Epistatic interaction between ERAP2 and HLA modulates HIV-1 adaptation and disease outcome in an Australian population.

A strong genetic predictor of outcome following untreated HIV-1 infection is the carriage of specific alleles of human leukocyte antigens (HLAs) that present viral epitopes to T cells. Residual variation in outcome measures may be attributed, in part, to viral adaptation to HLA-restricted T cell responses. Variants of the endoplasmic reticulum aminopeptidases (ERAPs) influence the repertoire of T cell epitopes presented by HLA alleles as they trim pathogen-derived peptide precursors to optimal lengths for antigen presentation, along with other functions unrelated to antigen presentation. We investigated whether ERAP variants influence HLA-associated HIV-1 adaptation with demonstrable effects on overall HIV-1 disease outcome. Utilizing host and viral data of 249 West Australian individuals with HIV-1 subtype B infection, we identified a novel association between two linked ERAP2 single nucleotide polymorphisms (SNPs; rs2248374 and rs2549782) with plasma HIV RNA concentration (viral load) (P adjusted = 0.0024 for both SNPs). Greater HLA-associated HIV-1 adaptation in the HIV-1 Gag gene correlated significantly with higher viral load, lower CD4+ T cell count and proportion; P = 0.0103, P = 0.0061, P = 0.0061, respectively). When considered together, there was a significant interaction between the two ERAP2 SNPs and HLA-associated HIV-1 adaptation on viral load (P = 0.0111). In a comprehensive multivariate model, addition of ERAP2 haplotypes and HLA associated adaptation as an interaction term to known HLA and CCR5 determinants and demographic factors, increased the explanatory variance of population viral load from 17.67% to 45.1% in this dataset. These effects were not replicated in publicly available datasets with comparably sized cohorts, suggesting that any true global epistasis may be dependent on specific HLA-ERAP allelic combinations. Our data raises the possibility that ERAP2 variants may shape peptide repertoires presented to HLA class I-restricted T cells to modulate the degree of viral adaptation within individuals, in turn contributing to disease variability at the population level. Analyses of other populations and experimental studies, ideally with locally derived ERAP genotyping and HLA-specific viral adaptations are needed to elucidate this further.

Adult orthodontic retreatment: A survey of patient profiles and original treatment failings.

INTRODUCTION: A growing number of adult patients are seeking orthodontic treatment. This research aimed to analyze the particulars of patients seeking retreatment and identify the causes of their original treatment failure. METHODS: An online questionnaire survey of adults seeking first-time orthodontic treatment (control) and retreatment (study) was conducted. Index of complexity, outcome, and need (ICON) scores were determined. Appraisal of treatment records was carried out to identify the causes of original treatment failure. RESULTS: No significant differences were found between retreatment adult patients and first-timers regarding reasons for seeking orthodontic treatment, malocclusion type, self-perception of malocclusion, level of self-motivation, willingness for surgery, expectations of treatment improvement and duration. The predominant reason for seeking treatment in both groups was for aesthetic concerns. Retreatment patients presented with lower ICON scores (39.4; standard error, 0.26) than the first-time patients (54.3; standard error, 0.23), P ≤0.001. The predominant reasons for original treatment failings were poor treatment, maturational changes, inadequate retention, shortcomings in diagnosis and treatment planning, and unfavorable growth. Other causes were related to transverse deficiency, secondary malocclusion (after periodontal breakdown), poor retention compliance, and temporomandibular joint degeneration. CONCLUSIONS: Adult orthodontic retreatment and first-time seekers' profiles are remarkably similar. Aesthetic concerns were the leading reasons patients sought treatment. ICON was not a useful proxy of patient profiles. Poor treatment was the chief reason for the failure of the original treatment. In terms of clinical significance, clinicians should be mindful of the patient profiles of retreatment seekers and vigilant about the possible causes of failings of orthodontic treatment to avoid suboptimal outcomes.

Hepatic iron loading in mice increases cholesterol biosynthesis.

UNLABELLED: Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up-regulated with increasing hepatic iron (R(2) between 0.602 and 0.164), including those of the rate-limiting enzyme, 3-hydroxy-3-methylglutarate-coenzyme A reductase (Hmgcr; R(2) = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R(2) = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R(2) = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol-regulatory element binding factor 2 (Srebf2; R(2) = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron. CONCLUSION: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity.

