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Biomedical subjects

M Mariani

Publications and source records attributed to M Mariani.

At least 163 records · Page 9Linked to original sources
PubMed1992

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.

Antibodies, Monoclonal↗
PubMed1992

Treatment of intracranial human glioblastoma by direct intratumoral administration of 131I-labelled anti-tenascin monoclonal antibody BC-2.

Ten patients with bulky brain glioblastoma, recurring after surgery, radiotherapy or chemotherapy, underwent direct intralesional radioimmunotherapy (RIT) using a monoclonal antibody (MAb), BC-2, raised against tenascin and labelled with 131I. Tenascin, the BC-2-recognized glycoprotein, is an antigen expressed by the stroma of malignant gliomas but not by normal cerebral tissue. Preliminary studies in animals have demonstrated the ability of anti-tenascin radiolabelled MAbs to detect and reduce tumours. A mean MAb dose of 1.93 mg (corresponding to 551.3 MBq of 131I) was injected directly into the tumour by means of a stereotaxic technique. Both systemic and local toxicity were negligible. After 24 hr, average tumour BC-2 uptake was 4.9% per gram and its effective half-life in neoplastic tissue was 66.5 hr: a mean radiation dose to target tissue of 36.48 cGy per MBq of injected 131I was delivered. Normal brain tissue and the major organs were spared. Most patients underwent multiple injections, reaching a cumulative tumour radiation ranging from 7,000 to 41,000 cGy. RIT failed to achieve any result in 4 of the 10 patients; in 3, the disease was stabilized; in the remaining 3, CT scan or NMR revealed 2 partial remission (greater than 50% reduction in tumour volume; PR) and I complete remission (CR). One patient with PR relapsed after II months; the other 2 patients were still maintaining their responses at the time of writing, 17 (CR) and 12 (PR) months after injection.

Adult↗
PubMed1992

Heterogeneity and phenotypic instability of chemotherapeutic and immunologic sensitivity in murine and human melanoma cell clones.

The aim of the present study was to examine the phenotypic heterogeneity of murine and human melanoma cell lines with particular reference to anticancer drug sensitivity, growth pattern and susceptibility to lysis by lymphokine (rIL2) activated killer (LAK cells). Clones selected for a different drug sensitivity were tested to evaluate the stability of such properties after different in vitro passages. A possible relationship between drug sensitivity and LAK susceptibility was also analyzed. The results indicated a high heterogeneity in murine and in human melanoma clones for all the parameters. However, drug sensitivity, which was stable although for only a few passages in an untreated human melanoma, was highly unstable in murine naturally or drug-induced resistant cells. Finally, whereas human drug-resistant clones were sensitive to lysis by LAK cells and an inverse correlation was found with the level of drug resistance, murine clones appeared to be LAK sensitive, and no correlation was found between the level of drug resistance and LAK sensitivity. Our data indicate a different stability in drug response of human and murine cells and a different behaviour of human and murine drug-resistant cells in response to LAK lysis.

Animals↗
PubMed1992

In vitro activity of novel sulphonic derivatives of distamycin A.

The successful growth of tumors is dependent on the process of vascularization elicited by the tumor itself. As confirmed by many authors, there is a correlation between the presence of factors that stimulate tumor growth and angiogenesis. One of the approaches we have explored to control angiogenesis has been to synthesize compounds able to complex growth factors. A number of sulphonated derivatives of distamycin A were found active in inhibiting the binding of bFGF and PDGF beta on Swiss 3T3 cells with ID50 values ranging between 142-587 microM for bFGF and 28-79 microM for PDGF beta. The effect of these new derivatives in inhibiting angiogenesis was initially explored in the chorioallantoic membrane assay. It was observed that the selected compounds were active in this model system at the concentration of 350 nm/pellet. These new molecules present low or no cytotoxic activity on M5076 murine reticulosarcoma cells, the ID50 values being higher than 60 microM after 72 h continuous exposure in vitro.

3T3 Cells↗
PubMed1992

Two-step tumour targetting in ovarian cancer patients using biotinylated monoclonal antibodies and radioactive streptavidin.

