Immune consequences of intraocular administration of modified adenoviral vectors.

To investigate the immune consequences of intraocular administration of modified adenoviral vectors, C57BL/6 normal and retinal degeneration C57BL/6 (rd/rd) mice were immunized with subcutaneous, subretinal, vitreal, or anterior chamber injections of replication-deficient adenovirus (AdV) containing the Escherichia coli beta-galactosidase gene (AdV-LacZ). Fourteen days after the initial inoculation, the animals were immune challenged with an injection of AdV-LacZ in the right ear pinna. Antigen-induced delayed type hypersensitivity (DTH) was measured by determining relative ear swelling. Normal C57BL/6 mice immunized with subretinal, vitreal, or anterior chamber injections did not demonstrate a DTH response. The rd/rd C57BL/6 mice injected in the anterior chamber with the viral construct also did not respond with DTH in a manner similar to normal mice responding to intraocular injection and subsequent challenge. However, the rd/rd C57BL/6 mice immunized by the subretinal or vitreal route did respond to immune challenge with a DTH response. Histologic examination of the eyes showed a lack of infiltration by inflammatory cells. Although these results suggest that the potential for immune consequences is reduced when modified adenoviral vectors are used in the normal ocular environment, these vectors used in the vitreal cavity of rd/rd animals may induce a systemic response to the vectors.

Metastatic and nonmetastatic models of retinoblastoma.

To generate animal models of retinoblastoma that closely resemble metastatic and nonmetastatic human disease for the purposes of examining tumor biology and developing alternate treatments, human retinoblastoma cell lines were injected into the vitreal cavities of immunodeficient mice. Two reproducible animal models with contrasting biological behaviors analogous to human retinoblastoma have been developed. The Y79 retinoblastoma model demonstrated specific tumor evolution similar to that seen in human invasive and metastatic disease. Y79 retinoblastoma cells formed intraocular tumors that were initially confined to the vitreal cavity. Tumors progressively invaded the retina, subretinal space, choroid, optic nerve head, and anterior chamber of the eye. Tumors progressed into the subarachnoid space and focally invaded the brain. Metastases were detected in the contralateral optic nerve. Large tumors developed extraocular extensions. The histology of the tumors showed a poorly differentiated pattern with high mitotic rate, foci of necrosis, and calcification. The WERI-Rb model more closely resembled nonmetastatic human retinoblastoma. WERI- Rb tumors were localized in the eye with only anterior choroidal invasion at late stages. To examine potential biological differences in vitro, the retinoblastoma cell lines were cocultured with adherent choroid cells or adherent glioma cells which represent the targets of invasive retinoblastoma in vivo. Consistent with the in vivo observations, Y79 cells but not WERI-Rb cells adhere specifically to both the choroidal and the glioma cell lines.

Mycophenolate mofetil, with cyclosporine and prednisone, reduces early rejection while allowing the use of less antilymphocytic agent induction and cyclosporine in renal recipients with delayed graft function.

Antilymphocytic agent induction (ALAI), with antithymocyte globulin or monoclonal antibody, is generally used in renal transplantation (TX) to spare renal allografts with poor initial function from the toxic effects of cyclosporine (CsA) and/or to augment immunosuppression (IS) in the patient at a high risk for early rejection. ALAI, unfortunately, increases the cost of TX and the risk to the patient, having been associated with many adverse side effects. An IS protocol, which results in a low incidence of early rejection while using less CsA and ALAI, is a worthwhile goal. We compare our experience with mycophenolate mofetil (MMF), CsA, and prednisone (MMFCP; n = 62) to our azathioprine (AZA), CsA, and prednisone (AZACP; n = 50) triple-drug IS, with and without ALAI. The patient characteristics for age, race, first TX, cadaveric donor, pediatric recipient, and dialysis in the first post-op week (DGF) were not different for the MMFCP versus AZACP groups. There were more females in the MMFCP group (51.6% versus 30.0%, p = 0.022). We report that rejection-free survival at 6 months (RF6) was better in the MMFCP versus AZACP group (83.9% versus 60.0%, p = 0.005). Less ALAI and CsA were used in the MMFCP patients. At 1 year, actuarial graft survival was 91.9% in the MMFCP group and 81.9% in the AZACP group (p = 0.116). Actuarial 1-year patient survivals were not different in the two patient groups. In the sub-population of patients with DGF, the RF6 in the MMFCP (n = 13) group was 92.3% versus 57.1% in the AZACP (n = 14) group (p = 0.041). The reduction in early rejection episodes in the patients on MMFCP with DGF was accomplished while using half as much ALAI and lower CsA doses and levels. The African-American recipient sub-population on MMFCP also demonstrated an improvement in RF6 while using less ALAI and CsA (78.6% versus 48.0%, p = 0.022). We conclude that the use of MMF-based triple-drug IS results in fewer rejection episodes while allowing for lower CsA levels and less ALAI, even in patients with delayed graft function.

