Abstract Background: Identifying patients at high risk for metachronous colorectal cancer metastasis is essential for improving prognosis. The balance between host immune regulation and tumor immune evasion ultimately permits or prevents cancer progression. Lymphocytic infiltration in colorectal cancer can predict long-term clinical outcomes. However, these lymphocytes' origin, subtype and function have yet to be adequately explored. We investigated the association between the development of metastasis and CD8+T cells expressing the integrin protein CD103, a marker of tissue-resident memory T cells. Methods: In this retrospective, case-control matched study, we conducted multiplex immunofluorescence staining of tumor samples from 124 patients with colorectal cancer. Tissue microarrays containing representative cores of the central tumor, invasive margin, and adjacent normal tissue were immunostained for CD3, CD8, CD103, pSMAD3, and cytokeratin, using tyramide signal amplification. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kruskal-Wallis one-way analysis of variance with subsequent Dunn’s test for pair-wise comparisons was performed using the Bonferroni method of multiple-comparison correction. Results: Central tumor and invasive margin CD3+ densities were associated with synchronous metastasis. Compared to non-metastatic patients, those who went on to develop metachronous metastasis (n=42) had a low proportion of intra-epithelial CD8+CD103+ T cells at the central tumor (non-metastatic: 32.8%, IQR 15.0-42.7%; metachronous metastasis: 12.9%, IQR 2.08-28.4%; P = 0.013). Similarly, patients with metachronous metastasis had a low proportion of stromal CD8+CD103+ T cells at the invasive margin (non-metastatic: 3.51%, IQR 1.83-6.55%; metachronous metastasis: 1.75%, IQR 0.62-3.72%; P = 0.019). On multivariate Cox regression of histological features, amongst patients with metastasis, significant overall survival prognostic markers included a low proportional CD8+CD103+ T cell infiltration (HR 2.41, 95%CI 1.16-5.01, P = 0.019), mucinous features (HR 4.28, 95%CI 1.98-9.24, P < 0.001), and the presence of perineural invasion (HR 2.75, 95%CI 1.28-5.89, P < 0.001). Conclusion: CD8+CD103+ lymphocytes may protect from the development of colorectal metastasis. Proportional CD8+CD103+ T cell infiltration is a promising prognostic marker in colorectal adenocarcinoma for the development of metachronous metastasis, and, amongst patients with metastasis, it is a prognostic marker of overall survival. Citation Format: Ryan Cohen, Tracey Lee-Pullen, Timothy Miller, Shadi Pirasteh, Cameron Platell, Katie Meehan, Kathy Fuller, Melanie McCoy. Tissue-resident cytotoxic T cell infiltration predicts metachronous colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5525.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

BACKGROUND:The prognostic value of tumor-associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co-expressing the immunoinhibitory molecule PD-L1 and their spatial relationship with CD8+ T-cells in patients treated for stage III colon cancer.METHODS:Tissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD-L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan-Meier estimates and Cox regression were used to assess survival.RESULTS:Intratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33-.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33-.83, P = .006), intratumoral CD11c+ PD-L1+ cell density (HR = .57, 95% CI .35-.92, P = .021), and stromal CD11c+ PD-L1+ cell density (HR = .48, 95% CI .30-.77, P = .003) on leading-edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+ PD-L1+ cell density in tumor epithelium and stromal compartments.CONCLUSION:Here we showed that PD-L1-expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically "hot" tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor-associated DC may help to further elucidate their prognostic value.

SOX2 (sex-determining region-Y homeobox-2) is a transcription factor essential for the maintenance of pluripotency and is also associated with stem-cell-like properties in preclinical cancer models. Our previous study on a cohort of stage III colon cancer patients demonstrated high SOX2+ cell densities were associated with poor prognosis. However, most patients were treated with adjuvant chemotherapy so the prognostic value of SOX2 could not be assessed independently from its value as a predictive marker for non-response to chemotherapy. This study aimed to assess whether SOX2 was a true prognostic marker or a marker for chemotherapy response in a historical cohort of patients, a high proportion of whom were chemotherapy-naïve. SOX2 immunostaining was performed on tissue micro-arrays containing tumor cores from 797 patients with stage II and III colorectal cancer. SOX2+ cell densities were then quantified with StrataQuest digital image analysis software. Overall survival was assessed using Kaplan–Meier estimates and Cox regression. It was found that high SOX2+ cell densities were not associated with poor overall survival. Furthermore, all patients had a significant improvement in survival after 5-fluorouracil (5-FU) treatment, irrespective of their SOX2+ cell density. Therefore, SOX2+ cell densities were not associated with prognosis or chemotherapy benefit in this study. This is in contrast to our previous study, in which most patients received oxaliplatin as part of their treatment, in addition to 5-FU. This suggests SOX2 may predict response to oxaliplatin treatment, but not 5-FU.

