Impact of Steroids on Natural Killer Cells Against Cytotoxicity and Hepatitis C Virus Replication.

BACKGROUND: Natural killer (NK) cells play important roles in killing tumor and virus-infected cells. Immunosuppression used after organ transplantation is thought to increase the risk of tumor recurrence and viral infections. However, the effect of immunosuppressive drugs on NK cells has not yet been clearly established. Therefore, we examined the effect of immunosuppression on NK cells. METHODS: NK cells were cultured for 7 days in the presence of interleukin-2 (100 U/mL) with or without the following immunosuppressive drugs: tacrolimus, cyclosporine A, corticosteroid (methylprednisolone [MP]), mycophenolate mofetil, and rapamycin. The effect of the drugs on NK cell activation was tested on the basis of the following: NK cell phenotype, NK cell proliferation, cytotoxicity against K562 cells, cytokine production by NK cells, and anti-hepatitis C virus (HCV) activity with HCV genomic replicon cells. RESULTS: NK cells showed relatively robust functions in the presence of tacrolimus and cyclosporine A. Mycophenolate mofetil and rapamycin significantly prevented only NK cell proliferation (P < .05). In contrast, MP significantly inhibited the proliferation, cytotoxicity, and anti-HCV effect (10.9%, 18.5%, and 1.9%, respectively) of NK cells. Furthermore, MP specifically inhibited the expression of NK cell activation markers and the production of interferon-γ (P < .05). CONCLUSIONS: Corticosteroids have distinct effects on NK cells, which may have important implications for NK cell function in cytotoxicity and HCV effect after transplantation.

Liver, pancreas and kidney transplantation for the treatment of Wolcott-Rallison syndrome.

We present the case of a child who underwent a combined liver, pancreas and double kidney transplant following complications of Wolcott-Rallison syndrome (WRS) a rare genetic disorder that causes infantile insulin-dependent diabetes mellitus (IDDM) and often death in childhood from fulminant liver and concomitant kidney failure. WRS is characterized clinically through infantile IDDM, propensity for liver failure following viral infections, bone dysplasia and growth failure and developmental delay. Fewer than 60 cases with WRS are reported in the literature, mostly from consanguineous parents. Future episodes of liver failure, the main contributor to the increased mortality in WRS, may be prevented through timely liver transplantation. To the best of our knowledge, transplantation has not been utilized to manage complications of WRS prior to this report.

Uterus transplantation model in sheep with heterotopic whole graft and aorta and cava anastomoses.

Uterine transplantation in the sheep model has been described as a partial or whole orthotopic graft from a living donor with vascular anastomoses. As an alternative to surrogate pregnancy or adoption uterus transplantation might be indicated for cases of infertility of uterine origin. The main complications might be rejection and thrombosis. The objective of this work was to develop a model of whole uterus transplantation that was applicable to the human setting, using grafts obtained from brain-dead donors, and suitable for immunologic and viability follow-up with a reduced risk of thrombosis. Two donors and 1 recipient were operated. The first graft was used for an anatomic study; the second was used for transplantation. The donor operation consisted of an en bloc harvest of the uterus, adnexa, and proximal vagina with the distal aorta and cava. After harvest the donor sheep was humanely killed. In the recipient ewe, heterotopic implantation was performed in the lower abdomen. An End-to-side anastomoses of aorta and cava were performed below the recipient's renal vessels. A cutaneous vaginal stoma was performed in the right lower quadrant. The recipient ewe was humanely killed for an autopsy study. The anatomy of uterine veins of the ewe differs from the human. The uterine and ovarian veins join, forming the utero-ovarian vein, which drains at the confluence of the common iliac to the cava. En bloc harvesting allows for rapid graft preparation, with vascular cuffs easily anastomosed with a low risk of thrombosis. The vaginal stoma seems appropriate to facilitate follow-up and graft biopsy. This approach can be a suitable experimental model applicable to humans using grafts from brain-dead donors.

Comparative analysis of T-cell depletion method for clinical immunotherapy-anti-hepatitis c effects of natural killer cells via interferon-γ production.

