Jean-Marie Pleau

Studies on the zinc binding site to the serum thymic factor

Gel filtration studies of 65Zn2+ binding to thymulin show that the nonapeptide can strongly bind one zinc metal ion. At pH 7.5, thymulin binds one zinc ion with an apparent affinity constant Kd of 5 +/- 2 X 10(-7) M. Binding is pH dependent. No binding is observed below pH 6.0. Ga3+, Al3+, Mn2+ and Cu2+ can compete with the binding of Zn2+ at pH 7.5. A good correlation between the competition potencies of metal ions used and the extent of biological activity of thymulin in the presence of these metal ions in an in vitro rosette assay is observed. Structural analogs of thymulin and non-thymulin-related peptides were used in a gel filtration technique to tentatively define the nature of amino acids present in the Zn2+-binding site of thymulin.

An enzyme immunoassay for synthetic thymulin

Thymulin, a metallononapeptide with the following aminoacid sequence: pyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-AsnOH is a thymic hormone involved in T cell differentiation requiring zinc to express biological activity as measured by the rosette assay. We established an enzyme immunoassay (EIA) for synthetic zinc-free thymulin with a thymulin-acetylcholinesterase conjugate as tracer and specific polyclonal rabbit antithymulin antibodies. The assay is performed as a classical competition assay in microtiter plates previously coated with mouse monoclonal IgG to rabbit IgG. A quantitative thymulin assay more sensitive than radioimmunoassays (RIAs) previously described was obtained with a sensitivity (IC50) of 32.5 +/- 5 pg/ml and a detection limit of 5 pg/ml. Analysis in the EIA of synthetic thymulin analogs showed that the minimal peptidic structure necessary for enzymatic tracer competition is the C-terminal part Lys3 to Asn9. It was also shown that the biologically active form of thymulin (zinc-bound) has the same immunoreactivity as zinc-free thymulin and that other thymic hormones, thymosin alpha 1 and thymopoietin II (or TP5) and unrelated short peptides do not cross-react with thymulin. These data demonstrate the specificity of this EIA for thymulin and show its suitability for application in biological fluids.

Monoclonal antibody against the serum thymic factor (FTS)

Hybrid cell lines secreting antibodies specific for synthetic serum thymic factor (FTS) were prepared by cell fusion and cloning techniques. Spleen cells from BALB/c mice immunized with BSA-coupled FTS were fused with P3-x63-Ag8.653 myeloma cells. Antibodies produced by these hybrids were screened in vitro for their ability to absorb the activity of synthetic FTS in a rosette assay and in vivo for their capacity to induce the disappearance of endogenous FTS. Subsequently, the clones selected were transferred intraperitoneally into BALB/c mice. Ascitic fluid was produced and used as a source of antibody. The monoclonal antibody was shown to bind specifically to thymic reticulo-epithelial cells, using an indirect immunofluorescence technique. Furthermore, once injected into normal mice, the antibody induced the disappearance of FTS from the serum and modified the azathioprine sensitivity of spleen rosette-forming cells for more than 3 weeks.

Extracellular matrix distribution and islet morphology in the early postnatal pancreas: anomalies in the non-obese diabetic mouse

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect.

We previously showed intrathymic alterations in non‐obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)‐4 and VLA‐5] and laminin (VLA‐6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA‐5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double‐negative cells, and more pronounced in mature CD4+ and CD8+ thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin‐containing transwell chambers. In situ, intra‐perivascular space thymocytes were VLA‐5‐negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA‐5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Antagonistic analogue of Serum Thymic Factor (FTS) interacting with the FTS cellular receptor

Abstract An analogue of facteur thymique serique (FTS), which was inactive in the rosette assay used to characterize FTS, has been shown to inhibit FTS effects on rosette-forming cells and to displace [3H]FTS from its specific cellular receptor.

Proapoptosis and antiapoptosis-related molecules during postnatal pancreas development in control and nonobese diabetic mice: relationship with innervation.

