Publications by authors named "Bruce K Patterson"

38 Publications

Disruption of CCR5 signaling to treat COVID-19-associated cytokine storm: Case series of four critically ill patients treated with leronlimab.

J Transl Autoimmun 2021 6;4:100083. Epub 2021 Jan 6.

Amarex Clinical Research, 20201 Century Blvd, Germantown, MD, 20874, USA.

Coronavirus disease 2019 (COVID-19) is associated with considerable morbidity and mortality. The number of confirmed cases of infection with SARS-CoV-2, the virus causing COVID-19 continues to escalate with over 70 million confirmed cases and over 1.6 million confirmed deaths. Severe-to-critical COVID-19 is associated with a dysregulated host immune response to the virus, which is thought to lead to pathogenic immune dysregulation and end-organ damage. Presently few effective treatment options are available to treat COVID-19. Leronlimab is a humanized IgG4, kappa monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). It has been shown that in patients with severe COVID-19 treatment with leronlimab reduces elevated plasma IL-6 and chemokine ligand 5 (CCL5), and normalized CD4/CD8 ratios. We administered leronlimab to 4 critically ill COVID-19 patients in intensive care. All 4 of these patients improved clinically as measured by vasopressor support, and discontinuation of hemodialysis and mechanical ventilation. Following administration of leronlimab there was a statistically significant decrease in IL-6 observed in patient A (p=0.034) from day 0-7 and patient D (p=0.027) from day 0-14. This corresponds to restoration of the immune function as measured by CD4+/CD8+ T cell ratio. Although two of the patients went on to survive the other two subsequently died of surgical complications after an initial recovery from SARS-CoV-2 infection.
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http://dx.doi.org/10.1016/j.jtauto.2021.100083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823045PMC
January 2021

SARS-CoV-2 Viral RNA Shedding for More Than 87 Days in an Individual With an Impaired CD8+ T Cell Response.

Front Immunol 2020 8;11:618402. Epub 2021 Jan 8.

Department of Emergency Medicine, Washington University School of Medicine, Saint Louis, MO, United States.

Prolonged shedding of viral RNA occurs in some individuals following SARS-CoV-2 infection. We perform comprehensive immunologic evaluation of one individual with prolonged shedding. The case subject recovered from severe COVID-19 and tested positive for SARS-CoV-2 viral RNA repeatedly as many as 87 days after the first positive test, 97 days after symptom onset. The subject did not have any associated rise in anti-Spike protein antibody titers or plasma neutralization activity, arguing against re-infection. This index subject exhibited a profoundly diminished circulating CD8+ T cell population and correspondingly low SARS-CoV-2-specific CD8+ T cell responses when compared with a cohort of other recovering COVID-19 subjects. CD4+ T cell responses and neutralizing antibody responses developed as expected in this individual. Our results demonstrate that detectable viral RNA shedding in the upper airway can occur more than 3 months following infection in some individuals with COVID-19 and suggest that impaired CD8+ T cells may play a role in prolonged viral RNA shedding.
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http://dx.doi.org/10.3389/fimmu.2020.618402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820941PMC
February 2021

Genome-wide DNA methylation profiling of peripheral blood reveals an epigenetic signature associated with severe COVID-19.

J Leukoc Biol 2021 Jan 19. Epub 2021 Jan 19.

Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, New York, New York, USA.

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic RNA virus causing coronavirus disease 2019 (COVID-19) in humans. Although most patients with COVID-19 have mild illness and may be asymptomatic, some will develop severe pneumonia, acute respiratory distress syndrome, multi-organ failure, and death. RNA viruses such as SARS-CoV-2 are capable of hijacking the epigenetic landscape of host immune cells to evade antiviral defense. Yet, there remain considerable gaps in our understanding of immune cell epigenetic changes associated with severe SARS-CoV-2 infection pathology. Here, we examined genome-wide DNA methylation (DNAm) profiles of peripheral blood mononuclear cells from 9 terminally-ill, critical COVID-19 patients with confirmed SARS-CoV-2 plasma viremia compared with uninfected, hospitalized influenza, untreated primary HIV infection, and mild/moderate COVID-19 HIV coinfected individuals. Cell-type deconvolution analyses confirmed lymphopenia in severe COVID-19 and revealed a high percentage of estimated neutrophils suggesting perturbations to DNAm associated with granulopoiesis. We observed a distinct DNAm signature of severe COVID-19 characterized by hypermethylation of IFN-related genes and hypomethylation of inflammatory genes, reinforcing observations in infection models and single-cell transcriptional studies of severe COVID-19. Epigenetic clock analyses revealed severe COVID-19 was associated with an increased DNAm age and elevated mortality risk according to GrimAge, further validating the epigenetic clock as a predictor of disease and mortality risk. Our epigenetic results reveal a discovery DNAm signature of severe COVID-19 in blood potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against SARS-CoV-2.
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http://dx.doi.org/10.1002/JLB.5HI0720-466RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013321PMC
January 2021