A network modeling approach to analysis of the Th2 memory responses underlying human atopic disease.

Complex cellular functions within immunoinflammatory cascades are conducted by networks of interacting genes. In this study, we employed a network modeling approach to dissect and interpret global gene expression patterns in allergen-induced Th cell responses that underpin human atopic disease. We demonstrate that a subnet of interconnected genes enriched for Th2 and regulatory T cell-associated signatures plus many novel genes is hardwired into the atopic response and is a hallmark of atopy at the systems level. We show that activation of this subnet is stabilized via hyperconnected "hub" genes, the selective disruption of which can collapse the entire network in a comprehensive fashion. Finally, we investigated gene expression in different Th cell subsets and show that regulatory T cell- and Th2-associated signatures partition at different stages of Th memory cell differentiation. Moreover, we demonstrate the parallel presence of a core element of the Th2-associated gene signature in bystander naive cells, which can be reproduced by rIL-4. These findings indicate that network analysis provides significant additional insight into atopic mechanisms beyond that achievable with conventional microarray analyses, predicting functional interactions between novel genes and previously recognized members of the allergic cascade. This approach provides novel opportunities for design of therapeutic strategies that target entire networks of genes rather than individual effector molecules.

Computed tomographic assessment of lung aeration at different positive end-expiratory pressures in a porcine model of intra-abdominal hypertension and lung injury.

BACKGROUND: Intra-abdominal hypertension (IAH) is common in critically ill patients and is associated with increased morbidity and mortality. High positive end-expiratory pressures (PEEP) can reverse lung volume and oxygenation decline caused by IAH, but its impact on alveolar overdistension is less clear. We aimed to find a PEEP range that would be high enough to reduce atelectasis, while low enough to minimize alveolar overdistention in the presence of IAH and lung injury. METHODS: Five anesthetized pigs received standardized anesthesia and mechanical ventilation. Peritoneal insufflation of air was used to generate intra-abdominal pressure of 27 cmH2O. Lung injury was created by intravenous oleic acid. PEEP levels of 5, 12, 17, 22, and 27 cmH2O were applied. We performed computed tomography and measured arterial oxygen levels, respiratory mechanics, and cardiac output 5 min after each new PEEP level. The proportion of overdistended, normally aerated, poorly aerated, and non-aerated atelectatic lung tissue was calculated based on Hounsfield units. RESULTS: PEEP decreased the proportion of poorly aerated and atelectatic lung, while increasing normally aerated lung. Overdistension increased with each incremental increase in applied PEEP. "Best PEEP" (respiratory mechanics or oxygenation) was higher than the "optimal CT inflation PEEP range" (difference between lower inflection points of atelectatic and overdistended lung) in healthy and injured lungs. CONCLUSIONS: Our findings in a large animal model suggest that titrating a PEEP to respiratory mechanics or oxygenation in the presence of IAH is associated with increased alveolar overdistension.

Influence of wild-type MLL on glucocorticoid sensitivity and response to DNA-damage in pediatric acute lymphoblastic leukemia.

BACKGROUND: Rearrangement of the mixed-lineage leukemia gene (MLL) is found in 80% of infant acute lymphoblastic leukemia (ALL) and is associated with poor prognosis and resistance to glucocorticoids (GCs). We have recently observed that GC resistance in T-ALL cell lines is associated with a proliferative metabolism and reduced expression of MLL. In this study we have further explored the relationship between MLL status and GC sensitivity. RESULTS: Negative correlation of MLL expression with GC resistance in 15 T-ALL cell lines was confirmed by quantitative RT-PCR. The absence of MLL-rearrangements suggested that this relationship represented expression of wild-type MLL. Analysis of MLL expression patterns revealed a negative relationship with cellular metabolism, proliferation and anti-apoptotic transcriptional networks. In silico analysis of published data demonstrated that reduced levels of MLL mRNA are associated with relapse and prednisolone resistance in T-ALL patients and adverse clinical outcome in children with MLL-rearranged ALL. RNAi knockdown of MLL expression in T-ALL cell lines significantly increased resistance to dexamethasone and gamma irradiation indicating an important role for wild-type MLL in the control of cellular apoptosis. CONCLUSIONS: The data suggests that reduced expression of wild-type MLL can contribute to GC resistance in ALL patients both with and without MLL-translocations.

