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R Lemieux

Publications and source records attributed to R Lemieux.

At least 37 records · Page 2Linked to original sources

An expectation maximization reconstruction algorithm for emission tomography with non-uniform entropy prior.

A Bayesian image reconstruction algorithm is proposed for emission tomography. It incorporates the Poisson nature of the noise in the projection data and uses a non-uniform entropy as an a priori probability distribution of the image in a maximum a posteriori (MAP) approach. The expectation maximization (EM) method was applied to find the MAP estimator. The Newton-Raphson numerical method whose convergence and positive solutions are proven, was used to solve the EM problem. The prior mean at iteration k was determined by smoothing the image obtained at iteration k-1. Comparisons between the ML and the MAP algorithm were carried out with a numerical phantom that contains a narrow valley region. The ML solution after 50 iterations was chosen as the initial solution for the MAP algorithm, since the global performance of the ML algorithm deteriorates with increasing number of iterations while its local performance in the valley region is always improving. The resulting algorithm is a compromise between ML who has the best local performance in the valley region and the MAP who has the best global performance.

Algorithms↗

Use of hu-IgG-SCID mice to evaluate the in vivo stability of human monoclonal IgG antibodies.

Human and in vitro modified mAbs such as humanized rodent mAbs and immunotoxins are now considered for a variety of applications in humans. The adequate in vivo stability of these Ig preparations is not easily predicted from in vitro studies and may be essential for many therapeutic applications. In this study, we report the development and characterization of an in vivo model for testing this parameter using SCID mice containing a physiological concentration of human IgG (hu-IgG-SCID). The model was tested with several IgG1 and IgG3 human mAbs reacting with the human Rh(D) red cell antigen. It is known that human IgG have a shorter half-life in SCID mice than in humans. However, our results showed that the half-life of IgG3 mAbs (1.5 +/- 0.5 days) was much shorter than the one of IgG1 mAbs (5.8 +/- 1.4 days), indicating that the relative stability of IgG1 and IgG3 human mAbs in hu-IgG-SCID mice is similar to the one previously reported in humans (21 days vs. 7 days respectively). The IgG catabolism rate in humans is known to be inversely proportional to serum IgG concentrations. Accordingly, the dilution of the mAbs in a large excess (200-fold) of human IgG was found to be an important parameter of the hu-IgG-SCID mouse model since much longer (3-4-fold) mAb half-lives were obtained in the presence of a lower dose or in the absence of co-injected human IgG. This study show the usefulness of this animal model for the evaluation of human antibody stability in an in vivo environment.

Animals↗

Type 2 T-cell-independent murine immune response to the human AB0 blood group antigens.

The human AB0 blood group determinants are simple carbohydrate structures which are widely distributed in nature. Much work has been done on the structure of the A and B antigens but little is known on the regulation of anti-A and anti-B immune responses. To develop a model system, we have characterized the AB0 immunity of normal Balb/c mice and found a significant level of serum natural anti-A but almost no anti-B. This finding and the known IgM predominance among immune anti-A produced in the Balb/c mouse indicate that the AB0 immunity of this mouse strain is comparable to the one of human blood group B individuals. Following immunization with human red blood cells, similar levels of anti-A were produced in normal and athymic Balb/c mice showing that the anti-A response is T cell independent. Furthermore, no anti-A or anti-B antibodies were produced in CBA/xid mice indicating a type 2 T-cell-independent immune response. These results may contribute to a better understanding of human AB0 responses and establish the mouse as a suitable model to study the immunobiology of AB0 antigens.

ABO Blood-Group System↗

Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system.

