Effect of Melissa officinalis supplementation on growth performance and meat quality characteristics in organically produced broilers.

1. A trial was conducted to study the effect of Melissa officinalis supplementation on organic broiler performance and meat chemical, microbiological, sensory and nutritional quality. 2. Male and female day-old Ross 308 chicks were fed on a standard commercial diet containing 0, 2.5, 5 or 10 g/kg feed ground M. officinalis for 84 d before slaughter. 3. Weight gain and feed conversion ratio were significantly improved in the broilers receiving either 5 or 10 mg M. officinalis/kg feed. 4. Inclusion of M. officinalis did not affect muscle chemical and fatty acid composition. 5. On the basis of microbiological and sensory experimental data and subsequent extension of meat shelf life, M. officinalis did not reduce the microbial populations of the meat, but was effective in limiting lipid oxidation.

p53 enhances the Delta-24 conditionally replicative adenovirus anti-glioma effect.

Previous studies have demonstrated that the conditionally replicative adenovirus Ad5Delta24 is a powerful cytolytic agent against glioma selectively affecting cells with a defective p16/Rb/E2F pathway. The p53 protein is also known to be an apoptotic factor for glioma cells. In this study, we examined the simultaneous delivery of the combination of exogenous p53 and Ad5Delta24 adenovirus in glioma cells. Infecting cells with low doses of adenovirus p53 and Ad5Delta24 resulted in an additive effect on cell death. The cell death induced by both agents was independent of the p53 status of cells. Flow cytometry revealed that the potent anti-tumor effect induced by the mixture of Ad5CMV-p53 and Ad5Delta24 adenoviruses was due to a combination of apoptosis and cell lysis. Our results indicate that Ad5CMV-p53 enhances the oncolytic effect of the Ad5Delta24 adenovirus, and the combination of adenovirus Ad5Delta24 and Ad5CMV-p53 may thus be a potential therapeutic tool for gliomas.

Transfer of E2F-1 to human glioma cells results in transcriptional up-regulation of Bcl-2.

Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-x(L) by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.

A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo.

Effective anti cancer strategies necessitate the use of agents that target tumor cells rather than normal tissues. In this study, we constructed a tumor-selective adenovirus, Delta24, that carries a 24-bp deletion in the E1A region responsible for binding Rb protein. Immunoprecipitation analyses verified that this deletion rendered Delta24 unable to bind the Rb protein. However, titration experiments in 293 cells demonstrated that the Delta24 adenovirus could replicate in and lyse cancer cells with great efficiency. Lysis of most human glioma cells was observed within 10 - 14 days after infection with Delta24 at 10 PFU/cell. In vivo, a single dose of the Delta24 virus induced a 66.3% inhibition (P<0.005) and multiple injections, an 83.8% inhibition (P<0.01) of tumor growth in nude mice. However, normal fibroblasts or cancer cells with restored Rb activity were resistant to the Delta24 adenovirus. These results suggest that the E1A-mutant Delta24 adenovirus may be clinically and therapeutically useful against gliomas and possibly other cancers with disrupted Rb pathway.

Novel approaches toward early diagnosis of islet allograft rejection.

BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

Co-expression of E2F-2 enhances the p53 anti-cancer effect in human glioma cells.

Gliomas are highly resistant to conventional treatment. Improved knowledge of the molecular defects of glioma cells offers new avenues for the development of gene therapy strategies. Transfer of the p53 gene has proven effective in suppressing proliferation in human glioma cell lines. However, several human glioma cell lines are resistant to p53-induced cell death. The E2F family of transcription factors are pivotal for the regulation of cell-cycle and cell-death related genes in gliomas. In the present study, we sought a more effective strategy for glioma treatment by examining the therapeutic potential of the simultaneous transfer of p53 and E2F-2 to gliomas. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated that the transfer of both p53 and E2F-2 induced cell death in D-54 MG, a p53-resistant glioma cell line. In addition, transfer of E2F-2 did not interfere with the apoptotic properties of exogenous wild-type p53 in U-251 MG cells. Finally, the expression of E2F-2 in D-54 MG cells suppressed the expression of the apoptotic molecule mdm-2 induced by exogenous p53 in these cells. These results show that co-expression of E2F-2 and p53 enhances the anti-cancer effect of p53 in gliomas.