Liver transplantation (LT) is a life-saving treatment for liver cirrhosis patients with hepatocellular carcinoma (HCC). However, 10%-20% HCC recurrence rate after LT is due to the immunosuppression inducing tumor growth. We recently reported a novel immunotherapy with donor liver natural killer (NK) cells to prevent HCC and hepatitis C virus (HCV) recurrence after LT. In this cell processing procedure, Muromonab-CD3 (Orthoclone OKT3, an anti-CD3 antibody) was added to the culture medium to deplete CD3(+) T cells to prevent graft-versus-host disease. However, the manufacture of OKT3 was discontinued in 2010, when other treatments with similar efficacy and fewer side effects became available. In this study, we examined alternative reagents for T-cell depletion-MACS GMP CD3 pure (GMP CD3), antithymocyte globulin, and alemtuzumab-for NK cell immunotherapy in the allogeneic setting. We observed that GMP CD3 showed exactly the same effects on liver mononuclear cells as OKT3, including activation of NK cells and depletion of T cells. Interestingly, binding of T-cell depletion antibodies to NK cells led to an anti-HCV effect via interferon-γ production. These results with the use of in vitro culture systems suggested that antibodies which produce T-cell depletion affected NK cell function.

Citrulline level is a potent indicator of acute rejection in the long term following pediatric intestinal/multivisceral transplantation.

Citrulline has been advocated as a marker for acute cellular rejection (ACR) in intestinal transplantation; however, its significance as a forewarning in the long-term follow-up remains unknown. This study aimed to investigate the association between citrulline levels and the grading of ACR to establish a cutoff point that accurately predicts ACR beyond 3 months posttransplant in the pediatric patient population. During a 16-year period (1995-2011), a total of 13 499 citrulline samples were prospectively collected from 111 consecutive pediatric intestinal/multivisceral transplant recipients: 2155 were obtained concurrently with intestinal biopsies. There were 185 ACR episodes observed among 74/111 (67%) patients (median follow-up: 4.4 years). Citrulline levels were inversely proportional to the severity of ACR. Negative predictive values for any type of ACR (cutoff, 20 µmol/L) and moderate/severe ACR (cutoff, 10 µmol/L) were 95% and 99%, respectively. When patients were divided according to graft size, diagnostic accuracy using the same cutoff was identical. Similarly, subgroup analysis by the timing of citrulline measurement prior to biopsy varying from 1 to 7 days demonstrated comparable results. Citrulline is a potent indicator as a danger signal for ACR, being an exclusionary, noninvasive biomarker with excellent negative predictive values in the long term after pediatric intestinal/multivisceral transplant.

MicroRNA signature of intestinal acute cellular rejection in formalin-fixed paraffin-embedded mucosal biopsies.

Despite continuous improvement of immunosuppression, small bowel transplantation (SBT) is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need to uncover novel insights that will lead to strategies to achieve better control of ACR. We hypothesized that particular miRNAs provide critical regulation of the intragraft immune response. The aim of our study was to identify miRNAs involved in intestinal ACR. We examined 26 small intestinal mucosal biopsies (AR/NR group; 15/11) obtained from recipients after SBT or multivisceral transplantation. We investigated the expression of 384 mature human miRNAs and 280 mRNAs associated with immune, inflammation and apoptosis processes. We identified differentially expressed 28 miRNAs and 58 mRNAs that characterized intestinal ACR. We found a strong positive correlation between the intragraft expression levels of three miRNAs (miR-142-3p, miR-886-3p and miR-132) and 17 mRNAs including CTLA4 and GZMB. We visualized these miRNAs within cells expressing CD3 and CD14 proteins in explanted intestinal allografts with severe ACR. Our data suggested that miRNAs have a critical role in the activation of infiltrating cells during intestinal ACR. These differences in miRNA expression patterns can be used to identify novel biomarkers and therapeutic targets for immunosuppressive agents.

Association between panel reactive antibodies and acute small bowel rejection: analysis of a series of 324 intestinal transplants.

