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Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex, multi-system, debilitating disability manifesting as severe fatigue and post-exertional malaise. The chronic dysfunctions in ME/CFS are increasingly recognized as significant health factors with potential parallels with ‘long COVID’. However, the etiology of ME/CFS remains elusive with limited high-resolution human studies. In addition, reliable biomarker-based diagnostics have not been well-established, but may assist in disease classification, particularly during different temporal phases of the disease. Here, we performed deep multi-‘omics (shotgun metagenomics of gut microbiota and plasma metabolomics) and clinical phenotyping of healthy controls (n=79) vs. two cohorts of ME/CFS patients – those with short-term disease (10y, n=79). Overall, ME/CFS was characterized by reduced gut microbiome diversity and richness with high heterogeneity, and depletion of sphingomyelins and short-chain fatty acids in the plasma. We found significant differences when stratifying by cohort; short-term ME/CFS was associated with more microbial dysbiosis, but long-term ME/CFS was associated with markedly more severe phenotypic and metabolic abnormalities. We identified a reduction in the gene-coding capacity (and relative abundance of butyrate producers) of microbial butyrate biosynthesis together with a reduction in the plasma concentration of butyrate, especially in the short-term group. Global co-association and detailed gene pathway correlation analyses linking the microbiome and metabolome identified additional potential biological mechanisms underlying host-microbiome interactions in ME/CFS, including bile acids and benzoate pathways. Finally, we built multiple state-of-the-art classifiers to identify microbes, microbial gene pathways, metabolites, and clinical features that individually or together, were most able to differentiate short or long-term MECFS, or MECFS vs. healthy controls. Taken together, our study presents the highest resolution, multi-cohort and multi-‘omics analysis to date, providing an important resource to facilitate mechanistic hypotheses of host-microbiome interactions in ME/CFS. more...

BackgroundGenomics-driven discoveries of microbial species have provided extraordinary insights into the biodiversity of human microbiota. In addition, a significant portion of genetic variation between microbiota exists at the subspecies, or strain, level. High-resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. High-throughput approaches are needed to acquire and identify the significant species- and strain-level diversity present in the oral, skin, and gut microbiome. Here, we describe and validate a streamlined workflow for cultivating dominant bacterial species and strains from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.ResultsOf total genera discovered by either metagenomic sequencing or culturomics, our cultivation pipeline recovered between 18.1–44.4% of total genera identified. These represented a high proportion of the community composition reconstructed with metagenomic sequencing, ranging from 66.2–95.8% of the relative abundance of the overall community. Fourier-Transform Infrared spectroscopy (FT-IR) was effective in differentiating genetically distinct strains compared with whole-genome sequencing, but was less effective as a proxy for genetic distance.ConclusionsUse of a streamlined set of conditions selected for cultivation of skin, oral, and gut microbiota facilitates recovery of dominant microbes and their strain variants from a relatively large sample set. FT-IR spectroscopy allows rapid differentiation of strain variants, but these differences are limited in recapitulating genetic distance. Our data highlights the strength of our cultivation and characterization pipeline, which is in throughput, comparisons with high-resolution genomic data, and rapid identification of strain variation. more...

Background. Inferior quality of biological material compromises data, slows discovery, and wastes research funds. The gut microbiome plays a critical role in human health and disease, yet little attention has been given to optimizing collection and processing methods of human stool. Methods. We collected the entire bowel movement from 2 healthy volunteers: one to examine stool sample heterogeneity and one to test stool sample handling parameters. Sequencing and bioinformatic analyses were used to examine the microbiome composition. Results. The microbiome profile varied depending on where the subsample was obtained from the stool. The exterior cortex of the stool was rich in specific phyla and deficient in others while the interior core of the stool revealed opposite microbiome profiles. Sample processing also resulted in varying microbiome profiles. Homogenization and stabilization at 4°C gave superior microbial diversity profiles compared to the fresh or frozen subsamples of the same stool sample. Bacterial proliferation continued in the fresh subsample when processed at ambient temperature. Bacteroidetes proliferated and Firmicutes diminished during the 30-minute processing of fresh sample. The frozen sample had good overall diversity but Proteobacteria diminished likely because of the freeze/thaw. Conclusions. The microbiome profile is specific to the section of the stool being sampled. Stool sample collection, homogenization, and stabilization at 4°C for 24 hours provides a “neat”, high-quality sample of sufficient quantity that can be banked into aliquots with nearly identical microbial diversity profiles. This collection pipeline is essential to accelerate our understanding of the gut microbiome in health and disease more...

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an example of a complex disease of unknown etiology. Multiple studies point to disruptions in immune functioning in ME/CFS patients as well as with specific genetic polymorphisms and alterations of the DNA methylome in lymphocytes. However, the association between DNA methylation and genetic background in relation to the ME/CFS is currently unknown. In this study we explored this association by characterizing the genomic (~4.3 million SNPs) and epigenomic (~480 thousand CpG loci) variability between populations of ME/CFS patients and healthy controls. We found significant associations of methylation states in T-lymphocytes at several CpG loci and regions with ME/CFS phenotype. These methylation anomalies are in close proximity to genes involved with immune function and cellular metabolism. Finally, we found significant correlations of genotypes with methylation phenotypes associated with ME/CFS. The findings from this study highlight the role of epigenetic and genetic interactions in complex diseases, and suggest several genetic and epigenetic elements potentially involved in the mechanisms of disease in ME/CFS. more...

Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating idiopathic disease characterized by unexplained fatigue that fails to resolve with sufficient rest. Diagnosis is based on a list of symptoms and exclusion of other fatigue-related health conditions. Despite a heterogeneous patient population, immune and hypothalamic-pituitary-adrenal (HPA) axis function differences, such as enhanced negative feedback to glucocorticoids, are recurring findings in ME/CFS studies. Epigenetic modifications, such as CpG methylation, are known to regulate long-term phenotypic differences and previous work by our group found DNA methylome differences in ME/CFS, however the relationship between DNA methylome modifications, clinical and functional characteristics associated with ME/CFS has not been examined. Methods We examined the DNA methylome in peripheral blood mononuclear cells (PBMCs) of a larger cohort of female ME/CFS patients using the Illumina HumanMethylation450 BeadChip Array. In parallel to the DNA methylome analysis, we investigated in vitro glucocorticoid sensitivity differences by stimulating PBMCs with phytohaemagglutinin and suppressed growth with dexamethasone. We explored DNA methylation differences using bisulfite pyrosequencing and statistical permutation. Linear regression was implemented to discover epigenomic regions associated with self-reported quality of life and network analysis of gene ontology terms to biologically contextualize results. Results We detected 12,608 differentially methylated sites between ME/CFS patients and healthy controls predominantly localized to cellular metabolism genes, some of which were also related to self-reported quality of life health scores. Among ME/CFS patients, glucocorticoid sensitivity was associated with differential methylation at 13 loci. Conclusions Our results indicate DNA methylation modifications in cellular metabolism in ME/CFS despite a heterogeneous patient population, implicating these processes in immune and HPA axis dysfunction in ME/CFS. Modifications to epigenetic loci associated with differences in glucocorticoid sensitivity may be important as biomarkers for future clinical testing. Overall, these findings align with recent ME/CFS work that point towards impairment in cellular energy production in this patient population. Electronic supplementary material The online version of this article (doi:10.1186/s12920-017-0248-3) contains supplementary material, which is available to authorized users. more...

Objective Wheat gluten and related proteins can trigger an autoimmune enteropathy, known as coeliac disease, in people with genetic susceptibility. However, some individuals experience a range of symptoms in response to wheat ingestion, without the characteristic serological or histological evidence of coeliac disease. The aetiology and mechanism of these symptoms are unknown, and no biomarkers have been identified. We aimed to determine if sensitivity to wheat in the absence of coeliac disease is associated with systemic immune activation that may be linked to an enteropathy. Design Study participants included individuals who reported symptoms in response to wheat intake and in whom coeliac disease and wheat allergy were ruled out, patients with coeliac disease and healthy controls. Sera were analysed for markers of intestinal cell damage and systemic immune response to microbial components. Results Individuals with wheat sensitivity had significantly increased serum levels of soluble CD14 and lipopolysaccharide (LPS)-binding protein, as well as antibody reactivity to bacterial LPS and flagellin. Circulating levels of fatty acid-binding protein 2 (FABP2), a marker of intestinal epithelial cell damage, were significantly elevated in the affected individuals and correlated with the immune responses to microbial products. There was a significant change towards normalisation of the levels of FABP2 and immune activation markers in a subgroup of individuals with wheat sensitivity who observed a diet excluding wheat and related cereals. Conclusions These findings reveal a state of systemic immune activation in conjunction with a compromised intestinal epithelium affecting a subset of individuals who experience sensitivity to wheat in the absence of coeliac disease. more...

Gulf War Illness (GWI) is a complex disorder affecting nervous, endocrine and immune regulation. Accordingly, we propose that GWI presents with a distinct pattern of immune signaling. To explore this we compared interaction patterns linking immune markers and their evolution during exercise. Blood was collected from 9 GWI and 11 control subjects prior to a Graded eXercise Test (GXT) (t₀), at peak effort (t₁) and 4 h post-exercise (t₂). Salivary cortisol and plasma, serum or culture supernatants were analyzed for concentrations of neuropeptide Y (NPY), IL-1α, IL-5, IL-6, IL-10, TNF-α, IFN-γ and soluble CD26 (sCD26). Immune cell populations were surface stained for CD19, CD2, CD3, CD4, CD8, CD26, CD56, CD16, and CD11a. Mutual information (MI) networks linking these immune markers were generated in each group at each time point. Graph theory was used to describe the evolution of each network's structure and identify potential nucleating points. Distinct in topology, GWI networks had more abundant connections but were less organized. NPY, IL-1α, TNF-α and CD2+/CD26+ nodes were better integrated in the GWI network at rest. Under effort (t₁) these differences were replaced by significant restructuring around nodes for CD19+ B cell population, IL-5, IL-6 and soluble CD26 concentrations. This pattern subsided post-exercise. Further analysis indicated that IL-1α and CD2+/CD26+ nodes strongly influenced this characteristic modulation of B and T cell network motifs. This potentially heightened lymphocyte and HPA axis responsiveness to IL-1 stimulation in the context of a mixed Th1:Th2 immune signature supports an autoimmune component in GWI etiology. more...

Chronic Fatigue Syndrome (CFS) is a complex illness affecting 4 million Americans for which no characteristic lesion has been identified. Instead of searching for a deficiency in any single marker, we propose that CFS is associated with a profound imbalance in the regulation of immune function forcing a departure from standard pre-programmed responses. To identify these imbalances we apply network analysis to the co-expression of 16 cytokines in CFS subjects and healthy controls. Concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12, 13, 15, 17 and 23, IFN-γ, lymphotoxin-α (LT-α) and TNF-α were measured in the plasma of 40 female CFS and 59 case-matched controls. Cytokine co-expression networks were constructed from the pair-wise mutual information (MI) patterns found within each subject group. These networks differed in topology significantly more than expected by chance with the CFS network being more hub-like in design. Analysis of local modularity isolated statistically distinct cytokine communities recognizable as pre-programmed immune functional components. These showed highly attenuated Th1 and Th17 immune responses in CFS. High Th2 marker expression but weak interaction patterns pointed to an established Th2 inflammatory milieu. Similarly, altered associations in CFS provided indirect evidence of diminished NK cell responsiveness to IL-12 and LT-α stimulus. These observations are consistent with several processes active in latent viral infection and would not have been uncovered by assessing marker expression alone. Furthermore this analysis identifies key sub-networks such as IL-2:IFN-γ:TNF-α that might be targeted in restoring normal immune function. more...

