Effect of dietary Astragalus Polysaccharide supplements on testicular miRNA expression profiles and enzymatic changes of breeder cocks.

Astragalus Polysaccharide (APS) is an important feed additive due to its immunomodulatory functions. Previous studies have proven that miRNAs play important roles in posttranscriptional gene regulation. Our goals were to identify differentially expressed miRNAs in testes in responses to APS dietary supplements and to find the effects of APS on breeder cock testes. We measured several enzymatic activities in testes and sperm samples and further generated miRNA expression profiles of testes from breeder cocks fed with control diets and extra APS. As a result, we found APS could increase testicular functional activities of marker enzymes. Meanwhile, there were 16 up-regulated and 17 down-regulated miRNAs in APS group, compared with the control group meeting the criteria of P-values < 0.05. Meanwhile, twelve differentially expressed miRNAs were validated by Mir-XTM miRNA RT-qPCR. Further GO and KEGG analyses of target genes for differentially expressed miRNAs revealed that some miRNAs may be involved in testicular nutrient metabolisms and NK cell mediated cytotoxicity pathway. Moreover, the effect of dietary APS supplements on NK cell mediated cytotoxicity pathway was also validated by RT-qPCR. Our results provided a novel insight into the effect of dietary APS supplements on testicular miRNA expression profiles and enzymatic changes of breeder cocks.

Elicitation of T cell responses to histologically unrelated tumors by immunization with the novel cancer-testis antigen, brother of the regulator of imprinted sites.

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4(+) T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8(+)-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.

Proliferation potential-related protein, an ideal esophageal cancer antigen for immunotherapy, identified using complementary DNA microarray analysis.

PURPOSE: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. EXPERIMENTAL DESIGN: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. RESULTS: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24-restricted CTLs. These CTLs, generated from HLA-A24-positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP-specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. CONCLUSIONS: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.

Physiological relevance of tumor necrosis factor in mediating macrophage-Leydig cell interactions.

Previously, we reported that testicular macrophages constitutively release tumor necrosis factor (TNF) in vitro and are unresponsive to bacterial endotoxins [lipopolysaccharides (LPS)]. These properties are not typical of other tissue macrophages. The goals of the present study were, therefore, to establish 1) if testicular macrophages also release TNF in vivo, and 2) if secretion of TNF in vitro is influenced by the isolation procedure. In vivo TNF production was assessed by assaying testicular interstitial fluid for TNF. Using the L929 cytotoxicity assay for TNF, we found that interstitial fluid contained a cytotoxic factor(s), but this bioactivity was not due to either authentic TNF or a TNF-like molecule acting through the TNF receptor. This was established by showing that 1) antibodies to TNF alpha and -beta could not neutralize interstitial fluid cytotoxicity; 2) interstitial fluid was cytotoxic to TNF-resistant L929 cells; and 3) there was no detectable TNF immunoreactivity in interstitial fluid, as measured by enzyme-linked immunosorbent assay. Therefore, we evaluated whether the release of TNF in vitro was induced by the isolation procedure, particularly by collagenase, which is used to free interstitial cells. Testicular macrophages obtained without the use of collagenase (agitation of testes in buffer) did not release TNF, but responded to the TNF-releasing effect of LPS. Exposure of peritoneal macrophages to collagenase resulted in constitutive TNF release in vitro and lack of responsiveness to LPS. There was no evidence that a non-TNF cytotoxic factor was released in the conditioned medium by any macrophage preparation. Taken together, our findings show that testicular macrophages do not constitutively release TNF, and collagenase has a significant activating effect on macrophages. Testicular macrophages will, however, release TNF when exposed to LPS, indicating that TNF could be a paracrine regulator of testicular steroidogenesis under pathological conditions.

Secretion of tumor necrosis factor alpha by testicular macrophages.

While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor alpha (TNF alpha) has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNF alpha. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNF alpha. The TNF alpha activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNF alpha but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNF alpha, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNF alpha activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNF alpha.

In vitro cell-mediated and complement-mediated cytotoxicity to murine testicular cells.

Male BALB/c mice at 8 to 14 weeks of age were divided into three groups: group 1 was immunized with an emulsion of testicular cells (TCs) (10(7)/mouse), complete Freund's adjuvant (CFA), and extract of Bordetella pertussis (BP); group 2 was given CFA and BP injections; and group 3 was given sterile saline injections. Suspensions of TCs and spleen cells (SCs) from each mouse were prepared 4 weeks after the first immunization for a cytotoxic T lymphocyte (CTL) assay. For the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) assays, TCs and SCs were prepared from normal male and female mice, respectively. Targets were labeled with Na251 Cr0(4). The interactions of targets (TCs) and effectors (SCs) were conducted at 32.5 degrees C (for CTL, ADCC, and ACC assays) or 37 degrees C (for ACC assay). In the CTL assay, SCs from group 1 and group 2 caused significantly more killing than those from group 3. Specific cytotoxicity in the ADCC assay was only detected in the serum (maximum specific lysis 47.65%) of one mouse. No other cytotoxicity was detectable in 61 serum samples from group 1 (n = 25), group 2 (n = 17), and group 3 (n = 19). In the ACC assay, no significant specific cytolysis was found at different incubation temperatures (32.5 and 37.0 degrees C) in 44 serum samples from the three groups. These results suggest that CTLs are important in the pathogenesis of experimental allergic orchitis (EAO). Adjuvant alone, probably because of breakdown of the blood-testis barrier, causes significant T lymphocyte cytotoxicity to TCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated cytotoxicity of bovine mononuclear cells to IBRV-infected cells: dependence on Sephadex G-10 adherent cells.

