The Impact and Effects of Host Immunogenetics on Infectious Disease Studies Using Non-Human Primates in Biomedical Research.

Understanding infectious disease pathogenesis and evaluating novel candidate treatment interventions for human use frequently requires prior or parallel analysis in animal model systems. While rodent species are frequently applied in such studies, there are situations where non-human primate (NHP) species are advantageous or required. These include studies of animals that are anatomically more akin to humans, where there is a need to interrogate the complexity of more advanced biological systems or simply reflect susceptibility to a specific infectious agent.

Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate.

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery.

Distinct characteristics of microglia from neurogenic and non-neurogenic regions of the human brain in patients with Mesial Temporal Lobe Epilepsy.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas.

Single-cell image analysis reveals over-expression of organic anion transporting polypeptides (OATPs) in human glioblastoma tissue.

Background: Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Whilst the role of the efflux transporters are well established in GBM, the expression and function of uptake transporters, such as the organic anion transporting polypeptide (OATP) family, are not well understood. OATPs possess broad substrate specificity that includes anti-cancer agents; therefore, we sought to investigate the expression of four OATP isoforms in human GBM cell types using patient tumor tissue.

Elucidating the cellular uptake mechanisms of heptamethine cyanine dye analogues for their use as an anticancer drug-carrier molecule for the treatment of glioblastoma.

The development of chemotherapies for glioblastoma is hindered by their limited bioavailability and toxicity on normal brain function. To overcome these limitations, we investigated the structure-dependent activity of heptamethine cyanine dyes (HMCD), a group of tumour-specific and BBB permeable near-infrared fluorescent dyes, in both commercial (U87MG) and patient-derived GBM cell lines. HMCD analogues with strongly ionisable sulphonic acid groups were not taken up by patient-derived GBM cells, but were taken up by the U87MG cell line.

The amyotrophic lateral sclerosis-linked protein TDP-43 regulates interleukin-6 cytokine production by human brain pericytes.

Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS.

Characterisation of PDGF-BB:PDGFRβ signalling pathways in human brain pericytes: evidence of disruption in Alzheimer's disease.

Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-β (PDGFRβ) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRβ signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB.

Routine culture and study of adult human brain cells from neurosurgical specimens.

When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens.

Identifying Neural Progenitor Cells in the Adult Human Brain.

The discovery, in 1998, that the adult human brain contains at least two populations of progenitor cells and that progenitor cells are upregulated in response to a range of degenerative brain diseases has raised hopes for their use in replacing dying brain cells. Since these early findings, the race has been on to understand the biology of progenitor cells in the human brain, and they have now been isolated and studied in many major neurodegenerative diseases. Before these cells can be exploited for cell replacement purposes, it is important to understand how to (1) locate them, (2) label them, (3) determine what receptors they express, (4) isolate them, and (5) examine their electrophysiological properties when differentiated.

Cytoprotective agent troxipide-cyanine dye conjugate with cytotoxic and antiproliferative activity in patient-derived glioblastoma cell lines.

Cytoprotective agents are mainly used to protect the gastrointestinal tract linings and in the treatment of gastric ulcers. These agents are devoid of appreciable cytotoxic or cytostatic effects, and medicinal chemistry efforts to modify them into anticancer agents are rare. A drug repurposing campaign initiated in our laboratory with the primary focus of discovering brain cancer drugs resulted in drug-dye conjugate 1, a combination of the cytoprotective agent troxipide and heptamethine cyanine dye MHI 148.

Single-cell image analysis reveals a protective role for microglia in glioblastoma.

Background: Microglia and tumor-associated macrophages (TAMs) constitute up to half of the total tumor mass of glioblastomas. Despite these myeloid populations being ontogenetically distinct, they have been largely conflated. Recent single-cell transcriptomic studies have identified genes that distinguish microglia from TAMs.

Cardiac glycosides target barrier inflammation of the vasculature, meninges and choroid plexus.

Neuroinflammation is a key component of virtually all neurodegenerative diseases, preceding neuronal loss and associating directly with cognitive impairment. Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain vasculature, surrounding meninges and the choroid plexus. We designed a high content screening system to target inflammation in human brain-derived cells of the blood-brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers.

Isolation and culture of functional adult human neurons from neurosurgical brain specimens.

The ability to characterize and study primary neurons isolated directly from the adult human brain would greatly advance neuroscience research. However, significant challenges such as accessibility of human brain tissue and the lack of a robust neuronal cell culture protocol have hampered its progress. Here, we describe a simple and reproducible method for the isolation and culture of functional adult human neurons from neurosurgical brain specimens, adult human neurons form a dense network and express a plethora of mature neuronal and synaptic markers.

PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproliferative activity in patient derived glioblastoma cell lines.

We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed strong cytotoxic activity with nanomolar potency (EC: 128 nM), which was a 780 fold improvement over rucaparib itself. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug for treatment for glioblastoma even though these cell lines were resistant to TMZ treatment.

Human in vitro systems for examining synaptic function and plasticity in the brain.

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue.

Vaccination of Macaques with DNA Followed by Adenoviral Vectors Encoding Simian Immunodeficiency Virus (SIV) Gag Alone Delays Infection by Repeated Mucosal Challenge with SIV.

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope.

The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines.

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC: 50.9 nM).

Environmental Surveillance Reveals Complex Enterovirus Circulation Patterns in Human Populations.

Background: Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates.

Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation.

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS.

Genetic diversity in the env V1-V2 region of proviral quasispecies from long-term controller MHC-typed cynomolgus macaques infected with SHIVSF162P4cy.

Intra-host evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) has been shown by viral RNA analysis in subjects who naturally suppress plasma viremia to low levels, known as controllers. However, little is known about the variability of proviral DNA and the inter-relationships among contained systemic viremia, rate of reservoir reseeding and specific major histocompatibility complex (MHC) genotypes, in controllers. Here, we analysed the proviral DNA quasispecies of the env V1-V2 region, in PBMCs and in anatomical compartments of 13 long-term controller monkeys after 3.

Detection of bovine viral diarrhoea virus nucleic acid, but not infectious virus, in bovine serum used for human vaccine manufacture.

Bovine viral diarrhoea virus (BVDV) is a cattle pathogen that has previously been reported to be present in bovine raw materials used in the manufacture of biological products for human use. Seven lots of trivalent measles, mumps and rubella (MMR) vaccine and 1 lot of measles vaccine from the same manufacturer, together with 17 lots of foetal bovine serum (FBS) from different vendors, 4 lots of horse serum, 2 lots of bovine trypsin and 5 lots of porcine trypsin were analysed for BVDV using recently developed techniques, including PCR assays for BVDV detection, a qRT-PCR and immunofluorescence-based virus replication assays, and deep sequencing to identify and genotype BVDV genomes. All FBS lots and one lot of bovine-derived trypsin were PCR-positive for the presence of BVDV genome; in contrast all vaccine lots and the other samples were negative.

Unique and shared inflammatory profiles of human brain endothelia and pericytes.

Background: Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed.

Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction.

Background: Brain pericytes ensheathe the endothelium and contribute to formation and maintenance of the blood-brain-barrier. Additionally, pericytes are involved in several aspects of the CNS immune response including scarring, adhesion molecule expression, chemokine secretion, and phagocytosis. In vitro cultures are routinely used to investigate these functions of brain pericytes, however, these are highly plastic cells and can display differing phenotypes and functional responses depending on their culture conditions.

Isolation of Vaccine-Like Poliovirus Strains in Sewage Samples From the United Kingdom.

Background: Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017.

Methods: Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation.

Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis.

Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuV) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuV associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines.