FHL1 promotes chikungunya and o'nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design.

Arthritogenic alphaviruses are positive-strand RNA viruses that cause debilitating musculoskeletal diseases affecting millions worldwide. A recent discovery identified the four-and-a-half-LIM domain protein 1 splice variant A (FHL1A) as a crucial host factor interacting with the hypervariable domain (HVD) of chikungunya virus (CHIKV) nonstructural protein 3 (nsP3). Here, we show that acute and chronic chikungunya disease in humans correlates with elevated levels of FHL1. We generated FHL1-/- mice, which when infected with CHIKV or o'nyong-nyong virus (ONNV) displayed reduced arthritis and myositis, fewer immune infiltrates, and reduced proinflammatory cytokine/chemokine outputs, compared to infected wild-type (WT) mice. Interestingly, disease signs were comparable in FHL1-/- and WT mice infected with arthritogenic alphaviruses Ross River virus (RRV) or Mayaro virus (MAYV). This aligns with pull-down assay data, which showed the ability of CHIKV and ONNV nsP3 to interact with FHL1, while RRV and MAYV nsP3s did not. We engineered a CHIKV mutant unable to bind FHL1 (CHIKV-ΔFHL1), which was avirulent in vivo. Following inoculation with CHIKV-ΔFHL1, mice were protected from disease upon challenge with CHIKV and ONNV, and viraemia was significantly reduced in RRV- and MAYV-challenged mice. Targeting FHL1-binding as an approach to vaccine design could lead to breakthroughs in mitigating alphaviral disease.

A booster regime of liposome-delivered live-attenuated CHIKV vaccine RNA genome protects against chikungunya virus disease in mice.

Mosquito-transmitted chikungunya virus (CHIKV) is the causal pathogen of CHIKV disease and is responsible for global epidemics of arthritic disease. CHIKV infection can lead to severe chronic and debilitating arthralgia, significantly impacting patient mobility and quality of life. Our previous studies have shown a live-attenuated CHIKV vaccine candidate, CHIKV-NoLS, to be effective in protecting against CHIKV disease in mice vaccinated with one dose. Further studies have demonstrated the value of a liposome RNA delivery system to deliver the RNA genome of CHIKV-NoLS directly in vivo, promoting de novo production of live-attenuated vaccine particles in vaccinated hosts. This system, designed to bypass live-attenuated vaccine production bottlenecks, uses CAF01 liposomes. However, one dose of CHIKV-NoLS CAF01 failed to provide systemic protection against CHIKV challenge in mice, with low levels of CHIKV-specific antibodies. Here we describe CHIKV-NoLS CAF01 booster vaccination regimes designed to increase vaccine efficacy. C57BL/6 mice were vaccinated with three doses of CHIKV-NoLS CAF01 either intramuscularly or subcutaneously. CHIKV-NoLS CAF01 vaccinated mice developed a systemic immune response against CHIKV that shared similarity to vaccination with CHIKV-NoLS, including high levels of CHIKV-specific neutralising antibodies in subcutaneously inoculated mice. CHIKV-NoLS CAF01 vaccinated mice were protected against disease signs and musculoskeletal inflammation when challenged with CHIKV. Mice given one dose of live-attenuated CHIKV-NoLS developed a long lasting protective immune response for up to 71 days. A clinically relevant CHIKV-NoLS CAF01 booster regime can overcome the challenges faced by our previous one dose strategy and provide systemic protection against CHIKV disease.

Anti-inflammatory actions of Pentosan polysulfate sodium in a mouse model of influenza virus A/PR8/34-induced pulmonary inflammation.

Introduction: There is an unmet medical need for effective anti-inflammatory agents for the treatment of acute and post-acute lung inflammation caused by respiratory viruses. The semi-synthetic polysaccharide, Pentosan polysulfate sodium (PPS), an inhibitor of NF-kB activation, was investigated for its systemic and local anti-inflammatory effects in a mouse model of influenza virus A/PR8/1934 (PR8 strain) mediated infection. Methods: Immunocompetent C57BL/6J mice were infected intranasally with a sublethal dose of PR8 and treated subcutaneously with 3 or 6 mg/kg PPS or vehicle. Disease was monitored and tissues were collected at the acute (8 days post-infection; dpi) or post-acute (21 dpi) phase of disease to assess the effect of PPS on PR8-induced pathology. Results: In the acute phase of PR8 infection, PPS treatment was associated with a reduction in weight loss and improvement in oxygen saturation when compared to vehicle-treated mice. Associated with these clinical improvements, PPS treatment showed a significant retention in the numbers of protective SiglecF+ resident alveolar macrophages, despite uneventful changes in pulmonary leukocyte infiltrates assessed by flow cytometry. PPS treatment in PR8- infected mice showed significant reductions systemically but not locally of the inflammatory molecules, IL-6, IFN-g, TNF-a, IL-12p70 and CCL2. In the post-acute phase of infection, PPS demonstrated a reduction in the pulmonary fibrotic biomarkers, sICAM-1 and complement factor C5b9. Discussion: The systemic and local anti-inflammatory actions of PPS may regulate acute and post-acute pulmonary inflammation and tissue remodeling mediated by PR8 infection, which warrants further investigation.

N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

The Delta SARS-CoV-2 Variant of Concern Induces Distinct Pathogenic Patterns of Respiratory Disease in K18-hACE2 Transgenic Mice Compared to the Ancestral Strain from Wuhan.

Compared to the original ancestral strain of SARS-CoV-2, the Delta variant of concern has shown increased transmissibility and resistance toward COVID-19 vaccines and therapies. However, the pathogenesis of the disease associated with Delta is still not clear. In this study, using K18-hACE2 transgenic mice, we assessed the pathogenicity of the Delta variant by characterizing the immune response following infection. We found that Delta induced the same clinical disease manifestations as the ancestral SARS-CoV-2, but with significant dissemination to multiple tissues, such as brain, intestine, and kidney. Histopathological analysis showed that tissue pathology and cell infiltration in the lungs of Delta-infected mice were the same as in mice infected with the ancestral SARS-CoV-2. Delta infection caused perivascular inflammation in the brain and intestinal wall thinning in K18-hACE2 transgenic mice. Increased cell infiltration in the kidney was observed in both ancestral strain- and Delta-infected mice, with no clear visible tissue damage identified in either group. Interestingly, compared with mice infected with the ancestral strain, the numbers of CD45+ cells, T cells, B cells, inflammatory monocytes, and dendritic cells were all significantly lower in the lungs of the Delta-infected mice, although there was no significant difference in the levels of proinflammatory cytokines between the two groups. Our results showed distinct immune response patterns in the lungs of K18-hACE2 mice infected with either the ancestral SARS-CoV-2 or Delta variant of concern, which may help to guide therapeutic interventions for emerging SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 variants, with the threat of increased transmissibility, infectivity, and immune escape, continue to emerge as the COVID-19 pandemic progresses. Detailing the pathogenesis of disease caused by SARS-CoV-2 variants, such as Delta, is essential to better understand the clinical threat caused by emerging variants and associated disease. This study, using the K18-hACE2 mouse model of severe COVID-19, provides essential observation and analysis on the pathogenicity and immune response of Delta infection. These observations shed light on the changing disease profile associated with emerging SARS-CoV-2 variants and have potential to guide COVID-19 treatment strategies.

Interleukin-17 contributes to Ross River virus-induced arthritis and myositis.

Arthritogenic alphaviruses are mosquito-borne viruses that are a major cause of infectious arthropathies worldwide, and recent outbreaks of chikungunya virus and Ross River virus (RRV) infections highlight the need for robust intervention strategies. Alphaviral arthritis can persist for months after the initial acute disease, and is mediated by cellular immune responses. A common strategy to limit inflammation and pathology is to dampen the overwhelming inflammatory responses by modulating proinflammatory cytokine pathways. Here, we investigate the contribution of interleukin-17 (IL-17), a cytokine involved in arthropathies such as rheumatoid arthritis, in the development RRV-induced arthritis and myositis. IL-17 was quantified in serum from RRV-infected patients, and mice were infected with RRV and joints and muscle tissues collected to analyse cellular infiltrates, tissue mRNA, cytokine expression, and joint and muscle histopathology. IL-17 expression was increased in musculoskeletal tissues and serum of RRV-infected mice and humans, respectively. IL-17-producing T cells and neutrophils contributed to the cellular infiltrate in the joint and muscle tissue during acute RRV disease in mice. Blockade of IL-17A/F using a monoclonal antibody (mAb) reduced disease severity in RRV-infected mice and led to decreased proinflammatory proteins, cellular infiltration in synovial tissues and cartilage damage, without affecting viral titers in inflamed tissues. IL-17A/F blockade triggered a shift in transcriptional profile of both leukocyte infiltrates and musculoskeletal stromal cells by downregulating proinflammatory genes. This study highlights a previously uncharacterized role for an effector cytokine in alphaviral pathology and points towards potential therapeutic benefit in targeting IL-17 to treat patients presenting with RRV-induced arthropathy.

Infectious Clones Produce SARS-CoV-2 That Causes Severe Pulmonary Disease in Infected K18-Human ACE2 Mice.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has caused extensive mortality and morbidity and wreaked havoc on socioeconomic structures. The urgent need to better understand SARS-CoV-2 biology and enable continued development of effective countermeasures is aided by the production of laboratory tools that facilitate SARS-CoV-2 research. We previously created a directly accessible SARS-CoV-2 toolkit containing user-friendly reverse genetic (RG) infectious clones of SARS-CoV-2. Here, using K18-human ACE2 (hACE2) mice, we confirmed the validity of RG-rescued SARS-CoV-2 viruses to reproduce the infection profile, clinical disease, and pathogenesis already established in mice infected with natural SARS-CoV-2 isolates, often patient derived. RG-rescued SARS-CoV-2-infected K18-hACE2 mice developed substantial clinical disease and weight loss by day 6 postinfection. RG-rescued SARS-CoV-2 was recovered from the lungs and brains of infected K18-hACE2 mice, and infection resulted in viral pneumonia with considerable changes in lung pathology, as seen previously with natural SARS-CoV-2 infection. In mice infected with RG-rescued SARS-CoV-2-mCherry, mCherry was detected in areas of lung consolidation and colocalized with clinically relevant SARS-CoV-2-assocated immunopathology. RG-rescued SARS-CoV-2 viruses successfully recapitulated many of the features of severe COVID-19 associated with the K18-hACE2 model of SARS-CoV-2 infection. With utility in vivo, the RG-rescued SARS-CoV-2 viruses will be valuable resources to advance numerous areas of SARS-CoV-2 basic research and COVID-19 vaccine development.IMPORTANCE To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations. In this study, we further prove the toolkit's value by demonstrating the in vivo utility of RG-rescued SARS-CoV-2 isolates. RG-rescued SARS-CoV-2 isolates reproduce disease signs and pathology characteristic of the K18-hACE2 mouse model of severe COVID-19 in infected mice. Having been validated as a model of severe COVID-19 previously using only natural SARS-CoV-2 isolated from patients, this is the first investigation of RG-rescued SARS-CoV-2 viruses in K18-hACE2 mice. The RG-rescued SARS-CoV-2 viruses will facilitate basic understanding of SARS-CoV-2 and the preclinical development of COVID-19 therapeutics.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX3CR1+ Macrophages in Tissue Repair.

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose.

Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.

Attenuation and Stability of CHIKV-NoLS, a Live-Attenuated Chikungunya Virus Vaccine Candidate.

Our previous investigation of the nucleolar localisation sequence (NoLS) of chikungunya virus (CHIKV) capsid protein demonstrated the role of capsid in CHIKV virulence. Mutating the NoLS of capsid in CHIKV led to the development of a unique live-attenuated CHIKV vaccine candidate, termed CHIKV-NoLS. CHIKV-NoLS-immunised mice developed long-term immunity from CHIKV infection after a single dose. To further evaluate CHIKV-NoLS attenuation and suitability as a vaccine, we examined the footpad of inoculated mice for underlying CHIKV-NoLS-induced immunopathology by histological and flow cytometric analysis. In comparison to CHIKV-WT-infected mice, CHIKV-NoLS-inoculated mice exhibited minimal inflammation and tissue damage. To examine the stability of attenuation, the plaque phenotype and replication kinetics of CHIKV-NoLS were determined following extended in vitro passage. The average plaque size of CHIKV-NoLS remained notably smaller than CHIKV-WT after extended passage and attenuated replication was maintained. To examine thermostability, CHIKV-NoLS was stored at 21 °C, 4 °C, -20 °C and -80 °C and infectious CHIKV-NoLS quantified up to 84 days. The infectious titre of CHIKV-NoLS remains stable after 56 days when stored at either -20 °C or -80 °C. Interestingly, unlike CHIKV-WT, the infectious titre of CHIKV-NoLS is not sensitive to freeze thaw cycles. These data further demonstrate preclinical safety and stability of CHIKV-NoLS.

Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation.

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus-a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.

Prediction of relapse in paediatric pre-B acute lymphoblastic leukaemia using a three-gene risk index.

Despite high cure rates 25% of children with acute lymphoblastic leukaemia (ALL) relapse and have dismal outcome. Crucially, many are currently stratified as standard risk (SR) and additional markers to improve patient stratification are required. Here we have used diagnostic bone marrow specimens from 101 children with pre-B ALL to examine the use of gene expression profiles (GEP) as predictors of long-term clinical outcome. Patients were divided into two cohorts for model development and validation based on availability of specimen material. Initially, GEP from 55 patients with sufficient material were analysed using HG-U133A microarrays, identifying an 18-gene classifier (GC) that was more predictive of outcome than conventional prognostic parameters. After feature selection and validation of expression levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR), a three-gene qRT-PCR risk index [glutamine synthetase (GLUL), ornithine decarboxylase antizyme inhibitor (AZIN), immunoglobulin J chain (IGJ)] was developed that predicted outcome with an accuracy of 89% in the array cohort and 87% in the independent validation cohort. The data demonstrate the feasibility of using GEP to improve risk stratification in childhood ALL. This is particularly important for the identification of patients destined to relapse despite their current stratification as SR, as more intensive front-line treatment options for these individuals are already available.

High expression of connective tissue growth factor in pre-B acute lymphoblastic leukaemia.

In recent years microarrays have been used extensively to characterize gene expression in acute lymphoblastic leukaemia (ALL). Few studies, however, have analysed normal haematopoietic cell populations to identify altered gene expression in ALL. We used oligonucleotide microarrays to compare the gene expression profile of paediatric precursor-B (pre-B) ALL specimens with two control cell populations, normal CD34(+) and CD19(+)IgM(-) cells, to focus on genes linked to leukemogenesis. A set of eight genes was identified with a ninefold higher average expression in ALL specimens compared with control cells. All of these genes were significantly deregulated in an independent cohort of 101 ALL specimens. One gene, connective tissue growth factor (CTGF, also known as CCN2), had exceptionally high expression, which was confirmed in three independent leukaemia studies. Further analysis of CTGF expression in ALL revealed exclusive expression in B-lineage, not T-lineage, ALL. Within B-lineage ALL approximately 75% of specimens were consistently positive for CTGF expression, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies using Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. These findings indicate that CTGF is secreted by pre-B ALL cells and may play a role in the pathophysiology of this disease.

Identification of novel molecular prognostic markers for paediatric T-cell acute lymphoblastic leukaemia.

In the last four decades the survival of patients with newly diagnosed childhood T-cell acute lymphoblastic leukaemia (T-ALL) has improved dramatically. In sharp contrast, relapsed T-ALL continues to confer a dismal prognosis. We sought to determine if gene expression profiling could uncover a signature of outcome for children with T-ALL. Using 12 patient specimens obtained before therapy started, we examined the gene expression profile by oligonucleotide microarrays. We identified three genes, CFLAR, NOTCH2 and BTG3, whose expression at the time of diagnosis accurately distinguished the patients according to disease outcome. These genes are involved in the regulation of apoptosis and cellular proliferation. The prognostic value of the three predictive genes was assessed in an independent cohort of 25 paediatric T-ALL patients using quantitative real-time reverse transcription polymerase chain reaction. Patients assigned to the adverse outcome group had a significantly higher cumulative incidence of relapse compared with patients assigned to the favourable outcome group (46% vs. 8%, P = 0.029). Five-year overall survival was also significantly worse in the patients assigned to the adverse outcome group (P = 0.0039). The independent influence of the 3-gene predictor was confirmed by multivariate analysis. Our study provides proof of principle that genome-wide expression profiling can detect novel molecular prognostic markers in paediatric T-ALL.

