The role of the oviduct environment in embryo survival.

CONTEXT: Declining fertility is an issue in multiple mammalian species. As the site of fertilisation and early embryo development, the oviduct plays a critical role in embryo survival, yet there is a paucity of information on how the oviduct regulates this process. AIMS: We hypothesised that differences in steroid hormone signalling and/or immune function would be observed in a model of poor embryo survival, the peripubertal ewe. METHODS: We examined expression of steroid hormones in systemic circulation, oviductal expression of oestrogen receptorαand genes important in steroid hormone signalling, and immune function in pregnant and cyclic peripubertal and adult ewes on day 3 after oestrus. KEY RESULTS: Concentrations of progesterone, but not oestradiol, were decreased in the peripubertal ewe compared to the adult ewe. Oestrogen receptorαprotein expression was increased in the peripubertal ewe, but pathway analysis of gene expression revealed downregulation of the oestrogen signalling pathway compared to the adult ewe. Differential expression of several genes involved in immune function between the peripubertal and adult ewe was consistent with an unfavourable oviductal environment in the peripubertal ewe lamb. Oestradiol concentration was positively correlated with the expression of multiple genes involved in the regulation of immune function. CONCLUSIONS: Differences in the immune environment of the oviduct, potentially linked to differential modulation by steroid hormones, may partially underly the poor fertilisation and early embryo survival observed in the peripubertal ewe. IMPLICATIONS: A unfavourable oviductal environment may play an important role in limiting reproductive success.

Effect of birth rank, and placentome subtype on expression of genes involved in placental nutrient transport in sheep.

Placental function is a key determinant of fetal growth and development that can be influenced by maternal and fetal environmental factors. The molecular mechanisms by which the placenta senses and responds to environmental cues are poorly understood. This exploratory study aimed to characterize the effect of birth rank (single vs. twin) and placentome morphologic subtype on expression of genes involved in nutrient transport, angiogenesis, immunity and stress response. Cotyledonary tissue was collected from type A, B and C placentomes from five single and six twin fetuses at 140 days of gestation. GLUT1 and GLUT3 were the most highly expressed genes consistent with the high demand for glucose to support fetal growth. Expression of BCKDHß and IGF-2 was 1.3- and 1.5-fold higher, respectively, and PCYT1A was 3-fold lower in singles compared to twins (P < 0.05) while no other differences in gene expression were observed between birth ranks. Expression of EAAT2 and LAT2 was higher while PCYT1A was lower in A compared to B type cotyledons. Expression of GUCY1B1/3 and IGF-1 was higher while CD98 and LAT2 were lower in type B compared to C cotyledons (P < 0.05). Compared to type C cotyledons, expression of EAAT2, IGF-1, IGF-2, LAT1 was higher, while TEK was lower in type A cotyledons. The effects of birth rank on placental gene expression in this study indicated that placental nutrient transport and/or function differs between single and twin pregnancies in sheep. Differences in gene expression between the placentome subtypes suggests that changes in placentome morphology are associated with shifts in amino acid transport and metabolism, oxidative stress and angiogenesis and/or blood flow. This study highlights that placental gene expression differs in response to birth rank and placentome morphologic subtype which suggests that both maternal and fetal factors may influence placental function in sheep. These associations provide insights into gene pathways for more targeted future investigations as well as potential adaptations to improve placental efficiency to support fetal growth in twin pregnancies.

Growth performance, antibody response, and mammary gland development in New Zealand dairy replacement bovine heifers fed low or high amounts of unpasteurized whole milk.