Coxiella burnetii is a macrophage-tropic, Gram-negative organism, which causes acute Q fever infection in humans. This zoonotic infection causes illness ranging from asymptomatic seroconversion to severe and protracted disease featuring hepatitis and pneumonia. Interactions between C. burnetii lipopolysaccharide (LPS) and host Toll-like receptors (TLR)-2 and -4 have been implicated in pathogen recognition, phagocytosis and signaling responses. Nonconservative single nucleotide polymorphisms in the coding regions of TLR-2 (Arg677Trp and Arg753Gln) and TLR-4 (Asp299Gly) have been found to correlate with mycobacterial infections and Gram-negative sepsis respectively. Associations between the TLR-2 and -4 polymorphisms, illness characteristics and immune response parameters were examined in subjects with acute Q fever (n=85) and comparison subjects with viral infections (n=162). No correlation was demonstrated between these polymorphisms and susceptibility to Q fever, illness severity or illness course. more...

Viral exposure—the complete history In addition to causing illness, viruses leave indelible footprints behind, because infection permanently alters the immune system. Blood tests that detect antiviral antibodies can provide information about both past and present viral exposures. Typically, such tests measure only one virus at a time. Using a synthetic representation of all human viral peptides, Xu et al. developed a blood test that identifies antibodies against all known human viruses. They studied blood samples from nearly 600 people of differing ages and geographic locations and found that most had been exposed to about 10 viral species over their lifetime. Despite differences in the rates of exposure to specific viruses, the antibody responses in most individuals targeted the same viral epitopes. Science , this issue 10.1126/science.aaa0698 more...

Chronic fatigue syndrome (CFS) is a debilitating illness characterized by multiple unexplained symptoms including fatigue, cognitive impairment and pain. People with CFS have no characteristic physical signs or diagnostic laboratory abnormalities, and the etiology and pathophysiology remain unknown. CFS represents a complex illness that includes alterations in homeostatic systems, involves multiple body systems and results from the combined action of many genes, environmental factors and risk-conferring behavior. In order to achieve understanding of complex illnesses, such as CFS, studies must collect relevant epidemiological, clinical and laboratory data and then integrate, analyze and interpret the information so as to obtain meaningful clinical and biological insight. This issue of Pharmacogenomics represents such an approach to CFS. Data was collected during a 2-day in-hospital study of persons with CFS, other medically and psychiatrically unexplained fatiguing illnesses and nonfatigued controls identified from the general population of Wichita, KS, USA. While in the hospital, the participants’ psychiatric status, sleep characteristics and cognitive functioning was evaluated, and biological samples were collected to measure neuroendocrine status, autonomic nervous system function, systemic cytokines and peripheral blood gene expression. The data generated from these assessments was made available to a multidisciplinary group of 20 investigators from around the world who were challenged with revealing new insight and algorithms for integration of this complex, high-content data and, if possible, identifying molecular markers and elucidating pathophysiology of chronic fatigue. The group was divided into four teams with representation from the disciplines of medicine, mathematics, biology, engineering and computer science. The papers in this issue are the culmination of this 6-month challenge, and demonstrate that data integration and multidisciplinary collaboration can indeed yield novel approaches for handling large, complex datasets, and reveal new insight and relevance to a complex illness such as CFS. more...

Acute infection is known to perturb psycho-neuroendocrine-immune (PNI) gene expression. Oligonucleotide microarrays were used to examine PNI gene expression in the peripheral blood of 13 subjects with infectious mononucleosis (IM). Novel peripheral blood gene expression activity was correlated with central-nervous-system-mediated symptoms including fatigue and sleep disturbance. Of note, expression of the MADS box transcription enhancer factor 2 polypeptide C (MEF2C) gene, previously implicated in skeletal muscle myogenesis, correlated with symptoms of musculo-skeletal pain and fatigue. Expression of the hypocretin/orexin receptor HCRTR2, which has been implicated in narcolepsy, correlated with sleep disturbance. And, VACHT, the vesicular acetylcholine transporter, was highly correlated with neurocognitive disturbance. The expression of both HCRTR2 and MEF2C in the peripheral blood was validated by reverse transcription PCR. Thus, investigation of the PNI response in peripheral blood may provide novel insights into the complex pathophysiology of centrally mediated disease states. more...

Molecular events occurring with high-risk human papillomavirus (HPV)-associated dysplastic differentiation of cervical epithelial cells are largely unknown. This study used differential display PCR to identify expression changes between nondifferentiating monolayer and differentiated organotypic (raft) cultures of W12 keratinocytes. These cells were originally derived from a clinical biopsy of HPV 16-positive dysplastic cervical epithelium and retain high-risk HPV 16 and the ability to differentiate, albeit with dysplastic morphology. Using this model system we identified 84 genes with changed expression during dysplastic differentiation. Most (70/84, approximately 80%) were down-regulated with differentiation, consistent with a restriction of expression during terminal differentiation. Twenty-two genes had no known function and 6 novel expressed sequence tags were identified among this group. Of the 62 genes with known functions, 25 belonged to transcription-, translation-, and posttranslation-related categories and 30 had functions associated with neoplastic initiation/progression, calcium signaling, epithelial differentiation, and structure remodeling. Some of the genes with altered expression identified in this model of dysplastic differentiation may be useful biomarkers for early detection of cervical neoplasia and other HPV-associated oropharyngeal and anogenital cancers. more...

To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray results. The results for both RNA extraction methods were comparable, although one method resulted in residual DNA that slightly affected the microarray results. As little as one microgram of total RNA could be used to synthesize a cDNA probe and resulted in a gene expression profile that was similar to one produced using 5 µg total RNA, even though the overall signal intensity was lower. These experiments illustrate that microarray technology holds great promise for the use of limited clinical samples in the diagnostic setting. more...

Peripheral blood is representative of many systemic processes and is an ideal sample for expression profiling of diseases that have no known or accessible lesion. Peripheral blood is a complex mixture of cell types and some differences in peripheral blood gene expression may reflect the timing of sample collection rather than an underlying disease process. For this reason, it is important to assess study design factors that may cause variability in gene expression not related to what is being analyzed. Variation in the gene expression of circulating peripheral blood mononuclear cells (PBMCs) from three healthy volunteers sampled three times one day each week for one month was examined for 1,176 genes printed on filter arrays. Less than 1% of the genes showed any variation in expression that was related to the time of collection, and none of the changes were noted in more than one individual. These results suggest that observed variation was due to experimental variability. more...

