Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

CD4+ CD25+ FOXP3+ regulatory T cells increase de novo in kidney transplant patients after immunodepletion with Campath-1H.

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3(+) regulatory T (Treg) cells. Flow cytometry demonstrated that CD4(+)CD25(+)FOXP3(+) lymphocytes increased significantly within the CD4(+) T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4(+)CD25(+)FOXP3(+) cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo.

The developmental outcome of children with antenatal mild isolated ventriculomegaly.

OBJECTIVE: To evaluate standardized developmental test performance of infants and children who as fetuses had mild isolated cerebral ventriculomegaly diagnosed by ultrasound. METHODS: Ultrasound records from 1990 to 1996 were searched for cases of mild isolated ventriculomegaly, and standardized developmental testing of the children was offered to their parents. Each consented child was matched to a normal antepartum subject with respect to sex, race, indication for ultrasound, and gestational age (+/- 2 weeks) at the time of ultrasound. Tests of cognitive, motor, and adaptive behavior were then administered by examiners blinded to the subjects' case or comparison status. RESULTS: Twenty-two cases and an equal number of matched comparison subjects completed the testing. The ventriculomegaly and comparison groups were similar with respect to parental age, maternal education, and household income. The ventriculomegaly subjects scored significantly lower than the comparison group on both the Bayley Scales of Infant Development: mental development index (88.95 versus 99.68, P = .017) and psychomotor development index (95.99 versus 103.95, P = .039). Eight of the 22 ventriculomegaly children were classified as developmentally delayed on the mental developmental index compared with one of 22 children in the comparison group (P = .021). Adaptive behavior skills, as measured by the Vineland Behavior Scales (99.64 versus 102.68), were not significantly different between the groups (P = .571). CONCLUSION: Mild isolated ventriculomegaly detected on antepartum sonographic examination is associated with a significant risk for developmental delay. Insofar as these children were judged to be completely normal at birth, our findings represent an important application of antepartum sonography for identifying infants who could be targeted for early childhood intervention.

Quantitative analysis of T cell activation: role of TCR/ligand density and TCR affinity.

(B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.

The anti-La response of a single MRL/Mp-lpr/lpr mouse: specificity for DNA and VH gene usage.

Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp-lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti-Sm monoclonals bound DNA and expressed the same V region genes as anti-DNA. In addition, many had multiple VHCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti-DNA response, we have analyzed the response to the La RNP. Six anti-La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single-stranded DNA, while antibodies from five hybridomas bound double-stranded DNA. Two hybridomas expressed a VH7183 gene which is used by members of two previously reported anti-DNA clones and two anti-Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti-La and anti-DNA responses in MRL/lpr mice, suggesting that cross-reactive anti-RNP and anti-DNA responses are a general feature of autoimmunity in this lupus model.

Molecular characterization of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. Differences from spontaneous anti-DNA in the content and location of VH CDR3 arginines.

Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.

Overlap of the anti-Sm and anti-DNA responses of MRL/Mp-lpr/lpr mice.

Autoantibodies specific for the Sm ribonucleoprotein are spontaneously produced in patients with SLE and in mice of the MRL mouse strains. We have previously reported the characterization of the clonality and V region gene use of 41 MRL/Mp-lpr/lpr (MRL/lpr)-derived B cell hybridomas selected for Sm binding. In this report, we show that many of the expressed V genes of these hybridomas are also expressed by anti-DNA hybridomas of MRL/lpr mice. Moreover, the anti-Sm hybridomas from nine clonal groups produce antibodies that bind ssDNA, and those of five clones produce antibodies that also bind dsDNA. Sm/DNA-specific hybridomas, but not Sm-only-specific hybridomas, have a higher than expected content of arginine residues in CDR3 of the H chain, similar to MRL/lpr hybridomas selected on the basis of DNA binding. One clone displays intraclonal differences in DNA binding, inasmuch as the most extensively mutated members produce antibodies that are able to bind dsDNA and have a higher affinity for ssDNA than the least mutated members of this clone. Thus, DNA appears to be a selecting Ag in this response. These data indicate an overlap in the anti-Sm and anti-DNA autoimmune responses in MRL mice that may have implications for the activation of anti-Sm B cells, and for defining the spectrum of Ag targeted in SLE.

V region gene analysis of anti-Sm hybridomas from MRL/Mp-lpr/lpr mice.

Anti-Sm autoantibodies are unique to SLE, but are present in only 25% of patients with this disease. This response also occurs at a similar frequency in mice of the autoimmune MRL strains. Previous analyses of the anti-Sm response in these mice indicate that its occurrence is controlled by stochastic events, and suggest that Sm is the driving Ag. To further elucidate the role of Ag in this response, and to test the hypothesis that the 25% incidence is due to a requirement for particular Ig gene rearrangements or somatic mutations, we have analyzed the specificity and V-region gene sequences of 41 anti-Sm B cell hybridomas derived from nine anti-Sm-positive MRL/Mp-lpr/lpr mice. The majority of hybridomas are specific for the D peptide of the Sm particle. Hybridomas of independent origin express unique VH/V kappa combinations with diverse junctional sequences and are variable in the extent of somatic mutation. Thus, the response does not appear to be dependent upon the occurrence of a rare Ig gene rearrangement or specific somatic mutation. The response exhibits restriction in JH and VH gene use, and in individual mice is oligoclonal, suggestive of Ag selection. In the few B cells for which mutations can be identified, the evidence for selection of mutant B lymphocytes, based on patterns of mutation, is ambiguous. However, there is remarkably little intraclonal diversity, suggesting that the overall mutation rates in these clones are low.

Effect of chemical ablation of myenteric neurons on neurotransmitter levels in the rat jejunum.

We have quantified neurotransmitter changes in the rat jejunum in which the myenteric neurons were ablated by serosal application of benzalkonium chloride. Within 2 days after benzalkonium chloride treatment, there was a 40% reduction in the activity of choline acetyltransferase, a specific marker for cholinergic neurons, and a 25% reduction in the amount of vasoactive intestinal peptide per centimeter length of jejunum. By 15 days, levels were comparable to those in control segments, and by 45 days after myenteric neuronal ablation, levels in treated tissues were twice those in controls. In contrast, we observed no reduction in the amount of leucine-enkephalin per centimeter length of jejunum at early times after benzalkonium chloride treatment, although by 45 days, levels of this neurotransmitter in treated segments of jejunum were more than twice those in controls. Significant increases in muscle thickness and tissue weight were also observed at 15, 30, and 45 days after myenteric neuronal ablation. Thus we have observed that in response to chemical ablation of myenteric neurons in the rat jejunum, there is a thickening of the smooth muscle layers and a compensatory increase in the production of certain neurotransmitters by the surviving neuronal elements in the gut.