Gene-based outcome prediction in multiple cohorts of pediatric T-cell acute lymphoblastic leukemia: a Children's Oncology Group study.

BACKGROUND: Continuous complete clinical remission in T-cell acute lymphoblastic leukemia (T-ALL) is now approaching 80% due to the implementation of aggressive chemotherapy protocols but patients that relapse continue to have a poor prognosis. Such patients could benefit from augmented therapy if their clinical outcome could be more accurately predicted at the time of diagnosis. Gene expression profiling offers the potential to identify additional prognostic markers but has had limited success in generating robust signatures that predict outcome across multiple patient cohorts. This study aimed to identify robust gene classifiers that could be used for the accurate prediction of relapse in independent cohorts and across different experimental platforms. RESULTS: Using HG-U133Plus2 microarrays we modeled a five-gene classifier (5-GC) that accurately predicted clinical outcome in a cohort of 50 T-ALL patients. The 5-GC was further tested against three independent cohorts of T-ALL patients, using either qRT-PCR or microarray gene expression, and could predict patients with significantly adverse clinical outcome in each. The 5-GC featured the interleukin-7 receptor (IL-7R), low-expression of which was independently predictive of relapse in T-ALL patients. In T-ALL cell lines, low IL-7R expression was correlated with diminished growth response to IL-7 and enhanced glucocorticoid resistance. Analysis of biological pathways identified the NF-kappaB and Wnt pathways, and the cell adhesion receptor family (particularly integrins) as being predictive of relapse. Outcome modeling using genes from these pathways identified patients with significantly worse relapse-free survival in each T-ALL cohort. CONCLUSIONS: We have used two different approaches to identify, for the first time, robust gene signatures that can successfully discriminate relapse and CCR patients at the time of diagnosis across multiple patient cohorts and platforms. Such genes and pathways represent markers for improved patient risk stratification and potential targets for novel T-ALL therapies.

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.

An overview of microbial food safety programs in beef, pork, and poultry from farm to processing in Canada.

Canada's vision for the agri-food industry in the 21st century is the establishment of a national food safety system employing hazard analysis and critical control point (HACCP) principles and microbiological verification tools, with traceability throughout the gate-to-plate continuum. Voluntary on-farm food safety (OFFS) programs, based in part on HACCP principles, provide producers with guidelines for good production practices focused on general hygiene and biosecurity. OFFS programs in beef cattle, swine, and poultry are currently being evaluated through a national recognition program of the Canadian Food Inspection Agency. Mandatory HACCP programs in federal meat facilities include microbial testing for generic Escherichia coli to verify effectiveness of the processor's dressing procedure, specific testing of ground meat for E. coli O157:H7, with zero tolerance for this organism in the tested lot, and Salmonella testing of raw products. Health Canada's policy on Listeria monocytogenes divides ready-to-eat products into three risk categories, with products previously implicated as the source of an outbreak receiving the highest priority for inspection and compliance. A national mandatory identification program to track livestock from the herd of origin to carcass inspection has been established. Can-Trace, a data standard for all food commodities, has been designed to facilitate tracking foods from the point of origin to the consumer. Although much work has already been done, a coherent national food safety strategy and concerted efforts by all stakeholders are needed to realize this vision. Cooperation of many government agencies with shared responsibility for food safety and public health will be essential.

Translating microarray data for diagnostic testing in childhood leukaemia.

BACKGROUND: Recent findings from microarray studies have raised the prospect of a standardized diagnostic gene expression platform to enhance accurate diagnosis and risk stratification in paediatric acute lymphoblastic leukaemia (ALL). However, the robustness as well as the format for such a diagnostic test remains to be determined. As a step towards clinical application of these findings, we have systematically analyzed a published ALL microarray data set using Robust Multi-array Analysis (RMA) and Random Forest (RF). METHODS: We examined published microarray data from 104 ALL patients specimens, that represent six different subgroups defined by cytogenetic features and immunophenotypes. Using the decision-tree based supervised learning algorithm Random Forest (RF), we determined a small set of genes for optimal subgroup distinction and subsequently validated their predictive power in an independent patient cohort. RESULTS: We achieved very high overall ALL subgroup prediction accuracies of about 98%, and were able to verify the robustness of these genes in an independent panel of 68 specimens obtained from a different institution and processed in a different laboratory. Our study established that the selection of discriminating genes is strongly dependent on the analysis method. This may have profound implications for clinical use, particularly when the classifier is reduced to a small set of genes. We have demonstrated that as few as 26 genes yield accurate class prediction and importantly, almost 70% of these genes have not been previously identified as essential for class distinction of the six ALL subgroups. CONCLUSION: Our finding supports the feasibility of qRT-PCR technology for standardized diagnostic testing in paediatric ALL and should, in conjunction with conventional cytogenetics lead to a more accurate classification of the disease. In addition, we have demonstrated that microarray findings from one study can be confirmed in an independent study, using an entirely independent patient cohort and with microarray experiments being performed by a different research team.