The use of peripheral B lymphocytes in the successful preparation of human monoclonal antibodies by hybridoma technology is highly dependent on lymphocyte activation procedures. We studied the ability of peripheral human B lymphocytes cultured in vitro and activated through their CD40 antigen (CD40 system) (Banchereau et al., 1991) to form antibody-secreting heterohybridomas after fusion with murine X63Ag8.653 myeloma cells. The frequency of antibody-secreting heterohybridomas formation was greatly increased (15 times) by culture of B cells in the CD40 system. The CD40 system offers many advantages over other procedures of B lymphocyte activation representing a significant technological advance in the preparation of human monoclonal antibodies by standard hybridoma technology.

Animals↗

Rapid apoptotic cell death of B-cell hybridomas in absence of gene expression.

Apoptosis is an active form of cell death which plays an important role in different biological processes. The induction of apoptosis usually requires de novo gene expression but has also been observed in certain types of cells in absence of gene expression or following a block of gene expression. We show here that inhibition of macromolecular synthesis induced the rapid apoptotic death of most antibody-secreting B-cell hybridomas. The effect was observed in presence of both protein synthesis (cycloheximide [CHX]) and transcription (actinomycin D [Act D]) inhibitors and was characterized by extensive degradation of nucleic acids (DNA and RNA) within 2-3 h of treatment. The CHX treatment not only severely impaired the proliferation of the cells but also resulted in the loss of cell viability (MTT assay) without the need of de novo gene expression. The susceptibility to apoptosis varied among different B-cell hybridomas and was inherited from the SP2/0 myeloma cell fusion partner. These results indicate the constitutive activation of a death program in B-cell hybridomas and its inhibition at a late stage by the continuous expression of gene(s) coding for short-lived protein(s). The occurrence of this phenomenon may well be related to the abundant and deregulated (translocation) expression, in this type of cells, of the c-myc gene which has recently been shown to be a potent inducer of apoptosis in growth-arrested fibroblasts.

Animals↗

Essential role of optimal protein synthesis in preventing the apoptotic death of cultured B cell hybridomas.

The monoclonal antibody productivity of cell culture systems is strongly dependent on the maintenance of hybridoma cell viability. We report that partial (< 50%) and transient (3 h) inhibition of protein synthesis by cycloheximide or deprivation of an essential amino acid induces apoptosis (programmed cell death) in B cell hybridomas. This unusual mechanism of apoptosis induction is likely to play a significant role in limiting cell viability in batch and perfusion cultures of hybridomas and emphasizes the importance of constantly maintaining a near optimal rate of macromolecular synthesis by optimization of all culture parameters. Inhibition of apoptosis in hybridomas by cell engineering and other technologies should permit, in the near future, a significant increase in the antibody productivity of existing cell culture systems.

Animals↗

Direct agglutination of weak D red cells by tetramolecular complexes containing monoclonal IgG anti-D.

The capacity of blood group antibodies to agglutinate red cells suspended in saline is largely dependent on the antibody isotype. The immunological cross-linking of IgG antibodies has previously been described as a means to increase the reactivity of IgG in many situations. We have prepared anti-D-containing complexes by blending a human IgG anti-D monoclonal antibody (mAb) and a murine anti-human IgG mAb. In standard red cell serology assays, the anti-D complexes exhibited a very high avidity and could agglutinate weak D-positive red cells in direct saline testing. These results indicate that potent saline hem-agglutinating reagents of RhD and eventually of other blood group specificities can be prepared from IgG mAbs.

Animals↗

Functional cooperation among human IgG-specific murine monoclonal antibodies for the detection of weak blood group antibodies in routine agglutination tests.

Seven monoclonal antibodies (MAbs) selected from a large panel of human IgG-specific murine MAbs were characterized serologically and studied for their ability to cooperate in routine antihuman globulin agglutination tests. In binding inhibition experiments, three of these MAbs were shown to bind simultaneously to immobilized human IgG molecules. Cooperation among these MAbs increased significantly the capacity of the individual MAbs to agglutinate red cells sensitized with weak blood group antibodies. These results demonstrate the usefulness of selected MAb blending for the preparation of potent antihuman IgG reagents from murine monoclonal antibodies.

