Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis

HSP72 is an important marker for various environmental stresses and diseases, and many researchers need to detect HSP72 levels in various cells. We have therefore developed an assay to monitor intracellular heat-shock protein 72 expression on a microfluidic Lab-on-a-chip platform. We established this method to detect HSP72 intracellularly by antibody staining with DNA counterstaining. The Lab-on-a-chip technology is simple and efficient when performing flow cytometric assays. By permeabilizing the cells for the delivery of antibodies, we were able to show HSP72 expression after 30 min heat-shock at 44 degrees C and then at various post-incubation times at 37 degrees C. We compared our method to a conventional flow cytometer and an enzyme immunoassay technique.     

Mutations in the WRN gene in mice accelerate mortality in a p53-null background

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.     

Cutting edge: sustained expansion of CD8+ T cells requires CD154 expression by Th cells in acute graft versus host disease

Brief treatment with alphaCD154 Ab has been shown to prevent acute graft versus host disease (aGvHD). We extend these data to show that in the absence of CD154 function, donor T cells are unable to expand or generate high level anti-host CTL activity. Using transgenic (Tg) alloreactive CD8+ T cells adoptively transferred into allogeneic recipients, we show that short-term expansion of the CD8+ Tg T cells occurred in the absence of Th cells, and this short-term expansion could be facilitated with an agonistic alphaCD40. While CD40 agonism could enhance short-term expansion, sustained expansion of CD8+ Tg T cells required bona fide CD154-expressing CD4+ alloreactive Th cells. While CD154 was necessary for CD8+ Tg T cell sustained expansion, IL-2 was also implicated as essential. These observations suggest alphaCD154 therapy in GvHD is effective because the treatment causes an abortive CD8 alloresponse leading to the exhaustion or deletion of alloreactive CD8+ clones preventing the development of disease.     

A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection

Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.     

Human Factor H-Related Protein 2 (CFHR2) Regulates Complement Activation

Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.     

Survival and movements of head-started Mojave desert tortoises

Head-starting is a conservation strategy in which young animals are protected in captivity temporarily before their release into the wild at a larger size, when their survival is presumably increased. The Mojave desert tortoise (Gopherus agassizii) is in decline, and head-starting has been identified as one of several conservation measures to assist in recovery. To evaluate the efficacy of indoor head-starting, we released and radio-tracked 68 juvenile tortoises from a 2015 cohort in the Mojave National Preserve, California, USA. We released 20 tortoises at hatching (control) in September 2015, and reared 28 indoors and 20 outdoors in predator-proof enclosures for 7 months before releasing them in April 2016. We monitored tortoises at least weekly after release until 27 October 2016, and documented survivorship, movement, and surface activity. We estimated survivorship by treatment and evaluated effects of treatment, proximity to a raven (Corvus corax) nest (predator) coincidentally established after release, distance moved between monitoring events, surface activity, and release size on individual fate in a generalized linear model. Although indoor head-start tortoises reached the size of 5–6-year-old wild tortoises by release at 7 months of age, survival did not differ significantly among the 3 treatment groups. Combined annual survival was 0.44 (95% CI = 0.34–0.58). Tortoises that were closer to an active raven nest were significantly more likely to die, as were those seen more often outside their burrows and active aboveground. Predicted estimates for short-term probability of survival approached 1.0 as distance from a raven nest exceeded approximately 1.6 km. Rearing treatment, movement distance, and body size were not significant predictors of fate over the 1-year monitoring period. Head-started tortoises released ≥1.6 km from areas of raven activity will likely have higher short-term survival. Population recovery through head-starting alone is unlikely to be successful if systemic ecosystem-level issues, such as habitat degradation and conditions that promote human-subsidized predators, are not ameliorated. © 2019 The Wildlife Society.