A proteome-wide analysis unveils a core Epstein-Barr virus antibody signature of classic Hodgkin lymphoma across ethnically diverse populations.

Epstein-Barr virus (EBV) is an oncogenic virus associated with various malignancies, including classical Hodgkin lymphoma (cHL). Despite its known association, the specific role of humoral immune response to EBV remains poorly characterized in cHL. To address this, we conducted a study using a custom protein microarray to measure the antibody responses in cHL patients and matched healthy controls recruited from an East-Asian hospital-based case-control study. We identified 16 IgG antibodies significantly elevated in EBV-positive cHL compared with controls, defining an "East-Asian antibody signature of EBV-positive cHL." We evaluated responses against these 16 antibodies in a distinct European population, leveraging data from our previous European cHL case-control study from the UK, Denmark, and Sweden. A subset of antibodies (14/16, 87.5%) from the "East-Asian antibody signature of EBV-positive cHL" exhibited significant associations with cHL in the European population. Conversely, we assessed the "European antibody signature of EBV-positive cHL" identified in our prior study which consisted of 18 EBV antibodies (2 IgA, 16 IgG), in the East-Asian population. A subset of these antibodies (15/18, 83.3%) maintained significant associations with cHL in the East-Asian population. This cross-comparison of antibody signatures underscores the robust generalizability of EBV antibodies across populations. Five anti-EBV IgG antibodies (LMP-1, TK, BALF2, BDLF3, and BBLF1), found in both population-specific antibody signatures, represent a "core signature of EBV-positive cHL." Our findings suggest that the antibody responses targeting these core EBV proteins reflect a specific EBV gene expression pattern, serving as potential biomarkers for EBV-positive cHL independent of population-specific factors.

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria.

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection' with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs.

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples.

Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.

Exposure to harmful housing conditions is common in children admitted to Wellington Hospital.

AIM: To measure the prevalence of exposure to potentially modifiable risk factors in the homes of children hospitalised in Wellington. METHODS: Parents/caregivers of all children admitted to Wellington Public Hospital during a two-week period in July 2012 completed a standardised questionnaire in a face-to-face interview. The questionnaire collected sociodemographic, health and housing condition data. RESULTS: We interviewed parents/caregivers of 106 children, of whom 72% were aged 0-4 years. Respiratory conditions were the most common cause of admission. One third of parents noticed dampness and mould in their house, 50% stated that their house was colder than they preferred during the past month, 20% lived in uninsulated houses, 20% lived in overcrowded houses, and 38% were exposed to second hand smoke (SHS). Compared to New Zealand European (NZE) children, the odds ratios (OR) for Pacific children living in cold and overcrowded houses and being exposed to SHS were 14.0 (95%CI 3.0-66.0), 10.8 (95%CI 2.6-44.1) and 16.0 (95%CI 4.8-55.5) respectively. OR for Maori children living in cold and overcrowded houses and being exposed to SHS were 3.0 (95%CI 1.0-9.0), 6.8 (95%CI 1.6-30.1) and 8.0 (95%CI 2.5-28.6) respectively, compared to NZE children. The OR for children from deprived neighbourhoods (NZDep2006 areas 7-10) living in cold and overcrowded houses and being exposed to SHS were 4.1 (95%CI 1.8-9.6), 5.7 (95%CI 1.9-17.0) and 4.1 (95%CI 1.6-9.6) respectively. CONCLUSIONS: Among children admitted to Wellington Hospital there is a high prevalence of exposure to cold, damp and overcrowded houses and many children are exposed to SHS. Maori and Pacific children and children living in socioeconomically deprived areas are more likely than others to be exposed to these potential risk factors for childhood hospitalisation. This audit of child admissions could be repeated to provide surveillance of modifiable risk factors. A shortened version of the questionnaire could be used to screen children to identify those with harmful exposures in their home environment, provided suitable intervention programmes can be established.