Amyloid-β Peptide-specific DARPins as a Novel Class of Potential Therapeutics for Alzheimer Disease

Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease.     

The Intramembrane Proteases Signal Peptide Peptidase-Like 2a and 2b Have Distinct Functions In Vivo

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.     

Low ERK Phosphorylation in Cancer-Associated Fibroblasts Is Associated with Tamoxifen Resistance in Pre-Menopausal Breast Cancer

Purpose

The aim of this study was to evaluate ERK phosphorylation as a stromal biomarker for breast cancer prognosis and tamoxifen treatment prediction within a randomized tamoxifen trial.

Patients and Methods

Tissue microarrays of two breast cancer cohorts including in total 743 invasive breast cancer samples were analyzed for ERK phosphorylation (pERK) and smooth muscle actin-alpha expression (SMAα) in cancer-associated fibroblasts (CAFs) and links to clinico-pathological data and treatment-predictive values were delineated.

Results

By analyzing a unique randomized tamoxifen trial including breast cancer patients receiving no adjuvant treatment we show for the first time that patients low in ERK phosphorylation in CAFs did not respond to tamoxifen treatment despite having estrogen-receptor alpha (ERα-positive tumors compared to patients with high pERK levels in CAFs (P = 0.015, multivariate Cox regression interaction analysis). In both clinical materials we further show a significant association between pERK and SMAα, a characteristic marker for activated fibroblasts. SMAα expression however was not linked to treatment-predictive information but instead had prognostic qualities.

Conclusion

The data suggests that the presence of a subpopulation of CAFs, defined by minimal activated ERK signaling, is linked to an impaired tamoxifen response. Thus, this report illustrates the importance of the stroma for monitoring treatment effects in pre-menopausal breast cancer.     

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

Abstract Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.     

Synthesis and Evaluation of a Peptide Targeted Small Molecular Gd-DOTA Monoamide Conjugate for MR Molecular Imaging of Prostate Cancer

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.     

Fast‐responding thermal‐death‐time tubes for the determination of thermal bacteria inactivation

The knowledge of thermal inactivation kinetics, usually expressed in terms of D‐ and z‐values, is of crucial importance for the design of sanitation and sterilization processes. In this study, we designed a simple, fast‐responding, and mechanically stable aluminum tube for inactivation measurements and compared these experiments with the successive‐sampling method at different temperatures. Up to 65°C, we determined a come‐up time of approximately 15 s for the tubes, which is lower than the corresponding values of other devices, presumably because of lower wall thickness, material properties, and a higher surface to volume ratio. D‐values of Escherichia coli calculated from tube inactivation experiments by first‐order kinetics were 370 s (56°C), 126 s (58°C), 53.2 s (60°C), 33.8 s (62°C), and 3.22 s (65°C), and the corresponding values determined with the successive‐sampling flask method were insignificantly different (417, 138, 48.6, and 29.1 s for 56, 58, 60, and 62°C, respectively). These data as well as those measured for Enterobacter cloacae, Pseudomonas putida, Serratia odorifera, and Yersinia rhodei were in close accordance with literature values.     

Rescue of melanocortin 4 receptor (MC4R) nonsense mutations by aminoglycoside-mediated read-through

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.     

La autoantigen suppresses IRES-dependent translation of the hepatitis A virus

The human RNA-binding protein La, is an essential trans-acting factor in IRES-dependent translation initiation of poliovirus, the prototypic picornavirus. For hepatitis A virus (HAV), an unusual member of this virus family, the role of host proteins in its inefficient translation and slow replication is unclear. Using small interfering RNA in vivo and purified La in vitro, we demonstrate for the first time that La suppresses HAV IRES-mediated translation and replication. We show that La binds specifically to distinct parts of the HAV IRES and that—unlike poliovirus—HAV proteinase 3C does not cleave La. The La-mediated suppression of HAV translation and stimulation of poliovirus translation implies unexpected mechanistic differences between viral IRES elements.     

Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica

Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 Å resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two α/β domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30° from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity.