Oliver Drognitz

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

CD8+ T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8+ T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8+CD161+ T cells in healthy donors and those with hepatitis C virus and defined a population of CD8+ T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor γ-t, P = 6 × 10−9; RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 × 10−7; IL-18 receptor, P = 4 × 10−6), and chemokine receptors (e.g., CCR6, P = 3 × 10−8; CXCR6, P = 3 × 10−7; CCR2, P = 4 × 10−7). CD161+CD8+ T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-γ and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8+ T cells in normal humans and a substantial fraction of tissue-infiltrating CD8+ T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

Immunodominance and functional alterations of tumor‐associated antigen‐specific CD8+ T‐cell responses in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor‐infiltration of naturally occurring CD8+ T‐cell responses targeting several tumor‐associated antigens (TAA). We used overlapping peptides spanning the entire alpha‐fetoprotein (AFP), glypican‐3 (GPC‐3), melanoma‐associated gene‐A1 (MAGE‐A1) and New York‐esophageal squamous cell carcinoma‐1 (NY‐ESO‐1) proteins and major‐histocompatibility‐complex‐class‐I‐tetramers specific for epitopes of MAGE‐A1 and NY‐ESO‐1 to analyze TAA‐specific CD8+ T‐cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon‐γ (IFN‐γ)‐producing CD8+ T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8+ T‐cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early‐stage HCC and associated with patient survival. After antigen‐specific expansion, TAA‐specific CD8+ T cells were detectable by tetramer staining but impaired in their ability to produce IFN‐γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA‐specific CD8+ T‐cell proliferation but did not restore IFN‐γ‐production. Conclusion: Naturally occurring TAA‐specific CD8+ T‐cell responses are present in patients with HCC and therefore constitute part of the normal T‐cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN‐γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA‐specific CD8+ T‐cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA‐specific CD8+ T‐cell responses. (Hepatology 2014;59:1415‐1426)

Impact of different crystalloid volume regimes on intestinal anastomotic stability.

Background:Anastomotic insufficiency still remains an unsolved problem in digestive surgery. Little clinical data, regarding the impact of perioperative volume management exist, which suggest lower complication rates in intestinal surgery under restrictive volume regimens. The aim of our study was to investigate the effect of the extent of intraoperative fluid administration with crystalloids on the stability of intestinal anastomoses. Material and Methods:Twenty-one rats were randomly assigned to 3 experimental groups (n = 7 rats/group): control group CO (9 mL kg−1 h−1 crystalloid infusion), volume restriction group V (−) (3 mL kg−1 h−1), and animals with volume overload V (+) (36 mL kg−1 h−1). After midline incision, all animals received the corresponding infusion for a 30-minute period. Infusion was continued for further 30 minutes whereas an end-to-end small bowel anastomosis was performed 15 cm proximal to the Bauhin valve with 8 nonabsorbable interrupted inverting sutures. At reoperation on the 4th postoperative day, the anastomotic segment was dissected and the bursting pressure [mmHg] was measured. As a second parameter for the quality of anastomotic healing, hydroxyproline concentration was examined with a spectrophotometric method [&mgr;g/g dry tissue]. Histologically, structural changes of the anastomotic segments were assessed by 2 pathologists. Data are given as mean ± SEM. Results:Anastomotic insufficiency was not seen in all animals. Bursting pressure of CO animals was 102 ± 8 mmHg. Bursting pressure was lowest in V (+) with high volume exposure at 77 ± 6 mmHg and significantly lower than V (−) (112 ± 9 mmHg; P = 0.01) whereas the difference compared with the CO group did not reach significant values. Hydroxyproline concentration in V (+) (64.4 &mgr;g/g dry tissue ± 7.7) was significantly lower compared with V (−) (91.7 &mgr;g/g dry tissue ± 9.1) animals (P < 0.05). In all animals with volume overload a marked submucosal edema was found. Conclusion:We could demonstrate for the first time in a systematic investigation, that the quantity of crystalloid infusion, applied intraoperatively, has a significant impact on functional (bursting pressure) and structural (hydroxyproline) stability of intestinal anastomoses in the early postoperative period. Because the stability and quality of an intestinal anastomosis have an impact on insufficiency rates, it should be noted that volume overload may have deleterious effects on anastomotic healing and postoperative complications in digestive surgery, possibly because of a marked bowel wall edema.

Long-term outcome of ABO-incompatible living donor kidney transplantation based on antigen-specific desensitization. An observational comparative analysis

BACKGROUNDnABO-incompatible living donor kidney transplantation based on specific conditioning has been successfully adopted by transplant centres worldwide. Excellent short-term results have been reported in small cohorts. However, long-term data and comparative analyses are still sparse. We report on the outcome of 40 consecutive ABO-incompatible living donor kidney transplant recipients and compare their clinical course to a control group of 43 ABO-compatible living donor transplant patients transplanted during the same time period.nnnMETHODSnThis is an observational single-centre analysis of 40 consecutive patients undergoing ABO-incompatible kidney grafting between April 2004 and April 2009, using a protocol of rituximab, antigen-specific immunoadsorption, intravenous immunoglobulin, basiliximab induction and oral triple immunosuppression with tacrolimus, mycophenolic acid and prednisone. Forty-three ABO-compatible kidney transplant recipients served as controls. The control group had also received basiliximab induction and an identical initial maintenance immunosuppression. The two groups were observed for an average of 39 and 19 months, respectively.nnnRESULTSnThere was a significantly higher incidence of lymphoceles requiring surgical revisions in the ABO-incompatible group. However, this surgical complication did not affect patient or graft survival. Mean serum creatinine, estimated glomerular filtration rate and proteinuria did not differ between the two groups. Furthermore, ABO-incompatible and ABO-compatible patients had the same incidence of humoral and cellular rejections. Despite a more aggressive induction therapy, no differences in infectious or malignant complications were observed.nnnCONCLUSIONSnABO-incompatible living donor kidney transplantation utilizing a combination of rituximab and antigen-specific immunoadsorption yielded results identical to ABO-compatible transplantation despite a significantly higher number of human leukocyte antigen mismatches.

ABO‐incompatible kidney transplantation using antigen‐specific immunoadsorption and rituximab: a single center experience

Abstract: Background: For years ABO‐incompatible kidney transplantations were preferentially performed in Japanese centers. In order to overcome the increased risk of humoral rejections, patients were treated with multiple sessions of plasmapheresis, intensified immunosuppressive therapy and splenectomy before transplantation. Despite good long‐term results regarding patient and organ survival rates, increased morbidity during the early post‐transplant period prevented a broad application of this method. Recently, a new protocol including the anti‐CD20‐antibody (Ab) rituximab and blood group‐specific immunoadsorption instead of splenectomy and plasmapheresis was published with excellent short‐term results.

High levels of C-reactive protein after simultaneous pancreas-kidney transplantation predict pancreas graft-related complications and graft survival

Background. Although pancreas graft-related complications are frequent after simultaneous pancreas-kidney transplantation (SPK), there are no parameters predicting the risk for these complications. Method. A two-center retrospective study was performed in 97 patients who underwent SPK to investigate the peak serum value of c-reactive protein (CRP) during the first 72 hr after SPK in view of graft-related complications and graft survival. Results. Mean peak CRP was 115.6±71.5 mg/L. Mean peak CRP was higher in patients needing relaparotomy (n=31) (136.4 vs. 105.8 mg/L, P =0.048), especially when postoperative bleeding was excluded (P =0.015); in patients with graft pancreatitis (P =0.03); and in patients with graft loss (n=19; P <0.001) compared with patients without these complications. With a cut-off of peak CRP at the level of mean plus 1 SD (187.05 mg/L), there was a significantly higher incidence of relaparotomies (P =0.01; bleedings excluded:P =0.003), graft pancreatitis (P =0.03), and pancreas graft loss (P <0.0001) in patients with high peak CRP compared with patients with low peak CRP. No differences were noticed with regard to rejection rate, mortality, and kidney graft loss. Conclusion. Our findings suggest that peak CRP is a helpful parameter in predicting pancreas graft-related complications and pancreas graft survival after SPK. Our results also stress the importance of early graft damage in pancreas transplantation.

