FVB/N mouse strain regulatory T cells differ in phenotype and function from the C57BL/6 and BALB/C strains.

Regulatory T cells (Treg) are vital to the maintenance of immune homeostasis. The genetic background of an inbred mouse strain can have a profound effect on the immune response in the animal, including Treg responses. Most Treg studies focus on animals created on the C57BL/6 or BALB/c background. Recent studies have demonstrated a difference in the phenotype and behavior of C57BL/6 and BALB/c Tregs. In this study, we have investigated the function of FVB/N Tregs compared to C57BL/6 and BALB/c. We observed that while FVB/N Tregs appear to suppress normally in a cell contact-dependent system, FVB/N Tregs are less capable of suppressing when regulation depends on the secretion of a soluble factor. FVB/N Tregs produce IL-10; however, TGF-ß was not detected in any culture from C57BL/6 or FVB/N. C57BL/6 Foxp3+ Tregs expressed more of the TGF-ß-related proteins glycoprotein-A repetitions predominant (GARP) and latency-associated peptide (LAP) on the cell surface than both FVB/N and BALB/c, but C57BL/6 Tregs expressed significantly less Ctse (Cathepsin E) mRNA. Each strain displayed different abilities of thymic Tregs (tTreg) to maintain Foxp3 expression and had a varying generation of induced Tregs (iTregs). In vitro generated FVB/N iTregs expressed significantly less GARP and LAP. These results suggest Tregs of different strains have varying phenotypes and dominant mechanisms of action for the suppression of an immune response. This information should be taken into consideration when Tregs are examined in future studies, particularly for therapeutic purposes in a genetically diverse population.

Altered T-Cell Balance in Lymphoid Organs of a Mouse Model of Colorectal Cancer.

The adenomatous polyposis coli (APC) gene is a known tumor suppressor gene, and mice with mutations in Apc (ApcMin/+) spontaneously form multiple intestinal neoplasms. In this model of human colorectal cancer (CRC), it has been reported that CD4+ T-cell-derived interleukin 17 (IL-17) promotes intestinal tumor development, but it is not known if the Apc mutation actually directly alters T-cell function and subsequently tumor immunosurveillance. To investigate the ApcMin/+ mutation on T-cell function, flow cytometric, histochemical, and immunofluorescent studies on both wild-type (Apc+/+) and ApcMin/+ mice were performed. We identified decreased levels of interferon gamma (IFN-γ+)IL-17+ double-positive CD4+ cells in the mesenteric lymph nodes and Peyer's patches of ApcMin/+ mice. In addition, altered levels of CD8+ cells, and changes in CD8+ production of IFN-γ and granzyme B were observed. These T-cell alterations did modify tumor immunosurveillance, as the adoptive transfer of splenocytes from ApcMin/+ animals into a chemically induced CRC model resulted in the inability to prevent epithelial dysplasia. These results suggest an altered T-cell balance in ApcMin/+ mice may disrupt intestinal homeostasis, consequently limiting intestinal tumor immunosurveillance.

Environmental-mediated intestinal homeostasis in neonatal mice.

BACKGROUND: Immunoglobulin A (IgA) plays a key role in coating luminal antigens and preventing translocation of harmful bacteria. The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor that when stimulated activates factors important for barrier function and intestinal homeostasis. We hypothesize that AhR signaling is critical for establishment of intestinal homeostasis in neonates. MATERIAL AND METHODS: Mice: C57BL/6 (B6) AhR+/+ wild type (WT), B6.AhR-/- Aryl-hydrocarbon receptor knockout (KO), and B6.AhR+/+ raised on an AhR ligand-free diet (AhR LF). Enzyme-linked immunosorbent assay was used to measure fecal and serum IgA levels. Bacterial translocation was measured by culturing the mesenteric lymph nodes. RESULTS: Two week old KO mice had significantly less fecal IgA compared with WT (and AhR LF, P value = 0.0393. The amount of IgA from the gastric contents of 2-wk-old mice was not significantly different. At age 8 wk, AhR LF mice had significantly less fecal IgA than WT and KO P value = 0.0077. At 2 wk, KO mice had significantly higher levels of bacterial translocation and at 8 wk AhR LF had significantly higher levels of bacterial translocation compared with WT. CONCLUSIONS: In neonatal mice, the lack of AhR signaling is associated with loss of intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation. In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity.

Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.

Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may provide a rationale for designing therapeutics targeting LSC-enriched cell populations.

Pathogenesis of necrotizing enterocolitis: modeling the innate immune response.

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant's intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice.

Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses. Cisplatin caused cell cycle arrest in a dose- and time-dependent manner that paralleled effects in vitro, and it readily penetrated into hypoxic tumor regions. Similar levels of Pt/cell were found in xenografts treated with oxaliplatin. Mass cytometry offers the unique capability to study the cellular uptake and anticancer effects of platinum-containing drugs at the single cell level in animal models, and it has the potential for application to samples obtained from cancer patients during treatment.

Consumption of acidic water alters the gut microbiome and decreases the risk of diabetes in NOD mice.

Infant formula and breastfeeding are environmental factors that influence the incidence of Type 1 Diabetes (T1D) as well as the acidity of newborn diets. To determine if altering the intestinal microbiome is one mechanism through which an acidic liquid plays a role in T1D, we placed non-obese diabetic (NOD)/ShiLtJt mice on neutral (N) or acidified H2O and monitored the impact on microbial composition and diabetes incidence. NOD-N mice showed an increased development of diabetes, while exhibiting a decrease in Firmicutes and an increase in Bacteroidetes, Actinobacteria, and Proteobacteria from as early as 2 weeks of age. NOD-N mice had a decrease in the levels of Foxp3 expression in CD4(+)Foxp3(+) cells, as well as decreased CD4(+)IL17(+) cells, and a lower ratio of IL17/IFNγ CD4+ T-cells. Our data clearly indicates that a change in the acidity of liquids consumed dramatically alters the intestinal microbiome, the presence of protective Th17 and Treg cells, and the incidence of diabetes. This data suggests that early dietary manipulation of intestinal microbiota may be a novel mechanism to delay T1D onset in genetically pre-disposed individuals.

An introduction to mass cytometry: fundamentals and applications.

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Maintenance of host leukocytes in peripheral immune compartments following lethal irradiation and bone marrow reconstitution: implications for graft versus host disease.

Bone marrow reconstitution is utilized as a tool for disease treatment and as a research technique to elucidate the function of bone marrow derived cells. Clinically successful engraftment is indicated by the development of a functioning immune repertoire. In research, reconstitution is considered successful if >85% of splenic leukocytes are of donor origins. Previous work suggests that splenic reconstitution may not be indicative of reconstitution in the mucosa. We sought to evaluate mucosal reconstitution in animals following a standard bone marrow eradication and reconstitution technique. Bone marrow was harvested from adult B6.SJL donor mice (CD45.1) and injected via either the retro-orbital or intraperitoneal route into lethally irradiated B6 (CD45.2) adult or neonatal recipients respectively. The expression of CD45 by flow cytometry was used to calculate reconstitution with respect to immune compartment and cell type. In reconstituted adult animals 93.2±1.5% of splenic leukocytes expressed the donor CD45.1 antigen thus meeting the standard definition of reconstitution, however only 58.6±13.6% of intestinal lamina propria lymphocytes and 52.4±16.0% of intestinal intraepithelial lymphocytes were of donor origin, confirming splenic reconstitution fails to represent peripheral immune reconstitution. T-cells in the gastrointestinal tract are the most poorly reconstituted, while B-cells appear to be almost universally replaced by donor cells. The inadequate mucosal reconstitution was not corrected by evaluating later time points or by performing the bone marrow transfer during the neonatal period. This demonstration that substantial host T-cells remain in the intestinal mucosa after a "successful" bone marrow transplantation should cause a re-evaluation of data from research bone marrow chimera experiments, as well as the mechanisms for complications after clinical bone marrow transplantation.

Murine P-glycoprotein deficiency alters intestinal injury repair and blunts lipopolysaccharide-induced radioprotection.