This study evaluated the influence of feeding low and high preweaning allowances of unpasteurized whole milk (MA) on intake, selected blood metabolites, antibody response, mammary gland growth, and growth of New Zealand (NZ) dairy heifers to 7 mo of age. At 10 ± 2 d of age (study day 0), group-housed (six·pen-1) heifer calves (Holstein-Friesian × Jersey) were allocated to low (4 L whole milk·calf-1·d-1; n = 7 pens) or high (8 L whole milk·calf-1·d-1; n = 7 pens) MA for the next 63 d. Calves were gradually weaned between days 63 ± 2 and 73 ± 2. Calves in each pen had ad-libitum access to clean water, pelleted calf starter, and chopped grass hay from day 1 to 91 ± 2 d. At 92 ± 2 d, all calves were transferred to pasture, grazed in a mob, and their growth and selected blood metabolites were measured until day 209. All animals were weighed weekly during the indoor period (to day 91) and then at days 105, 112, 128, 162, 184, and 209. Skeletal growth measurements and blood samples to analyze selected metabolites were collected at the start of the experiment, weaning, and then postweaning on day 91, and day 201. Specific antibodies against Leptospira and Clostridia were quantified in weeks 7, 13, and 27. Mammary glands were scanned using ultrasonography at the start of the experiment, weaning, and day 201. Feeding high vs. low amounts of MA increased the preweaning growth in heifer calves (P = 0.02) without negatively affecting postweaning average daily gain (ADG) (P = 0.74). Compared with heifers fed with low MA, high MA fed heifers had a greater increase in antibodies against Leptospira and Clostridia by 13 wk of age (P = 0.0007 and P = 0.06, respectively). By 27 wk of age, the antibody response was the same in heifers offered low or high MA. There was no effect of MA on the total size of the mammary gland, measured by ultrasonography, at weaning and 7 mo of age. However, the greater MA was associated with more mammary parenchyma (P = 0.01) and less mammary fat pad (P = 0.03) in back glands at 7 mo of age compared with heifers fed lower MA. In conclusion, feeding a high vs. a low amount of unpasteurized whole milk increased the preweaning growth of New Zealand replacement heifers without negatively affecting their ADG during postweaning under grazing conditions. Feeding more (8 vs. 4 L·d-1) unpasteurized whole milk positively affected antibody responses early in life and mammary gland composition by 7 mo of age in dairy heifers reared for pasture-based dairy systems.

This study evaluated the effect of unpasteurized whole milk allowance on intake, antibody response, mammary gland growth, and growth performance of heifers until 7 mo of age. Feeding greater (8 L·d−1) vs. lower (4 L·d−1) milk allowance to heifer calves increased preweaning body weight without having any negative effect on postweaning growth under grazing. Heifers fed high milk allowance had significantly better antibody responses against Leptospira and Clostridia by 3 mo of age and had more mammary parenchyma (potential milk making tissue), and less mammary fat pad (supporting tissue) by 7 mo of age.

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes.

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R2 = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R2 = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R2 = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786 as a fusion ('MAP fusion protein particle'). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Effect of an injectable trace mineral supplement on the immune response of dairy calves.

On a spring calving, pastoral dairy farm, the first 40 heifer calves born after calving mid-point (50% of the herd calved) were blood sampled within 24 h. Thirty were selected, using stratified randomisation to form two equal groups (treatment and control) with the same distribution of serum total protein, copper, selenium, zinc, and manganese concentrations, age and breed. From the remaining 10 calves, five were randomly selected into a sentinel group to assess field exposure to Salmonella spp. All calves received two injections of a killed vaccine containing Salmonella spp. antigens at 2 and 6 weeks of age. Concurrently, the treatment group were injected with 1 mL/50 kg trace mineral supplement (TMS) containing 40 mg zinc, 10 mg manganese, 5 mg selenium, 15 mg copper per mL. Sentinel animals received no injections. All animals were bled from 2 to 9 weeks for assay of immune function. At three and four weeks, white blood cells from TMS calves had an increased percentage of cells phagocytosing (effect size = 9.36 and 4.35) and increased number of bacteria ingested per cell (effect size = 0.93 and 1.52). No differences were detected in gamma interferon response (effect size <0.15) or Salmonella sp. antibody titres (effect size <0.20).

Far-off and close-up feeding levels affect immunological performance in grazing dairy cows during the transition period.

