Biomedical subjects

L Polito

Publications and source records attributed to L Polito.

At least 19 recordsLinked to original sources

PubMed2005

Occupational sensitization to ribosome-inactivating proteins in researchers.

BACKGROUND: Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur. OBJECTIVE: Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs. METHODS AND RESULTS: We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated. CONCLUSION: We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.

Adult↗

PubMed2004

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine.

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC(50) values of 0.1-0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

Antibodies, Monoclonal↗

PubMed2004

Expression of CTLA-4 in nonhuman primate lymphocytes and its use as a potential target for specific immunotoxin-mediated apoptosis: results of in vitro studies.

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Animals↗

PubMed2004

Immunotoxins and other conjugates: pre-clinical studies.

A wide variety of conjugates containing RIPs, of either chemical or recombinant type, have been made and tested against dangerous cells in vitro and in animal models. Many of these pre-clinical studies will be reviewed here dividing them on the basis of the target cell and the surface molecule specifically recognized.

Animals↗

PubMed2002

Transferrin toxin but not transferrin receptor immunotoxin is influenced by free transferrin and iron saturation.

BACKGROUND: Cytotoxic agents can be targeted successfully to cancer cells. The efficacy of such novel and potent anticancer strategies may be influenced by variables of iron metabolism. METHODS: The in vitro cytotoxicity against glioma cells of transferrin (Tf)-based targeted toxins was compared with that of alpha-transferrin receptor (TfR)-immunotoxin. RESULTS: Of four Tf-based targeted toxins, Tf-gelonin, Tf-pokeweed antiviral protein, Tf-momordin and Tf-saporin, inhibitory concentration 50% values against glioma-derived cell lines HS683 and U251, ranged from [4.8 +/- 1.5] x 10(-10) m for Tf-saporin to [26.9 +/- 15.3] x 10(-10) m for Tf-gelonin in [(3)H]-leucine incorporation assays. Tf-saporin and alpha-TfR-saporin-immunotoxin had similar efficacy, even in the more quantitative clonogenic assay (4-5 log kill with 1 x 10(-9) m) using the myeloma cell line RPMI 8226 and glioma cell line U251. However, on RPMI 8226, the efficacy of Tf-saporin 1 x 10(-9) m was reduced by 90% in the presence of 150 microg mL(-1)(=20% of normal plasma value) competing diferric transferrin, whereas the efficacy of the corresponding immunotoxin was affected only marginally. In addition, the efficacy of Tf-based conjugates will depend on their iron saturation state. Iron desaturation of Tf-saporin was demonstrated by [(59)Fe]-labelling, subsequent CM-Sepharose chromatography and SDS-PAGE. Desaturation led to virtually complete loss of affinity for the transferrin receptor, as determined by flow cytometry, which could be largely restored upon resaturation. CONCLUSION: Transferrin-based toxin conjugates are strongly influenced by the presence of free transferrin and the iron saturation state. The corresponding alpha-transferrin receptor-immunotoxin does not show these disadvantages, has similar efficacy and should be preferred for further experiments.

Antibodies, Monoclonal↗

PubMed2002

The Mediet Project.

Preliminary evidence from a case control study of healthy postmenopausal women living in Palermo, Sicily, is presented to investigate the potential impact of a traditional Mediterranean diet on the risk of developing breast cancer. Of the 230 women who fulfilled specific eligibility criteria, 115 were enrolled in the study based on serum testosterone values equal to or greater than the median population value (0.14 microg/ml). Women were then individually randomized into a diet intervention (n = 58) and a control (n = 55) group. Women in the intervention group attended a weekly "cooking course" for 1 year, being trained by professional chefs in the correct use of the natural ingredients of the traditional Mediterranean diet, including whole cereals, legumes, seeds, fish, cruciferous vegetables, and many others. The intervention group was subsequently instructed to follow the learned diet at home, while the control group was only advised to increase the consumption of fruits and vegetables, as recommended by WHO. The following measures were taken at the beginning, middle, and end of the study: (a) fasting blood and 12-hour urine samples to assay defined hormonal endpoints; (b) height, weight, and circumference of the waist and hip; and (c) a food frequency and computerized 24-hour dietary recall questionnaire. After 1 year, both the control and the intervention groups showed satisfactory compliance rates (81 and 85%, respectively). In addition, preliminary results so far obtained reveal an unequivocal trend towards weight loss, a strong reduction in cholesterol levels, and a psychophysical feeling of well-being by women adopting the Mediterranean diet. The study is currently ongoing to verify the association of changes in serum and urine hormone levels and breast cancer risk in the intervention group.