P-glycoprotein (P-gp) has been reported to increase stem cell proliferation and regulate apoptosis. Absence of P-gp results in decreased repair of intestinal epithelial cells after chemical injury. To further explore the mechanisms involved in the effects of P-gp on intestinal injury and repair, we used the well-characterized radiation injury model. In this model, injury repair is mediated by production of prostaglandins (PGE(2)) and lipopolysaccharide (LPS) has been shown to confer radioprotection. B6.mdr1a(-/-) mice and wild-type controls were subjected to 12 Gy total body X-ray irradiation and surviving crypts in the proximal jejunum and distal colon were evaluated 3.5 days after irradiation. B6.mdr1a(-/-) mice exhibited normal baseline stem cell proliferation and COX dependent crypt regeneration after irradiation. However, radiation induced apoptosis was increased and LPS-induced radioprotection was blunted in the C57BL6.mdr1a(-/-) distal colon, compared to B6 wild-type controls. The LPS treatment induced gene expression of the radioprotective cytokine IL-1α, in B6 wild-type controls but not in B6.mdr1a(-/-) animals. Lipopolysaccharid-induced radioprotection was absent in IL-1R1(-/-) animals, indicating a role for IL-1α in radioprotection, and demonstrating that P-gp deficiency interferes with IL-1α gene expression in response to systemic exposure to LPS.

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry.

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.

Helicobacter felis--associated gastric disease in microbiota-restricted mice.

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis-specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.

Critical role for P-glycoprotein expression in hematopoietic cells in the FVB.Mdr1a(-/-) model of colitis.

OBJECTIVE: P-glycoprotein (P-gp), the functional product of the multidrug resistance gene (MDR), is a transmembrane protein that extrudes substrates from the intracellular environment. P-gp is expressed on the apical surface of epithelial cells and on cells from the hematopoietic lineage. Human MDR polymorphisms have been associated with the increased risk of inflammatory bowel disease, and FVB/N animals deficient in mdr1a expression develop spontaneous colitis. Previous studies using adult bone marrow chimeras indicated that colitis development in this animal model was contingent on P-gp deficiency in radiation-resistant epithelial cells; however, the use of adult animals may mask the role of hematopoietic immune cells in colitis initiation, due to preexisting epithelial abnormalities. SUBJECTS AND METHODS: To assess the importance of P-gp expression in intestinal epithelial and hematopoietic-derived cells on colitis induction in FVB.mdr1a(-/-) animals, we developed a neonatal model of bone marrow reconstitution. FVB/N and FVB.mdr1a(-/-) adult and neonatal animals were lethally irradiated and reconstituted with bone marrow from FVB/N or FVB.mdr1a(-/-) donors. Animals were observed for 20 weeks. RESULTS: Adult FVB/N animals deficient in P-gp expression in hematopoietically derived immune cells developed colitis similar to adult animals deficient in P-gp expression in radiation-resistant epithelial/stromal cells. Neonatal animals deficient in P-gp expression in hematopoietically derived immune cells developed a more histologically significant colitis than those deficient in P-gp expression in epithelial tissue. CONCLUSIONS: The use of a neonatal model of bone marrow reconstitution has revealed a critical role for P-gp expression in hematopoietically derived immune cells in colitis development in the FVB.mdr1a(-/-) model.

Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

A self-propagating matrix metalloprotease-9 (MMP-9) dependent cycle of chronic neutrophilic inflammation.

BACKGROUND: Chronic neutrophilic inflammation is a poorly understood feature in a variety of diseases with notable worldwide morbidity and mortality. We have recently characterized N-acetyl Pro-Gly-Pro (Ac-PGP) as an important neutrophil (PMN) chemoattractant in chronic inflammation generated from the breakdown of collagen by the actions of MMP-9. MMP-9 is present in the granules of PMNs and is differentially released during inflammation but whether Ac-PGP contributes to this ongoing proteolytic activity in chronic neutrophilic inflammation is currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing isolated primary blood PMNs from human donors, we found that Ac-PGP induces significant release of MMP-9 and concurrently activates the ERK1/2 MAPK pathway. This MMP-9 release is attenuated by an inhibitor of ERK1/2 MAPK and upstream blockade of CXCR1 and CXCR2 receptors with repertaxin leads to decreased MMP-9 release and ERK 1/2 MAPK activation. Supernatants obtained from PMNs stimulated by Ac-PGP generate more Ac-PGP when incubated with intact collagen ex vivo; this effect is inhibited by an ERK1/2 pathway inhibitor. Finally, clinical samples from individuals with CF demonstrate a notable correlation between Ac-PGP (as measured by liquid chromatography-tandem mass spectrometry) and MMP-9 levels even when accounting for total PMN burden. CONCLUSIONS/SIGNIFICANCE: These data indicate that ECM-derived Ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK, highlighting for the first time a matrix-derived chemokine (matrikine) augmenting its generation through a discrete receptor/intracellular signaling pathway. These findings have notable implications to the development unrelenting chronic PMN inflammation in human disease.