A new method for intraperitoneal tumour targetting in ovarian cancer using biotinylated monoclonal antibodies (MoAb) and radioactive streptavidin is described. Fifteen patients with histologically documented ovarian carcinoma were injected intraperitoneally with 2 mg of biotinylated MoAb MOv18, followed 3-5 days later by 100-150 micrograms of indium-111 streptavidin, at the specific activity of 280-370 MBq/mg in 500 ml of normal saline. No toxicity was observed. Tumours were imaged from 2 to 48 h after radioactivity injection by recording both planar and single photon emission tomography (SPET) data. All patients underwent surgery 1-8 days later (mean 3 days) after scanning. The resected tumour and normal tissue radioactivity were measured. On the day of surgery, the tumour to normal tissue ratio was 9:1 (range 3:1-30:1) and 45:1 (range 12:1-120:1) for intra- and extraperitoneal samples, respectively. The mean tumor to blood ratio was 14:1 (range 4:1-30:1). The injected dose (i.d.) per gram of tumour was 0.112 (range 0.01-0.3) for recurrences and 0.05 for primary tumour (range 0.005-0.2). Over 24-48 h 14% i.d. (range 8-18% i.d.) was found in the urine, 14% i.d. (range 6-29% i.d.) in the blood and 63% i.d. (range 56-70% i.d.) was still in the peritoneal cavity. These preliminary clinical data suggest that this two-step strategy may be superior to the conventional approach (radiolabelled antibodies) for intraperitoneal radioimmunolocalization and radioimmunotherapy of ovarian cancer.

Bacterial Proteins↗
PubMed1992

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice.

We prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin. The monoclonal antibody AR-3 was cross-linked to ricin through a thioether bond. Further studies showed that the immunoconjugate suppressed the tumour growth of HT-29 cells in intraperitoneally grafted nude mice, without showing any undesirable ricin toxicity. In this work, to demonstrate the therapeutic activity of the AR-3-ricin conjugate injected into mice bearing subcutaneous tumour, we first evalauted its pharmacokinetic behaviour and biodistribution. The behaviour of the immunoconjugate injected intravenously was almost intermediate between that of the antibody and ricin. Moreover, when the immunotoxin was intravenously administered to nude mice bearing subcutaneous tumour, no therapeutic effects appeared, in accordance with the relatively low permeability of the immunotoxin from the blood to the skin. In contrast, peritumoral treatment produced a strong reduction of the neoplastic nodules without substantial regrowth of the malignant cells. This result was also achieved when the immunotoxin treatment was performed on a well-established tumour. This finding was strictly related to the specifcity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.

Adenocarcinoma↗
PubMed1992

Energy metabolism in myocardial stunning.

We investigated the effect of reversible ischemia, leading to persistent contractile dysfunction (stunning), on myocardial energy metabolism. The balance of energy metabolism is expressed by the phosphorylation state of cytosolic nucleotides. This variable cannot be measured directly because of nucleotide compartmentation, but in the isolated heart it can be estimated by the release of purine catabolites. We have previously shown that increased energy consumption or impaired energy production cause purine release to increase, while primary reduction in energy consumption has the opposite effect. Isolated working rat hearts were reperfused after 10 min of global ischemia, measuring hemodynamic variables, tissue high energy phosphate compounds and purine release. In post-ischemic recovery, aortic flow and minute work decreased to 82 +/- 3% and 77 +/- 4% of control, adenine nucleotide pool was reduced by 4.6 mumol/g dry wt, phosphocreatine to creatine ratio increased significantly and purine release decreased to 42 +/- 6% (P < 0.01). The rate of purine salvage, as evaluated by the incorporation of exogenous 3H-adenosine and 14C-hypoxanthine into tissue nucleotides, was much lower than net purine release, and was unchanged after ischemia and reperfusion. The adenine nucleotide pool could be depleted to the same extent as in the stunned myocardium by prolonged (60 min) aerobic perfusion. In this group the hemodynamic variables were unchanged and purine release averaged 87 +/- 9% of control (P = NS). In other experiments prolonged perfusion was combined with preload reduction in order to decrease energy demand. This protocol reproduced the effects of ischemia-reperfusion: aortic flow and minute work averaged 79 +/- 4% and 73 +/- 9% of control, adenine nucleotide depletion was 4.4 mumol/g dry wt and purine release decreased to 38 +/- 5% (P < 0.01). Our findings support the view that stunning is not due to adenine nucleotide depletion or to impairment in energy production, which would cause purine release to increase, but rather to primary reduction in energy utilization.

Animals↗
PubMed1992

Inhibitory effect of suramin on receptor binding and cytotoxic activity of tumor necrosis factor alpha.

The effect of suramin on the binding of human Tumor Necrosis Factor alpha (huTNF alpha) to specific cell-surface receptors as well as on its cytotoxic activity in vitro was investigated. Suramin inhibited both activities in a dose-dependent manner. Experiments designed to discriminate if suramin exerted its inhibitory activity on the ligand or on the receptor showed that the ligand (huTNF alpha) was the most likely target for suramin in this system. These results may explain, in part, the immunosuppressive activities of suramin that have been observed in vivo and suggest that suramin could be useful in those disease states in which hyperproduction of huTNF alpha has been shown to play a pathogenic role.

Cell Survival↗
PubMed1992

Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells.

The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.

