Functional effects of haemoglobin can be rescued by haptoglobin in an in vitro model of subarachnoid haemorrhage.

During subarachnoid haemorrhage, a blood clot forms in the subarachnoid space releasing extracellular haemoglobin (Hb), which causes oxidative damage and cell death in surrounding tissues. High rates of disability and cognitive decline in SAH survivors are attributed to loss of neurons and functional connections during secondary brain injury. Haptoglobin sequesters Hb for clearance, but this scavenging system is overwhelmed after a haemorrhage. Whilst exogenous haptoglobin application can attenuate cytotoxicity of Hb in vitro and in vivo, the functional effects of sub-lethal Hb concentrations on surviving neurons and whether cellular function can be protected with haptoglobin treatment remain unclear. Here we use cultured neurons to investigate neuronal health and function across a range of Hb concentrations to establish the thresholds for cellular damage and investigate synaptic function. Hb impairs ATP concentrations and cytoskeletal structure. At clinically relevant but sub-lethal Hb concentrations, we find that synaptic AMPAR-driven currents are reduced, accompanied by a reduction in GluA1 subunit expression. Haptoglobin co-application can prevent these deficits by scavenging free Hb to reduce it to sub-threshold concentrations and does not need to be present at stoichiometric amounts to achieve efficacy. Haptoglobin itself does not impair measures of neuronal health and function at any concentration tested. Our data highlight a role for Hb in modifying synaptic function in surviving neurons, which may link to impaired cognition or plasticity after SAH and support the development of haptoglobin as a therapy for subarachnoid haemorrhage.

Neurofilament light predicts neurological outcome after subarachnoid haemorrhage.

To improve outcome prediction following subarachnoid haemorrhage (SAH), we sought a biomarker integrating early brain injury and multiple secondary pathological processes in a prospective study of 42 non-traumatic SAH patients and 19 control individuals. Neurofilament light (NF-L) was elevated in CSF and serum following SAH. CSF and serum NF-L on Days 1-3 post-SAH strongly predicted modified Rankin score at 6 months, independent of World Federation of Neurosurgical Societies (WFNS) score. NF-L from Day 4 onwards also had a profound impact on outcome. To link NF-L to a SAH-specific pathological process, we investigated NF-L's relationship with extracellular haemoglobin. Most CSF haemoglobin was not complexed with haptoglobin, yet was able to be bound by exogenous haptoglobin i.e. haemoglobin was scavengeable. CSF scavengeable haemoglobin was strongly predictive of subsequent CSF NF-L. Next, we investigated NF-L efflux from the brain after SAH. Serum and CSF NF-L correlated positively. The serum/CSF NF-L ratio was lower in SAH versus control subjects, in keeping with glymphatic efflux dysfunction after SAH. CSF/serum albumin ratio was increased following SAH versus controls. The serum/CSF NF-L ratio correlated negatively with the CSF/serum albumin ratio, indicating that transfer of the two proteins across the blood-brain interface is dissociated. In summary, NF-L is a strong predictive marker for SAH clinical outcome, adding value to the WFNS score, and is a promising surrogate end point in clinical trials.

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.

Haemoglobin causes neuronal damage in vivo which is preventable by haptoglobin.

After subarachnoid haemorrhage, prolonged exposure to toxic extracellular haemoglobin occurs in the brain. Here, we investigate the role of haemoglobin neurotoxicity in vivo and its prevention. In humans after subarachnoid haemorrhage, haemoglobin in cerebrospinal fluid was associated with neurofilament light chain, a marker of neuronal damage. Most haemoglobin was not complexed with haptoglobin, an endogenous haemoglobin scavenger present at very low concentration in the brain. Exogenously added haptoglobin bound most uncomplexed haemoglobin, in the first 2 weeks after human subarachnoid haemorrhage, indicating a wide therapeutic window. In mice, the behavioural, vascular, cellular and molecular changes seen after human subarachnoid haemorrhage were recapitulated by modelling a single aspect of subarachnoid haemorrhage: prolonged intrathecal exposure to haemoglobin. Haemoglobin-induced behavioural deficits and astrocytic, microglial and synaptic changes were attenuated by haptoglobin. Haptoglobin treatment did not attenuate large-vessel vasospasm, yet improved clinical outcome by restricting diffusion of haemoglobin into the parenchyma and reducing small-vessel vasospasm. In summary, haemoglobin toxicity is of clinical importance and preventable by haptoglobin, independent of large-vessel vasospasm.

Development of an intravenous immunoglobulin with improved safety and functional activity.

The development and properties of a liquid intravenous immunoglobulin (Gammaplex(®)), of high purity, stability and functional activity, is described. Virus and TSE reduction by specific steps in the process were evaluated by spiking studies using small-scale models. The removal of procoagulant activity was determined using immunochemical and functional activity assays. Neutralisation and opsonic activity were used to demonstrate the functional activity of the IgG. The final low pH formulated product was stable at room temperature and was of high purity and functional activity. Three dedicated virus inactivation steps, i.e. solvent detergent, low pH and virus filtration, were shown to be effective. When combined with the B + I ethanol precipitation step, this gave a total reduction of >21 to >24 log for the enveloped and >10 to >13 log for the non-enveloped viruses tested. Several steps in the process were shown to contribute to TSE removal using scrapie. Potential procoagulant activity including Factor XI/XIa, was reduced to very low/undetectable levels in the final product. A new high purity liquid IVIG product has been developed, of high purity and good functional activity and stability. The process includes various steps for the removal of pathogens and procoagulant activity.

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.

Virus inactivation in albumin by a combination of alkali conditions and high temperature.

Non-enveloped viruses such as HAV and B19 are of potential concern in plasma products. In the case of albumin, pasteurisation at 60 °C for 10 h is generally used for virus inactivation. However this procedure is only partially effective against some non-enveloped viruses. Using a range of non-enveloped viruses i.e. HAV, SV40, CPV, treatment at a high pH of about 9.5 and a temperature of 60 °C for 10 h was found to be effective for virus inactivation. These extreme conditions caused no increase in aggregate composition of the albumin. In addition the albumin composition was stable over a period of at least 6 months. The ligand binding properties of the albumin, as determined using the dye phenol red, were also not affected by this treatment. This procedure has the potential for increasing the spectrum of viruses inactivated by the 60 °C pasteurisation step.