Safety profile and effects on the peripheral immune response of fecal microbiota transplantation in clinically healthy dogs.

BACKGROUND: Fecal microbiota transplantation (FMT) is increasingly used for gastrointestinal and extra-gastrointestinal diseases in veterinary medicine. However, its effects on immune responses and possible adverse events have not been systematically investigated. HYPOTHESIS/OBJECTIVES: Determine the short-term safety profile and changes in the peripheral immune system after a single FMT administration in healthy dogs. ANIMALS: Ten client-owned, clinically healthy dogs as FMT recipients, and 2 client-owned clinically healthy dogs as FMT donors. METHODS: Prospective non-randomized clinical trial. A single rectal enema of 5 g/kg was given to clinically healthy canine recipients. During the 28 days after FMT administration, owners self-reported adverse events and fecal scores. On Days 0 (baseline), 1, 4, 10, and 28 after FMT, fecal and blood samples were collected. The canine fecal dysbiosis index (DI) was calculated using qPCR. RESULTS: No significant changes were found in the following variables: CBC, serum biochemistry, C-reactive protein, serum cytokines (interleukins [IL]-2, -6, -8, tumor necrosis factor [TNF]-α), peripheral leukocytes (B cells, T cells, cluster of differentiation [CD]4+ T cells, CD8+ T cells, T regulatory cells), and the canine DI. Mild vomiting (n = 3), diarrhea (n = 4), decreased activity (n = 2), and inappetence (n = 1) were reported, and resolved without intervention. CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal microbiota transplantation did not significantly alter the evaluated variables and recipients experienced minimal adverse events associated with FMT administration. Fecal microbiota transplantation was not associated with serious adverse events, changes in peripheral immunologic variables, or the canine DI in the short-term.

Dynamic evaluation of blood immune cells predictive of response to immune checkpoint inhibitors in NSCLC by multicolor spectrum flow cytometry.

Introduction: Immune checkpoint inhibitors (ICIs) only benefit a subset of cancer patients, underlining the need for predictive biomarkers for patient selection. Given the limitations of tumor tissue availability, flow cytometry of peripheral blood mononuclear cells (PBMCs) is considered a noninvasive method for immune monitoring. This study explores the use of spectrum flow cytometry, which allows a more comprehensive analysis of a greater number of markers using fewer immune cells, to identify potential blood immune biomarkers and monitor ICI treatment in non-small-cell lung cancer (NSCLC) patients. Methods: PBMCs were collected from 14 non-small-cell lung cancer (NSCLC) patients before and after ICI treatment and 4 healthy human donors. Using spectrum flow cytometry, 24 immune cell markers were simultaneously monitored using only 1 million PBMCs. The results were also compared with those from clinical flow cytometry and bulk RNA sequencing analysis. Results: Our findings showed that the measurement of CD4+ and CD8+ T cells by spectrum flow cytometry matched well with those by clinical flow cytometry (Pearson R ranging from 0.75 to 0.95) and bulk RNA sequencing analysis (R=0.80, P=1.3 x 10-4). A lower frequency of CD4+ central memory cells before treatment was associated with a longer median progression-free survival (PFS) [Not reached (NR) vs. 5 months; hazard ratio (HR)=8.1, 95% confidence interval (CI) 1.5-42, P=0.01]. A higher frequency of CD4-CD8- double-negative (DN) T cells was associated with a longer PFS (NR vs. 4.45 months; HR=11.1, 95% CI 2.2-55.0, P=0.003). ICIs significantly changed the frequency of cytotoxic CD8+PD1+ T cells, DN T cells, CD16+CD56dim and CD16+CD56- natural killer (NK) cells, and CD14+HLDRhigh and CD11c+HLADR + monocytes. Of these immune cell subtypes, an increase in the frequency of CD16+CD56dim NK cells and CD14+HLADRhigh monocytes after treatment compared to before treatment were associated with a longer PFS (NR vs. 5 months, HR=5.4, 95% CI 1.1-25.7, P=0.03; 7.8 vs. 3.8 months, HR=5.7, 95% CI 169 1.0-31.7, P=0.04), respectively. Conclusion: Our preliminary findings suggest that the use of multicolor spectrum flow cytometry helps identify potential blood immune biomarkers for ICI treatment, which warrants further validation.

A distinct subpopulation of leukemia initiating cells in acute precursor B lymphoblastic leukemia: quiescent phenotype and unique transcriptomic profile.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 µm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. METHODS: Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. RESULTS: Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. CONCLUSION: These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Anti-Cancer Activity of PAK4/NAMPT Inhibitor and Programmed Cell Death Protein-1 Antibody in Kidney Cancer.

