A toolbox for the longitudinal assessment of healthspan in aging mice.

The number of people aged over 65 is expected to double in the next 30 years. For many, living longer will mean spending more years with the burdens of chronic diseases such as Alzheimer's disease, cardiovascular disease, and diabetes. Although researchers have made rapid progress in developing geroprotective interventions that target mechanisms of aging and delay or prevent the onset of multiple concurrent age-related diseases, a lack of standardized techniques to assess healthspan in preclinical murine studies has resulted in reduced reproducibility and slow progress. To overcome this, major centers in Europe and the United States skilled in healthspan analysis came together to agree on a toolbox of techniques that can be used to consistently assess the healthspan of mice. Here, we describe the agreed toolbox, which contains protocols for echocardiography, novel object recognition, grip strength, rotarod, glucose tolerance test (GTT) and insulin tolerance test (ITT), body composition, and energy expenditure. The protocols can be performed longitudinally in the same mouse over a period of 4-6 weeks to test how candidate geroprotectors affect cardiac, cognitive, neuromuscular, and metabolic health.

Murine glomerular mesangial cell uptake of apoptotic cells is inefficient and involves serum-mediated but complement-independent mechanisms.

An increased number of apoptotic bodies have been detected in glomeruli of non-nephritic kidneys of C1q-deficient mice. In these mice an in vivo impaired uptake of apoptotic cells by peritoneal macrophages was also demonstrated. Here we investigated whether C1q plays a role in the in vitro clearance of apoptotic cells by glomerular mesangial cells. Phagocytosis was assessed using a novel flow cytometric assay that was validated by immunofluorescence studies. The uptake of apoptotic cells by mesangial cells, measured as percentage of mesangial cells ingesting apoptotic cells, was approximately 25%, 10% and 10% for a T cell lymphoma line (RMA), thymocytes and neutrophils, respectively. The uptake reached a plateau phase after 3 h, was specific for apoptotic cells and was mediated by serum but not by complement components C1q or C3. The phagocytosis of apoptotic cells was significantly inhibited by Arg-Gly-Asp-Ser (RGDS), a peptide capable of blocking the interaction of thrombospondin with CD36 or the vitronectin receptor. Pretreatment of the mesangial cells with dexamethasone (200 nm) but not with LPS increased the uptake markedly. These findings indicate that murine mesangial cells are capable of taking up syngeneic apoptotic cells, although much less efficiently than professional phagocytic cells. They also show that serum proteins other than complement components mediate the removal of apoptotic cells by murine mesangial cells in vitro.

Accurate intracellular localization of HLA-DM requires correct spacing of a cytoplasmic YTPL targeting motif relative to the transmembrane domain.

HLA-DM is an MHC class II-related heterodimer that is targeted to lysosomal compartments by a tyrosine-based signal YTPL, present in the cytoplasmic tail of the beta chain. Similar signals in other proteins control transport to different intracellular locations and can be recognized at several sorting sites within the cell including the trans-Golgi network, the plasma membrane and the early or sorting endosome. Therefore, in addition to recognizing the basic tyrosine motif, the sorting machinery must be sensitive to additional features associated with these elements. Here we show that efficient trafficking of HLA-DM to lysosomal compartments is dependent upon the proximity of its tyrosine motif to the transmembrane domain. Constructs in which the spacing is altered are rapidly internalized but are expressed at the cell surface. We conclude that the spacing of the HLA-DMB-encoded tyrosine motif relative to the transmembrane domain is an important feature controlling DM sorting in endosomes.

Linkage between rheumatoid arthritis susceptibility and the presence of HLA-DR4 and DR beta allelic third hypervariable region sequences in southern Chinese persons.

OBJECTIVE: To analyze HLA-DR and DQ associations with rheumatoid arthritis (RA) in patients from southern China. METHODS: In 66 patients and 45 controls, restriction fragment length polymorphism studies were performed using DRB, DQA, and DQB probes, and DRB allele-specific typing of polymerase chain reaction-amplified DRB DNA: RESULTS: The frequency of HLA-DR4 was significantly increased among RA patients (42.4% versus 17.8%). Increased frequencies of the DQA3 allele (77.8% versus 48.9%) and the DQB1*0302 allele (71.0% versus 46.3%), which are in linkage disequilibrium with DR4, were also found. Oligonucleotide typing showed that the amino acid sequence LLEQRRAA, spanning amino acid positions 67-74 of the DR beta molecule, was found in 19 of 49 patients and 5 of 32 controls. The main DR4 allelic subtypes found in the population were DRB1*0404 and DRB1*0405, both of which carried the sequence. There was no difference in subtype distribution between patients and controls. CONCLUSION: Chinese RA patients have an increased frequency of HLA-DR4 alleles which possess the same DRB third allelic hypervariable sequence shown to be associated with susceptibility in Caucasian RA patients.