Cell by cell immuno- and cancer marker profiling of non-small cell lung cancer tissue: Checkpoint marker expression on CD103+, CD4+ T-cells predicts circulating tumor cells.

Transl Oncol 2021 Jan 18;14(1):100953. Epub 2020 Nov 18.

IncellDx Inc, 1541 Industrial Road, San Carlos, CA, United States. Electronic address:

Non-small cell lung cancer (NSCLC) has a poor prognosis. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization and stained with a total 25 immune, cancer markers and DNA content dye and analyzed with high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. We investigated a total of 74 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes (CTLA-4, LAG3, TIM3, PD-1), within the CD103CD4 T lymphocyte subset. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44E-cadherin cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103CD4 T cells that could potentially be predictive of CTCs and targets of therapy.
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http://dx.doi.org/10.1016/j.tranon.2020.100953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683336PMC
January 2021

CCR5 inhibition in critical COVID-19 patients decreases inflammatory cytokines, increases CD8 T-cells, and decreases SARS-CoV2 RNA in plasma by day 14.

Int J Infect Dis 2021 Feb 10;103:25-32. Epub 2020 Nov 10.

Vaccine & Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health & Science University, Portland, OR, USA.

Objective: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now a global pandemic. Emerging results indicate a dysregulated immune response. Given the role of CCR5 in immune cell migration and inflammation, we investigated the impact of CCR5 blockade via the CCR5-specific antibody leronlimab on clinical, immunological, and virological parameters in severe COVID-19 patients.

Methods: In March 2020, 10 terminally ill, critical COVID-19 patients received two doses of leronlimab via individual emergency use indication. We analyzed changes in clinical presentation, immune cell populations, inflammation, as well as SARS-CoV-2 plasma viremia before and 14 days after treatment.

Results: Over the 14-day study period, six patients survived, two were extubated, and one discharged. We observed complete CCR5 receptor occupancy in all donors by day 7. Compared with the baseline, we observed a concomitant statistically significant reduction in plasma IL-6, restoration of the CD4/CD8 ratio, and resolution of SARS-CoV2 plasma viremia (pVL). Furthermore, the increase in the CD8 percentage was inversely correlated with the reduction in pVL (r = -0.77, p = 0.0013).

Conclusions: Our study design precludes clinical efficacy inferences but the results implicate CCR5 as a therapeutic target for COVID-19 and they form the basis for ongoing randomized clinical trials.
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http://dx.doi.org/10.1016/j.ijid.2020.10.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654230PMC
February 2021

Disruption of the CCL5/RANTES-CCR5 Pathway Restores Immune Homeostasis and Reduces Plasma Viral Load in Critical COVID-19.

medRxiv 2020 May 5. Epub 2020 May 5.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is now pandemic with nearly three million cases reported to date. Although the majority of COVID-19 patients experience only mild or moderate symptoms, a subset will progress to severe disease with pneumonia and acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. Emerging results indicate a dysregulated immune response characterized by runaway inflammation, including cytokine release syndrome (CRS), as the major driver of pathology in severe COVID-19. With no treatments currently approved for COVID-19, therapeutics to prevent or treat the excessive inflammation in severe disease caused by SARS-CoV-2 infection are urgently needed. Here, in 10 terminally-ill, critical COVID-19 patients we report profound elevation of plasma IL-6 and CCL5 (RANTES), decreased CD8+ T cell levels, and SARS-CoV-2 plasma viremia. Following compassionate care treatment with the CCR5 blocking antibody leronlimab, we observed complete CCR5 receptor occupancy on macrophage and T cells, rapid reduction of plasma IL-6, restoration of the CD4/CD8 ratio, and a significant decrease in SARS-CoV-2 plasma viremia. Consistent with reduction of plasma IL-6, single-cell RNA-sequencing revealed declines in transcriptomic myeloid cell clusters expressing IL-6 and interferon-related genes. These results demonstrate a novel approach to resolving unchecked inflammation, restoring immunologic deficiencies, and reducing SARS-CoV-2 plasma viral load via disruption of the CCL5-CCR5 axis, and support randomized clinical trials to assess clinical efficacy of leronlimab-mediated inhibition of CCR5 for COVID-19.
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http://dx.doi.org/10.1101/2020.05.02.20084673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277012PMC
May 2020

Using adaptive genetic algorithms combined with high sensitivity single cell-based technology to detect bladder cancer in urine and provide a potential noninvasive marker for response to anti-PD1 immunotherapy.

