Physiological effects of dual target DBS in an individual with Parkinson's disease and a sensing-enabled pulse generator.

INTRODUCTION: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or globus pallidus (GP) is an established therapy for Parkinson's disease (PD). Novel DBS devices can record local field potential (LFP) physiomarkers from the STN or GP. While beta (13-30 Hz) and gamma (40-90 Hz) STN and GP LFP oscillations correlate with PD motor severity and with therapeutic effects of treatments, STN-GP interactions in electrophysiology in patients with PD are not well characterized. METHODS: Simultaneous bilateral STN and GP LFPs were recorded in a patient with PD who received bilateral STN-DBS and GP-DBS. Power spectra in each target and STN-GP coherence were assessed in various ON- and OFF-levodopa and DBS states, both at rest and with voluntary movement. RESULTS: OFF-levodopa and OFF-DBS, beta peaks were present at bilateral STN and GP, coincident with prominent STN-GP beta coherence. Levodopa and dual-target-DBS (simultaneous STN-DBS and GP-DBS) completely suppressed STN-GP coherence. Finely-tuned gamma (FTG) activity at half the stimulation frequency (62.5 Hz) was seen in the STN during GP-DBS at rest. To assess the effects of movement on FTG activity, we recorded LFPs during instructed movement. We observed FTG activity in bilateral GP and bilateral STN during contralateral body movements while on GP-DBS and ON-levodopa. No FTG was seen with STN-DBS or dual-target-DBS. CONCLUSION: Dual-target-DBS and levodopa suppressed STN-GP coherence. FTG throughout the basal ganglia was induced by GP-DBS in the presence of levodopa and movement. This bilateral STN-FTG and GP-FTG corresponded with the least severe bradykinesia state, suggesting a pro-kinetic role for FTG.

A2B Adenosine Receptor Enhances Chemoresistance of Glioblastoma Stem-Like Cells under Hypoxia: New Insights into MRP3 Transporter Function.

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Medicinal Immunoglobulin G Products (2020) Show High Infectivity Neutralizing Activity Against Seasonal Influenza Virus Strains Selected for Future Vaccines (2020-2022).

Immunoglobulin (Ig)G medicinal products manufactured in 2020 were tested for infectivity neutralization and hemagglutination inhibition against World Health Organization-selected influenza strains included in worldwide vaccines 2020-2022. The IgG batches (from US plasma) showed potent activity. Intravenous immunoglobulin could potentially add to therapies for serious influenza cases in immunocompromised patients. Further study is warranted.

Effective presence of antibodies against common human coronaviruses in immunoglobulin medicinal products.

BACKGROUND: Immunoglobulin products (for intravenous, intramuscular and subcutaneous administration) prepared from geographically diverse plasma pools were tested for activity against common human coronaviruses (HCoVs). Products from plasma obtained from Germany, Czech Republic, Slovak Republic, USA and Spain were tested for antibodies to common HCoVs: 229E, OC43, NL63 and HKU1. As these products are manufactured from pooled plasma from thousands of donors, the antibodies therein are representative of HCoV exposure in the population at large. METHODS: Immunoglobulin products were tested for antibodies to four common HCoVs by enzyme-linked immunosorbent assays (ELISAs). Neutralization assays were conducted using HCoV-229E cultured on to MRC5 cells. RESULTS: ELISAs showed that when expressed as specific activity (anti-HCoV activity/mg immunoglobulin), similar activity against the four common HCoVs was seen across the immunoglobulin products regardless of concentration or geographic origin. Highest anti-HCoV activity was seen against HCoV-229E, followed by HCoV-OC43, HCoV-NL63 and HCoV-HKU1. The neutralization assays showed similar potency for two immunoglobulin products prepared by different processes. CONCLUSIONS: To the authors' knowledge, this is the first demonstration of antibodies to common HCoVs in immunoglobulin products. These results may explain the cross-reactivity seen with pre-pandemic immunoglobulin products and severe acute respiratory syndrome coronavirus-2, and contribute to differences in severity of illness between patients.

Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Hyperimmune Immunoglobulin Demonstrates Potent Neutralization and Antibody-Dependent Cellular Cytotoxicity and Phagocytosis Through N and S Proteins.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) vaccinations have provided a significant reduction in infections, effective COVID-19 treatments remain an urgent need. METHODS: Functional characterization of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hyperimmune immunoglobulin (hIG) from human convalescent plasma was performed by different virus neutralization methodologies (plaque reduction, virus-induced cytotoxicity, median tissue culture infectious dose [TCID50] reduction, and immunofluorimetry) at different laboratories using geographically different SARS-CoV-2 isolates (USA [1], Italy [1], and Spain [2]; 2 containing the D614G mutation). Neutralization capacity against the original Wuhan SARS-CoV-2 strain and variants (D614G mutant, B.1.1.7, P.1, and B.1.351) was evaluated using a pseudovirus expressing the corresponding spike (S) protein. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) was also evaluated. RESULTS: All SARS-CoV-2 isolates were potently neutralized by hIG as shown by all 4 methodologies. Wild-type SARS-CoV-2 and variants were effectively neutralized using the pseudovirus. The hIG (IgG type) induced ADCC and ADCP against SARS-CoV-2 N and S proteins but not E protein. Very low concentrations (25-100 µg IgG/mL) were required. A potent effect was triggered by antibodies in hIG solutions against the SARS-CoV-2 S and N proteins. CONCLUSIONS: Beyond neutralization, IgG Fc-dependent pathways may play a role in combatting SARS-CoV-2 infections using COVID-19 hIG. This could be especially relevant for the treatment of more neutralization-resistant SARS-CoV-2 variants.

Cross-neutralization activity against SARS-CoV-2 is present in currently available intravenous immunoglobulins.

Background: Cross-reactivity against human coronaviruses with Flebogamma® DIF and Gamunex®-C, two available intravenous immunoglobulins (IVIG), has been reported. In this study, these IVIG were tested for neutralization activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). Materials & methods: Neutralization capacity of lots of IVIG manufactured prior to COVID-19 pandemic was assessed against these viruses in cell culture. Infectivity neutralization was quantified by percent reduction in plaque-forming units and/or cytopathic/cytotoxic methods. Results: All IVIG preparations showed neutralization of SARS-CoV-2 isolates. All IVIG lots produced neutralization of SARS-CoV. No IVIG preparation showed significant neutralizing activity against MERS-CoV. Conclusion: The tested IVIG contain antibodies with significant in vitro cross-neutralization capacity against SARS-CoV-2 and SARS-CoV, but not MERS-CoV. These preparations are currently under evaluation as potential therapies for COVID-19.

Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics: 20 years' experience of the plasma protein manufacturers.

BACKGROUND: Nanofiltration entails the filtering of protein solutions through membranes with pores of nanometric sizes that have the capability to effectively retain a wide range of viruses. STUDY DESIGN AND METHODS: Data were collected from 754 virus validation studies (individual data points) by Plasma Protein Therapeutics Association member companies and analyzed for the capacity of a range of nanofilters to remove viruses with different physicochemical properties and sizes. Different plasma product intermediates were spiked with viruses and filtered through nanofilters with different pore sizes using either tangential or dead-end mode under constant pressure or constant flow. Filtration was performed according to validated scaled-down laboratory conditions reflecting manufacturing processes. Effectiveness of viral removal was assessed using cell culture infectivity assays or polymerase chain reaction (PCR). RESULTS: The nanofiltration process demonstrated a high efficacy and robustness for virus removal. The main factors affecting nanofiltration efficacy are nanofilter pore size and virus size. The capacity of nanofilters to remove smaller, nonenveloped viruses was dependent on filter pore size and whether the nanofiltration process was integrated and designed with the intention to provide effective parvovirus retention. Volume filtered, operating pressure, and total protein concentration did not have a significant impact on the effectiveness of virus removal capacity within the investigated ranges. CONCLUSIONS: The largest and most diverse nanofiltration data collection to date substantiates the effectiveness and robustness of nanofiltration in virus removal under manufacturing conditions of different plasma-derived proteins. Nanofiltration can enhance product safety by providing very high removal capacity of viruses including small non-enveloped viruses.

Currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens.

Aim: There is a critical need for effective therapies that are immediately available to control the spread of COVID-19 disease. Material & methods: Gamunex®-C and Flebogamma® DIF (Grifols) intravenous immunoglobulin (IVIG) products were tested using ELISA techniques for antibodies against several antigens of human common betacoronaviruses that may crossreact with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Results: Both IVIGs showed consistent reactivity to components of the tested viruses. Positive crossreactivity was seen in SARS-CoV, middle east respiratory syndrome-CoV and SARS-CoV-2. For SARS-CoV-2, positive reactivity was observed at IVIG concentrations ranging from 100 µg/ml with Gamunex-C to 1 mg/ml with Flebogamma 5% DIF. Conclusion: Gamunex-C and Flebogamma DIF contain antibodies reacting against SARS-CoV-2 antigens. Studies to confirm the utility of IVIG preparations for COVID-19 management may be warranted.

Pituitary Apoplexy: Results of Surgical and Conservative Management Clinical Series and Review of the Literature.

OBJECTIVE: Pituitary apoplexy is associated with visual, cranial nerve, and endocrine dysfunction. In this article, the results of surgical and conservative management of pituitary apoplexy in a single center are evaluated and a review of the literature is presented. METHODS: A retrospective analysis was made of patients with pituitary apoplexy who underwent surgery or conservative management at our center between January 2007 and June 2017. Surgery was typically selected for patients who presented with acute deterioration of visual status and/or level of consciousness. Patients with no visual field deficit and those who had medical contraindications to undergo a surgical procedure because of previous comorbidities typically had conservative treatment. Baseline characteristics and clinical and radiologic outcomes were reviewed. A review of the literature (1990-2018) was performed according to PRISMA guidelines. Studies comparing the results of conservative and surgical management were identified. Visual, cranial nerve, and endocrine outcomes and tumor recurrence were analyzed. RESULTS: Forty-nine patients (73.1%) were managed surgically and 18 (26.9%) conservatively. After careful case selection, patients underwent surgical or conservative treatment. Patients who underwent conservative treatment had fewer visual deficits. At diagnosis, visual deficit (38.8% vs. 75.5%; P = 0.008) and cranial nerve palsy (27.7% vs. 51%; P = 0.058) were less common in the conservative group. Conservative and surgical treatments had similar visual and cranial nerve improvement rates (75% vs. 58.3%, P = 0.63 and 75% vs. 69.2%, P = 1.0, respectively). In the conservative group, tumor shrinkage was observed in 76.4% of cases. The systematic review retrieved 11 studies. No significant difference between conservative and surgical treatment for clinical outcomes (visual field recovery, odds ratio [OR], 1.45; 95% confidence interval [CI], 0.72-2.92; cranial nerve recovery, OR, 2.30; 95% CI, 0.93-5.65; and hypopituitarism, OR, 1.05; 95% CI, 0.64-1.74) or tumor recurrence (OR, 0.68; 95% CI, 0.20-2.34) was observed. CONCLUSIONS: A tailored approach to pituitary apoplexy, one that does not include an absolute need for surgery, is appropriate. Conservative management is appropriate in selected patients presenting without visual deficits.

Relationship between infarct size and serum uric acid levels during the acute phase of stroke.

