Hanaa Nafady-Hego

Factors affecting operational tolerance after pediatric living-donor liver transplantation: impact of early post-transplant events and HLA match*

Pediatric recipients of living‐donor liver transplants (LDLT) can often discontinue immunosuppression (IS). We examined factors affecting development of operational tolerance (OT), defined as off IS for >1 year, in this population. A historic cohort analysis was conducted in 134 pediatric primary semi‐allogeneic LDLT. Multivariate logistic regression analysis was used. The frequency of peripheral regulatory T cells (Tregs) was determined at >10 years post‐Tx by FACS analysis. IS was successfully discontinued in 84 tolerant patients (Gr‐tol), but not in 50 intolerant patients (Gr‐intol). The Gr‐intol consisted of 24 patients with rejection (Gr‐rej) and 26 with fibrosis of grafts (Gr‐fib). The absence of early rejection [odds ratio (OR) 2.79, 95% CI 1.11–7.02, P = 0.03], was a positive independent predictor, whereas HLA‐A mismatch (0.18, 0.03–0.91, P = 0.04) was a negative predictor. HLA‐DR mismatches did not affect OT. The Treg frequency was significantly decreased in Gr‐intol (4.9%) compared with Gr‐tol (7.6%) (P = 0.003). There were increased levels of tacrolimus in the first week in Gr‐Tol (P = 0.02). Although HLA‐B mismatch (8.73, 1.09–70.0, P = 0.04) was a positive independent predictor of OT, its clinical significance remains doubtful. In this large cohort of pediatric LDLT recipients, absence of early rejection, HLA‐A match and the later predominance of Tregs are factors associated with OT.

The generation of donor-specific CD4+CD25++CD45RA+ naive regulatory T cells in operationally tolerant patients after pediatric living-donor liver transplantation.

Background. CD4+CD25++CD45RA+ cells (naïve regulatory T cells [naïve-Tregs]) have been identified as a functionally premature form of CD4+CD25+++CD45RA− cells (conventional-Tregs). However, their contribution to transplant tolerance remains to be elucidated. Method. We examined the frequency and the function of conventional and naive-Tregs in the peripheral blood derived from operationally tolerant patients after pediatric living-donor liver transplant (Gr-tol). The data were compared with those of patients who were unable to be weaned off immunosuppression due to rejection (group-intolerance [Gr-intol]), patients in the process of weaning immunosuppression (Gr-weaning) and healthy volunteers (group-healthy volunteers [Gr-vol]). Results. In Gr-tol, the frequency of conventional-Tregs was significantly higher than that in Gr-vol and tended to be higher than that in Gr-intol. The frequency of naive-Tregs was significantly decreased in Gr-intol versus those in Gr-tol, -weaning, and -vol. In mixed lymphocyte reactions, donor-specific hyporesponsiveness of CD4+ cells was observed only in Gr-tol but not in the other groups. Depletion of conventional or naive-Tregs from CD4+ cells demonstrated that the suppressive properties of donor antigen-reactive conventional and naïve-Tregs were upregulated compared with those of third-party antigen-reactive conventional and naïve-Tregs in Gr-tol only. Conclusions. This is the first report providing detailed evidence that donor-specific naïve-Tregs were generated and their suppressive properties were upregulated in the peripheral blood of tolerant patients, whereas their frequency was downregulated in intolerant patients. Therefore, we speculate that not only conventional-Tregs play a role in Tx tolerance but also the role of naïve-Tregs is critical.

Intragraft Vδ1 γδ T cells with a unique T-cell receptor are closely associated with pediatric semiallogeneic liver transplant tolerance.