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

With the use of cancer immunotherapy drugs targeting the T cell inhibitory programmed death-1 (PD-1) signalling pathway on the rise, the ability to select patients most likely to benefit is becoming increasingly important. While there is currently no biomarker to definitively predict response, expression of programmed death ligand 1 (PD-L1) by the tumour and/or associated immune cells remains one of the best predictors of efficacy.1Sunshine J. Taube J.M. PD-1/PD-L1 inhibitors.Curr Opin Pharmacol. 2015; 23: 32-38Crossref PubMed Scopus (381) Google Scholar Consequently, anti-PD-1/PD-L1 drugs have been developed in parallel with companion or complementary diagnostic assays for evaluating PD-L1 expression. There are now multiple PD-L1 immunohistochemistry (IHC) assays available, which use different antibody clones, have different recommended staining protocols and different cut-off points for positivity. International collaborative evaluations (such as the American Association for Cancer Research-initiated Blueprint PD-L1 IHC Assay Comparison Project2International Association for the Study of Lung Cancer. Preliminary Results Comparing PD-L1 IHC Diagnostic Assays in Lung Cancer Released. 5 May 2016; cited 22 Sep 2016. https://www.iaslc.org/articles/preliminary-results-comparing-pd-l1-ihc-diagnostic-assays-lung-cancer-released.Google Scholar) are underway to compare the performance of these assays for diagnostic use. However, there is currently little information available to inform the selection of a suitable PD-L1 IHC assay for use in research studies. We aimed to develop a sensitive and reliable manual IHC protocol for detecting PD-L1 on human formalin fixed, paraffin embedded (FFPE) colorectal cancer (CRC) tissues in a research setting. We reviewed publicly available information on antibodies commonly used to detect PD-L1 on FFPE tumour tissues with IHC (Supplementary Table 1, Appendix A). We selected three monoclonal antibodies to compare, based on their commercial availability and quality of tumour cell membrane staining in published images: E1L3N (Cell Signaling Technology, USA), 28-8 (Abcam, UK) and 22C3 (Dako, Denmark). We evaluated the effect of using different polymer-based detection methods and antigen retrieval solutions on assay performance, and then compared the optimised manual staining protocols to a validated automated assay (SP263, Roche, Switzerland; performed on the Ventana Benchmark Ultra, Ventana, USA). Of note, 28-8 and 22C3 are the antibodies used in the US Food and Drug Administration (FDA)-approved complementary diagnostic test for nivolumab and companion diagnostic test for pembrolizumab, respectively. We describe the optimisation process and the assay conditions we have selected for our subsequent research studies. Detailed description of the reagents and methods used is given in the Supplementary Methods (Appendix A). Optimal dilutions of the E1L3N, 28-8 and 22C3 antibodies were determined using placental tissue and then used to compare three polymer-based detection methods: (1) MACH 3 HRP Polymer Detection (‘MACH 3’; Biocare Medical, USA), (2) Novolink Polymer Detection System (‘Novolink’, Leica Biosystems, Germany), and (3) Envision FLEX+ System (‘Envision+’, Dako, Denmark). The three antibody clones produced a comparable membranous staining pattern on CRC tissue, indicating specificity for membrane-bound PD-L1, as previously reported.3Sun W.Y. Lee Y.K. Koo J.S. Expression of PD-L1 in triple-negative breast cancer based on different immunohistochemical antibodies.J Transl Med. 2016; 14: 173Crossref PubMed Scopus (90) Google Scholar, 4Smith J. Robida M.D. Acosta K. et al.Quantitative and qualitative characterization of Two PD-L1 clones: SP263 and E1L3N.Diagn Pathol. 2016; 11: 44Crossref PubMed Scopus (63) Google Scholar However, staining intensity varied depending on the clone and detection system used (Fig. 1A). MACH 3 detection resulted in weak E1L3N staining and failed to detect 22C3 and 28-8 staining. The Envision+ detection method generated the strongest staining for E1L3N and 28-8, whereas 22C3 staining, albeit weaker than E1L3N and 28-8 staining, was better with Novolink detection. The SignalStain Boost Rabbit-HRP detection reagent (Cell Signaling Technology) was also tested for the rabbit monoclonal antibodies, E1L3N and 28-8. Again, staining with E1L3N was stronger than with 28-8 (data not shown). Using the Envision+ detection method, we then compared antigen retrieval using a CINtec Histology kit Tris-EDTA pH 9.0 solution (‘CINtec solution’, Roche, Australia), to two lower pH retrieval solutions: sodium citrate pH 6.0 (‘Citrate solution’) and Dako Target Retrieval pH 6.1 (‘Dako solution’). The Dako solution is a modified citrate buffer recommended for use with 28-8 and 22C3 by their respective manufacturers. Treatment with the Citrate solution resulted in the weakest PD-L1 staining for all antibody clones (Fig. 1B). Weaker staining was shown for 22C3 than for E1L3N and 28-8 with all three retrieval solutions. The Dako solution provided the strongest staining intensity for 22C3, and therefore was selected for use with this clone. This combination of Dako solution and Envision+ detection also produced stronger 22C3 staining than the Dako solution with the Novolink system (Fig. 1A), or the CINtec solution with the Novolink system (data not shown). The CINtec solution was selected for use with E1L3N and 28-8 due to a marginal increase in staining intensity compared to the Dako solution. These data suggest that the choice of detection system and antigen retrieval solution both significantly affected staining intensity. We then directly compared our protocol for each antibody (and the manufacturer-recommended assay conditions), with PD-L1 staining using SP263 on the Ventana automated platform (Table 1). All four antibodies produced a similar membranous staining pattern, however staining intensity differed (Fig. 2A; Supplementary Fig. 1, Appendix A). E1L3N produced marginally stronger and more complete cell membrane staining (with slightly lower background) than 28-8, which in turn stained stronger than 22C3 under these manual assay conditions. This difference in staining intensity between antibodies was also observed in other tissue types, including tonsil, lung and small intestine (Supplementary Fig. 2, Appendix A). In addition to tumour PD-L1 expression, expression by TAIC was also detected using each of the antibodies tested (Fig. 2B).Table 1IHC assay conditionsConditionsPD-LI antibodySP263E1L3N28-822C3Manufacturer recommended Dilution/application timeReady-to-use/64 min1:200/60 min1:500/60 min1:50/30 min Antigen retrieval systemCC1 pH 8.0Tris-EDTA pH 9.0Dako citrate pH 6.1Dako Citrate pH 6.1 DetectionOptiViewSignalStainNovolinkEnvision+ with DAB enhancerIn-house-optimised Dilution/application timeN/D1:100/60 mins1:250/60 mins1:50/60 mins Antigen retrieval systemN/DCINtec Tris-EDTA pH 9.0CINtec Tris-EDTA pH 9.0Dako Citrate pH 6.1 DetectionN/DEnvision+Envision+Envision+N/D, not done; CC1, cell conditioning 1. Open table in a new tab N/D, not done; CC1, cell conditioning 1. Finally, we used IHC reference standards consisting of cell lines with a defined negative, low, intermediate, or strong PD-L1 protein expression to confirm detection of a dynamic range of PD-L1 expression. All three antibody clones produced comparable staining of reference cell lines (Supplementary Fig. 3, Appendix A). Whilst the reference standards provide a useful tool for testing assay performance, staining on archival clinical material may be affected by factors beyond the control of the researcher, such as sample preparation and processing methods or storage conditions. Therefore, optimisation of assays for specific applications is important. In summary, the choice of IHC protocol significantly affects PD-L1 staining intensity, highlighting the importance of carefully optimising assay components for specific applications. In our hands and for our specific application, E1L3N generally produced the strongest staining and was least affected by changes in assay conditions. Since E1L3N reacts with a different part of the PD-L1 protein (the intracellular region) to 28-8 and 22C3 (the extracellular domains),5Mahoney K.M. Sun H. Liao X. et al.PD-L1 antibodies to its cytoplasmic domain most clearly delineate cell membranes in immunohistochemical staining of tumor cells.Cancer Immunol Res. 2015; 3: 1308-1315Crossref PubMed Scopus (105) Google Scholar, 6Phillips T. Simmons P. Inzunza H.D. et al.Development of an automated PD-L1 immunohistochemistry (IHC) assay for non-small cell lung cancer.Appl Immunohistochem Mol Morphol. 2015; 23: 541-549Crossref PubMed Scopus (150) Google Scholar it is perhaps not surprising that the clones differ in their sensitivity to changes in antigen retrieval and signal detection methods. The protocol we have selected for use in our subsequent research studies involving FFPE CRC tissue is the E1L3N antibody with pH 9.0 EDTA-based epitope retrieval solution (CINtec solution) and a linker-amplified detection system (Envision+). We found this combination to give consistent strong staining with minimal background signal. However, given the heterogeneous expression of PD-L1 in different tumour types,7Scognamiglio G. De Chiara A. Di Bonito M. et al.Variability in immunohistochemical detection of programmed death ligand 1 (PD-L1) in cancer tissue types.Int J Mol Sci. 2016; 17: E790Crossref PubMed Scopus (33) Google Scholar optimal assay conditions will likely depend on variables such as tissue type, storage conditions and processing protocols. While we have focussed on optimising an assay for use in research studies, our findings are highly clinically relevant. Since tumour PD-L1 expression is usually taken into account when deciding on suitability for PD-1/PD-L1-targeted therapy, variations in PD-L1 staining caused by technical rather than biological variation could have important consequences for patients. Funding was provided by the Tonkinson Foundation for Colorectal Cancer Research. The authors state that there are no conflicts of interest to disclose.

