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Data on Mesothelin expression in human normal and cancerous tissues is controversial. We employed immunohistochemistry on a tissue microarray from 599 pancreatic cancers and 12 large tissue sections of pancreatitis. Mesothelin expression was highest in pancreatic adenocarcinomas (89%) and adenocarcinomas of the ampulla Vateri (79%), infrequent in pancreatitis and absent in 6 acinus cell carcinomas and normal pancreas. Mesothelin expression was unrelated to pathological tumor stage, grade, metastasis, and tumor infiltrating CD8+ lymphocytes. In conclusion, pancreatic cancer may be ideally suited for putative anti-mesothelin therapies, and mesothelin may represent a suitable biomarker for pancreatic cancer diagnosis, especially on small biopsies.

BACKGROUND Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. METHODS More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. RESULTS At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p

Introduction: Mucin 5AC (MUC5AC) belongs to the family of secreted gel-forming mucins. It is physiologically expressed in some normal mucin producing epithelial cells but also in pancreatic, ovarian, and colon cancer cells. The role of MUC5AC expression in cancer is not fully understood. This study was designed to explore the role of MUC5AC for pancreatic cancer progression, its association to microsatellite instability, and its diagnostic utility. Methods: Mucin 5AC expression was studied immunohistochemically in a tissue microarray (TMA) from 532 pancreatic cancers, 61 cancers of the ampulla Vateri, six acinar cell carcinomas and 12 large sections of pancreatitis. Results: Mucin 5AC staining was interpretable in 476 of 599 (79%) arrayed cancers. Staining was completely absent in normal pancreas and pancreatitis, but frequent in pancreatic cancer. Membranous and cytoplasmic MUC5AC expression was most common in pancreatic adenocarcinomas (71% of 423), followed by carcinomas of the ampulla Vateri (43% of 47), and absent in six acinar cell carcinomas. Mucin 5AC expression was unrelated to tumor phenotype (tumor stage, tumor grade, lymph node, and distant metastasis), and microsatellite instability in ductal adenocarcinomas and carcinomas of the ampulla Vateri. Conclusion: Our study indicates that MUC5AC is an excellent biomarker for pancreatic cancer diagnosis, especially to support the sometimes-difficult diagnosis on small biopsies. Mucin 5AC expression is unrelated to pancreatic cancer aggressiveness.

The transmembrane channel protein DOG1 (Discovered on GIST1) is normally expressed in the gastrointestinal interstitial cells of Cajal and also in gastrointestinal stroma tumors arising from these cells. However, there is also evidence for a relevant role of DOG1 expression in colorectal cancers. This study was undertaken to search for associations between DOG1 expression and colon cancer phenotype and key molecular alterations.A tissue microarray containing samples from more than 1,800 colorectal cancer patients was analyzed by immunohistochemistry.DOG1 immunostaining was detected in 503 (30.2%) of 1,666 analyzable colorectal cancers and considered weak in 360 (21.6%), moderate in 78 (4.7%), and strong in 65 (3.9%). Strong DOG1 immunostaining was associated with advanced pT stage (p=0.0367) and nodal metastases (p=0.0145) but these associations were not retained in subgroups of 1,135 mismatch repair proficient and 86 mismatch repair deficient tumors. DOG1 positivity was significantly linked to several molecular tumor features including mismatch repair deficiency (p=0.0034), BRAF mutations (p0.0001), nuclear p53 accumulation (p=0.0157), and PD-L1 expression (p=0.0199) but unrelated to KRAS mutations and the density of tumor infiltrating CD8 positive lymphocytes.Elevated DOG1 expression is frequent in colorectal cancer and significantly linked to important molecular alterations. However, DOG1 overexpression is largely unrelated to histopathological parameters of cancer aggressiveness and may thus not serve as a prognostic parameter for this tumor entity.

Mucin 5AC (MUC5AC) is a secreted gel-forming mucin expressed by several epithelia. In the colon, MUC5AC is expressed in scattered normal epithelial cells but can be abundant in colorectal cancers. To clarify the relationship of MUC5AC expression with parameters of tumor aggressiveness and mismatch repair deficiency (dMMR) in colorectal cancer, a tissue microarray containing 1812 colorectal cancers was analyzed by immunohistochemistry. MUC5AC expression was found in 261 (15.7%) of 1,667 analyzable colorectal cancers. MUC5AC expression strongly depended on the tumor location and gradually decreased from proximal (27.4% of cecum cancers) to distal (10.6% of rectal cancers; p p p

Background Microsatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking. Methods To study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Results In six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (p Conclusions Our data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.

