C13orf31 (FAMIN) is a central regulator of immunometabolic function.

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.

Blood identified and quantified in formalin fixed paraffin embedded lung sections using eosin fluorescence.

Eosin Y is a common stain in histology. Although usually used for colourimetric imaging where the dye is used to stain pink/red a range of structures in the tissue, Eosin Y is also a fluorochrome, and has been used in this manner for decades. In this study our aim was to investigate the fluorescence properties of the dye to enable quantification of structures within formalin-fixed paraffin-embedded (FFPE) tissue sections. To do this, FFPE sections of hamster tissue were prepared with haematoxylin and eosin Y dyes. Spectral detection on a confocal laser scanning microscope was used to obtain the fluorescence emission spectra of the eosin Y under blue light. This showed clear spectral differences between the red blood cells and congealed blood, compared to the rest of the section. The spectra were so distinct that it was possible to discern these in fluorescence and multi-photon microscopy. An image analysis algorithm was used to quantify the red blood cells. These analyses could have broad applications in histopathology where differentiation is required, such as the analysis of clotting disorders to haemorrhage or damage from infectious disease.

Utility of reference materials for Zika Virus nucleic acid testing.

The emergence of Zika virus (ZIKV) in the Americas has resulted in increased nucleic acid amplification testing (NAT) of clinical samples and blood donations. New molecular diagnostic assays have been developed resulting in a corollary requirement for ZIKV reference material. To address this we have produced and calibrated two African lineage ZIKV reference materials: a highly concentrated secondary standard (NIBSC: 16/110) and a lower concentration external quality control (QC) reagent (NIBSC: 16/124) and compared their performance in three ZIKV NAT assays in relation with the First International Standard (IS) for Zika Virus NAT assays (PEI: 11468/16). In summary the African lineage ZIKV reference materials were detected by all three assays. The ZIKV lineage did not affect the performance of the secondary standard. The external QC reagent (16/124) was detected by all three assays highlighting its suitability for use as a low positive control to monitor assay performance on a regular basis. The relative potency of 16/110 to the IS was 5.49E+06IU/mL (95% CI: 1.46E+06-2.06E+07) and 16/124 to 16/110 was 8.36E+03 (95% CI: 7.83E+03-8.92E+03). The global availability of African lineage ZIKV reference materials will facilitate standardization of ZIKV molecular diagnostic assays between and within laboratories whilst preserving the IS.

Convalescent human plasma candidate reference materials protect against Crimean-Congo haemorrhagic fever virus (CCHFV) challenge in an A129 mouse model.

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is spread by infected ticks or direct contact with blood, tissues and fluids from infected patients or livestock. Infection with CCHFV causes severe haemorrhagic fever in humans which is fatal in up to 83 % of cases. CCHFV is listed as a priority pathogen by the World Health Organization (WHO) and there are currently no widely-approved vaccines. Defining a serological correlate of protection against CCHFV infection would support the development of vaccines by providing a 'target threshold' for pre-clinical and clinical immunogenicity studies to achieve in subjects and potentially obviate the need for in vivo protection studies. We therefore sought to establish titratable protection against CCHFV using pooled human convalescent plasma, in a mouse model. Convalescent plasma collected from seven individuals with a known previous CCHFV virus infection were characterised using binding antibody and neutralisation assays. All plasma recognised nucleoprotein and the Gc glycoprotein, but some had a lower Gn glycoprotein response by ELISA. Pooled plasma and two individual donations from convalescent donors were administered intraperitoneally to A129 mice 24 h prior to intradermal challenge with CCHFV (strain IbAr10200). A partial protective effect was observed with all three convalescent plasmas characterised by longer survival post-challenge and reduced clinical score. These protective responses were titratable. Further characterisation of the serological reactivities within these samples will establish their value as reference materials to support assay harmonisation and accelerate vaccine development for CCHFV.

Pathogenesis of Rift Valley Fever Virus in a BALB/c Mouse Model Is Affected by Virus Culture Conditions and Sex of the Animals.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease in livestock and humans. Whilst initially restricted to the African continent, recent spread to the Arabian Peninsula has highlighted the likelihood of entry into new regions. Due to the absence of a regulatory-approved human vaccine, work is ongoing to develop and assess countermeasures. As such, small animal models play a pivotal role in providing information on disease pathogenesis and elucidating which intervention strategies confer protection. To develop and establish the BALB/c mouse model, we challenged mice with RVFV grown from two separate cell lines: one derived from mosquitoes (C6/36) and the other mammalian derived (Vero E6). Following infection, we assessed the clinical course of disease progression at days 1 and 3 post-challenge and evaluated viral tropism and immune analytes. The results demonstrated that RVFV infection was affected by the cell line used to propagate the challenge virus, with those grown in insect cells resulting in a more rapid disease progression. The lowest dose that caused uniform severe disease remained the same across both virus preparations. In addition, to demonstrate reproducibility, the lowest dose was used for a subsequent infection study using male and female animals. The results further demonstrated that male mice succumbed to infection more rapidly than their female counterparts. Our results establish an RVFV mouse model and key parameters that affect the course of disease progression in BALB/c mice.