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls. Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls. Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the diffrentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers. more...

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays. more...

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band ( P = 0.001) and to detect an HPV type ( P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails. more...

We have optimized conditions for the chemiluminescent analysis of gene expression using high-density filter arrays (HDFAs). High sensitivity and specificity were achieved by optimizing cDNA probe synthesis, hybridization, and detection parameters. The chemiluminescent expression profile reflected expected differences in the transcripts isolated from different sources (placenta and keratinocytes). We estimated the detection limit for low-abundance message to be 1-15 transcripts per cell, a sensitivity rivaling that reported for microarray formats and exceeding that reported for autoradiographic HDFAs. The method allows for short exposure times and reuse of probe. It should be equally applicable to techniques such as differential screening of cDNA libraries and differential display PCR. more...

Post-treatment Lyme disease symptoms (PTLDS) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) have several clinical features in common, including fatigue, musculoskeletal pain, and cognitive difficulties (Gaudino et al., 1997). Immunologic mechanisms have been suspected to play a role in both PTLDS and ME/CFS. However, biomarkers for the two conditions are currently lacking, creating a barrier to better understand them. In a previous study published in BBI, we developed a semi-quantitative immunoblot assay to compare antibody reactivity to neural antigens in a group of PTLDS patients and controls (Chandra et al., 2010). Inclusion/exclusion criteria were previously described (Chandra et al., 2010). We found a significantly increased level and frequency of IgG anti-neural antibody reactivity in the PTLDS patients (41 of 83; 49.4%) both in comparison to patients who had been treated for Lyme disease previously but did not have residual symptoms (5 of 27; 18.5%) and to healthy controls who never had Lyme disease (3 of 20; 15.0%) (p more...

Background: ThinPrep is a fluid-based technique for collection and processing of cytologic specimens. The present study was designed to determine whether the collection solution preserved RNA for molecular analysis. Methods and Results: Cervical cancer cell lines and cord blood lymphocytes were used to test the efficacy of various protocols for fixation, storage, and extraction of RNA. Total RNA was extracted and analyzed by denaturing gel electrophoresis. Preserved cells stored for 24 hours at room temperature or 4°C had intact 28S and 18S ribosomal RNA. Both cellular and viral messenger RNAs were amplified from preserved samples by reverse transcription polymerase chain reaction (RT-PCR). Viral messenger RNA (mRNA) could be detected in a mixture of preserved cells containing 10% human papillomavirus (HPV) positive cells. RNA preservation in clinical samples was adequate for RT-PCR of cellular mRNA. Conclusions: Both experimental samples and clinical samples collected in the preservation media had intact total RNA. Amplification of both cellular and HPV mRNA was successful. more...

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues. more...

Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis, is a complex multifactorial disease that is characterized by the persistent presence of fatigue and other particular symptoms for a minimum of 6 months. Symptoms fail to dissipate after sufficient rest and have major effects on the daily functioning of CFS sufferers. CFS is a multi-system disease with a heterogeneous patient population showing a wide variety of functional disabilities and its biological basis remains poorly understood. Stable alterations in gene function in the immune system have been reported in several studies of CFS. Epigenetic modifications have been implicated in long-term effects on gene function, however, to our knowledge, genome-wide epigenetic modifications associated with CFS have not been explored. We examined the DNA methylome in peripheral blood mononuclear cells isolated from CFS patients and healthy controls using the Illumina HumanMethylation450 BeadChip array, controlling for invariant probes and probes overlapping polymorphic sequences. Gene ontology (GO) and network analysis of differentially methylated genes was performed to determine potential biological pathways showing changes in DNA methylation in CFS. We found an increased abundance of differentially methylated genes related to the immune response, cellular metabolism, and kinase activity. Genes associated with immune cell regulation, the largest coordinated enrichment of differentially methylated pathways, showed hypomethylation within promoters and other gene regulatory elements in CFS. These data are consistent with evidence of multisystem dysregulation in CFS and implicate the involvement of DNA modifications in CFS pathology. more...

To determine the clinical relevance of human papillomavirus (HPV) integration and E2 function suggested by in vitro studies, we investigated 50 patients with HPV 16-positive primary cervical carcinoma (stage Ib-IV) diagnosed and treated at one institution. The physical state of HPV was determined by colorimetric in situ hybridization and was not found to vary by stage. Overall, 62% of tumors had integrated HPV, 16% had episomal and 22% had both integrated and episomal. The E1/E2 region was evaluated by 8 separate polymerase chain reactions, which resulted in overlapping products. There was no significant variation in ability to amplify the E1/E2 region with stage. E1/E2 amplification correlated with physical state. Nearly all tumors with episomal or mixed HPV 16 DNA amplified all 8 E1/E2 fragments. Half of the tumors with integrated HPV 16 DNA failed to amplify one or more E1/E2 fragments. Disruptions were most frequent in the E2 region. For all 46 patients receiving curative therapy, the Kaplan-Meier estimate of disease-free survival was determined for those whose primary tumors had amplifiable E2 compared with those lacking one or more E2 DNA fragments. Disruption of E2 was associated with significantly shortened disease-free survival. Int. J. Cancer 74:50–56. © 1997 Wiley-Liss, Inc. more...