The first successful renal transplant was carried out more than five decades ago between identical twins. At these early days, acute rejection was the limiting factor. Due to tremendous progress in immunosuppressive therapy and surgical technique, today, renal transplantation is the gold standard therapy for patients with end-stage renal disease. In fact, in comparison with chronic hemodialysis, renal transplantation offers an increase in quality of life while reducing comorbidities associated with dialysis treatment. Despite numerous beneficial achievements, no further improvement regarding patient outcome can be observed over the last two decades. Graft survival rates remain unchanged. The leading causes for graft loss are chronic allograft nephropathy and death with functioning graft. This might be related to a constant increase of the proportion of donors presenting extended donor criteria as well as a more liberal acceptance of candidates for a renal transplant. In the near future, one has to focus more closely on the posttransplant patient care to minimize factors associated with chronic allograft damage. These include post-transplant diabetes, hyperlipidemia, high blood pressure, cytomegalovirus infection, etc.

Background Combined lung and liver transplantation (Lu-LTx) is a therapeutic option for selected patients with coexisting lung and liver disease. For several reasons, Lu-LTx is performed in few centers and information about the technical issues, posttransplant management and long-term outcomes associated with this procedure is limited. Methods We analyzed data from 13 consecutive patients who underwent combined Lu-LTx at Hannover Medical School (Hannover, Germany) between April 1999 and December 2003. The main indications were cystic fibrosis, alpha1-proteinase inhibitor deficiency and portopulmonary hypertension. All patients had advanced cirrhosis and severe pulmonary disease manifestation. Results Ten patients received a sequential double Lu-LTx, one patient received a single Lu-LTx, one received a double lung and split liver transplantation, and one received an en-bloc heart-lung and liver transplantation. Immunosuppression was based on cyclosporine in a triple/quadruple regimen. Postoperative surgical complications occurred in eight patients. There were two perioperative deaths; two patients died during the first year on day 67 and 354, respectively, and one patient died at month 53. The overall patient survival rates at 1, 3, and 5 years were 69%, 62%, and 49%, respectively. Conclusion Combined Lu-LTx is a therapeutic option for highly selected patients with end-stage lung and liver disease with acceptable long-term outcome.

Occult hepatitis B is defined as the persistence of hepatitis B virus (HBV) DNA in persons without HBV surface antigen (HBsAg). The primary site for HBV persistence in persons with occult hepatitis B is considered to be the liver. We provide virological and immunological evidence for long-term persistence of HBV, even after removal of the infected liver, in 25 consecutive, randomly selected liver transplant recipients who tested positive for anti-HBV core antigen (anti-HBcAg) and negative for HBsAg at the time of transplantation. Furthermore, in a cohort of 101 anti-HBcAg-positive/HBsAg-negative patients, 2 showed clinical HBV reactivation after transplantation. Thus, these data indicate that a long-term extrahepatic HBV reservoir exists, which is relevant not only for liver transplantation but also for other types of transplantations, including bone marrow grafting.

With continuously rising survival rates following renal transplantation, health-related quality of life (HQOL) of long-term transplant survivors becomes increasingly important.Recipients more than 15 years after successful renal transplantation were studied retrospectively. HQOL in 139 long-term transplant recipients was assessed using the SF-36 and the disease-specific kidney transplant questionnaire (KTQ-25).Long-term transplant recipients revealed satisfactory HQOL that was comparable to the healthy population in four of eight SF-36 categories (role physical, social functioning, role emotional and mental health). Other SF-36 categories such as physical functioning, physical pain, general health, and vitality were reduced. Among the study population, disease-specific HQOL was comparable or even improved to that of patients awaiting transplantation. In contrast to retired or unemployed patients, employed recipients revealed a highly significant improved HQOL in numerous SF-36 categories such as physical functioning (P0.001), physical pain (P0.001), general health (P0.001), vitality (P0.001), social functioning (P0.005), and mental health (P0.001), as well as for the KTQ-dimensions physical symptoms (P0.001), fatigue (P0.001), uncertainty/fear (P0.01), and emotions (P0.05). Other factors positively correlating with improved HQOL in certain dimensions were living situation, systolic blood pressure, and recipient age.More than 15 years after renal transplantation, recipients present satisfactory HQOL comparable to the general healthy population or at least to pretransplant patients. Vocational rehabilitation following renal transplantation is of highest importance among long-term survivors and is associated with improved HQOL.

