C/EBP homologous protein drives pro-catabolic responses in chondrocytes

Introduction

Excess C/EBP homologous protein (CHOP) expression is one feature of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Here, we focused on CHOP expression and function in chondrocytes.

Methods

We studied human knee osteoarthritis (OA) cartilage, bovine chondrocytes cultured in alginate and subjected to sub-lethal biomechanical injury, and knee chondrocytes of human autopsy donors. We performed siRNA knockdown and transfection.

Results

UPR activation was increased in human knee OA cartilage in situ, and in biomechanically injured cultured chondrocytes in vitro. In normal human chondrocytes, CHOP “gain of function” sensitized chondrocytes to IL-1β induced nitric oxide (NO) and matrix metalloproteinase (MMP)-3 release without inducing these responses by itself. Excess CHOP expression, by itself, induced superoxide production and apoptosis. Conversely, siRNA knockdown of CHOP and the UPR-specific mediator X-box binding protein (XBP1) inhibited NO release by >80% (P <0.0005) in response to IL-1β, and blunted MMP-3 release, whereas there were only minimal effects of the UPR mediator GRP78 on these responses. The anti-inflammatory metabolic “super-regulator” AMP kinase (AMPK) is known to limit UPR activation in vascular muscle cells. Here, CHOP supported the capacity of IL-1β to suppress AMPK activity in chondrocytes. We also observed that inhibition of AMPK activity promoted an increase in chondrocyte CHOP expression. Conversely, pharmacologic activation of AMPK by 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) blunted chondrocyte CHOP expression in response to biomechanical injury.

Conclusions

Biomechanical injury and IL-1 signaling stimulate UPR activation in chondrocytes. CHOP mediates chondrocyte catabolic and apoptotic responses to IL-1β, and does so partly by inhibiting AMPK activity. Conversely, development of excess CHOP activity is limited by AMPK activity in chondrocytes. Our findings suggest a mechanism for potential chondroprotection by AICAR and other AMPK activators. The work is of translational relevance for OA, since several drugs that activate AMPK are already in the clinic for arthritis (for example, allosteric AMPK activators sodium salicylate and high dose aspirin, and methotrexate, which activates AMPK by generating AICAR).     

Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts

Purpose

To investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts.

Methods and Materials

Lens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2α, spliced XBP1, ATF6, ATF4 and p-IRE1α in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts.

Results

In the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group.

Conclusions

The UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts.     

UPR Activation and the Down–Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium

Purpose

To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.

Methods and Materials

The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.

Results

In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.

Conclusion

These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract.     

ATF6alpha Promotes Astroglial Activation and Neuronal Survival in a Chronic Mouse Model of Parkinson’s Disease

Accumulating evidence suggests a crucial role for the unfolded protein response (UPR) in Parkinson’s disease (PD). In this study, we investigated the relevance of the UPR in a mouse model of chronic MPTP/probenecid (MPTP/P) injection, which causes severe and persistent degeneration of dopaminergic neurons. Enhanced activation of the UPR branches, including ATF6α and PERK/eIF2α/ATF4, was observed after MPTP/P injections into mice. Deletion of the ATF6α gene accelerated neuronal degeneration and ubiquitin accumulation relatively early in the MPTP/P injection course. Surprisingly, astroglial activation was strongly suppressed, and production of the brain-derived neurotrophic factor (BDNF) and anti-oxidative genes, such as heme oxygenase-1 (HO-1) and xCT, in astrocytes were reduced in ATF6α −/− mice after MPTP/P injections. Decreased BDNF expression in ATF6α −/− mice was associated with decreased expression of GRP78, an ATF6α-dependent molecular chaperone in the ER. Decreased HO-1 and xCT levels were associated with decreased expression of the ATF4-dependent pro-apoptotic gene CHOP. Consistent with these results, administration of the UPR-activating reagent tangeretin (5,6,7,8,4′-pentamethoxyflavone; IN19) into mice enhanced the expression of UPR-target genes in both dopaminergic neurons and astrocytes, and promoted neuronal survival after MPTP/P injections. These results suggest that the UPR is activated in a mouse model of chronic MPTP/P injection, and contributes to the survival of nigrostriatal dopaminergic neurons, in part, through activated astrocytes.     