Twenty-year graft survival and graft function analysis by a matched pair study between pediatric en bloc kidney and deceased adult donors grafts.

Background. Pediatric en bloc kidney grafts, especially those from donors aged younger than 12 months, are still regarded controversially with respect to long-term graft survival and function as well as the postoperative development of serious hypertension and proteinuria. Patients and Methods. This retrospective single-center study analyzed 78 pediatric en bloc kidney grafts transplanted between October 1989 and December 2008. Mean donor age was 15 months in the pediatric en bloc kidney donor group and 37.8 years in the matched pair group. The mean follow-up period was 9.3 years (range, 1-19 years). Statistical analysis was performed using the Kaplan-Meier test for patient and graft survival. Continuous variables were compared using independent sample t test. Results. Graft survival for the pediatric donors after 1, 5, and 10 years were 83.1%, 76.0%, 73.9% and for the matched pair control group 89.6%, 78.7%, and 57.8%, respectively. Serum creatinine levels after 1, 5, and 10 years were 1.0, 0.8, 1.1 mg/dL and for the matched pair control group 1.5, 1.7, and 1.6 mg/dL, respectively. No significant long-term differences were detected between the study cohort groups with respect to the postoperative development of hypertension and proteinuria. Conclusion. Overall, pediatric en bloc kidney grafts are well suited to extend the scarce kidney donor pool in experienced centers because of a superior long-term outcome for graft survival and function in comparison with deceased adult kidney grafts. Special attention has to be paid to the substantial higher initial graft loss rate during the first postoperative year.

Chemotherapy, Liver Injury, and Postoperative Complications in Colorectal Liver Metastases

BackgroundSystemic chemotherapy (CTx) is increasingly used before surgery for colorectal liver metastases (CRC-LM). However, CTx may cause liver injury like steatosis, steatohepatitis, and sinusoidal injury which may be associated with postoperative morbidity. Some recent data have even shown an increased mortality in patients with CTx-associated steatohepatitis. We, therefore, analyzed our recent experience with potential hepatic injury and its association with CTx and morbidity in patients undergoing surgery for CRC-LM.MethodsFrom 2001 to 2007, 179 patients underwent primary liver resection for CRC-LM. Sufficient non-tumorous liver parenchyma could be re-evaluated for this study in 102 patients. In these 102 patients (66% male, median age 62xa0years, median BMI 26, 8% diabetics (IDDM)), liver injury was classified using established criteria for steatosis and sinusoidal dilatation (SD) and then compared with preoperative CTx and postoperative outcome. Fifty-eight percent of the operations were (extended) hemihepatectomies (ExtRes), 42% segmental or wedge resections (LimRes). Before resection, 66% had received CTx (33% FU-based (FU), 19% oxaliplatin-based (Oxa), 12% irinotecan-based (Iri), and 3% Oxa+Iri). The interval between CTx and surgery was always ≥4xa0weeks.ResultsMortality was 3/102 (2.9%). Any complication occurred in 48%, hepatic insufficiency in 5.9%, and liver-related complications in 24%. Hepatic steatosis >20% was found in 37% (half of them with steatosis >50%). BMI correlated with the frequency of steatosis. Steatosis >20% was more frequent in patients with preoperative chemotherapy but did not depend on the chemotherapy regimen. No relevant risk factor for grades 2 and 3 SD was found. The specific use of Oxa or Iri did not significantly correlate with hepatic injury. Neither a CTx per se nor the different CTx regimens nor the extent of hepatic injury showed any negative influence on mortality, complication rates, or hepatic insufficiency. Patients with IDDM had a higher mortality (25% vs 1% without IDDM; pu2009<u20090.02), increased complication rate (75% vs 46%; pu2009=u20090.11), a higher rate of hepatic insufficiency (25% vs 4%; pu2009<u20090.02), and more liver related complications (50% vs 21%; pu2009=u20090.06). Patients undergoing ExtRes had a higher overall (pu2009<u20090.01) and liver-related (pu2009=u20090.05) complication rate compared to LimRes. None of the 34 patients with preoperative Oxa or Iri died or developed hepatic insufficiency.ConclusionsIn our experience, hepatic injury (steatosis) was influenced by BMI and by preoperative CTx. Neither preoperative CTx nor liver injury increased perioperative morbidity. Patients with IDDM were at a rather high perioperative risk.