Background T-cell receptor V&dgr;2 &ggr;&dgr; T cells (V&dgr;2 cells) participate in host defense, whereas V&dgr;1 &ggr;&dgr; T cells (V&dgr;1 cells) may regulate immune responses. V&dgr;1 cells appear to play a role in fetomaternal tolerance and our aim was to examine their role in liver transplant tolerance. Methods To determine whether V&dgr;1 cells increase within accepted grafts after semiallogeneic pediatric liver transplantation, the V&dgr;1/V&dgr;2 ratio was assessed at the transcriptional level and the complementarity-determining region 3 loop of the &dgr; chain of V&dgr;1 cells was sequenced in biopsies from immunosuppression-free (n=6) or almost free (n=3) liver transplant recipients, referred to as group tolerance (Gr-Tol; n=9). The results were compared with biopsies from grafts of recipients on maintenance immunosuppression due to concern of rejection (Gr-IS; n=11). Chronically rejected grafts (Gr-CR; n=6) and normal livers (Gr-NL; n=8) were also examined. Results The V&dgr;1/V&dgr;2 ratio was the highest in Gr-Tol (0.07±0.06) compared with Gr-IS (0.03±0.02; P=0.04), Gr-CR (0.01±0.02; P=0.008), and Gr-NL (0.02±0.04; P=0.01). There was an identical complementarity-determining region 3 sequence (100% homologous) among all recipients in Gr-Tol, which was dominant in six of nine recipients. This sequence was not seen in Gr-IS or Gr-CR, although it was observed in five of six normal livers. Conclusions A unique V&dgr;1-bearing T-cell clone accumulates within accepted human liver grafts. It might be useful as a biomarker of tolerance and the identification of its ligand might aid in the development of a novel strategy for tolerance induction.

Pattern of bacterial and fungal infections in the first 3 months after pediatric living donor liver transplantation: An 11-year single-center experience†‡

Infection after pediatric living donor liver transplantation (LDLT) is a major cause of morbidity and mortality. Here, we sought to determine the incidence, timing, location, and risk factors for bacterial and fungal infections. We retrospectively investigated infection for 3 postoperative months in 345 consecutive pediatric patients (56.2% were females) who underwent primary LDLT at Kyoto University Hospital, Japan. A total of 179 patients (51.9%) developed at least 1 bacterial and/or fungal infection episode, with an infection rate of 2.5 per patient. The predominant infection site was the surgical site (52%). Most of the bacterial and fungal infection occurred within the first month. Enterococcus species followed by multidrug‐resistant Pseudomonas aeruginosa and methicillin‐resistant Staphylococcus aureus were the predominant bacterial pathogens. All fungal isolates were Candida species. Prolonged preoperative hospital stay more than 7 days (P = 0.025) and bile leak (P = 0.047) were independent predictors of bacterial infection. Preoperative ascites (P = 0.009) and prolonged insertion of intravascular catheters (P = 0.001) independently predicted fungal infections. Bacterial and fungal infections were responsible for 42.9% of the causes of death in our study. To avoid bacterial and fungal infections after LDLT, broader‐spectrum prophylaxis to cover the range of organisms seen in these infections should be considered as a more favorable treatment regimen to prevent prophylaxis failure, especially for patients with a preoperative hospital stay more than 7 days or operative complications in the form of a bile leak. Early drain removal and prophylactic antifungal drugs should be considered for patients with preoperative ascites. Cooperation between attending physicians and infectious disease physicians can improve the outcome of patients after LDLT. Liver Transpl 17:976–984, 2011.

Minimal but essential doses of immunosuppression: a more realistic approach to improve long-term outcomes for pediatric living-donor liver transplantation.

