Biomedical subjects

I Moraru

Publications and source records attributed to I Moraru.

At least 19 recordsLinked to original sources

PubMed1994

Two inositol 1,4,5-trisphosphate binding sites in rat basophilic leukemia cells: relationship between receptor occupancy and calcium release.

Quantal calcium release is a novel paradigm for second messenger signal transduction which provides spatial and temporal control of calcium release from intracellular stores by inositol 1,4,5-trisphosphate (InsP3). We have proposed a mechanism to account for this phenomenon [Kindman, L. A., & Meyer, T. (1993) Biochemistry 32, 1270-1277], which hypothesized the existence of five channels, each with a different affinity for InsP3. As a direct test of this hypothesis, InsP3 binding to microsomes from RBL cells was examined under conditions similar to those used for calcium release. Scatchard analyses performed under a variety of conditions indicates the presence of high affinity (KD = 0.9 +/- 0.3 nM) and low affinity (KD = 47 +/- 5 nM) InsP3 binding sites. The low affinity sites are more prevalent, constituting 82 +/- 5% of the total. Both sites are identified in the presence and absence of MgATP. Moreover, both sites are selective for InsP3 over InsP4, through high concentrations of InsP4 displace InsP3 from each site (with inhibition constants of 16 and 267 nM InsP4, respectively). The relative abundance of the two InsP3 binding sites is Ca2+ dependent. An increase in Ca2+ from 0.1 to 0.5 microM results in the apparent conversion of a portion of the low affinity sites into high affinity sites into high affinity sites. Ca2+ (0.5 microM) also increased the KD of the low affinity InsP3 binding site. Given the presence of both high and low affinity InsP3 binding sites, two simple mathematical models describing both the kinetics of calcium release and quantal calcium release from RBL cells were developed.(ABSTRACT TRUNCATED AT 250 WORDS)

2,3-Diphosphoglycerate↗

PubMed1990

Hairy cell leukemia. Immunological study.

The study has been performed on peripheral blood and splenic malignant cells from 16 patients with hairy cell leukaemia (HCL). The cell surface markers were identified by rosette techniques and using monoclonal antibodies (m Ab). The surface markers' expression of the hairy cells (HC) varied. The E receptors, the T-cell antigens, the HLA-DR antigens and smIgG were either expressed or not according to the affected organ, the progress of illness, or the treatment. The surface pattern changed sometimes in the same patient during the progress of illness. These observations demonstrate that HCL is a unique disease with malignant cells characterized by a marked variability of the cell surface markers. To demonstrate the ability of hairy cells to bind labile smIgG, the cells were studied by affinity chromatography on SpA-Sepharose 6MB and by ES-rosette assay. The percent of cells bound on SpA-Sepharose varied between 6% and 66%, representing the hairy cells with labile-bound smIgG. With affinity chromatography it was also possible to separate the hairy cells with a special phenotype: T3+ T4+ T8+ T11+ surface membrane labile-bound IgG+ (11gG+) FcR+, HLA-DR+ EACD+ (Ripley rosette forming cells), resembling a normal subset of large granular lymphocytes (LGL). The percentage of these cells varied between 60% and 86% of the bound cells. These observations suggest that in HCL, the malignant transformation might involve a common progenitor for the B, T and LGL lineages, the hairy cell being a hybrid type of malignant cell. Its main immunological peculiarity is the marked mobility of the surface membrane structures and hence the lability (plasticity) of the surface markers' expression.

Adult↗

PubMed1990

Treatment of murine EL4 leukemia in ascitic form with anti-Thy 1.2 specific immunotoxins.

C57BL/6 mice with EL4 leukemia cells in ascitic form were intraperitoneally treated with ricin A chain-multivalent antibody immunotoxins. The immunotoxins containing rabbit IgG anti-Thy 1.2 antibodies complemented by protein A of Staphylococcus aureus were able to interact specifically with the target cells and to induce an antitumor effect as revealed by an increase in survival time of the mice. No apparent secondary effects consecutive to a cytotoxic action on the normal Thy 1.2 antigen bearing cells were observed with the immunotoxin doses used.

Animals↗

PubMed1989

Experimental model for testing the efficiency of immunotoxins administered in vivo: evaluation of two ricin A-chain--multivalent antibody immunotoxins.

