Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis.

The main limitations to umbilical cord blood (UCB) transplantation (UCBT) in adults are delayed engraftment, poor immunological reconstitution and high rates of non-relapse mortality (NRM). Double UCBT (DUCBT) has been used to circumvent the issue of low cell dose, but acute GVHD remains a significant problem. We describe our experience in 32 subjects, who underwent DUCBT after reduced-intensity conditioning with fludarabine/melphalan/antithymocyte globulin and who received sirolimus and tacrolimus to prevent acute GVHD. Engraftment of neutrophils occurred in all patients at a median of 21 days, and platelet engraftment occurred at a median of 42 days. Three subjects had grade II-IV acute GVHD (9.4%) and chronic GVHD occurred in four subjects (cumulative incidence 12.5%). No deaths were caused by GVHD and NRM at 100 days was 12.5%. At 2 years, NRM, PFS and OS were 34.4, 31.2 and 53.1%, respectively. As expected, immunologic reconstitution was slow, but PFS and OS were associated with reconstitution of CD4(+) and CD8(+) lymphocyte subsets, suggesting that recovery of adaptive immunity is required for the prevention of infection and relapse after transplantation. In summary, sirolimus and tacrolimus provide excellent GVHD prophylaxis in DUCBT, and this regimen is associated with low NRM after DUCBT.

HLA-C mismatch is associated with inferior survival after unrelated donor non-myeloablative hematopoietic stem cell transplantation.

HLA-C matching is an important determinant of outcome after myeloablative unrelated donor (URD) hematopoietic stem cell transplantation. However, its importance in non-myeloablative stem cell transplantation (NST) is not known. We report a retrospective analysis of 111 patients who underwent URD NST, of whom 78 were 10/10 matched at HLA-A, B, C, DRB1, DQB1 and 33 were mismatched at one or more HLA-C antigen/allele (24 HLA-C only; nine HLA-C+other locus mismatch). Patients were conditioned with busulfan (0.8 mg/kg/day i.v. x 4 days) and fludarabine (30 mg/m(2)/day i.v. x 4 days). Graft-versus-host disease prophylaxis included cyclosporine/prednisone- or tacrolimus/mini-methotrexate-based regimens. HLA-C disparity did not impair engraftment. Median marrow donor chimerisms were >or=90% donor at day+30 and +100 in both groups. Overall survival at 2 years was 30% in HLA-C-mismatched and 51% in 10/10-matched patients (P=0.008). In Cox regression, HLA-C mismatch was an independent predictor of death (hazard ratio 1.85, P=0.04). Treatment-related mortality was higher in the HLA-C-mismatched group: 48 versus 16% (P=0.0001). Cumulative relapse incidence was 35% in the HLA-C-mismatched and 55% in the 10/10-matched cohort, P=0.09. HLA-C mismatch is associated with inferior survival after URD NST.

Disentangling the perturbational effects of amino acid substitutions in the DNA-binding domain of p53.

The spectrum of somatic cancer-associated missense mutations in the human TP53 gene was studied in order to assess the potential structural and functional importance of various intra-molecular properties associated with these substitutions. Relating the observed frequency of particular amino acid substitutions in the p53 DNA-binding domain to their expected frequency, as calculated from DNA sequence-dependent mutation rates, yielded estimates of their relative clinical observation likelihood (RCOL). Several biophysical properties were found to display significant covariation with RCOL values. Thus RCOL values were observed to decrease with increasing solvent accessibility of the substituted residue and with increasing distance from the p53 DNA-binding and Zn2+ -binding sites. The number of adverse steric interactions introduced by an amino acid replacement was found to be positively correlated with its RCOL value, irrespective of the magnitude of the interactions. A gain in hydrogen bond number was found to be only half as likely to come to clinical attention as mutations involving either a reduction or no change in hydrogen bond number. When the difference in potential energy between the wild-type and mutant DNA-binding domains was considered, RCOL values exhibited a minimum around changes of zero. Finally, classification of mutated residues in terms of their protein/solvent environment yielded, for somatic p53 mutations, RCOL values that resembled those previously determined for inherited mutations of human factor IX causing haemophilia B, suggesting that similar mechanisms may be responsible for the mutation-related perturbation of biological function in different protein folds.

Exploration of compact protein conformations using the guided replication Monte Carlo method.

We have studied the use of a new Monte Carlo (MC) chain generation algorithm, introduced by T. Garel and H. Orland [(1990) Journal of Physics A, Vol. 23, pp. L621-L626], for examining the thermodynamics of protein folding transitions and for generating candidate C(alpha) backbone structures as starting points for a de novo protein structure paradigm. This algorithm, termed the guided replication Monte Carlo method, allows a rational approach to the introduction of known "native" folded characteristics as constraints in the chain generation process . We have shown this algorithm to be computationally very efficient in generating large ensembles of candidate C(alpha) chains on the face centered cubic lattice, and illustrate its use by calculating a number of thermodynamic quantities related to protein folding characteristics. In particular, we have used this static MC algorithm to compare such temperature-dependent quantities as the ensemble mean energy, ensemble mean free energy, the heat capacity, and the mean-square radius of gyration. We also demonstrate the use of several simple "guide fields" for introducing protein-specific constraints into the ensemble generation process. Several extensions to our current model are suggested, and applications of the method to other folding related problems are discussed.

Molecular defects in CRM+ factor VII deficiencies: modelling of missense mutations in the catalytic domain of FVII.

The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families. Four missense mutations located in the catalytic domain of factor VII were found. The previously reported 304Arg-->Gln substitution was present in the homozygous and heterozygous forms, with different polymorphic haplotypes, thus demonstrating that it is recurrent and frequent in the Italian population. The 310Cys-->Phe substitution was found in the homozygous form and in the compound heterozygous condition with the nonsense mutation 356Trp-->stop. Two missense mutations, 298Met-->Ile and 342Gly-->Arg, were found in the homozygous and in the heterozygous condition respectively. Molecular heterogeneity was further increased by finding of the 353Arg-->Gln polymorphism in the doubly heterozygous condition with the 304 and 342 mutations. Plausible explanations for loss of FVII function were found by inspecting a model of the serine protease domain of factor VIIa. Inefficient activation of the catalytic site is predicted for 298Met-->Ile. 342Gly-->Arg would directly distort the geometry of the 'oxyanion hole' preventing formation of a substrate enzyme intermediate. 310Cys-->Phe is predicted to have an adverse effect on tissue factor interaction. These mutations point to important regions of the factor VII molecule.

A proposed molecular model for the carboxy terminus of HIV-1 gp120 showing structural features consistent with the presence of a T-cell alloepitope.

A computer graphics molecular model of the C terminus of gp120 of HIV has been constructed using predicted secondary structure based on homologies with proteins for which X-ray crystallographic data have been published. The model shows sequences known to be important in CD4 binding in close proximity to regions with a high probability of forming alpha helical and beta strand motifs. The orientation adopted by these domains approximates to the known 3D structure of HLA-A2 alpha 2 chain without constraints based on HLA-A2 as a template being introduced. The model may therefore represent an energetically favourable conformation for a part of gp120 which mimics the binding domain for the T-cell receptor on MHC molecules. Recognition of gp120 as an alloepitope in high affinity association with CD4 would explain many of the sequelae of acquired immune deficiency on HIV infection.