INTRODUCTION: Uric acid has gained considerable attention as a potential neuroprotective agent in stroke during the last decades, however, its role in the pathophysiology of ischemic stroke remains poorly understood. A serial evaluation of uric acid levels during the acute phase of stroke and its association with infarct size on magnetic resonance imaging is lacking. METHODS: We present a cohort study of 31 patients with ischemic stroke who were not candidates for thrombolysis according to current criteria at the time. We performed daily measurements of serum uric acid and total antioxidant capacity of plasma during the first week after symptoms onset and 30 days after. Infarct size was determined in the acute phase by a DWI sequence and the final infarct size with a control MRI (FLAIR) at day 30. RESULTS: Uric acid significantly decreases between days 2 to 6 compared to day 1, after adjustment by sex, age and DWI at diagnosis, with a nadir value at 72h. A mixed model analysis showed a negative association between DWI at diagnosis and uric acid evolution during the first week after stroke. Moreover, multivariable linear regression of uric acid values during follow up on DWI volumes demonstrated that DWI volume at diagnosis is negatively associated with uric acid levels at day 3 and 4. There were no significant associations between total antioxidant capacity of plasma and DWI at diagnosis, or FLAIR at any point. DISCUSSION: Patients with larger infarcts exhibited a significant decrease in serum uric acid levels, accounting for a more prominent reactive oxygen species scavenging activity with subsequent consumption and decay of this antioxidant. The different kinetics of total antioxidant capacity of plasma and serum uric acid levels suggests a specific role of uric acid in the antioxidant response in ischemic stroke.

Ethmoid Meningoencephalocele in a Patient with Cerebrofacial Arteriovenous Metameric Syndrome.

BACKGROUND: Skull base meningoencephaloceles are a rare condition, frequently secondary to traumatic or iatrogenic causes. Cerebrofacial arteriovenous metameric syndrome (CAMS) is characterized by the presence of retinal, facial, and cerebral arteriovenous malformations (AVMs) with metameric distribution. To our knowledge, this is the first reported case associating these 2 conditions. CASE DESCRIPTION: A 45-year-old woman previously diagnosed with CAMS type 2 presented with a long history of cerebrospinal fluid (CSF) rhinorrhea. Magnetic resonance imaging and digital subtraction angiography demonstrated a right-sided facial and orbital AVM extending posteriorly along the optic tract into the suprasellar cistern, and a right-sided meningoencephalocele protruding into the olfactory recess and ethmoid sinus. An extended endoscopic endonasal approach was performed to resect the meningoencephalocele and to repair the CSF leak without complications. CONCLUSIONS: We report the unusual association between the development of a meningoencephalocele and a metameric syndrome, and comment on clinical implications in the management of this patient.

Human mesenchymal stem cells maintain their phenotype, multipotentiality, and genetic stability when cultured using a defined xeno-free human plasma fraction.

BACKGROUND: Mesenchymal stem cells (MSCs) show promising characteristics for their use in advanced therapy medicinal products. However, there are some unresolved concerns, such as the use of animal components for their expansion. In this study we assessed the suitability of a xeno-free supplement for cell culture (SCC) derived from human plasma, to culture and expand human MSCs (hMSCs) from different origins. Characteristics of viable cultured hMSCs such as genetic stability, phenotype and multipotentiality were qualitatively evaluated. METHODS: hMSCs from adipose tissue (AT), bone marrow (BM) and umbilical cord (UC) and supplier sources (commercial/non-commercial) were used. After hMSCs expansion in a xeno-free medium, classical hMSCs markers were studied by immunocytochemistry, and genetic stability was tested by classic karyotyping. The capacity of hMSCs to differentiate into adipogenic, osteogenic, and chondrogenic cells in differentiation media was assessed using different staining. Different lots of SCC were used to assure consistency between batches. RESULTS: All hMSCs tested maintained their morphology and adherence to plastic during their expansion, and preserved their genetic stability, phenotype and differentiation potential. No differences were observed when using different lots of SCC. Moreover, the proliferation rate, evaluated as population doubling time (PDT) of commercial BM and AT hMSCs, was higher in the xeno-free medium than in the control media provided by the suppliers of the cells (PDT of 4.6 for BM-hMSC and 6.4 for AT-hMSC in xeno-free medium, and 7.0 and 14.7 respectively in the commercial media). UC-hMSCs PDT was similar in all the media tested. When using non-commercial BM-hMSCs, PDT was lower in the xeno-free medium, but reverted to the control level with the addition of growth factors. CONCLUSIONS: SCC-containing medium can be a feasible xeno-free alternative to expand hMSCs for advanced therapies.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

INTRODUCTION: Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. METHODS: SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. RESULTS: SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. CONCLUSIONS: The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Novel therapeutic strategies for traumatic brain injury: acute antioxidant reinforcement.