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response.

Ureaplasma spp. are a common vaginal microorganism causally linked to inflammation-driven preterm birth (PTB). The nature of the immune response to Ureaplasma spp. may influence PTB risk. This study sought to define maternal T cell cytokine responses to in vitro stimulation with Ureaplasma parvum serovar 3 (UpSV3) in vaginally colonised (UP+) and non-colonised (UP) pregnant women. Whole blood flow cytometry demonstrated an increase (p=0.027) in the baseline frequency of IFN gamma-positive CD3(+)CD4(-)(CD8(+)) T cells in UP+ women. UpSV3 stimulation resulted in a significant and specific increase (p =0.001) in the frequency of IFN gamma-positive CD3(+)CD4(-)(CD8(+)) T cells, regardless of vaginal colonisation status. UpSV3 stimulation also increased the frequency of IFN gamma-positive CD3(+)CD4(+) T cells, particularly in the UP+ group (p = 0.003). This is the first published study to examine T cell responses to Ureaplasma spp. exposure. Future appropriately-powered studies are needed to assess whether insufficient priming or a loss of tolerance to Ureaplasma spp. is occurring in UP+ women at risk of PTB. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

We hypothesised that circulating monocytes of women with vaginal colonisation with Ureaplasma spp., genital microorganisms known to cause inflammation-driven preterm birth, would elicit a tolerised cytokine response to subsequent in vitro Ureaplasma parvum serovar 3 (UpSV3) stimulation. Using multi-parameter flow cytometry, we found no differences with regard to maternal colonisation status in the frequency of TNF-α-, IL-6-, IL-8- and IL-1β-expressing monocytes in response to subsequent UpSV3 stimulation (P > 0.10 for all cytokines). We conclude that vaginal Ureaplasma spp. colonisation does not specifically tolerise monocytes of pregnant women towards decreased responses to subsequent stimulation.

