Efficiency clustering for low-density microarrays and its application to QPCR

<p>Abstract</p> <p>Background</p> <p>Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.</p> <p><b>Results</b></p> <p>Our results indicate that QPCR primer-pairs differ significantly both in reliability and efficiency. They can only be used in an array format if the raw data (so called CT values for real-time QPCR) are transformed to take these differences into account. We developed a novel method to obtain efficiency-adjusted CT values. We introduce transformed confidence intervals as a novel measure to identify unreliable primers. We introduce a robust clustering algorithm to combine efficiencies of groups of probes, and our results indicate that using <it>n </it>< 10 cluster-based mean efficiencies is comparable to using individually determined efficiency adjustments for each primer pair (<it>N </it>= 96-1024).</p> <p><b>Conclusions</b></p> <p>Careful estimation of primer efficiency is necessary to avoid significant measurement inaccuracies. Transformed confidence intervals are a novel method to assess and interprete the reliability of an efficiency estimate in a high throughput format. Efficiency clustering as developed here serves as a compromise between the imprecision in assuming uniform efficiency, and the computational complexity and danger of over-fitting when using individually determined efficiencies.</p

Stress hos kjønnscellefri atlantisk laks (Salmo Salar L.) Effekter av transport og sjøvannseksponering på sentrale faktorer i hypothalamus-hypofyse- interrenale (HPI)-aksen

Genetisk innblanding fra rømt oppdrettslaks er i dag vurdert av noen som den største trusselen mot ville norske laksestammer. Selv enkelthendelser vil ha en stor risiko for masserømming da en merd kan inneholde opptil 200 000 individ. Samtidig er kjønnsmodning blitt et økende problem særlig knyttet til resirkuleringsanlegg (RAS). Kjønnsmodning bringer med seg en rekke velferdsmessige utfordringer som redusert vekst, immunrespons og osmoregulatorisk kapasitet. Derfor vil en effektiv og velfungerende steriliseringsmetode kunne bidra til å løse flere viktige utfordringer både for miljø, men også for nye og nåværende produksjonsmetoder. Eksisterende steriliseringsmetoder som for eksempel triploidisering krever imidlertid en spesialtilpasset produksjon og resulterer i redusert ytelse hos den sterile fisken. Nye metoder for sterilisering av laks er derfor nødvendig. Gjennom å fjerne kjønnscellelinjen på embryonalstadiet har det blitt utviklet en ny metode for sterilisering av atlantisk laks (Salmo salar). Fjerning av kjønnscellelinjen vil imidlertid kunne påvirke viktige endokrine og fysiologiske mekanismer, og dermed fiskens produksjonsegenskaper. For at en slik steriliseringsmetode skal kunne benyttes i kommersiell sammenheng må en nøye evaluere eventuelle effekter på viktige fysiologiske systemer og prosesser. I akvakultur vil en rekke produksjonsrelaterte hendelser (flytting, sortering, vaksinering, smolt transport, avlusing etc.) kunne resultere i en fysiologisk stressrespons hos fisken. Disse fysiologiske forandringene skjer hyppig i oppdrett hvor svak fisk som oftest dør før produksjonssyklusen er over. I dette forsøket ble laksen holdt ved kontinuerlig lys og varierende temperatur (6-12ºC) fra startfôring 06.03.2017 til 28.08.2018. Smoltifisering ble indusert via en standard protokoll brukt ved HiT. Videre ble en gruppe fisk uten kjønnsceller og en kontrollgruppe med intakte kjønnsceller kjørt gjennom en simulert transport etterfulgt av full sjøvannseksponering i 23 timer. Prøveuttaket ble fordelt over noen dager hvor faktorer som kortisol, osmolalitet, klorid og genuttrykk i HPI-aksen ble undersøkt. Kortisol, osmolalitet og klorid ble målt under alle tre uttakene, mens genuttrykk crf1a og crf1b i hjernen, pomca og pomcb i hypofysen og gr1, gr2, mc2r og 11bhsd2 i hodenyren ble målt pre-stress og post sjøvannseksponering. Resultatene viser med hensyn på plasma kortisol at kjønnscellefri-fisk kanskje presterer enda bedre enn kontrollfisk etter 23 timer på sjøvann. Genuttrykk for steril- og kontrollfisk viser liten til ingen forskjell mellom gruppene, foruten om hannfisken hvor nivåene av crf1a og crf1b er signifikant økt hos kontroll pre-stress og crf1b og 11bhsd2 etter 23 timer med sjøvannseksponering. Resultatene fra dette forsøket viser at det i praksis er små forskjeller i håndtering av stress og sjøvannstoleranse mellom kjønnscellefri og fertil atlantisk laks

