Isolation of Double Negative αβ T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants^1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function⁷ due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney⁸ in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d α-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.     

SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG‐initiated polyGln and a polyAla protein expressed by repeat‐associated non‐ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady‐state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.     

Incidence of severe acute renal failure in adults: results of a community based study.

OBJECTIVE--To determine the age related incidence of severe acute renal failure in adults in two health districts in England. DESIGN--Prospective study of patients identified as having severe acute renal failure within a two year period; subsequent monitoring of outcome for a further two years. SETTING--Two health districts in Devon. SUBJECTS--Those adults in a population of 444,971 who developed severe acute renal failure (serum creatinine concentration > 500 mumol/l) for the first time during two years, with subsequent fall of the serum creatinine concentration below the index value. MAIN OUTCOME MEASURES AND RESULTS--125 adults (140 per million total population yearly, 172 per million adults) developed severe acute renal failure, of whom 90 (72%) were over 70. Age related incidence rose from 17 per million yearly in adults under 50 to 949 per million yearly in the 80-89 age groups. In 31 patients (25%) the cause was prostatic disease, which was related to a good prognosis (84% (26) alive at three months). Overall survival was 54% (67) at three months and 34% (42) at two years and was not significantly age related. 18 per million total population yearly (22 per million adult population) received acute dialysis. Referral rate for specialised opinion was 51 per million total population yearly with an estimated appropriate referral rate of 70 per million per year. CONCLUSIONS--The incidence of severe acute renal failure in the community is at least twice as high as the incidence reported from renal unit based studies. Prostatic disease, a preventable and treatable problem, is the most common cause. Survival figures indicate that age alone should not be a bar to specialist referral or treatment.     

Microbial community composition and dolomite formation in the hypersaline microbial mats of the Khor Al-Adaid sabkhas,Qatar

Extremophiles - The Khor Al-Adaid sabkha in Qatar is among the rare extreme environments on Earth where it is possible to study the formation of dolomite—a carbonate mineral whose origin...