Urol Oncol 2020 03 27;38(3):77.e9-77.e15. Epub 2019 Sep 27.

Incelldx, Inc, San Carlos, CA 94070.

Objective: To use adaptive genetic algorithms (AGA) in combination with single-cell flow cytometry technology to develop a noninvasive test to detect bladder cancer.

Materials And Methods: Fifty high grade, cystoscopy confirmed, superficial bladder cancer patients, and 15 healthy donor early morning urine samples were collected in an optimized urine collection media. These samples were then used to develop an assay to distinguish healthy from cancer patients' urine using AGA in combination with single-cell flow cytometry technology. Cell recovery and test performance were verified based on cystoscopy and histology for both bladder cancer determination and PD-L1 status.

Results: Bladder cancer patients had a significantly higher percentage of white blood cells with substantial PD-L1 expression (P< 0.0001), significantly increased post-G1 epithelial cells (P < 0.005) and a significantly higher DNA index above 1.05 (P < 0.05). AGA allowed parameter optimization to differentiate normal from malignant cells with high accuracy. The resulting prediction model showed 98% sensitivity and 87% specificity with a high area under the ROC value (90%).

Conclusions: Using single-cell technology and machine learning; we developed a new assay to distinguish bladder cancer from healthy patients. Future studies are planned to validate this assay.
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http://dx.doi.org/10.1016/j.urolonc.2019.08.019DOI Listing
March 2020

Concordance of PD-L1 Expression Detection in Non-Small Cell Lung Cancer (NSCLC) Tissue Biopsy Specimens Between OncoTect iO Lung Assay and Immunohistochemistry (IHC).

Am J Clin Pathol 2018 Aug;150(4):346-352

TriCore Reference Laboratories, Albuquerque, NM.

Objectives: We report on the validity of a fully quantitative technology to determine tumor cells' PD-L1 expression compared with a standard immunohistochemical (IHC) assay in non-small cell lung cancer.

Methods: Nineteen fresh tissue specimens were processed into single-cell suspensions using the IncellPREP Kit. Cells were treated with the OncoTect iO Lung Assay, which quantitatively assessed tumor-infiltrating lymphocytes (TILs), DNA content, and PD-L1 expression on diploid and aneuploid tumor populations.

Results: Comparison of the OncoTect iO Lung Assay with IHC revealed a concordance of 95% overall (negative percent agreement, 97%; positive percent agreement, 89%). PD-L1 expression varied depending on the DNA content. The number of TILs and antigen-presenting cells (APCs) was significantly decreased in tumor compared with normal lung tissue.

Conclusions: The nonsubjective OncoTect iO Lung Assay has been shown to be at least as accurate and sensitive as IHC for the detection of PD-L1 expression while providing additional information to guide treatment.
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http://dx.doi.org/10.1093/ajcp/aqy063DOI Listing
August 2018

Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4 T cells.

Nat Med 2017 Jul 29;23(7):885-889. Epub 2017 May 29.

Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, USA.

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4 T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4 T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.
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http://dx.doi.org/10.1038/nm.4347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505781PMC
July 2017

Quantification of PD-L1 and PD-1 expression on tumor and immune cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry.

Cancer Immunol Immunother 2016 11 26;65(11):1317-1323. Epub 2016 Aug 26.

IncellDx, Inc., 1700 El Camino Real, Menlo Park, CA, 94027, USA.

Objective: We report a truly quantitative technology for PD-L1 expression in non-small cell lung cancer (NSCLC). In addition, we present a non-enzymatic technology that creates a cell suspension from fresh tumor tissue so that either fine-needle aspiration (FNA) or fresh tissue can be used in this assay.

Methods: Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, California) was performed on 4-mm punch biopsies. An FNA was taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, and PD-L1 and then stained with DAPI to identify intact, single cells, and to analyze cell cycle.

