Diane J. Pidwell

Vision Improvement in Retinal Degeneration Patients by Implantation of Retina Together with Retinal Pigment Epithelium

PURPOSE To demonstrate efficacy and safety of the implantation of neural retinal progenitor cell layers (sheets) with its retinal pigment epithelium (RPE) in retinitis pigmentosa (RP) and dry age-related macular degeneration (AMD) patients with 20/200 or worse vision in the surgery eye. DESIGN Interventional nonrandomized clinical trial. METHODS Ten patients (six RP, four AMD) received retinal implants in one eye and were followed in a phase II trial conducted in a clinical practice setting. Early Treatment Diabetic Retinopathy Study (EDTRS) was the primary outcome measure. All implant recipients and nine of 10 tissue donors were deoxyribonucleic acids typed. RESULTS Seven patients (three RP, four AMD) showed improved EDTRS visual acuity (VA) scores. Three of these patients (one RP, two AMD) showed improvement in both eyes to the same extent. Vision in one RP patient remained the same, while vision in two RP patients decreased. One RP patient has maintained an improvement in vision from 20/800 to 20/200 ETDRS for more than five years; at the six-year examination, it was still maintained at 20/320 while the nonsurgery eye had deteriorated to hand motion vision. This patient also showed a 22.72% increase in light sensitivity at five years compared to microperimetry results at two years; the other patients showed no improved sensitivity. Although no match was found between donors and recipients, no rejection of the implanted tissue was observed clinically. CONCLUSIONS Seven (70%) of 10 patients showed improved VA. This outcome provides clinical evidence of the safety and beneficial effect of retinal implants and corroborates results in animal models of retinal degeneration.

Transplantation of intact sheets of fetal neural retina with its retinal pigment epithelium in retinitis pigmentosa patients.

PURPOSE To show the safety of transplanting sheets of fetal neural retina together with its retinal pigment epithelium (RPE) to patients with retinitis pigmentosa. DESIGN Interventional case series. METHODS Sheets of fetal neural retina and RPE were transplanted together into the subretinal space near the fovea unilaterally in the eyes of five patients with retinitis pigmentosa who had only light perception in both eyes. The patients were followed for 6 months. The main outcome measures were tissue typing of both donors and recipients, fluorescein angiography, multifocal electroretinogram (mfERG) testing, and clinical examination. No immunosuppressive medications were given. RESULTS No evidence of rejection was observed. Up to 6 months there was no evidence of tissue disintegration, retinal edema, or scarring. There was no change in vision both by Snellen acuity and with mfERGs. Growth of the transplant was noted in two of five patients at 6 months vs. 2 weeks. All patients typed were HLA mismatched with donor tissue. CONCLUSIONS This study indicates that fetal retina can be transplanted together with its RPE and survive for at least 6 months without evidence of rejection. However, no improvements in vision were observed, possibly due to the severe retinal degeneration of the patients.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

Background. Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. Methods. [ACI→WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. Results. [ACI→WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4±2.8 days after limb transplantation. [ACI→WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. Conclusions. Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI→WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Composite tissue allotransplantation in chimeric hosts part II. A clinically relevant protocol to induce tolerance in a rat model

Background. We and others have shown that mixed allogeneic chimerism induces donor-specific tolerance to composite tissue allografts across major histocompatibility complex barriers without the need for immunosuppression. However, a delay period between bone marrow transplantation and limb allotransplantation is required, making such protocols impractical for clinical application. This study eliminates this delay period in a rat hind limb allotransplantation model by performing mixed allogeneic chimerism induction and transplantation “simultaneously.” Methods. Group 1 included controls in which naïve Wistar Furth (WF) hosts received ACI hind limbs. Group 2 included (ACI→WF) chimeras that received limbs from third-party donors (Fisher), and group 3 included chimeras that received irradiated (1,050 cGy) ACI limbs. In group 4, WF hosts conditioned with 950 cGy received irradiated (1,050 cGy) ACI limbs followed by infusion of 100×106 ACI T-cell–depleted bone marrow cells and immunotherapy (tacrolimus and mycophenolate mofetil) for 28 days. Group 5 animals received the same treatment as group 4 animals without immunotherapy. Results. The rats in groups 1 and 2 rejected their limbs within 10 days. Only one rat in group 4 survived to the end of the study. Groups 3 and 5 demonstrated long-term limb survival without rejection or graft-versus-host disease. High levels of donor chimerism (>80%) were achieved and maintained throughout the study. Mixed lymphocyte reaction assays in both groups revealed donor-specific hyporesponsiveness with vigorous third-party reactivity. Conclusions. This study demonstrated that infusion of donor bone marrow cells into conditioned hosts immediately after limb transplantation results in stable mixed chimerism, robust tolerance, and reliable limb allograft survival.

