Antonio Concetti

Prognostic relevance of altered Fas (CD95)‐system in human breast cancer

The Fas ligand (FasL) and its receptor Fas (APO‐1 or CD95) are members, respectively, of the tumor necrosis factor family that, upon interaction with each other, play a key role in the initiation of one apoptotic pathway. Faulty regulation of the Fas system has been described in a variety of human tumors with different histogenetic origin. Here, we describe the expression and distribution of Fas receptor and ligand pair antigens in surgical samples collected from a cohort of 186 patients bearing breast neoplasms (45 benign and 141 malignant lesions). Immunoperoxidase staining of formalin‐fixed tissues showed that 91.1% of benign lesions expressed Fas, which was present in only 56.7% of malignant tumors. On the other hand, FasL was found positive in 22.2% of benign neoplasms and up‐regulated in in situ as well as invasive carcinomas (53.9%). Moreover, in malignant tumors, the expression of receptor and ligand antigens appeared to be inversely related. When these findings were correlated with pathological parameters of prognostic relevance, a significant association was observed between FasL and the presence of metastatic lymph nodes and larger tumor size while Fas expression correlated to node‐negative status and smaller tumor size. Patients with Fas positive tumors exhibited longer disease‐free survival than those with Fas‐negative carcinoma while FasL did not influence patient outcome. These relationships indicate that benign and malignant mammary lesions are characterized by differential cellular expression of Fas and FasL and suggest that a neoplastic Fas negative/FasL positive phenotype may be linked to breast cancer progression. Int. J. Cancer (Pred. Oncol.) 89:127–132, 2000.

Genetic immunization against neu/erbB2 transgenic breast cancer

Abstract erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice, like some breast cancer patients erbB2+X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Th1 phenotype by DNA immunization. Intramuscular injections of neuNT plasmid drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms.

Autoantibody to p185erbB2/neu oncoprotein by vaccination with xenogenic DNA.

Abstract The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4+ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year.

Isolation of the mating-inducing factor of the ciliate Euplotes☆

Numerous strains of different mating types of the marine ciliate Euplotes raikovi have been found to be autonomous excreters into the surrounding medium of specific mating-inducing factors (gamones) (Luporini, P et al., J exp zool 226 (1983) 1 [9]). The gamone from the mating type represented by strain 13 has been isolated and identified as a glycoprotein with a molecular weight (MW) of about 12 kD and a pI of 4. It has been termed euplomone r 13. At a concentration of 3 X 10(-12) M, euplomone r 13 specifically induces cells of a complementary mating type to unite in conjugation within 2 h.

Antibody and CD8+ T Cell Responses against HER2/neu Required for Tumor Eradication after DNA Immunization with a Flt-3 Ligand Fusion Vaccine

Purpose: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. Experimental Design: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. Results: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell–mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in naïve hosts. Conclusions: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

BACKGROUND We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.

Cyanide dissociation from the hemoglobin of Parascaris equorum

The reduction of cyanomethemoglobin by dithionite leads to the appearance of an intermediate, the complex of cyanide with ferrous hemoglobin, whose dissociation is easily followed in a stopped flow apparatus. This reaction was studied in the hemoglobin from the parasitic nematode Parascaris equorum, whose extremely high oxygen affinity is due to a very low dissociation rate. The rate of cyanide dissociation from ferrous Parascaris hemoglobin is not so dramatically different from that of other hemoglobins and myoglobins. Other features of the reaction are: (i) the rate constant of cyanide release is pH independent, an observation which is agreement with the possible absence of the distal histidine, given the mechanism suggested in a previous study (Bellelli, A., Antonini, G., Brunori, M, Springer, B.A. and Sligar, S.G. (1990) J. Biol. Chem. 265, 18898-18901), and (ii) the time-course shows no kinetic cooperativity. The structural basis of the extremely high oxygen affinity of Parascaris hemoglobin cannot be explained on the basis of the results here reported. This study also confirms that, even though cyanide binding to ferrous hemoglobins is controlled by distal interactions, the functional behaviour of this ligand is characteristic and differs from the behaviour of oxygen.

Molecular stability of DNA typing short tandem repeats in the mammary tree of patients with breast cancer.

Archival pathologic specimens are a rich source for the studies of hereditary diseases, cancer genetics, and identification cases in forensic science. In this study, the intraindividual consistency of eight identifying microsatellite polymorphisms (i.e., HMTH01, vWFA31, F13A, MITMH26, FES-FPS, CD4, TPOX, CSF1PO)in a cohort of 40 patients with invasive breast carcinoma were analyzed. Nests of cancer and adjacent morphologically normal ductal–lobular structures (TDLUs) were microdissected as discrete regions from hematoxylin-eosin–stained slides. As controls for each case, DNA templates were prepared from TDLUs located in nontumor quadrants and from unaffected breast skin. Over 1,400 carefully controlled PCR reactions were reviewed, and no evidence was found for microsatellite mismatches among intraindividual cancer and control DNAs. The negative results, supported by validation experiments, strongly argue that alterations of simple repeats are rare somatic events during the onset and progression of breast cancer.This study suggests that PCR artifacts may be a relevant cause of misdiagnosis of microsatellite instability in human sporadic cancer.

Ligand-dependent behavior of the hemoglobin from the ascarid Parascaris equorum

Abstract Hemoglobin from the ascarid Parascaris equorum is made of eight identical subunits, each containing protoheme as a prosthetic group. The thermodynamic and kinetic properties of the binding of this protein with three ligands, i.e., oxygen, carbon monoxide and ethyl isocyanide, have been investigated. The reaction with ethyl isocyanide is characterized by a hyperbolic binding curve ( n = 1), whereas binding of CO displays a weak but definite cooperativity ( n = 1.3). Displacement experiments suggest that the noncooperative binding of ethyl isocyanide occurs fully in the high-affinity state, related to a very early quaternary switch. All three ligands display binding kinetics similar to those of T-state HbA. Dissociation rate constants for CO and ethyl isocyanide fall in the same range observed for other hemoglobins. On the other hand, oxygen affinity is much higher than in human HbA and, similarly to Ascaris lumbricoides Hb, it has to be attributed to an unusually slow dissociation rate constant. In this paper it is shown that such a slow kinetic process is not affected by: (i) pH; (ii) guanidine-HCl; (iii) a large excess of p -chloromercuribenzoate; (iv) the lifetime of the oxy-Hb complex.