Traumatic brain injury (TBI) is the most important cause of disability in individuals under the age of 45 years and thus represents a significant social and economic burden. Evidence strongly suggests that oxidative stress is a cornerstone event leading to and propagating secondary injury mechanisms such as excitotoxicity, mitochondrial dysfunction, apoptosis, autophagy, brain edema, and inflammation. TBI has defied conventional approaches to diagnosis and therapy development because of its heterogeneity and complexity. Therefore, it is necessary to explore alternative approaches to therapy development for TBI. The aim of this review is to present a therapeutic approach for TBI, taking into account the evidence supporting the role for oxidative stress in the pathophysiological processes of secondary brain injury. The role of agents such as mitochondria-targeted antioxidants (melatonin and new mitochondria-targeted antioxidants), nicotinamide adenine dinucleotide phosphate (NADPH) inhibitors (antioxidant vitamins and apocynin), and other compounds having mainly antioxidant properties (hydrogen-rich saline, sulforaphane, U-83836E, omega-3, and polyphenols) is covered. The rationale for innovative antioxidant therapies based on current knowledge and particularly the most recent studies regarding this field is discussed. Particular considerations and translational potential of new TBI treatments are examined and a novel therapeutic proposal for TBI is presented.

Robustness of nanofiltration for increasing the viral safety margin of biological products.

In this study, the virus-removal capacity of nanofiltration was assessed using validated laboratory scale models on a wide range of viruses (pseudorabies virus; human immunodeficiency virus; bovine viral diarrhea virus; West Nile virus; hepatitis A virus; murine encephalomyocarditis virus; and porcine parvovirus) with sizes from 18 nm to 200 nm and applying the different process conditions existing in a number of Grifols' plasma-derived manufacturing processes (thrombin, α1-proteinase inhibitor, Factor IX, antithrombin, plasmin, intravenous immunoglobulin, and fibrinogen). Spiking experiments (n = 133) were performed in process intermediate products, and removal was subsequently determined by infectivity titration. Reduction Factor (RF) was calculated by comparing the virus load before and after nanofiltration under each product purification condition. In all experiments, the RFs were close to or greater than 4 log10 (>99.99% of virus elimination). RF values were not significantly affected by the process conditions within the limits assayed (pH, ionic strength, temperature, filtration ratio, and protein concentration). The virus-removal capacity of nanofiltration correlated only with the size of the removed agent. In conclusion, nanofiltration, as used in the manufacturing of several Grifols' products, is consistent, robust, and not significantly affected by process conditions.

Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities.

Stroke is the second leading cause of death, after ischemic heart disease, and accounts for 9% of deaths worldwide. According to the World Health Organization [WHO], 15 million people suffer stroke worldwide each year. Of these, more than 6 million die and another 5 million are permanently disabled. Reactive oxygen species [ROS] have been implicated in brain injury after ischemic stroke. There is evidence that a rapid increase in the production of ROS immediately after acute ischemic stroke rapidly overwhelm antioxidant defences, causing further tissue damage. These ROS can damage cellular macromolecules leading to autophagy, apoptosis, and necrosis. Moreover, the rapid restoration of blood flow increases the level of tissue oxygenation and accountsfor a second burst of ROS generation, which leads to reperfusion injury. Current measures to protect the brain against severe stroke damage are insufficient. Thus, it is critical to investigate antioxidant strategies that lead to the diminution of oxidative injury. The antioxidant vitamins C and E, the polyphenol resveratrol, the xanthine oxidase [XO] inhibitor allopurinol, and other antioxidant strategies have been reviewed in the setting of strokes. This review focuses on the mechanisms involved in ROS generation, the role of oxidative stress in the pathogenesis of ischemic stroke, and the novel therapeutic strategies to be tested to reduce the cerebral damage related to both ischemia and reperfusion.