Results: Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r  - 0.8) for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1 % cutoff for positivity in the tumor cell population and a 1 % cutoff of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive. Ten of 12 lung tumor samples were concordant while 2 were discordant. PD-L1 expression by flow cytometry varied widely (1.2-89.4 %) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population. Fine, unequivocal quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue.
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http://dx.doi.org/10.1007/s00262-016-1889-3DOI Listing
November 2016

Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood.

J Clin Microbiol 2015 Jan 22;53(1):60-6. Epub 2014 Oct 22.

IncellDx, Inc., Menlo Park, California, USA

Plasma viral load and CD4 counts are effective for clinical monitoring, but they do not give a full representation of HIV-1 quasispecies in cellular reservoirs, the major repository of replication-competent HIV-1 in infected individuals. We sought to develop a diagnostic system that might stimulate the replication-competent HIV-1 reservoirs for enhanced clinical monitoring, including selection of antiretroviral regimens. Whole-blood samples from 45 HIV-infected individuals were collected into 1 ViraStim HIV-1 activation tube and 1 EDTA tube. Samples were tested for viral load and cell type-specific HIV-1 replication. Further, 7 matched activated/nonactivated samples were sequenced using the Trugene HIV-1 genotyping kit. The percentage of patients with replication-competent virus in peripheral blood mononuclear cells (PBMCs) varied, depending on the baseline plasma viral load in the EDTA tubes. Six out of 24 patients with a starting plasma viral load of <20 copies/ml (cp/ml), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml, and 8 out of 13 patients with starting viral loads of >1,000 all showed increases in viral replication of >5-fold. These increases came from cellular reservoirs in blood as determined by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). When resistance genotypes in plasma from activation tubes were compared to those from EDTA tubes for 7 patients, all patients showed additional mutations in the activation tube, while 3 patients demonstrated additional genotypic resistance determinants. We show that HIV-1 viral replication can be stimulated directly from infected whole blood. The sequencing results showed that 3 of 7 cases demonstrated additional drug resistance following stimulation.
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http://dx.doi.org/10.1128/JCM.02539-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4290926PMC
January 2015

Identification and characterization of bacterial vaginosis-associated pathogens using a comprehensive cervical-vaginal epithelial coculture assay.

PLoS One 2012 15;7(11):e50106. Epub 2012 Nov 15.

Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America.

Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050106PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499514PMC
April 2013

Analysis of multiple cell reservoirs expressing unspliced HIV-1 gag-pol mRNA in patients on antiretroviral therapy.

Future Virol 2012 Aug;7(8):819-832

LabCorp Clinical Trials, Advanced Cytometric Applications, Brentwood, TN, USA ; IncellDx Inc., Menlo Park, CA, USA.

AIMS: Longitudinal percentage change of eight HIV-1 gag-pol mRNA cellular reservoirs from HIV-infected subjects on antiretroviral therapy was ascertained by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). MATERIALS #ENTITYSTARTX00026; METHODS: Serial peripheral blood mononuclear cells were taken from three subjects with treatment success, limited response and viral breakthrough plasma viral load (PVL) profiles. SUSHI was carried out on monocytes, macrophages, CD4(+) cells and naive, memory and activated T-cell reservoirs followed with broad light scatter flow cytometry. RESULTS: All gag-pol(+) reservoirs declined in the treatment success patient and similar to PVL. Only some gag-pol(+) reservoirs responded similarly to PVL for the limited treatment patient, and most gag-pol(+) reservoirs increased 16 weeks prior to PVL breakthrough in the viral breakthrough patient. CONCLUSION: SUSHI measures changes in a wide range of gag-pol(+) reservoirs in response to antiretroviral therapy.
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http://dx.doi.org/10.2217/fvl.12.69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486786PMC
August 2012

Study of HIV-1 transmission across cervical mucosa to tonsil tissue cells using an organ culture.

Am J Reprod Immunol 2013 Jan 18;69(1):52-63. Epub 2012 Oct 18.

Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Problem: SIV model indicates that upon traversing the cervicovaginal mucosa, SIV/SIV-infected cells migrate to regional lymph nodes where active replication occurs prior to systemic virus dissemination. The purpose of the study is to develop a model to study early HIV-1 transmission events that occur after crossing the cervical mucosa into regional lymph nodes.

Methods Of Study: We developed an organ culture model combining intact cervical tissue explants and tonsil tissue cells as the surrogate draining lymphoid tissue. Viral replication was measured by HIV-1 p24 production, quantification of viral DNA and viral RNA expression in tonsil cells.