Correlations of siblings' and mothers' utilisation of primary and hospital health care: a record linkage study in Western Australia.

A relationship between maternal and child use of general practitioners (GPs) has been shown to exist for some time, however, the reasons for this relationship are not clear and the extent to which this relationship extends to tertiary care is unknown. The aim of this study was to examine the relationships between the utilisation of health care by siblings and mothers over a 14 year period. A retrospective cohort study of 756 mothers and their 1494 children up to age 14 years was conducted in three general practices in Western Australia. Medicare claims and hospital morbidity records for 1984-1997 were linked using deterministic and probabilistic matching. Generalised Estimating Equations and correlations were used to examine the relationships between the utilisation of primary and hospital health care by family members. Significant correlations were found between hospital admissions of all participants and their GP visits, specialist visits, pathology and diagnostic imaging combined and hospital length of stay. There was a strong association between siblings' use of GPs. A child's rate of GP attendance increased with that of its mother. There was a weak but significant relationship between siblings' use of hospitals, and a child's hospital admission rate increased with that of its mother. It is concluded that there is a strong relationship between siblings' use of GPs and a weaker but still significant association between the hospital admissions of siblings. As expected, there were strong associations between mother and child visits to GPs. There was also an association between a mother's use of hospital and that of her children. This finding reduces the plausibility that the relationships found between utilisation of health care by siblings and mothers can be explained entirely by behavioural factors, and suggests the presence of intergenerational correlation of morbidity.

Pretravel Health Advice Among Australians Returning From Bali, Indonesia: A Randomized Controlled Trial Protocol.

BACKGROUND: The effect of pretravel health advice (PTHA) on travel-related illness rates is poorly understood, and to date there are no published randomized controlled trials evaluating the impact of PTHA outcomes. OBJECTIVE: This study aims to determine the effect of an online PTHA intervention on travel-related illness rates in Western Australians visiting Bali, Indonesia. METHODS: Western Australian travelers to Bali will be recruited online before departure and will be randomly allocated to an intervention or control group by computer algorithm. The intervention in this study is a short animated video, with accompanying text, containing PTHA relevant to Bali. An online posttravel survey will be administered to all participants within two weeks of their return from Bali. The primary outcome is the difference in self-reported travel-related illness rates between control and intervention groups. Secondary outcomes include the difference in risk prevention behaviors and health risk knowledge between the control and intervention groups. Further secondary outcomes include whether individuals in the control group who sought external PTHA differ from those who did not with respect to risk prevention behaviors, health risk knowledge, and health risk perception, as well as the rate of self-reported travel-related illness. RESULTS: The study began recruitment in September 2016 and will conclude in September 2017. Data analysis will take place in late 2017, with results disseminated via peer-reviewed journals in early 2018. CONCLUSIONS: This will be the first randomized controlled trial to examine the effect of a novel PTHA intervention upon travel-related illness. In addition, this study builds upon the limited existing data on the effectiveness of PTHA on travel-related illness. CLINICALTRIAL: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12615001230549; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=369567 (Archived by WebCite at http://www.webcitation.org/6m0G7xJg1).

Health Seeking Behaviours and Knowledge of Infectious Disease Risks in Western Australian Travellers to Southeast Asian Destinations: An Airport Survey.

