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L Callegaro

Publications and source records attributed to L Callegaro.

At least 37 records · Page 2Linked to original sources
PubMed1991

Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli.

The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl-B-D-thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse-phase high performance liquid chromatography. The amino-terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS-PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expression and purification of human CNTF.

Base Sequence↗
PubMed1991

Differences in induction of c-fos transcription by cholera toxin-derived cyclic AMP and Ca2+ signals in astrocytes and 3T3 fibroblasts.

The B subunit of cholera toxin, a protein which binds specifically to membrane ganglioside GM1, is known to affect cell growth and differentiation. To investigate the mechanism of these cellular responses at the nuclear level, we used the induction of c-fos in astrocytes and 3T3 fibroblasts as a model. Northern blot analysis showed that treatment with B subunit provokes a rapid and transient expression of c-fos mRNA, independent of a measurable increase in cyclic AMP. The B subunit signal, which is mediated by Ca2+, was compared to cholera toxin and other agents which increase intracellular cyclic AMP levels. In transient transfection assays of astrocytes and fibroblasts, functional analysis of c-fos promoter deletions was used to identify the elements involved in transcriptional activation by B subunit. In astrocytes, the DNA region including the serum response element and the cyclic AMP response element (CRE) are equally required, whereas 3T3 cells require only the CRE for maximal induction. A synergistic effect of signal transduction was mediated by calcium and cyclic AMP on the CRE, being positive in 3T3 cells and negative in astrocytes. Diverse regulatory elements may be thus involved in responses of different cell types to the same extracellular signal. Furthermore, a single regulatory element (CRE) can integrate both calcium and cyclic AMP signals in the control of gene expression.

Animals↗
PubMed1991

A simple method to detect contaminating peptides and amino acids in large-scale ganglioside preparations.

A reverse-phase liquid chromatography method was developed to analyse the presence of contaminating peptides and amino acids in large-scale monosialoganglioside preparations. Samples were hydrolysed under controlled conditions and derivatized with phenylisothiocyanate. PTC-amino acids were then separated and identified by HPLC. The sensitivity of the method allowed detection of a least 50 pmoles of amino acid per mg of ganglioside with excellent reproducibility and little or no interference of by-products derived from hydrolysis of the glycosphingolipid. The response was consistently linear for each amino acid within the examined analytical range. The ease and speed of performance make this method a useful tool for rapidly monitoring large-scale extraction and purification processes of biologicals obtained from natural sources.

Amino Acid Sequence↗
PubMed1990

Chemical residues of ganglioside molecules involved in interactions with lymphocyte surface targets leading to CD4 masking and inhibition of mitogenic proliferation.

Human lymphocyte CD4 becomes undetectable in the presence of exogenous gangliosides (CD4 masking), as originally described by Offner et al. (J. Immunol. 1987. 139: 3295). CD4 masking is apparently due to in situ rearrangement of the glycoprotein; since no direct binding of ganglioside to CD4 could be demonstrated, it was suggested that the effect could be mediated by interactions with other, as yet unidentified, surface structures. To gain insight into the structural requirements of the interaction(s) that leads to CD4 masking, we assayed the effects of a battery of gangliosides and of ganglioside derivatives on (a) CD4 masking; (b) cholera toxin binding (as a well known ganglioside-protein interaction) and (c) inhibition of lymphocyte mitogenic proliferation (as a second ganglioside interaction with a lymphocyte surface target). Our results indicate that the three interactions are distinctly different, since ganglioside chemical groups which are essential for one of the interactions are irrelevant for the others, and lead to the conclusion that gangliosides can interact with lymphocyte surface targets in a number of ways, causing a number of independent biological effects.

CD4 Antigens↗
PubMed1990

Large scale purification and immunological characterization of human placental nerve growth factor.

Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which cross-reacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF.

Antibodies↗
PubMed1989

Immunoscintigraphy of adenocarcinomas by means of radiolabeled F(ab')2 fragments of an anti-carcinoembryonic antigen monoclonal antibody: a multicenter study.