Suicide gene therapy for treatment of retinoblastoma in a murine model.

Children presenting with large retinoblastomas are currently treated by enucleation. As most patients are young children, the long-term repercussions of such surgery are often devastating. Subsequent radiation or chemotherapy, although effective in managing residual tumor, greatly increase the probability of the development of second malignancies later in life. Smaller tumors can sometimes be managed with local cryo- or laser surgery, thus saving the eye. The hypothesis that gene therapy could be used to reduce the tumor size sufficiently to allow local control was tested using a murine model of retinoblastoma. Y79Rb human retinoblastoma cells can be killed in vitro when transduced with an adenoviral vector containing the herpes simplex thymidine kinase gene (AdV-TK) followed by treatment with the prodrug ganciclovir. Intravitreal injections of Y79Rb cells in immunodeficient mice produce an aggressive, metastatic murine model of retinoblastoma. When these murine retinoblastomas were transduced in vivo with AdV-TK and the animals treated with intraocular injections of ganciclovir, 70% showed a complete ablation of detectable tumor. Treated animals had a significant prolongation of progression-free survival as compared with untreated controls. Gene therapy effectively reduced the tumor burden in this murine model of retinoblastoma. Thus gene therapy, in conjunction with local surgical control, may provide an effective alternative to enucleation, systemic chemotherapy, or radiotherapy for treatment of large, nonmetastatic retinoblastomas in children.

Distal revascularization-interval ligation for limb salvage and maintenance of dialysis access in ischemic steal syndrome.

PURPOSE: Traditional options for treating ischemic steal syndrome related to a functioning dialysis access graft or fistula include banding or ligation. Unfortunately, these techniques usually result in inconsistent limb salvage, loss of a functional access, or both. We report our experience with an alternative method of limb revascularization that eliminates steal while maintaining continuous dialysis access. METHODS: Patients who had critical limb ischemia and functioning arteriovenous fistulae (AVF) underwent color-flow duplex scanning, digital photoplethysmography, and arteriography. Arterial ligation distal to the AVF origin eliminated the steal physiologic mechanism while arterial bypass grafting from above to below the AVF revascularized the extremity (distal revascularization-interval ligation [DRIL] procedure). RESULTS: From March 1994 through December 1996, 21 patients with functioning extremity AVFs presented with critical ischemia and steal syndrome. Eleven patients had chronic ischemia with rest pain, paresthesias, or ulcerations related to nine native fistulae (six brachiocephalic, two basilic vein transpositions, one radiocephalic) and two prosthetic bridge grafts (one upper arm, one lower extremity). Acute ischemia developed in 10 patients related to three native fistulae (two brachiocephalic, one radiocephalic) and seven prosthetic bridge grafts (three forearm, three lower extremity, one upper arm). All 21 patients were treated with the DRIL technique. Three of these patients required treatment for ischemia at the time of AVF construction. Nineteen of 21 bypass procedures were performed with autogenous vein, including nine brachial-brachial, three brachial-radial, two radial-radial, two brachial-ulnar, one popliteal-popliteal, one femoral-popliteal, and one femoral-peroneal. Polytetrafluoroethylene grafts were used for one external iliac-popliteal bypass graft and one axillary-brachial bypass graft. Limb salvage and maintenance of a functional fistula were achieved in 100% and 94%, respectively, at 18 months by life-table analysis. CONCLUSION: The DRIL technique reliably restores antegrade flow to the ischemic limb, eliminates the potential pathway for the steal physiologic mechanism, and maintains continuous dialysis access in these difficult patients.

Cloning and analysis of the cDNA encoding the rod G-protein transduction alpha, beta1 and gamma1 subunits from the canine retina.

The canine (Canis familiaris) retinal rod transducin (G(T)) alpha, beta1 and gamma1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G(T alpha) differed by 5 amino acids from the corresponding human sequence, whereas beta1 and gamma1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.