Results: In this combined organ culture model, transmission of cell-free and cell-associated R5- and X4-tropic HIV-1 through the cervical mucosa to tonsilar cells was observed as determined by HIV-1 p24 in culture supernatant, and the presence of HIV-1 proviral DNA, HIV-1 p24 gag protein in CD4(+) , CD11c(+) , CD68(+) cells, and expression of HIV-1 mRNA expressing CD45RO(+)  CD4 T cells in tonsil cells. Furthermore, co-receptor usage of HIV-1 in tonsil cells correlated with inoculating virus tropism.

Conclusions: Our combined cervix-tonsil organ culture could serve as an experimental model to study the earliest stages of HIV-1 transmission through cervicovaginal mucosa to its proximal lymph nodes.
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http://dx.doi.org/10.1111/aji.12018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862563PMC
January 2013

Leukemia inhibitory factor downregulates human papillomavirus-16 oncogene expression and inhibits the proliferation of cervical carcinoma cells.

Infect Dis Obstet Gynecol 2011 4;2011:463081. Epub 2011 Jun 4.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.

The constitutive proliferation and resistance to differentiation and apoptosis of neoplastic cervical cells depend on sustained expression of human papillomavirus oncogenes. Inhibition of these oncogenes is a goal for the prevention of progression of HPV-induced neoplasias to cervical cancer. SiHa cervical cancer cells were transfected with an HPV-16 promoter reporter construct and treated with leukemia inhibitory factor (LIF), a human cytokine of the interleukin 6 superfamily. SiHa and CaSki cervical cancer cells were also assessed for proliferation by MTT precipitation, programmed cell death by flow cytometry, and HPV E6 and E7 expression by real-time PCR. LIF-treated cervical cancer cells showed significantly reduced HPV LCR activation, reduced levels of E6 and E7 mRNA, and reduced proliferation. We report the novel use of LIF to inhibit viral oncogene expression in cervical cancer cells, with concomitant reduction in proliferation suggesting re-engagement of cell-cycle regulation.
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http://dx.doi.org/10.1155/2011/463081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124004PMC
October 2011

Quantification of intracellular HPV E6/E7 mRNA expression increases the specificity and positive predictive value of cervical cancer screening compared to HPV DNA.

Gynecol Oncol 2011 Jan 14;120(1):89-93. Epub 2010 Oct 14.

Department of Pathology and Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94304, USA.

Objectives: Current methods for HPV screening rely on the detection of L1 DNA from high risk genotypes (HRHPV). These assays have very high negative predictive values (~99%), however, the specificity and positive predictive value of HPV DNA tests for pre-cancerous and cancerous lesions (CIN 2+) is less than 50%. The purpose of this study was to compare HPV DNA with intracellular HPV E6, E7 mRNA quantification in an effort to improve the performance of cervical cancer screening.

Methods: Liquid-based cervical cytology specimens collected in either PreservCyt or SurePath were processed for routing cytology, HPV HRDNA detection by Hybrid Capture 2 and HPV E6, E7 mRNA quantification in cells using the same sample. We analyzed a total of 2049 samples including 73 with CIN 2, CIN 3, or squamous cell carcinoma by biopsy and 1694 samples from women with normal cytology.

Results: The positive predictive value of HPV E6, E7 mRNA quantification in cells for CIN2+ was 78% which was greater than HPV DNA alone (43%). The specificity of HPV E6, E7 mRNA quantification was 96% based on normal cytology compared to 82% for HC2 while the specificity of HPV E6, E7 mRNA quantification based on CIN 2- histology was 85% compared to 35% for HC2.

Conclusions: With similar sensitivity and greater specificity/positive predictive value, HPV E6, E7 mRNA quantification in cells is an improvement over HPV DNA for cervical cancer screening.
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http://dx.doi.org/10.1016/j.ygyno.2010.09.013DOI Listing
January 2011

Simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI) in HIV-1 disease monitoring.

Methods Mol Biol 2010 ;659:337-46

Department of Pathology, Stanford University Medical School, Stanford, CA, USA.

The field of virology is undergoing a revolution as diagnostic tests and new therapies are allowing clinicians to treat, monitor, and predict outcomes of viral diseases. The majority of these techniques, however, destroy the factory of viral production and the information inherent in the reservoir - the cell. In this chapter, we describe a technique that combines cell surface immunophenotyping (to unequivocally identify cell types) and ultrasensitive fluorescence in situ hybridization (U-FISH) for HIV-1 to detect productively infected cells. Identification of virus and host (cells) allows earlier detection of changes in viral production and viral suppression but most importantly allows clinicians to monitor response to anti-viral therapy on a cell-by-cell and tissue-by-tissue basis taking into account the fact that the human body consists of very different, distinct compartments with unique selection pressures exerted on the viral life cycle.
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http://dx.doi.org/10.1007/978-1-60761-789-1_26DOI Listing
December 2010

Diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting.