Disruption of paraoxonase 3 impairs proliferation and antioxidant defenses in human A549 cells and causes embryonic lethality in mice.

We had shown previously that paraoxonase 3 (PON3), a putative circulating antioxidant, was systemically upregulated in late-gestation rat, sheep, and human fetuses. Our overarching hypothesis is that preterm human infants are delivered with low levels of PON3 and that this contributes to a state of oxidative stress. We sought to determine whether absence of Pon3 was associated with reduced neonatal viability in mice and studied the offspring from crosses between Pon3(+/-) mice. The number of Pon3(-/-) animals at E10.5 and E17.5 was significantly lower than the expected 25% (9.3 and 7.9% respectively, P < 0.001). On the first day of postnatal life, this was reduced further (2.4%, significantly less than the proportion in fetal life, P = 0.04). Pon3(+/-) animals had lower body and placental weights than wild-type littermates at E17.5, an effect that was independent of the parent of origin of the mutant allele. We then studied the effect of PON3 knockdown in a human cell line, A549. Stable knockdown of PON3 using short-hairpin RNA reduced cell proliferation in 21% oxygen. We then studied the effect of transient knockdown of PON3 using short interfering RNA (siRNA) in the same cell line in low (2%) or ambient (21%) oxygen. Knockdown of PON3 using siRNA reduced total antioxidant capacity in 21% (P = 0.008) but not 2% oxygen. We conclude that the absence of Pon3 in mice resulted in increased rates of early fetal and neonatal death. Knockdown of PON3 in human cells reduced cell proliferation and total antioxidant capacity.

Mucosal Responses to Zika Virus Infection in Cynomolgus Macaques.

Zika virus (ZIKV) cases continue to be reported, and no vaccine or specific antiviral agent has been approved for the prevention or treatment of infection. Though ZIKV is primarily transmitted by mosquitos, cases of sexual transmission and prolonged viral RNA presence in semen have been reported. In this observational study, we report the mucosal responses to sub-cutaneous and mucosal ZIKV exposure in cynomolgus macaques during acute and late chronic infection. Subcutaneous challenge induced a decrease in the growth factor VEGF in colorectal and cervicovaginal tissues 100 days post-challenge, in contrast to the observed increase in these tissues following vaginal infection. This different pattern was not observed in the uterus, where VEGF was upregulated independently of the challenge route. Vaginal challenge induced a pro-inflammatory profile in all mucosal tissues during late chronic infection. Similar responses were already observed during acute infection in a vaginal tissue explant model of ex vivo challenge. Non-productive and productive infection 100 days post-in vivo vaginal challenge induced distinct proteomic profiles which were characterized by further VEGF increase and IL-10 decrease in non-infected animals. Ex vivo challenge of mucosal explants revealed tissue-specific modulation of cytokine levels during the acute phase of infection. Mucosal cytokine profiles could represent biosignatures of persistent ZIKV infection.

SARS-CoV-2 Virus-like Particles Produced by a Single Recombinant Baculovirus Generate Anti-S Antibody and Protect against Variant Challenge.

Coronavirus Disease 2019 (COVID-19), caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous ~100 nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein and reduced virus shedding and protected against disease associated weight loss following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant). Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.

Early Isolates of SARS-CoV-2 Result in Different Pathogenesis in the Transduced Mouse Model of COVID-19.

