Autophagy profiling in single cells with open source CellProfiler-based image analysis.

Single cell-based analysis of macroautophagy/autophagy is largely achieved through the use of fluorescence microscopy to detect autophagy-related proteins that associate with autophagic membranes and therefore can be quantified as fluorescent puncta. In this context, an automated analysis of the number and size of recognized puncta is preferable to a manual count, because more reliable results can be generated in a short time. Here we present a method for open source CellProfiler software-based analysis for quantitative autophagy assessments using GFP-tagged WIPI1 (WD repeat domain, phosphoinositide interacting 1) images acquired with Airyscan or confocal laser-scanning microscopy. The CellProfiler protocol is provided as a ready-to-use software pipeline, and the creation of this pipeline is detailed in both text and video formats. In addition, we provide CellProfiler pipelines for endogenous SQSTM1/p62 (sequestosome 1) or intracellular lipid droplet (LD) analysis, suitable to assess forms of selective autophagy. All protocols and software pipelines can be quickly and easily adapted for the use of alternative autophagy markers or cell types, and can also be used for high-throughput purposes.Abbreviations: AF Alexa Fluor ATG autophagy related BafA1 bafilomycin A1 BSA bovine serum albumin DAPI 4,6-diamidino-2-phenylindole DMEM Dulbecco's modified Eagle's medium DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid EBSS Earle's balanced salt solution FBS fetal bovine serum GFP green fluorescent protein LD lipid droplet LSM laser scanning microscope MAP1LC3B microtubule associated protein 1 light chain 3 beta MTOR mechanistic target of rapamycin kinase PBS phosphate-buffered saline PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3 SQSTM1 sequestosome 1 TIFF tagged image file format U2OS U-2 OS cell line WIPI WD repeat domain, phosphoinositide interacting.

The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

Human WIPI ß-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.

Vaccine delivery by polymeric vehicles in the mouse reproductive tract induces sustained local and systemic immunity.

Design of easily administered vaccines to protect the female reproductive tract against STIs such as HIV, HPV and HSV is a major step in improving world health standards. However, the effect of immunization routes and regimens (prime/boost) on immune response is not well-understood. Here, we present a systematic study of vaccine delivery by different routes and prime/boosting regimens to produce a robust humoral immune response in the reproductive tract. A model antigen, ovalbumin (OVA), was delivered orally or intranasally via polymer particles, and intravaginally via polymer disks to female mice. Repeated prime/boost at a single site result in high OVA-specific antibody levels in the serum for mice immunized orally (IgA) and invaginally (IgA and IgG) after 3 months. Vaginal antibody titers were the highest for mice immunized by intravaginal routes. Vaginal boosting following intranasal or oral priming did not appear to offer similar advantages to those primed intravaginally. Systemic immunization with OVA in Freund's adjuvant produced robust serum IgG levels, but little serum IgA or antibodies in the vaginal washings. All immunization schemes produced a significant level of IgG in the intestinal mucosa, with the exception of nasal priming followed by intravaginal boost with slow-releasing disks. In contrast, only immunization by nasal priming and intravaginal boost with fast-releasing disks was able to achieve significantly high intestinal IgA titers.

Outcomes of early switching from intravenous to oral antibiotics on medical wards.

OBJECTIVES: To evaluate outcomes following implementation of a checklist with criteria for switching from intravenous (iv) to oral antibiotics on unselected patients on two general medical wards. METHODS: During a 12 month intervention study, a printed checklist of criteria for switching on the third day of iv treatment was placed in the medical charts. The decision to switch was left to the discretion of the attending physician. Outcome parameters of a 4 month control phase before intervention were compared with the equivalent 4 month period during the intervention phase to control for seasonal confounding (before-after study; April to July of 2006 and 2007, respectively): 250 episodes (215 patients) during the intervention period were compared with the control group of 176 episodes (162 patients). The main outcome measure was the duration of iv therapy. Additionally, safety, adherence to the checklist, reasons against switching patients and antibiotic cost were analysed during the whole year of the intervention (n = 698 episodes). RESULTS: In 38% (246/646) of episodes of continued iv antibiotic therapy, patients met all criteria for switching to oral antibiotics on the third day, and 151/246 (61.4%) were switched. The number of days of iv antibiotic treatment were reduced by 19% (95% confidence interval 9%-29%, P = 0.001; 6.0-5.0 days in median) with no increase in complications. The main reasons against switching were persisting fever (41%, n = 187) and absence of clinical improvement (41%, n = 185). CONCLUSIONS: On general medical wards, a checklist with bedside criteria for switching to oral antibiotics can shorten the duration of iv therapy without any negative effect on treatment outcome. The criteria were successfully applied to all patients on the wards, independently of the indication (empirical or directed treatment), the type of (presumed) infection, the underlying disease or the group of antibiotics being used.

Intravenous opiate maintenance in a cohort of injecting drug addicts.

BACKGROUND: Drug addiction is a major problem in many societies. The opiate maintenance program includes a prospective cohort of injecting drug addicts, treated with opiates such as heroin intravenously. An important aim of this program is to keep patients under medical supervision in order to reduce the health hazards associated with illicit drug consumption. In this paper we report the performance of this drug policy in terms of retention and analyse treatment withdrawals and hospitalisations. METHODS: Treatment retention was assessed using the Kaplan-Meier method (treatment withdrawals were defined as the event of interest). We analysed factors associated with treatment withdrawal using Cox regression analysis. In addition, we analysed hospitalisations occurring during the study period. RESULTS: Of 175 patients included in the study, 76 withdrew from the study. Of these, 29 were transferred to a substitution program. The 3-year probability of remaining in the study was 61.7% (95%CI: 54.8-69.4%). Risk of treatment withdrawal was independently associated with age (hazard ratio 0.88 [95%CI: 0.82-0.95]) and years of intravenous drug use (hazard ratio 1.11 [95%CI: 1.04-1.18]). No association was found between risk of treatment withdrawal and HIV-, Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) serostatus. The percentages of HIV, HBV and HCV seroconversions observed during the study period were 0.7, 16.2 and 23.3%, respectively. A total of 84 hospitalisations were recorded in 49 patients, mainly due to infectious diseases. Hospitalised patients were not more likely to withdraw from the program. CONCLUSIONS: Retention is high in the intravenous opiate maintenance program and favours the continuation of this drug policy. Individuals with a shorter history of injecting drug use and of older age are more likely to continue intravenous opiate maintenance treatment.