F(ab')2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody F023C5, determined to be more suitable than intact IgG and Fab fragments for immunoscintigraphy, were labeled with 131I or conjugated to DTPA for instant 111In-labeling, and administered i.v. (2-3 mCi/0.5 mg) to 509 patients in 11 nuclear medicine departments: 284 patients had gastrointestinal adenocarcinomas, 204 had nongastrointestinal adenocarcinomas and 21 were control; serum CEA was elevated in 169 patients, normal in 115, and not determined in 225. The following results were obtained: (a) no adverse reactions; (b) tumor imaging in 324 patients (in particular, in 81.5% CEA-seropositive and in 69.0% CEA-seronegative patients); (c) no significant difference in sensitivity among the results of the 11 departments; (d) no significant difference in overall sensitivity between 131I-and 111In-labeled immunoradiopharmaceuticals; (e) the fraction of documented lesions imaged was 73.3% in CEA-seropositive and 53.7% in CEA-seronegative patients; (f) the detection of liver metastases was hampered, particularly when using the 111In-labeled reagent, by nonspecific radioactivity uptake; (g) the major cause of negative immunoscintigraphy results was a lack of CEA in tumor lesions, as documented by immunohistochemistry; (h) lesion size is also important since the sensitivity was 64% for lesions up to 2 cm in diameter and 84% for larger lesions; (i) many "unexpected" radiolocalizations were recorded. Most were identified as occult tumor lesions. In 35 patients, this finding contributed to the early detection of tumor recurrences.

Adenocarcinoma↗
PubMed1989

Improved method for purification of human platelet factor 4 by affinity and ion-exchange chromatography.

A simple method for the reproducible purification of human platelet factor 4 (PF4) is described. PF4 is obtained in a highly purified form from platelet concentrate by utilizing a combination of affinity and FPLC ion-exchange chromatography. In every instance, after elution from heparin affinity and cation exchange chromatography, SDS gel electrophoresis reveals a single band attributable to PF4. Moreover, the amino acid composition of PF4 isolated by this method is compatible with that described for a PF4 cDNA clone and with other reported PF4 analysis. The purified protein is used to study in vitro the affinity of PF4 for several glycosaminoglycans (GAGs), by measuring the fluorescence of each PF4-GAG complex bound to fluorescamine. PF4 affinity for GAGs is as follows: heparin greater than heparan sulphate much greater than dermatan sulphate.

Amino Acids↗
PubMed1989

Lack of suppression by gangliosides of humoral or cellular immunity in vivo.

Gangliosides inhibit the proliferative responses of murine and human lymphocytes in vitro, and it has been suggested that they are immunosuppressive in vivo, although no in vivo studies have been performed. In view of the use of gangliosides to treat patients with a variety of neurological disorders, experiments were undertaken to evaluate the possible immunomodulatory effects of gangliosides in vivo. BALB/c mice were injected 5 days a week with buffer, mixed brain gangliosides, or GM1 ganglioside at dosage levels of 30 mg/kg, 60 mg/kg or 90 mg/kg. After 30 days of treatment, the mice were immunized with keyhole limpet hemocyanin or pneumococcal type III polysaccharide and the ganglioside treatment was continued. No differences between treated and control groups were noted in either the magnitude or duration of the antibody response. No differences between groups were noted in the proliferative responses of splenic mononuclear cells to concanavalin A or allogeneic antigens after 9-10 weeks of treatment, nor in the generation of cytotoxic effector cells after 90 days of treatment. Thus, despite the well-documented immunosuppressive effects of gangliosides in vitro, no evidence for a suppressive effect on humoral or cellular immunity in vivo was obtained in these studies.

Animals↗
PubMed1989

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats.

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.

Animals↗
PubMed1989

CD4 modulation and inhibition of HIV-1 infectivity induced by monosialoganglioside GM1 in vitro.