During the peripartum period, dairy cows often have signs of inflammation. Various stresses, including infectious and metabolic diseases, have been discussed as causative for this inflammation. In this study, expression profiles for 17 immune markers were measured in whole blood preparations from 78 dairy cows over a time frame starting 1 wk before calving to 4 wk after calving. Additionally, the effects of far-off and close-up feeding on immune function of dairy cows during the peripartum period were investigated. Cows were assigned to 1 of 2 feeding levels in late lactation to achieve a low and high BCS at the time of dry-off (approximately 4.25 and 5.0 on a 10-point scale). Following dry-off, both herds were managed to achieve a BCS of 5.0 one month before calving; this involved controlled feeding (i.e., maintenance) and over-feeding of ME during the far-off dry period. Within each far-off feeding-level treatment, cows were offered 65, 90, or 120% of their precalving ME requirements for 3 wk precalving in a 2 × 3 factorial arrangement. Analysis of gene expression profiles from blood cells revealed effects of time indicating that the transition cow's immune system counteracts the peripartum inflammation, whereas later postcalving it becomes activated to provide protection against postpartum infections. Far-off feeding affected (P < 0.05) the expression of 2 of the investigated genes at calving. Interleukin-6 (IL-6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in unstimulated, peripheral leukocytes were lower (P < 0.05) in animals from the Far-Off_Over-fed group compared with the Far-Off_Control-fed group. Close-up feeding had several effects on gene expression, indicating that immune function in Feed120 animals was distinct from the Feed90 and Feed65. In conclusion, feeding management precalving becomes an important intervention to ensure immunocompetence at and after calving.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

Grazing dairy cows had decreased interferon-γ, tumor necrosis factor, and interleukin-17, and increased expression of interleukin-10 during the first week after calving.

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n=29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n=39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n=19; untreated control n=20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.

Comparison of gene expression of immune mediators in lung and pulmonary lymph node granulomas from cattle experimentally infected with Mycobacterium bovis.

The cellular infiltrates and macrophage activation pathways may differ in granulomas found in the lungs and pulmonary lymph nodes of cattle infected with Mycobacterium bovis. The aim of this study was to compare the histopathology and gene expression profiles of cytokines and immune mediators for cattle which had these lesions in both sites. Ten Friesian-cross, 15-16 month old cattle were challenged intratracheally with 5 × 10(3)CFU of virulent M. bovis and killed and necropsied at 28 weeks after infection. Seven animals were found to have gross TB granulomas in both their lungs and pulmonary lymph nodes (PLN) and these lesions were fully encapsulated with central necrosis and mineralisation. Neutrophil infiltration was clearly involved in granuloma in lung whereas neutrophils were limited in lesions of PLN. Comparisons were made of immune mediators from these two sites from the same animals as well as those between lesioned PLN tissues and non-lesioned prescapular lymph nodes (PSLN). Gene expressions of the immune mediators were normalised using a housekeeping gene (U1), a monocyte/macrophage marker (CD14) and a common leucocyte marker (CD45). mRNA expression of IFN-γ, IL-17A, IRF5(1) and arginase 1 (Arg1) was significantly up-regulated in lung compared to that for PLN (p<0.05), while mRNA expression of IFN-γ, IL-12p40, TNF-α and iNOs for PLN was significantly higher than that for PSLN (p<0.05). In addition, IL-10 mRNA expression was significantly higher for lung compared to PLN when normalised for CD45 (p<0.05). The results suggested that the stronger proinflammatory immune response in the lesioned lung may be a consequence of enhanced expression of IRF5 promoting IFN-γ and IL-17 production. In contrast, Arg1 expression in the lungs could facilitate the infection through competing with iNOs for l-arginine, preventing generation of nitric oxide for clearance of M. bovis infection.

Reversal of myeloid cell-mediated immunosuppression in patients with metastatic renal cell carcinoma.

