A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Cross-reactivity of antibodies to different rumen methanogens demonstrated using immunomagnetic capture technology.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

Making the most of high-dimensional cytometry data.

High-dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low- to high-dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high-dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high-dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high-dimensional experiments to maximize quality data collection.

CellTrace Violet™ inhibits equine lymphocyte proliferation.

CellTrace Violet™ is a commonly used fluorescent dye used with flow cytometry to identify cell proliferation. Activated equine lymphocytes were examined using flow cytometry, microscopy and tritiated thymidine proliferation assays. CellTrace Violet™ was incorporated into the equine lymphocytes effectively. Equine lymphocytes proliferated when activated with pokeweed mitogen, but did not proliferate when previously stained with CellTrace Violet™. Serial dilutions of CellTrace Violet™ did not eliminate the inhibition of activated lymphocytes. Equine lymphocyte viability was greater than 90 % for both stained and unstained cells. Based on these data, CellTrace Violet™ is not recommended for the assessment of lymphocyte proliferation in equine cells. The mechanism of inhibition of equine lymphocyte proliferation by CellTrace Violet™ is unknown.

Consumption of an Anthocyanin-Rich Extract Made From New Zealand Blackcurrants Prior to Exercise May Assist Recovery From Oxidative Stress and Maintains Circulating Neutrophil Function: A Pilot Study.

Aim: To evaluate blackcurrant anthocyanin-rich extract (BAE) consumption on time- and dose-dependent plasma anthocyanin bioavailability and conduct a pilot study to explore the potential effect of BAE in promoting recovery from exercise-induced oxidative stress, and maintenance of circulating neutrophil function. Methods: Time- and dose-dependent blackcurrant anthocyanin bioavailability was assessed using LC-MS in 12 participants over 6 h after the ingestion of a placebo or BAE containing 0.8, 1.6, or 3.2 mg/kg total anthocyanins. In a separate pilot intervention exercise trial, 32 participants consumed either a placebo or 0.8, 1.6, or 3.2 mg/kg BAE (8 individuals per group), and then 1 h later performed a 30 min row at 70% VO2max. Blood was collected during the trial for oxidative, antioxidant, inflammatory, and circulating neutrophil status. Results: Consumption of BAE caused a time- and dose-dependent increase in plasma anthocyanins, peaking at 2 h after ingestion of 3.2 mg/kg BAE (217 ± 69 nM). BAE consumed 1 h prior to a 30 min row had no effect on plasma antioxidant status but hastened the recovery from exercise-induced oxidative stress: By 2 h recovery, consumption of 1.6 mg/kg BAE prior to exercise caused a significant (P < 0.05) 34 and 32% decrease in post-exercise plasma oxidative capacity and protein carbonyl levels, respectively, compared to placebo. BAE consumption prior to exercise dose-dependently attenuated a small, yet significant (P < 0.01) transient 13 ± 2% decline in circulating neutrophils observed in the placebo group immediately post-exercise. Furthermore, the timed consumption of either 1.6 or 3.2 mg/kg BAE attenuated a 17 ± 2.4% (P < 0.05) decline in neutrophil phagocytic capability of opsonised FITC-Escherichia coli observed 6 h post-exercise in the placebo group. Similarly, a dose-dependent increase in neutrophil surface expression of complement receptor-3 complex (CR3, critical for effective phagocytosis of opsonised microbes), was observed 6 h post-exercise in both 1.6 and 3.2 mg/kg BAE intervention groups. Conclusions: Consumption of BAE (>1.6 mg/kg) 1 h prior to exercise facilitated recovery from exercise-induced oxidative stress and preserved circulating neutrophil function. This study provides data to underpin a larger study designed to evaluate the efficacy of timed BAE consumption on post-exercise recovery and innate immunity.

Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species.

Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21.

Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoid-like reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and even nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, which facilitates tumor progression.

Long-term deposition of inhaled antigen in lung resident CD11b-CD11c+ cells.