Two immunotoxins, the ricin A chain-multivalent hybrid antibody (750 kDa) and the complex A chain-staphylococcal protein A-rabbit IgG antibody (370 kDa), were prepared. A simple method was elaborated to test the immunotoxins' efficiency in selectively killing target cells in tumor-bearing mice. The target cell (murine EL4 leukemia) was coated with a xenogenic molecule by a method conserving its ability to proliferate and kill the inoculated animals. When the challenged animals were treated with these immunotoxins, which were specific for the antigenic molecule coating the tumor cells, survival time was lengthened compared with that of untreated animals, corresponding to a proportion of over 90% cells killed. This demonstrates the efficiency of the immunotoxins and the validity of the method elaborated.

Animals↗

PubMed1989

A particular case of large granular lymphocytes lymphoma.

A patient with non-cutaneous lymphocytic lymphoma with leukaemic spread is reported. The large majority of the peripheral blood mononuclear cells formed rosettes with sheep erythrocytes (E), had receptors for the Fc portion of IgG (Fc gamma R) and displayed an unusual enzymatic profile. Part of these cells had also labile bound surface membrane IgG. The leukaemic cells were morphologically characterized as large granular lymphocytes (LGL) and consisted of two different types of cells: lymphoid cells and monocytoid cells. The histopathological diagnosis was T-cell lymphoma, pleomorphic type, diffuse. Despite their morphological heterogeneity, all the proliferative cells had the same immunological phenotype, showed normal ADCC activity but no NK activity. After three months, without specific treatment, the surface pattern changed: the cells resembled mature LGL and the NK activity increased. The cytochemical study of the leukaemic cells revealed an enzymatical peculiarity: besides an enzymatic profile characteristic for relatively mature LGL, the cells also displayed peroxidase activity. This unusual aspect--the expression by an individual malignant cell of markers believed to be restricted to a single cellular lineage--might be interpreted as a lineage infidelity or lineage promiscuity. The observation that this lymphoma developed in a patient with a long history of hypogammaglobulinaemia is of particular interest.

Adult↗

PubMed1988

Use of glutaraldehyde-treated erythrocytes as indicator cells for the identification of the Clq component on the macrophage membrane by a rosette assay.

Glutaraldehyde-treated sheep red blood cells (EGA) form rosettes with macrophages of various origin. The binding of EGA to cell surface is mediated by the membrane associated Clq as demonstrated by the inhibition of EGA rosette formation either after treatment of macrophages with anti-Clq serum or after treatment of EGA with Clq. The results clearly demonstrate that the EGA-binding receptor of macrophage is the Clq component.

Aldehydes↗

PubMed1988

The Fc receptor for IgA expression and affinity on lymphocytes and macrophages.

The interaction between the Fc receptor for IgA (FcR alpha) and human secretory IgA (sIgA) was studied in a heterologous system by using rabbit alveolar macrophages or splenic lymphocytes. The capacity of FcR alpha-bearing cells to interact with the ligand via the Fc region was demonstrated. The proportion of FcR alpha-bearing lymphocytes and macrophages was found to be 11 and 17%, respectively. The cell-bound radiolabelled ligand showed a release half-time of 70 min for lymphocytes and 45 min for macrophages. The equilibrium constant (K) and the maximum number of ligand molecules bound per cell (n) in mean values was 5.2 +/- 0.05 X 10(8) M-1 and 3.3 +/- 0.15 X 10(5) molecules per cell, respectively. Increased percentages of FcR alpha-bearing cells and of ligand density per cell were obtained after stimulation by lung inflammation. A significant increase of the K value was recorded with macrophages from rabbits with lung acute inflammation (mean value 7.3 +/- 0.21 + 10(8) M-1) while the increase with lymphocytes was not significant. The calculation method of K and n is discussed.

Animals↗

PubMed1988

Protein A vectorized toxins--II. Preparation and "in vitro" cytotoxic effect of protein A-ricin A chain conjugate on antibody coated human tumour cells.