Cloning of the cDNA encoding rod photoreceptor cGMP-phosphodiesterase alpha and gamma subunits from the retinal degenerate Labrador retriever dog.

The nucleotide (nt) sequence of the cDNA encoding the retinal rod cyclic 3'5'-GMP phosphodiesterase (PDE) alpha and gamma subunits from two strains of dogs-(i) Labrador Retrievers homozygous for autosomally recessively inherited rod-cone degeneration and (ii) the wild-type Beagle-are reported. Cloning of these subunits was accomplished by polymerase chain reaction using retinal cDNA libraries as templates. The nt sequence of alpha PDE predicts a 861-amino-acid polypeptide which is 97.7 per cent and 96.9 per cent identical to the bovine and human counterparts, respectively. PDE gamma encodes an 87-amino-acid polypeptide differing from bovine and murine gamma subunits by only one amino acid. Since no differences were found between these two strains of dogs, the cause of the Labrador Retriever's degeneration remains to be determined.

Lymphoblastic lymphoma following prenatal exposure to phenytoin.

PURPOSE: Oncogenesis has been associated with prenatal exposure to phenytoin, concomitant with or independent of the fetal hydantoin syndrome. The majority of reported cases have been embryonal tumors of neural crest origin and have occurred in the first 3 years of life. PATIENTS AND METHODS: We report a boy who was exposed to phenytoin throughout gestation and later developed T-lymphocyte lymphoblastic lymphoma, a previously unreported malignancy associated with in utero phenytoin exposure. Previously reported cases of neoplasia occurring after such exposure are tabulated. CONCLUSION: The actual transplacental oncogenic potential of phenytoin and the epidemiology of this association are poorly understood. Phenytoin-induced alterations in lymphocyte-mediated immunosurveillance or oxidative metabolic clearance may be etiologic. Inquiry into prenatal phenytoin exposure should be done in any child who develops cancer, especially those who develop a rare tumor or present with a more common tumor at an unusually young age. Continued documentation of such cases will advance the understanding of phenytoin-associated transplacental oncogenesis.

Elevation of cGMP with normal expression and activity of rod cGMP-PDE in photoreceptor degenerate labrador retrievers.

Cyclic guanosine 3',5'-monophosphate (cGMP) levels were determined in retinas from a strain of Labrador Retrievers with inherited retinal dystrophy manifesting at early stages of retinal differentiation. The cGMP contents of dystrophic retinas of dogs from 1 to 4 months of age (n = 7) were significantly higher (p = 0.001) than in age-matched controls of the same breed (n = 11). Ultrastructure along the vertical retinal meridian was studied in developing retinas and findings were related to those of age-matched wild-type controls of the same breed. Slow central to peripheral progression of degeneration was observed in affected dogs. No differences were found in total cGMP-phosphodiesterase (PDE) activity, in PDE subunit composition as determined by Western blotting of 2-month-old homozygote affected retinas, or in the amino acid sequence deduced from the nucleotide sequence of the PDE beta-subunit as compared to controls. This model of photoreceptor degeneration thus is the first case of an apparent abnormality of cGMP metabolism that is not associated with a defect in the PDE catalytic subunits, and it is also the first reported model not associated with severe developmental abnormalities and rapid degeneration.

Lead- and calcium-mediated inhibition of bovine rod cGMP phosphodiesterase: interactions with magnesium.

Previously we showed that cGMP hydrolysis in rat whole retinal homogenates exhibited a dose-dependent inhibition following developmental lead exposure and a concentration-dependent inhibition with direct Pb2+ exposure. Additionally, developmental lead exposure resulted in a dose-dependent increase in retinal cGMP and rod Ca2+ levels. To determine whether Pb2+ or Ca2+ directly inhibited the rod-specific cGMP phosphodiesterase (PDE) and to examine the kinetic mechanism of this inhibition, purified bovine rod cGMP PDE was assayed in the presence of varying concentrations of cGMP, and Mg2+, Pb2+, and/or Ca2+. Increasing concentrations of the substrate, cGMP, resulted in a shift of the Pb2+ and Ca2+ concentration-response curves to the left, indicating a decrease in the half-maximal inhibitory concentrations of Pb2+ from nanomolar to picomolar levels. Increasing concentrations of the cofactor, Mg2+, resulted in a shift of the Pb2+ and Ca2+ concentration-response curves to the right, indicating a decrease in the inhibition of PDE activity by Pb2+ or Ca2+. A plot of 1/velocity vs 1/Mg2+ as a function of Pb2+ revealed that picomolar concentrations of Pb2+ competitively inhibited PDE relative to millimolar concentrations of Mg2+. Consistent with this finding, Mg2+ reversed the Pb(2+)-induced inhibition of PDE. Our recent kinetic analysis showed that Mg2+ and cGMP bind at interacting sites on the PDE in a random order. The present results reveal that Pb2+ may bind at the same site but with 4-6 log units higher affinity than Mg2+, thus preventing the hydrolysis of cGMP. These findings provide a novel mechanism for understanding the Pb(2+)-induced inhibition of cGMP PDE. These results may have implications for other enzymes using Mg2+ as a cofactor and suggest that Mg2+ may be useful in these situations for reversing the inhibition by Pb2+.