INTRODUCTION: Panel reactive antibodies (PRA) to class I and II HLA molecules have been associated with acute kidney graft rejection, but their role in small bowel transplantation has not been characterized. METHODS: Since 1994, 324 SBT, alone or as multivisceral transplantation (MVT), have been performed in 286 patients. Routine and surveillance biopsies were performed to rule out or confirm acute rejection (AR), and PRA quantification was performed at varying intervals. We obtained data from 110 patients and 651 PRA measurements. While AR grade (mild to severe, grades 1-3) was determined by histopathological analysis, the status of no AR was determined also by clinical data. When biopsy samples or PRA measurements were frequent around an AR episode within periods of 7 days, the highest value was used. RESULTS: A comparison could be made between 259 instances in which there was a PRA measurement and simultaneous rejection evaluation. Positive PRA showed association with AR (P < 0.001). The positive and negative predictive values were 44% and 79%, respectively. No correlation was found in the severity of rejection. CONCLUSION: The presence of increased levels of PRA is a risk factor of rejection in small bowel transplantation. Alloantibody-mediated injury to the graft contributes frequently to acute rejection of small bowel, and it is associated with cell-mediated immunity in variable proportion.

Citrulline evaluation in bowel transplantation.

INTRODUCTION: The purpose of this study was to evaluate the correlation of plasma citrulline and rejection episodes in intestinal transplantation. METHODS: From January 2007 until present, we performed citrulline assays on our small bowel patients. We investigated the correlation of these assays with the rejection status of the patients. The rejection status of the graft was defined based on graft biopsies. RESULTS: Of 5195 citrulline samples, average serum citrulline levels decreased significantly when the patients presented a rejection episode. We found the following: no rejection, 17.38 microm/L; mild rejection, 13.05 microm/L; moderate rejection, 7.98 microm/L; and severe rejection, 6.05 microm/L. Our current emphasis is to determine the predictive power of citrulline with other biomarkers versus as a separate and isolated measurement. CONCLUSIONS: In our study, citrulline levels correlated significantly with the rejection status of the graft. Serial follow-up of the patients using this assay may alert us to the possibility of increased alloreactivity and rejection episodes.

International grading scheme for acute cellular rejection in small-bowel transplantation: single-center experience.

A standardized grading scheme for the assessment of acute cellular rejection (ACR) in small-intestine transplantation was proposed in 2003 at the Eighth International Small Intestinal Transplantation Symposium. We have implemented the current grading scheme for ACR in small-bowel transplantation since October 2003. The pathologic diagnoses of those small-intestine biopsy samples, including ACR grade and other supplementary findings were evaluated. A total of 3484 small intestine biopsy samples from 155 patients were available for evaluation in this study. Frequency of grades 0, indeterminate, 1, 2, and 3 acute cellular rejection was 33.9%, 49.1%, 12.6%, 3.7%, and 0.8%, respectively. Duration of ACR episode strongly correlated with grade of ACR episode (P < .001). Other supplementary findings included acute vascular rejection component, 2.2%; increase in lymphoplasmacytic infiltrate in lamina propria, 15.7%; mucosal fibrosis, 0.4%; and regenerative changes, 0.3%. Our data substantiate that this grading system is reliable and useful for clinical decision making in bowel transplantation. We suggest that an assessment and quantification of supplementary findings be considered a component of the International Pathology Grading Scheme.

Immediate antibody-mediated (hyperacute) rejection in small-bowel transplantation and relationship to cross-match status and donor-specific C4d-binding antibodies: case report.

BACKGROUND: The role of preformed donor-specific antibodies (DSAs) as a barrier to isolated intestinal transplantation (ITx) remains ambiguous; thus, a positive cross-match has not been a contraindication to ITx. OBJECTIVE: To report the case of a patient with Crohn's disease who underwent ITx and developed immediate antibody-mediated rejection on reperfusion of the allograft. METHODS: Percent reactive antibody testing was performed using pretransplantation serum samples and at transplantation using bead-based assays (Luminex, Luminex Corp, Austin, Tex) and flow cytometry solid-phase assays (FlowPRA single-antigen beads (One Lambda, Inc, Canoga Park, Calif). Serologic tests, flow cytometry cross-matching, and flow cytometry assays of C4d-binding serum antibodies were also performed. Histologic and immunofluorescent analysis of biopsy specimens was performed. RESULTS: HLA typing revealed no sharing of class I or II antigens between donor and recipient. Pretransplantation donor-specific antibodies (DSA) were present at transplantation. Cross-matching (performed during surgery) was positive for class I and II by serologic testing and flow cytometry. After reperfusion, the graft immediately developed severe ischemic injury and arteritis on mucosal biopsy specimens, with immunoglobulin deposition. The DSA C4d binding antibodies were also present. After intense immunosuppression and plasmapheresis, the graft and the biopsy histologic findings showed marked improvement (day 2). By day 7 posttransplantation, patient and graft status were stable. The patient has remained clinically stable for more than a year after transplantation. CONCLUSIONS: Pretransplant DSA in ITx can be a risk factor for immediate (hyperacute) but potentially reversible antibody-mediated rejection. Thus, pretransplantation DSA and cross-match results are critical components to be considered in patients awaiting or undergoing ITx.