Following intranasal inoculation of cattle with infectious bovine rhinotracheitis virus (IBRV) mononuclear cells that produced a genetically unrestricted cytotoxic response against IBRV-infected, but not against uninfected cells, were present in peripheral blood. Cytotoxicity was detected between 6 and 14 days after primary infection in a 20 h, but not in a 5 h, 51Cr-release assay. Cytotoxic activity was present in peripheral blood mononuclear cells from infected and subsequently hyperimmunized cattle for a considerably longer time. Neither natural cytotoxicity, antibody-dependent cell cytotoxicity, nor antibody produced during the assay was responsible for the cytotoxicity. However, cytotoxicity was dependent upon an adherent mononuclear cell that was partially removed by passage over nylon wool and completely removed by passage over Sephadex G-10.

Inability to detect a K cell in bovine peripheral blood leukocytes.

Antibody-dependent cellular cytotoxicity to viral-infected cells, chicken red blood cells, and tumor cells was tested using different effector cell populations: polymorphonuclear cells, mononuclear cells, and mononuclear cells separated into adherent and nonadherent populations by Sephadex G-10. Polymorphonuclear cells were the most efficient mediators of antibody-dependent cellular cytotoxicity against most targets, although a combination of G-10 adherent and polymorphonuclear cells was more efficient in killing infectious bovine rhinotracheitis virus-infected cells than either single cell population. Removal of G-10 adherent cells from the mononuclear cell population removed all antibody-dependent cellular cytotoxicity from that population, indicating the lack of a typical K cell in bovine peripheral blood.

Antithymocyte globulin reacts with many normal human cell types.

Antithymocyte globulin (ATG) is frequently effective therapy for aplastic anemia. Its mechanism of action is often assumed to be upon a lymphocyte inhibitor of hematopoiesis. However, specificity for T lymphocytes would not be anticipated from consideration of the method of preparing ATG. In fact, using flow microfluorometry and fluorescence immunohistochemistry, we have found that ATG binds to virtually all circulating lymphocytes, granulocytes, and platelets, as well as to bone marrow cells. Extensive absorption of ATG with either granulocytes or lymphocytes does not eliminate reactivity with the opposite cells, indicating that ATG recognizes some distinct antigens on each cell type. Treatment of cells with ATG blocks the binding of monoclonal antibodies directed against either lymphocyte differentiation or histocompatibility antigens. ATG also binds to visceral tissues, including thymus and testis cell membranes and the nuclear and cytoplasmic components of tonsil, kidney, liver, breast, lung, and intestine. In vitro cytotoxicity of ATG was demonstrated for both T and non- T lymphocytes and platelets. Despite its name, ATG is not specific for a particular cell type, and it would be premature to conclude that its effect is mediated through a specific lymphocyte population.

In vitro production of the factors effective in the transfer of experimental autoimmune aspermatogenesis.

We studied the biological effects of the factors released into the culture medium by the lymphocytes from animals sensitized with testicular antigen. The supernatants from cultures of these lymphocytes were active in vitro in the migration inhibition tests because they transferred the sensitivity to normal spleen cells. In vivo, they transferred aspermatogenesis in the allogeneic and xenogeneic system. The results suggest a specific effect on the behaviour of macrophages rather than a direct cytotoxic effect of the active factors.

Embryonic antigens and growth of murine fibrosarcomata.

The amount of embryonic antigens (EA) was estimated in 13 BALB/c fibrosarcomata by in vitro cell mediated cytotoxicity of anti-embryo spleen cells and by quantitative absorption of an anti-embryo antiserum. A direct relationship between amount of EA and tumour growing capacity was found. EA were detected also on fast dividing testicular cells. It is suggested that EA expression on tumour cells is related to a cell membrane function controlling mitosis rather than to a function specifically related to the neoplastic status. Tumour take of low doses of 2 EA-bearing sarcomata was found to be enhanced in anti-embryo immune BALB/c mice in comparison with that in normal and anti-fibroblast immune mice.

Serum induced lymphoid cell mediated cytotoxicity to human transitional cell carcinomas of the genitourinary tract.

In some serums of patients with transitional cell carcinoma (TCC), a factor is present which induces lymphocytes from most donors with or without TCC to become cytotoxic against TCC-derived target cells. The induced cytotoxicity was directed against target cells derived from TCC's of the renal pelvis, ureter, and urinary bladder, but not against cells derived from normal kidney, bladder, testis, or skin or from renal cell carcinoma. Cytotoxicity occurred without complement but did not occur without effector cells.

Rejection of tumor cells in vitro.

The emergence of lymphoblast-like cells, capable of rapidly destroying tumor cells, was observed in primary cultures of an antigenic sarcoma transplantable in strain 13 guinea pigs. It is likely that these cytotoxic cells represent the progeny of lymphocytes sensitive to tutmor antigens that had infiltrated the tumor tissue.