Quality and safety of bovine clones and their products.

A multidisciplinary research programme was developed to get a scientific expertise for the quality assessment of products obtained from cloned livestock. Thirty-seven bovine Holstein female clones of five different genotypes and their products were analysed in comparison with 38 control animals obtained by conventional artificial insemination and raised under the same conditions at the same experimental farm. Animal evaluation included over 150 criteria and more than 10 000 measurements to check the physiological status and health over a 3-year period. All the parameters studied were in the normal range for age and breed, but some significant differences were detected between clone and control groups in terms of delayed onset of puberty in clones, higher neutrophil counts in haematology or lower biochemical plasma concentrations of gamma glutamyl transferase. Milk and meat analyses were conformable to expected values. We, however, found some differences in fatty acid (FA) composition of milk and muscle suggesting a possible deviation in lipid metabolism as assessed by higher delta-9 desaturase activity indexes in both milk and muscles from clones compared with controls. Repeated muscle biopsies in the semitendinosus muscle of the same animals demonstrated a higher oxidative activity in muscle of young clones (8 months of age) compared with controls, suggesting a delayed muscle maturation in clones. Nutritional evaluation of milk and meat using the rat feeding trials did not show any difference between clone and control products for food intake, growth rate, body composition of the rats, nor for possible allergenicity. Possible reactivation of bovine endogenous retroviruses (BERVs) was analysed and compared between normal and cloned cattle. As expected, these BERV sequences are not transcribed and no RNA was detected in the blood of clones, donor animals or controls; therefore, it may be assumed that the sanitary risk associated with BERV sequences is not higher in cattle derived from somatic nuclear transfer than in cattle born from conventional reproduction. Our results confirm that the quality and safety of products (milk and meat) from adult and clinically healthy cloned cattle is globally similar to normal animals. However, from a strictly biological point of view, the slightly delayed maturation we observed in the muscle of clones together with some marginal differences identified in FA composition of both muscle and milk, point to the need for more refined analysis to totally exclude any risks from the consumption of those products.

Peptide fragments released from Phe-caseinomacropeptide in vivo in the rat.

The aim of this study was to investigate the pharmacokinetics of bovine Phe-caseinomacropeptide (Phe-CMP) in the rat after oral administration. This polypeptide was monophosphorylated and mainly nonglycosylated: Phe-CMP-1P. During gastrointestinal digestion and absorption, Phe-CMP-1P was degraded. Intact Phe-CMP-1P and CMP-1P were rapidly released from the stomach. In contrast, partial hydrolysis by pancreatic enzymes was observed. In vitro hydrolysis by brush-border membrane vesicles also indicated that the peptide was degraded. In the blood, "CMP-immunoreactive material" appeared rapidly, reaching a maximum level of 5.5 microg/ml at 60 min.

Bovine lactoferrin induces both mucosal and systemic immune response in mice.

Lactoferrin (Lf) is a milk iron-binding glycoprotein that plays a role in iron transport and acts as both a bacteriostatic and a growth modulating agent. The aim of this study was to investigate the nature of immune responses induced by repeated oral administration of bovine milk Lf in mice. Groups of ten female BALB/c mice were fed daily for 4 weeks with two doses of protein antigen: a low (0.05 mg/g body weight per d) or high (1 mg/g body weight per d) dose of Lf, or water as a control. A fourth group was immunized intramuscularly with 0.01 mg Lf in complete Freund's adjuvant. Anti-Lf IgA and IgG were detected in the intestinal fluid and serum of mice given Lf. Total immunoglobulins were higher in the intestinal fluid in Lf groups than in the control group. No difference could be detected in the serum. IgA and IgG secretion was enhanced in Peyer's patches and spleen from Lf-fed mice, in comparison with controls. [3H]thymidine uptake into Peyer's patch and spleen cells from both control and Lf-fed mice was enhanced by 75 micrograms Lf/ml in vitro, but Lf groups had a greater proliferation rate than the control group. These findings suggested that Lf could act as an immunostimulating factor on the mucosal immune system and that activation of the mucosal immune system is dependent on the ability of Lf to bind to the intestinal mucosa.

Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells.

