Corrigendum: Case Series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequalae of COVID (PASC).

[This corrects the article DOI: 10.3389/fmed.2023.1122529.].

Case series: Maraviroc and pravastatin as a therapeutic option to treat long COVID/Post-acute sequelae of COVID (PASC).

Post-acute sequelae of COVID (PASC), or long COVID, is a multisystem complication of SARS-CoV-2 infection that continues to debilitate millions worldwide thus highlighting the public health importance of identifying effective therapeutics to alleviate this illness. One explanation behind PASC may be attributed to the recent discovery of persistent S1 protein subunit of SARS-CoV-2 in CD16+ monocytes up to 15 months after infection. CD16+ monocytes, which express both CCR5 and fractalkine receptors (CX3CR1), play a role in vascular homeostasis and endothelial immune surveillance. We propose targeting these receptors using the CCR5 antagonist, maraviroc, along with pravastatin, a fractalkine inhibitor, could disrupt the monocytic-endothelial-platelet axis that may be central to the etiology of PASC. Using five validated clinical scales (NYHA, MRC Dyspnea, COMPASS-31, modified Rankin, and Fatigue Severity Score) to measure 18 participants' response to treatment, we observed significant clinical improvement in 6 to 12 weeks on a combination of maraviroc 300 mg per oral twice a day and pravastatin 10 mg per oral daily. Subjective neurological, autonomic, respiratory, cardiac and fatigue symptoms scores all decreased which correlated with statistically significant decreases in vascular markers sCD40L and VEGF. These findings suggest that by interrupting the monocytic-endothelial-platelet axis, maraviroc and pravastatin may restore the immune dysregulation observed in PASC and could be potential therapeutic options. This sets the framework for a future double-blinded, placebo-controlled randomized trial to further investigate the drug efficacy of maraviroc and pravastatin in treating PASC.

Immune-Based Prediction of COVID-19 Severity and Chronicity Decoded Using Machine Learning.

Expression of CCR5 and its cognate ligands have been implicated in COVID-19 pathogenesis, consequently therapeutics directed against CCR5 are being investigated. Here, we explored the role of CCR5 and its ligands across the immunologic spectrum of COVID-19. We used a bioinformatics approach to predict and model the immunologic phases of COVID so that effective treatment strategies can be devised and monitored. We investigated 224 individuals including healthy controls and patients spanning the COVID-19 disease continuum. We assessed the plasma and isolated peripheral blood mononuclear cells (PBMCs) from 29 healthy controls, 26 Mild-Moderate COVID-19 individuals, 48 Severe COVID-19 individuals, and 121 individuals with post-acute sequelae of COVID-19 (PASC) symptoms. Immune subset profiling and a 14-plex cytokine panel were run on all patients from each group. B-cells were significantly elevated compared to healthy control individuals (P<0.001) as was the CD14+, CD16+, CCR5+ monocytic subset (P<0.001). CD4 and CD8 positive T-cells expressing PD-1 as well as T-regulatory cells were significantly lower than healthy controls (P<0.001 and P=0.01 respectively). CCL5/RANTES, IL-2, IL-4, CCL3, IL-6, IL-10, IFN-γ, and VEGF were all significantly elevated compared to healthy controls (all P<0.001). Conversely GM-CSF and CCL4 were in significantly lower levels than healthy controls (P=0.01). Data were further analyzed and the classes were balanced using SMOTE. With a balanced working dataset, we constructed 3 random forest classifiers: a multi-class predictor, a Severe disease group binary classifier and a PASC binary classifier. Models were also analyzed for feature importance to identify relevant cytokines to generate a disease score. Multi-class models generated a score specific for the PASC patients and defined as S1 = (IFN-γ + IL-2)/CCL4-MIP-1ß. Second, a score for the Severe COVID-19 patients was defined as S2 = (IL-6+sCD40L/1000 + VEGF/10 + 10*IL-10)/(IL-2 + IL-8). Severe COVID-19 patients are characterized by excessive inflammation and dysregulated T cell activation, recruitment, and counteracting activities. While PASC patients are characterized by a profile able to induce the activation of effector T cells with pro-inflammatory properties and the capacity of generating an effective immune response to eliminate the virus but without the proper recruitment signals to attract activated T cells.

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

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.

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

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.

Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) up to 15 Months Post-Infection.

The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. That non-classical monocytes may be a source of inflammation in PASC warrants further study.

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.

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 CD103+CD4+ 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 CD44+E-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 CD103+CD4+ T cells that could potentially be predictive of CTCs and targets of therapy.

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

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.

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

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.

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

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.

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.

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.

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

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.

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

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.

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.

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 2 - 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.

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

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.

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

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.

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

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 ß-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 ß-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.

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

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.

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

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.

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

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.