Effects of Organ Preservation, Ischemia Time and Caspase Inhibition on Apoptosis and Microcirculation in Rat Pancreas Transplantation

This study was undertaken to examine the impact of ischemia‐reperfusion (I/R) injury on microcirculation and apoptosis in experimental pancreas transplantation. Pancreatic grafts were subjected to different preservation solutions and cold ischemia times (CITs): University of Wisconsin (UW), 6‐h CITs (group U6); UW, 18‐h CITs (group U18); normal saline, 6‐h CITs (group S6); and normal saline, 6‐h CITs with Z‐Asp‐2,6‐dichlorobenzoyloxymethylketone (pan‐caspase inhibitor; group S6 & CI). Nontransplanted animals served as controls. At 1‐ and 2‐h reperfusion microcirculation was assessed by means of intravital microscopy. Apoptosis was detected by in situ nick end‐labeling method (TUNEL) at 2‐h reperfusion. Deterioration of microcirculation was lowest in group U6 and highest in groups S6 and S6 & CI compared with controls. The apoptotic index (cells per high power fields) of groups U6, U18 and S6 correlated well with functional capillary density (r=− 0,70, p < 0.0001) and leucocyte sticking (r= 0,69, p < 0.0001) at 1‐h reperfusion. Caspase inhibition had no impact on microcirculation but significantly reduced AI compared with group S6 (p < 0.001). These data suggest that pancreatic I/R injury‐induced apoptotic cell death well predicts the extent microcirculatory impairment. Caspase inhibition might be a promising strategy in reducing I/R injury in pancreas transplantation.

Characterization of microcirculatory disturbance in a novel model of pancreatic ischemia-reperfusion using intravital fluorescence-microscopy.

Introduction Microcirculatory disturbances caused by ischemia–reperfusion injury (IRI) are the crucial hallmarks of pancreatitis following pancreas transplantation. Aims To develop a novel rodent model of normothermic in situ ischemia of a pancreatic tail-segment that simulates the clinical situation of pancreas transplantation by flushing the organ via an inserted microcatheter and thus enables selective treatment of the organ via this access. Methodology Four experimental groups were investigated (n = 7 Wistar rats/group): sham animals without ischemia and dissection of the pancreas; control animals with dissection of a pancreatic tail segment pedunculated on the splenic vessels and flushing od this segment with saline via a microcatheter; and two groups of animals treated like controls with a pancreatic ischemia time of 1 hour or 2 hours. With use of intravital epifluorescence microscopy, the microcirculatory damage was characterized by investigation of functional capillary density (FCD) and leukocyte adherence in postcapillary venules (LAV) before ischemia and during a reperfusion time of 2 hours. Dry:wet ratio determinations, light microscopy, and electron microscopic investigations were performed to characterize the histologic organ damage. Results FCD decreased significantly (p < 0.05) 2 hours after reperfusion in the groups of 1-hour (−29.21%) and 2-hour ischemia (−42.73%), in comparison with baseline values. LAV increased significantly (p < 0.05), 4.3- and 5.8-fold, after 1-hour and 2-hour ischemia during the observation time. The histologic damage was similar to posttransplantation pancreatitis in humans 1 hour after reperfusion. In sham and control animals these alterations were not significant. Conclusions The rodent in situ model of pancreatic IRI showed standardized microcirculatory damage dependent on the ischemia time. Offering the possibility of selective treatment by the direct artery access to the ischemic pancreatic area, the model enables investigations of questions related to human pancreas transplantation.