Protein A of Staphylococcus aureus was covalently bound to reduced ricin A chain toxin by N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate consisting mainly of one molecule of protein A bound to two molecules of A chains (Mr 107,000) was purified by tandem affinity chromatography on ConA-Sepharose 4B and IgG-Sepharose 4B. The purified protein A-A chain conjugate was able to bind and kill human lymphoma cells coated either with monoclonal mouse IgG2a anti-kappa antibody or with polyclonal rabbit anti-kappa antibody. The cytotoxic activity of protein A-A chain conjugate in conjunction with either mouse or rabbit anti-kappa antibodies was 10 times higher than that of rabbit IgG anti-mouse IgG coupled with A chain on Daudi cells coated with mouse anti-kappa antibody and 100 times higher than that of rabbit anti-kappa antibody coupled with A chain on non-coated Daudi cells. The cytotoxic effect of protein A-A chain conjugate on antibody-coated Daudi cells (9 x 10(-12) M) was comparable with that of ricin toxin on non-coated Daudi cells (2 x 10(-12) M). The results recommend the use of protein A-ricin A chain toxin conjugate as a unique specific toxin for the "in vitro" killing of antibody-coated target cells.

Burkitt Lymphoma↗

PubMed1988

In vivo follow-up of the cytotoxic effect of protein A--ricin toxin conjugate, on antibody-coated target cells.

Antibodies of the IgG class, specifically interacted with H-2 antigens of murine leukemia EL4 cells, were used to bind the ricin toxin covalently linked to protein A of Staphylococcus aureus. The toxin thus complexed, introduced in the cytoplasm by endocytosis, was able to kill the leukemic cells inoculated in animals. The interaction of immunotoxin with the leukemic cells was performed in vitro using one, two or three treatments and the cytotoxic effect on the target cells was followed up in vivo. The time interval between immunotoxin treatments was indicated by the membrane turn-over study of EL4 cells coated with specific antibodies in their monomeric form, complexed by protein A or interacted with protein A--ricin toxin conjugate. A proportion of 99.8% cells killed was obtained after three treatments.

Animals↗

PubMed1988

In vivo follow up of the cytotoxic effect of ricin toxin vectorized by multivalent hybrid antibody on target cells.

A multivalent hybrid antibody complex composed of two IgG molecules specific for ricin toxin and two specific for the H-2 antigens of murine leukemia EL4 cells, cross-linked by SpA, was used as vector of the toxin to the target cells. The high affinity of the hybrid antibody for the specific antigens achieved an efficient attachment to the EL4 cell membrane and binding of ricin toxin; this high-molecular-weight complex, introduced by endocytosis into the leukemic cell cytoplasm, was able to specifically deliver the toxin to the target cells. The effect of multivalent hybrid antibody-vectorized toxin was followed up in vivo. This method enabled determination of the proportion of killed cells (over 90% after a single treatment of leukemic cells or about 99% after double treatment). The presence of a low proportion of tumoral cells maintaining their proliferative capacity is discussed.

Animals↗

PubMed1987

Binding and cytotoxic effect of ricin toxin on multivalent hybrid antibody-coated target cells.

Multivalent hybrid antibodies with dual specificity were prepared by cross-linking with protein A two antibodies of different specificity, one against ricin toxin and the other against the H-2 antigens of murine leukemia EL4 cells. This bifunctional antibody specifically attached to EL4 cells was able to capture ricin toxin (RcA2) molecules with an affinity 20 times higher than that of the galactose-containing glycoproteins of the cell surface (nonspecific binding). The hybrid-bound RcA2 gained access into the target cell cytoplasm by endocytosis and blocked [3H]thymidine incorporation as efficiently as free RcA2 does on nontreated EL4 cells (3.3 X 10(-11) M). These results indicate that multivalent hybrid antibody, easy to prepare in purified form and endowed with high affinity for both target cell and ricin toxin, may be utilized efficiently for the specific delivery of toxins to target cells.

Animals↗

PubMed1986

Protein A vectorized toxins--I. Preparation and properties of protein A-ricin toxin conjugates.

Protein A of Staphylococcus aureus (mol. wt 43,000) was covalently bound to ricin toxin (mol. wt 60,000) by N-succinimidyl 3-(2-pyridyldithio) propionate. The conjugate consisting of one molecule of protein A bound to one molecule of ricin toxin (mol. wt 100,000) was purified by successive affinity chromatographies on IgG-Sepharose 4B and ConA-Sepharose 4B. The purified protein A-ricin toxin conjugate was able to bind and kill IgG antibody coated leukemia EL4 cells leaving unaffected EL4 cells not coated with antibody. The cytotoxic efficacy of the conjugate was comparable to that of nonconjugated ricin toxin. The results recommend the use of protein A-ricin toxin conjugate as a "universal" specific toxin for the "in vitro" killing of various antibody-coated target cells.

