Skin grafts from genetically modified α-1,3-galactosyltransferase knockout miniature swine: A functional equivalent to allografts.

Burn is associated with a considerable burden of morbidity worldwide. Early excision of burned tissue and skin grafting of the resultant wound has been established as a mainstay of modern burn therapy. However, in large burns, donor sites for autologous skin may be limited. Numerous alternatives, from cadaver skin to synthetic substitutes have been described, each with varying benefits and limitations. We previously proposed the use of genetically modified (alpha-1,3-galactosyl transferase knockout, GalT-KO) porcine skin as a viable skin alternative. In contrast to wild type porcine skin, which has been used as a biologic dressing following glutaraldehyde fixation, GalT-KO porcine skin is a viable graft, which is not susceptible to loss by hyperacute rejection, and undergoes graft take and healing, prior to eventual rejection, comparable to cadaver allogeneic skin. In the current study we aimed to perform a detailed functional analysis of GalT-KO skin grafts in comparison to allogeneic grafts for temporary closure of full thickness wounds using our baboon dorsum wound model. Grafts were assessed by measurement of fluid loss, wound infection rate, and take, and healed appearance, of secondary autologous grafts following xenograft rejection. Comparison was also made between fresh and cryopreserved grafts. No statistically significant difference was identified between GalT-KO and allogeneic skin grafts in any of the assessed parameters, and graft take and function was not adversely effected by the freeze-thaw process. These data demonstrate that GalT-KO porcine grafts are functionally comparable to allogeneic skin grafts for temporary closure of full thickness wounds, and support their consideration as an alternative to cadaver allogeneic skin in the emergency management of large burns.

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts.

The emergence of skin-containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin-resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II-mismatched chimeras were tolerant of VCAs. MHC class I-mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient-type CD8+ lymphocytes. Systemic donor-specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Validation of multisociety combined task force definitions of abnormal disk morphology.

BACKGROUND AND PURPOSE: The multisociety task force descriptively defined abnormal lumbar disk morphology. We aimed to use their definitions to provide a higher level of evidence for the validation of MR imaging in the evaluation of this pathology in patients who have undergone diskectomy by retrospectively classifying their preoperative MRI. MATERIALS AND METHODS: This retrospective, institutional review board-approved study included 54 of 86 consecutive patients (47 men; average age, 44 years) enrolled in an ongoing prospective trial of surgically treated lumbar disk herniation who had preoperative MRI and documented intraoperative classification of the abnormal disk as protrusion, extrusion, or sequestration by the treating surgeon. Preoperative MRI was classified by 2 blinded radiologists; discrepancies were resolved by a third reader. Statistical analysis of interobserver agreement and imaging compared with surgical findings was performed. RESULTS: The readers disagreed on only 1 of the 54 cases. The third reader resolved the disagreement. Eight protrusions and 46 extrusions were found on imaging, with no sequestrations. At surgery, there were 13 protrusions and 40 extrusions, with 2 of the extrusions also containing sequestrations; the remaining case had only sequestration. There were 16 discrepancies between imaging and surgery, resulting in 70% agreement. CONCLUSIONS: This study, which was intended to validate the multisociety combined task force definitions of abnormal disk morphology by using MR imaging with a surgical criterion standard, found 70% agreement between imaging diagnosis and surgical findings. Although reasonable, this finding highlights differences that often exist between intraoperative and preoperative imaging findings of lumbar disk herniation.

Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation.

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Vascularized composite allograft tolerance across MHC barriers in a large animal model.

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

DNA sequence-dependent folding determines the divergence in binding specificities between Maf and other bZIP proteins.

Maf family transcription factors are atypical basic region-leucine zipper (bZIP) proteins that contain a variant basic region and an ancillary DNA-binding region. These proteins recognize extended DNA sequence elements flanking the core recognition element bound by canonical bZIP proteins. We have investigated the causes for the differences in DNA recognition between Maf and other bZIP family proteins through studies of Maf secondary structure, trypsin sensitivity, binding affinity, dissociation rate and DNA contacts. Our results show that specific DNA binding by Maf is coupled to a conformational change involving both the basic and ancillary DNA-binding regions that depends on the extended DNA sequence elements. Two basic region amino acid residues that differ between Maf and canonical bZIP proteins facilitate the conformational change required for Maf recognition of the extended elements. Nucleotide base contacts made by Maf differ from those made by canonical bZIP proteins. Taken together, our results suggest that the unusual DNA binding specificity of Maf family proteins is mediated by concerted folding of structurally unrelated DNA recognition motifs.

Lanosterol analogs: dual-action inhibitors of cholesterol biosynthesis.