A transduced mouse model of SARS-CoV-2 infection was established using Balb/c mice. This was achieved through the adenovirus-vectored delivery of the hACE2 gene, to render the mice transiently susceptible to the virus. The model was characterised in terms of the dissemination of hACE2 receptor expression, the dissemination of three SARS-CoV-2 virus variants in vivo up to 10 days following challenge, the resulting histopathology and the clinical signs induced in the mice. In transduced mice, the infection was short-term, with a rapid loss in body weight starting at day 2 with maximum weight loss at day 4, followed by subsequent recovery until day 10. The induced expression of the hACE2 receptor was evident in the lungs, but, upon challenge, the SARS-CoV-2 virus disseminated beyond the lungs to spleen, liver and kidney, peaking at day 2 post infection. However, by day 10 post infection, the virus was undetectable. The lung histopathology was characterised by bronchial and alveolar inflammation, which was still present at day 10 post infection. Transduced mice had differential responses to viral variants ranking CVR-Glasgow 1 > Victoria-1 > England-2 isolates in terms of body weight loss. The transduced mouse model provides a consistent and manipulatable model of SARS-CoV-2 infection to screen viral variants for their relative virulence and possible interventions.

Intrinsic host susceptibility among multiple species to intranasal SARS-CoV-2 identifies diverse virological, biodistribution and pathological outcomes.

SARS-CoV-2 exhibits a diverse host species range with variable outcomes, enabling differential host susceptibility studies to assess suitability for pre-clinical countermeasure and pathogenesis studies. Baseline virological, molecular and pathological outcomes were determined among multiple species-one Old World non-human primate (NHP) species (cynomolgus macaques), two New World NHP species (red-bellied tamarins; common marmosets) and Syrian hamsters-following single-dose, atraumatic intranasal administration of SARS-CoV-2/Victoria-01. After serial sacrifice 2, 10 and 28-days post-infection (dpi), hamsters and cynomolgus macaques displayed differential virus biodistribution across respiratory, gastrointestinal and cardiovascular systems. Uniquely, New World tamarins, unlike marmosets, exhibited high levels of acute upper airway infection, infectious virus recovery associated with mild lung pathology representing a host previously unrecognized as susceptible to SARS-CoV-2. Across all species, lung pathology was identified post-clearance of virus shedding (antigen/RNA), with an association of virus particles within replication organelles in lung sections analysed by electron microscopy. Disrupted cell ultrastructure and lung architecture, including abnormal morphology of mitochondria 10-28 dpi, represented on-going pathophysiological consequences of SARS-CoV-2 in predominantly asymptomatic hosts. Infection kinetics and host pathology comparators using standardized methodologies enables model selection to bridge differential outcomes within upper and lower respiratory tracts and elucidate longer-term consequences of asymptomatic SARS-CoV-2 infection.

Developmental control of the Nlrp6 inflammasome and a substrate, IL-18, in mammalian intestine.

The inflammasome is a multiprotein complex whose formation is triggered when a NOD-like receptor binds a pathogen ligand, resulting in activated caspase-1, which converts certain interleukins (IL-1ß, IL-18, and IL-33) to their active forms. There is currently no information on regulation of this system around the time of birth. We employed transcript profiling of fetal rat intestinal and lung RNA at embryonic days 16 (E16) and 20 (E20) with out-of-sample validation using quantitative RT-PCR. Transcript profiling and quantitative RT-PCR demonstrated that transcripts of core components of the NOD-like receptor Nlrp6 inflammasome (Nlrp6, Pycard, Caspase-1) and one of its substrates, IL-18, were increased at E20 compared with E16 in fetal intestine and not lung. Immunohistochemistry demonstrated increased Pycard in intestinal epithelium. Western blotting demonstrated that IL-18 was undetectable at E16, clearly detectable at E20 in its inactive form, and detectable postnatally in both its inactive and active form. Dramatic upregulation of IL-18 was also observed in the fetal sheep jejunum in late gestation (P = 0.006). Transcription factor binding analysis of the rat array data revealed an overrepresentation of nuclear transcription factor binding sites peroxisome proliferator-activated receptor γ (PPAR-γ) and retinoid X receptor-α and chicken ovalbumin upstream promoter transcription factor 1 in the region 1,000 bp upstream of the transcription start site. Rosiglitazone, a PPAR-γ agonist, more than doubled levels of NLRP6 mRNA in human intestinal epithelial (Caco2) cells. These observations provide the first evidence, to our knowledge, linking activity of PPAR-γ to expression of a NOD-like receptor and adds to a growing body of evidence linking pattern recognition receptors of the innate immune system and intestinal colonization.

Potential human immunotherapeutics for plague.