Adult↗

PubMed2001

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Abatacept↗

PubMed2001

Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation.

The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.

Amino Acid Sequence↗

PubMed2000

In vitro anti-tumour activity of anti-CD80 and anti-CD86 immunotoxins containing type 1 ribosome-inactivating proteins.

Immunotoxins specific for the CD80 and CD86 antigens were prepared by linking three type 1 ribosome-inactivating proteins (RIPs), namely bouganin, gelonin and saporin-S6, to the monoclonal antibodies M24 (anti-CD80) and 1G10 (anti-CD86). These immunotoxins showed a specific cytotoxicity for the CD80/CD86-expressing cell lines Raji and L428. The immunotoxins inhibited protein synthesis by target cells with IC50s (concentration causing 50% inhibition) ranging from 0.25 to 192 pmol/l as RIPs. The anti-CD80 immunotoxins appeared 1-2 log more toxic for target cells than the anti-CD86 ones. Immunotoxins containing saporin and bouganin induced apoptosis of target cells. The toxicity for bone marrow haemopoietic progenitors of these conjugates was also evaluated. Bouganin and related immunotoxins at concentrations up to 100 nmol/l did not significantly affect the recovery of committed progenitors or of more primitive cells. The saporin-containing immunotoxins at concentrations >/= 1 nmol/l showed some toxicity on colony-forming unit cells (CFU-C). The expression of the CD80 and CD86 molecules is prevalently restricted to antigen-presenting cells and is also strong on Hodgkin and Reed-Sternberg cells in Hodgkin's disease. Present results suggest that immunotoxins targeting type 1 ribosome-inactivating proteins to these antigens could be considered and further studied for the therapy of Hodgkin's disease or other CD80/CD86-expressing tumours.

Antibodies, Monoclonal↗

PubMed2000

Polynucleotide: adenosine glycosidase activity of immunotoxins containing ribosome-inactivating proteins.

Polynucleotide:adenosine glycosidases (rRNA N-glycosidases, EC 3.2.2.22, more commonly known as ribosome-inactivating proteins, RIP) are a numerous family of plant and bacterial enzymes, shown to release also adenine from DNA in vitro. They are well suited for the preparation of specifically toxic conjugates with several carriers, including monoclonal antibodies (immunotoxins). Here we show that (i) immunotoxins containing various PNAG (dianthin, gelonin, momordin I, PAP-S, PDS-2, ricin A-chain, saporin-L1, saporin-S6) all act on DNA; (ii) activity on DNA in vitro is less compromised by disulphide linkage to antibody than is inhibition of cell-free protein translation; and (iii) specific cytotoxicity of immunotoxin does not correlate with substrate specificity.

Adenine↗

PubMed1999

Preparation of highly purified momordin II without ribonuclease activity.

Momordin II, a ribosome-inactivating protein from Momordica charantia seeds, was purified by a procedure involving a series of chromatographies on S-Sepharose, Sephadex G-50, CM-Sepharose, and Red Sepharose columns. Highly purified momordin II inhibited cell-free protein synthesis, released adenine from rat liver ribosomes and from DNA, and had no RNase activity.

Adenine↗

PubMed1998

Evaluation of immunotoxins containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OM124): in vitro and in vivo studies.

Immunotoxins were prepared with three ribosome-inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6, linked to the anti-CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22-expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC50 (concentration causing 50% inhibition) ranging from < 5 x 10(-15) to 7.6 x 10(-11) M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5 x 10(-14) to 5 x 10(-8)M, with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 x 10[-8] M). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10(-9) M. Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3 x 10[-6]), when 60% of the animals remained tumour-free.