As the number of Australians engaging in short-term international travel increases, so does the opportunity for importing overseas-acquired infectious diseases. This study aimed to determine knowledge of infectious disease risks and pre-travel health advice (PTHA) seeking behaviour among Western Australians travelling to Bali, Indonesia or Thailand. Passengers departing from Perth International Airport were invited to participate in a self-administered survey. The survey determined PTHA seeking behaviour, knowledge of specific disease risks, and expected disease-prevention behaviours abroad. Multivariate regression modelling was used to assess demographic and travel-related factors associated with seeking PTHA. Responses from 1334 travellers were analysed. The proportion correctly identifying specific overseas disease risks ranged from 27% to 98%. High levels of planned disease-preventive behaviours were reported; however only 32% of respondents sought PTHA for their trip, most commonly from friends/family (15%) or a GP (14%). Many travellers (87%) made online travel purchases, but few (8%) used the Internet to source PTHA. WA travellers to Bali and Thailand were unlikely to seek PTHA and knowledge varied regarding infectious disease risks associated with travel. High rates of internet use when planning travel may provide an opportunity for destination-specific health promotion messaging and should be explored.

Transcriptome analysis of human ageing in male skin shows mid-life period of variability and central role of NF-κB.

Age is well-known to be a significant factor in both disease pathology and response to treatment, yet the molecular changes that occur with age in humans remain ill-defined. Here, using transcriptome profiling of healthy human male skin, we demonstrate that there is a period of significantly elevated, transcriptome-wide expression changes occurring predominantly in middle age. Both pre and post this period, the transcriptome appears to undergo much smaller, linear changes with increasing age. Functional analysis of the transient changes in middle age suggest a period of heightened metabolic activity and cellular damage associated with NF-kappa-B and TNF signaling pathways. Through meta-analysis we also show the presence of global, tissue independent linear transcriptome changes with age which appear to be regulated by NF-kappa-B. These results suggest that aging in human skin is associated with a critical mid-life period with widespread transcriptome changes, both preceded and proceeded by a relatively steady rate of linear change in the transcriptome. The data provides insight into molecular changes associated with normal aging and will help to better understand the increasingly important pathological changes associated with aging.

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.

Benefits of routine immunizations on childhood survival in Tari, Southern Highlands Province, Papua New Guinea.

BACKGROUND: Non-specific beneficial as well as deleterious effects of childhood immunizations have been reported in areas of high mortality. This study aimed to determine the effects of diphtheria-tetanus-whole-cell-pertussis (DTP), BCG, hepatitis B, and measles vaccines on mortality in the highlands of Papua New Guinea (PNG). METHODS: Demographic events for children born in 1989-1994 who were under monthly demographic surveillance in Tari were recorded from birth until age 2 years, out-migration, death, or the end of the study period. Data on BCG, hepatitis B, DTP, measles and pneumococcal polysaccharide vaccination were collected monthly from clinic records. To allow for different characteristics of immunized and non-immunized children, analysis included conditioning on a propensity score for vaccination, adjusting for differences in children's background characteristics. RESULTS: In all, 101/3502 children (3%) who had at least one vaccine died between ages 29 days and 24 months were compared to 112/546 (21%) who had none. BCG was associated with lower mortality in the 1-5 month age group (hazard ratio [HR] = 0.17, 95% CI: 0.09, 0.34), measles vaccine with lower mortality at age 6-11 months (HR = 0.42, 95% CI: 0.17, 1.01), and pneumococcal polysaccharide vaccine with lower mortality at age 12-23 months (HR = 0.42, 95% CI: 0.19, 0.93). One or more doses of DTP was associated with lower overall mortality (HR = 0.27, 95% CI: 0.16, 0.44), particularly in the 1-5 month age group (HR = 0.19, 95% CI: 0.10, 0.34), and also in those who had had prior BCG (HR = 0.45, 95% CI: 0.22, 0.91). CONCLUSION: Routine immunizations are effective in reducing overall mortality in young children in an area of high mortality. In particular, DTP, whether considered separately or in addition to BCG, was associated with a lowering of overall mortality, in contrast to findings reported from Guinea-Bissau.

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.

Drug-gene modeling in pediatric T-cell acute lymphoblastic leukemia highlights importance of 6-mercaptopurine for outcome.