Predictors of Acute Rejection After Lung Transplantation

BACKGROUND Acute rejection (AR) after lung transplantation (LTx) impacts survival and quality of life. The objective of this study, therefore, was to identify risk factors for AR after LTx, focusing on donor- and recipient-specific factors, operative variables, and immunologic issues, including pretransplant panel-reactive antibody (PRA) levels, and donor-recipient human leukocyte antigen (HLA) mismatch. METHODS From March 1996 to November 2007, 481 adults undergoing LTx had 3237 serial transbronchial biopsy specimens that were evaluated for perivascular rejection (grade A0 to A4). Longitudinal analysis was used to characterize the prevalence of rejection grade and influence of donor, recipient, technical, and immunologic variables. RESULTS AR was highest (54%≥A1) in the first 2 months after LTx, decreased at 6 months (16%≥A1), then remained steady. Prevalence of AR at any time was dominated by donor-specific factors of young age (p<0.0001), blunt trauma (p=0.008), and nonblack race (p=0.012) and by recipient class II PRA exceeding 10% (p=0.005). AR within 2 months was associated with HLA mismatch at the DR locus (p=0.0006) and use of non-O blood-group donors (p=0.008). AR at 4 years and longer after LTx was associated with HLA mismatch at the B locus (p=0.01). CONCLUSIONS Only a few recipient and operative factors were identified for AR after LTx. Moderately sensitized recipients identified by class II PRA exceeding 10% and those with HLA mismatches at the B and DR loci appear to be more susceptible to AR; however, such immunologic variations appear to be well controlled with current donor selection and immunosuppression protocols. The impact of donor-specific variables on AR is surprisingly strong and warrants closer inspection.

Lack of killer immunoglobulin‐like receptor 2DS2 (KIR2DS2) and KIR2DL2 is associated with poor responses to therapy of recurrent hepatitis C virus in liver transplant recipients

Killer immunoglobulin‐like receptors (KIRs) expressed on natural killer and natural killer T cells are involved in activation of these cells and can influence antiviral immunity in the liver. This study investigated the association between KIR genetic diversity and sustained virologic response (SVR) to Peginterferon and Ribavirin (Peg/RBV) therapy in liver transplant (LT) recipients with hepatitis C virus (HCV) recurrence. We tested KIR genotypes in 44 HCV‐infected LT recipients treated with Peg/RBV for 48 weeks. Patients were categorized as having KIR genotypes A/A or B/x and analyzed for association with SVR. Fifteen of 44 (34%) patients had SVR. Only 2 of 18 (11%) who lacked KIR2DS2/KIR2DL2 achieved SVR compared to 13 of 26 (50%) who carried these two genes (odds ratio: 8.0, 95% confidence interval: 1.5‐42.0, P = 0.008). The association between lack of KIR2DS2/KIR2DL2 and SVR remained significant after exclusion of 10 patients with non‐genotype 1 HCV. No correlation was found with other activating or inhibitory KIR genes. Absence of KIR2DS2 and/or KIR2DL2 is associated with failure of Peg/RBV therapy in patients with recurrent HCV after LT. These findings support the role of natural killer and natural killer T cells in HCV clearance after LT and might be generalizable to treatment of HCV infection outside the setting of LT. Liver Transpl 15:1557–1563, 2009.