Animals↗
PubMed1992

Production and characterization of murine mAbs to the extracellular domain of human neu oncogene product GP185HER2.

The oncogene HER-2/neu encodes a transmembrane glycoprotein of 185 kDa (gp185HER-2) with tyrosine-kinase activity. Gene amplification and high levels of expression of gp185HER-2 have been found to correlate with poor clinical outcome in breast and ovarian carcinomas. Employing a somatic cell hybrid fusion protocol, which yields a high frequency production of hybridomas, we have analyzed the extent of the murine immune response to the gp 185 extracellular domain. In a single fusion experiment, using as immunogen NIH 3T3 cells expressing high levels of a transfected human HER-2 gene, we have generated mAbs, mainly of IgG1 isotype, displaying high affinity (10(7)-10(10) mol/L) to gp 185. Analysis of the epitope specificity has allowed the identification of five distinct groups of spatially related epitopes, each provided with different immunodominancy, and all resistant to formalin fixation. The use of inhibitor of N-linked glycosylation tunicamicyn has demonstrated that the mAbs bind to epitopes localized in the protein core of gp185HER-2. Because recent reports have shown that gp185HER-2 has a restricted expression in normal tissues and is homogenously detectable in metastatic foci of gp 185 + primary tumors, antibodies to this macromolecule, in addition to their prognostic value, may represent reagents for in vitro and in vivo diagnostic applications, as well as for the development of therapeutic strategies.

Animals↗
PubMed1992

Cardiac A2 adenosine receptors--influence of ischaemia.

OBJECTIVE: The aim was to detect cardiac A2 adenosine receptors through radioligand binding, and to assess the effect of ischaemia on these receptors. METHODS: Isolated working rat hearts were subjected either to aerobic perfusion or to global ischaemia. A membrane fraction was prepared from ventricular tissue, and 3H-5'-N-ethylcarboxamide adenosine (NECA) binding was determined in the presence of N6-cyclopentyl adenosine (CPA). A2 binding was calculated as the fraction of NECA binding displaced by 100 microM CPA but not displaced by 50 nM CPA. RESULTS: Analysis of A2 NECA binding according to single binding site model yielded Kd = 22.0 nM, Bmax = 34.0 fmol.mg-1 in control hearts; Kd = 49.7 nM, Bmax = 44.3 fmol.mg-1 in hearts subjected to 30 min ischaemia (p < 0.05 for difference in Kd). In the control group a two site model provided a significantly (p < 0.05) better fit (Kd = 5.6 and 183.7 nM, Bmax = 9.5 and 64.4 fmol.mg-1 for the high and low affinity sites respectively). The high affinity component of A2 NECA binding disappeared in the presence of the GTP analogue guanyl-5'-yl imidodiphosphate, suggesting the existence of multiple coupling states of the receptor. In the ischaemic group no significant improvement in data fitting was obtained with the two site model. CONCLUSIONS: The results provide evidence of the existence of cardiac A2 adenosine receptors. Ischaemia modifies receptor properties and appears to affect chiefly the high affinity component of A2 binding, possibly by preventing receptor interaction with membrane G proteins.

Adenosine↗
PubMed1992

Beta-endorphin concentration in the femoral and spermatic venous blood of males affected by varicocele.

Several studies in the rat indicate that opioid peptides produced in the testicular interstitial compartment can affect events in the tubular compartment. For example, it is thought that some specific functions of Sertoli cells, such as androgen-binding protein production are decreased by a paracrine mechanism. In this study ACTH beta-endorphin and cortisol were measured in the femoral and spermatic venous blood drawn from 18 patients affected by varicocele during catheterization for venous occlusion. The results showed the absence of a significant secretory gradient of beta-endorphin in the human testis and also demonstrated that this opioid is circulating at picomolar concentrations within human testis under stress conditions.

Adrenocorticotropic Hormone↗
PubMed1992

HLA haplotypes and susceptibility to Graves' disease.

Graves' disease is a polygenic disease in which the HLA cluster could play a role. The purpose of our study is to identify HLA haplotypes in a family with closely related susceptibility to Graves' disease and foresee the risk of disease in the youngest daughter. The family studied had included the father (47 years), mother (46 years) and 3 daughters (18, 17 and 13 years). The mother and 2 eldest daughters were affected by Graves' disease. HLA-A, -B, -C, -DR and -DQ were performed with standard microlymphotoxicity techniques. A mother's role in passing susceptibility to Graves' disease to daughters is undisputed; it seems to be due to the B35 HLA allele. Also, the third daughter (at 15 years) has an HLA B35 allele, and actually has an incipient humoral hyperthyroidism.