Two monoclonal antibodies directed to the V antigen of Yersinia pestis have been tested for protective efficacy in a murine model of bubonic plague. Mice were infected with a current clinical isolate from Madagascar, designated Y. pestis 10-21/S. Mab7.3, delivered to mice intra-periteoneally at either 24 h prior to, or 24 h post-infection, was fully protective, building on many studies which have demonstrated the protective efficacy of this Mab against a number of different clinical isolates of Y. pestis. Mab 29.3, delivered intra-peritoneally at either -24 h or +24 h, protected 4/5 mice in either condition; this has demonstrated the protective efficacy of this Mab in vivo for the first time. These results add to the cumulative data about Mab7.3, which is currently being humanized and highlight its potential as a human immunotherapeutic for plague, which is an enduring endemic disease in Madagascar and other regions of Africa, Asia, and South America.

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Towards quantification of protective antibody responses by passive transfer of the 1st WHO International Standard for Ebola virus antibody in a guinea pig model.

Ebola virus (EBOV) represents a major concern to global health due to the unpredictable nature of outbreaks. Infection with EBOV can cause a severe viral haemorrhagic fever with no licensed vaccine or treatment, restricting work with live EBOV to Containment/Biosafety Level 4 facilities. Whilst the magnitude of recent outbreaks has provided an impetus for vaccine and antiviral development, establishing the efficacy of candidate vaccine materials relies on EBOV challenge models and advanced human trials should outbreaks occur and where logistics and funding allow. To address these hurdles in vaccine development, we investigated whether a recently established serological reference standard, the 1st WHO International Standard for Ebola virus antibody, could be used to provide a quantifiable correlate of immune protection in vivo. Dilutions of the International Standard were inoculated into naïve guinea pigs 24 h before challenge with a lethal dose of Ebola virus. Only subjects receiving the highest dose of the International Standard exhibited evidence of delayed progression. Due to it being a WHO established reagent and available globally upon request, this standard allows for effective comparisons of data between laboratories and may prove valuable to select the candidate vaccines that are most likely to confer humoral immune protection ensuring the most promising candidates progress into efficacy studies.

Passive immunisation of convalescent human anti-Zika plasma protects against challenge with New World Zika virus in cynomolgus macaques.

Zika virus (ZIKV) causes neurological complications in susceptible individuals, highlighted in the recent South American epidemic. Natural ZIKV infection elicits host responses capable of preventing subsequent re-infection, raising expectations for effective vaccination. Defining protective immune correlates will inform viral intervention strategies, particularly vaccine development. Non-human primate (NHP) species are susceptible to ZIKV and represent models for vaccine development. The protective efficacy of a human anti-ZIKV convalescent plasma pool (16/320-14) developed as a candidate reference material for a WHO International Standard was evaluated in macaques. Convalescent plasma administered to four cynomolgus macaques (Macaca fascicularis) intra-peritoneally 24 hrs prior to sub-cutaneous challenge with 103 pfu ZIKVPRVABC59 protected against detectable infection, with absence of detectable ZIKV RNA in blood and lymphoid tissues. Passively immunised anti-ZIKV immunoglobulin administered prior to time of challenge remained present only at very low levels 42 days post-challenge. Absence of de novo antibody responses in passively immunised macaques indicate sterilising immunity compared with naïve challenge controls that exhibited active ZIKV-specific IgM and IgG responses post-challenge. Demonstration that the presence of convalescent anti-ZIKV at levels of 400 IU/mL neutralising antibody protects against virus challenge provides a scientific framework for development of anti-ZIKV vaccines and facilitates regulatory approval.

WHO international standard for anti-rubella: learning from its application.

The WHO international standard for anti-rubella was first established in the 1960s when clinical diagnostics were in their infancy. Since the endorsement of the first international standard for anti-rubella IgG (RUBI-1-94), new rubella vaccines have been developed and global coverage of rubella vaccination has increased. Methods used to measure concentrations of anti-rubella IgG have also evolved to rapid, high-throughput binding assays, which have replaced often cumbersome and highly technical functional assays. During this timeframe, the protective concentration of antibody was set at 10 IU/mL by extrapolation of functional assay correlates; however, the subpopulation of antibodies within a polyclonal serum that confer protection remained undefined. Anti-rubella assays have variable formats, including antigens used, such that the same clinical sample tested on different assays can report different values with potentially devastating consequences, such as recommending to terminate pregnancy. WHO convened a meeting of experts in the rubella field to discuss the use of RUBI-1-94 and the potential future role of this international standard. The main conclusions of this meeting questioned the appropriateness of 10 IU/mL as the cutoff for protection and acknowledged the continuing role of RUBI-1-94 as a reference preparation to address analytical sensitivity and assay variation.

High susceptibility, viral dynamics and persistence of South American Zika virus in New World monkey species.