The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase.

To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 mumol/L [3H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but not to the rod in bovine retina. ROS-1 immunoadsorbed approximately 80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of approximately 87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.

Effects of magnesium on cyclic GMP hydrolysis by the bovine retinal rod cyclic GMP phosphodiesterase.

Knowledge of the kinetics of the rod cyclic GMP phosphodiesterase is essential for understanding the kinetics and gain of the light response. Therefore, the interactions between Mg2+, cyclic GMP, and purified, trypsin-activated bovine rod cyclic GMP phosphodiesterase (EC 3.1.4.17) were examined. The effects of Mg2+ and of cyclic GMP on the rod phosphodiesterase activity were mutually concentration-dependent. Formation of a free Mg-cyclic GMP complex is unlikely due to its high dissociation constant (Kd = 19 mM). Plots of 1/velocity versus 1/[cyclic GMP] as a function of [Mg2+] and 1/velocity versus 1/[Mg2+] as a function of [cyclic GMP] intersected to the left of the 1/velocity axis. This is consistent with the formation of a ternary complex between the phosphodiesterase, Mg2+, and cyclic GMP. A competitive inhibitor of the phosphodiesterase relative to cyclic GMP, 3-isobutyl-1-methylxanthine, non-competitively inhibited the enzyme relative to Mg2+, Pb2+, a competitive inhibitor of the phosphodiesterase relative to Mg2+ [D. Srivastava, R.L. Hurwitz and D. A. Fox (1995) Toxicol. Appl. Pharmacol, in the press] non-competitively inhibited the enzyme relative to cyclic GMP. Collectively these results are suggestive of a rapid equilibrium random binding order of Mg2+ and cyclic GMP to the rod phosphodiesterase.

Lead-induced alterations in rod-mediated visual functions and cGMP metabolism: new insights.

Long-term scotopic (rod-mediated) visual deficits following developmental lead exposure occur in monkeys and hooded rats. This report describes and summarizes previous ERG and biochemical findings, presents new biochemical data aimed at determining the mechanism of inhibition of lead on rod cGMP-PDE, presents an integratory framework for understanding the ERG and cGMP results and speculates on the implications of the present data. A- and b-wave voltage-log intensity and latency-log intensity functions, generated from single-flash ERGs in fully dark-adapted rats, revealed that low and moderate level lead exposure caused decreases in absolute sensitivity and amplitude, and increases in latency. Rod- and cone-mediated flicker fusion frequency measures revealed selective rod deficits in temporal resolution. In addition, the slope of the increment threshold function was decreased, but only at scotopic adapting backgrounds, and dark adaptation was delayed. Prior exposure to lead produced a dose-response inhibition of retinal cGMP-phosphodiesterase (PDE) resulting in an increase in cGMP in dark-adapted and light-adapted states and an increase in the calcium content of rods. In vitro experiments with adult rat retinas incubated with 10(-9) to 10(-4) M Pb2+ revealed a concentration-dependent inhibition of cGMP-PDE which suggested that Pb2+ directly inhibited the rod cGMP-PDE. This was confirmed in experiments conducted with isolated, purified, trypsin-activated bovine rod cGMP-specific PDE exposed to 5 x 10(-8) to 10(-4) M Pb2+. The cGMP data are entirely consistent with the observed ERG changes. The ERG data is relevant to low level pediatric lead poisoning since rat rods are similar to human rods. Finally, since a lesion in the gene that codes for a cAMP-PDE leads to defective learning and memory in the Drosophila dunce flies, it is possible that lead-induced alterations in cyclic nucleotide phosphodiesterases contribute to the long-term CNS deficits produced by developmental lead exposure.

