aizhong Hu

Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study.

Campath‐1H, an anti‐CD52 monoclonal antibody, was used as induction therapy (40 mg i.v. total dose) in 29 primary human renal transplants, and the patients were maintained on rapamycin monotherapy (levels 8–15 ng/mL) post‐transplant. Campath‐1H profoundly depletes lymphocytes long‐term and more transiently depletes B cells and monocytes. All patients are alive and well at 3–29 months of follow up. One graft was lost because of rejection. There have been no systemic infections and no malignancies. Eight of 29 patients have experienced rejection, which was successfully treated in seven of eight patients. Five of these patients had pathological evidence of a humoral component of their rejection. Seven of the 29 patients were converted to standard triple therapy on account of rejection. Rapamycin was generally well tolerated in that there were no significant wound‐healing problems; two lymphoceles required surgical drainage; and most patients were treated with a lipid‐lowering agent. Flow crossmatch testing post‐transplant revealed evidence of alloantibody in two patients tested with previous combined cellular and humoral rejection. Biopsies have shown no chronic allograft nephropathy to date. In view of the relatively high incidence of early humoral rejection, we plan to modify the immunosuppressive regimen in subsequent pilot studies. This clinical trial provides insight into the use of Campath‐1H induction in combination with rapamycin maintenance monotherapy.

FN18-CRM9 immunotoxin promotes tolerance in primate renal allografts

BACKGROUND Transplant tolerance, rather than immunity, may be favored in the setting of a lower mature lymphoid mass in the recipient induced by anti-T cell agents. A novel immunosuppressive agent, FN18-CRM9, known to specifically kill T cells with great potency, was evaluated in a transplant model. METHODS In order to ablate recipient T cells, the immunotoxin FN18-CRM9 was administered to rhesus monkey recipients of MHC-mismatched renal allografts. Donor lymphocytes were injected intrathymically into some animals. RESULTS All monkeys with T-cell depletion by immunotoxin had prolonged allograft survival, and tolerance confirmed by skin grafting has been confirmed in five of six long-surviving recipients. CONCLUSIONS In this clinically relevant model, profound but transient T-cell depletion by a single agent substantially promotes tolerance.

Elevation of CXCR3-Binding Chemokines in Urine Indicates Acute Renal-Allograft Dysfunction

A noninvasive urinary test that diagnoses acute renal allograft dysfunction would benefit renal transplant patients. We aimed to develop a rapid urinary diagnostic test by detecting chemokines. Seventy‐three patients with renal allograft dysfunction prompting biopsy and 26 patients with stable graft function were recruited. Urinary levels of CXCR3‐binding chemokines, monokine induced by IFN‐γ (Mig/CXCL9), IFN‐γ‐induced protein of 10 kDa (IP‐10/CXCL10), and IFN‐inducible T‐cell chemoattractant (I‐TAC/CXCL11), were determined by a particle‐based triplex assay. IP‐10, Mig and I‐TAC were significantly elevated in renal graft recipients with acute rejection, acute tubular injury and BK virus nephritis. Using 100 pg/mL as the threshold level, both IP‐10 and Mig had diagnostic value (sensitivity 86.4%; specificity 91.3%) in differentiating acute graft dysfunction from other clinical conditions. In terms of monitoring the response to antirejection therapy, this urinary test is more sensitive and predictive than serum creatinine. These results indicate that this rapid test is clinically useful.

T-lymphocyte Alloresponses of Campath-1h-treated Kidney Transplant Patients

Background. Kidney transplant patients given Campath-1H (Alemtuzumab) immunodepletion therapy and long-term rapamycin monotherapy have excellent graft survival and function at three years. As an initial step in understanding the characteristics of repopulated T lymphocytes in these patients, we performed several assays to assess alloreactivity. Methods. We measured T-cell responses using CFSE-labeled recipient lymphocytes in a direct one-way MLR, and also analyzed the kinetics of expression of IFN-γ. We examined the T-cell responses of Campath-treated transplant patients on monotherapy versus those treated with anti-CD25 (Basiliximab) induction therapy and maintenance immunosuppression consisting of cyclosporine A, mycophenolate mofetil, and steroids. Results. On average, proliferative responses to donor antigen were equal between Campath and control groups. However, the Campath group displayed a greater response to third party compared to donor antigen (CD3+ P=0.04, CD4+ P=0.07, CD8+ P<0.01), whereas the control group did not display a greater response to third party (CD3+ P=0.69, CD4+ P=0.72, CD8+ P=0.60). Interestingly, more Campath patients (4 of 15) than control patients (0 of 8) displayed donor specific unresponsiveness as gauged by IFN-γ expression and T-cell proliferation (P=0.15). Conclusions. These studies suggest that Campath-1H in conjunction with rapamycin monotherapy retains intact immune responses to third party alloantigen, yet may promote hyporesponsiveness to donor antigen.