J Infect Chemother 2010 Jun 27;16(3):155-61. Epub 2010 Feb 27.

East-West Diagnostics/Theranostics, San Francisco, CA, USA.

The lack of sensitivity of rapid immunoassays in detecting the novel 2009 H1N1 influenza virus infection has led to recommendations on influenza diagnostic testing for clinicians treating patients as well as advising clinicians on testing decisions. Studies have also shown that rapid immunoassays for seasonal influenza virus show considerable variability in performance characteristics, based on age of patient, prevalence of disease, course of infection, and the quality of the kit used. While public health authorities are currently focused on influenza virus diagnostics, a lack of sensitivity of rapid immunoassays for other viral respiratory pathogens has been widely reported, such as the very limited value of rapid immunoassays for the detection of respiratory syncytial virus in adults. In light of the lack of sensitivity of diagnostic tests for suspected 2009 H1N1 influenza virus infection, as well as their variable performance characteristics for seasonal influenza virus, a number of recommendations have been made by public health authorities advising clinicians on the need for clinical judgment as an important part of testing and treatment decisions as well as reliance on local epidemiologic and surveillance data. With the availability of new molecular methodologies that are user-friendly and allow the front-line physician as well as hospital infection control programs to significantly improve respiratory viral diagnostics, there is a need to carefully determine the most optimal diagnostic testing methodology based on the clinical setting. This review will describe the historical, current, and changing dynamics of respiratory virus infection diagnostics.
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http://dx.doi.org/10.1007/s10156-010-0035-yDOI Listing
June 2010

Determination of hepatitis C virus-infected, monocyte lineage reservoirs in individuals with or without HIV coinfection.

J Infect Dis 2009 Sep;200(6):947-54

Department of Pathology, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, 3375 Hillview Avenue, Palo Alto, CA 94304, USA.

Because previous reports found an association between hepatitis C virus (HCV) coinfection and progression of human immunodeficiency virus (HIV) disease, we investigated whether HIV and HCV may reciprocally influence viral replication in monocyte lineage cells in vivo. Using a novel technique called simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI), we rapidly and unequivocally identified HCV reservoirs in peripheral blood from HCV-infected individuals with and without HIV coinfection. We found that HCV infects both CD14(+), CD16(+)(+) monocytic cells and CD14(+)(+), CD16(+)(+) monocytic cells but not CD14(+)(+), CD16- cells in individuals infected with HCV with or without HIV coinfection. To address these HCV tropism differences, we found that the HCV receptor CD81 is highly expressed on CD14(+), CD16(+)(+) and CD14(+)(+), CD16(+)(+) cells but not on monocytes (CD14(+)(+), CD16-). These findings have important implications for the diagnosis and treatment of HCV infection, mother-to-child transmission of HCV, and possible virus-virus interactions in HCV-HIV coinfected individuals.
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http://dx.doi.org/10.1086/605476DOI Listing
September 2009

Evaluation of the NanoChip 400 system for detection of influenza A and B, respiratory syncytial, and parainfluenza viruses.

J Clin Microbiol 2008 May 5;46(5):1724-7. Epub 2008 Mar 5.

Clinical Laboratories, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, USA.

The NanoChip400 system uses multiplex PCR chemistry and electronic microarray detection of influenza A and B viruses; respiratory syncytial viruses A and B; and human parainfluenza virus types 1, 2, and 3. The results obtained with the NanoChip 400 system were compared with those obtained by direct fluorescent-antibody staining (DFA) and real-time PCR with 122 and 130 specimens, respectively. Concordance between DFA and NanoChip 400 system was obtained for 106 of 122 (86.9%) specimens. On the basis of discrepancy analysis with specimens available for confirmatory real-time PCR testing, the sensitivity and specificity of the NanoChip 400 were 98.6% and 100%, respectively. With respect to specimens previously tested by real-time PCR, the NanoChip 400 system demonstrated a sensitivity of 91.1% and a specificity of 100%. The NanoChip 400 system provides clinical laboratories with a practical, rapid, and sensitive method for the detection of common respiratory viruses.
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http://dx.doi.org/10.1128/JCM.01947-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395092PMC
May 2008

Rapid, high throughput determination of cervical cytology specimen adequacy using a capillary-based cytometer.

