Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody.

BACKGROUND: CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS: To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS: The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.

Long-term anti-CD154 dosing in nephritic mice is required to maintain survival and inhibit mediators of renal fibrosis.

The CD154/CD40 pathway is required for the development and progression of disease in a variety of autoimmune model systems. We have demonstrated previously that long-term anti-CD154 treatment of nephritic (SWRxNZB)F1 mice prolonged survival and preserved kidney function. Herein we ask if long-term treatment is required and further characterize the protective effect on renal pathology by examining alpha-smooth muscle actin, collagen and TGF-beta1 expression in renal tissue. The effects of anti-CD154 on brain and heart inflammation are also examined. Three dosing strategies of anti-CD154 mAb were compared in SNF1 mice that exhibited moderate or severe nephritis: (1) weekly for 6 weeks; (2) monthly; (3) weekly for 6-12 weeks followed by monthly dosing. Proteinuria, serum anti-DNA, anti-CD154 pharmacokinetics and serum soluble CD154 analyses were performed. Anti-CD154 treatment of moderate disease increased survival across all regimens, although weekly followed by monthly maintenance dosing proved most efficacious. This regime also inhibited renal alpha-smooth muscle actin and collagen deposition. Only the most aggressive anti-CD154 treatment protocol increased survival in severely nephritic mice. Long-term anti-CD154 treatment significantly inhibits key mediators of kidney fibrosis and is required to maximize survival and renal function. Potential reasons for differential therapeutic efficacy in moderately vs severely nephritic mice are discussed.

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

Role of CD154 in the secondary immune response: the reduction of pre-existing splenic germinal centers and anti-factor VIII inhibitor titer.

Using the murine model of hemophilia A, we have examined the role of CD154 in the secondary immune response to factor VIII (FVIII). We previously reported that repeated i.v. injection of FVIII in hemophilia A mice induces a T cell-dependent anti-FVIII antibody formation. Herein, blocking of CD154 by a monoclonal antibody in FVIII-primed hemophilia A mice resulted in the disappearance of pre-existing spleen germinal centers (GC) in the white pulp within 24 h of treatment. Moreover, further expansion of GC in response to FVIII challenge was completely inhibited. In parallel, anti-FVIII antibody titers were markedly reduced and T cell responses to FVIII were abolished. The rapid disappearance of the GC after anti-CD154 treatment was not accompanied by increased B cell apoptosis; instead B cells accumulated in the peripheral zone of the splenic white pulp. Interestingly, repeated exposure to FVIII with anti-CD154 antibody administration blocked anti-FVIII antibody formation but failed to induce long-lasting unresponsiveness. Our data demonstrate that the CD40-CD154 interaction is critical for B cell homeostasis and the secondary immune response to FVIII. For potential clinical application, the data also suggest that therapies targeting the CD154 molecule may be useful for the treatment of high titer FVIII inhibitors in hemophilia A.

The early detection and secondary prevention of alcoholism in France.

The conceptual foundation and structural development of a major secondary prevention program established in France to screen, diagnose and treat persons in the prodromal stages of alcoholism are described. Also discussed are the application and validity of a simple examination procedure used to identify alcoholics in this program.

[Use of the measurement of gamma-glutamyltranspeptidase activity in serum to determine the success of cures for alcohol detoxification (author's transl)]. / Emploi de la mesure de la gamma-glutamyltranspeptidase sérique pour contrôler le succes des cures de désintoxication anti-alcoolique

44 patients were studied during a 2-year period following a cure for alcohol detoxification. 29 patients (group A) did not start drinking again while 15 relapsed less than one year after their cure (group R). The average gamma GT activity (m) in mU per ml serum was, in group A, 148 (standard deviation, S.D. 184) at the beginning of the cure, 21 (S.D. 14) after one year and 19 (S.D. 13) after 2 years. During the same period there was no significant decrease in group R (cure: m 140, S.D. 161: 1 year: m 166, S.D. 164; 2 years: m 162, S.D. 163). On the other hand, all the subjects of group A who had a high gamma GT activity (greater than 29 mU/ml) at the start of the cure showed a decrease after one year.