Drugs which suppress hepatic cholesterol biosynthesis are important therapeutic tools for lowering serum cholesterol, a major risk factor in coronary heart disease. With the goal of developing molecules that will effectively shut down cholesterol biosynthesis in hepatic tissue but allow for the buildup of the isoprenes needed for the biosynthesis of polyisoprenes other than sterols, we have designed and evaluated a series of lanosterol analogs to act as dual-action inhibitors of cholesterol biosynthesis. These sterols were predicted to act as competitive inhibitors of lanosterol 14alpha-methyl demethylase (P-450DM) and as partial suppressors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in the pathway. Compounds which have been identified as dual-action inhibitors of cholesterol biosynthesis include analogs of the intermediates generated during the removal of the 14alpha-methyl group of lanosterol by P-450DM, aminolanosterols with the amine nitrogen placed in the vicinity of C-32, and lanosterol analogs with a ketone or oxime functionality at C-15. While some dual-action inhibitors require an active P-450DM for suppression of HMGR activity, others do not. The inability of some compounds to suppress HMGR activity in cells which lack P-450DM activity suggests either that these compounds require P-450DM for conversion to an active metabolite which then suppresses HMGR activity, or that they cause the accumulation of the natural demethylation intermediates resulting in the suppression of HMGR activity. Lanosterol analogs, in contrast to 25-hydroxycholesterol, do not inhibit transcription of the HMGR gene. Rather, they inhibit translation of the HMGR mRNA, and in most cases also accelerate the degradation of enzyme protein. The potential pharmacological utility of cholesterol biosynthesis inhibitors may be determined at least in part by their effects on LDL receptor (LDLR) activity. The transcriptional regulator 25-hydroxycholesterol suppresses both HMGR and LDLR activities, while the post-transcriptional regulatory lanosterol analogs exhibit a more desirable profile, lowering HMGR levels without suppressing LDLR expression, and in some cases actually enhancing cellular LDL metabolism. Lanosterol analogs which function as dual-action inhibitors of cholesterol biosynthesis promise to be useful not only as tools for dissecting the cellular regulation of cholesterol metabolism, but also as models for the development of safe, effective hypocholesterolemic agents.

DNA bending determines Fos-Jun heterodimer orientation.

Heterodimeric transcription factors can bind to palindromic recognition elements in two opposite orientations with potentially distinct effects on transcriptional activity. We have determined the orientation of Fos-Jun binding at different AP-1 sites using a novel gel-based fluorescence resonance energy transfer assay. The orientation preference of heterodimer binding varied over a greater than 10-fold range. Single base pair substitutions that alter bending of flanking sequences reversed the orientation of heterodimer binding. Single amino acid substitutions that reduce the difference in DNA bending between Fos and Jun also reduced the orientation preference. Consequently, indirect read-out mediated by differences in DNA structure can contribute to the structural organization of nucleoprotein complexes.

Molecular basis of cooperative DNA bending and oriented heterodimer binding in the NFAT1-Fos-Jun-ARRE2 complex.

Cooperative DNA binding by transcription factors that bind to separate recognition sites is likely to require bending of intervening sequences and the appropriate orientation of transcription factor binding. We investigated DNA bending in complexes formed by the basic region-leucine zipper domains of Fos and Jun with the DNA binding region of nuclear factor of activated T cells 1 (NFAT1) at composite regulatory elements using gel electrophoretic phasing analysis. The NFAT1-Fos-Jun complex induced a bend at the ARRE2 site that was distinct from the sum of the bends induced by NFAT1 and Fos-Jun separately. We designate this difference DNA bending cooperativity. The bending cooperativity was directed toward the interaction interface between Fos-Jun and NFAT1. We also examined the influence of NFAT1 on the orientation of Fos-Jun heterodimer binding using a novel fluorescence resonance energy transfer assay. The interaction with NFAT1 could reverse the orientation of Fos-Jun heterodimer binding to the ARRE2 site. The principal determinants of both cooperative DNA bending and oriented heterodimer binding were localized to three amino acid residues at the amino-terminal ends of the leucine zippers of Fos and Jun. Consequently, interactions between transcription factors can remodel promoters by altering DNA bending and the orientation of heterodimer binding.

The Drosophila gene asteroid encodes a novel protein and displays dosage-sensitive interactions with Star and Egfr.

The asteroid gene of Drosophila was found to lie within 189 bp of Star. Asteroid cDNA clones were isolated and sequenced and a single putative open reading frame was identified that encodes a novel protein of 815 amino acids with a calculated molecular mass of 93 kilodaltons. Using cDNA probes, asteroid transcripts were localized to the proliferative tissues of embryos and to the mitotically active tissue anterior to the morphogenetic furrow in eye imaginal discs. Ribonuclease protection assays identified a mutation of asteroid that acts as a dominant enhancer of Star mutations and also enhances the Ellipse mutation, EgfrE1. Based on these data, a model for asteroid gene function in EGF receptor signaling is presented.

Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein.