A recombinant human erythropoietin (rH-EPO) was obtained from the culture supernatants of human B-lymphoblastoid cells transfected by the human EPO gene. rH-EPO was purified by a two-step method based on immunoaffinity and ion exchange chromatography. The first step was achieved by an anti-EPO monoclonal antibody (Mab). This Mab, immobilized on Sepharose 4B, allowed a 410-fold purification of the protein. The second step consisted of ion exchange chromatography on DEAE Sephacel. The combination of these two steps results in a highly purified rH-EPO with a global yield of about 50%; the specific activity of the protein was 176,000 IU/A280. The NMR spectrum was characteristic for a well structured, single-conformation protein. The purified protein was analyzed by SDS-PAGE and isoelectric focusing. The biological activity of purified rH-EPO was measured in vivo, by the incorporation of 59Fe into red blood cells (RBC) of polycythemic mice and in vitro by the proliferative response of an EPO-dependent cell line. The purified protein expressed in lymphoblastoid cells of human origin had the same biological activity as that of urinary EPO and rH-EPO produced in other mammalian cells.

Quantitation of immunoglobulin classes and subclasses of autoantibodies bound to red cells in patients with and without hemolysis.

The concentration of IgG, IgA, and IgM, as well as IgG subclasses, was measured by an enzyme-linked immunosorbent assay in autoantibodies eluted from red cells (RBCs); the number of molecules of each isotype per RBC was calculated. Three groups were analyzed: Group 1 included 23 patients with autoimmune hemolytic anemia (AIHA) associated with warm autoantibodies of IgG class; Group 2 included 11 patients without anemia but with a positive direct antiglobulin test (DAT); Group 3 included 10 healthy DAT-negative subjects. The mean number of IgG molecules per RBC in Group 1 (920) was about three times that in Group 2 (306) and about 17 times that in Group 3 (54). The range of RBC-bound IgG showed an overlap between the two groups of patients. The mean number of IgM and IgA molecules per RBC was low in the three groups. IgG1 predominated in all groups except in two patients with AIHA, in whom IgG3 made up at least 50 percent of total IgG. The mean number of IgG1, IgG2, and IgG4 molecules per RBC in Group 1 was about three times that in Group 2, whereas the mean number of IgG3 molecules per RBC was 10 times as high (p < 0.001). It follows that IgG3 was more common in patients of Group 1, but it was also detected in patients of Group 2.

Intrathecal synthesis of IgG subclasses in multiple sclerosis and in acquired immunodeficiency syndrome (AIDS).

Serum and cerebrospinal fluid (CSF) of 50 neurological patients (24 multiple sclerosis (MS), ten acquired immunodeficiency syndrome (AIDS) and 16 other neurological diseases (OND)) and ten controls were analyzed by enzyme-linked immunosorbent assay (ELISA) for IgG subclass quantification and for the calculation of intrathecal synthesis (ITS). Total IgG was determined by two methods: electroimmunodiffusion (EID) and ELISA. A highly significant correlation was established between both methods. The existence of ITS was proved by the IgG/albumin ratio, the IgG index, Tourtellotte's formula, and Schuller's formula. In AIDS patients all IgG subclasses showed an increase in the CSF, whereas in sera only the IgG1 was significantly increased. CSF of MS patients showed a predominant increase of IgG1 whereas no significant modification of IgG subclasses was observed in sera. In most of the AIDS patients there was an ITS of IgG1, IgG3 and IgG4, but rarely (3/10) IgG2. In contrast, a polyclonal ITS of IgG was exceptional (1/24) in MS patients. No significant correlation could be established between clinical data and IgG subclass ITS in MS. The variations of each IgG subclass in serum and in ITS were not significantly correlated. Measurement of each IgG subclass and calculation of ITS seems essential in order to analyze any subclass antibody repertory inside the central nervous system.

Enzyme immunoassays for detection of malarial antigens in human plasmas by Plasmodium falciparum monoclonal antibodies.