Sleep deprivation induces the unfolded protein response in mouse cerebral cortex

Little is known about the molecular mechanisms underlying sleep. We show the induction of key regulatory proteins in a cellular protective pathway, the unfolded protein response (UPR), following 6 h of induced wakefulness. Using C57/B6 male mice maintained on a 12:12 light/dark cycle, we examined, in cerebral cortex, the effect of different durations of prolonged wakefulness (0, 3, 6, 9 and 12 h) from the beginning of the lights-on inactivity period, on the protein expression of BiP/GRP78, a chaperone and classical UPR marker. BiP/GRP78 expression is increased with increasing durations of sleep deprivation (6, 9 and 12 h). There is no change in BiP/GRP78 levels in handling control experiments carried out during the lights-off period. PERK, the transmembrane kinase responsible for attenuating protein synthesis, which is negatively regulated by binding to BiP/GRP78, is activated by dissociation from BiP/GRP78 and by autophosphorylation. There is phosphorylation of the elongation initiation factor 2alpha and alteration in ribosomal function. These changes are first observed after 6 h of induced wakefulness. Thus, prolonging wakefulness beyond a certain duration induces the UPR indicating a physiological limit to wakefulness.     

Intraluminal Blockade of Cell-Surface CD74 and Glucose Regulated Protein 78 Prevents Substance P-Induced Bladder Inflammatory Changes in the Rat

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to α1-inhibitor III (A1-I3; a member of the α2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes.

Methodology/Principal Findings

Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control).GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells.

Conclusions/Significance

GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Time of Progression to Osteopenia/Osteoporosis in Chronically HIV-Infected Patients: Screening DXA Scan

Background

Algorithms for bone mineral density (BMD) management in HIV-infected patients are lacking. Our objective was to assess how often a dual-energy x-ray absorptiometry (DXA) scan should be performed by assessing time of progression to osteopenia/osteoporosis.

Methods

All DXA scans performed between 2000 and 2009 from HIV-infected patients with at least two DXA were included. Time to an event (osteopenia and osteoporosis) was assessed using the Kaplan–Meier method. Strata (tertiles) were defined using baseline minimum T scores. Differences between strata in time to an event were compared with the log-rank test.

Results

Of 391 patients (1,639 DXAs), 49.6% had osteopenia and 21.7% osteoporosis at their first DXA scan. Of the 112 (28.6%) with normal BMD, 35.7% progressed to osteopenia; median progression time was 6.7 years. These patients were stratified: “low-risk" (baseline minimum T score >−0.2 SD), “middle-risk" (between −0.2 and −0.6 SD), and “high-risk" (from −0.6 to −1 SD); median progression time to osteopenia was 8.7, >7.2, and 1.7 years, respectively (p<0.0001). Of patients with osteopenia, 23.7% progressed to osteoporosis; median progression time was >8.5 years. Progression time was >8.2 years in “low-risk" tertile (T score between −1.1 and −1.6 SD), >8.5 years in “middle-risk" (between −1.6 and −2), and 3.2 years in “high-risk" (from −2 to −2.4) (p<0.0001).

Conclusions

Our results may help to define the BMD testing interval. The lowest T score tertiles would suggest recommending a subsequent DXA in 1–2 years; in the highest tertiles, ≥6 years. Early intervention in patients with bone demineralization could reduce fracture–related morbidity/mortality.     

Influence of the Time of Day and Fasting Duration on Glucose Level following a 1-Hour, 50-Gram Glucose Challenge Test in Pregnant Women

Background

Previous studies have shown that the time of day (TD) of glucose measurement and the fasting duration (FD) influence the glucose levels in adults. Few studies have examined the effects of the TD and FD on the glucose level following a 1-hour, 50-gram glucose challenge test (GCT) in pregnant women in screening for or diagnosing gestational diabetes mellitus (GDM). The objective of this study was to investigate the influence of the TD (morning, afternoon, night) and the FD (the time of the last food ingestion as follows: ≤1 hour, 1–2 hours, and >2 hours) by examining their combined effects on the glucose levels following a 50-gram GCT in pregnant women.

Methods and Results

We analyzed the data of 1,454 non-diabetic pregnant Taiwanese women in a prospective study. Multiple linear regression and multiple logistic regression were used to estimate the relationships between the 9 TD-FD groups and the continuous and binary glucose levels (cut-off at 140 mg/dL) following a 50-gram GCT, after adjusting for maternal age, nulliparity, pre-pregnancy body mass index, and weight gain. Different TD and FD groups were associated with variable glucose responses to the 50-gram GCT, some of which were significant. The estimate coefficients (β) of the TD-FD groups “night, ≤1 hr” and “night, 1–2 hr” revealed significantly lower glucose concentrations [β (95% confidence interval [CI]): −6.46 (−12.53, −0.38) and −6.85 (−12.50, −1.20)] compared with the “morning, >2 hr” group. The TD-FD groups “afternoon, ≤1 hr” and “afternoon, 1–2 hr” showed significantly lower odds ratios (OR) of a positive GCT; the adjusted ORs (95% CI) were 0.54 (0.31–0.95) and 0.58 (0.35–0.96), respectively.

