Detection of high molecular weight amyloid protein complexes in patient sera using Biological On-Line Tracer Sedimentation (BOLTS)

The systemic amyloidoses are a rare but deadly class of protein folding disorders with significant unmet diagnostic and therapeutic needs. The current model for symptomatic amyloid progression includes a causative role for soluble toxic aggregates as well as for the fibrillar tissue deposits. Although much research is focused on elucidating the potential mechanism of aggregate toxicity, evidence to support their existence in vivo has been limited. We report the use of a technique we have termed biological on-line tracer sedimentation (BOLTS) to detect abnormal high-molecular-weight complexes (HMWCs) in serum samples from individuals with systemic amyloidosis due to aggregation and deposition of wild-type transthyretin (senile systemic amyloidosis, SSA) or monoclonal immunoglobulin light chain (AL amyloidosis). In this proof-of-concept study, HMWCs were observed in 31 of 77 amyloid samples (40.3%). HMWCs were not detected in any of the 17 nonamyloid control samples subjected to BOLTS analyses. These findings support the existence of potentially toxic amyloid aggregates and suggest that BOLTS may be a useful analytic and diagnostic platform in the study of the amyloidoses or other diseases where abnormal molecular complexes are formed in serum

Translating intracarotid artery transplantation of bone marrow-derived NCS-01 cells for ischemic stroke: Behavioral and histological readouts and mechanistic insights into stem cell therapy

The present study used in vitro and in vivo stroke models to demonstrate the safety, efficacy, and mechanism of action of adult human bone marrow-derived NCS-01 cells. Coculture with NCS-01 cells protected primary rat cortical cells or human neural progenitor cells from oxygen glucose deprivation. Adult rats that were subjected to middle cerebral artery occlusion, transiently or permanently, and subsequently received intracarotid artery or intravenous transplants of NCS-01 cells displayed dose-dependent improvements in motor and neurological behaviors, and reductions in infarct area and peri-infarct cell loss, much better than intravenous administration. The optimal dose was 7.5 × 106 cells/mL when delivered via the intracarotid artery within 3 days poststroke, although therapeutic effects persisted even when administered at 1 week after stroke. Compared with other mesenchymal stem cells, NCS-01 cells ameliorated both the structural and functional deficits after stroke through a broad therapeutic window. NCS-01 cells secreted therapeutic molecules, such as basic fibroblast growth factor and interleukin-6, but equally importantly we observed for the first time the formation of filopodia by NCS-01 cells under stroke conditions, characterized by cadherin-positive processes extending from the stem cells toward the ischemic cells. Collectively, the present efficacy readouts and the novel filopodia-mediated mechanism of action provide solid lab-to-clinic evidence supporting the use of NCS-01 cells for treatment of stroke in the clinical setting

Detection of high-molecular-weight amyloid serum protein complexes using biological on-line tracer sedimentation

The systemic amyloidoses are a rare, but deadly class of protein folding disorders with significant unmet diagnostic and therapeutic needs. The current model for symptomatic amyloid progression includes a causative role for soluble toxic aggregates as well as for the fibrillar tissue deposits. Although much research is focused on elucidating the potential mechanism of aggregate toxicity, evidence to support their existence in vivo has been limited. We report the use of a technique we have termed Biological On-Line Tracer Sedimentation (BOLTS) to detect abnormal high molecular weight complexes (HMWCs) in serum samples from individuals with systemic amyloidosis due to aggregation and deposition of wild-type transthyretin (senile systemic amyloidosis, SSA) or monoclonal immunoglobulin light chain (AL amyloidosis). In this proof-of-concept study, HMWCs were observed in 31 out of 77 amyloid samples (40.3%). HMWCs were not detected in any of the 17 non-amyloid control samples subjected to BOLTS analyses. These findings support the existence of potentially toxic amyloid aggregates, and suggest that BOLTS may be a useful analytic and diagnostic platform in the study of the amyloidoses or other diseases where abnormal molecular complexes are formed in serum

Spleen Participation in Partial MHC Class II Construct Neuroprotection in Stroke

Pathological progression of stroke in the peripheral and central nervous systems (PNS and CNS) is characterized by multiple converging signalling pathways that exacerbate neuroinflammation-mediated secondary cell death. This creates a need for a novel type of immunotherapy capable of simultaneously lowering the synergistic inflammatory responses in the PNS and CNS, specifically the spleen and brain. Previously, we demonstrated that partial major histocompatibility complex (MHC) class II constructs can be administered subcutaneously to promote histological and behavioural effects that alleviate common symptoms found in a murine model of transient stroke. This MHC class II manipulates T cell cytokine expression in both PNS and CNS, resulting in dampened inflammation. In our long-standing efforts towards translational research, we recently demonstrated that a potent next generation mouse-based partial MHC class II construct named DRmQ (DRa1L50Q-mMOG-35-55) similarly induces neuroprotection in stroke rats, replicating the therapeutic effects of the human homolog as DRhQ (DRa1L50Q-human (h)MOG-35-55) in stroke mice. Our preclinical studies showed that DRmQ reduces motor deficits, infarct volume and peri-infarct cell loss by targeting inflammation in this second species. Moreover, we provided mechanistic support in both animal studies that partial MHC class II constructs effectively modulate the spleen, an organ which plays a critical role in modulating secondary cell death. Together, these preclinical studies satisfy testing the constructs in two stroke models, which is a major criterion of the Stroke Therapy Academic Industry Roundtable (STAIR) criteria and a key step in effectively translating this drug to the clinic. Additional translational studies, including dose-response and larger animal models may be warranted to bring MHC class II constructs closer to the clinic