Patients relapsing with T-cell acute lymphoblastic leukemia (T-ALL) face a dismal outcome. The aim of this study was to identify new markers of drug resistance and clinical response in T-ALL. We measured gene expression and drug sensitivity in 15 pediatric T-ALL cell lines to find signatures predictive of resistance to 10 agents used in therapy. These were used to generate a model for outcome prediction in patient cohorts using microarray data from diagnosis specimens. In three independent T-ALL cohorts, the 10-drug model was able to accurately identify patient outcome, indicating that the in vitro-derived drug-gene profiles were clinically relevant. Importantly, predictions of outcome within each cohort were linked to distinct drugs, suggesting that different mechanisms contribute to relapse. Sulfite oxidase (SUOX) expression and the drug-transporter ABCC1 (MRP1) were linked to thiopurine sensitivity, suggesting novel pathways for targeting resistance. This study advances our understanding of drug resistance in T-ALL and provides new markers for patient stratification. The results suggest potential benefit from the earlier use of 6-mercaptopurine in T-ALL therapy or the development of adjuvants that may sensitize blasts to this drug. The methodology developed in this study could be applied to other cancers to achieve patient stratification at the time of diagnosis.

Interactions between acute lymphoblastic leukemia and bone marrow stromal cells influence response to therapy.

The cure rate for pediatric patients with B precursor acute lymphoblastic leukemia (pre-B ALL) is steadily improving, however relapses do occur despite initial response to therapy. To identify links between drug resistance and gene deregulation we used oligonucleotide microarray technology and determined in 184 pre-B ALL specimen genes differentially expressed compared to normal CD34(+) specimens. We identified 20 signature genes including CTGF, BMP-2, CXCR4 and IL7R, documented to regulate interactions in the bone marrow. We recorded remarkably similar levels of expression in three independent patient cohorts, and found distinct patterns in cytogenetically defined subgroups of pre-B ALL. The canonical pathways that were affected are involved in inter- and intra-cellular communication, regulating signaling within the microenvironment. We tested experimentally whether interaction with stromal cells conferred protection to four drugs used in current ALL therapy, and demonstrated that bone marrow stromal cells significantly influenced resistance to vincristine and cytosine arabinoside. Compounds designed to block the identified cellular interactions within the bone marrow microenvironment are expected to mobilise the leukemic cells and make them more accessible to contemporary antileukemic agents. The data provide novel insight into the pathobiology of ALL and indicate new therapeutic targets for patients with ALL.

Classification and regression tree and spatial analyses reveal geographic heterogeneity in genome wide linkage study of Indian visceral leishmaniasis.

BACKGROUND: Genome wide linkage studies (GWLS) have provided evidence for loci controlling visceral leishmaniasis on Chromosomes 1p22, 6q27, 22q12 in Sudan and 6q27, 9p21, 17q11-q21 in Brazil. Genome wide studies from the major focus of disease in India have not previously been reported. METHODS AND FINDINGS: We undertook a GWLS in India in which a primary ∼10 cM (515 microsatellites) scan was carried out in 58 multicase pedigrees (74 nuclear families; 176 affected, 353 total individuals) and replication sought in 79 pedigrees (102 nuclear families; 218 affected, 473 total individuals). The primary scan provided evidence (≥2 adjacent markers allele-sharing LOD≥0.59; nominal P≤0.05) for linkage on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X, with peaks at 6p25.3-p24.3 and 8p23.1-p21.3 contributed to largely by 31 Hindu families and at Xq21.1-q26.1 by 27 Muslim families. Refined mapping confirmed linkage across all primary scan families at 2q12.2-q14.1 and 11q13.2-q23.3, but only 11q13.2-q23.3 replicated (combined LOD = 1.59; P = 0.0034). Linkage at 6p25.3-p24.3 and 8p23.1-p21.3, and at Xq21.1-q26.1, was confirmed by refined mapping for primary Hindu and Muslim families, respectively, but only Xq21.1-q26.1 replicated across all Muslim families (combined LOD 1.49; P = 0.0045). STRUCTURE and SMARTPCA did not identify population genetic substructure related to religious group. Classification and regression tree, and spatial interpolation, analyses confirm geographical heterogeneity for linkages at 6p25.3-p24.3, 8p23.1-p21.3 and Xq21.1-q26.1, with specific clusters of families contributing LOD scores of 2.13 (P = 0.0009), 1.75 (P = 0.002) and 1.84 (P = 0.001), respectively. CONCLUSIONS: GWLS has identified novel loci that show geographical heterogeneity in their influence on susceptibility to VL in India.