The screening of blood donors for the detection of dangerous disease carriers is a mandatory requirement for blood transfusion centers. Enzyme immunoassay (EIA) is a suitable method for the examination of large populations. We describe a sandwich EIA allowing the detection of soluble malarial antigens in plasma using 11 mouse monoclonal antibodies. Among the 121 combinations tested, 2 were selected for their sensitivity and specificity. Both were applied to plasmas of (a) acute patients, (b) people living in malarious areas, (c) blood donors at risk (travelers), and (d) sedentary blood donors without risk. With 1 of the 2 combinations, the percentage of positive answers was 68.4% (n = 38) for a, 62.6% (n = 206) for b, 4.5% (n = 398) for c, and 0.8% (n = 485) for d; with the other combination, the percentage of positive answers was 68.4% for a, 46.1% for b, 1.5% for c, and 0% for d. Using 2 combinations simultaneously, the positive results were 94.7% for a, 70.4% for b, 5% for c, and 0.8% for d. The 2 assays are complementary and the pair can be used for maximum Plasmodium falciparum antigen recognition in prospective donors.

[Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method]. / Estimation quantitative du taux des sous-classes d'IgG sériques par une méthode immunoenzymatique.

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in "ELISA" and immunoprecipitation technics.

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.

Renal deposits of lipoprotein-immunoglobulin complexes in Plasmodium chabaudi-infected mice.

Lipoprotein metabolism is altered and immunoglobulin-lipoprotein complexes (Ig-Lp) are formed during malaria infection (1-5). Ig-Lp were detected in the sera of Plasmodium chabaudi-infected mice 9 days post-infection (1 or 2 days after parasitemia had peaked at about 50%) and reached a maximum on day 13 (when the parasitemia had decreased to less than 1%). Renal glomerular deposits of IgM were first detected at day 3 and were heavy from day 9 to day 29; deposits of IgG and low density lipoprotein (LDL) were present from days 9 to 62, and were more dense from days 22 to 29; deposits of C3 were observed from day 13 to day 29. Apoprotein B component was found in heparin eluates of kidneys on day 10, 14, and 29. Fractionated Ig-Lp, as well as whole sera from day-13 infected mice, were injected into uninfected mice that developed LDL glomerular deposits only when pre-treated with histamine. LDL glomerular deposits were also observed after i.v. injection of day-29 sera (containing free anti-lipoprotein antibody) into day-7 infected mice, but not when a mixture of day-29 and day-7 sera was injected into normal recipient mice. LDL glomerular deposits, however, were observed when recipient mice were treated with the Plasmodium-derived Insoluble Material (PDIM) 3 days before the injection of the day-29-day-7 sera mixture or day-13 serum. Two hours after the i.v. injection of 125I-Ig-Lp, the radioactivity of the kidneys was higher in histamine-treated, PDIM-treated, and P. chabaudi-infected mice than in controls. The clearance of 125I-Ig-Lp was higher in infected and in PDIM-treated mice than in controls. We suggest that the glomerular deposit of Ig-Lp that occurs during P. chabaudi infection requires an enhancing factor such as PDIM that is released during infection.

[Anti-plasmodium falciparum monoclonal antibodies. Preliminary results]. / Anticorps monoclonaux anti-Plasmodium falciparum. Résultats préliminaires.

P. falciparum grown in culture was used to immunize mice and to obtain hybridomas. Two hybrids were selected secreting high titers of monoclonal antibodies directed against the cytoplasm of erythrocytic forms of the Plasmodium and not against the membrane of normal or parasitized erythrocytes. Such antibodies are of potential value in the immuno-diagnosis of malaria.

[Purification of a beta lipoprotein fraction rich in Lp(a)]. / Purification d'une fraction de bêta lipoprotéines riche en Lp (a)

A Lp (a) rich fraction was prepared from heparin/manganese chloride precipitated beta-lipoproteins, by using DEAE cellulose chromatography and gel filtration on Sepharose 4 B.

Lp (a) blood types. Comparison of immunological testing with results of polyacrylamide gel electrophoresis.

The results of serum beta-lipoproteins typing of 100 random human sera for Lp (a) antigen by the standard immunological method were compared with those of polyacrylamide gel electrophoresis which may detect an Lp (a) band migrating more slowly than the bulk of LDL. This band was found in all the 33 Lp (a+) but also in 9 of the 67 Lp (a-) sera examined.