Abdominal wall transplantation: surgical and immunologic aspects.

Abdominal wall transplantation is a type of composite tissue allograft that can be utilized to reconstitute the abdominal domain of patients undergoing intestinal transplantation. We have presented herein combined experience and long-term follow-up results of a series of abdominal wall transplants performed at 2 institutions. A total of 15 abdominal wall transplants from cadaveric donors were performed in 14 patients at the end of intestinal transplant surgery or, in 2 cases, a few days after the primary intestinal transplant. The vascular supply was through the inferior epigastric vessels, from the iliac vessels in 12 cases and via a microsurgical technique in 3 cases. Immunosuppression consisted of induction with alemtuzumab and maintenance treatment with tacrolimus monotherapy. Two grafts lost to vascular thrombosis were removed. Five patients are still alive, although all deaths were unrelated to the abdominal wall transplant. There were 3 episodes of abdominal wall graft rejection, treated with steroids; the abdominal wall graft and the intestinal grafts experienced rejection independent from each other. In summary, abdominal wall transplantation is a feasible technique for recipients of intestinal or multivisceral transplants, when the closure of the abdominal cavity by primary intention is technically impossible.

Hepatic gas gangrene following orthotopic liver transplantation: three cases treated with re-transplantation and a review of the literature.

Gas gangrene is a rare and devastating infectious process that can occur after liver transplantation, most often following hepatic artery thrombosis. We here report 3 cases of gas gangrene following orthotopic liver transplantation. Blood cultures were positive for Clostridium clostridiiforme in one case. In 2 other cases liver tissue from explanted specimens was positive for Enterobacter cloacae. Ultrasound demonstrated hepatic artery thrombosis and computed tomography imaging revealed diffuse liver necrosis with gas formation in each case. All 3 patients were successfully treated with a combination of antibiotics and emergent re-transplantation. We review previously published cases of gas gangrene after liver transplant and emphasize the importance of hepatic artery thrombosis in the development of this syndrome as well as the frequent involvement of non-clostridial organisms. Early diagnosis and aggressive combined medical and surgical treatment including re-transplantation are essential for successful treatment of these rare and catastrophic infections.

Analysis of acute cellular rejection episodes in recipients of primary intestinal transplantation: a single center, 11-year experience.

Intestinal transplantation has evolved over the years with major improvements in patient and graft survival. Acute cellular rejection of the intestine, however, still remains one of the most challenging aspects of postoperative management. We analyzed retrospectively collected data from 209 recipients of primary intestinal grafts at our institution over the past 11 years. A total of 290 episodes of biopsy-proven rejection requiring clinical treatment were analyzed. Rejection episodes doubled in length, on average, with each increasing grade (mild, moderate, severe). We observed increased incidence of overall rejection and particularly severe rejection in recipients of isolated intestinal and liver-intestine grafts in comparison with multivisceral grafts. Two rejection history variables had a significant negative impact on graft survival: the occurrence of a severe rejection episode and a rejection episode lasting >or=21 days. The lower incidence rate of severe rejection in recipients of multivisceral grafts might be due to a combination of increased donor lymphatic tissue and larger load of donor-derived immune competent cells present in the graft. The development of more effective monitoring and treatment protocols to prevent the occurrence of severe and/or lengthy rejection episodes is of critical importance for intestinal graft survival.

An association of lower serum citrulline levels within 30 days of acute rejection in patients following small intestine transplantation.

