Biomarkers in the diagnosis and study of psychogenic nonepileptic seizures: A systematic review.

OBJECTIVE: Video electroencephalography (vEEG) is the gold-standard method for diagnosing psychogenic nonepileptic seizures (PNES), but such assessment is expensive, unavailable in many centers, requires prolonged hospitalization, and many times is unable to capture an actual seizure episode. This paper systematically reviews other non-vEEG candidate biomarkers that may facilitate both diagnosis and study of PNES as differentiated from epileptic seizures (ES). METHODS: PubMed database was searched to identify articles between 1980 and 2015 (inclusion: adult PNES population with or without controls, English language; exclusion: review articles, meta-analyses, single case reports). RESULTS: A total of 49 studies were examined, including neuroimaging, autonomic nervous system, prolactin, other (non-prolactin) hormonal, enzyme, and miscellaneous marker studies. Functional MRI studies have shown PNES is hyperlinked with dissociation and emotional dysregulation centers in the brain, although conflicting findings are seen across studies and none used psychiatric comparators. Heart rate variability suggests increased vagal tone in PNES when compared to ES. Prolactin is elevated in ES but not PNES, although shows low diagnostic sensitivity. Postictal cortisol and creatine kinase are nonspecific. Other miscellaneous biomarkers (neuron specific enolase, brain derived neurotropic factor, ghrelin, leptin, leukocytosis) showed no conclusive evidence of utility. Many studies are limited by lack of psychiatric comparators, size, and other methodological issues. CONCLUSION: No single biomarker successfully differentiates PNES from ES; in fact, PNES is only diagnosed via the negation of ES. Clinical assessment and rigorous investigation of psychosocial variables specific to PNES remain critical, and subtyping of PNES is warranted. Future investigational and clinical imperatives are discussed.

The effects of aging on dopaminergic neurotransmission: a microPET study of [11C]-raclopride binding in the aged rodent brain.

Rodent models are frequently used in aging research to investigate biochemical age effects and aid in the development of therapies for pathological and non-pathological age-related degenerative processes. In order to validate the use of animal models in aging research and pave the way for longitudinal intervention-based animal studies, the consistency of cerebral aging processes across species needs to be evaluated. The dopaminergic system seems particularly susceptible to the aging process, and one of the most consistent findings in human brain aging research is a decline in striatal D2-like receptor (D2R) availability, quantifiable by positron emission tomography (PET) imaging. In this study, we aimed to assess whether similar age effects can be discerned in rat brains, using in vivo molecular imaging with the radioactive compound [(11)C]-raclopride. We observed a robust decline in striatal [(11)C]-raclopride uptake in the aged rats in comparison to the young control group, comprising a 41% decrement in striatal binding potential. In accordance with human studies, these results indicate that substantial reductions in D2R availability can be measured in the aged striatal complex. Our findings suggest that rat and human brains exhibit similar biochemical alterations with age in the striatal dopaminergic system, providing support for the pertinence of rodent models in aging research.

Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth.

FMS-like tyrosine kinase 3 (FLT3) receptor is highly expressed in an array of hematological malignancies including approximately 90% of acute myelogenous leukemia (AML). Ligand stimulation of the receptor promotes the survival and proliferation of leukemia cells. Strategies targeting FLT3 using monoclonal antibodies may therefore constitute an effective therapeutic approach for these leukemia. Towards this, we selected a naïve antibody phage display library on both recombinant FLT3 receptor protein and FLT3-expressing leukemia cells using a tailored selection scheme that was designed to isolate antagonistic phage antibodies that not only interfere with receptor/ligand binding but also trigger receptor internalization upon cell surface binding. Phage antibodies were screened first for their ability to bind to cell surface receptor and induce receptor internalization, followed by their activity in blocking ligand-receptor interaction and neutralizing ligand-stimulated receptor activation and cell proliferation. We identified three fully human antibodies, EB10, A2IN, and D4-3, which bound specifically to both soluble and cell surface-expressed FLT3. All three antibodies were shown to be internalized upon binding to cell surface-expressed receptor in a time-dependent fashion. EB10 and D4-3 blocked ligand binding to the receptor with IC(50)s of 14 and 7 nM, respectively. Further, EB10 and D4-3 inhibited FLT3 ligand-induced receptor phosphorylation and cell proliferation in EOL-1 leukemia cells. Taken together, these results suggest that both EB10 and D4-3 may represent excellent therapeutic candidates for the treatment of FLT3-expressing human leukemia, both as unmodified antibodies and as conjugates of cytotoxic agents.

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor.

Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.

An efficient route to the production of an IgG-like bispecific antibody.

Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human kappa chain (C(L)) and the first constant domain of human heavy chain (C(H1)), to form two polypeptides, (scFv)(1)-C(L) and (scFv)(2)-C(H1)-C(H2)-C(H3), respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)(4)-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)(4)-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)(4)-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)(4)-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.

Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2.

Bispecific antibody (BsAb) technology has been successfully used as a means to construct novel antibody (Ab) molecules with increased avidity for binding, by combining two Ab or their fragments directed against different epitopes within the same antigen. Using two single chain antibodies (scFv) isolated from a phage display library, we have constructed a bispecific diabody directed against two different epitopes on the extracellular domain (ECD) of human vascular endothelial growth factor receptor 2 (VEGFR2), the kinase-insert domain-containing receptor (KDR). Neither of the parent scFv blocks KDR/VEGF interactions or inhibits VEGF-induced receptor activation. The diabody binds to KDR with an affinity that is 1.5- to 3-fold higher than its parent scFv, mainly due to a much slower dissociation rate (k(off)), which is approximately 17- to 26-fold slower than that of the individual scFv. In addition, the diabody binds simultaneously to, and thus cross-links, the two epitopes on the receptor(s). It is rather unexpected that the diabody effectively blocked KDR/VEGF interactions, and inhibited both VEGF-induced activation of the receptor and mitogenesis of human endothelial cells. Taken together, our results suggest that the diabody is most likely to exert its effect through steric hindrance and/or causing major conformational changes of the receptor. This is the first report on the construction of a bispecific diabody with acquired novel antagonistic activity.

Inhibition of vascular endothelial growth factor induced mitogenesis of human endothelial cells by a chimeric anti-kinase insert domain-containing receptor antibody.

The kinase insert domain-containing receptor (KDR) is the human vascular endothelial growth factor (VEGF) receptor responsible for the mitogenic and angiogenic effects of VEGF. There is much experimental evidence to suggest that the VEGF/KDR pathway plays an important role in tumor angiogenesis, a process essential for tumor growth and metastasis. Here we produced a chimeric anti-KDR antibody (IgG1), c-p1C11, from a single chain (scFv) antibody isolated from a phage display library. C-p1C11 binds specifically to the extracellular domain of soluble as well as cell-surface expressed KDR. It effectively blocks VEGF-KDR interaction and inhibits VEGF-stimulated activation of KDR and MAP kinases p44/p42 of human endothelial cells. Furthermore, c-p1C11 efficiently neutralizes VEGF-induced mitogenesis of human endothelial cells. Our results suggest that antibodies against KDR have potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.