Human papillomaviruses (HPVs) are associated with at least 80% of cervical carcinomas and are classified as high-risk or low-risk based on whether or not they are commonly found in cervical cancers. The high-risk HPVs have early gene products (E6 and E7) that immortalize human keratinocytes and are at least partially responsible for causing cervical carcinoma. E6 and E7 from the high-risk viruses interact strongly with the tumor suppressors p53 and Rb; those from the low-risk HPVs do not. Transformation involves a multi-step process and requires additional factors besides high-risk HPV infection. High-risk HPVs are capable of immortalizing primary human keratinocytes in tissue culture, but such cells become transformed only after certain chromosomal changes take place, possibly having to do with oncogene activation. The DNA of high-risk HPVs is frequently (if not always) integrated into the genome of cancer cells; it is normally episomal in premalignant lesions. Integration disrupts the E2 and E5 genes and viral gene regulation. Cells containing integrated viral DNA show excessively high levels of E6 and E7. While there is some conflicting evidence, it appears that the p53 and Rb tumor-suppressor genes are more frequently mutated in HPV-negative tumors than they are in HPV-positive tumors, suggesting that for tumor formation to proceed the p53 and Rb proteins must be inactivated either by interaction with the viral proteins or by mutation. The presence of an activated oncogene in a cell lacking functional p53 or Rb may then be sufficient to cause tumor progression. more...

Epidemiologic studies show an association between infection with human immunodeficiency virus type 1 (HIV-1) and human papillomavirus (HPV) associated anogenital disease. To investigate possible molecular mechanisms of HIV-1 modulation of HPV expression, we studied the effect of an HIV-1 regulatory protein, tat1, on gene expression directed by the upstream regulatory region (URR) of HPV type 16 (HPV 16). HPV 16 URR-directed chloramphenicol acetyltransferase (CAT) expression driven by the native HPV 16 promoter (P97) was increased in the presence of tat1 alone. Tat1 also reversed E2-mediated repression of P97-directed CAT expression. E2 mediated CAT expression with URR constructs containing the SV40 promoter was enhanced when tat1 and E2 were cotransfected. Using a cervical carcinoma cell line (SiHa), E2 enhancement of URR-directed gene expression was elevated in the presence of extracellular tat1 or during cocultivation with HIV-1-infected cells. These results show HIV can modulate HPV gene expression in cell culture and that the increased rate of HPV-associated cervical disease in asymptomatic HIV-seropositive women may result from HPV-HIV molecular interactions. more...

Background The association between HIV infection and invasive cervical cancer that has been reported may reflect differential prevalence of human papillomavirus (HPV) infection or uncontrolled confounding. We conducted a case-control study in a West African population to assess the relationship between HIV infection and invasive cervical cancer, taking into account HPV infection and other potential risk factors for cervical cancer. Methods Women with invasive cervical cancer (cases) or normal cervical cytology (controls) were recruited in a hospital-based case-control study in Abidjan, Côte d'Ivoire. Odds ratios and 95% confidence intervals (CI) were estimated in logistic regression analyses controlling for important cofactors. Results HIV infection was noted in 22/132 (16.7%) cases and 10/120 (8.3%) controls (p = 0.048). High-risk HPV infection was detected in cervical tumor samples from 89.4% of case-participants and in cervical cytology samples in 31.1% of control-participants. In logistic regression analysis, HIV infection was associated with cervical cancer in women with HPV (OR 3.4; 95% CI 1.1-10.8). Among women aged ≤ 40 years, risk factors for cervical cancer were high-risk HPV infection (OR 49.3; 95% CI 8.2-295.7); parity > 2 (OR 7.0; 95% CI 1.9-25.7) and HIV infection (OR 4.5; 95% CI 1.5-13.6). Among women aged > 40 years, high-risk HPV infection (OR 23.5; 95% CI 9.1-60.6) and parity > 2 (OR 5.5; 95% CI 2.3-13.4), but association with HIV infection was not statistically significant. Conclusions These data support the hypothesis that HIV infection is a cofactor for cervical cancer in women with HPV infection, and, as in all populations, the need for promoting cervical screening in populations with high prevalence of HIV infection. more...

Background Gulf War Illness (GWI) remains a serious health consequence for at least 11,000 veterans of the first Gulf War in the early 1990s. Our understanding of the health consequences that resulted remains inadequate, and this is of great concern with another deployment to the same theater of operations occurring now. Chronic immune cell dysfunction and activation have been demonstrated in patients with GWI, although the literature is not uniform. We exposed GWI patients and matched controls to an exercise challenge to explore differences in immune cell function measured by classic immune assays and gene expression profiling. Methods This pilot study enrolled 9 GWI cases identified from the Department of Veterans Affairs GWI registry, and 11 sedentary control veterans who had not been deployed to the Persian Gulf and were matched to cases by sex, body mass index (BMI) and age. We measured peripheral blood cell numbers, NK cytotoxicity, cytokines and expression levels of 20,000 genes immediately before, immediately after and 4 hours following a standard bicycle ergometer exercise challenge. Results A repeated-measures analysis of variance revealed statistically significant differences for three NK cell subsets and NK cytotoxicity between cases and controls (p < 0.05). Linear regression analysis correlating NK cell numbers to the gene expression profiles showed high correlation of genes associated with NK cell function, serving as a biologic validation of both the in vitro assays and the microarray platform. Intracellular perforin levels in NK and CD8 T-cells trended lower and showed a flatter profile in GWI cases than controls, as did the expression levels of the perforin gene PRF1. Genes distinguishing cases from controls were associated with the glucocorticoid signaling pathway. Conclusion GWI patients demonstrated impaired immune function as demonstrated by decreased NK cytotoxicity and altered gene expression associated with NK cell function. Pro-inflammatory cytokines, T-cell ratios, and dysregulated mediators of the stress response (including salivary cortisol) were also altered in GWI cases compared to control subjects. An interesting and potentially important observation was that the exercise challenge augments these differences, with the most significant effects observed immediately after the stressor, possibly implicating some block in the NK and CD8 T-cells ability to respond to "stress-mediated activation". This has positive implications for the development of laboratory diagnostic tests for this syndrome and provides a paradigm for exploration of the immuno-physiological mechanisms that are operating in GWI, and similar complex syndromes. Our results do not necessarily elucidate the cause of GWI, but they do reveal a role for immune cell dysfunction in sustaining illness. more...