Agglutination Tests↗

Production and characterization of anti-DNA-RNA monoclonal antibodies and their application in Listeria detection.

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, cDNA-mRNA and phi X174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs were isolated. The 20D3 MAb, generated with the phi X174 DNA-RNA hybrid, showed association constants of 1.05 x 10(12), 2.12 x 10(10), and 1.68 x 10(7) for the antigens phi X174 DNA-RNA, cDNA-mRNA, and poly(rA)-poly(dT), respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 x 10(5), 5 x 10(12), and 7.1 x 10(8) for the above-described antigens, respectively. With the 20D3 MAb, an immunoassay was developed for the detection of Listeria DNA-RNA hybrids. In brief, a biotinylated rRNA gene probe specific for the genus Listeria was hybridized with rRNA in the solution phase. The hybrids thus formed were then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-target hybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specific for the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.

Animals↗

A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.

Amino Acid Sequence↗

Negative effect of multiple antigen injections on the yield of murine monoclonal antibodies obtained by hybridoma technology.

One of the critical steps in the preparation of monoclonal antibodies is the obtainment, by in vivo immunization and boost, of the maximal number of antigen-activated B lymphocytes. In hybridoma laboratories, the common procedure is to immunize a group of mice with several antigen injections and use the mouse showing the highest serum antibody titre for the fusion experiment. The observation that the use of mice hyperimmunized with human red blood cells failed to yield a high number of monoclonal antibodies, led us to study the effect of multiple antigen injection prior to the fusion experiment. The results obtained showed that the maximal yields of monoclonal antibodies were obtained using mice that had received only one or two antigen injections while the mice immunized with three antigen injections consistently yielded at least a three-fold reduction in the number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction of a tolerance/suppression state which can prevent the final activation step. These results point out to the importance of avoiding the hyperimmunization of mice for the preparation of a high number of monoclonal antibodies by standard hybridoma technology.

Animals↗

Tolerance to intravenous nitroglycerin in patients with congestive heart failure: role of increased intravascular volume, neurohumoral activation and lack of prevention with N-acetylcysteine.

To better understand the mechanism of nitrate tolerance in patients with congestive heart failure, 13 patients received a 24 h infusion of nitroglycerin (1.5 micrograms/kg body weight per min) with or without N-acetylcysteine (225 mg/kg per 24 h). The infusions were separated by a 24 h nitrate-free interval. By the end of the nitroglycerin infusion, mean arterial pressure had returned to baseline values and there was a significant increase in ventricular filling pressures and systemic vascular resistance compared with values after 1 h of treatment. The simultaneous infusion of N-acetylcysteine had no effect on these changes. Although a strict fluid restriction of 1.5 liters/day was maintained for 1 week before and throughout the study, after 24 h of nitroglycerin infusion there was a significant and similar degree of hemodilution whether nitroglycerin was infused alone (9.1 +/- 4.3%) or with N-acetylcysteine (8.7 +/- 4.1%). This hemodilution corresponded to an increase in intravascular volume of 745 +/- 382 ml, most of which occurred during the 1st h. Plasma renin activity increased and plasma atrial natriuretic peptide decreased during the infusion. The results of this study suggest that nitrate tolerance is multifactorial. In addition to the previously described pharmacologic tolerance to the effect of nitroglycerin on vascular smooth muscle, a capillary fluid shift from the extravascular to intravascular space appears to be involved, especially during the 1st h of the infusion. A third mechanism, reflex neurohumoral activation, also seems to contribute to the genesis of nitroglycerin tolerance.

Acetylcysteine↗

Noninvasive evaluation of supraventricular tachycardias.

In this article we discuss the role of noninvasive methods in evaluation of supraventricular tachycardias. The limitation of Holter monitoring and exercise testing is discussed. A significant portion of the article is devoted to the role of esophageal recording, body surface potential mapping, and phase image analysis, areas that are often underutilized but that have potential in the diagnosis of supraventricular tachycardias.