The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shut-off mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg305), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.

Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region.

Members of the Rho subfamily of GTP-binding proteins contain a region of amino acid sequence (residues 122-134) that is absent from other Ras-like proteins and is termed the Rho insert region. To address the functional role of this domain, we have constructed a Cdc42Hs/Ras chimera in which loop 8 from Ha-Ras was substituted for the region in Cdc42Hs that contains the 13-amino acid insert region. Our data indicate that the insert region of Cdc42Hs is not essential for its interactions with various target/effector molecules or for interactions with the guanine nucleotide exchange factor, Dbl, or the Cdc42 GTPase-activating protein (GAP). However, the regulation of GDP dissociation and GTP hydrolysis on Cdc42Hs by the Rho GDP-dissociation inhibitor (GDI) is extremely sensitive to changes in the insert region, such that a Cdc42Hs/Ha-Ras chimera that lacks this insert is no longer susceptible to a GDI-induced inhibition of GDP dissociation and GTP hydrolysis. The insensitivity to GDI activity is not due to the inability of the GDI molecule to bind to the Cdc42Hs/Ha-Ras chimera, and in fact, the GDI is fully capable of stimulating the release of this chimera from membranes.

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun.

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos-Jun-AP-1 complex may contribute to its functional versatility at different promoters.

Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3.

The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies. The protein can be purified on a nickel affinity resin due to a hexa-His tag that is incorporated onto the amino terminus of the domain. PBD binds to Cdc42Hs in a guanine nucleotide-dependent manner as demonstrated by a novel fluorescence assay that takes advantage of the spectroscopic properties of N-methylanthraniloyl (Mant)-guanine nucleotides. Ionic strength has little effect on the affinity of PBD for Cdc42Hs, but alkaline pH values tend to weaken the interaction. We have shown that the inhibition of the GTPase activity of Cdc42Hs, as well as a previously undescribed inhibition of guanine nucleotide dissociation, is mediated by the PBD portion of the mPAK-3 molecule. These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleotide domain. We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotide binding site, had any effect on PBD binding. Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold decrease in affinity. Finally, the catalytic domain of the GTPase activating protein, Cdc42-GAP, was shown to inhibit PBD binding in a competitive manner, indicating that this target molecule and the negative regulator (GAP) bind to overlapping sites on the Cdc42Hs molecule.

Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.

The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of approximately 32 A for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.

Potency ranking of methemoglobin-forming agents.

This study represents the first systematic attempt to rank methemoglobin-forming agents. It is a quantitative potency ranking study utilizing linear regression analysis of dose-response data for comparative purposes. Six agents that are direct-acting and eight that require bioactivation were tested for their ability to induce methemoglobin formation in Dorset sheep erythrocytes under defined in vitro conditions. The agents were then ranked according to three complementary methods based on the slope of the linear regression, the calculated dose expected to induce a given amount of methemoglobin formation and the calculated percentage methemoglobin response induced by 1 mmol l-1 of the agent. The direct-acting agents, ranked from most to least potent inducers of methemoglobin formation, are: p-dinitrobenzene > o-dinitrobenzene > copper = nitrite > chlorite > chlorate. The ranking from most to least potent inducers of the bioactivated agents are: alpha-naphthol > p-nitroaniline > m-nitroaniline, o-nitroaniline > p-nitrotoluene = aniline > m-nitrotoluene = o-nitrotoluene. The ranking procedures are discussed and issues of interindividual variation and agent-specific sensitivities are addressed.

The influence model of sponsorship. A Catholic hospital is now part of the "Mayo Clinic".

Saint Marys Hospital was founded in Rochester, MN, in 1889. Constructed by the Sisters of St. Francis, it was staffed by physician members of the local Mayo family. The Mayo practice grew into an association of many physicians and medical residents who later began to staff Rochester Methodist Hospital also; the three healthcare institutions became collectively known as the "Mayo Clinic." By the mid-1980s, billing was so complex for the three still-independent facilities that their leaders decided to integrate more formally. This was done in three phases and resulted in the creation of a single institution known as the Mayo Medical Center. From Saint Marys' standpoint, the facilitating document in this process was a "Sponsorship Agreement" whose purpose was to maintain the sponsor's interests and obligations in the integrated structure. A Sponsorship Board was created to continue the hospital's Catholic tradition, including maintaining its chaplaincy, chapels, religious symbols, and special funds. The Sponsorship Board views the new environment as a special challenge. Its members know that Catholic sponsorship: Comforts patients, who realize they are in the hands of people motivated by the Christian ethic Creates an atmosphere in which patients and their families can seek the spiritual support that often aids healing Strengthens a sense of community among physicians, hospital staff, and administrators The Sponsorship Board hopes the sponsor's influence may come to affect the whole Mayo Medical Center, bringing patients, family members, and staff an "added dimension" of care.