Background and aims: Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear. Methods: We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining. Results: We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls. Conclusions: We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

Kidney transplantation (KTx) remains a challenging procedure in small children. This study presents our centre results. From 1983 to 2004, 40 of 442 paediatric KTx were performed in children with a body weight

Transmission of cancer is a fatal risk in organ transplantation. We present a case of incidental renal carcinoma in a kidney obtained from a living donor. A 56-yr-old father was evaluated for donation for his 28-yr-old daughter. An MRT scan revealed two cysts in the right kidney. Right-sided donor nephrectomy and subsequent transplantation was performed. The wall of the prominent cyst was partially excised prior to transplantation. Histology revealed a high-grade renal clear cell carcinoma 10 d after transplantation. Following careful evaluation the recipient underwent partial nephrectomy. Immunosuppression was switched to rapamune. The graft function remained stable. Donor and recipient are without evidence of tumor recurrence 1 yr after transplantation. Our policy to obtain the kidney presenting anatomical variations proved to be beneficial for the donor. In case of transmission of cancer partial resection preserving graft function might be justified.

In this study we focused on the quality of life and satisfaction of living kidney donors comparing traditional lumbar (LDN) and mini-incision donor nephrectomy (MIDN). From May 1996 to December 2002, 174 donor nephrectomies including 127 cases of LDN and 47 cases of MIDN were performed. Donors were evaluated using the SF-36 quality-of-life survey as well as a questionnaire dealing with donors' attitude towards kidney donation, financial burdens, pain, cosmetic satisfaction and duration of sick leave. Our donors achieved comparable or even higher scores in all the SF-36 categories in comparison to the general US population. Following MIDN, quality of life tended to be superior compared to that of LDN donors; however, statistical significance was reached only in one of the eight categories. Duration of sick leave following surgery was in favor of MIDN compared to LDN donors. Statistically significant differences favoring MIDN were observed regarding postoperative hospital stay and cosmetic satisfaction. The procedure would be again undergone by 94 of LDN and 97% of MIDN donors. Open-donor nephrectomy is a safe and cost-effective procedure. Introduction of the here-described MIDN has led to comparable or even improved results compared to LDN.

At present, it is frequently accepted to expand the organ pool for liver transplantation (LTx) by including livers from critical donors. From 1990 to June 2002 a total of 1,208 LTx were performed. Of those, 67 livers from donors older than 60 years were transplanted to 66 patients, including re-LTx in eight patients. Fourteen patients had malignant diseases (21%). Ten patients had a high urgency status (15%). Median donor age was 65 years (range 61-80 years). Primary graft function was observed in 84%. Patient survival rate at 1 and 5 years was 79% and 62%, and graft survival was 68% and 53%, respectively. No difference was observed in LTx with livers from donors younger than 60 years. Fifteen graft losses occurred during the study. Surgical complications were observed in 23 patients (34%). The outcome of LTx with livers from donors older than 60 years is satisfactory and is comparable to results of LTx with livers from donors younger than 60 years. The frequency of vascular complications and cholestasis syndrome is not increased.

Background/Aims : Adult bone marrow contains progenitors capable of generating hepatocytes. Here a new liver failure model is introduced to assess whether bone marrow-derived progeny contribute to liver regeneration after acute hepatotoxic liver failure. Methods : Retrorsine was used to inhibit endogenous hepatocyte proliferation, before inducing acute liver failure by carbon tetrachloride. Bone marrow chimeras were generated before inducing liver failure to trace bone marrow-derived cells. Therefore, CD45 and major histocompatibility complex (MHC) class I dimorphic rat models were applied. Results : Early after acute liver failure a multilineage inflammatory infiltrate was observed, mainly consisting of granulocytes. In long-term experiments small numbers of CD90 + /CD45 − cells of donor origin occurred in clusters associated with portal triads. Bone marrow cell infusion was not able to enhance liver regeneration. Cellular hypertrophy was the predominant way of liver mass regeneration in models applying retrorsine. Conclusions : Retrorsine pretreatment did not affect sensitivity for carbon tetrachloride. A multilineage inflammatory infiltrate was observed in rats whether pretreated with retrorsine or not. Few donor cells co-expressing CD90 (THY 1) were present in recipient livers, which may resemble donor-derived hematopoietic progenitors or oval cells. No other donor cells within liver parenchyma were detected. This is in contrast to other cell infusion models of acute cell death.

The discussion of compensating for shortages of cadaveric donation with increased living donation often reveals differences between the Scandinavian countries and Germany. Possible adoption of Scandinavian structures to improve the rate of living donations in Germany warrants analysis of the actual differences between these two regions. Close examination reveals that significantly higher rates of living donation are achieved only in Sweden and Norway. In Norway, a frequently postulated negative effect on cadaveric donation due to very high rates of living donation could not be confirmed. In contrast to Germany and as a consequence of Norwegian geography, kidney transplantation has been regarded in Norway as the first-line therapy for endstage renal disease for more than 35 years. Living donation has since been actively pursued and is traditionally the transplantation of first choice. In Germany, living donation is still regarded as the second choice after cadaveric donation, due to legal regulations. Significant improvements in living donation frequencies could be achieved there by adopting the active Norwegian approach to living donor identification.