BACKGROUND: Kidney cancer (or renal cell carcinoma, RCC) is the sixth most common malignancy in the United States and is increasing in incidence. Despite new therapies, including targeted therapies and immunotherapies, most RCCs are resistant to treatment. Thus, several laboratories have been evaluating new approaches to therapy, both with single agents as well as combinations. Although we have previously shown efficacy of the dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitor KPT-9274, and the immune checkpoint inhibitors (CPI) have shown utility in the clinic, there has been no evaluation of this combination either clinically or in an immunocompetent animal model of kidney cancer. METHODS: In this study, we use the renal cell adenocarcinoma (RENCA) model of spontaneous murine kidney cancer. Male BALB/cJ mice were injected subcutaneously with RENCA cells and, after tumors were palpable, they were treated with KPT-9274 and/or anti-programmed cell death 1 (PDCD1; PD1) antibody for 21 days. Tumors were measured and then removed at animal euthanasia for subsequent studies. RESULTS: We demonstrate a significant decrease in allograft growth with the combination treatment of KPT-9274 and anti-PD1 antibody without significant weight loss by the animals. This is associated with decreased (MOUSE) Naprt expression, indicating dependence of these tumors on NAMPT in parallel to what we have observed in human RCC. Histology of the tumors showed substantial necrosis regardless of treatment condition, and flow cytometry of antibody-stained tumor cells revealed that the enhanced therapeutic effect of KPT-9274 and anti-PD1 antibody was not driven by infiltration of T cells into tumors. CONCLUSIONS: This study highlights the potential of the RENCA model for evaluating immunologic responses to KPT-9274 and checkpoint inhibitor (CPI) and suggests that therapy with this combination could improve efficacy in RCC beyond what is achievable with CPI alone.

Composition of the Intranuclear Inclusions of Fragile X-associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55-200 CGG repeats) in the 5' noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.

Toward Cell Type-Specific In Vivo Dual RNA-Seq.

Dual RNA-seq has emerged as a genome-wide expression profiling technique, simultaneously measuring RNA transcript levels in a given host and its pathogen during an infection. Recently, the method was transferred from cell culture to in vivo models of bacterial infections; however, specific host cell-type resolution has not yet been achieved. Here we present a detailed protocol that describes the application of Dual RNA-seq to murine colonocytes isolated from mice infected with the enteric pathogen Salmonella Typhimurium. At day 5 after oral infection, the mice were humanely euthanized, their colons extracted, and colonocytes isolated and fixed. Upon antibody staining of cell type-specific surface markers, the fraction of Salmonella-invaded colonocytes was collected by fluorescence-activated cell sorting based on a fluorescent signal emitted by the internalized bacteria. Total RNA was extracted from cells enriched by this method, and ribosomal transcripts from host and microbial cells were removed prior to cDNA synthesis and library generation. We compared different protocols for library preparation and discuss their respective advantages and caveats when applied to minute RNA amounts that constitute an inherent challenge for in vivo transcriptomics. Our results introduce an ultralow input protocol that holds promise for cell type-specific in vivo Dual RNA-seq for charting gene expression of a bacterial pathogen within its respective in vivo niche, along with the consequent host response.

Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development.

RATIONALE: Quantifying cellular proteins in ventricular myocytes (MCs) is challenging due to tissue heterogeneity and the variety of cell sizes in the heart. In post-weaning cardiac ontogeny, rod-shaped MCs make up the majority of the cardiac mass while remaining a minority of cardiac cells in number. Current biochemical analyses of cardiac proteins do not correlate well the content of MC-specific proteins to cell type or size in normally developing tissue. OBJECTIVE: To develop a new large-particle fluorescent-activated cell sorting (LP-FACS) strategy for the purification of adult rod-shaped MCs. This approach is developed to enable growth-scaled measurements per-cell of the MC proteome and sarcomeric proteins (i.e. myosin heavy chain (MyHC) and alpha-actin (α-actin)) content. METHODS AND RESULTS: Individual cardiac cells were isolated from 21 to 94days old mice. An LP-FACS jet-in-air system with a 200-µm nozzle was defined for the first time to purify adult MCs. Cell-type specific immunophenotyping and sorting yielded ≥95% purity of adult MCs independently of cell morphology and size. This approach excluded other cell types and tissue contaminants from further analysis. MC proteome, MyHC and α-actin proteins were measured in linear biochemical assays normalized to cell numbers. Using the allometric coefficient α, we scaled the MC-specific rate of protein accumulation to growth post-weaning. MC-specific volumes (α=1.02) and global protein accumulation (α=0.94) were proportional (i.e. isometric) to body mass. In contrast, MyHC and α-actin accumulated at a much greater rate (i.e. hyperallometric) than body mass (α=1.79 and 2.19 respectively) and MC volumes (α=1.76 and 1.45 respectively). CONCLUSION: Changes in MC proteome and cell volumes measured in LP-FACS purified MCs are proportional to body mass post-weaning. Oppositely, MyHC and α-actin are concentrated more rapidly than what would be expected from MC proteome accumulation, cell enlargement, or animal growth alone. LP-FACS provides a new standard for adult MC purification and an approach to scale the biochemical content of specific proteins or group of proteins per cell in enlarging MCs.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1ß secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1ß promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Immune Activation and HIV-Specific CD8(+) T Cells in Cerebrospinal Fluid of HIV Controllers and Noncontrollers.