Profiling for KSHV and Other Herpesviruses In Vivo In Clinical Specimens

The 1994 discovery of Kaposi Sarcoma-associated herpesvirus (KSHV) was an important finding, especially as the sharp rise in HIV infections in the early 1990's led to an explosive Kaposi Sarcoma (KS) epidemic in men who have sex with men. This cancer-causing herpesvirus is the causative agent of three diseases in addition to KS: Multicentric Castlemen Disease (MCD), Primary Effusion Lymphoma (PEL), and the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). By understanding viral gene expression in human patients, we hope to better understand KSHV infection and its diseases, with the hope to shed light on novel therapeutic techniques. In order to quantify KSHV viral gene expression, I devised methods to profile human clinical specimens using high-throughput qPCR. This technique was used to profile microRNA expression and was published as a methods video journal in the Journal of Visualized Experiments (Chugh, Tamburro, and Dittmer, 2010). The accompanying manuscript is found in the Appendix chapter. This same high-throughput technique was employed in Chapter II to determine KSHV viral load and to determine expression of viral kinases, and in Chapter III to determine KSHV gene expression profiles. In Chapter II, we hypothesized that HIV patients with newly diagnosed KS in Malawi could potentially benefit from treatment with the antiherpesviral drug Ganciclovir (GCV). This was addressed through a pilot study with the AIDS Malignancy Consortium (AMC). By using qPCR to assess gene expression of two viral kinases critical to drug responsiveness, we found evidence that 78% of patients express a viral kinase and would potentially benefit from this therapy. This finding is important, as it indicates that patients can be stratified for treatment based on kinase expression in a tumor biopsy, which will maximize potential effectiveness and limit waste of drugs in a resource limited setting. This work will lead to upcoming treatment studies in this population. Next, we used whole-genome KSHV transcriptome profiling to understand whether all KS patients from the Malawian cohort express similar gene expression profiles or if subgroups can be distinguished. Our results indicate the gene expression profiles vary significantly between patients. Specifically, two subpopulations were discovered- those with and those without high levels of genome-wide transcription. Patients with high transcription levels demonstrated significantly higher CD4 counts and KSHV viremia in plasma, although HIV viremia, number of tumors, or size of biopsied lesion were not significantly associated with KSHV transcription. This work is described in Chapter III. In Chapter IV, we employed high-throughput DNA sequencing as a method to diagnose co-infections in an end-stage AIDS patient. This was published in the Journal Virology (Tamburro et al. 2012). The patient had minimal KS skin lesions despite having one of the highest viral loads on record- leading to the diagnosis of the newly described condition KSHV Inflammatory Cytokine Syndrome (KICS). Additionally, sequencing uncovered a co-infection with HHV6a, which may have accounted for Systemic Inflammatory Response Syndrome (SIRS) that ultimately led to the patient's death. Notable to the research community, this work resulted in the first complete sequence of KSHV using direct methods, rather than relying on pre-sequencing cloning or cell passage methods.Doctor of Philosoph

Cytokine and Chemokine Responses in Human Herpesvirus 8 Infection of Monocyte Derived Dendritic Cells and B Lymphocytes