The addition of monosialoganglioside GM1 to serum-free culture medium efficiently and specifically inhibited CD4 antigen expression on normal T lymphocytes from peripheral blood or thymus as well as on cells from H9 and Molt-3 lines; other molecules such as CD3, CD2 and CD8 were not affected. Subsequent addition of fetal calf serum or bovine and human serum albumin blocked GM1 action on CD4 expression, most likely through the formation of ganglioside-albumin complexes. Removal of GM1 from the medium was followed by the prompt reappearance of CD4 on the cell surface. GM1 treatment of H9 and Molt-3 cells greatly reduced HIV-1 infectivity, which was evaluated by reverse transcriptase activity levels in culture supernatants and p24 detection on target cells. GM1 also inhibited syncytial formation in Molt-3 cells even when treatment was initiated 24h after infection. The GM1 effect on HIV-1 infectivity, however, was not long-lasting since removal of the compound was followed by a rapid increase in viral replication, probably due to CD4 re-expression and HIV-1 propagation from a few initially infected cells.

CD4 Antigens↗
PubMed1988

Immunoscintigraphy of adenocarcinomas by means of 111In-labelled F(ab')2 fragments of anti-CEA monoclonal antibody F023C5.

F(ab')2 fragments of F023C5, an anti-CEA monoclonal antibody, were conjugated to diethylenetriamine pentaacetic acid (DTPA) and converted into a ready to use reagent for instant 111In-labelling. The resulting 111In radiopharmaceutical (2-3 mCi/0.5 mg of F(ab')2 fragments) was administered intravenously and tested for its ability to image (at 48-72 h after administration) 31 primary and 85 metastatic carcinoma lesions in 70 adenocarcinoma patients (26 gastrointestinal, 18 breast and 26 lung tumor patients) whose serum CEA was elevated in 43 cases and normal in the other 27. The results were as follows: no adverse reactions or evidence of toxicity were observed in any of the patients. positive immunoscintigraphy results were obtained in 78% CEA-seropositive and in 64% seronegative patients. the fraction of documented lesions which was imaged was 69% in CEA-seropositive patients and 39% in seronegative patients. several 'unexpected' radiolocalizations were imaged; 30/34 were confirmed as (previously occult) tumour lesions in follow-up studies. In 6 patients, the immunoscintigraphic demonstration of previously occult lesions contributed to the early detection of tumour recurrences. the major limitation of the method is the non-specific radioactivity accumulation in the liver, which prevents the detection of liver metastases. in CEA-seropositive patients the fraction of imaged extra-hepatic tumour lesions is over 80%. the major cause of negative immunoscintigraphy results is lack of CEA in the tumour lesion, as demonstrated by immunohistochemistry of surgically removed tumours.

Adenocarcinoma↗
PubMed1987

Immunoscintigraphy of colorectal carcinoma with F (ab')2 fragments of anti-CEA monoclonal antibody.

A monoclonal antibody to carcinoembryonic antigen (CEA) (F023C5), belonging to IgG1 class, was obtained by cell fusion technique. Preliminary screening on different tissues was performed with immunoperoxidase staining, which showed good specificity of the antibody for gastric and colorectal carcinomas. F(ab')2 fragments were subsequently prepared and labeled with 131I and 111In. After immunoreactivity check the radiopharmaceuticals were injected intravenously. Sixteen patients with 22 primary or secondary localizations of colorectal carcinoma were studied following the recommendations of the ethical Committee of the Istituto Nazionale Tumori, Milan, Italy. Serial scans were performed after injection of the two radioactive reagents. In vivo pharmacokinetics of the compound was studied. Radioactivity level in surgical specimens was measured, and immunostaining was performed. All tumors were found to express the antigen. Eleven out of 12 tumor localizations of the gastrointestinal tract and three out of ten liver metastases were imaged. Specificity of tumor uptake was assessed by simultaneous injection of an irrelevant antibody.

Antibodies, Monoclonal↗
PubMed1986

Search for HTLV-I and LAV/HTLV-III antibodies in serum and CSF of multiple sclerosis patients.

In order to confirm the findings on the presence of antibodies against human T-lymphotropic retroviruses in subjects affected by multiple sclerosis we studied paired serum and CSF samples from 32 MS patients. Both ELISA and Western blot procedures were employed to detect antibodies against HTLV-I and LAV/HTLV-III antigens. Negative results were obtained in all samples examined, except one which was reactive to HTLV-I in ELISA but not in Western blot.