Cytometry B Clin Cytom 2008 Mar;74(2):133-6

Invirion Diagnostics LLC, Oak Brook, Illinois 60523, USA.

Despite the fact that compact instruments dedicated to counting cells are readily available especially in hematology, liquid-based cervical cytology specimen adequacy is still determined by pathologist review following the preparation of slides. More than 100 liquid-based cervical cytology specimens collected in ThinPrep vials were processed into slides for diagnosis and assessment of specimen adequacy using the Bethesda 2001 criteria. Residual liquid (100 muL) was transferred to a single well of a 96-well plate and run in a capillary cytometer (Guava Technologies, Hayward, CA) with forward scatter and side scatter detectors. We determined that the sensitivity and specificity of this assay for unsatisfactory samples was 100% and 97%, respectively, compared with cytopathologic examination. The difference in the concentration of ectocervical cells/muL (EPU) between samples determined to be unsatisfactory by virtue of less than an estimated 5,000 cells per slide and satisfactory samples were statistically significant (P< 0.001). Here, we report the use of this rapid specimen adequacy assay on a small, capillary based, personal cytometer. Use of this instrument reduced the amount of sample required by >90% and reduced the average per sample assay time from 120 s to 10 s compared with flow cytometry.
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http://dx.doi.org/10.1002/cyto.b.20385DOI Listing
March 2008

Patterns of known and novel small RNAs in human cervical cancer.

Cancer Res 2007 Jul;67(13):6031-43

Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA.

Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-0561DOI Listing
July 2007

Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.

J Clin Microbiol 2007 Jul 9;45(7):2340-3. Epub 2007 May 9.

Department of Biochemistry and Biophysics, University of California, San Francisco at Mission Bay, office BH403C, QB3 building, 1700 4th Street, San Francisco CA 94158, USA.

A pan-virus DNA microarray (Virochip) was used to detect a human metapneumovirus (hMPV) strain associated with a critical respiratory tract infection in an elderly adult with chronic lymphocytic leukemia. This infection had previously eluded diagnosis despite extensive microbiological testing for possible etiologic agents. The patient's hMPV strain did not grow in viral culture, and only one of five specific reverse transcription-PCR assays for hMPV was positive.
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http://dx.doi.org/10.1128/JCM.00364-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933008PMC
July 2007

Leukemia inhibitor factor (LIF) inhibits HIV-1 replication via restriction of stat 3 activation.

AIDS Res Hum Retroviruses 2007 Mar;23(3):398-406

Center for Infectious Medicine, Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden.

Leukemia inhibitor factor (LIF) has been shown to potently inhibit HIV-1 replication in vitro and in human organ explant cultures. Furthermore, LIF activates the Jak/Stat signaling pathway with which many viruses, including HIV-1, interfere. We used CXCR4 and the LIF signaling receptor (gp130)-expressing cMAGI cells transfected with CD4, CCR5, and HIV-LTR-beta-galactosidase as a model system to investigate the potential involvement of Stat proteins in the anti-HIV-1 effect of LIF. Pretreatment with recombinant human (rh)LIF resulted in a significantly reduced uptake of HIV-1(BaL) , HIV-1(LAI), and SIVmac251 viral particles without affecting uptake of murine leukemia retroviral particles. HIV-1(BaL), HIV-1(LAI), as well as rhLIF selectively induced phosphorylation of Stat 3 but not Stat 1 or Stat 5. However, treatment of cMAGI cells with rhLIF prior to HIV-1 infection downregulated the HIV-1-mediated Stat 3 phosphorylation. In addition, peripheral blood mononuclear cells (PBMCs) transfected with Stat 3 siRNA prior to HIV-1(LAI) or HIV-1(BaL) infection produced significantly less HIV-1 p24 antigen as compared to nontransfected HIV-1(LAI) and HIV-1(BaL)-infected PBMCs. Thus, the Jak/Stat signaling pathway is important for the HIV-1 replication life cycle and rhLIF excerts its anti-HIV-1 activity by disrupting this signaling cascade.
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http://dx.doi.org/10.1089/aid.2006.0100DOI Listing
March 2007

Proinflammatory and type 1 cytokine expression in cervical mucosa during HIV-1 and human papillomavirus infection.