Human herpesvirus-8 induces a wide range of inflammatory immune mediators known to contribute to its associated cancer, Kaposi’s Sarcoma (KS). Soluble immune mediators, such as cytokines, chemokines and growth factors produced during HHV-8 infection have been associated with tumor-cell proliferation, angiogenesis and vascular permeability. We sought to determine immune mediator production by two antigen presenting cells (APC) that are susceptible to HHV-8 infection, i.e., monocyte derived Dendritic cells (MDDC) and B lymphocytes. Dendritic cells abundantly express the HHV-8 receptor, type II C-type lectin, DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) resulting in viral entry, whereas only a small percentage of activated B cells express DC-SIGN in vitro. Despite this, HHV-8 infection of MDDC results in an abortive replicative cycle, whereas full-lytic cycle replication occurs in the B cells. I hypothesized that immune mediators produced by HHV-8 infected APC are unique between cell types and that HHV-8 infects a subset of B cells and initiates cytokine and chemokine production that contributes to HHV-8 replication, viral dissemination and initiation of KS and HHV-8 lymphomas. I used a cytometric bead array to determine cytokine and chemokine production in B cells and MDDC, as well as qRT-PCR, TCID50 assay and flow cytometry to determine HHV-8 replication in B cells. I identified significant differences in the quality and quantity of cytokine and chemokine profiles of HHV-8 infected APC. MDDC produced significant levels of MCP-1, MIP-1α, MIP-1β, RANTES, IP-10 and IL-10, while B cells produced significant levels of MIP-1α, MIP-1β, IL-6, TNF-α and IL-8. HHV-8 lytic replication in B cells resulted in polyfunctional immune mediator activity that may contribute to viral replication and proliferation of target cell populations in HHV-8 related cancers. The importance of this work was demonstrated by the detection of B cell-produced cytokines and chemokines in HHV-8/HIV co-infected individuals who developed KS. This is the first extensive, multiparameter, longitudinal study of HHV-8 infection of B cells and immune mediators in development of KS. This study provides novel targets for vaccine development and treatment options for KS, which could have great implications on Public Health

CD8 T Lymphocyte Responses to Human Herpesvirus 8 Lytic and Latency Proteins

Human herpesvirus 8 (HHV-8) is a lymphotropic gammaherpesvirus that causes Kaposi's sarcoma (KS), a vascular tumor of spindle-shaped endothelial cells. The importance of determining effective prevention and treatment for HHV-8 infection is evident in that KS continues to be the most common cancer among HIV-1 and AIDS patients. It is postulated that CD8+ T cell responses play an important role in controlling HHV-8 infection and preventing KS. However, there are minimal data supporting the role for T cell immunity in control of HHV-8 infection. Therefore, I investigated CD8+ T cell responses to HHV-8 lytic and latency proteins. I used our dendritic cell-based system in an IFN-γ ELISPOT to identify novel epitopes in healthy, HHV-8 seropositive individuals, then further assessed new epitopes for immune mediators using polychromatic flow cytometry. I also used multimer complexes to directly detect HHV-8-specific CD8+ T cells in blood. I investigated the effect of regulatory T cells (Treg) on these anti-HHV-8 T cell responses by depleting samples of Treg. I then applied these assays to patients in the Multicenter AIDS Cohort Study to longitudinally investigate the role of these responses during the progression to KS. Through these studies, I identified 10 novel HLA A*0201-restricted epitopes, which activated both monofunctional and polyfunctional CD8+ T cells producing various combinations of immune mediators. Responses were lower over many years prior to development of KS. Although CD8+ T cell IFN-γ responses were modest, a low but consistent percent of HHV-8-specific CD8+ T cells were present in blood, suggesting a functional down-regulation of this response. In support of this, removal of Treg enhanced CD8+ T cell responses to HHV-8 epitopes, and numbers of Treg increased prior to KS development. Overall, these data support that T cell responses, in frequency, magnitude and quality, are essential for the control of HHV-8 infection to prevent disease development. They also indicate that these antiviral T cell responses are in part controlled by Treg. Involvement of CD8+ T cells and Treg in control of HHV-8 infection has public health significance through implications for understanding the immunopathogenesis of HHV-8 needed for prevention and treatment of KS