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Dominant thalassemia-like phenotypes associated with mutations in exon 3 of the beta-globin gene.

Mutations producing beta-thalassemia reach individual gene frequencies greater than .01 in malarial-endemic regions because beta-thalassemia trait individuals have increased genetic fitness over that of normal individuals. Exon 3 of the beta-globin gene has been relatively spared as a site of common beta-thalassemia mutations. Frameshifts caused by the loss of a single nucleotide and nonsense mutations produce beta-thalassemia trait when they occur in exons 1 and 2. In contrast, they usually produce chronic hemolytic anemia when present in exon 3. Certain missense mutations in exon 3 produce unstable globins and thalassemia intermedia with hemolysis in heterozygotes. Here we report two new mutations in exon 3 of the beta-globin gene. One is a single nucleotide deletion in codon 109 in a 78-year-old Lithuanian with chronic hemolytic anemia and features of thalassemia. It leads to an abnormal globin (beta Manhattan) that is elongated to 156 amino acids. The second is a CAG-CGG missense mutation at codon 127 that causes a Gln----Pro substitution (beta Houston) and a thalassemia intermedia with hemolysis in three generations of a British-American family. Although the clinical phenotypes of these two patients differed little, differences in globin-synthetic ratios were significant, presumably reflecting differences in the ability of each abnormal beta-globin to form alpha beta dimers. The paucity of high-frequency exon 3 mutations and their worldwide distribution is likely attributable to their phenotypic severity and loss of increased genetic fitness vis-a-vis malaria.

Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel.

We have determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel by analysis of cDNA clones and amplified DNA. The open reading frames predicted polypeptides of 690 and 683 residues exhibiting 88% sequence similarity. Sequence comparison indicated that the rod channels consist of a variable 90-residue N-terminal region, a short highly charged segment rich in lysine and glutamate, and a 540-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (59%) of the visual cGMP-gated channel to the olfactory cAMP-gated channel established the existence of a family of cyclic nucleotide-gated ion channel genes. RNA blot analysis revealed transcripts of 3.2 kilobases (kb) in human, mouse, and dog, 3.2, 4.6, and 5.2 kb in bovine, and 3.6 kb in fish. The human channel gene was mapped by polymerase chain reaction of somatic cell hybrid DNAs to chromosome 4 (p14-q13) near the centromere. The mouse channel gene locus (Cncg) was mapped by interspecific backcross haplotype analysis 0.9 centimorgan proximal of the Kit locus on chromosome 5.

Transmission of human immunodeficiency virus type 1 from a seronegative organ and tissue donor.

BACKGROUND: Since 1985, donors of organs or tissues for transplantation in the United States have been screened for human immunodeficiency virus type 1 (HIV-1), and more than 60,000 organs and 1 million tissues have been transplanted. We describe a case of transmission of HIV-1 by transplantation of organs and tissues procured between the time the donor became infected and the appearance of antibodies. The donor was a 22-year-old man who died 32 hours after a gunshot wound; he had no known risk factors for HIV-1 infection and was seronegative. METHODS: We reviewed the processing and distribution of all the transplanted organs and tissues, reviewed the medical histories of the donor and HIV-1-infected recipients, tested stored donor lymphocytes for HIV-1 by viral culture and the polymerase chain reaction, and tested stored serum samples from four organ recipients for HIV-1 antigen and antibody. RESULTS: HIV-1 was detected in cultured lymphocytes from the donor. Of 58 tissues and organs obtained from the donor, 52 could be accounted for by the hospitals that received them. Of the 48 identified recipients, 41 were tested for HIV-1 antibody. All four recipients of organs and all three recipients of unprocessed fresh-frozen bone were infected with HIV-1. However, 34 recipients of other tissues--2 receiving corneas, 3 receiving lyophilized soft tissue, 25 receiving ethanol-treated bone, 3 receiving dura mater treated with gamma radiation, and 1 receiving marrow-evacuated, fresh-frozen bone--tested negative for HIV-1 antibody. Despite immunosuppressive chemotherapy, HIV-1 antibody appeared between 26 and 54 days after transplantation in the three organ recipients who survived more than four weeks. CONCLUSIONS: Although rare, transmission of HIV-1 by seronegative organ and tissue donors can occur. Improvements in the methods used to screen donors for HIV-1, advances in techniques of virus inactivation, prompt reporting of HIV infection in recipients, and accurate accounting of distributed allografts would help to reduce further this already exceedingly low risk.