Sirolimus for rescue and primary immunosuppression in transplanted children receiving tacrolimus.

AIMS: The role of sirolimus (SRL) as a rescue agent (n=42) and as a component of primary immunosuppression (n=8) was evaluated in a mixed population of 50 transplanted children receiving tacrolimus (liver: 26, heart: 5, intestinal: 5, liver-intestine: 9, lung: 1, bone marrow: 1, liver-kidney: 1, multivisceral: 1). Rescue indications for tacrolimus (TAC) failure were recurrent acute rejection and acute rejection complicating withdrawal of immunosuppression in posttransplant lymphoproliferative disorder (PTLD). Rescue indications for TAC toxicity were nephrotoxicity, pancreatitis, seizures, hypertrophic cardiomyopathy, and graft-versus-host disease. RESULTS: Mean age at rescue was 11.5 years and mean follow-up was 204 (range 18-800) days. As primary immunosuppression, SRL+TAC prevented early acute rejection in 7/8 children. The indication for rescue resolved in 33/42 children. In children with TAC toxicity, this was associated with decrease in TAC doses by 50%, significant improvements in renal function, and continuing decline in Epstein-Barr virus (EBV) viral load in PTLD patients. Serious adverse events led to discontinuation of SRL in 9/42 rescue patients, 3 of them also experienced acute rejection. Three additional children also experienced acute rejection on SRL therapy (overall incidence 6/50, 12%). Pharmacokinetic analysis in the first week of SRL administration suggested a short half-life (11.8+/-5.5 hr, n=21). CONCLUSIONS: SRL and reduced-dose TAC may achieve adequate immunosuppression without compromising renal function or enhancing EBV viremia significantly.

In vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: pathogenic autoreactivity requires permissive light chains.

Lymphocyte antigen receptors are promising targets for immune intervention strategies in disorders marked by repertoire skewing or expansion of lymphocyte subsets. Appropriate application of immune receptor modulation is predicated on understanding the role of a particular receptor in pathogenesis and disease regulation. The VHB/W16 gene, restricted to mice carrying the j haplotype for the J558 family, is overexpressed by murine lupus anti-DNA Ig. This gene is also expressed recurrently among nephritogenic anti-DNA Ig recovered from several autoimmune strains, suggesting that cells expressing this pathogenic receptor are positively selected during disease progression. To explore the extent and mechanisms by which Ig H chains expressing this gene contribute to autoimmunity, an Ig H chain gene was engineered for in vitro and in vivo recombination studies. Site-directed mutagenesis generated unique restriction sites to link PCR-amplified V region (VDJ) cDNA to previously isolated genomic fragments containing Ig regulatory and signal sequences. The new 3 kb VDJ gene was then ligated to a 9 kb fragment encoding the IgM constant region. Transfection of H chain loss variant myeloma with the complete 12 kb construct, termed 238H-Cmicro, resulted in secretion of intact Ig pairing 238H-Cmicro, with a lambda L chain; however, transfectant Ig lacked autoreactivity and pathogenicity. Introduction of the 238H-Cmicro H chain as a transgene onto the non-autoimmune C57BL/6 background resulted in abundant B cell surface expression of 238H-Cmicro, however, four transgenic Ig recovered by fusion of LPS-stimulated splenocytes and formed by combination of 238H-Cmicro, with endogenous kappa chains do not bind DNA or laminin. These results indicate that the antigen binding sites encoded by this disease-associated gene and/or H chain must associate with permissive L chains to specify autoimmunity. The 238H-Cmicro, transgenic model should prove useful in dissecting the in vivo fate of 238H-Cmicro, L combinations that produce pathogenic autoreactive receptors and in evaluating receptor-targeted interventions.

Anti-idiotypic monoclonal Ig specific for an anti-laminin Ig heavy chain transgene variable region.

Techniques currently available to obtain anti-idiotypic reagents reactive with a single chain of a lymphocyte antigen receptor rely on immunization with intact soluble or cell-bound Ig or T-cell receptors. Ready recovery of single-chain-specific monoclonal antibodies (MAbs) depends on the presence of an immunodominant epitope on the desired chain and chance recovery of the responding clone. Here we present a method to maximize recovery of an Ig heavy-chain-specific anti-idiotypic Ig, using sequential immunization with MAbs expressing the H chain V region in combination with different H chain isotypes and with different light chains. The latter was produced by in vitro transfection of an H-chain-loss variant myeloma cell line with a transgene construct expressing the Ig H chain V region of interest. Sequential immunization may be a useful strategy to enhance selection of anti-Id reagents reactive with single chain-specific epitopes.

Anti-laminin reactivity and glomerular immune deposition by in vitro recombinant antibodies.

Growing evidence suggests that recombinatorial events prior to antigen contact can generate pathogenic autoantibodies in the nonautoimmune individual, thus providing potential disease mediators if conditions arise that permit bypass of tolerance and activation of autoreactive lymphocytes. To examine the disease potential of selected germline antibody genes, Ig were created de novo by in vitro recombination of Ig H and L chains. H chain loss variant (i.e., L-chain only) cell lines were transfected with a DNA construct encoding the variable region and regulatory sequences (LamH) of a nephrotropic murine lupus anti-laminin Ig, and the resultant Ig were examined for in vitro antigen reactivity and in vivo glomerular immune deposition. The results indicate that two light chains, LamL (Vk8, Jk5) and 238L (Vk4, Jk5), expressing unrelated germline V1 genes, combine with LamH to generate Ig that bind basement membrane laminin in vitro, diverge in their capacity to bind ssDNA, and produce two distinct patterns of glomerular immune deposits in vivo: dense mesangial matrix (LamH/LamL) and dramatic linear glomerular basement membrane (LamH/238L) deposits. The Ig genes used by both LamH and 238L are present in nonautoimmune mice as well as in lupus-prone strains. We conclude that certain unmutated Ig genes can contribute to multiple distinct disease associated specificities, including binding to intrinsic kidney antigens, and that mutation is not essential to generate these Ig. Collectively, these observations suggest that pathogenic autoantibodies can be generated in the normal preimmune repertoire by random recombinatorial and somatic events in the absence of mutation.

Galloway-Mowat syndrome of abnormal gyral patterns and glomerulopathy.

The combination of microcephaly, gyral abnormalities, developmental delay, and a glomerulopathy constitutes a recognizable syndrome. The inheritance is autosomal recessive. Additional abnormalities may include seizures, minor facial anomalies, and hiatal hernia. Onset of proteinuria often occurs in the first 3 months of life, but always before age 3 years. A uniform pattern of renal histologic changes has not been found. There is no effective treatment for the neurologic or renal manifestations of this condition. The prognosis is extremely poor; every patient but one has died before age 5 1/2 years. Antenatal diagnosis may be possible.