REFERENCES 1. World Health Organization. Control of leishmaniasis. Geneva: World Health Organization; 2007. Available at: http://apps.who.int/gb/ ebwha/pdf_files/WHA60/A60_10-en.pdf. Accessed March 25, 2010. 2. Herwalt BL. Leishmaniasis. Lancet 1999; 354: 1191. 3. Antinori S, Cascio A, Parravicini C, et al. Leishmaniasis among organ transplant recipients. Lancet Infect Dis 2008; 8: 191. 4. Badaro R, Jones TC, Carvalho EM, et al. New perspectives on a subclinical form of visceral leishmaniasis. J Infect Dis 1996; 154: 1003. 5. Campos-Varela I, Len O, Castells L, et al. Visceral leishmaniasis among liver transplant recipients: An overview. Liver Transplant 2008; 14: 1816. 6. Murray HW, Berman JD, Davies CR, et al. Advances in leishmaniasis. Lancet 2005; 366: 1561. 7. Horber FF, Lerut JP, Reichen J, et al. Visceral leishmaniasis after orthotopic liver transplantation: Impact of persistent splenomegaly. Transpl Int 1993; 6: 55. 8. Hernandez-Perez J, Yebra-Bango M, JimenezMartinez E, et al. Visceral leishmaniasis (kala-azar) in solid organ transplantation: Report of five cases and review. Clin Infect Dis 1999; 29: 918. 9. Halkic N, Ksontini R, Scholl B, et al. Recurrent cytomegalovirus disease, visceral leishmaniasis, and Legionella pneumonia after liver transplantation: A case report. Can J Anaesth 2004; 51: 84. 10. Tsiodras S, Zafiropoulou R, Giotakis J, et al. Deep sinus aspergillosis in a liver transplant recipient successfully treated with a combination of caspofungin and voriconazole. Transplant Infect Dis 2004; 9: 37. 11. Basset D, Faraut F, Marty P, et al. Visceral leishmaniasis in organ transplant recipients: 11 new cases and review of the literature. Microbes Infect 2005; 7: 1370. 12. Ozcan D, Seckin D, Allahverdiyev AM, et al. Liver transplant recipient with concomitant cutaneous and visceral leishmaniasis. Pediatr Transplant 2007; 11: 228. 13. Murray HW. Treatment of visceral leishmaniasis (Kala-Azar): A decade of progress and future approaches. Int J Infect Dis 2000; 4: 158. 14. Clemente WT, Couto CA, Ribeiro DD, et al. An atypical course of visceral leishmaniasis (Kala-azar) in a liver transplant recipient. Transplantation 2007; 83: 368. 15. Brasil, Ministério da Saúde. Leishmaniose Visceral Grave. Brasília, 2006. Available at: http:// portal.saude.gov.br/portal/arquivos/pdf/ manual_lv_grave_nc.pdf. Accessed March 18, 2010. 16. Brasil, Ministério da Saúde. Leishmaniose visceral. Recomendações clínicas para redução da letalidade. Brasília, 2009. 17. Oliveira CMC, Oliveira MLMB, Andrade SCA, et al. Visceral leishmaniasis in renal transplant recipients: Clinical aspects, diagnostic problems, and response to treatment. Transplant Proc 2008; 40: 755. 18. López-Vélez R, Videla S, Márquez M, et al. Spanish HIV-leishmania study group. Amphotericin B lipid complex versus no treatment in the secondary prophylaxis of visceral leishmaniasis in HIV-infected patients. J Antimicrob Chemother 2004; 53: 540. 19. Fernández-Guerrero ML, Robles P, Rivas P, et al. Visceral leishmaniasis in immunocompromised patients with and without AIDS: A comparison of clinical features and prognosis. Acta Trop 2004; 90: 11.

Augmented damage of islets by impaired exocrine acinar cells undergoing apoptosis that is possibly converted to necrosis during isolation

Islet damage attributed to impaired exocrine cells during pancreas preservation and isolation procedure remains elusive, although released exocrine enzymes could directly damage islets. The aim of this study is to investigate the cellular mechanisms associated with exocrine cells and their possible impact on the islet cell survival and function after isolation. Mouse pancreata were stored in cold University of Wisconsin preservation solution for 0, 24 and 48 h and incubated with or without collagenase at 37℃ for 15 min. During preservation, the percentage of exocrine cells with necrosis, which means impaired cellular membrane that allows intracellular enzymes to be released, remains low (

Utility of CD127 combined with FOXP3 for identification of operational tolerance after liver transplantation