Spleen participation in partial MHC class II construct neuroprotection in stroke

Pathological progression of stroke in the peripheral and central nervous systems (PNS and CNS) is characterized by multiple converging signalling pathways that exacerbate neuroinflammation-mediated secondary cell death. This creates a need for a novel type of immunotherapy capable of simultaneously lowering the synergistic inflammatory responses in the PNS and CNS, specifically the spleen and brain. Previously, we demonstrated that partial major histocompatibility complex (MHC) class II constructs can be administered subcutaneously to promote histological and behavioural effects that alleviate common symptoms found in a murine model of transient stroke. This MHC class II manipulates T cell cytokine expression in both PNS and CNS, resulting in dampened inflammation. In our long-standing efforts towards translational research, we recently demonstrated that a potent next generation mouse-based partial MHC class II construct named DRmQ (DRa1L50Q-mMOG-35-55) similarly induces neuroprotection in stroke rats, replicating the therapeutic effects of the human homolog as DRhQ (DRa1L50Q-human (h)MOG-35-55) in stroke mice. Our preclinical studies showed that DRmQ reduces motor deficits, infarct volume and peri-infarct cell loss by targeting inflammation in this second species. Moreover, we provided mechanistic support in both animal studies that partial MHC class II constructs effectively modulate the spleen, an organ which plays a critical role in modulating secondary cell death. Together, these preclinical studies satisfy testing the constructs in two stroke models, which is a major criterion of the Stroke Therapy Academic Industry Roundtable (STAIR) criteria and a key step in effectively translating this drug to the clinic. Additional translational studies, including dose-response and larger animal models may be warranted to bring MHC class II constructs closer to the clinic

Spleen Participation in Partial MHC Class II Construct Neuroprotection in Stroke

Pathological progression of stroke in the peripheral and central nervous systems (PNS and CNS) is characterized by multiple converging signalling pathways that exacerbate neuroinflammation-mediated secondary cell death. This creates a need for a novel type of immunotherapy capable of simultaneously lowering the synergistic inflammatory responses in the PNS and CNS, specifically the spleen and brain. Previously, we demonstrated that partial major histocompatibility complex (MHC) class II constructs can be administered subcutaneously to promote histological and behavioural effects that alleviate common symptoms found in a murine model of transient stroke. This MHC class II manipulates T cell cytokine expression in both PNS and CNS, resulting in dampened inflammation. In our long-standing efforts towards translational research, we recently demonstrated that a potent next generation mouse-based partial MHC class II construct named DRmQ (DRa1L50Q-mMOG-35-55) similarly induces neuroprotection in stroke rats, replicating the therapeutic effects of the human homolog as DRhQ (DRa1L50Q-human (h)MOG-35-55) in stroke mice. Our preclinical studies showed that DRmQ reduces motor deficits, infarct volume and peri-infarct cell loss by targeting inflammation in this second species. Moreover, we provided mechanistic support in both animal studies that partial MHC class II constructs effectively modulate the spleen, an organ which plays a critical role in modulating secondary cell death. Together, these preclinical studies satisfy testing the constructs in two stroke models, which is a major criterion of the Stroke Therapy Academic Industry Roundtable (STAIR) criteria and a key step in effectively translating this drug to the clinic. Additional translational studies, including dose-response and larger animal models may be warranted to bring MHC class II constructs closer to the clinic

Valence and anion binding of bovine ribonuclease A between pH 6 and 8

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis–Tris propane over a pH range of 6.00–8.00. The results of data analysis using a Debye–Hückel–Henry model, compared with expectations based on pKA values, are consistent with the binding of two chlorides by RNase A. The decreased protein valence resulting from anion binding contributes 2–3 kJ/mol to protein stabilization. This work demonstrates the utility of first-principle valence determinations to detect protein solution properties that might otherwise remain undetected

Eyeballing Stroke: Blood Flow Alterations in the Eye and Visual Impairments Following Transient Middle Cerebral Artery Occlusion in Adult Rats

Middle cerebral artery occlusion in rodents remains a widely used model of ischemic stroke. Recently, we reported the occurrence of retinal ischemia in animals subjected to middle cerebral artery occlusion, owing in part to the circulatory juxtaposition of the ophthalmic artery to the middle cerebral artery. In this study, we examined the eye hemodynamics and visual deficits in middle cerebral artery occlusion-induced stroke rats. The brain and eye were evaluated by laser Doppler at baseline (prior to middle cerebral artery occlusion), during and after middle cerebral artery occlusion. Retinal function-relevant behavioral and histological outcomes were performed at 3 and 14 days post-middle cerebral artery occlusion. Laser Doppler revealed a typical reduction of at least 80% in the ipsilateral frontoparietal cortical area of the brain during middle cerebral artery occlusion compared to baseline, which returned to near-baseline levels during reperfusion. Retinal perfusion defects closely paralleled the timing of cerebral blood flow alterations in the acute stages of middle cerebral artery occlusion in adult rats, characterized by a significant blood flow defect in the ipsilateral eye with at least 90% reduction during middle cerebral artery occlusion compared to baseline, which was restored to near-baseline levels during reperfusion. Moreover, retinal ganglion cell density and optic nerve depth were significantly decreased in the ipsilateral eye. In addition, the stroke rats displayed eye closure. Behavioral performance in a light stimulus-mediated avoidance test was significantly impaired in middle cerebral artery occlusion rats compared to control animals. In view of visual deficits in stroke patients, closely monitoring of brain and retinal perfusion via laser Doppler measurements and examination of visual impairments may facilitate the diagnosis and the treatment of stroke, including retinal ischemia