Carly Hamilton

Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies

Gastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host–pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host–pathogen interactions may help to identify novel prevention and control strategies.

Interactions between natural killer cells and dendritic cells favour T helper1-type responses to BCG in calves

Vaccination of neonatal calves with BCG induces a significant level of protection from infection with Mycobacterium bovis, the causative agent of bovine tuberculosis. Since neonatal vaccination of humans with BCG induces activation of NK cells, and young calves have high circulating numbers of these cells, we hypothesised that NK cells are important in the protective response to BCG. Furthermore, since NK cells play a role in shaping adaptive immune responses through interactions with DCs, we investigated the interactions between NK cells and DCs in the context of BCG. DCs infected with BCG expressed significantly higher levels of MHC class II and the co-stimulatory molecules CD40 and CD80, alongside augmented production of the Th1 polarising cytokine IL-12, when compared with uninfected DCs. Following in vitro co-culture with BCG-infected DCs, NK cells increased their expression of the activatory molecule CD25, with preferential activation of the CD2− NK cell subset. NK cell effector function, as measured by production of IFN-γ, was also significantly enhanced following co-culture with BCG-infected DCs. This study provides novel evidence to demonstrate that NK cells phenotypically and functionally mature after interactions with DCs in the context of BCG. Furthermore, through the production of IFN-γ and IL-12 by NK cells and DCs respectively, this interaction may drive protective Th1-type immune responses to Mycobacteria.

Development of in vitro enteroids derived from bovine small intestinal crypts

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host–pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or “mini gut”, cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host–pathogen interactions in the bovine small intestine can be studied.

Frequency and phenotype of NK cells and NK cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2+ CD25lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2− CD25hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2− NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2− subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.