Metastable Tolerance to Rhesus Monkey Renal Transplants Is Correlated with Allograft TGF-β1+CD4+ T Regulatory Cell Infiltrates

Approaches that prevent acute rejection of renal transplants in a rhesus monkey model were studied to determine a common mechanism of acceptance. After withdrawal of immunosuppression, all 14 monkeys retained normal allograft function for >6 mo. Of these, nine rejected their renal allograft during the study, and five maintained normal function throughout the study period. The appearance of TGF-β1+ interstitial mononuclear cells in the graft coincided with a nonrejection histology, whereas the absence/disappearance of these cells was observed with the onset of rejection. Analysis with a variety of TGF-β1-reactive Abs indicated that the tolerance-associated infiltrates expressed the large latent complex form of TGF-β1. Peripheral leukocytes from rejecting monkeys lacking TGF-β1+ allograft infiltrates responded strongly to donor Ags in delayed-type hypersensitivity trans-vivo assays. In contrast, allograft acceptors with TGF-β1+ infiltrates demonstrated a much weaker peripheral delayed-type hypersensitivity response to donor alloantigens (p < 0.01 vs rejectors), which could be restored by Abs that either neutralized active TGF-β1 or blocked its conversion from latent to active form. Anti-IL-10 Abs had no restorative effect. Accepted allografts had CD8+ and CD4+ interstitial T cell infiltrates, but only the CD4+ subset included cells costaining for TGF-β1. Our data support the hypothesis that the recruitment of CD4+ T regulatory cells to the allograft interstitium is a final common pathway for metastable renal transplant tolerance in a non-human primate model.

Noninvasive detection of acute and chronic injuries in human renal transplant by elevation of multiple cytokines/chemokines in urine.

Background. Injuries in kidney transplant is currently diagnosed by needle biopsy. A noninvasive test that sensitively detects these injuries would benefit the patients. Methods. Urine samples were collected from healthy controls and kidney transplant recipients. Urine samples were screened first with an antibody array consisting of 120 chemokines and cytokines and then with a multiplex beads assay. Representative parameters, including macrophage inflammatory protein-1Δ, osteoprotegerin, monokine induced by interferon-γ (IFN), and IFN-γ–induced protein of 10 kDa, were simultaneously determined by a quadruplex assay in urine samples from 84 patients with renal allograft injury, 29 patients with stable graft function, and 19 healthy individuals. Results. Twenty-three cytokines/chemokines were found to be elevated in urine samples of patients with acute rejection by the antibody array. The second round of screening confirmed that 11 of the 23 parameters were elevated in the patients but not in the healthy controls. Induced protein of 10 kDa and monokine induced by IFN-γ were significantly elevated in urine samples of patients with acute renal injury, and macrophage inflammatory protein-1Δ and osteoprotegerin were significantly elevated in patients with both acute and chronic renal injuries. The combination of the four parameters had a high positive detection rate (97.6%) for renal transplant injury and could differentiate between acute and chronic injury. Conclusion. These results might indicate that the present multiplex assay provides a basis to establish a noninvasive method for the diagnosis and monitoring of renal transplant injury.

Monotherapy with the novel human anti-CD154 monoclonal antibody ABI793 in rhesus monkey renal transplantation model.

Background. This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys. Methods. Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice. Results. Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses. Conclusions. ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.