Atrial Flutter↗

Increased proportion of B cell hybridomas secreting monoclonal antibodies of desired specificity in cultures containing macrophage-derived hybridoma growth factor (IL-6).

The addition of macrophage feeder cells or conditioned medium has been shown to increase the yield of murine hybridomas obtained after the fusion of myeloma cells and activated B lymphocytes. It has been shown recently that the conditioned medium contains a growth factor (HGF) active on newly formed hybridomas and that the human HGF is similar to B cell stimulatory factor 2 which can induce the synthesis of antibodies in transformed B cells. We have compared in several fusion experiments the stimulatory effects of HGF both on the yield of hybridomas and on the number of antibody-secreting hybridomas. The results obtained clearly showed that while the stimulatory effect of HGF on the yield of growth-positive wells was variable and sometimes barely detectable, the proportion of growth-positive wells containing monoclonal antibodies was consistently much higher in the HGF-containing cultures. These results suggest that the majority of the antibody-secreting newly formed hybridomas are sensitive to HGF and indicate that HGF is a very useful culture supplement for the generation of a high number of antibody-producing hybridomas even if it may not increase significantly the yield of viable hybridomas.

Animals↗

New methodology for specific inhalation challenges with occupational agents in powder form.

Numerous agents in powder form (wood dust, flour, antibiotics, drugs, etc.) can cause occupational asthma. The diagnosis is generally confirmed by specific inhalation challenges in a special challenge room. There are several pitfalls to the procedure: 1) subjects may be exposed to high concentrations of particles; the concentrations of particles may be higher than the threshold limit value-short term exposure level (TLV-STEL), possibly resulting in severe or irritant reactions; 2) the exposure is erratic. To overcome these problems, a new device for aerosolization of powders has been developed. This apparatus consists of three parts: a particles generator, an aerosol delivery system connected to an orofacial mask, and monitors - a photometer and a cascade impactor. Information on the concentration and size distribution of inhaled particles can therefore be obtained. We performed specific inhalation challenges with this apparatus on 20 subjects suspected of having occupational asthma. The concentration of particles was generally below or close to the TLV-STEL and information was obtained on the proportion of particles with a diameter less than 10 mu. Falls in forced expiratory volume in one second (FEV1) were progressive with each increase in the duration of exposure. The five subjects who had negative reactions to exposure were asked to tip the relevant product from one tray to another in the traditional realistic way. All had negative responses. We conclude that this new procedure offers advantages over the traditional method as it gives information on the concentration and size distribution of inhaled particles and makes the drawing of a dose-response curve possible, both of which may improve the safety and accuracy of the test.

Adult↗

Characterization of higher avidity monoclonal antibodies produced by murine B-cell hybridoma variants selected for increased antigen binding of membrane Ig.

Somatic mutation in the Ig genes plays a major role in the increase of antibody affinity observed in secondary immunologic responses. It has been shown that the mechanism responsible for the high rate of somatic mutation in the Ig genes was active not only in normal B lymphocytes but also in B-cell hybridomas secreting mAb. Also, it has been reported that B-cell hybridomas were positive for membrane Ig of the same specificity as the secreted mAb. The presence of membrane Ig suggested that somatic variants secreting mAb of higher affinity could be selected by the increased capacity of these hybridoma cells to bind immobilized Ag. This hypothesis was tested with hybridoma cells secreting an IgM mAb reacting with the A Ag of the ABO blood group system. In two selection experiments, we have isolated several variant cell lines secreting mAb of increased avidity for the A Ag under similar IgM concentrations. Biochemical characterization of one of the variant mAb indicated that the mutation responsible for the increased avidity has occurred in the heavy chain gene. The method developed may have profound implications for the diagnostic and therapeutic use of mAb and will permit the study, in an in vitro system, of the role of somatic mutations in antibody diversity.

Animals↗