Conclusions

Our findings demonstrate the importance of standardizing the TD and FD for the 1-hour, 50-gram GCT. In screening for and diagnosing GDM, the TD and FD are modifiable factors that should be considered in clinical practice and epidemiological studies.     

Longitudinal Associations between Self-Rated Health and Performance-Based Physical Function in a Population-Based Cohort of Older Adults

Background

Although self-rated health (SRH) and performance-based physical function (PPF) are both strong predictors of mortality, little research has investigated the relationships between them. The objective of this study was to evaluate longitudinal, bi-directional associations between SRH and PPF.

Methods

We evaluated longitudinal associations between SRH and PPF in 3,610 adults aged 65–89 followed for an average of 4.8 (standard deviation [SD]: 4.4) years between 1994 and July 2011 in the Adult Changes in Thought study, a population-based cohort in the Seattle area. SRH was assessed with a single-item question in the ACT study. Participants were asked at each evaluation to rate their health as “excellent”, “very good”, “good”, “fair”, or “poor” in response to the question “In general, how would you rate your health at this time”. PPF scores (ranging from 0–16, with higher indicating better performance) included walking speed, chair rises, grip strength, and balance.

Results

At the baseline visit, participants averaged 74.5 (SD: 5.8) years of age and 2,115 (58.6%) were female. In multivariable linear mixed models, PPF declined with age, with more rapid decreases associated with very good, good, and fair (vs. excellent) baseline SRH. Adjusted annual change in PPF was −0.17 points (95% confidence interval [CI]: −0.19, −0.15) for individuals with excellent baseline SRH and −0.21 points (95% CI: −0.22, −0.19) for participants with fair SRH. In multivariable generalized linear mixed models, lower baseline PPF quartiles were associated with lower odds of excellent/very good/good SRH at age 75, however, differences between baseline PPF quartiles diminished with age.

Conclusions

These results suggest that less than excellent SRH predicts decline in physical functioning, however, poor physical functioning may not predict change in SRH in a reciprocal fashion. SRH provides a simple assessment tool for identifying individuals at increased risk for decline in physical function.     

Safety of Onartuzumab in Patients with Solid Tumors: Experience to Date from the Onartuzumab Clinical Trial Program

Background

Onartuzumab, a recombinant humanized monovalent monoclonal antibody directed against MET, the receptor for the hepatocyte growth factor, has been investigated for the treatment of solid tumors. This publication describes the safety profile of onartuzumab in patients with solid tumors using data from the global onartuzumab clinical development program.

Methods

Adverse event (AE) and laboratory data from onartuzumab phase II/III studies were analyzed and coded into standardized terms according to industry standards. The severity of AEs was assessed using the NCI Common Toxicity Criteria, Version 4. Medical Dictionary for Regulatory Activities (MedDRA) AEs were grouped using the standardized MedDRA queries (SMQs) “gastrointestinal (GI) perforation”, “embolic and thrombotic events, venous (VTE)”, and “embolic and thrombotic events, arterial (ATE)”, and the Adverse Event Group Term (AEGT) “edema.” The safety evaluable populations (patients who received at least one dose of study treatment) for each study were included in this analysis.

Results

A total of 773 onartuzumab-treated patients from seven studies (phase II, n = 6; phase III, n = 1) were included. Edema and VTEs were reported in onartuzumab-treated patients in all seven studies. Edema events in onartuzumab arms were generally grade 1–2 in severity, observed more frequently than in control arms and at incidences ranging from 25.4−65.7% for all grades and from 1.2−14.1% for grade 3. Hypoalbuminemia was also more frequent in onartuzumab arms and observed at frequencies between 77.8% and 98.3%. The highest frequencies of all grade and grade ≥3 VTE events were 30.3% and 17.2%, respectively in onartuzumab arms. The cumulative incidence of all grade ATE events ranged from 0−5.6% (grade ≥3, 0−5.1%) in onartuzumab arms. The frequency of GI perforation was below 10% in all studies; the highest estimates were observed in studies with onartuzumab plus bevacizumab for all grades (0−6.2%) and grade ≥3 (0−6.2%).

Conclusions

The frequencies of VTE, ATE, GI perforation, hypoalbuminemia, and edema in clinical studies were higher in patients receiving onartuzumab than in control arms; these are considered to be expected events in patients receiving onartuzumab.     

Unusually High Incidence of Paediatric Coeliac Disease in Sweden during the Period 1973 – 2013

Objective

The prevalence of coeliac disease in Sweden during the “epidemic period” (1984−1996) was one of the highest in the world. The aim of this study was to assess the coeliac disease incidence in our region over the 41-year period, and how diagnostic activity and diagnostic accuracy were affected by the introduction of antibody testing. We also looked into how patients with mild enteropathy were evaluated.