Background The pancreatic anastomosis is still the Achilles heel in partial pancreatoduodenectomy (PPD). Methods This study describes retrospectively a series of 441 patients who underwent standard or extended PPD and reconstruction by either pancreatogastrostomy or pancreatojejunostomy over a period of 13 years (1988–2000). Results Reconstruction of the pancreatic remnant was achieved by pancreatogastrostomy in 250 patients (56·7 per cent) and by pancreatojejunostomy in 191 patients (43·3 per cent). The leakage rate of the pancreatic anastomosis was 2·8 per cent after pancreatogastrostomy versus 12·6 per cent after pancreatojejunostomy (P < 0·001), whereas other surgical complications (bile leakage, haemorrhage, pancreatitis) were identical in the two groups. The leakage rate after standard PPD with or without vascular reconstruction was 2·0 per cent (four of 205 patients) after pancreatogastrostomy and 11·5 per cent (18 of 156) after pancreatojejunostomy (P < 0·001); following extended PPD it was 6·7 per cent (three of 45) after pancreatogastrostomy and 17·1 per cent (six of 35) after pancreatojejunostomy. The mortality rate due to leakage was 1·6 per cent (four of 250 patients) after pancreatogastrostomy versus 5·2 per cent (ten of 191) after pancreatojejunostomy (P = 0·037). Conclusion Pancreatogastrostomy is a safe and reliable method of reconstruction after PPD that may be associated with a lower leakage and mortality rate than pancreatojejunostomy.

Background. Donor lymphocytes infused after organ transplantation can have strong immunoregulatory effects. Application of such protocols for transplant tolerance induction in a clinical setting will, however, require combination of specific immunomodulatory strategies with nonspecific immunosuppressive medication for safety reasons. The aim of this study was to analyze the effects of immunosuppressive treatment on tolerance induction protocols by posttransplantation donor lymphocyte infusion. Methods. The interaction of postoperative donor leukocyte infusion with different types, dosage, and timing of immunosuppressive drugs were studied in a rat model of heart transplantation. Results. Tolerance could be achieved if donor cell infusion was combined with delayed, but not immediate, low-dose cyclosporine treatment, and this was associated with activation and apoptosis of host lymphocytes. In contrast, combinations with an antibody against the interleukin 2 receptor led to long-term graft survival but severe chronic rejection, and combinations with high-dose cyclosporine or sirolimus led to acute rejection. Conclusions. Postoperative donor leukocyte infusion is a potential way for tolerance induction, but the type, dose, and timing of medication are highly critical for its efficacy.

Background Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. Methods Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. Results Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism 60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. Conclusions Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.

Background: Although the transplantation of solid organs and cellular grafts is a clinical routine, the morbidity and mortality associated with immunosuppression is significant. This could be avoided by the induction of donor-specific tolerance. To develop targeted antirejection strategies and regimens to induce donor-specific tolerance, cell populations in the recipient-mediating rejection of solid organ and cellular grafts must be defined. In this study we examined the role of αβ-TCR + cells in the rejection of allogeneic heart grafts, by use of knockout (KO) mice deficient in the production of αβ-TCR + T cells. Methods: C57BL/6-Tcrb tmlMom (αβ-KO) and C57BL6/J (B6) recipient mice were transplanted with B10.BR/SgSnJ (B10.BR) or BALB/c heart allografts. Animals also received bone marrow from normal B10.BR donors, followed by donor-specific or third-party heart transplants. Results: Naive B6 control mice rejected B10.BR and BALB/c grafts within 16 days. In striking contrast, B10.BR and BALB/c heart allografts were indefinitely accepted in unmanipulated αβ-KO mice. The immune responsiveness was restored after bone marrow transplantation from normal donors. After bone marrow transplantation major histocompatibility–disparate BALB/c third-party heart grafts were rejected, whereas donor-specific grafts were still accepted. Conclusions: αβ-TCR + T cells play a nonredundant role in the rejection of heart allografts in mice. Bone marrow chimerism is associated with donor-specific transplantation tolerance. (Surgery 1999;126:121-6.)

