Bridget McLaughlin

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 1β) 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.

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.

Characterization of equine E‐selectin

Expression of E‐selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during inflammation, yet E‐selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E‐selectin genes derived from activated cultures of purified equine (horse), cervid (black‐tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E‐selectin are similar, whereas human and equine E‐selectin are more closely related to each other than to the ruminant molecules. Human E‐ and P‐selectin‐specific monoclonal antibodies that also recognize equine E‐selectin were identified and used to characterize its expression. Expression of E‐selectin was more readily induced by lipopolysaccharide treatment in equine ECs than in human ECs and supported adhesion and activation of neutrophils, consistent with the extreme sensitivity of horses to endotoxaemia and septic shock.

Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell—Derived Neural Stem Cells

AbstractmicroRNAs (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-/CD29Low/CD24High. This relatively pure and viable subpopulation expressed the neuronal marker β 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 β 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.

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.

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 [IFNγ]) 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.

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.

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-1β 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-1β 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.

Nanoparticle Targeted Therapy Against Childhood Acute Lymphoblastic Leukemia

The goal of our project is to develop a unique ligand-conjugated nanoparticle (NP) therapy against childhood acute lymphoblastic leukemia (ALL). LLP2A, discovered by Dr. Kit Lam, is a high-affinity and high-specificity peptidomimetic ligand against an activated α4β1 integrin. Our study using 11 fresh primary ALL samples (10 precursor B ALL and 1 T ALL) showed that childhood ALL cells expressed activated α4β1 integrin and bound to LLP2A. Normal hematopoietic cells such as activated lymphocytes and monocytes expressed activated α4β1 integrin; however, normal hematopoietic stem cells showed low expression of α4β1 integrin. Therefore, we believe that LLP2A can be used as a targeted therapy for childhood ALL. The Lam lab has developed novel telodendrimer-based nanoparticles (NPs) which can carry drugs efficiently. We have also developed a human leukemia mouse model using immunodeficient NOD/SCID/IL2Rγ null mice engrafted with primary childhood ALL cells from our patients. LLP2A-conjugated NPs will be evaluated both in vitro and in vivo using primary leukemia cells and this mouse model. NPs will be loaded first with DiD near infra-red dye, and then with the chemotherapeutic agents daunorubicin or vincristine. Both drugs are mainstays of current chemotherapy for childhood ALL. Targeting properties of LLP2A-conjugated NPs will be evaluated by fluorescent microscopy, flow cytometry, MTS assay, and mouse survival after treatment. We expect that LLP2A-conjugated NPs will be preferentially delivered and endocytosed to leukemia cells as an effective targeted therapy.