Adolescent↗
PubMed1986

Multicenter study of immunoscintigraphy with radiolabeled monoclonal antibodies in patients with melanoma.

A multicenter study was performed to analyze the efficacy of 99mTc- and 111In-labeled F(ab')2 fragments of monoclonal antibody (MoAb) 225.28S (reactive with a high molecular weight melanoma associated antigen) to radioimage malignant lesions in patients with melanoma. A total of 254 melanoma patients, carrying 412 documented melanoma lesions, were studied in 10 nuclear medicine departments. A total of 377 lesions were visualized in 206 patients; in particular (a) 250 of 412 known lesions were visualized in 159 of 191 patients known to carry melanoma lesions; (b) 95 occult lesions were visualized in 61 patients of the same group; and (c) 32 lesions were visualized in 15 of 63 patients without diagnosed lesions. The melanomic nature of 101 of 127 radioimaged occult lesions was confirmed by clinical criteria and/or by additional laboratory investigations. These results indicate that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S can provide clinically useful information. Analysis of the variables influencing the outcome of immunoscintigraphy with 99mTc- and 111In-labeled F(ab')2 fragments of MoAb 225.28S confirmed the role of size, anatomic site, and level of high molecular weight melanoma associated antigen in melanoma lesions. Such analysis also showed, for the first time, the influence (a) of the isotope used to radiolabel the antibody fragments and (b) of the clinical stage of the patients. The present study has shown good agreement in the results obtained by the 10 nuclear medicine departments, suggesting that immunoscintigraphy with radiolabeled F(ab')2 fragments of MoAb 225.28S is a reliable procedure.

Antibodies, Monoclonal↗
PubMed1985

HBeAg/anti-HBe determination with a new monoclonal immunoradiometric assay.

The performance of two assays for the HBeAg/anti-HBe system associated with hepatitis B virus infection, using monoclonal (EBK-Sorin) and polyclonal (HBe-Abbott) antibodies, has been compared. The results on a random population (1,000 samples) demonstrated for the monoclonal reagent a higher sensitivity, without any loss in specificity, and with the further advantage of the use of lower radioactivity levels. The proportion of positives and negatives obtained with the two kits was found to remain unchanged in the case of HBeAg, while a markedly larger percentage of positives (7% higher) was detected for anti-HBe using the monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1985

Imaging with 131I-labeled monoclonal antibodies to a high-molecular-weight melanoma-associated antigen in patients with melanoma: efficacy of whole immunoglobulin and its F(ab')2 fragments.

In vitro experiments selected optimal conditions to radiolabel with 131I the whole immunoglobulin and F(ab')2 fragments of the monoclonal antibody (MoAb) 225.28S to a high-molecular-weight melanoma-associated antigen (HMW-MAA). Injection of the radiolabeled whole immunoglobulin and F(ab')2 fragments of the MoAb 225.28S into eight patients with melanoma resulted in the accumulation of radioactivity in 10 of 18 metastases. This localization is specific because of the close relationship between detection of HMW-MAA in lesions by immunohistochemical techniques and outcome of immunoscintigraphy and because of the different distribution in tumors and adjacent tissues of radiolabeled F(ab')2 fragments of MoAb 225.28S compared with 99mTc-pertechnetate and with radiolabeled F(ab')2 fragments of MoAb 4C4 to hepatitis B surface antigen. F(ab')2 fragments are superior to whole immunoglobulins to perform immunoscintigraphy, since they markedly reduce the background in bone marrow, liver, and spleen. The sensitivity of the procedure allows the detection of lesions with a diameter of at least 1.5 cm and is influenced by the level of the HMW-MAA in lesions and by their anatomical site.

Adult↗
PubMed1983

Applications of bioreactors in medicine.

The use of immobilized enzymes, antibodies or reactive proteins was recently considered as a promising approach for detoxification purposes. The appropriate solutions of some specific problems were described and discussed. An hollow fiber haemodialyzer with a membrane surface area of 0.6 m2 was used as standard support for L-asparaginase immobilization. Hemoperfusion columns based on modified agarose beads were also manufactured for the immobilization of antiphenobarbital antibodies.

Animals↗