Aims First, to investigate the prognostic significance of T cell subsets and RANK/RANKL signalling in the primary tumour of locally advanced breast cancer (LABC) and metastatic breast cancer patients. Second, to investigate the predictive significance of a relationship between clinical response to the anti-RANKL monoclonal antibody denosumab and immunopathological features of the tumour cells. Methods Multiplex immunohistochemistry (IHC; Biocare) is used to assess the marker pairs CD4/CD8, RANK/PR and RANKL/FOXP3 in 84 patients in full-face serial sections and 60 patients in tissue microarrays with LABC or metastatic breast cancer. Aperio ImageScope software and ImageJ digital cell-counting algorithms are used to quantify total marker-positive cells as a percentage of total cells. Results Statistical analysis of data collected so far shows an independent positive trend between the presence of FOXP+ T cells or CD4+ cells, but not CD8+ T cells, and shorter disease free survival (DFS) and overall survival (OS) (p = 0.089). Discussion Previous research on T cell infiltration and breast cancer outcomes has produced conflicting data. Our data demonstrate evidence on a large, targeted cohort on the relevance of the presence of different T cell subsets and outcome. This may represent a novel diagnostic tool. First, to investigate the prognostic significance of T cell subsets and RANK/RANKL signalling in the primary tumour of locally advanced breast cancer (LABC) and metastatic breast cancer patients. Second, to investigate the predictive significance of a relationship between clinical response to the anti-RANKL monoclonal antibody denosumab and immunopathological features of the tumour cells. Multiplex immunohistochemistry (IHC; Biocare) is used to assess the marker pairs CD4/CD8, RANK/PR and RANKL/FOXP3 in 84 patients in full-face serial sections and 60 patients in tissue microarrays with LABC or metastatic breast cancer. Aperio ImageScope software and ImageJ digital cell-counting algorithms are used to quantify total marker-positive cells as a percentage of total cells. Statistical analysis of data collected so far shows an independent positive trend between the presence of FOXP+ T cells or CD4+ cells, but not CD8+ T cells, and shorter disease free survival (DFS) and overall survival (OS) (p = 0.089). Previous research on T cell infiltration and breast cancer outcomes has produced conflicting data. Our data demonstrate evidence on a large, targeted cohort on the relevance of the presence of different T cell subsets and outcome. This may represent a novel diagnostic tool.

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze–thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators. © 2014 International Society for Advancement of Cytometry

Background Maternal fish oil supplementation during pregnancy has been associated with altered infant immune responses and a reduced risk of infant sensitization and eczema.Objective To examine the effect of early postnatal fish oil supplementation on infant cellular immune function at 6 months of age in the context of allergic disease.Methods In a double-blind randomized controlled trial (ACTRN12606000281594), 420 infants of high atopic risk received fish oil [containing 280 mg docosahexaenoic acid (DHA) and 110 mg eicosapentanoic acid (EPA)] or control oil daily from birth to 6 months. One hundred and twenty infants had blood collected at 6 months of age. Fatty acid levels, induced cytokine responses, T cell subsets and monocyte HLA-DR expression were assessed at 6 months of age. Infant allergies were assessed at 6 and 12 months of age.Results DHA and EPA levels were significantly higher in the fish oil group and erythrocyte arachidonic acid (AA) levels were lower (all P < 0.05). Infants in the fish oil group had significantly lower IL-13 responses (P = 0.036) to house dust mite (HDM) and higher IFN gamma (P = 0.035) and TNF (P = 0.017) responses to phytohaemaglutinin (PHA). Infants with relatively high DHA levels had lower Th2 responses to allergens including lower IL-13 to beta-lactoglobulin (BLG) (P = 0.020), and lower IL-5 to BLG (P = 0.045).Conclusions and clinical relevance Postnatal fish oil supplementation increased infant n-3 polyunsaturated fatty acid (PUFA) levels and associated with lowered allergen-specific Th2 responses and elevated polyclonal Th1 responses. Our results add to existing evidence of n-3 PUFA having immunomodulatory properties that are potentially allergy-protective.