Complement activation resulting in significant increases of C4a split product may be a marker of postexertional malaise in individuals with chronic fatigue syndrome (CFS). This study focused on identification of the transcriptional control that may contribute to the increased C4a in CFS subjects after exercise. We used quantitative reverse-transcription polymerase chain reaction to evaluate differential expression of genes in the classical and lectin pathways in peripheral blood mononuclear cells (PBMCs). Calibrated expression values were normalized to the internal reference gene peptidylpropyl isomerase B (PPIB), the external reference gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), or the geometric mean (GM) of the genes ribosomal protein, large, P0 (RPLP0) and phosphoglycerate kinase 1 (PGK1). All nine genes tested, except mannose-binding lectin 2 (MBL2), were expressed in PBMCs. At 1 hour postexercise, C4, mannan-binding lectin serine protease 2 (MASP2) and ficolin 1 (FCN1) transcripts were detected at higher levels (or = 2-fold) in at least 50% (4 of 8) of CFS subjects and were detected in 88% (7 of 8) CFS subjects when subjects with overexpression of either C4 or MASP2 were combined. Only an increase in the MASP2 transcript was statistically significant (PPIB, P = 0.001; GM, P = 0.047; rbcL, P = 0.045). This result may be due to the significant but transient downregulation of MASP2 in control subjects (PPIB, P = 0.023; rbcL, P = 0.027). By 6 hours postexercise, MASP2 expression was similar in both groups. In conclusion, lectin pathway responded to exercise differentially in CFS than in control subjects. MASP2 down-regulation may act as an antiinflammatory acute-phase response in healthy subjects, whereas its elevated level may account for increased C4a and inflammation-mediated postexertional malaise in CFS subjects. more...

Background Systems biologic approaches such as Weighted Gene Co-expression Network Analysis (WGCNA) can effectively integrate gene expression and trait data to identify pathways and candidate biomarkers. Here we show that the additional inclusion of genetic marker data allows one to characterize network relationships as causal or reactive in a chronic fatigue syndrome (CFS) data set. Results We combine WGCNA with genetic marker data to identify a disease-related pathway and its causal drivers, an analysis which we refer to as "Integrated WGCNA" or IWGCNA. Specifically, we present the following IWGCNA approach: 1) construct a co-expression network, 2) identify trait-related modules within the network, 3) use a trait-related genetic marker to prioritize genes within the module, 4) apply an integrated gene screening strategy to identify candidate genes and 5) carry out causality testing to verify and/or prioritize results. By applying this strategy to a CFS data set consisting of microarray, SNP and clinical trait data, we identify a module of 299 highly correlated genes that is associated with CFS severity. Our integrated gene screening strategy results in 20 candidate genes. We show that our approach yields biologically interesting genes that function in the same pathway and are causal drivers for their parent module. We use a separate data set to replicate findings and use Ingenuity Pathways Analysis software to functionally annotate the candidate gene pathways. Conclusion We show how WGCNA can be combined with genetic marker data to identify disease-related pathways and the causal drivers within them. The systems genetics approach described here can easily be used to generate testable genetic hypotheses in other complex disease studies. more...

BackgroundGenomic profiling of peripheral blood reveals altered immunity in chronic fatigue syndrome (CFS) however interpretation remains challenging without immune demographic context. The object of this work is to identify modulation of specific immune functional components and restructuring of co-expression networks characteristic of CFS using the quantitative genomics of peripheral blood.MethodsGene sets were constructed a priori for CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes and CD16+ neutrophils from published data. A group of 111 women were classified using empiric case definition (U.S. Centers for Disease Control and Prevention) and unsupervised latent cluster analysis (LCA). Microarray profiles of peripheral blood were analyzed for expression of leukocyte-specific gene sets and characteristic changes in co-expression identified from topological evaluation of linear correlation networks.ResultsMedian expression for a set of 6 genes preferentially up-regulated in CD19+ B cells was significantly lower in CFS (p = 0.01) due mainly to PTPRK and TSPAN3 expression. Although no other gene set was differentially expressed at p < 0.05, patterns of co-expression in each group differed markedly. Significant co-expression of CD14+ monocyte with CD16+ neutrophil (p = 0.01) and CD19+ B cell sets (p = 0.00) characterized CFS and fatigue phenotype groups. Also in CFS was a significant negative correlation between CD8+ and both CD19+ up-regulated (p = 0.02) and NK gene sets (p = 0.08). These patterns were absent in controls.ConclusionDissection of blood microarray profiles points to B cell dysfunction with coordinated immune activation supporting persistent inflammation and antibody-mediated NK cell modulation of T cell activity. This has clinical implications as the CD19+ genes identified could provide robust and biologically meaningful basis for the early detection and unambiguous phenotyping of CFS. more...

This work investigates the significance of changes in association patterns linking indicators of neuroendocrine and immune activity in patients with chronic fatigue syndrome (CFS). Gene sets preferentially expressed in specific immune cell isolates were integrated with neuroendocrine data from a large population-based study. Co-expression patterns linking immune cell activity with hypothalamic–pituitary–adrenal (HPA), thyroidal (HPT) and gonadal (HPG) axis status were computed using mutual information criteria. Networks in control and CFS subjects were compared globally in terms of a weighted graph edit distance. Local re-modeling of node connectivity was quantified by node degree and eigenvector centrality measures. Results indicate statistically significant differences between CFS and control networks determined mainly by re-modeling around pituitary and thyroid nodes as well as an emergent immune sub-network. Findings align with known mechanisms of chronic inflammation and support possible immune-mediated loss of thyroid function in CFS exacerbated by blunted HPA axis responsiveness. more...