Transplant nephrectomy after allograft failure is associated with allosensitization.

OBJECTIVES To evaluate the effect of transplant nephrectomy (TN) on the percentage of panel reactive antibody (%PRA) and donor-specific antibody (DSA) levels in patients with renal allograft failure. METHODS The records of patients with failed kidney transplants, who had undergone TN from 2000 to 2007, were reviewed. The pre- and post-TN serum samples were available for analysis from 31 patients. Human leukocyte antigen typing and the %PRA was measured in these patients using standard serologic techniques. The pre- and post-TN patient serum samples were evaluated for DSA levels using solid phase assays and single antigen beads. The pre- and post-TN measurements of the %PRA and DSA levels were compared using the Wilcoxon signed rank test, and the associated clinical variables were identified on multivariate regression analysis. RESULTS The mean %PRA increased from 33.4 to 75.6 for class I antigens (P < .001) and from 38.9 to 60.6 (P = .002) for class II antigens in patients before and after TN, respectively. This increase was associated with an increase in the mean human leukocyte antigen class I and class II DSA levels from 33,518 molecular equivalents of soluble fluorochrome (MESF) to 121,457 MESF (P < .001) and from 45,459 MESF to 126,968 MESF (P < .001), respectively. Regression analysis showed that rejection episodes and an interval from graft failure to TN of <10 months were associated with greater increases in the mean %PRA (P < .001) and mean DSA levels (P = .02). CONCLUSIONS The results of the present study have confirmed that the %PRA increases after TN in patients with renal allograft failure, and sensitization occurs after TN, with an increase in DSA levels. Rejection episodes and early TN after graft failure might result in a greater degree of sensitization.

In vitro stimulation of human endothelial cells by sera from a subpopulation of high-percentage panel-reactive antibody patients.

We have studied a serum activity that enhances in vitro ICAM-1 expression by human endothelial cells (EC) and report that this activity can be found in approximately 8% of pretransplant serum samples from individuals with a history of high %PRA. Hence, most high %PRA sera lack this activity, and, furthermore, mixing these negative sera does not result in an active serum pool. In patients with active serum, the ICAM-1 enhancing activity is found only sporadically, despite the continuous detection of endothelial-reactive antibodies. Absorption of Ig from a high %PRA serum reduced ICAM-1 enhancing activity, as well as endothelial-reactive antibodies. However, enhancing activity can sometimes be observed in sera that lack detectable endothelial-reactive antibodies, and none of several patient sera with defined MHC class I-specific alloantibodies displayed ICAM-1 enhancing activity. Together, these data suggest that ICAM-1 enhancing activity may not necessarily be mediated by anti-MHC alloantibodies. In addition to influencing this expression, ICAM-1 active patient sera also influence EC expression of VCAM-1 and MHC class I, but not MHC class II molecules, a pattern that is similar to that stimulated by TNFα. However, coincubation of EC with active serum plus soluble TNF receptor did not block the endothelial phenotypic changes, despite the ability of the soluble receptor to completely abrogate endothelial changes induced by TNFα. IFNγ also increases endothelial ICAM-1 expression, but has response kinetics different from that of active serum. Interestingly, brief treatment of endothelial cells with IFNγ greatly increased the amount of IgG bound from the active sera by EC. We conclude that some pretransplant patients occasionally express an activity in their serum that influences EC expression of several adhesion molecules, including ICAM-1, VCAM-1, and MHC class I. This activity may be associated with alloanti-bodies, but is independent of MHC class I-reactive antibodies, circulating TNFα, or IFNγ. The relevance of a serum-borne component capable of activating EC is discussed.

The Histocompatibility Laboratory in Clinical Transplantation

Cross-matching and histocompatibility testing constitute the backbone of successful kidney and pancreas transplantation. This remains even more germane in the clinical arena given the growing recognition of sensitization and antibody-mediated rejection as therapeutic challenges. This chapter covers the basic underpinnings of tissue typing and the growing complexity presented by the bewildering array of assays that are available. The implications of newer antibody detection techniques on clinical transplantation and organ allocation are discussed.