An in vivo model of hyperacute rejection: characterization and evaluation of the effect of transgenic human complement inhibitors

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2h of perfusion with human plasma and after 1 day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1, IL-1, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2h of perfusion, as an early event. GOT and GPT values increase drammatically after 2h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.     

Fmoc-protected iminosugar modified asparagine derivatives as building blocks for glycomimetics-containing peptides

CSF114(Glc) is the first synthetic Multiple Sclerosis Antigenic Probe able to identify autoantibodies in a statistically significant number of Multiple Sclerosis patients. The beta-turn conformation of this glucopeptide is fundamental for a correct presentation of the epitope Asn(Glc). To verify the influence of sugar mimics in antibody recognition in Multiple Sclerosis, we synthesized Fmoc-protected Asn derivatives containing alkaloid-type sugar mimics. The corresponding glycomimetics-containing peptide derivatives of the CSF114-type sequence were tested in competitive and solid-phase non-competitive ELISA on Multiple Sclerosis patients' sera.     

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4⁺CD25⁺FOXP3⁺ circulating cells (CD4 T_reg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4⁺ T_reg cells rapidly decreased after primary tumour debulking, while CD8⁺CD25⁺FOXP3⁺ (CD8 T_reg) cells are not detectable in peripheral blood. Similar results on CD4 T_reg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T_reg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β₁ and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8⁺ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T_reg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.     

Impact of Bt Corn on Rhizospheric and Soil Eubacterial Communities and on Beneficial Mycorrhizal Symbiosis in Experimental Microcosms

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.     

Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

DNA heterogeneity and genetic control of tumorigenesis in nicotiana tumorous and nontumorous genotypes

The possible relevance of changes in amounts of highly repetitive DNA sequences for plant differentiation and dedifferentiation processes has been suggested in several cases. Data are lacking however on (1) the genetic control of these phenomena and (2) cause-effect relationships between DNA amplification and specific ontogenetic patterns. The present study was carried out on a Nicotiana genetic system consisting of the tumorous amphidiploid N glauca X N langsdorffii, a nontumorous mutant of it, their F₁, and a backcross to the tumorous parent. Backcross segregation ratios were shown to be compatible with a “single gene” hypothesis, the F₁ plant being nontumorous but showing a low percentage of tumors induced by wounds, 6-azauracil or X-rays. In vitro studies of excised pith tissue grown on Linsmaier and Skoog medium for different periods of time showed the presence, confirmed by cytological analyses, of amplification of highly repetitive sequences only in the nontumorous stock, as judged by reassociation experiments in the first 24–96 hours of culture. CsCl analytical ultracentrifugation of those sequences showed the appearance in the same stock of a heavy DNA satellite (density = 1.721 gm/ml), whose presence was also confirmed by derivative melting curves. Amplification seemed to be essential for the initiation of cell division, which was completely inhibited in the nontumorous genotype and partially influenced in the F₁ by incorporation during the critical period (24–96 hours of the primary explant) of 5-bromo-2′-deoxy-uridine. The results are discussed in terms of an hypothesis of an integrated gene-controlled, hormone-mediated regulatory system of cell proliferation involving changes in target repetitive DNA sequences.     

Hyperglucosylated adhesin‐derived peptides as antigenic probes in multiple sclerosis: Structure optimization and immunological evaluation

Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide–antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N‐linked β‐d ‐glucopyranosyl moieties (N‐Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C‐terminal portion HMW1(1205–1526) were essential for high‐affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347–1354) fragment bearing one or two N‐Glc respectively on Asn‐1349 and/or Asn‐1352. The N‐glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC₅₀ in the range 10^?8–10^?10 M by competitive indirect enzyme‐linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di‐N‐glucosylated adhesin peptide Ac‐KAN (Glc)VTLN (Glc)TT‐NH₂ as the shortest sequence able to inhibit high‐avidity interaction with N‐Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)‐ and circular dichroism (CD)‐based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N‐glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.     

Immobilization of matrix metalloproteinase 8 (MMP-8) for online drug screening

Matrix metalloproteinase 8 (MMP-8) has been reported to have a key role in several pathologic conditions, like heart diseases, osteoarthritis, multiple sclerosis, and various other inflammatory conditions. Therefore, there is a great interest regarding the development of MMP-8 selective inhibitors. In the recent years, immobilized enzyme reactors (IMERs) proved to be an efficient alternative to solution-based assays. Besides the recycling of the enzyme, IMER approach allows a simple way to determine affinity data and thus the ranking of inhibiting potency of the compounds under study, especially when coupled to MS. In this study, the immobilization of MMP-8 was investigated in terms of type of support, kinetic parameters, storage and pH stability. Epoxy activated silica resulted the best matrix for the preparation of an immobilized enzyme reactor (IMER) containing human MMP-8. The IMER was successfully used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments.     

Highlights at the gate of tryptophan catabolism: a review on the mechanisms of activation and regulation of indoleamine 2,3-dioxygenase (IDO), a novel target in cancer disease

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of Kynurenine pathway along the major route of Tryptophan catabolism. The scientific interest in the enzyme has been growing since the observations of the involvement of IDO in the mechanisms of immune tolerance and in the mechanisms of tumor immuno-editing process. In view of this latter observation, in particular, preclinical studies of small molecule inhibitors of the enzyme have indicated the feasibility to thwart the immuno-editing process and to enhance the efficacy of current chemotherapeutic agents, supporting the notion that IDO is a novel target in cancer disease. This review covers the structural and conformational aspects of substrate recognition by IDO, including the catalytic mechanism and the so-far puzzling mechanisms of enzyme activation. Furthermore, we discuss the recent advances of medicinal chemistry in the field of IDO inhibitors.     

Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.