Background Mixed allogeneic bone marrow chimerism induces tolerance to solid organ grafts. Although we previously reported that partially ablative conditioning with 700 cGy of total body irradiation (TBI) is sufficient to allow for bone marrow engraftment in mice, we determined that a minimum of 1000 cGy was required in the rat. Because T cells and NK cells are critical in bone marrow graft rejection, our purpose was to examine whether targeting of radioresistant NK cells and/or T cells in the recipient hematopoietic microenvironment would reduce the TBI dose required for engraftment of allogeneic rat bone marrow. Methods Wistar Furth rats received either anti-NK3.2.3 monoclonal antibodies on days -3 and -2, anti-lymphocyte serum on day -5, a combination of both or no pretreatment. TBI was performed on day 0 and rats were reconstituted with 100x10(6) T cell-depleted bone marrow cells from ACI donors. Results Engraftment of T cell-depleted rat bone marrow was readily achieved in animals conditioned with 1000 cGy TBI alone (12/12) and the level of donor chimerism averaged 89%. At 900 cGy TBI alone only one of eight recipients engrafted. In striking contrast, 11 of 12 animals pretreated with anti-NK monoclonal antibodies and irradiated with 900 cGy showed donor chimerism at a mean level of 41%. No further enhancement of bone marrow engraftment could be achieved when recipients were pretreated with antilymphocyte serum alone or antilymphocyte serum plus anti-NK monoclonal antibodies. Mixed allogeneic chimeras exhibited stable multilineage chimerism and donor-specific tolerance to subsequent cardiac allografts. Conclusion Specific targeting of radioresistant host NK cells allows for a significant reduction of the TBI dose required for allogeneic bone marrow engraftment.

Bone marrow chimerism may solve two major limitations in the transplantation of solid organs and cellular grafts: (1) the requirement for life-long immunosuppressive therapy, and (2) acute and chronic rejection. When untreated bone marrow is transplanted into major histocompatibility complex (MHC)-disparate rats, lethal graft-vs-host disease (GVHD) occurs in the majority of recipients. T-cell depletion using anti-CD3 and anti-CD5 monoclonal antibody (mAb) to avoid GVHD led to an increased occurrence of failure of engraftment. We previously identified a cellular population in mouse bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across complete MHC barriers. In light of the fact that facilitating cells have a CD8+/CD3+/TCR- phenotype and mostly coexpress CD5, we evaluated in this study whether T-cell depletion of rat bone marrow using anti-alphabetaTCR mAb would retain engraftment potential yet avoid GVHD. T-cell depletion of bone marrow was performed using anti-alphabetaTCR mAb and immunomagnetic beads. Recipients were conditioned with 1100 or 1000 cGy of total body irradiation and reconstituted with 100 x 10(6) T-cell depleted (TCD) MHC- and minor antigen-disparate bone marrow cells. Animals were monitored clinically and histologically for GVHD. Chimerism was assessed by flow cytometry. Immunomagnetic bead depletion resulted in a reduction of T cells from 1.92%+/-0.21% to 0.10%+/-0.04% of total bone marrow. T-cell depletion did not remove facilitating cells (CD8+/alphabetaTCR-/gammadeltaTCR-/NK3.2.3-) from bone marrow. Further, the engraftment potential of TCD bone marrow was not affected, as 100% of animals engrafted and high levels of donor chimerism were detectable. Animals reconstituted with TCD bone marrow showed no clinical evidence of GVHD and histology revealed none to minimal changes, whereas recipients transplanted with untreated bone marrow succumbed to severe lethal GVHD. T-cell depletion using antialphabetaTCR mAb and immunomagnetic beads selectively removes T cells from the bone marrow graft while sparing facilitating cells that are required for engraftment of allogeneic bone marrow across MHC barriers. Moreover, the cells required for engraftment of HSC do not produce GVHD.

We have previously identified a cellular population in murine bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex (MHC) barriers without causing graft-versus-host disease. Here we investigated the effect of flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of facilitating cells (FC) and HSC into peripheral blood (PB). Mice were injected with FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or both in combination. The number of FC (CD8+/βTCR−/γδTCR−) and HSC (lineage−/Sca-1+/c-kit+) was assessed daily by flow cytometry. Lethally irradiated allogeneic mice were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF showed a highly significant synergy on the mobilization of FC and HSC. The peak efficiency for mobilization of FC (21-fold increase) and HSC (200-fold increase) was reached on day 10. Our data further suggest that the proliferation of FC and HSC induced by FL in addition to the mobilizing effect mediated by G-CSF might be responsible for the observed synergy of both growth factors. Finally, the engraftment potential of PBMC mobilized with FL and G-CSF or FL alone was superior to PBMC obtained from animals treated with G-CSF alone. Experiments comparing the engraftment potential of day 7 and day 10 mobilized PBMC indicate that day 10, during which both FC and HSC reached their maximum, might be the ideal time point for the collection of both populations. © 1998 by The American Society of Hematology.

