Profile of chicken cytokines induced by lipopolysaccharide is modulated by dietary alpha-tocopheryl acetate.

Previously, we found that 25 to 50 IU/kg of dietary vitamin E (VE) had very different immunoregulatory effects than high VE levels (200 IU/kg), and we hypothesized that this difference was due to different cytokine profiles. Chicks were fed 0, 30, or 200 IU/kg supplemental VE and percentages of CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- lymphocytes, and the ratio of CD4+/CD8+ lymphocytes was determined. The expression of chicken splenic interleukin-1beta (IL-1beta), myelomonocytic growth factor (MGF), interferon (IFN-gamma), and transforming growth factor-beta (TGFbeta) mRNA was determined by reverse transcription (RT)-PCR after intravenous injection of lipopolysaccharide (LPS). Due to a tendency for increased CD4-CD8+ lymphocytes at 30 IU/kg VE (P=0.072), the CD4+/CD8+ ratio was significantly lower for 30 IU/kg VE compared with 0 IU/kg VE (P=0.041). The VE dose of 200 IU/kg decreased the constitutive (prior to LPS) expression of TGFbeta. The LPS caused an increase in IL-1beta, MGF, and IFNgamma expression at all VE concentrations and had no effect on IL-2 and TGFbeta mRNA expression. Dietary VE decreased MGF mRNA (P=0.049) in a dose-dependent manner but had no effect on the expression of other cytokines. The decreased expression of MGF could explain the immunomodulatory effect of VE in inflammation.

Relationship between the level of dietary vitamin E and the immune response of broiler chickens.

The relationship between the dietary level of vitamin E (VE) and the immune response of broilers was studied in three experiments. Immunity was assessed as antibody production to infectious bronchitis virus (IBV), SRBC, and Brucella abortus (BA) antigens, mitogenic response to phytohemagglutinin A (PHA) and concanavalin A (Con A), cutaneous basophil hypersensitivity (CBH) to PHA, and lipopolysaccharide induction of acute-phase proteins (APP) and heterophilia. A range of VE (0, 10, 17.5, 25, 37.5, 50, 100, and 200 IU/kg) levels were supplemented to a basal diet (corn-soy) containing 10.2 IU of VE/kg. We found a dose-dependent increase in antibody production in response to attenuated IBV between 0 and 25 IU/kg of supplemented VE and no further increase at higher levels. Antibody levels to SRBC were higher in birds supplemented with 50 IU of VE/kg compared to those supplemented with 0 or 200 IU/kg of VE. Antibody production in response to BA antigens was not influenced by VE. Mitogenic responses were suppressed by supplemented VE in Experiment 1 for PHA (25 IU/kg diet) and Con A (25 and 50 IU/kg diets). CBH and APP levels were not affected by VE. Heterophilia was lowest at 50 IU/kg 6 h after lipopolysaccharide injection (Experiment 1). Our study showed that moderate (25 to 50 IU/kg) levels of VE supplementation were most immunomodulatory and that high levels were less effective.

Apoptosis-related gene and protein expression in human lymphoma xenografts (Raji) after low dose rate radiation using 67Cu-2IT-BAT-Lym-1 radioimmunotherapy.

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to 67Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, 67Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 micrograms Lym-1, or 335-500 microCi 67Cu on less than 400 micrograms Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-beta 1 and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. 67Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after 67Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with 67Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.

Divergence of the inflammatory response in two types of chickens.

We compared inflammatory responses to lipopolysaccharide (LPS) injection in laying type (Brown Nick) to broiler type (Avian x Avian) chicks. Rectal temperature was measured at 0, 1, 2, 4, 6, 12, and 24h after LPS injection (0, 0.1, 0.3, 0.6, 1, 2.5, or 5mg/kg bw). In layers, rectal temperature increased from 41.31+/-0.19 degrees C to a maximum 42.27+/-0.41 degrees C at 4h after 1mg/kg LPS. Relative to layers, the febrile response in broilers was considerably lower, delayed in onset, and required higher levels of LPS (5mg/kg). Proliferation of spleen cells from un-injected chicks in response to LPS, PHA, and Con A was evaluated in vitro. IFNgamma, TGFbeta(2), MGF and IL-1beta relative to beta-actin mRNA expression were analyzed in spleen cells stimulated with LPS. Splenocytes from layers had a higher proliferative response to LPS (P=0.045), but lower proliferative response to PHA (P=0.004) and Con A (P=0.004) than broilers. Expression of mRNA for MGF, IL-1beta and IFNgamma was lower in broilers than in layers (P<0.001). Reduced production of the pro-inflammatory cytokines in broilers could have resulted from the observed increased production of the immunosuppressive cytokine TGFbeta(2.) These differences in cytokine expression may explain the blunted febrile response in broilers compared to layers. Because the acute phase response of inflammation causes decreased food intake, the blunted inflammatory response of broilers may permit faster growth.

Decreased C-MYC and BCL2 expression correlates with methylprednisolone-mediated inhibition of Raji lymphoma growth.

Methylprednisolone (MP) and related corticosteroids are a fundamental part of regimens used to treat lymphoma and leukemia. In many of these malignancies, oncogenic activation of C-MYC and BCL2 is seen. Abnormalities of the tumor suppressor p53, which exerts growth-suppressing and apoptosis-enhancing functions through the transcriptional regulation of downstream genes including CDKN1, GADD45, and BCL2, are also often found. The goal was to determine the modulation of expression of the oncogenes (C-MYC and BCL2), the p53 pathway described above, and the apoptosis marker TGF-beta 1 in the human Raji lymphoma following MP treatment. Raji xenografts were grown in nude mice and growth curves characterized by sequential measurement. Mice were treated daily for 8 days with MP. Tumors were harvested untreated, or at 1 or 8 days after cessation of MP treatment, and the RNA was extracted. RT-PCR was used to determine the level of mRNA expression of the genes. Tumor growth was greatly reduced in the MP-treated mice. Gene expression levels for C-MYC and BCL2 were reduced at 1 day following MP and approached control levels 8 days after MP treatment. Expression levels of p53, CDKN1, and GADD45 were moderately and coordinately decreased at 1 day after cessation of MP treatment and remained repressed a week later. TGF-beta 1 exhibited no change in expression levels. These results suggest that decreased expression of C-MYC and BCL2 may play a role in the molecular events that initiate and are responsible for the growth inhibition of Raji lymphoma xenografts by MP.