Methods

In the county of Östergötland in Sweden, 2790 paediatric patients were investigated for suspected coeliac disease between 1973 and 2013. Notes were scrutinised for data on sex, age, histopathological reports and final diagnosis. For comparative purposes this period was divided into three sub-periods (1973−1983, 1984−1996 and 1997−2013) named pre-epidemic, epidemic and post-epidemic.

Results

Coeliac disease diagnosis was received by 1,030 patients. The peak incidence rate, 301 cases/100,000 in 1994 for the age group 0−1.9 years is the highest figure ever reported. The other age groups, 2−4.9, 5−14.9, and 15−17.9 years, also had high incidence rates. After the 1984−1996 “epidemic period” the incidence decreased for the youngest group but continued to increase for the other groups. The cumulative incidence at 18 years-of-age for children born during the epidemic reached 14 cases/1000 births, the highest figure hitherto reported. Diagnostic activity differed significantly between the three sub-periods (p<0.001) increasing gradually from 1984 and reaching a peak value of 0.87 in 2012. Cases of mild enteropathy were more frequently regarded as non-coeliac disease cases, decreasing significantly in the “post-epidemic” period (p<0.001).

Conclusions

The incidence rate and cumulative incidence of coeliac disease were possibly the highest ever reported. Changes in diagnostic activity and accuracy could not be attributed to the introduction of new antibody tests, possibly because of other changes e.g. variations in the symptoms at presentation and improved knowledge of the disease among parents and health professionals.     

Oxidative stress-mediated aldehyde adduction of GRP78 in a mouse model of alcoholic liver disease: functional independence of ATPase activity and chaperone function

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.     

Outcomes for patients with the same disease treated inside and outside of randomized trials: a systematic review and meta-analysis

Background:

It is unclear whether participation in a randomized controlled trial (RCT), irrespective of assigned treatment, is harmful or beneficial to participants. We compared outcomes for patients with the same diagnoses who did (“insiders”) and did not (“outsiders”) enter RCTs, without regard to the specific therapies received for their respective diagnoses.

Methods:

By searching the MEDLINE (1966–2010), Embase (1980–2010), CENTRAL (1960–2010) and PsycINFO (1880–2010) databases, we identified 147 studies that reported the health outcomes of “insiders” and a group of parallel or consecutive “outsiders” within the same time period. We prepared a narrative review and, as appropriate, meta-analyses of patients’ outcomes.

Results:

We found no clinically or statistically significant differences in outcomes between “insiders” and “outsiders” in the 23 studies in which the experimental intervention was ineffective (standard mean difference in continuous outcomes −0.03, 95% confidence interval [CI] −0.1 to 0.04) or in the 7 studies in which the experimental intervention was effective and was received by both “insiders” and “outsiders” (mean difference 0.04, 95% CI −0.04 to 0.13). However, in 9 studies in which an effective intervention was received only by “insiders,” the “outsiders” experienced significantly worse health outcomes (mean difference −0.36, 95% CI −0.61 to −0.12).

Interpretation:

We found no evidence to support clinically important overall harm or benefit arising from participation in RCTs. This conclusion refutes earlier claims that trial participants are at increased risk of harm.When people are asked to participate in a randomized controlled trial (RCT), it is natural for them to ask several questions in return. How safe are these treatments? How many extra visits and tests must I undergo? Will the researchers keep my family doctor informed about what’s going on? What outcomes are to be measured, and do they include ones that are of interest to me as a patient?These multiple questions can be summarized as follows: Would I fare better being treated within the trial (as an “insider”) or in routine clinical care outside it (as an “outsider”)? Patients may ask this question in 1 of 2 ways. The first is highly specific: “Am I better off receiving this specific treatment as an insider or as an outsider?” Alternatively, they might ask a more general question: “Am I better off having my illness managed, regardless of the specific treatment I would receive, as an insider or as an outsider?” These questions are highly appropriate, and both deserve to be asked and answered,^1,2 especially given that nonsystematic reviews have suggested a possible “inclusion benefit” from participating in trials.³These 2 specific patient questions are analogous to those posed by researchers asking whether treatments do more good than harm when applied under “ideal” circumstances (in explanatory trials) or in the “real world” of routine health care (in pragmatic trials). Vist and colleagues answered the explanatory question when their earlier review⁴ found no advantage or disadvantage from receiving the same treatment inside or outside an RCT. Left unanswered, however, was the broader, more pragmatic question. In our experience, trial participants are often offered new, as-yet-untested treatments that would not be available to them outside the trial. This review looks at the dilemma faced by these patients, which needs to be addressed before general conclusions can be drawn about trial safety.     

CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

Background

Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.

Methods

Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.

Results

We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.

Conclusions

Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.     

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.