The Role of NK cell - Dendritic Cell Interactions during BCG Vaccination

Background: Polyvalent vaccination represents a recent attempt to improve the effectiveness of lung cancer immunotherapy. This study aimed to investigate whether a gene expression pattern of tumor-associated antigens (TAA) would exist indicating that their use will be most appropriate for the polyvalent vaccination of Caucasian non-small cell lung carcinoma (NSCLC) patients. We examined the concomitant expression of genes belonging to different TAA families for which expression frequencies either have never been detected in NSCLC or vary widely in the literature. Methods: Tumor material from 23 patients with NSCLC (12 adenocarcinomas, 8 squamous cell carcinomas, 3 bronchoalveolar carcinomas) was examined. mRNA transcripts were detected for 5 genes of the survivin family, 5 MAGE-A genes as well as the genes of human telomerase reverse transcriptase (hTERT) and p53, by the use of quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) or semiquantitative RT-PCR. Results: 15/23 (65%) and 8/23 (35%) tumor samples were found expressing 6-11 and 2-5 out of the 12 examined TAAs, respectively, at levels >1% of the testis reference sample. The most prevalent TAA patterns observed were those of survivin standard (survivinstd)/survivin-2B expressed by 22/23 (95.5%) tumor samples and of survivin-std/survivin-2B/hTERT expressed by 19/23 (82.5%) tumor samples. The expression levels of the survivin-std gene strongly positively correlated to those of the survivin-2B (p=0.001) and the hTERT genes (p=0.031). The number of concomitantly expressed genes was found to be positively correlated to the age of the patients (p=0.001) and the tumor size (p=0.048). Conclusion: This study provides evidence that, in Caucasian patients with NSCLC, highly prevalent expression patterns of TAA genes, predominantly of overexpressed TAAs, do exist. This result implies that the combined use of these TAA could help in designing more effective NSCLC immunotherapeutic protocols.Purpose/Objective: Rheumatoid arthritis (RA) is a chronic autoimmune disease that leads to joint destruction. The recruitment of effectors cells, including monocytes to the joint space is an important step in RA progression and is mediated by chemokines (Ch) and their receptors (ChR). MicroRNAs are a recently discovered class of posttranscriptional regulators. Many members of the miR family are implicated in the regulation of cell movement and migration. Our previous study showed miR-155 is upregulated in RA synovial fluid (SF) monocytes suggesting that this miR may be involved in activation of these cells, including their migration into joint space. We hypothesized that miR-155 could regulates migration of monocytes in RA by modulating the expression of the chemokine and chemokine receptor system. Materials and methods: Peripheral blood (PB) CD14+ cells from healthy controls (HC) and RA patients were transfected with miR-155 mimic or scramble mimic using N-TER nanoparticles and cultured for 48 h. TaQman Low Density Array and multiplex assay was used to evaluate ChR expression and Ch production, respectively. Similar analysis was carried out on bone marrow monocytes (BMM) from miR-155-/- and WT mice. In addition, absolute copy numbers of miR- 155 transcripts in PB and SF CD14+ of RA and HC were assessed by QPCR. Results: PB and SF monocytes in RA patients showed higher copy number of miR-155 compared to HC. Overexpression of miR-155 in HC and RA monocytes did not affect the production of CCL2, CCL7, CCL21, CXCL5, CXCL8, CXCL7, CXCL10 and CX3CL1. In contrast, overexpression of miR-155 induced the production of chemokines such as CCL4, CCL5 and CCL22 in RA monocytes and CCL3 in both RA and HC. Analysis of chemokine receptors in BMM of miR-155-/- and WT mice revealed significantly higher levels of CCR1, CCR2, CCR5 and CXCR4 in miR-155 deficient cells suggesting that miR-155 can act as a negative regulator of these receptors in homeostatic state. As expected, TLR-4 ligand significantly suppressed expression of these receptors in both WT and miR-155-/- cells. Analysis of 3’UTRs of Ch/ ChR (TargetScan) suggests that miR-155 is likely interfering with signaling pathways implicated in Ch/ChR system expression. Conclusions: Deregulation of miR-155 in RA monocytes can contribute to the production of pro-inflammatory chemokines by these cells and to their accumulation at sites of inflammation.Purpose/Objective: Sphingosine kinase (SPHKs), SphK1 and SphK2, have been identified to phosphorylate sphingosine into sphingosine-1- phosphate (S1P). They are involved in a wide variety of cellular responses. S1P acts via S1P Receptors, S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, all of which can be bound and activated specifically by S1P. A defect either in S1P signalling or S1PRs has been associated with many pathologies. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by high levels of proinflammatory cytokine production. Elevated SPHK1, S1P, and S1P1 have been reported in RA synovium. S1P signalling via S1P1 promotes synoviocyte proliferation, increases COX-2 expression and prostaglandin E2 production. This study comprehensively evaluated expression of SPHK1/2 and S1PRs in RA patients compare to healthy controls (HC) and osteoarthritis (OA) in peripheral blood (PB) and synovial tissues, respectively. Materials and methods: mRNA and protein expression of SPHK1/2 and SIPRs were examined in neutrophils, monocytes and T lymphocytes of peripheral blood of 10 HC and RA patients, who met the diagnostic criteria of 2010 ARC / EULAR by QPCR and FACS, respectively. Competitive ELISA assessed SIP in serum of RA patients with remission and relapse and HC. We also performed SPHK 1/2 and SIPRs immunohistochemistry in synovial tissue from 4 RA/ OA patients. Results: S1P was three times high in RA than those observed in HC, also was statistically higher in RA patient with relapse than remission. Intracellular expression of hSPHK1 in RA patients, with opposed to HC, was up regulated 1.4-folds in monocytes and T- lymphocytes with significance expression in CD4T cells. hS1P1 and hS1P3 exhibited a similar expression were up-regulated in neutrophils, while, hS1P5 was statistical high in T cells. In contrast, hS1P4 was down regulated in all sorted cells particularly in CD4T cells. As opposed to OA synovial tissue, RA synovial tissues were strongly positive for hSPHK1 and hS1P1, 3 expressions. Quantitative analysis showed, SPHK1 and hS1P3 are expressed in lining, sub lining and vascular endothelial layer, while hS1P1 expressed mainly in lining and sub lining layers of the RA synovial tissue compared with OA. Conclusions: These results suggest that SPHKs/S1P and its S1PRs might play a role in RA pathogenesis. The clinical significance of S1P as a biomarker for disease activity deserves further attention.