The activity of vesicular stomatitis viruses was monitored on 3 dairy farms in Costa Rica. Antibody levels were measured and clinical disease monitored in 165 cattle during a 20 month period (1986-1988). Vesicular stomatitis New Jersey (VS NJ) virus was shown to be enzootic on these farms by a 94.2% prevalence of neutralizing antibody; this did not vary significantly between herds. The mean prevalence of antibody to vesicular stomatitis Indiana (VS IN) virus was 15.2%, but was significantly higher in 1 herd. A total of 25 cases (annual incidence rate of 9%) of clinical vesicular stomatitis (VS) was reported. VS NJ virus was identified as the causal agent by detection of VS NJ virus antigens by the complement fixation test. VS NJ virus was isolated in 11 cases. All episodes of disease occurred between November and January, the beginning of the dry season. Most animals maintained stable neutralizing antibody titers throughout the study, and all diseased animals were previously seropositive to VS NJ virus. A total of 31 animals with neutralizing antibodies to VS NJ virus had a VS NJ virus-specific IgM response, and 6 animals had IgM responses that persisted for as long as 6 months. There was no relation between IgM responses and clinical disease occurrence. VS NJ virus persisted predominantly as a subclinical infection in cattle throughout the year in enzootic areas of Costa Rica. The humoral response did not prevent reinfection with VS NJ virus. more...

Background: A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. Methods: Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. Results: Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. Conclusions: This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected. Published 2007 Wiley-Liss, Inc. more...

Chronic fatigue syndrome (CFS) is a debilitating disorder of unknown etiology with no known lesions, diagnostic markers or therapeutic intervention. The pathophysiology of CFS remains elusive, although abnormalities in the central nervous system (CNS) have been implicated, particularly hyperactivity of the serotonergic (5-hydroxytryptamine; 5-HT) system and hypoactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Since alterations in 5-HT signaling can lead to physiologic and behavioral changes, a genetic evaluation of the 5-HT system was undertaken to identify serotonergic markers associated with CFS and potential mechanisms for CNS abnormality. A total of 77 polymorphisms in genes related to serotonin synthesis (TPH2), signaling (HTR1A, HTR1E, HTR2A, HTR2B, HTR2C, HTR3A, HTR3B, HTR4, HTR5A, HTR6, and HTR7), transport (SLC6A4), and catabolism (MAOA) were examined in 137 clinically evaluated subjects (40 CFS, 55 with insufficient fatigue, and 42 non-fatigued, NF, controls) derived from a population-based CFS surveillance study in Wichita, Kansas. Of the polymorphisms examined, three markers (-1438G/A, C102T, and rs1923884) all located in the 5-HT receptor subtype HTR2A were associated with CFS when compared to NF controls. Additionally, consistent associations were observed between HTR2A variants and quantitative measures of disability and fatigue in all subjects. The most compelling of these associations was with the A allele of -1438G/A (rs6311) which is suggested to have increased promoter activity in functional studies. Further, in silico analysis revealed that the -1438 A allele creates a consensus binding site for Th1/E47, a transcription factor implicated in the development of the nervous system. Electrophoretic mobility shift assay supports allele-specific binding of E47 to the A allele but not the G allele at this locus. These data indicate that sequence variation in HTR2A, potentially resulting in its enhanced activity, may be involved in the pathophysiology of CFS. more...

Microarrays are a powerful laboratory tool for the simultaneous assessment of the activity of thousands genes. Remarkable advances in biological sample collection, preparation and automation of hybridization have enabled the application of microarray technology to large, population-based studies. Now, microarrays have the potential to serve as screening tools for the detection of altered gene expression activity that might contribute to diseases in human populations. Reproducible and reliable microarray results depend on multiple factors. In this chapter, biological sample parameters are introduced that should be considered for any microarray experiment. Then, the microarray technology that we have successfully applied to limited biological sample from all our molecular epidemiology studies is detailed. This reproducible and reliable approach for using microarrays should be applicable to any biological questions asked. more...

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease characterized by intense and debilitating fatigue not due to physical activity that has persisted for at least 6 months, post-exertional malaise, unrefreshing sleep, and accompanied by a number of secondary symptoms, including sore throat, memory and concentration impairment, headache, and muscle/joint pain. In patients with post-exertional malaise, significant worsening of symptoms occurs following physical exertion and exercise challenge serves as a useful method for identifying biomarkers for exertion intolerance. Evidence suggests that intestinal dysbiosis and systemic responses to gut microorganisms may play a role in the symptomology of ME/CFS. As such, we hypothesized that post-exertion worsening of ME/CFS symptoms could be due to increased bacterial translocation from the intestine into the systemic circulation. To test this hypothesis, we collected symptom reports and blood and stool samples from ten clinically characterized ME/CFS patients and ten matched healthy controls before and 15 minutes, 48 hours, and 72 hours after a maximal exercise challenge. Microbiomes of blood and stool samples were examined. Stool sample microbiomes differed between ME/CFS patients and healthy controls in the abundance of several major bacterial phyla. Following maximal exercise challenge, there was an increase in relative abundance of 6 of the 9 major bacterial phyla/genera in ME/CFS patients from baseline to 72 hours post-exercise compared to only 2 of the 9 phyla/genera in controls (p = 0.005). There was also a significant difference in clearance of specific bacterial phyla from blood following exercise with high levels of bacterial sequences maintained at 72 hours post-exercise in ME/CFS patients versus clearance in the controls. These results provide evidence for a systemic effect of an altered gut microbiome in ME/CFS patients compared to controls. Upon exercise challenge, there were significant changes in the abundance of major bacterial phyla in the gut in ME/CFS patients not observed in healthy controls. In addition, compared to controls clearance of bacteria from the blood was delayed in ME/CFS patients following exercise. These findings suggest a role for an altered gut microbiome and increased bacterial translocation following exercise in ME/CFS patients that may account for the profound post-exertional malaise experienced by ME/CFS patients. more...