The central nervous system (CNS) is an important target of HIV, and cerebrospinal fluid (CSF) can provide a window into host-virus interactions within the CNS. The goal of this study was to determine whether HIV-specific CD8(+) T cells are present in CSF of HIV controllers (HC), who maintain low to undetectable plasma viremia without antiretroviral therapy (ART). CSF and blood were sampled from 11 HC, defined based on plasma viral load (VL) consistently below 2,000 copies/ml without ART. These included nine elite controllers (EC, plasma VL <40 copies/ml) and two viremic controllers (VC, VL 40-2,000 copies/ml). All controllers had CSF VL <40 copies/ml. Three comparison groups were also sampled: six HIV noncontrollers (NC, VL >10,000 copies/ml, no ART); seven individuals with viremia suppressed due to ART (Tx, VL <40 copies/ml); and nine HIV-negative controls. CD4(+) and CD8(+) T cells in CSF and blood were analyzed by flow cytometry to assess expression of CCR5, activation markers CD38 and HLA-DR, and memory/effector markers CD45RA and CCR7. HIV-specific CD8(+) T cells were quantified by major histocompatibility complex class I multimer staining. HIV-specific CD8(+) T cells were detected ex vivo at similar frequencies in CSF of HC and noncontrollers; the highest frequencies were in individuals with CD4 counts below 500 cells/µl. The majority of HIV-specific CD8(+) T cells in CSF were effector memory cells expressing CCR5. Detection of these cells in CSF suggests active surveillance of the CNS compartment by HIV-specific T cells, including in individuals with long-term control of HIV infection in the absence of therapy.

The immunologic effects of maraviroc intensification in treated HIV-infected individuals with incomplete CD4+ T-cell recovery: a randomized trial.

The CCR5 inhibitor maraviroc has been hypothesized to decrease T-cell activation in HIV-infected individuals, but its independent immunologic effects have not been established in a placebo-controlled trial. We randomized 45 HIV-infected subjects with CD4 counts <350 cells per mm(3) and plasma HIV RNA levels <48 copies per mL on antiretroviral therapy (ART) to add maraviroc vs placebo to their regimen for 24 weeks followed by 12 weeks on ART alone. Compared with placebo-treated subjects, maraviroc-treated subjects unexpectedly experienced a greater median increase in % CD38+HLA-DR+ peripheral blood CD8+ T cells at week 24 (+2.2% vs -0.7%, P = .014), and less of a decline in activated CD4+ T cells (P < .001). The % CD38+HLA-DR+ CD4+ and CD8+ T cells increased nearly twofold in rectal tissue (both P < .001), and plasma CC chemokine receptor type 5 (CCR5) ligand (macrophage-inflammatory protein 1ß) levels increased 2.4-fold during maraviroc intensification (P < .001). During maraviroc intensification, plasma lipopolysaccharide declined, whereas sCD14 levels and neutrophils tended to increase in blood and rectal tissue. Although the mechanisms explaining these findings remain unclear, CCR5 ligand-mediated activation of T cells, macrophages, and neutrophils via alternative chemokine receptors should be explored. These results may have relevance for trials of maraviroc for HIV preexposure prophylaxis and graft-versus-host disease. This trial was registered at www.clinicaltrials.gov as #NCT00735072.

Impact of highly active antiretroviral therapy initiation on CD4(+) T-cell repopulation in duodenal and rectal mucosa.

