Vaccination with p53 peptide-pulsed dendritic cells is associated with disease stabilization in patients with p53 expressing advanced breast cancer; monitoring of serum YKL-40 and IL-6 as response biomarkers

p53 Mutations are found in up to 30% of breast cancers and peptides derived from over-expressed p53 protein are presented by class I HLA molecules and may act as tumor-associated epitopes in cancer vaccines. A dendritic cell (DC) based p53 targeting vaccine was analyzed in HLA-A2+ patients with progressive advanced breast cancer. DCs were loaded with 3 wild-type and 3 P2 anchor modified HLA-A2 binding p53 peptides. Patients received up to 10 sc vaccinations with 5 x 10(6) p53-peptide loaded DC with 1-2 weeks interval. Concomitantly, 6 MIU/m(2) interleukine-2 was administered sc. Results from a phase II trial including 26 patients with verified progressive breast cancer are presented. Seven patients discontinued treatment after only 2-3 vaccination weeks due to rapid disease progression or death. Nineteen patients were available for first evaluation after 6 vaccinations; 8/19 evaluable patients attained stable disease (SD) or minor regression while 11/19 patients had progressive disease (PD), indicating an effect of p53-specific immune therapy. This was supported by: (1) a positive correlation between p53 expression of tumor and observed SD, (2) therapy induced p53 specific T cells in 4/7 patients with SD but only in 2/9 patients with PD, and (3) significant response associated changes in serum YKL-40 and IL-6 levels identifying these biomarkers as possible candidates for monitoring of response in connection with DC based cancer immunotherapy. In conclusion, a significant fraction of breast cancer patients obtained SD during p53-targeting DC therapy. Data encourage initiation of a randomized trial in p53 positive patients evaluating the impact on progression free survival.     

Weak population genetic differentiation in the most numerous Arctic seabird,the little auk

Quantifying patterns of genetic diversity and differentiation among populations of Arctic birds is fundamental for understanding past and ongoing population processes in the Arctic. However, the genetic differentiation of many important Arctic species remains uninvestigated. Here, phylogeography and population genetics were examined in the worldwide population of a small seabird, the little auk (dovekie, Alle alle)—the most numerous avian species of the Arctic ecosystem. Blood samples or feathers were collected from 328 little auks (325 from the nominate subspecies and 3 from the A. a. polaris) in nine main breeding aggregations in the northern Atlantic and one location from the Pacific Ocean. The mtDNA haplotypes of the two subspecies were not segregated into separate groups. Also, no genetic structure was found within the nominate race based on microsatellite markers. The level of genetic differentiation among populations was low yet significant (mean F _ST = 0.005). Some pairwise F _ST comparisons revealed significant differences, including those involving the most distant Pacific colony as well as among some Atlantic populations. Weak population differentiation following the model of isolation by distance in the little auk is similar to the patterns reported in other high-Arctic bird species, indicating that a lack of distinct genetic structure is a common phenomenon in the Arctic avifauna.     

Complex Changes in von Willebrand Factor-Associated Parameters Are Acquired during Uncomplicated Pregnancy

Background

The coagulation protein von Willebrand Factor (VWF) is known to be elevated in pregnancy. However, the timing and nature of changes in VWF and associated parameters throughout pregnancy are not well understood.

Objectives

To better understand the changes in VWF provoked by pregnancy, we studied VWF-associated parameters in samples collected over the course of healthy pregnancies.

Methods

We measured VWF antigen (VWF:Ag), VWF propeptide (VWFpp), Factor VIII (FVIII), and ADAMTS13 activity in samples collected from 46 women during pregnancy and at non-pregnant baseline. We also characterized pregnant vs. non-pregnant VWF multimer structure in 21 pregnancies, and performed isoelectric focusing (IEF) of VWF in two pregnancies which had samples from multiple trimesters.

Results

VWF:Ag and FVIII levels were significantly increased during pregnancy. ADAMTS13 activity was unchanged. VWFpp levels increased much later in pregnancy than VWF:Ag, resulting in a progressive decrease in VWFpp:Ag ratios. FVIII:VWF ratios also decreased in pregnancy. Most pregnancies exhibited a clear loss of larger VWF multimers and altered VWF triplet structure. Further evidence of acquired VWF qualitative changes in pregnancy was found in progressive, reversible shifts in VWF IEF patterns over gestation.