PURPOSE: Tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of biological therapy for renal cell carcinoma. Here we evaluate the role of CD33 myeloid-derived suppressor cells (MDSC) in the regulation of T-cell responses in renal cell carcinoma patients. We also examine effect of all-trans-retinoic acid (ATRA) on MDSC-mediated immune suppression. EXPERIMENTAL DESIGN: CD33-positive myeloid cells were isolated from the peripheral blood of renal cell carcinoma patients with magnetic beads and tested in vitro for their ability to inhibit T-cell responses. T-cell function was evaluated using ELISPOT and CTL assays. RESULTS: MDSC isolated from renal cell carcinoma patients, but not from healthy donors, were capable of suppressing antigen-specific T-cell responses in vitro through the secretion of reactive oxygen species and nitric oxide upon interaction with CTL. MDSC-mediated immune suppression and IFN-gamma down-regulation was reversible in vitro by exposing cells to the reactive oxygen species inhibitors. Moreover, ATRA was capable of abrogating MDSC-mediated immunosuppression and improving T-cell function by direct differentiation into antigen-presenting cell precursors. CONCLUSIONS: These results may have significant implications regarding the future design of active immunotherapy protocols that may include differentiation agents as part of a multimodal approach to renal cell carcinoma immunotherapy.

Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.

LOXL4 is a selectively expressed candidate diagnostic antigen in head and neck cancer.

Selective up-regulation of the mRNA of LOXL4, a member of the LOX matrix amine oxidase family, significantly correlated with lymph node metastases and higher tumour stages in head and neck squamous cell carcinomas (HNSCC). To evaluate the diagnostic and prognostic value of the protein we produced an antibody specific for LOXL4 and assessed the expression in 317 human HNSCC specimens. The LOXL4 protein was detected in 92.7% of primary tumours, in 97.8% of lymph node metastases and in affected oral mucosa with high-grade dysplasia, but was absent in various non-neoplastic tissues of the head and neck. TNM categories and overall survival did not link to grades of immunoreactivity. Studies in cultured primary hypopharyngeal HTB-43 carcinoma cells detected perinuclear and cell surface expression of LOXL4, but no nuclear localisation. Therefore, its interactive SRCR-domains and catalytic activity combined with tumour cell specific expression and cell surface associated location indicate multiple functions in tumour cell adhesion and interactions with the extracellular matrix. Our data suggest that LOXL4 is useful both as tumour marker and target in the treatment of HNSCC.

Vaccination strategy to target lysyl oxidase-like 4 in dendritic cell based immunotherapy for head and neck cancer.

Overexpression of lysyl oxidase (LOX) is associated with the invasive potential of metastatic breast and head and neck cancer (HNC) cells and reduced metastasis-free and overall survival. Recently, we have demonstrated up-regulation of a new member of the LOX family, lysyl oxidase-like 4 (LOXL4), in invasive HNC revealed a significant correlation between LOXL4 expression and local lymph node metastases and higher tumour stages. The objective of this study was to examine whether cellular LOXL4 may provide an effective target for cell-meditated immunotherapy in invasive tumours associated with LOXL4 overexpression. As a feasibility study we expressed LOXL4 mRNA in immature dendritic cells derived from human peripheral blood mononuclear cells (PBMC). LOXL4 protein expression was ascertained using Western blotting and immunocytochemistry with polyclonal rabbit anti-LOXL4 antibody. The successfully transfected immature dendritic cells (DCs) were induced to mature with GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, and PGE2, and then used to stimulate T cell enriched non-adherent fraction of PBMC. LOXL4 specific T cell stimulation induced cytotoxic T lymphocyte (CTL) response was monitored using IFN-gamma secretion from the non-adherent PBMC fraction exposed to mature, LOXL4 transfected DCs acting as the antigen presenting target cells. LOXL4-DC stimulated T cells produced higher IFN-gamma secretion compared to unstimulated T cells and T cells stimulated with untransfected DCs, in the presence of the pan-DR-epitope (PADRE). These initial results demonstrated the potential for LOXL4-transfected DCs to serve as efficient tumour vaccine and support their suitability as a vaccination strategy applicable to cancer patients with tumour specific up-regulation of LOXL4.

A dendritic cell based hybrid cell vaccine generated by electrofusion for immunotherapy strategies in HNSCC.