OBJECTIVE: The objective of this study was to assess the effects of HAART initiation on CD4(+) T-cell repopulation and T-cell immune activation in rectal and duodenal mucosa. DESIGN: The effects of HAART on the gastrointestinal tract remain controversial, and studies have reached different conclusions regarding its effectiveness at restoring mucosal CD4(+) T cells depending upon time of initiation, duration of treatment and gastrointestinal tract region studied. METHODS: We obtained blood, rectal biopsies and duodenal biopsies from 14 chronically infected individuals at baseline and at 4-9 months post-HAART initiation. We examined CD4(+) T-cell frequencies in blood, rectum and duodenum at both time points, and performed a detailed assessment of CD4(+) T-cell phenotype, immune activation marker expression and HIV-specific CD8(+) T-cell responses in blood and rectal mucosa. RESULTS: CD4(+) T-cell percentages increased significantly in blood, rectal and duodenal mucosa after 4-9 months of HAART (P = 0.02, 0.0005, 0.0002), but remained lower than in uninfected controls. HIV-specific CD8(+) T-cell responses in blood and rectal mucosa declined following HAART initiation (P = 0.0015, 0.021). CD8(+) T-cell coexpression of CD38 and HLA-DR in blood and mucosa, as well as plasma sCD14, declined significantly. CD28 expression on blood and mucosal CD8(+) T cells increased, whereas programmed death receptor-1 expression on blood HIV-specific CD4(+) and CD8(+) T cells decreased. CONCLUSION: Within the first months of HAART, limited CD4(+) T-cell reconstitution occurs in small and large intestinal mucosa. Nevertheless, decreased immune activation and increased CD28 expression suggest rapid immunological benefits of HAART despite incomplete CD4(+) T-cell reconstitution.

Role of miRNAs in neuronal differentiation from human embryonic stem cell-derived neural stem cells.

microRNAs (miRNAs) are important modulators in regulating gene expression at the post-transcriptional level and are therefore emerging as strong mediators in neural fate determination. Here, by use of the model of human embryonic stem cell (hESC)-derived neurogenesis, miRNAs involved in the differentiation from neural stem cells (hNSC) to neurons were profiled and identified. hNSC were differentiated into the neural lineage, out of which the neuronal subset was enriched through cell sorting based on select combinatorial biomarkers: CD15-/CD29(Low)/CD24(High). This relatively pure and viable subpopulation expressed the neuronal marker ß III-tubulin. The miRNA array demonstrated that a number of miRNAs were simultaneously induced or suppressed in neurons, as compared to hNSC. Real-time PCR further validated the decrease in levels of miR214, but increase in brain-specific miR7 and miR9 in the derived neurons. For functional studies, hNSC were stably transduced with lentiviral vectors carrying specific constructs to downregulate miR214 or to upregulate miR7. Manipulation of either miR214 or miR7 did not affect the expression of ß III-tubulin or neurofilament, however miR7 overexpression gave rise to enhanced synapsin expression in the derived neurons. This indicated that miR7 might play an important role in neurite outgrowth and synapse formation. In conclusion, our data demonstrate that miRNAs function as important modulators in neural lineage determination. These studies shed light on strategies to optimize in vitro differentiation efficiencies to mature neurons for use in drug discovery studies and potential future clinical applications.

Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms.

Cellular immune responses elicited by vaccination are complex and require polychromatic analysis to accurately characterize the phenotype and function of rare, responding cells. Technical challenges and a lack of instrument standardization between research sites have limited the application of polychromatic cytometry in multicenter clinical trials. Two previously developed six-color T cell subset immunophenotyping reagent panels deliberately designed to accommodate three additional low frequency functional measurements were compared for their reproducibility of staining across three different flow cytometers. We repeatedly measured similar T cell subset frequencies between the two reagent panels and across the three different cytometers. Spectral overlap reduced sensitivity in two of the three open measurement channels (PE [IL-2] and APC [IFN gamma]) for one reagent combination, particularly in subsets with low cytokine expression. There was no significant interassay variation for measurements across instrument platforms. Careful panel design will identify reagent combinations that minimize spectral spillover into channels reserved for cytokine measurement and comparable results can be achieved using different cytometers, however, it is important to establish standardized quality control procedures for each instrument to minimize variation between cytometers.

Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach.

Polychromatic flow cytometry offers the unprecedented ability to investigate multiple antigens per cell. Unfortunately, unwanted spectral overlaps and compensation problems increase when more than four colors are used, but these problems can be minimized if staining combinations are chosen carefully. We used an empiric approach to design, test and identify six-color T cell immunophenotyping reagent panels that can be expanded to include three or more functional or other markers in the FITC, PE, and APC channels without significant spectral limitations. Thirty different six-color T cell surface antigen reagent panels were constructed to identify major T cell subsets and maturational subtypes as defined by CCR7 and CD45RA expression, while excluding monocytes, B and non-viable cells. Staining performance of each panel was compared on cryopreserved cells from a single healthy donor recorded on a multiparameter cell sorter. Ten of the thirty reagent panels offered reliable resolution of T cell major and maturational surface markers. Of these, two panels were selected that showed the least spectral overlap and resulting background increase in the FITC, PE, and APC channels. These channels were left unoccupied for inclusion of additional phenotypic or functional markers, such as cytokines. Careful reagent titration and testing of multiple candidate panels are necessary to ensure quality results in multiparametric measurements.

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle, sheep, and deer.

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.