Background. The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance. Methods. C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting. Results. One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80×10 6 Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks. Conclusion. The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.

Reconstitution of lethally irradiated mice with a mixture of mouse and rat bone marrow cells (mouse + rat→mouse) results in mixed xenogeneic chimerism and donor-specific tolerance. The current study demonstrates that mouse and rat T lymphocytes that have developed in xenogeneic chimeras are restricted to Ag presentation by mouse, but not rat, APC. Restriction to host Ags results in functional immunocompetence with generation of antiviral cytotoxic activity in vivo, within and across species barriers. These data demonstrate for the first time that the host thymus is sufficient to support development and positive selection of functional cross-species T lymphocytes. The superior immunocompetence, as compared with fully xenogeneic (rat→mouse) chimeras, may prove to be of significant benefit in the clinical application of xenotransplantation to solid organ transplantation and immune reconstitution for AIDS.

Xenotransplantation is a newly evolving field. A renewed interest has emerged coincidentally with the shortage of donor organs for transplantation. Bone marrow (BM) chimerism has been suggested as a potential strategy to induce tolerance to xenografts and control the immune response across a species barrier. Bone marrow transplantation (BMTx) displays unique features compared to solid-organ transplantation or transplantation of other cellular grafts. To achieve engraftment of the pluripotent hematopoietic stem cell, which generates all lineages of the hematolymphopoietic system, conditioning of the recipient (usually a combination of irradiation and cytoablative chemotherapy) is required. Once engraftment is achieved, graft function is stable and rejection does not occur, even without immunosuppression. On the other hand, the graft itself is able to generate an immune response against the host, resulting in graft-versus-host disease (GVHD). A newly recognized advantage to xenotransplantation is species-specific disease resistance. In terms of BMTx, important questions arise: Can xenogeneic BM generate a competent immune response across species barriers? Will cross-species GVHD occur? What are the possible applications to humans? This review addresses these questions. Problems emerging from xenogeneic BMTx are summarized and strategies for their solution discussed.

Background Endoscopic treatment options for postsurgical intrathoracic leaks include injection of fibrin glue, clip application, and stent placement. Endoscopic vacuum-assisted closure (E-VAC) may be an effective treatment option. Objective To demonstrate that E-VAC is an effective endoscopic treatment option for closure of major intrathoracic postsurgical leaks. Design and Setting A prospective, single-center study at an academic medical center. Patients Eight consecutive patients with major intrathoracic postsurgical leaks. Interventions Endoscopic placement of transnasal draining tubes, armed with a size-adjusted sponge at their distal end, in the necrotic anastomotic cavities, followed by continuous suction. Sponge and drainage were changed twice weekly. Patients were followed-up for 193 ± 137 days. Main Outcome Measurement Successful leak closure. Results Successful closure of leaks was achieved in 7 of 8 patients (88%) after a mean of 23 ± 8 days. A median of 7 endoscopic interventions was necessary. No major treatment-associated short-term or long-term (follow-up, 193 ± 137 days) complications were noted. Limitations Small sample size, single-center study, and lack of randomization. Conclusion E-VAC is an effective endoscopic treatment modality for major postsurgical intrathoracic leaks. (This study is registered at Clinicaltrials.gov, identifier NCT00876551.)

With continuously rising survival rates following orthotopic liver transplantation (OLT), health-related quality of life (HRQOL) of transplant recipients becomes increasingly important. Recipients more than 15 years after OLT were studied retrospectively. HRQOL in 104 adult liver transplant recipients surviving more than 15 years after OLT was assessed using the German Version of the 36-Item Health Survey (SF-36). Liver transplant recipients surviving more than 15 years after OLT scored lower in all categories of SF-36 revealing a poor HRQOL in comparison to the German reference population. A statistical significance was reached in almost all SF-36 categories with the exceptions of mental health and bodily pain, where our study population scored similarly to the reference population. Job rehabilitation after OLT had a positive effect on HRQOL. Patients who returned to their job during the first year after OLT scored significantly higher in the SF-36 categories of physical functioning and role physical. Marital status and the immunosuppression used didn't affect HRQOL as there was no statistical significance reached in any of the comparisons performed. More than 15 years after OLT, long-term survivors present a poor HRQOL comparable to the reference population. Occupational rehabilitation was the only factor shown to positively influence long-term HRQOL.