Loss of cell surface expression of CD127 on CD4(+)CD25(++) regulatory T-cells (Tregs) may be a useful marker to efficiently isolate Tregs. As FOXP3 was specifically used to identify Tregs, combining these two markers could give better identification for patient with operational tolerance (OT) after liver transplantation. To testify this mixed lymphocyte reaction (MLR), the function of circulating CD4(+)CD25(++)CD127(dim) cells (CD127(dim) cells) was examined in immunosuppression (IS)-free pediatric recipients after liver transplantation (LTx) (group operational tolerance: OT) (Gr-tol n=25) compared to recipients who could not stop IS due to clinically overt rejection (group intolerance) (Gr-intol n=18), recipients who were weaning IS (Gr-weaning n=11) and age-matched healthy volunteers (Gr-vol n=11). In addition, the frequencies of CD127(dim) cells vs CD4(+)CD25(++)CD127(dim)FOXP3(+) (CD127(dim)FOXP3(+)) cells were compared in these four groups by FACS analyses. Our results showed that The proliferation of CD4 cells to donor antigens was reduced compared to third-party antigens only in Gr-tol (P=0.022) but not in other groups (P=NS). Depletion of CD127(dim) cells resulted in a donor antigen-specific abrogation of this MLR hyporesponsiveness in Gr-tol (P<0.001) but not other groups (P=NS). This implied that CD127 efficiently isolated donor antigen-specific Tregs. The frequencies of CD127(dim) cells were significantly lower in Gr-intol (5.2%±1.9%) compared to those in Gr-tol (7.8%±1.8%) (P<0.001) as were the frequencies of CD127(dim) FOXP3(+) cells (Gr-tol: 5.4%±1.7% vs Gr-intol: 2.9%±1.0%, P<0.001). Of interest, there were fewer CD127(dim)FOXP3(+) cells in Gr-intol (2.9%±1%) than in Gr-weaning (5.1%±1.8%) (P=0.002), but no difference in CD127(dim) cells (Gr-intol: 5.2%±1.9% vs Gr-weaning: 6.7%±2.0%) (NS). Thus, combining FOXP3 with CD127 for phenotype analysis demonstrated an unequivocal difference between Gr-intol and Gr-weaning that was not detected by CD127 alone. In conclusion CD127 was a useful surface marker to isolate donor-antigen-specific-Tregs in OT after LTx. The additive effect of its combination with FOXP3 is important in phenotypical Treg analyses of OT patients.

Outcome of Hepatitis B Virus Infection After Living-Donor Liver Transplant: A Single-center Experience Over 20 Years.

OBJECTIVES Despite living-donor liver transplant being a life-saving therapy for patients with hepatitis B virus with or without hepatocellular carcinoma, outcomes for patients with these diseases are worse. Hepatitis B virus recurrence or relapse of hepatocellular carcinoma can result in subsequent graft loss or patient death. In this study, we discuss the postoperative outcomes of patients with hepatitis B virus infection after living-donor liver transplant. MATERIALS AND METHODS We retrospectively analyzed 125 patients with hepatitis B virus-related end-stage liver disease, comparing results with 1228 control patients who had other pathologies, including hepatitis C virus, combined hepatitis B virus and hepatitis C virus, and neither virus. RESULTS Survival rates of patients with hepatitis B virus did not differ from the control groups (P > .05). Patients with concurrent hepatitis B virus and hepatocellular carcinoma were significantly older (P < .0001), had critical status (P < .0001), had chronic underlying pathology (P = .001), lower graft-to-recipient body weight ratio (P = .047), needed more intraoperative plasma transfusion, and experienced more rejection episodes than those without hepatocellular carcinoma. Of interest, in 5 patients who had hepatitis B virus recurrence after living-donor liver transplant, Model for End-Stage Liver Disease score was significantly higher than those who did not have recurrence (P = .015). In addition, 2 patients had hepatocellular carcinoma recurrence in the form of peritoneal metastasis, with both patients having high preoperative alpha-fetoprotein levels. CONCLUSIONS Our study provides details on long-term outcomes of patients with hepatitis B virus infection who had undergone living-donor liver transplant. Based on our results, we suggest that prolonged antiviral prophylactic therapy in the form of hepatitis B immunoglobulin with either lamivudine or entecavir be considered for patients who associated with risk factors to prevent postoperative recurrence.