of limitations in the detection of these bacterial contaminants. Besides monocyte activation tests of cytokine release, a number of bio-assays using stably TLR-transfected cell lines have been developed. Here, we compared the efficacy of TLR2- and TLR4-transfected cells to detect bacterial contamination with the responses of monocytes exposed to the same icodextrin samples. In contrast to monocyte models of cytokine release, we found that TLR2- and TLR4-transfected cell lines are highly sensitive to detect little PGN and LPS contaminations in the presence of icodextrin. With the intent to increase PGN reactivity, mutanolysin was used to generate soluble fragments in icodextrin samples. We found that such an enzymatic treatment led to an enhanced response of TLR2-transfected cells, even though parental icodextrin samples were poorly reactive. Altogether, these findings indicate that the use of TLR2- and TLR4-transfected cell lines is a valuable approach for helping to the decision of selecting icodextrin batches for peritoneal dialysis.

Impact of disease-causing mutations on TMEM165 subcellular localization, a recently identified protein involved in CDG-II.

TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1▵. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.