In clinical pancreas transplantation the choice of preservation solution may have an impact on graft pancreatitis. Experience with histidine-tryptophan-ketoglutarate (HTK) is still limited whereas University of Wisconsin (UW) solution is currently the preferred perfusate worldwide.The aim of this study was to analyze our experience with HTK in pancreas transplantation.In a retrospective analysis, data from 95 primary simultaneous pancreas-kidney transplantations were reviewed. The use of HTK (n=48) and UW (n=47) solution was stratified into two groups.Patient/graft survival and early graft function were compared.No significant differences between 1, 3 and 12 month patient survival (HTK: 97.9%, 97.9%, and 95.7% vs. UW: 95.7%, 89.4%, and 89.4%, respectively), and pancreas graft survival (HTK: 87.5%, 87.5%, and 85.4% vs. UW: 87.0%, 82.6%, and 82.6%, respectively) were detected. Higher values for peak lipase were observed on day 1 in the HTK group (not reaching significance: P=0.131) whereas no differences were noted for amylase and C-reactive protein.HTK is clinically comparable to UW. Both solutions have been shown to be safe for pancreas preservation. Successful pancreas transplantation depends on many factors such as donor and recipient factors, but skilled organ procurement techniques, organ preservation, and transplant experience in this field is mandatory. The choice of organ preservation solution is only one point in this context.

In renal transplantation, clinical decisions are based primarily on the Banff classification of biopsies. However, the incorporation of 'minor or nonspecific' cellular infiltrates into the Banff classification and their interpretation is uncertain. We analyzed 833 protocol and 306 indicated biopsies to test whether such infiltrates are harmless or whether they have a bearing on outcomes. We characterized morphology, localization and cellular composition of infiltrates, and correlated these findings to the Banff classification and allograft outcome. We found that protocol biopsies had the same prevalence of infiltrates as indication biopsies (87% vs. 87%). Diffuse cortical infiltrates, the hallmark of cellular rejection were more common in indication biopsies and related to tubulitis and a rise in serum creatinine. However, in biopsies with cellular rejection according to Banff criteria, we observed an increase in all infiltrate types (specific and nonspecific) and all cell types (T cells, B cells, histiocytes). The only predictor of allograft function outcome was persistent inflammation in sequential biopsies, irrespective of type, localization and composition of the cellular infiltrates. As detected by sequential biopsies, persistence of any inflammation including those infiltrates currently not considered by the Banff classification should be regarded as a morphological correlate of ongoing allograft damage.

Sinusoidal obstruction syndrome (SOS) is a specific complication of hematopoietic stem cell transplantation (HSCT) that can lead to substantial morbidity and treatment-related mortality. Heparin is frequently used as prophylaxis of and defibrotide as therapy for mild to moderate SOS. In severe cases of SOS these therapies are often ineffective, and orthotopic liver trans-plantation (OLT) may be the only option. Reports in the literature about the outcome of liver transplantation for SOS are contradictory. We describe our second case of OLT after HSCT. The patient died of intracranial hemorrhage 2 weeks after liver transplantation with good initial organ function. In the first case at our center, however, the patient survived more then 8 years. The reported short- to medium-range survival rate for OLT following HSCT is approximately 50%. On the basis of the experience at our center and the findings in a review of the literature, we developed a rational approach to the selection for liver transplantation of patients with life-threatening liver dysfunction after marrow transplantation.

Background Currently, many centers perform laparoscopic donor nephrectomy (DN). We studied the outcome of donors and recipients following open DN using either flank incision (ODN) or mini-incision (MIDN). Methods Data of 196 living kidney donors were recorded prospectively. In 127 cases ODN and 69 cases MIDN were performed. Results Demographic details of donors were comparable for both groups. The left kidney was procured in 58% for ODN and in 64% for MIDN. Multiple arteries were more frequently present when MIDN (11% vs. 28%) was performed. The mean operating time was 129 min for ODN and 133 min for MIDN. Early complications occurred in 7% following ODN and in 4% following MIDN. Late complications were observed in 21% after ODN and 1% after MIDN. The mean hospital stay was significantly longer following ODN compared with MIDN (7.5 vs. 6.4 days). The primary graft function rate was 97% in both groups. One-year graft survival was 97% after ODN and 100% after MIDN. Conclusions Results following MIDN are superior to those following ODN. Even in case of multiple renal vessels MIDN can be safely applied. In comparison with laparoscopic DN advantages of MIDN may be reduced costs, shorter operating time, and comparable cosmetic results.

