Mononuclear but not polymorphonuclear phagocyte depletion increases circulation times and improves mammary tumor-homing efficiency of donor bone marrow-derived monocytes

Tumor associated macrophages are an essential part of the tumor microenvironment. Consequently, bone marrow-derived monocytes (BMDMs) are continuously recruited to tumors and are therefore seen as ideal delivery vehicles with tumor-targeting properties. By using immune cell depleting agents and macroscopic in vivo fluorescence imaging, we demonstrated that removal of endogenous monocytes and macrophages (but not neutrophils) leads to an increased tumor accumulation of exogenously administered BMDMs. By means of intravital microscopy (IVM), we confirmed our macroscopic findings on a cellular level and visualized in real time the migration of the donor BMDMs in the tumors of living animals. Moreover, IVM also revealed that clodronate-mediated depletion drastically increases the circulation time of the exogenously administered BMDMs. In summary, these new insights illustrate that impairment of the mononuclear phagocyte system increases the circulation time and tumor accumulation of donor BMDMs

Squaric Ester-Based, pH-Degradable Nanogels:Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.</p

Non-classical ProIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages

Hyperprolactinemia in adults with Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a rare neurodevelopmental genetic disorder typically characterized by body composition abnormalities, hyperphagia, behavioural challenges, cognitive dysfunction, and hypogonadism. Psychotic illness is common, particularly in patients with maternal uniparental disomy (mUPD), and antipsychotic medications can result in hyperprolactinemia. Information about hyperprolactinemia and its potential clinical consequences in PWS is sparse. Here, we present data from an international, observational study of 45 adults with PWS and hyperprolactinemia. Estimated prevalence of hyperprolactinemia in a subset of centres with available data was 22%, with 66% of those related to medication and 55% due to antipsychotics. Thirty-three patients were men, 12 women. Median age was 29 years, median BMI 29.8 kg/m2, 13 had mUPD. Median prolactin was 680 mIU/L (range 329–5702). Prolactin levels were higher in women and patients with mUPD, with only 3 patients having severe hyperprolactinemia. Thyroid function tests were normal, 24 were treated with growth hormone, 29 with sex steroids, and 20 with antipsychotic medications. One patient had kidney insufficiency, and one a microprolactinoma. In conclusion, severe hyperprolactinemia was rare, and the most common aetiology of hyperprolactinemia was treatment with antipsychotic medications. Although significant clinical consequences could not be determined, potential negative long-term effects of moderate or severe hyperprolactinemia cannot be excluded. Our results suggest including measurements of prolactin in the follow-up of adults with PWS, especially in those on treatment with antipsychotic

Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals

We conduct a genome-wide association study (GWAS) of educational attainment (EA) in a sample of ~3 million individuals and identify 3,952 approximately uncorrelated genome-wide-significant single-nucleotide polymorphisms (SNPs). A genome-wide polygenic predictor, or polygenic index (PGI), explains 12-16% of EA variance and contributes to risk prediction for ten diseases. Direct effects (i.e., controlling for parental PGIs) explain roughly half the PGI's magnitude of association with EA and other phenotypes. The correlation between mate-pair PGIs is far too large to be consistent with phenotypic assortment alone, implying additional assortment on PGI-associated factors. In an additional GWAS of dominance deviations from the additive model, we identify no genome-wide-significant SNPs, and a separate X-chromosome additive GWAS identifies 57