South American Zika virus (ZIKV) recently emerged as a novel human pathogen, linked with neurological disorders. However, comparative ZIKV infectivity studies in New World primates are lacking. Two members of the Callitrichidae family, common marmosets (Callithrix jacchus) and red-bellied tamarins (Saguinus labiatus), were highly susceptible to sub-cutaneous challenge with the Puerto Rico-origin ZIKVPRVABC59 strain. Both exhibited rapid, high, acute viraemia with early neuroinvasion (3 days) in peripheral and central nervous tissue. ZIKV RNA levels in blood and tissues were significantly higher in New World hosts compared to Old World species (Macaca mulatta, Macaca fascicularis). Tamarins and rhesus macaques exhibited loss of zonal occludens-1 (ZO-1) staining, indicative of a compromised blood-brain barrier 3 days post-ZIKV exposure. Early, widespread dissemination across multiple anatomical sites distant to the inoculation site preceded extensive ZIKV persistence after 100 days in New and Old World lineages, especially lymphoid, neurological and reproductive sites. Prolonged persistence in brain tissue has implications for otherwise resolved human ZIKV infection. High susceptibility of distinct New World species underscores possible establishment of ZIKV sylvatic cycles in primates indigenous to ZIKV endemic regions. Tamarins and marmosets represent viable New World models for ZIKV pathogenesis and therapeutic intervention studies, including vaccines, with contemporary strains.

Simvastatin treatment prolongs the survival of scrapie-infected mice.

Statins, drugs that decrease cholesterol biosynthesis, are known to reduce the formation of the disease-associated isoform of the prion protein (PrP) in neuroblastoma cells in vitro. In this study, we report the effects of simvastatin, a clinically approved statin that penetrates the brain, on mice infected with the ME7 strain of scrapie. The decline in motor functions associated with scrapie infection was delayed in mice receiving (1 mg/kg) simvastatin, a dosage used to treat hypercholesterolemia in humans. Simvastatin treatment also significantly prolonged the survival times of infected mice (193 vs. 183 days). These results indicate that low-dosage simvastatin treatment affects the progression of experimental scrapie, and supports the concept that statin treatment may influence the prion pathogenesis.

Interferon-gamma increases neuronal death in response to amyloid-beta1-42.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder characterized by a progressive cognitive impairment, the consequence of neuronal dysfunction and ultimately the death of neurons. The amyloid hypothesis proposes that neuronal damage results from the accumulation of insoluble, hydrophobic, fibrillar peptides such as amyloid-beta1-42. These peptides activate enzymes resulting in a cascade of second messengers including prostaglandins and platelet-activating factor. Apoptosis of neurons is thought to follow as a consequence of the uncontrolled release of second messengers. Biochemical, histopathological and genetic studies suggest that pro-inflammatory cytokines play a role in neurodegeneration during Alzheimer's disease. In the current study we examined the effects of interferon (IFN)-gamma, tumour necrosis factor (TNF)alpha, interleukin (IL)-1beta and IL-6 on neurons. METHODS: Primary murine cortical or cerebellar neurons, or human SH-SY5Y neuroblastoma cells, were grown in vitro. Neurons were treated with cytokines prior to incubation with different neuronal insults. Cell survival, caspase-3 activity (a measure of apoptosis) and prostaglandin production were measured. Immunoblots were used to determine the effects of cytokines on the levels of cytoplasmic phospholipase A2 or phospholipase C gamma-1. RESULTS: While none of the cytokines tested were directly neurotoxic, pre-treatment with IFN-gamma sensitised neurons to the toxic effects of amyloid-beta1-42 or HuPrP82-146 (a neurotoxic peptide found in prion diseases). The effects of IFN-gamma were seen on cortical and cerebellar neurons, and on SH-SY5Y neuroblastoma cells. However, pre-treatment with IFN-gamma did not affect the sensitivity to neurons treated with staurosporine or hydrogen peroxide. Pre-treatment with IFN-gamma increased the levels of cytoplasmic phospholipase A2 in SH-SY5Y cells and increased prostaglandin E2 production in response to amyloid-beta1-42. CONCLUSION: Treatment of neuronal cells with IFN-gamma increased neuronal death in response to amyloid-beta1-42 or HuPrP82-146. IFN-gamma increased the levels of cytoplasmic phospholipase A2 in cultured neuronal cells and increased expression of cytoplasmic phospholipase A2 was associated with increased production of prostaglandin E2 in response to amyloid-beta1-42 or HuPrP82-146. Such observations suggest that IFN-gamma produced within the brain may increase neuronal loss in Alzheimer's disease.