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7(a) mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7(a) mAb treatment was performed in LEW.1W (RT1(u) RT7(a)) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)-congenic BM transplantation making use of a diallelic polymorphism (RT7(a)/RT7(b)) of rat CD45 was applied. Following injection of anti-RT7(a) mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7(a) mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1(u) RT7(b)) and transplantation of BM from LEW.1W rats pretreated with anti-RT7(a) mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7(b)/RT7(a)), very few RT7(a)-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7(a) mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7(a) mAb efficiently depletes early rat hematopoietic stem cells.

Donor lymphocytes infused after organ transplantation can have strong immunoregulatory effects. Application of such protocols for transplant tolerance induction in a clinical setting will, however, require combination of specific immunomodulatory strategies with nonspecific immunosuppressive medication for safety reasons. The aim of this study was to analyze the effects of immunosuppressive treatment on tolerance induction protocols by posttransplantation donor lymphocyte infusion.The interaction of postoperative donor leukocyte infusion with different types, dosage, and timing of immunosuppressive drugs were studied in a rat model of heart transplantation.Tolerance could be achieved if donor cell infusion was combined with delayed, but not immediate, low-dose cyclosporine treatment, and this was associated with activation and apoptosis of host lymphocytes. In contrast, combinations with an antibody against the interleukin 2 receptor led to long-term graft survival but severe chronic rejection, and combinations with high-dose cyclosporine or sirolimus led to acute rejection.Postoperative donor leukocyte infusion is a potential way for tolerance induction, but the type, dose, and timing of medication are highly critical for its efficacy.

Small children represent a challenging patient group in kidney transplantation (KTx). The aim of this study was to analyze patient and donor data influencing outcome in children that weighed15 kg.Sixty-eight kidneys were transplanted in 64 children that weighed15 kg. In 44 cases, kidneys came from cadaveric donors (CAD) and in 24 cases from living-related donors (LRD). Grafts were placed transperitoneally via midline incision (n=16) or extraperitoneally to the iliac fossa (n=52). Vascular anastomoses were routinely performed to the aorta and vena cava even when the extraperitoneal approach was used.Vascular thrombosis was observed in two (3%), urinary leaks in five (7%), and stenosis in four (6%) patients. In six children receiving organs from adults to the iliac fossa, wound closure was performed using an absorbable mesh to avoid organ compression. Initial graft function occurred in 60 cases (88%). Frequency of initial graft function was significantly higher after KTx from LRD (100%) compared with CAD (82%). The 1-, 5-, and 10-year patient survival was 93%, 91%, and 91%, respectively, and the 1-, 5-, and 10-year graft survival was 92%, 85%, and 85%, respectively. There was no significant difference in patient and graft survival when KTx from LRD and CAD were compared. Within the CAD group, graft survival was improved using kidneys from donors12 years compared with younger donors.Despite size discrepancy between recipients and grafts, KTx is feasible in children that weigh15 kg by using an improved surgical technique even when adult organs are placed to the iliac fossa.

PURPOSE: Tumor recurrence is the major limitation of long-term survival after liver transplantation for hepatocellular carcinoma (HCC) or fibrolamellar carcinoma (FLC). Understanding tumor-biologic characteristics is important for selection of patients and for development of adjuvant therapeutic strategies. PATIENTS AND METHODS: The study included 69 patients who underwent potentially curative liver transplantation for HCC/FLC and survived for more than 150 days; minimum follow-up was 33 months. Frequency, localization, and timing of recurrence were analyzed and compared with primary tumor and patient characteristics. RESULTS: Tumor recurrence was observed in 39 patients at 67 locations. Hematogenous spread was the major route of tumor recurrence (87%), and the most frequent sites were the liver (62%), lung (56%), and bone (18%). Parameters associated with recurrence were absence of cirrhosis, tumor size greater than 5 cm, more than five nodules, vascular infiltration, and International Union Against Cancer (UICC) stage IVA. Selective intrahepatic recurrence was found in nine patients (23%); it was associated with highly differentiated tumors, lack of vascular infiltration, and male sex. Recurrence at multiple sites was found predominantly in young patients (≤ 40 years) and for multicentric (> 5) primary tumors. Recurrences were observed within a wide time range after transplantation (43 to 3,204 days; median, 441 days); late recurrences (> 1,000 days, n = 8) were associated with highly differentiated or fibrolamellar tumors and low UICC stages. Surgical treatment was the only therapeutic option associated with prolonged survival after recurrence. CONCLUSION: In transplant recipients, hepatocellular carcinomas vary considerably in their pattern and kinetics of metastases. Tumor cells may persist in a dormant state for long time periods before giving rise to clinical metastases. Surgical treatment of recurrence should be considered whenever possible.