Animals↗

PubMed1986

Modulation of IgG effector functions by a monovalent fragment of staphylococcal protein A.

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.

Animals↗

PubMed1986

The "in vivo" effect of protein A on tumor growth.

The antitumor effect of two purified preparations of protein A obtained from Staphylococcus aureus strain Cowan 1 or the mutant strain A676 was investigated. This study using different doses of SpA (5 or 10 mg/animal) i.p. administered was performed in lymphosarcoma bearing rats at certain intervals of time after tumor transplantation. The treatment with SpA deriving from A676 strain which was started at the same time with the tumor transplantation led to a significant increase in the latency period of tumor growth and the prolongation of survival time of tumor bearing rats. Attempts to use a higher dose of SpA and also a smaller tumor inoculum did not improve the results with regard to inhibition of lymphosarcoma growth in inbred R rats.

Animals↗

PubMed1985

Radioimmunoassay of IgG and IgM anti-albumin autoantibodies in human serum.

An antigen-coated plate radioimmunoassay used to detect autoantibodies of IgG and IgM classes against human serum albumin is described. It comprises 3 steps: (a) adsorption of glutaraldehyde-polymerized human serum albumin on the wells of a plastic plate, (b) incubation of the serum to be tested with the antigen in the wells, and (c) the addition of radiolabelled antibodies against human IgG or IgM. The method has been used to estimate the level of anti-albumin autoantibodies (AAA) belonging to IgG or IgM classes in the sera of normal subjects and patients with liver diseases.

Autoantibodies↗

PubMed1985

Mutual inhibition of the binding of Clq and protein A to rabbit IgG immune complexes.

A complex of rabbit IgG antibody with horseradish peroxidase covalently linked to Sepharose 4B was used as an insoluble immune complex for studying the binding of complement factor C1q protein A from Staphylococcus aureus, and its IgG-binding fragments AB and B, to rabbit IgG. It was shown that protein A (mol. wt approx. 42,000) and fragments AB and B (mol. wts approx. 14,000 and 7000, respectively) inhibited the binding of C1q to insoluble immune complex at 4 degrees C. However, at 37 degrees C fragment B did not inhibit this binding. On the other hand, C1q, when bound to an insoluble immune complex, almost completely blocked the binding of protein A and fragment B at both temps. The higher affinity of C1q for its CH2-binding site than of fragment B for its CH2-binding site may explain the displacement of the latter from the CH2 domain. The mutual inhibition of the binding of C1q and protein A (and its smaller fragments) indicates that the binding sites for C1q and protein A are closely located in the CH2 domain.

Animals↗

PubMed1985

Detection of human lymphocyte subpopulations by using monoclonal antibodies and protein A-coated erythrocytes.

The results obtained by using the protein A-rosette assay for human peripheral blood lymphocytes treated with a series of monoclonal antibodies were very close to the estimates obtained by indirect fluorescence. The percentages of T3-, T4-, T8- and DR-positive cells (T3 = T lymphocytes, T4 = helper T lymphocytes, T8 = suppressor T lymphocytes, DR = B, L and null cells) in normal and pathological peripheral blood were found to vary within the limits reported by many other authors using different techniques for the identification of cells binding the specific monoclonal antibody.

Animals↗

PubMed1984

The isomyosins of rabbit middle ear muscles.

Isomyosins of the two middle ear muscles of rabbit have been separated by electrophoresis in non-dissociating conditions. Tensor tympani has four isomyosins called MM1 to MM4 by order of decreasing mobility. Stapedius has two isomyosins, similar to MM1 and MM4. MM1 and MM2 have the same mobility than FM2 and FM3, fast isomyosins observed in psoas. MM4 has the same mobility than IM, a slow isomyosin observed in extensor digitorum longus. MM3 is absent in soleus, psoas or extensor digitorum longus. Slow myosin (SM) of soleus and fast myosin (FM1) of psoas are not observed in middle ear muscles.

Animals↗