Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry.

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

Detection of early-stage lung cancer in sputum using automated flow cytometry and machine learning.

BACKGROUND: Low-dose spiral computed tomography (LDCT) may not lead to a clear treatment path when small to intermediate-sized lung nodules are identified. We have combined flow cytometry and machine learning to develop a sputum-based test (CyPath Lung) that can assist physicians in decision-making in such cases. METHODS: Single cell suspensions prepared from induced sputum samples collected over three consecutive days were labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. RESULTS: Automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83-0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89-0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71-0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. CONCLUSION: CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules < 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis. Trial registration ClinicalTrials.gov ID: NCT03457415; March 7, 2018.

Sputum analysis by flow cytometry; an effective platform to analyze the lung environment.

Low dose computed tomography (LDCT) is the standard of care for lung cancer screening in the United States (US). LDCT has a sensitivity of 93.8% but its specificity of 73.4% leads to potentially harmful follow-up procedures in patients without lung cancer. Thus, there is a need for additional assays with high accuracy that can be used as an adjunct to LDCT to diagnose lung cancer. Sputum is a biological fluid that can be obtained non-invasively and can be dissociated to release its cellular contents, providing a snapshot of the lung environment. We obtained sputum from current and former smokers with a 30+ pack-year smoking history and who were either confirmed to have lung cancer or at high risk of developing the disease. Dissociated sputum cells were counted, viability determined, and labeled with a panel of markers to separate leukocytes from non-leukocytes. After excluding debris and dead cells, including squamous epithelial cells, we identified reproducible population signatures and confirmed the samples' lung origin. In addition to leukocyte and epithelial-specific fluorescent antibodies, we used the highly fluorescent meso-tetra(4-carboxyphenyl) porphyrin (TCPP), known to preferentially stain cancer (associated) cells. We looked for differences in cell characteristics, population size and fluorescence intensity that could be useful in distinguishing cancer samples from high-risk samples. We present our data demonstrating the feasibility of a flow cytometry platform to analyze sputum in a high-throughput and standardized matter for the diagnosis of lung cancer.

Quality-Controlled Sputum Analysis by Flow Cytometry.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs. The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

Treatment of Corneal Infections Utilizing an Ocular Wound Chamber.

Purpose: To demonstrate that the ocular wound chamber (OWC) can be used for the treatment of bacterial keratitis (BK). Methods: A blepharotomy was performed on anesthetized, hairless guinea pigs to induce exposure keratopathy 72 hours before corneal wound creation and Pseudomonas aeruginosa inoculation. Twenty-four hours postinoculation, eyes were treated with an OWC filled with 500 µL 0.5% moxifloxacin hydrochloride ophthalmic solution (OWC), 10 µL 0.5% moxifloxacin hydrochloride drops (DROPS) four times daily, or not treated (NT). White light, fluorescein, and spectral domain optical coherence tomography (SD-OCT) images; ocular and periocular tissues samples for colony-forming units (CFU) quantification; and plasma samples were collected at 24 and 72 hours posttreatment. Results: White light, fluorescein, and SD-OCT imaging suggests OWC-treated eyes are qualitatively healthier than those in DROPS or NT groups. At 24 hours, the median number of CFUs (interquartile range) measured was 0 (0-8750), 150,000 (106,750-181,250), and 8750 (2525-16,000) CFU/mL for OWC, NT, and DROPS, respectively. While 100% of NT and DROPS animals remained infected at 24 hours, only 25% of OWC-treated animals showed infection. Skin samples at 24 hours showed infection percentages of 50%, 75%, and 0% in DROPS, NT, and OWC groups, respectively. OWC-treated animals had higher moxifloxacin plasma concentrations at 24 and 72 hours than those treated with drops. Conclusions: OWC use resulted in a more rapid decrease of CFUs when compared to DROPS or NT groups and was associated with qualitatively healthier ocular and periocular tissue. Translational Relevance: The OWC could be used clinically to continuously and rapidly deliver antimicrobials to infected ocular and periocular tissues, effectively lowering bacterial bioburdens and mitigating long-term complications.

Efficacy of Intravenous Hydroxocobalamin for Treatment of Sodium Methanethiolate Exposure in a Swine Model (Sus scrofa) of Severe Methanethiol Toxicity.

INTRODUCTION: Methanethiol is a highly toxic chemical present in crude oil and natural gas. At high concentrations, methanethiol causes metabolic acidosis, seizures, myocardial infarction, coma, and death. Occupational Health and Safety Administration lists methanethiol as a potential terrorist weapon. Methanethiol blocks the electron transport chain, resulting in lactic acidosis and acidemia. There is no specific treatment for methanethiol. Our objective was to measure the efficacy of intravenous (IV) hydroxocobalamin (HOC) versus no treatment (control) in methanethiol-induced apnea in a swine model. METHODS: Sixteen anesthetized swine received IV sodium methanethiolate to apnea and were randomized to receive either IV HOC or no treatment. Physiologic and laboratory parameters were monitored throughout the study. Power analysis indicated that 8 animals per group would be sufficient to find a moderate effect (f = 0.24) with 2 groups, α = 0.05, and 80% power. RESULTS: Both groups were similar in baseline characteristics. Following treatment, the HOC group had significantly higher heart rate and blood pressure at 5-10 minutes post-apnea, higher systemic vascular resistance at 5 minutes post-apnea, higher tidal volume, higher end-tidal carbon dioxide, and lower end-tidal oxygen 10-15 minutes post-apnea compared with controls. None of the animals survived to the end of the study (60 minutes). The Kaplan-Meier survival curves were significantly different between cohorts (log-rank p = 0.0321), with the HOC group surviving longer than controls (32.4 ± 7.3 vs. 25.8 ± 1.0 minutes). CONCLUSIONS: In our model of intravenous methanethiolate poisoning, IV HOC administration resulted in a transient improvement in vital signs and prolonged time to death; however, it did not improve survival.

