Spontaneous increase of DNA turnover in murine systemic lupus erythematosus

DNA synthesis and release was studied in unstimulated splenocytes of strains of mice known to develop spontaneous systemic lupus erythematosus (SLE)-like disease and in non-SLE age- and sex-matched strains as well. Newly synthesized DNA was measured as total acid-insoluble radioactive material present in cell pellet plus supernatant of unstimulated 0–72 h cell cultures [³H]thymidine-pulsed, whereas DNA release was measured as amount of acid-precipitable radioactivity found in supernatant of those cultures. In all strains known to develop spontaneous murine SLE the amount of newly synthesized DNA was 1.3–2.1-fold increased when compared to normal strains studied concomitantly. Furthermore, a significant increase in DNA release into medium, unrelated to cell viability, was observed in those strains as well. These observations clearly demonstrate different metabolic rates of synthesis and release of DNA in murine SLE. This difference suggests the existance of an underlying mechanism responsible for extracellular DNA abundancy, which may be important for the formation of circulating DNA-anti-DNA immune complexes.     

Biological effects of cyclosporin A: A new antilymphocytic agent

The fungus metabolite cyclosporin A is a small peptide acting as a novel antilymphocytic agent. It strongly depressed appearance of both direct and indirect plaque-forming cells and produced a clear dose-dependent inhibition of haemagglutinin formation in mice upon oral administration. Skin graft rejection in mice and graft-versus-host disease in mice and rats were considerably delayed by cyclosporin A which also prevented the occurrence of paralysis in rats with experimental allergic encephalomyelitis. This compound was not only highly effective in preventing development of Freund's adjuvant arthritis, but in addition improved the symptoms in rats with established arthritis, although it is inactive in acute inflammation. This new agent contrasts with other immunosuppressives and cytostatic drugs in its weak myelotoxicity. Experimental evidence suggests that cyclosporin A, rather than being cytostatic or lympholytic, affects an early stage of mitogenic triggering of the immunocompetent lymphoid cell.     

Lack of L-selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by Mel-14 antibody treatment

The process of mononuclear cell extravasation from the blood into the islets of Langerhans in nonobese diabetic (NOD) mice is dependent on the expression of a set of molecules, most of which remain to be defined. The observation that vascular addressins are expressed in inflamed islets raises the issue of the involvement of one of their ligands, L-selectin, in the pathogenesis of autoimmune diabetes. Treatment of NOD females with Mel-14, an antibody specific for L-selectin, reduced the spontaneous development of both insulitis and diabetes. Pretreatment of diabetic donors with Mel-14 decreased the capacity of their splenocytes to transfer the disease. However, the treatment of recipients had no effect on the transfer of diabetes by untreated diabetogenic splenocytes. To reconcile these apparently conflicting results, we fractionated spleen T cells from diabetic mice according to L-selectin expression. Diabetogenic cells were found only in the L-selectin subpopulation. Thus, diabetogenic cells in adult mice share phenotypic characteristics with activated/memory cells, and enter the pancreas using L-selectin-independent migratory pathways.     

Functional proteomics mapping of a human signaling pathway

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.     

Gammadelta T cells and interleukin-6 levels could provide information regarding the progression of human renal allograft

We have determined the percentage of alphabeta and gammadelta T cells by flow cytometry as well as serum interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) levels by enzyme-linked immunosorbent assay in kidney allograft recipients with acute, chronic or stable graft evolution. The percentage of CD4 and CD8 T cells in transplanted patients was lower than in the control group (P < 0.001) with the exception of CD8 gammadelta T cells from patients with stable evolution (P > 0.05). The serum levels of IL-6 and sIL-6R in acute and chronic rejection were higher than in the controls (P < 0.05). No differences in IL-6 levels were observed between the stable evolution and the control groups (P > 0.05). The levels of sIL-6R were higher in stable evolution patients than in the controls (P < 0.05) and no differences were observed between the chronic and stable evolution patients (P > 0.05). IL-6 decreased in patients with a favourable evolution, increased in those with an increased renal dysfunction and was maintained when the renal dysfunction was not modified. These results suggest that gammadelta T cells could participate in renal allograft maintenance and that IL-6 but not sIL-6R serum levels may provide a prognostic marker for measuring the evolution of kidney allograft.     

Strain-Dependent Migration of CD4 and CD8 Lymphocyte Subsets to Lymph Nodes in NOD (Nonobese Diabetic) and Control Mice

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.     

Does oxygen free radical increased formation explain long term complications of diabetes mellitus?

Oxygen free radicals (OFR) can form by reaction of glycated proteins with molecular oxygen. We hypothesize that this mechanism operates in tissues of diabetic patients when their content of glycated proteins is significantly increased. OFR are harmful to polyunsaturated fatty acids of lipid membranes, proteins, sugars and DNA. The most significant complications of diabetes, for example polyneuritis, retinopathy, microangiopathy, perforating ulcers, impaired healing, may depend on the excessive production of OFR by glycated proteins. Clues to these effects may be deduced from the decrease of glutathione stores in red blood cells, and the increases of lipid peroxidation and malondialdehyde formation, all of which have been documented to occur in the course of diabetes mellitus.     

Congenital posterior cervical spine malformation due to biallelic c.240‐4T>G RIPPLY2 variant: A discrete entity

The clinical and radiological spectrum of spondylocostal dysostosis syndromes encompasses distinctive costo‐vertebral anomalies. RIPPLY2 biallelic pathogenic variants were described in two distinct cervical spine malformation syndromes: Klippel–Feil syndrome and posterior cervical spine malformation. RIPPLY2 is involved in the determination of rostro‐caudal polarity and somite patterning during development. To date, only four cases have been reported. The current report aims at further delineating the posterior malformation in three new patients. Three patients from two unrelated families underwent clinical and radiological examination through X‐ray, 3D computed tomography and brain magnetic resonance imaging. After informed consent was obtained, family‐based whole exome sequencing (WES) was performed. Complex vertebral segmentation defects in the cervico‐thoracic spine were observed in all patients. WES led to the identification of the homozygous splicing variant c.240‐4T>G in all subjects. This variant is predicted to result in aberrant splicing of Exon 4. The current report highlights a subtype of cervical spine malformation with major atlo‐axoidal malformation compromising spinal cord integrity. This distinctive mutation‐specific pattern of malformation differs from Klippel–Feil syndrome and broadens the current classification, defining a sub‐type of RIPPLY2‐related skeletal disorder. Of note, the phenotype of one patient overlaps with oculo‐auriculo‐vertebral spectrum disorder.     

Inhibition of tumor cell proliferation by type IV collagen requires increased levels of cAMP

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).