BACKGROUND:Previous studies have demonstrated that reduced T-cell protein kinase C zeta (PKCζ) expression is associated with allergy development in infants born to atopic mothers. This study examined whether this relationship extends to a general population and addressed the basis for the association.METHODS:A flow cytometry assay was developed for the measurement of T-cell PKCζ levels in PBMC, cord blood mononuclear cell and whole blood. Cord blood T-cell PKCζ levels were measured in 135 neonates, and allergic disease was evaluated by skin prick test and clinical examination at 12 months of age.RESULTS:Allergic children (particularly those with eczema) had significantly lower neonatal T-cell PKCζ expression than nonallergic children (P < 0.001). PKCζ levels predicted allergic disease with optimal specificity of 86% and sensitivity of 54%. The sensitivity was increased in the children of allergic mothers, who had significantly lower PKC levels than the children of nonallergic mothers. Cord blood PKCζ levels did not affect T-cell maturation in culture as assessed by CD45RA/RO expression, but low PKCζ expression was associated with reduced capacity for IFNγ production by matured T cells. Low cord blood PKC expression was further associated with increased IL-13 responses at 6 months.CONCLUSIONS:The findings suggest a potential role for the use of PKCζ levels in cord blood T cells as a presymptomatic test to predict allergy risk in children, particularly offspring of allergic mothers, and that the basis of this relationship is related to cytokine patterns in mature T cells.

In mice, CD49f(hi) mammary stem cells (MaSCs) asymmetrically divide to generate CD49f(+) committed progenitor cells that differentiate into CD49f(-) phenotypes of the milk-secreting tissue at the onset of pregnancy. We show CD49f(+) primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy-associated hormones (PAH) compared with CD49f(+) cells from nonlactating tissue. Differentiation of CD49f(+) PMEC in extracellular matrix produces CD49f(-) luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f(-) luminal cells, while receptor activator of nuclear factor (NF)-κB ligand (RANKL) specifically stimulates division of basal CD49f(+) cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self-renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH.

Maternal fish oil supplementation during pregnancy has been associated with altered infant immune responses and a reduced risk of infant sensitization and eczema. To examine the effect of early postnatal fish oil supplementation on infant cellular immune function at 6 months of age in the context of allergic disease. In a double-blind randomized controlled trial (ACTRN12606000281594), 420 infants of high atopic risk received fish oil [containing 280 mg docosahexaenoic acid (DHA) and 110 mg eicosapentanoic acid (EPA)] or control oil daily from birth to 6 months. One hundred and twenty infants had blood collected at 6 months of age. Fatty acid levels, induced cytokine responses, T cell subsets and monocyte HLA-DR expression were assessed at 6 months of age. Infant allergies were assessed at 6 and 12 months of age. DHA and EPA levels were significantly higher in the fish oil group and erythrocyte arachidonic acid (AA) levels were lower (all P  < 0.05). Infants in the fish oil group had significantly lower IL-13 responses ( P  = 0.036) to house dust mite (HDM) and higher IFNγ ( P  = 0.035) and TNF ( P  = 0.017) responses to phytohaemaglutinin (PHA). Infants with relatively high DHA levels had lower Th2 responses to allergens including lower IL-13 to β-lactoglobulin (BLG) ( P  = 0.020), and lower IL-5 to BLG ( P  = 0.045). Postnatal fish oil supplementation increased infant n-3 polyunsaturated fatty acid (PUFA) levels and associated with lowered allergen-specific Th2 responses and elevated polyclonal Th1 responses. Our results add to existing evidence of n-3 PUFA having immunomodulatory properties that are potentially allergy-protective.