Animals↗

PubMed1998

Different sensitivity of CD30+ cell lines to Ber-H2/saporin-S6 immunotoxin.

The in vitro sensitivity of cells to a Ber-H2(anti-CD30)/saporin-S6 immunotoxin has been investigated. The CD30+ cell lines, K562, L428 and L540, were used to study cell binding, uptake and degradation of the immunotoxin. K562 cells were less sensitive than L428 and L540 cells to the immunotoxin by approximately one order of magnitude. The difference in cytotoxicity correlated with the intracellular accumulation and with the ratio of degraded over total internalized Ber-H2/saporin-S6, regardless of the immunotoxin binding to the cells. After 6 h incubation, the less sensitive K562 cells (i) accumulated only one third and one tenth of the immunotoxin accumulated by the more sensitive L428 and L540 cells, respectively, and (ii) degraded two thirds of the internalized protein versus one third degraded by either L428 or L540 cells. Ammonium chloride and chloroquine reduced the cytotoxicity of the immunotoxin towards K562 but not to L540 cells. This effect correlated with the increment of immunotoxin catabolism by K562 cells in the presence of chloroquine. In conclusion, uptake alone of an immunotoxin by target cells is not sufficient to assure its efficacy which might also depend on intracellular routing. Only a cytotoxicity test may be really predictive.

Antineoplastic Agents↗

PubMed1997

Molecular expression of 17 beta hydroxysteroid dehydrogenase types in relation to their activity in intact human prostate cancer cells.

In the present study we have inspected estrogen metabolism in cultured human prostate cancer cells (LNCaP, DU145, PC3), in relation to the expression of mRNAs for different 17 beta hydroxysteroid dehydrogenase (17 beta HSD) enzymes (from 1 to 4). Using an intact cell analysis, we have compared precursor degradation and product formation after incubation of cells with physiological amounts of radioactive E2 or estrone (E1) for 24-72 h and subsequent reverse-phase high performance liquid chromatography analysis. The LNCaP and DU145 cells only partly converted E2 to E1 (26 and 13% at 72 h, respectively), giving rise to an appreciable production of E2 from E1 (nearly 20% in all cases). Conversely, PC3 cells revealed a massive E2 oxidation to E1 (up to 90% by 72 h) and a scant formation of E2 (<2%) from E1. In addition, an appreciable formation of 16 alpha OHE1 was seen in either PC3 (11%) or DU145 (5%) cells. respectively using E2 or E1 as precursor. All three cell lines exhibited marked amounts of 17 beta HSD4 mRNA species, whilst even greater amounts of 17 beta HSD2 transcript were found in PC3 cells only. No mRNA for either 17 beta HSD1 or 17 beta HSD3 could be detected in any cell line. The present evidence indicates that pathways of estrogen metabolism are distinctly governed in prostate cancer cells depending on their endocrine status, being associated with a differential expression of mRNA for different 17 beta HSD enzymes.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity.

Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAI), and a new lectin-related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome-inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell-free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non-toxic type 2 (two chain) RIP. APA and SNAI had much less activity, and BDA and GNA did not inhibit protein synthesis.

3T3 Cells↗

PubMed1997

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd.

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

3T3 Cells↗

PubMed1997

Immunoconjugates made of an anti-EGF receptor monoclonal antibody and type 1 ribosome-inactivating proteins from Saponaria ocymoides or Vaccaria pyramidata.

The present paper describes two immunoconjugates consisting of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), named Mint5, covalently linked to the type 1 ribosome-inactivating proteins (RIPs) ocymoidine (Ocy) and pyramidatine (Pyra) from Saponaria ocymoides and Vaccaria pyramidata respectively. Both antibody and toxins are shown to retain their respective biological properties upon chemical conjugation. The immunoconjugates exert specific inhibition of EGFR expressing target cell proliferation and protein synthesis in in vitro assays and also inhibit the growth of grafted human tumour cells in nude mice.

Animals↗

PubMed1997

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration.

Antibodies, Monoclonal↗