In vitro cytotoxicity of nelarabine, clofarabine and flavopiridol in paediatric acute lymphoblastic leukaemia.

The in vitro efficacies of three new drugs--clofarabine (CLOF), nelarabine (NEL) and flavopiridol (FP) - were assessed in a panel of acute lymphoblastic leukaemia (ALL) cell lines. The 50% inhibitory concentration (IC50) for CLOF across all lines was 188-fold lower than that of NEL. B-lineage, but not T-lineage lines, were >7-fold more sensitive to CLOF than cytosine arabinoside (ARAC). NEL IC50 was 25-fold and 113-fold higher than ARAC in T- and B-lineage, respectively. T-ALL cells were eightfold more sensitive to NEL than B-lineage but there was considerable overlap. FP was more potent in vitro than glucocorticoids and thiopurines and at doses that recent phase I experience predicts will translate into clinical efficacy. Potential cross-resistance of CLOF, NEL and FP was observed with many front-line ALL therapeutics but not methotrexate or thiopurines. Methotrexate sensitivity was inversely related to that of NEL and FP. Whilst NEL was particularly effective in T-ALL, a subset of patients with B-lineage ALL might also be sensitive. CLOF appeared to be marginally more effective in B-lineage than T-ALL and has a distinct resistance profile that may prove useful in combination with other compounds. FP should be widely effective in ALL if sufficient plasma levels can be achieved clinically.

The gene expression signature of relapse in paediatric acute lymphoblastic leukaemia: implications for mechanisms of therapy failure.

Despite significant improvements in the treatment of childhood acute lymphoblastic leukaemia (ALL), the prognosis for relapsing patients remains poor. The aim of this study was to generate a transcriptional profile of relapsed ALL to increase our understanding of the mechanisms involved in therapy failure. RNA was extracted from 11 pairs of cryopreserved pre-B ALL bone marrow specimens taken from the same patients at diagnosis and relapse, and analysed using HG-U133A microarrays. Relapse specimens overexpressed genes that are involved with cell growth and proliferation, in keeping with their aggressive phenotype. When tested in 72 independent specimens of pre-B ALL and T-ALL, the identified genes could successfully differentiate between diagnosis and relapse in either lineage, indicating the existence of relapse mechanisms common to both. These genes have functions relevant for oncogenesis, drug resistance and metastasis, but are not related to classical multidrug-resistance pathways. Increased expression of the top-ranked gene (BSG) at diagnosis was significantly associated with adverse outcome. Several chromosomal loci, including 19p13, were identified as potential hotspots for aberrant gene expression in relapsed ALL. Our results provide evidence for a link between drug resistance and the microenvironment that has previously only been considered in the context of solid tumour biology.

Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate?

BACKGROUND: The use of microarray technology to assess gene expression levels is now widespread in biology. The validation of microarray results using independent mRNA quantitation techniques remains a desirable element of any microarray experiment. To facilitate the comparison of microarray expression data between laboratories it is essential that validation methodologies be critically examined. We have assessed the correlation between expression scores obtained for 48 human genes using oligonucleotide microarrays and the expression levels for the same genes measured by quantitative real-time RT-PCR (qRT-PCR). RESULTS: Correlations with qRT-PCR data were obtained using microarray data that were processed using robust multi-array analysis (RMA) and the MAS 5.0 algorithm. Our results indicate that when identical transcripts are targeted by the two methods, correlations between qRT-PCR and microarray data are generally strong (r = 0.89). However, we observed poor correlations between qRT-PCR and RMA or MAS 5.0 normalized microarray data for 13% or 16% of genes, respectively. CONCLUSION: These results highlight the complementarity of oligonucleotide microarray and qRT-PCR technologies for validation of gene expression measurements, while emphasizing the continuing requirement for caution in interpreting gene expression data.

Gene expression levels in small specimens from patients detected using oligonucleotide arrays.

Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.