INTRODUCTION: In a prospective protocol we studied whether serum citrulline level within 30 days of an acute rejection was predictive of the episode. METHODS: An acute rejection episode was defined as the date of occurrence of any biopsy-proven rejection in which treatment was initiated until two successive biopsies showed no further rejection. We compared the mean citrulline level based on values determined within 30 days of the start of an acute rejection episode with the mean citrulline level measured on the same patient during a rejection-free period. Serum citrulline measurements were available immediately prior to the occurrence of rejection for 22 patients who experienced 37 episodes. RESULTS: For the 12 episodes of mild rejection, the mean serum citrulline level +/- SE (standard error) was 15.0 + 2.3 micromol/L prior to rejection and 18.8 +/- 2.4 micromol/L during the rejection-free periods. A paired t test of the mean differences was not significant (P = 17). For the 25 episodes of moderate or severe rejection, the mean serum citrulline level was 12.4 +/- 1.1 micromol/L before rejection and 18.8 +/- 2.0 micromol/L during the rejection-free periods. A paired t test of the mean difference was statistically significant (P = .002). CONCLUSIONS: Although further study of citrulline as a marker for the early detection of acute rejection episodes is needed, our hope is that its use will help to prevent some of these early episodes from evolving into full-blown moderate or severe grades of rejection.

Intestinal transplantation with alemtuzumab (Campath-1H) induction for adult patients.

BACKGROUND: Alemtuzumab (Campath-1H [C1H]) is a humanized monoclonal antibody directed against the CD 52 antigen that is present on the surface of T cells, B cells, natural killer cells and monocytes. We studied its application in intestinal transplantation. METHODS: This is a retrospective review of adult patients who underwent intestinal transplantation between December 1994 and May 2005. Group 1: non-C1H group (n = 39); group 2: C1H group (n = 37). C1H was administered as an induction immunosuppression in four doses (0.3 mg/kg), or in two doses (30 mg/kg). Tacrolimus levels were maintained at low level (5-10 ng/dL). No maintenance steroids were given. RESULTS: One-year survival of group 1 and group 2 patients were 57% and 70%, respectively. This difference is not statistically significant. Of 37 patients in group 2, 21 are alive. The incidence of rejection was lower in group 2 (P < .005). Average current tacrolimus level is 6.97 +/- 3.98 ng/dL. Seventeen patients (81%) are steroid free, and 15 (71%) are maintained solely on tacrolimus. There was no graft versus host disease in group 2. CONCLUSIONS: Our preliminary data suggest that C1H can provide effective immunosuppression for intestinal transplantation. Incidence of rejection was less with this regimen using low maintenance tacrolimus and minimal steroids.

The impact of nonalcoholic fatty liver disease and the metabolic syndrome on progression of fibrosis in patients with recurrent HCV after liver transplantation.

Nonalcoholic fatty liver disease (NAFLD) and the metabolic syndrome (MS) have been shown to play a role in disease progression and response to therapy in patients with chronic hepatitis C virus (HCV) infection. The primary objective of this study was to evaluate the impact of coexisting NAFLD and MS on the progression of fibrosis in patients with recurrent HCV treated with interferon (IFN)/ribavirin after orthotopic liver transplantation (OLT). From 1998 to 2004, a total of 418 patients underwent OLT in our center for HCV-related cirrhosis. Thirty-five patients with recurrent HCV on IFN/ribavirin treatment, who had at least 2 posttransplant liver biopsies at least 6 months apart, were included in the study. Patients who had MS at the time of their first posttransplant biopsy were identified. The first and last posttransplant biopsies were assessed for the presence and severity of NAFLD, grade of inflammation, and stage of fibrosis. The fibrosis progression rate (FPR) was calculated and expressed in fibrosis units per month (FU/mo). Among 35 patients, 34% were diagnosed with NAFLD in the first posttransplant biopsy. The mean FPR was 0.05+/-0.16 FU/mo in the presence of NAFLD compared to 0.07+/-0.10 FU/mo in its absence (P=.68) and 0.03+/-0.06 FU/mo in the presence of MS versus 0.10+/-0.15 FU/mo in its absence (P=.06). When FPR values were divided into two categories of <0.16 FU/mo or >or=0.16 FU/mo (below/above the 25% upper quartile) or <0.08 FU/mo or >or=0.08 FU/mo (below/above the 50% upper quartile), there was no correlation between FPR categories and the presence of NAFLD with or without MS, only MS, or the absence of both in the first liver transplant biopsy (P=.13). Coexisting NAFLD or MS had no significant effect on the progression of fibrosis after OLT in patients with treated hepatitis C after OLT.