In this study we report the characterization of a population of lung resident CD11b(-)CD11c(+) cells that are able to take up inhaled antigen and retain it for extended periods of time. Ovalbumin conjugated to fluorescein-isothiocyanate (FITC-OVA) administered intranasally to mice was taken up by two main populations of cells in the lung, a migratory CD11c(+)CD11b(+) population consisting of dendritic cells (DC), which rapidly transported antigen to the draining lymph node (LN), and a resident CD11b(-)CD11c(+) population that retained engulfed antigen without apparently degrading it for up to 8 wk after administration. The FITC(+)CD11b(-)CD11c(+) cells did not migrate to draining LN at a detectable rate, and did not up-regulate expression of costimulatory molecules in response to LPS treatment. FITC(+)CD11b(-)CD11c(+) cells were found in the lung and bronchoalveolar lavage fluid, and their distribution was compatible with macrophages. Although FITC(+)CD11b(-)CD11c(+) cells expressed the DC marker DEC205 and other molecules associated with antigen-presenting cell function, they did not induce proliferation of antigen-specific CD4(+) T cells in vitro or acute cytokine production by activated CD4(+) T cells in vivo. Thus, FITC(+)CD11b(-)CD11c(+) cells appear to represent an intermediate cell type sharing properties with DC and macrophages. These cells may have a role in modulating the responses of lung resident T cells to inhaled antigens.

5,6-Dimethylxanthenone-4-acetic acid treatment of a non-immunogenic tumour does not synergize with active or passive CD8+ T-cell immunotherapy.

The chemotherapeutic drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) inhibits intratumoural blood flow, causing hypoxia, haemorrhagic necrosis, vascular collapse and tumour cell death. Production of TNF-alpha and IFN is also induced, causing local inflammation and activation of immune cells including CD8+ T cells. We used the tumour cell line LL-LCMV, which expresses the gp33 epitope of lymphocytic choriomeningitis virus in a non-immunogenic form, to investigate whether tumour cell death caused by treatment with DMXAA may improve the success of tumour immunotherapy mediated by CD8+ T cells. Treatment with DMXAA was effective at reducing the size of LL-LCMV tumours. However, compared to normal mice, tumour reduction was no more marked or sustained in mice carrying high numbers of naive, tumour-specific CD8+ T cells. The antitumour effect of activated CD8+ T cells was also not affected by DMXAA treatment. Tumour-specific CD8+ T cells activated in vivo by immunization with dendritic cells and specific tumour peptide antigen, or generated in vitro and adoptively transferred into tumour-bearing mice by i.v. injection, did not improve or sustain the reduction in tumour size induced by DMXAA treatment. We conclude that the presence of high numbers of naive CD8+ T cells, or immunotherapies leading to CD8+ T-cell activation, do not synergize with the tumour cell death and local inflammation induced by DMXAA treatment. It is possible that this lack of synergism may result from both treatments inducing activation of CD8+ T cells and that treatments that activate different populations of immune cells may achieve better success.

IL-4 deficiency does not impair the ability of dendritic cells to initiate CD4+ and CD8+ T cell responses in vivo.

Several reports have described a role of IL-4 in dendritic cell function. We have examined the number and phenotype of dendritic cells from C57Bl/6 wild-type and IL-4-/- mice, and compared their ability to induce T cell immune responses in vivo and in vitro. We observed that the number of dendritic cells in the spleens and lymph nodes of IL-4-/- mice is comparable to the number found in wild-type mice. In addition, the expression of maturation markers such as MHC II, CD40, CD80 and CD86, and of differentiation markers such as CD4, CD8 and CD11b, was also comparable in the two populations. Splenic wild-type and IL-4-/- dendritic cells were both able to present antigen to T cell receptor transgenic CD4+ or CD8+ T cells in culture. When pulsed with antigen in vitro and then injected subcutaneously into C57BL/6 host mice, both populations of dendritic cells were able to induce the division of T cell receptor transgenic CD4+ or CD8+ T cells in vivo. This was the case regardless of whether the antigen used in these experiments was a low or a high affinity T cell receptor ligand. Similarly, both populations of dendritic cells were able to activate antigen-specific cytotoxic T cell responses and initiate tumor-protective immune responses in vivo. We conclude that IL-4-/- and wild-type dendritic cells have a comparable ability to initiate T cell immune responses when in an IL-4-sufficient environment.