Tolerance of Vascularized Islet-Kidney Transplants in Rhesus Monkeys.

We previously reported that transplantation (Tx) of prevascularized donor islets as composite islet-kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cell (HSC) Tx in an attempt to induce tolerance in nonhuman primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IK Tx. HSC Tx was performed after mobilization and leukapheresis of the donors and conditioning of the recipients with total body irradiation, T cell depletion, and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IK Tx was performed either 20-22 (n = 3) or 208 (n = 1) days after HSC Tx. All animals accepted IKs without rejection. All recipients required >20 U/day insulin before IK Tx to maintain <200 mg/dL, whereas after IK Tx, three animals required minimal doses of insulin (1-3 U/day) and one animal was insulin free. These results constitute a proof-of-principle that this IK tolerance strategy may provide a cure for both end-stage renal disease and diabetes without the need for immunosuppression.

Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead Box Protein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Evidence for a gene controlling the induction of transplantation tolerance.

Class I mismatched kidney transplantation in Massachusetts General Hospital MHC-defined miniature swine has been studied extensively as a model for induction of systemic allograft tolerance. In a large series of juvenile swine, long-term graft acceptance has been observed consistently following a 12-day course of cyclosporine. It was therefore surprising when three of five recipients in one of our studies rejected their grafts. Examination of the origins of the rejecting animals revealed that they were derived from a subline of the SLA(dd) miniature swine herd that was intentionally being inbred toward full homozygosity and had been inbred for eight generations prior to these experiments. A blinded study of additional class I mismatched renal transplants into animals from this subline confirmed the genetic basis of this rejection. We present here preliminary evidence suggesting that a likely explanation for this phenomenon is that the rejectors in this subline are homozygous for a recessive mutant allele of a gene normally involved in the induction of tolerance. Subsequent studies will be directed toward identification and characterization of the gene(s) involved, since existence of a similar genetic locus in humans might have implications for assessing an individual's likelihood of graft rejection versus tolerance induction prior to organ transplantation.

Food supplements and European regulation within a precautionary context: a critique and implications for nutritional, toxicological and regulatory consistency.

In this paper, we review European legislation in the field of micronutrient food supplements and find it wanting. It is shown that the precautionary principle, embedded in European food legislation, pre-empts innovative developments in this field. In view of the scientific advances in micronutrients research, we subsequently critique the precautionary perspective and propose a novel outlook on micronutrients food supplements regulation. However, this requires a transition from the "survival" approach of the current deficiency-related RDAs to a "health-optimization" approach of a n(ew)-RDA. Genomic integrity is central in this envisioned transition.

Identification of point mutations in mixtures by capillary electrophoresis hybridization.

We have developed a rapid method for unambiguous identification and mutant fraction determination of individual mutants in mixtures of DNA sequence variants each differing by one or a few nucleotides. This method has applications to such diverse areas as interpretation of mutational spectra, screening of populations for polymorphisms and identification of species in environmental mixtures. In our approach, a mixture of unknown sequences labeled with a fluorescent dye is combined with a set of predetermined sequences (standards) representing the variants to be assayed. Labeling the standards with another dye allows the two sets of variants to be measured independently. Using constant denaturing capillary electrophoresis, the sequence variants are separated as individual peaks on the basis of differential melting equilibria. The unknown sequence variants are initially identified based on co-migration with particular standards. This preliminary identification is verified by hybridization of the unknown variants with the co-migrating standards within the capillary. We demonstrate the use of capillary electrophoresis hybridization to dissect complex mutational spectra of human cells in culture.

Mitochondrial mutational spectra in human cells and tissues.

We have found that human organs such as colon, lung, and muscle, as well as their derived tumors, share nearly all mitochondrial hotspot point mutations. Seventeen hotspots, primarily G --> A and A --> G transitions, have been identified in the mitochondrial sequence of base pairs 10,030-10,130. Mutant fractions increase with the number of cell generations in a human B cell line, TK6, indicating that they are heritable changes. The mitochondrial point mutation rate appears to be more than two orders of magnitude higher than the nuclear point mutation rate in TK6 cells and in human tissues. The similarity of the hotspot sets in vivo and in vitro leads us to conclude that human mitochondrial point mutations in the sequence studied are primarily spontaneous in origin and arise either from DNA replication error or reactions of DNA with endogenous metabolites. The predominance of transition mutations and the high number of hotspots in this short sequence resembles spectra produced by DNA polymerases in vitro.

Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis.

A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in a human B-cell line TK6 and T-cells from four individuals. A T --> C transition at position 16519 in the origin/membrane attachment site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G --> A transition at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful for the rapid screening of polymorphisms in a large number of samples.

Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 the N-ras gene by constant denaturant capillary electrophoresis (CDCE).

We used high fidelity PCR and constant denaturant capillary electrophoresis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] to separate wild type and different mutant N-ras exon 1 and 2 sequences. The set of plasmids containing N-ras cDNA, wild type or mutant sequences representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerase in a limited cycle polymerase chain reaction. One of the primers used for the amplification of each exon included a 40 nucleotide GC rich sequence that created high and low melting domains. The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were run on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wild type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a partially melted double stranded fragment. The denaturation and reannealing of wild type and mutant fragments together created wild type/mutant heteroduplexes. All the heteroduplexes were well resolved from wild type homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has resulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.

Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis.

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.