Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system

Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.     

SOBP is mutated in syndromic and nonsyndromic intellectual disability and is highly expressed in the brain limbic system

Intellectual disability (ID) affects 1%-3% of the general population. We recently reported on a family with autosomal-recessive mental retardation with anterior maxillary protrusion and strabismus (MRAMS) syndrome. One of the reported patients with ID did not have dysmorphic features but did have temporal lobe epilepsy and psychosis. We report on the identification of a truncating mutation in the SOBP that is responsible for causing both syndromic and nonsyndromic ID in the same family. The protein encoded by the SOBP, sine oculis binding protein ortholog, is a nuclear zinc finger protein. In mice, Sobp (also known as Jxc1) is critical for patterning of the organ of Corti; one of our patients has a subclinical cochlear hearing loss but no gross cochlear abnormalities. In situ RNA expression studies in postnatal mouse brain showed strong expression in the limbic system at the time interval of active synaptogenesis. The limbic system regulates learning, memory, and affective behavior, but limbic circuitry expression of other genes mutated in ID is unusual. By comparing the protein content of the +/jc to jc/jc mice brains with the use of proteomics, we detected 24 proteins with greater than 1.5-fold differences in expression, including two interacting proteins, dynamin and pacsin1. This study shows mutated SOBP involvement in syndromic and nonsyndromic ID with psychosis in humans.     

Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells

Background

In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential.

Methodology/Principal Findings

Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types.

Conclusions/Significance

These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.     

High dietary advanced glycation end products are associated with poorer spatial learning and accelerated Aβ deposition in an Alzheimer mouse model

There is growing evidence of the involvement of advanced glycation end products (AGEs) in the pathogenesis of neurodegenerative processes including Alzheimer's disease (AD) and their function as a seed for the aggregation of Aβ, a hallmark feature of AD. AGEs are formed endogenously and exogenously during heating and irradiation of foods. We here examined the effect of a diet high in AGEs in the context of an irradiated diet on memory, insoluble Aβ₄₂, AGEs levels in hippocampus, on expression of the receptor for AGEs (RAGE), and on oxidative stress in the vasculature. We found that AD‐like model mice on high‐AGE diet due to irradiation had significantly poorer memory, higher hippocampal levels of insoluble Aβ₄₂ and AGEs as well as higher levels of oxidative stress on vascular walls, compared to littermates fed an isocaloric diet. These differences were not due to weight gain. The data were further supported by the overexpression of RAGE, which binds to Aβ₄₂ and regulates its transport across the blood–brain barrier, suggesting a mediating pathway. Because exposure to AGEs can be diminished, these insights provide an important simple noninvasive potential therapeutic strategy for alleviating a major lifestyle‐linked disease epidemic.     

The Application of MRI for Depiction of Subtle Blood Brain Barrier Disruption in Stroke

The development of imaging methodologies for detecting blood-brain-barrier (BBB) disruption may help predict stroke patient''s propensity to develop hemorrhagic complications following reperfusion. We have developed a delayed contrast extravasation MRI-based methodology enabling real-time depiction of subtle BBB abnormalities in humans with high sensitivity to BBB disruption and high spatial resolution. The increased sensitivity to subtle BBB disruption is obtained by acquiring T1-weighted MRI at relatively long delays (~15 minutes) after contrast injection and subtracting from them images acquired immediately after contrast administration. In addition, the relatively long delays allow for acquisition of high resolution images resulting in high resolution BBB disruption maps. The sensitivity is further increased by image preprocessing with corrections for intensity variations and with whole body (rigid+elastic) registration. Since only two separate time points are required, the time between the two acquisitions can be used for acquiring routine clinical data, keeping the total imaging time to a minimum.A proof of concept study was performed in 34 patients with ischemic stroke and 2 patients with brain metastases undergoing high resolution T1-weighted MRI acquired at 3 time points after contrast injection. The MR images were pre-processed and subtracted to produce BBB disruption maps. BBB maps of patients with brain metastases and ischemic stroke presented different patterns of BBB opening. The significant advantage of the long extravasation time was demonstrated by a dynamic-contrast-enhancement study performed continuously for 18 min. The high sensitivity of our methodology enabled depiction of clear BBB disruption in 27% of the stroke patients who did not have abnormalities on conventional contrast-enhanced MRI. In 36% of the patients, who had abnormalities detectable by conventional MRI, the BBB disruption volumes were significantly larger in the maps than in conventional MRI.These results demonstrate the advantages of delayed contrast extravasation in increasing the sensitivity to subtle BBB disruption in ischemic stroke patients. The calculated disruption maps provide clear depiction of significant volumes of BBB disruption unattainable by conventional contrast-enhanced MRI.     

The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa

Several proteases are secreted by Pseudomonas aeruginosa including elastase, an abundantly secreted neutral zinc-metalloprotease. Elastase (encoded by lasB) is first synthesized with a relatively large propeptide (18 kDa) domain. Here, we present evidence that this propeptide functions as an intramolecular chaperone (IMC) essential for proper maturation of elastase into a hydrolytically active enzyme. An altered elastase allele (lasB6) that encoded an elastase precursor with a precise propeptide deletion was expressed in Escherichia coli, and disrupted cells contained only inactive elastase. However, co-expression of an allele (lasB7) expressing the propeptide as an independent, non-covalently linked protein rescued about one-third of the hydrolytic activity when compared with that obtained with wild-type lasB. Thus, the propeptide was essential for elastase activity and so defined elastase as an IMC-containing protease. We examined the possibility that the propeptide of elastase also plays a role in the localization of the mature protein past the outer bacterial membrane. Expression of lasB6 in P. aeruginosa (lasBΔ) in the absence of the propeptide resulted in production of inactive elastase that accumulated within the cell and was not secreted to the culture medium. When lasB7 co-expressed the non-covalently linked propeptide in the same cell with lasB6, efficient secretion was restored and active elastase was then found in the supernatant. Thus, the propeptide was needed for secretion of the mature protein as well as enzymatic activity. This chaperone-like activity of the propeptide appears to involve a direct interaction between the mature and propeptide sequences, and evidence for this was obtained by demonstrating that the non-covalently attached 18 kDa propeptide was co-precipitated with elastase using elastase antibodies. These results are consistent with a hypothesis that the propeptide domain acts as an IMC to control both enzymatic activity and competence for secretion.     

Coordinate expression of the endogenous p53 gene in beta cells of transgenic mice expressing hybrid insulin-SV40 T antigen genes.

The expression of p53 has been evaluated during oncogenesis of the pancreatic beta cells in transgenic mice harboring hybrid insulin-SV40 T antigen genes. Significant levels of p53 are detected in all cells expressing large T antigen. In contrast, the protein is undetectable in normal beta cells. There is a complete correspondence between the onset of expression of T antigen and the appearance of the endogenous p53 protein. In tumors, the two proteins are found in a complex. In addition, free uncomplexed T antigen is detected in every cell which expresses the transgene. These results are consistent with the participation of p53 in T antigen-induced tumorigenesis in vivo. The early appearance of p53 in all beta cells expressing large T cannot readily explain the progression of a small fraction of these cells into solid tumors.     

Baculovirus p35 increases pancreatic beta-cell resistance to apoptosis

beta-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in beta-cells exposed to cytokines and hIAPP, as well as the effects of p35 on beta-cell function. The p35 gene was introduced into betaTC-tet cells, a differentiated murine beta-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted beta-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from beta-cell damage caused by hIAPP in type 2 diabetes.     

Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts

Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-alpha decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-alpha diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is down-regulated by TNF-alpha in a concentration- and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-alpha does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.