OBJECTIVE: Hybrid cells generated from dendritic cells (DC) and tumor cells provide tumor-associated antigens (TAA) in a polyvalent mode and therefore they have aroused interest in cancer immunotherapy. The present study was designed to investigate the hybrid cell generation and optimize its implementation for a TAA-target treatment of head and neck squamous cell carcinoma (HNSCC). METHODS: Hybrid cells from mature DC and laryngeal carcinoma cell line UTSCC-19A were generated by electrofusion. Fusion efficiency and viability were determined by flow cytometry, light and fluorescence microscopy analyses. RESULTS: The gradual electrofusion process constituted real human tumor and dendritic cell hybrids characterized by polynuclear cells and double staining as a result of overlay of red (HLA-DR:R-PE) and green (HEA:FITC) fluorescence. Furthermore, analyses have proven viability of fusion results, and factors influencing fusion yield were determined. CONCLUSION: Physical fusion of mature dendritic cells with laryngeal carcinoma cells provides a dendritic cell based hybrid cell vaccine as a quantitative prerequisite for anti-cancer vaccination. Specific cytotoxic T-lymphocytes need to be induced before hybrid cell application in clinical studies.

Electrofusion generates diverse DC-tumour cell hybrids for cancer immunotherapy.

BACKGROUND: Hybrid cells generated from dendritic cells (DC) and tumour cells provide tumour-associated antigens (TAA) in a polyvalent mode. The present study was designed to investigate the hybrid cell generation by dendritic cells and different tumour cell lines to establish an electrofusion protocol with an optimal fusion setting. MATERIALS AND METHODS: Hybrid cells from mature DC and tumour cells were generated by electrofusion. Fusion efficiency was determined by flow cytometry, as well as by light and fluorescence microscopy analyses. RESULTS: The gradual electrofusion process constituted different human dendritic cell tumour cell hybrids of high diversity depending on electrical and non-electrical parameters. Factors influencing fusion frequency were determined by specific cell staining with mAbs, FACS analysis and trypan blue dye exclusion. CONCLUSION: Increased fusion efficiency was associated with reduced viability. The protocol presented in this work might be helpful for future fusion studies as a prerequisite for comparable in vitro and human vaccination trials.

Generation of cytotoxic responses in mice and human individuals against hematological malignancies using survivin-RNA-transfected dendritic cells.

Survivin is a member of the inhibitors of apoptosis family and is overexpressed in many types of human cancers, making it an attractive target for T cell-based immunotherapeutic strategies. Recently, HLA-A2-binding peptides derived from the survivin protein were identified as capable of inducing specific T cell responses in cancer patients. Here we demonstrate that human survivin-specific CTLs generated from PBMC by stimulation with autologous dendritic cells transfected with survivin-RNA were cytotoxic for a range of hemopoietic malignant cell lines and primary tumor cells isolated from patients with acute myeloid leukemia. We also show that vaccination of mice with survivin-RNA-transfected dendritic cells leads to long term resistance to challenge by a survivin-expressing lymphoma, demonstrating the potential of survivin as a tumor rejection Ag. Our data provide evidence for the use of survivin as a target structure for immunotherapeutic strategies against hematological neoplasms.

Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells.

Autologous dendritic cells transfected with total renal tumor RNA have been shown to be potent stimulators of CTLs and antitumor immunity in vitro. A Phase I trial was conducted to evaluate this strategy for feasibility, safety, and efficacy to induce tumor-specific T-cell responses in subjects with metastatic renal cell carcinoma. Renal tumor RNA-transfected dendritic cells were administered to 10 evaluable study patients with no evidence of dose-limiting toxicity or vaccine-related adverse effects including autoimmunity. In six of seven evaluable subjects, expansion of tumor-specific T cells was detected after immunization. The vaccine-induced T-cell reactivities were directed against a broad set of renal tumor-associated antigens, including telomerase reverse transcriptase, G250, and oncofetal antigen, but not against self-antigens expressed by normal renal tissues. Although most patients underwent secondary therapies after vaccination, tumor-related mortality of the study subjects was unexpectedly low with only 3 of 10 patients dying from disease after a mean follow-up of 19.8 months. These data provide a scientific rationale for continued clinical investigation of this polyvalent vaccine strategy in the treatment of metastatic renal cell carcinoma and, potentially, other cancers.