J Acquir Immune Defic Syndr 2007 May;45(1):9-19

Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

Suppression of immune activation and increased inflammation are prevalent during viral infection. To investigate the role of inflammation in HIV transmission, we studied the infectious and inflammatory milieu in cervical mucosa from HIV-1- and human papillomavirus (HPV)-coinfected and HPV-monoinfected women. The numbers of cytokine-, chemokine-, and p24-expressing cells were determined using in situ imaging analysis and intracellular staining of p24 antigen. Significantly higher expression of the proinflammatory cytokines, interleukin (IL)-1alpha/beta, was seen in cervical tissue from HIV/HPV-coinfected as compared with HPV-monoinfected tissues, whereas IL-2- and interferon (IFN)-gamma-expressing cells were higher in HPV-monoinfected tissues. IL-10 was low in both groups, whereas IL-4 was significantly higher in HPV-monoinfected and HIV/HPV-coinfected tissues than in HIV/HPV-negative controls. RANTES and macrophage inflammatory protein (MIP)-1beta but not MIP-1alpha were significantly higher in the genital tract of HIV/HPV-coinfected as compared with HPV-monoinfected individuals and controls. HIV/HPV-coinfected tissues had a higher level of human leukocyte antigen D-related (HLA-DR)-expressing dendritic cells (DCs). There was a positive correlation between the number of CD4(+) and CD8(+) T cells as well as CD1a, IL-1alpha, and RANTES expression and p24 antigen-expressing cells in the HIV/HPV-coinfected tissues. These findings suggest the persistence of immune activation and inflammation in the genital tract of women with HPV monoinfection and in HIV-infected women coinfected with HPV.
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http://dx.doi.org/10.1097/QAI.0b013e3180415da7DOI Listing
May 2007

Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression.

Blood 2007 Jun 1;109(12):5380-9. Epub 2007 Mar 1.

AIDS Immunopathogenesis Unit and the Division of Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy.

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.
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http://dx.doi.org/10.1182/blood-2006-08-042556DOI Listing
June 2007

p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia.

Am J Surg Pathol 2007 Jan;31(1):33-43

Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.

In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16 can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established. We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16 IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16 IHC. In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16 IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16 reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16 reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV. In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16 was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16 reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16 reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means. Overall, p16 IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.
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http://dx.doi.org/10.1097/01.pas.0000213347.65014.eeDOI Listing
January 2007

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.

Clin Infect Dis 2006 Oct 1;43(8):e71-6. Epub 2006 Sep 1.

Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.

A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.
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http://dx.doi.org/10.1086/507896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108001PMC
October 2006

Use of frozen-thawed cervical tissues in the organ culture system to measure anti-HIV activities of candidate microbicides.

AIDS Res Hum Retroviruses 2006 May;22(5):419-24

Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

Cervical tissue-based organ culture system has been used to test the cytotoxicity and antiviral activity of microbicides. One of the problems of using current organ culture methods for routine microbicide testing is the need to continually obtain fresh tissue, which can be limited in access and supply. Use of frozen tissue, stored when available and thawed when needed, would alleviate the need for constant access to new tissue. This study was designed to explore the possibility of using frozen-thawed cervical tissue to test microbicides for their anti-HIV activity. We provided biochemical, histological, and quantitative immunohistochemical data to demonstrate the integrity of the frozen-thawed organ culture system. Significant levels of HIV-1 mucosal transmission were noted with both fresh and frozen-thawed tissue, regardless of the coreceptor usage of the virus isolate. Furthermore, candidate microbicides UC781, beta-cyclodextrin, and octylglycerol inhibited HIV-1 transmission across the mucosa of frozen-thawed tissues with a level of efficiency similar to that of fresh tissues. Therefore, frozen-thawed cervical tissue in the organ culture system provides a practical and convenient model to screen topical microbicides for their ability to block sexual transmission of HIV-1, and reduces the problems associated with procurement of the numerous tissues required for evaluation and comparison of microbicide candidates among different laboratories.
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http://dx.doi.org/10.1089/aid.2006.22.419DOI Listing
May 2006

A comparison study of different PCR assays in measuring circulating plasma epstein-barr virus DNA levels in patients with nasopharyngeal carcinoma.

Clin Cancer Res 2005 Aug;11(16):5700-7

Department of Radiation Oncology, Stanford University, Stanford, California 94305-5407, USA.

Purpose: To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance.

Experimental Design: Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables.

Results: Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002).

Conclusions: The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.
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http://dx.doi.org/10.1158/1078-0432.CCR-05-0648DOI Listing
August 2005