Conclusions

These data support a new view of pregnancy in which VWF can acquire qualitative changes associated with advancing gestational age. Modeling supports a scenario in which both increased VWF production and doubling of the VWF half-life would account for the data observed. We propose that gestation induces a prolongation in VWF survival, which likely contributes to increased total VWF levels and altered VWF structure.     

Post-Stroke Mortality,Stroke Severity,and Preadmission Antipsychotic Medicine Use – A Population-Based Cohort Study

Background and Purpose

It has been suggested that antipsychotic medication may be neuroprotective and may reduce post-stroke mortality, but studies are few and ambiguous. We aimed to investigate the post-stroke effects of preadmission antipsychotic use.

Methods

We conducted a nationwide, population-based cohort study of 81,143 persons admitted with stroke in Denmark from 2003–2010. Using Danish health care databases, we extracted data on preadmission use of antipsychotics and confounding factors. We examined the association between current, former, and never use of antipsychotics and stroke severity, length of hospital stay, and 30-day post-stroke mortality using logistic regression analysis, survival analysis, and propensity score matching.

Results

Current users of antipsychotics had a higher risk of severe or very severe stroke on The Scandinavian Stroke Scale than never users of antipsychotics (adjusted odds ratios, 1.43; 95% CI, 1.29–1.58). Current users were less likely to be discharged from hospital within 30 days of admission than never users (probability of non-discharge, 27.0% vs. 21.9%). Antipsychotics was associated with an increased 30-day post-stroke mortality among current users (adjusted mortality rate ratios, 1.42; 95% CI, 1.29–1.55), but not among former users (adjusted mortality rate ratios, 1.05; 95% CI, 0.98–1.14).

Conclusions

Preadmission use of antipsychotics was associated with a higher risk of severe stroke, a longer duration of hospital stay, and a higher post-stroke mortality, even after adjustment for known confounders. Antipsychotics play an important role in the treatment of many psychiatric conditions, but our findings do not support the hypothesis that they reduce stroke severity or post-stroke mortality.     

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

The 26 S proteasome is a large proteolytic machine, which degrades most intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to Rpn11, a subunit of the regulatory complex of the human 26 S proteasome. Txnl1 is abundant, metabolically stable, and widely expressed and is present in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about-250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26 S proteasome. eEF1A1 is therefore a likely physiological substrate. In response to knockdown of Txnl1, ubiquitin-protein conjugates were moderately stabilized. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms.Degradation of proteins in eukaryotic cells plays a pivotal role in the regulation of several important processes, including cell division, antigen presentation, and signal transduction (1). Most intracellular proteins are degraded by the 26 S proteasome, a 2.5-MDa protease complex composed of more than 30 different subunits (2).To become degraded, proteins are typically first conjugated to a chain of ubiquitin moieties. This reaction is catalyzed by ubiquitin ligases. The ubiquitin chains lend the proteins affinity for the 26 S proteasome (3). For efficient degradation, certain ubiquitylated proteins are shuttled to the 26 S proteasome by substrate recruiting factors, such as Rad23, Dsk2, and eEF1A (4, 5).The 26 S proteasome is composed of two stable subcomplexes, the proteolytically active 20 S core and 19 S regulatory complexes, which bind to one or both ends of the cylindrical 20 S core particle (6). The regulatory complexes first recognize the ubiquitylated substrates (3), before the substrates are deubiquitylated (7, 8), unfolded (9, 10), and translocated into the 20 S particle for degradation.Although the 26 S proteasome has been known for more than 20 years (11), novel subunits and cofactors have been described recently (12, 13). Here we report another novel proteasome-associated protein, Txnl1 (thioredoxin-like protein 1), that associates directly with the proteasome subunit Rpn11. Txnl1 exhibits thioredoxin activity and targets eEF1A1 in vivo. Previous reports have shown that eEF1A1 transfers misfolded nascent proteins from the ribosome to the 26 S proteasome for degradation (5, 14, 15). Accordingly, ubiquitin-protein conjugates were stabilized upon knockdown of Txnl1 expression. Txnl1 therefore directly links protein reduction and proteolysis, two major intracellular protein quality control mechanisms.     

A β2-microglobulin cleavage variant fibrillates at near-physiological pH

β₂-microglobulin (β₂m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β₂m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native β₂m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β₂m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β₂m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β₂m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β₂m. This may explain the apparent lack of detectable amounts of the variant β₂m in extracts of amyloid from DRA patients.