Update on distance and velocity requirements for community ambulation.

PURPOSE: The purposes of this study were to provide an update to the ambulatory distance requirements for community ambulation and to update gait speed performance and requirements at intersections. METHODS: Distances were measured at 9 types of sites using a rolling measuring device in accordance with the protocol set forth by Lerner-Frankiel and associates. The 9 types of sites were supermarkets, drug stores, banks, department stores, post offices, medical offices, superstores, club warehouses, and hardware stores. Gait speed allotted by crosswalk signals as well as the gait speeds of individuals through crosswalks were recorded. Qualitative observations of the pedestrians' age (older - 65 years; younger < 65 years) and sex were also noted. RESULTS: Distances were measured at 141 different establishments. The shortest mean distance requirement was found in the medical offices at 65.82 (32.28) m. Club warehouses had the longest mean distance requirement at 676.82 (159.36) m. The mean gait speed used by the pedestrians (N = 139) was 1.32 (0.31) m/s while the mean speed necessary as set by the crosswalk signals was 0.49 (0.20) m/s. All of the individuals observed were able to cross the street within the allotted time and with adequate speed. The gait speeds met the normative data established for age and sex as well as data reported for slower older adults and some with incomplete spinal cord injury. CONCLUSIONS: Distance requirements for full community ambulation may need to be increased to 600 m or more. Gait speed requirements at crosswalks in the communities measured are set to accommodate the gait speed capabilities of older pedestrians who attempt crossing at controlled intersections.

Highly multiparametric analysis by mass cytometry.

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.

Role of postnatal acquisition of the intestinal microbiome in the early development of immune function.

OBJECTIVES: Therapy with broad-spectrum antibiotics is a common practice for premature infants. This treatment can reduce the biodiversity of the fecal microbiota and may be a factor in the cause of necrotizing enterocolitis. In contrast, probiotic treatment of premature infants reduces the incidence of necrotizing enterocolitis. We hypothesized that 1 mechanism for these observations is the influence of bacteria on postnatal development of the mucosal immune system. MATERIALS AND METHODS: Expression of immune molecules and microbial sensors was investigated in the postnatal mouse gastrointestinal tract by real-time polymerase chain reaction. Subsequently, 2-week-old specific pathogen-free and microbial-reduced (MR; antibiotic treated) mice were compared for immune molecule and microbial sensor expression, mesenteric lymph node T-cell numbers and activation, intestinal barrier function/permeability, systemic lymphocyte numbers, and T-cell phenotype commitment. RESULTS: Toll-like receptor 2, 4, and 5 expression was highest in 2-week-old specific pathogen-free mice, and this expression was decreased in MR mice. There was no difference in intestinal tight-junctional function, as evaluated by fluorescein isothiocyanate-dextran uptake, but MR mice had increased bacterial translocation across the intestinal epithelial barrier. MR mice had significantly fewer splenic B cells and mesenteric lymph node CD4+ T cells, but there were normal numbers of splenic T cells. These systemic T cells from MR mice produced more interleukin-4 and less interferon-gamma and IL-17, indicative of maintenance of the fetal, T-helper cell type 2 phenotype. CONCLUSIONS: The present study shows that intestinal commensal microbiota have an influence on early postnatal immune development. Determining specific bacteria and/or bacterial ligands critical for this development could provide insight into the mechanisms by which broad-spectrum antibiotics and/or probiotic therapy influence the development of the mucosal immune system and mucosal-related diseases.

Metal-Containing Polystyrene Beads as Standards for Mass Cytometry.

We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.