BACKGROUND Infectious mononucleosis (IM) commonly triggers a protracted postinfective fatigue syndrome (PIFS) of unknown pathogenesis. METHODS Seven subjects with PIFS with 6 or more months of disabling symptoms and 8 matched control subjects who had recovered promptly from documented IM were studied. The expression of 30,000 genes was examined in the peripheral blood by microarray analysis in 65 longitudinally collected samples. Gene expression patterns associated with PIFS were sought by correlation with symptom factor scores. RESULTS Differential expression of 733 genes was identified when samples collected early during the illness and at the late (recovered) time point were compared. Of these genes, 234 were found to be significantly correlated with the reported severity of the fatigue symptom factor, and 180 were found to be correlated with the musculoskeletal pain symptom factor. Validation by analysis of the longitudinal expression pattern revealed 35 genes for which changes in expression were consistent with the illness course. These genes included several that are involved in signal transduction pathways, metal ion binding, and ion channel activity. CONCLUSIONS Gene expression correlates of the cardinal symptoms of PIFS after IM have been identified. Further studies of these gene products may help to elucidate the pathogenesis of PIFS. more...

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest. more...

Background Exfoliated cervical cells are used in cytology-based cancer screening and may also be a source for molecular biomarkers indicative of neoplastic changes in the underlying tissue. However, because of keratinization and terminal differentiation it is not clear that these cells have an mRNA profile representative of cervical tissue, and that the profile can distinguish the lesions targeted for early detection. Results We used whole genome microarrays (25,353 unique genes) to compare the transcription profiles from seven samples of normal exfoliated cells and one cervical tissue. We detected 10,158 genes in exfoliated cells, 14,544 in the tissue and 7320 genes in both samples. For both sample types the genes grouped into the same major gene ontology (GO) categories in the same order, with exfoliated cells, having on average 20% fewer genes in each category. We also compared microarray results of samples from women with cervical intraepithelial neoplasia grade 3 (CIN3, n = 15) to those from age and race matched women without significant abnormalities (CIN1, CIN0; n = 15). We used three microarray-adapted statistical packages to identify differential gene expression. The six genes identified in common were two to four fold upregulated in CIN3 samples. One of these genes, the ubiquitin-conjugating enzyme E2 variant 1, participates in the degradation of p53 through interaction with the oncogenic HPV E6 protein. Conclusion The findings encourage further exploration of gene expression using exfoliated cells to identify and validate applicable biomarkers. We conclude that the gene expression profile of exfoliated cervical cells partially represents that of tissue and is complex enough to provide potential differentiation between disease and non-disease. more...

As pathways of communication between the nervous, endocrine, and immune systems are identified, the importance of the interplay of these systems for health and well-being is increasingly recognized. In this study, we created a comprehensive database of 1622 genes likely to be involved in synthetic, biochemical, and regulatory psycho-neuroendocrine-immune (PNI) pathways. Expression of 1058 of these genes was detected in the peripheral blood by querying both a peripheral blood-specific expressed sequence tag (EST) database and a peripheral blood database generated from microarray evaluation of 30,000 genes. Several neural and endocrine genes were expressed in the peripheral blood including hormone receptors, a hormone-responsive transcription factor, and neurotransmitter receptors. These findings document the expression of nervous and endocrine genes in the peripheral blood that have previously only been characterized in the respective system tissues, and indicate that the blood is a rich source of information that should help in deciphering the communication between the mind and the body. more...

We used differential-display PCR of peripheral blood mononuclear cells (PBMCs) to search for candidate biomarkers for chronic fatigue syndrome (CFS). PBMCs were collected from a subject with CFS and an age- and sex-matched control before and 24 h after exercise. RNA expression profiles were generated using 46 primer combinations, and the similarity between the individuals was striking. Differentially expressed bands were excised, reamplified, and sequenced, yielding 95 nonredundant sequences, of which 50 matched to known gene transcripts, 38 matched to genes with unknown functions, and 7 had no similarity to any database entry. Most (86%) of the differences between the two subjects were present at baseline. Differential expression of ten genes was verified by real-time reverse-transcription PCR: five (cystatin F, MHC class II, platelet factor 4, fetal brain expressed sequence tag, and perforin) were downregulated, and the remaining five genes (cathepsin B, DNA polymerase epsilon4, novel EST PBMC191MSt, heparanase precursor, and ORF2/L1 element) were upregulated in the subject with CFS. Many of these genes have known functions in defense and immunity, thus supporting prior suggestions of immune dysregulation in the pathogenesis of CFS. Differential-display PCR is a powerful tool for identification of candidate biomarkers. Investigation of these markers in samples from well-designed epidemiological studies of CFS will be required to determine the validity of these candidate biomarkers. The real-time reverse-transcription PCR assays that we developed for assay of these biomarkers will facilitate high-throughput testing of these additional samples. more...

The clinical utility of human papillomavirus (HPV) testing continues to be the focus of much debate. The clear epidemiologic link of HPV infection with the development of cervical intraepithelial neoplasia and invasive cervical cancers. more...

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-->3' direction; the appropriate template for any gene expression analysis. more...

Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™) in identifying multiple pathogens. Methods Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. Results Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. Conclusions This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens. more...

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 microg of purified poly(A)+ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 microg total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 microg total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard. more...

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays. more...

OBJECTIVE: To explore whether HIV types 1 and 2 and CD4 cell count affect cervical neoplasia independent of human papillomavirus (HPV) in women with high or low numbers of sexual partners residing in Abidjan, Cote d'Ivoire. METHODS: The study population and methods are described in the companion paper. Additional methods include a Papanicolaou smear for cytological diagnosis and statistical analysis. RESULTS: In maternal women, both HIV-1 and high risk HPV were significant independent risk factors for squamous intraepithelial lesions (SIL) (adjusted odds ratio (OR) 11.0 (95% CI 1.1-112) and 5.4 (1.5-18.8), respectively). Only high levels of HPV DNA in the lavage were associated with SIL (OR 13.2 (3.6-47.8)) in the maternal group. In female sex workers, high risk HPV was significantly associated with SIL (OR 23.7 (4.4-126)); HIV seropositivity was not. Any positive level (high or low amounts) of HPV DNA was significantly associated with SIL in sex workers (ORs 15.9 (3.3-76) and 12.7 (3.6-44), respectively). There was no association of SIL with CD4 cell counts < or = 500 x 10(6)/l in HIV seropositive women from either group. CONCLUSION: HPV or HIV-1 infection independently affect cervical neoplasia in women with low numbers of sex partners. more...