Adolescent↗
PubMed1992

Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

Intratumor heterogeneity for susceptibility to cytotoxic T lymphocytes (CTL)-mediated lysis represents a major obstacle to cancer adoptive immunotherapy. To overcome the heterogeneity observed in terms of susceptibility of target cells to cell-mediated lysis, in this study we used two purified bispecific monoclonal antibodies (bsmAbs) that recognize molecules expressed by cytotoxic effector cells (CD3 and IgG Fc receptorial molecules), as well as one high molecular weight melanoma-associated antigen (HMW-MAA). The ability of these reagents to enhance or induce a relevant in vitro cytotoxic activity by a CTL clone (CTL 49) isolated from PBL of a melanoma patient was tested on a large panel of autologous and allogeneic melanoma cell lines and clones. Functional studies revealed that the CTL 49 clone lysed all the HMW-MAA+ tumor lines in the presence of bsmAbs and that these reagents affected the target lysis in a cooperative fashion. The effectiveness of bsmAbs in overcoming the heterogeneous susceptibility of human melanoma cells to cell-mediated lysis may find practical implications in cancer adoptive immunotherapy.

Animals↗
PubMed1992

Effect of gallopamil on cardiac sarcoplasmic reticulum.

We investigated the effect of gallopamil on cardiac sarcoplasmic reticulum (SR) function. Heavy SR was prepared from bovine ventricular muscle. Oxalate-supported calcium uptake was stimulated by gallopamil at concentrations ranging from 10 to 300 nM, whereas higher concentrations were ineffective. Peak stimulation averaged 25-30% of control calcium uptake and was observed at free calcium concentrations ranging from 1 to 6 microM. Calcium uptake is actually the difference between active calcium transport by SR calcium-adenosine triphosphate (calcium-ATPase), and passive efflux through SR calcium-release channels. In the presence of 300 microM of ryanodine, a blocker of SR channels, calcium uptake increased by 43% under control conditions, but not further stimulation was produced by gallopamil. SR calcium-ATPase was not affected by gallopamil. Similar results were obtained when oxalate-supported calcium uptake was determined with use of unfractionated homogenate obtained from rat hearts. We conclude that gallopamil acts on SR calcium-release channels and reduces the probability of channel opening and/or channel conductivity. The dose-response curve is bell shaped, and the maximum effect, which corresponds to 65% of the maximum effect of ryanodine, is achieved at therapeutic concentrations. Such action might contribute to the beneficial effect of gallopamil in the treatment of myocardial ischemia.

Animals↗
PubMed1992

[Variations in the purine metabolism of the reperfused heart].

Isolated hearts were subjected to 30 min of aerobic perfusion followed by 10 min of global normothermic ischemia and 40 min of reperfusion. We determined the release of purine catabolites (adenosine, inosine, hypoxanthine, xanthine, uric acid) and the incorporation of exogenous 3H-adenosine and 14C-hypoxanthine into cellular nucleotides. Ischemia-reperfusion produced remarkable reduction in the release of purine catabolites, with no significant variation in the incorporation of adenosine and hypoxanthine.

Analysis of Variance↗
PubMed1992

[Spectrum analysis of heart rate variability in obstructive hypertrophic myocardiopathy. Evidence of altered autonomic function].

Altered sympathetic activity may play an important role in the pathogenesis of hypertrophic obstructive cardiomyopathy (HOCM). Spectral analysis of heart rate variability was employed to assess the sympatho-vagal function and balance in 18 patients with HOCM (11 males, 7 females, mean age 42 years, range 19-59) and in 15 healthy control subjects (9 males, 6 females, mean age 44 years, range 18-65). Electrocardiographic recordings obtained both at rest and during 60 degrees passive tilt, were digitized and analyzed by fast Fourier transform in order to obtain the power spectrum of heart rate variability. The low-frequency band (LF: 0.05-0.17 Hz) and the high-frequency band (HF: 0.18-0.34) of power spectrum were considered as indexes of sympathetic and vagal activities respectively. A semiquantitative two-dimensional echocardiographic score (SES) was used to assess the entity of myocardial hypertrophy whereas the entity of the intraventricular gradient was determined by continuous wave Doppler. Low-frequency band at rest was slightly but significantly reduced in HOCM group with respect to controls (35.2 +/- 2.0 vs 45.0 +/- 2.5 nu, respectively; p < 0.01), whereas the HF band and the LF/HF ratio were not different in the 2 groups. During tilt, control subjects showed a significant reduction of the HF band (-35%, p < 0.001), an increase in the LF band (+36%, p < 0.001) and a sharp increase in the LF/HF ratio (+105%, p < 0.001). On the contrary the baroreflex increase in the LF band and LF/HF ratio during tilt was markedly blunted, or even reverted, in patients with HOCM (-9%, NS and +5%, NS, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