Adenovirally-mediated transfer of E2F-1 potentiates chemosensitivity of human glioma cells to temozolomide and BCNU.

The therapeutic efficacy of standard cancer treatments such as chemotherapy may be improved if they are combined with gene-therapy. Less than 30% of patients with glioblastoma multiforme respond to adjuvant chemotherapy. Actively dividing cells are generally more sensitive to chemotherapy than are non-dividing cells. To determine whether forced cell-cycle progression selectively sensitizes tumor cells to alkylating agents, we examined the effects of overexpressing the E2F-1 protein (a positive regulator of cell-cycle progression) on the sensitivity of two malignant human glioma cell lines, U-251 MG and D-54 MG, to BCNU and temozolomide. Treating these cells with 20-35 microM BCNU or 20-30 microM temozolomide resulted in 50% growth inhibition (IC50) within 4 or 6 days, respectively. By contrast, cells that were first induced to overexpress E2F-1 protein by infection with an adenoviral vector had IC50s that were 37-50% lower. Conversely, transferring the cyclin-dependent kinase inhibitors p16 and p21 to the cells, also by adenoviral infection, produced 3 to 4-fold increases in chemoresistance. Cell-cycle analyses showed that the combination of E2F-1 overexpression and treatment with BCNU or temozolomide increased the proportion of cells in S phase, but the combination of p16 or p21 overexpression and drug treatment reduced the proportion of cells in S phase. These observations suggest that overexpression of genes that positively control cell-cycle progression may be useful for increasing the sensitivity of glioma cells to alkylating agents.

Genetic abnormalities in oligodendroglial and ependymal tumours.

Oligodendroglial and ependymal tumours are not the most common glial neoplasms; however, they are important subtypes of gliomas with different tumour biologies. Cytogenetic information has suggested that losses of chromosomes 1 p and 19 q are the most frequent genetic alterations in oligodendroglial tumours. Combined loss of these chromosomes has been associated with better chemotherapeutic response and prolonged overall survival. Loss of chromosome 22 is a well defined abnormality in ependymomas. In addition, deletion of chromosome 6 q may be another frequent chromosomic aberration in paediatric ependymomas.

No specific reactivity to E. coli glutamic acid decarboxylase from sera of newly-diagnosed insulin dependent diabetic patients.

The 65 kD isoform of Glutamic Acid Decarboxylase (GAD), is one of the major autoantigens in human type 1 diabetes mellitus. This enzyme shares amino acid identity, in select regions already determined as antigenic with its counterpart from E. coli. We tested the reactivity of diabetic and normal sera and an E. coli GAD-specific monoclonal antibody (2D9) to E. coli GAD by solid phase and competition ELISA, as well as immunoblotting to check for cross-reactivity of autoantibodies to the two antigens. Specific antibodies for E. coli GAD are present in diabetics and normal subjects without any differences in frequency and titer. The reactivity of such antibodies in ELISA could be blocked in a dose-dependent manner by the addition of excess antigen in the liquid phase. Furthermore, the monoclonal antibody against E. coli GAD does not recognise human recombinant GAD65 in an ELISA. We conclude that there is no basis for cross-reactivity between the two antigens, and antibody reactivity to GAD65 in man cannot arise from cross-reactivity to the E. coli enzyme.

The destructive action of IL-1alpha and IL-1beta in IDDM is a multistage process: evidence and confirmation by apoptotic studies, induction of intermediates and electron microscopy.

Using the rat beta-cell RIN-5AH insulinoma line as a means for studying insulin-dependent diabetes mellitus (IDDM), it is shown that interleukin-1 (IL-1) induces beta-cell damage initiated by early apoptotic signals. This action is demonstrated by DNA fragmentation, as assessed by specific BrdU labeling, surface expression of Fas and nitric oxide (NO) production. In addition, the interplay between NO and Fas is shown, while scanning electron microscopy (SEM) confirms apoptosis by revealing the degree and type of cellular damage which, in the case of IL-1alpha, can be reversed by an inhibitor to NO synthesis. Apoptosis is also reconfirmed by transmission electron microscopy (TEM) by observing condensed nuclear chromatin after IL-1 exposure. Thus, treatment of insulinoma cells with IL-1alpha and IL-1beta seems to initiate a number of signals, including PKC activation as published previously, that ultimately lead to beta-cell destruction. Each IL-1 isoform, however, definitely follows a different pathway of action.