Zoom endoscopic monitoring of small bowel allograft rejection.

BACKGROUND: The small bowel has been successfully transplanted in patients with irreversible intestinal failure. This report aims to describe endoscopic monitoring of small bowel rejection. METHODS: A magnification endoscope (zoom endoscope) was used in this study. In the first part of the study (October 1998 to March 2000, 271 endoscopy sessions), the specific endoscopic findings that correlated with rejection were determined. An analysis then was performed on data from the second period (March 2001 to November 2002, 499 sessions) to evaluate the zoom endoscope's accuracy in monitoring rejection. RESULTS: Specific endoscopic findings of rejection found in the first period included background erythema, villous congestion, blunted villous tip, and shortened villous height. When the rejection was successfully treated, endoscopic appearance returned to normal. On the basis of these findings, five endoscopic criteria (villous shortening, villous blunting, background erythema, villous congestion, and mucosal friability) were used to score endoscopic sessions in the second period. Endoscopic diagnosis of rejection was compared with histology. Adult patients showed a sensitivity of 45%, a specificity of 98%, a positive predictive value of 82%, and a negative predictive value of 88%. In pediatric patients, these values were, respectively, 61%, 84%, 57%, and 86%. On 59 distinct occasions (30 in period 1 and 29 in period 2) in which the results were endoscopy negative yet biopsy positive (mild) for rejection, we elected not to treat these rejections on the basis of clinical evaluation, and 58 (98%) resolved without further therapy. CONCLUSIONS: With the use of magnification, endoscopy is a useful tool for monitoring acute rejection in the small bowel allograft.

The impact of Campath 1H induction in adult liver allotransplantation.

BACKGROUND: We report our experience with Campath 1H in adult liver allotransplantation. METHODS: Between December 2001 and February 2004, 77 patients underwent liver transplantation using Campath 1H induction and low-dose maintenance tacrolimus immunosuppression. The control group consisted of 50 patients with similar baseline characteristics and the same eligibility criteria, transplanted under our standard Tacrolimus/steroids regimen. Hepatitis C patients were excluded from the study. RESULTS: Patient and graft survival were similar for both groups. The incidence of rejection was significantly lower in the Campath vs the control group (51% vs 65% at 12 months, P = .009). Tacrolimus trough levels and conversion from Tacrolimus or the addition of other immunosuppressive drugs due to nephrotoxicity were also significantly lower in the Campath 1H group. CONCLUSION: Campath 1H induction with low-dose Tacrolimus maintenance immunosuppression is an effective regimen in reducing acute rejection in adult liver transplantation, while maintaining lower tacrolimus levels and less nephrotoxicity than our conventional immunosuppressive regimen.

Abrogation of the alloreactive responses of cadaveric donor intestinal lymphocytes by intraoperative Campath-1H exposure.

A number of recent reports in clinical and experimental intestinal transplantation have suggested that the donor lymphocytes present in and around the gastrointestinal system are potent mediators of graft-versus-host (GvH) reactivity and that GvH responses may contribute to posttransplant morbidity. Therefore, we have tested the proliferative and cytotoxic capabilities of gut-associated lymphocytes from cadaveric donors obtained prerevascularization (pre-r) and about 6 hours postrevascularization (post-r) in recipients pretreated with Campath-1H antibody (alemtuzumab). Mixed lymphocyte reactions (MLR) were performed with lymphoid cells isolated from intestinal epithelial mucosa, lamina propria, and lymph nodes. The pre-r lymphocytes responded strongly to both the recipient and third-party alloantigenic stimulating cells. However, similar preparations from the post-r samples responded in MLR at significantly lower levels (P < .01). This post-r decrease in responsiveness was not observed in similar lymphocyte samples obtained from donors of recipients not treated with Campath-1H. Both the pre-r and post-r samples had similar flow cytometric profiles, suggesting that there was no receptor loss in these lymphoid tissues by the short-term 6-hour exposure to Campath-1H given to the recipient. Conversely, in preliminary experiments where the donor were treated with Campath-1H, it was observed that very few lymphocytes could be obtained from intestinal tissues (n = 3). These results suggested that Campath-1H treatment of the recipient could bring about a drastic reduction in an otherwise strong GvH reactivity by the donor intestinal immune cells.