Obesity-Induced Changes in T-Cell Metabolism Are Associated With Impaired Memory T-Cell Response to Influenza and Are Not Reversed With Weight Loss.

BACKGROUND: Obesity is an independent risk factor for increased influenza mortality and is associated with impaired memory T-cell response, resulting in increased risk of infection. In this study, we investigated if weight loss would restore memory T-cell response to influenza. METHODS: Male C57BL/6J mice were fed either low-fat or high-fat diet to induce obesity. Once obesity was established, all mice received primary infection with influenza X-31. Following a recovery period, we switched half of the obese group to a low-fat diet to induce weight loss. Fifteen weeks after diet switch, all mice were given a secondary infection with influenza PR8, and memory T-cell function and T-cell metabolism were measured. RESULTS: Following secondary influenza infection, memory T-cell subsets in the lungs of obese mice were decreased compared to lean mice. At the same time, T cells from obese mice were found to have altered cellular metabolism, largely characterized by an increase in oxygen consumption. Neither impaired memory T-cell response nor altered T-cell metabolism was reversed with weight loss. CONCLUSION: Obesity-associated changes in T-cell metabolism are associated with impaired T-cell response to influenza, and are not reversed with weight loss.

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Genotyping human papillomaviruses: development and evaluation of a comprehensive DNA microarray.

GOALS: To define the analytical and clinical performance of a human papillomavirus (HPV) custom-designed microarray targeting the HPV L1 gene for viral genotyping. METHODS: Microarray probes were designed by cataloging the genome sequence of all 120 known HPV types to generate tiling probes using eArray® software against the unique L1 capsid gene segments targeted by MY09/11 and FAP59/64 primers. The microarray (1 slide×8 arrays×60K features) synthesized in situ by inkjet printing was tested using synthetic type-specific HPV DNA and existing HPV DNA from cervical cytology. The synthetic HPV L1 segments (genotypes 6, 11, 16, 18, 31, 33, 35, 45, 53, 58, 66, 73, 83) were manufactured from sequences stored in the NCBI taxonomy database. Using the hybridization patterns of the synthetic HPV DNA as the Support Vector Machine classifier, HPV DNA from patient samples were genotyped and compared to antecedent DNA sequencing/BLAST® results for concordance. RESULTS: 16 cytology-derived HPV DNA samples and 13 synthetic type-specific HPV DNA samples were tested singly, in duplicate, or in combination on 40 arrays. The synthetic HPV DNA hybridization patterns were found to be uniquely distinctive to serve well as a classifier of unknown HPV-containing specimens. For the 16HPV DNA+ samples classified, 15 were concordant with DNA sequencing results. In 6/16 (38%) samples, the microarray hybridization pattern revealed ≥2 concurrent HPV infections. CONCLUSION: The novel "HPV Array" was sensitive and specific for detecting single and multiple infections. This proof-of-principle project demonstrated the accuracy and advantages of microarray technology for HPV genotyping.

Genomic loss of tumor suppressor miRNA-204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization.

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

Risk factors influencing antibody responses to Kaposi's sarcoma-associated herpesvirus latent and lytic antigens in patients under antiretroviral therapy.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) seropositivity and lytic antibody titer are predictors for Kaposi's sarcoma. METHODS: We examined demographic, viral, and immunologic factors that influence KSHV latent and lytic antibodies in HIV-infected patients. RESULTS: Detection rate of KSHV latent but not lytic antibodies was lower in patients with CD4 cells/mm3 less than 200 than greater than 200 (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.11-0.61) and CD8 cells/mm3 less than 400 than greater than 400 (OR, 0.26; 95% CI, 0.07-0.67). Overall seropositivity rate was higher in patients with CD4 cells/mm3 less than 200 than greater than 200 (OR, 2.34; 95% CI, 1.37-4.02) and HIV copies/mL greater than 400 than less than 400 (OR, 1.70; 95% CI, 1.09-2.65). Lytic antibody level was inversely correlated with CD4 count (P < 0.001). Lytic seropositivity (OR, 2.47; 95% CI, 1.35-4.50) and antibody level (adjusted difference mean optical density, 0.324; 95% CI, 0.16-0.46) were higher in patients with HIV infection greater than 15 than less than 15 years. Hispanics had higher lytic seropositivity rate (OR, 1.71; 95% CI, 1.07-2.73) and antibody level (adjusted difference mean optical density, 0.111; 95% CI, 0.03-0.18) than non-Hispanics. CONCLUSIONS: Lower CD4 and CD8 counts impair antibody response to KSHV latent antigens. Immune deterioration, long-term HIV infection, and Hispanic status are risk factors for Kaposi's sarcoma predictors.