Urine Osteoprotegerin and Monocyte Chemoattractant Protein-1 in Lupus Nephritis

Objective. Renal biopsy is the “gold standard” to determine renal activity in systemic lupus erythematosus (SLE), but it is expensive, invasive, and carries risk. Osteoprotegerin (OPG) is produced by the heart, lungs, kidney, and bone. Monocyte chemoattractant protein-1 (MCP-1), a chemotactic cytokine, is involved in the progression of glomerular and tubulointerstitial injury. We investigated both urine OPG and MCP-1 as potential biomarkers for lupus nephritis. Methods. Our subjects, 87 patients with SLE (88% women; 48% African American, 41% Caucasian, 11% other), mean age 44 years, were followed monthly to quarterly. Urinary OPG (pg/ml) and MCP-1 (pg/ml) were measured (Luminex MAP bead assay). Results. OPG concentrations were strongly associated with global disease activity and with both renal activity on a visual analog scale (VAS) (p = 0.0006) and renal disease activity descriptors of the SELENA SLEDAI, including hematuria (p = 0.001) and a positive anti-dsDNA (p = 0.013). MCP-1 was also associated with the renal VAS (p = 0.032), renal disease activity descriptors of SELENA SLEDAI, including hematuria (p = 0.027), and with a positive anti-dsDNA (p = 0.016). We also examined the relationship between the biomarkers and having a urine protein to creatinine ratio (pr/cr) ≥ 0.5. Among patients with medium or high OPG, 46% had urine pr/cr ≥ 0.5, compared to only 23% among those with low OPG (p = 0.032). The 2 biomarkers were strongly correlated with each other (Spearman correlation coefficient 0.77, p < 0.0001). Conclusion. The lack of availability of urine biomarkers has hampered development of new therapies for lupus nephritis. Urine MCP-1 and OPG were both associated with measures of lupus renal disease activity. Medium or high levels of OPG were predictive of a urine protein/creatinine ratio of ≥ 0.5. Further study, including longitudinal assessment and correlation with concurrent renal biopsies, is necessary before this assay can be used in the routine clinic setting.

Tolerance and near-tolerance strategies in monkeys and their application to human renal transplantation

Summary: Studies in non‐human primates to evaluate tolerance strategies in organ transplantation have led to innovation in human transplantation. The two strategies we have studied in detail in non‐human primates are T‐cell depletion by anti‐CD3 immunotoxin and co‐stimulation blockade. Each of these strategies has been extended into early human trials in renal transplantation. The results of these human and non‐human primate studies are summarized. Continued progress in better and safer immunosuppressive methods remains closely linked to research using non‐human primates. However, there has not been a one‐to‐one correspondence between efficacy in the primate and efficacy in humans. Rather, principles can be derived from non‐human primate studies that can be extended into human trials with the knowledge that regimens will likely differ in humans compared to non‐human primates.

Immunotoxin-treated rhesus monkeys: a model for renal allograft chronic rejection.

Background. Unlike acute and hyperacute rejection, chronic rejection (CR) still constitutes a poorly understood process. The onset is insidious, occurs in a period of months to years and, because the pathophysiology is not well understood, is untreatable. A reliable large-animal model for renal allograft CR is needed and has not been reported in the literature yet. Methods. CR biopsy changes were studied in major histocompatibility complex-mismatched renal allografts performed in nine rhesus monkeys that received CD3 T-lymphocyte depletion therapy with immunotoxin on the day of the transplantation (n=7) or 7 days before transplant (n=2). Results. Mean graft survival time was 613.77 days. Biopsy changes of CR were identified as soon as 84 days after transplant (mean, 336 days; range, 84–896 days). Most of the experimental animals had severe interstitial fibrosis, tubular atrophy, chronic transplant glomerulopathy, and chronic vascular rejection changes at the time of necropsy. A significant positive correlation between the severity of CR and the degree of CD68+ macrophage infiltrate of renal parenchyma and the degree of anemia and serum creatinine level elevations were also observed. Conclusions. Our findings are similar to those seen in human renal chronic allograft nephropathy, but in contrast, our model excludes all the nonimmune factors associated with chronic allograft nephropathy, including donor disease, injury from prolonged preservation, drug toxicity, and underlying recipient disease. Immunotoxin-treated rhesus monkeys emerge as an outstanding animal model for assisting us in understanding the pathophysiology of CR.