The mouse pancreas, an immature organ at birth, reaches its adult size and morphology after weaning (3 weeks of age). Around this time, apoptotic phenomena and various types of macrophages are normally present. During development, Fas–Fas ligand (FasL) interactions are known to play a role in apoptotic events involved in tissue remodeling and elimination of damaged cells, and macrophages are routinely observed near apoptotic cells. Apoptosis and Fas–FasL interactions are also thought to be involved in the pathogenesis of autoimmune diseases, particularly type 1 diabetes (T1D). Therefore, we used early postnatal mouse pancreata from three control strains (C57BL/6, DBA/2, BALB/c) and from two strains with the nonobese diabetic (NOD)–related genetic background (the spontaneous T1D NOD model and the lymphocyte-deficient NODscid strain) to study apoptotic phenomena together with the molecular and immunohistochemical expression of proapoptosis (Fas, FasL) and antiapoptosis (Bcl-2) proteins. First, although no major difference in the numbers of total pancreatic apoptotic cells was noted among strains, significantly more FasL+ expression was detected immunohistochemically in mice with the NOD genetic background than in control pancreata from birth to 1 month of age. Second, FasL+, Fas+, and Bcl-2+ structures seemed to be associated with innervation, regardless of the strain and age. Third, in control and NOD strains, nerves (identified by immunohistochemical labeling of peripherin or neurofilament 200), were often observed in periductular and peri-insular areas. Finally, some peripherin-positive nerves expressed the interferon-inducible protein-10 chemokine, and various types of macrophages were found to be in close proximity. These data highlight an overlooked, innervation-related aspect of normal mouse postnatal pancreas development with possible implications in T1D pathogenesis.

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

The interaction of the synthetic serum thymic factor (FTS, facteur thymique sérique) with a plasma membrane preparation of human T lymphocytes from the lymphoblastoid T cell line 1301 was studied using 3H-labelled FTS (specific activity 120 Ci/mmol). The binding is temperature dependent and function of the concentration of both 3H-labelled FTS and membrane proteins. At 37 degrees C, using 1 nM of 3H-labelled FTS as steady state is observed within 80 min. The binding is reversible, specific and saturable. Scatchard analysis reveals the existence of at least two binding sites with respective Kd of the order of 0.516 +/- 0.2 nM and 110 +/- 27.8 nM with concentration of 0.186 +/- 0.045 pmol and 2.026 +/- 0.367 pmol per mg of membrane protein.

Nucleotide sequences of variable regions of an human anti-acetylcholine receptor autoantibody derived from a myasthenic patient

Autoantibodies against the acetylcholine receptor (AChR) are involved in the neuromuscular dysfunction associated with myasthenia gravis (MG). We determined the nucleotide and the deduced amino acid sequences of the heavy and light chains for one human monoclonal anti-AChR autoantibody, derived from peripheral blood lymphocytes obtained from one MG patient. The heavy and light chain (VH and V lambda) genetic elements used in this autoantibody are respectively or closely related to HHG19 and Humlv117 germline structure. In addition, the expressed JH, J lambda and D segments differ from their described germline structures. This diversity could reflect allelic variation. The large number of differences found in VH, VL and D genetic elements when compared with their most closely related germline structures allowed us to conclude that we have characterized new VH and VL genes and we could not identify univocally somatic mutations. However, the analysed autoantibody, an IgG specific for the alpha chain of the AChR, revealed the presence of N addition segments on both sides of the D region and we may postulate that this antibody was the result of an antigen driven phenomenon.

Evidence for antigen driven selection in two monoclonal auto-antibodies derived from nonobese diabetic mice

The nonobese diabetic (NOD) mouse is a model of human type I diabetes. This diabetes is due to massive infiltration of the pancreatic beta cell of islets by autoreactive T cells (insulitis) followed by the destruction of insulin-producing cells. Circulating autoantibodies are also detected, notably against glutamic acid decarboxylase, peripherin and insulin. Two monoclonal autoantibodies directed against insulin and peripherin were obtained by fusing NOD spleen and myeloma cells. We report here the nucleotide sequence of the genes encoding for the V regions of these two antibodies. Somatic mutations were identified by comparing the light chain nucleotide sequence of one of these autoantibodies with its germline counterpart precursor established from NOD mice after PCR gene amplification. The other one displays N additions on both sides of the D region. These results strongly suggest that both autoantibodies have undergone diversification, either N additions or somatic mutations, and therefore present structural features of antibodies derived from animals immunized against exogenous antigens.