Evaluating full-thickness skin grafts in intraperitoneal onlay mesh position versus onlay position in mice.

BACKGROUND: Importance: Hernia surgery requires reinforcement material with few side effects when used in the intraperitoneal position. Autologous skin grafting may meet this requirement, but animal experiments are obligatory before being applied in humans. OBJECTIVE: To compare survival and effects of isogeneic full-thickness skin grafts in the intraperitoneal onlay mesh (IPOM) position in mice, with a control group using the onlay position. Primary end point was graft survival and secondary end point adhesion formation and inflammation through NF-κB activity. METHODS: Design: Intervention study with 8-week follow-up in accordance with ARRIVE criteria, performed between 2015 and 2016. SETTING: Animal laboratory. PARTICIPANTS: Transgenic C57BL/6 mice with isogeneic background were used. Recipients were female wild-type phenotype mice >3 mo (n = 24). Donors were male or female mice >7 mo, with phenotype-positive for the luciferase gene (n = 20) or positive for NF-κB-luciferase gene (n = 4). INTERVENTION: Full-thickness skin was grafted in the IPOM position and compared with grafts in the onlay position as controls. Survival was evaluated by regular longitudinal postoperative luminescence imaging over 8 wk. Adherence formation was evaluated macroscopically after sacrifice. Inflammation of full-thickness skin grafts in IPOM position of NF-κB mice was evaluated in four additional mice. Main outcome and measure: Survival of grafts, evaluated by luminescence. RESULTS: Ten animals received grafts in the IPOM position, and 10 in the onlay position as controls. Graft survival after 8 wk was 100% (20/20). Average luminescence at the end of the 8-week period was 999,597 flux (min 162,800, max 2,521,530) in the IPOM group (n = 10) and 769,708 flux (min 76,590, max 2,164,080) in the onlay control group (n = 10). No adhesions requiring sharp dissection (Jenkins' scale >2) were seen. Four animals with grafts in the IPOM position showed peak inflammation (NF-κB activity) 5 d after surgery subsiding toward the end of follow-up. CONCLUSIONS: Full-thickness skin survives as well in the IPOM position as in the onlay control position, and few adherences develop. Further studies are required to fully characterize the tissue remodeling and repair processes associated with IPOM skin grafting. The result is relevant in the search for alternative reinforcement materials to be used in complex hernia surgery in humans.

Multiple transmissions of chromosomally integrated human herpesvirus-6 in one family.

Chromosomally integrated human herpesvirus-6 (ciHHV-6) can be transmitted from parent to child or via allogeneic hematopoietic cell transplantation (HCT). We report a case of ciHHV-6 transmitted via syngeneic HCT, and vertically across 3 generations. ciHHV-6 was transmitted from a parent to the patient and her identical twin, and from the patient to her son. The patient underwent syngeneic HCT as rescue from chemotherapy-induced aplasia during which ciHHV-6 was re-transmitted to her, this time from her identical twin. This is the first report, to our knowledge, of a patient acquiring ciHHV-6 once via germline from a parent and again via syngeneic HCT from an identical twin.

Fibrosis in small syngeneic rat liver grafts because of damaged bone marrow stem cells from chronic alcohol consumption.

A patient with liver failure due to chronic and acute alcohol abuse under consideration for an urgent liver transplant shortly after stopping alcohol may have residual abnormalities that threaten transplant success, particularly for a small graft. To address this, we studied a model in which reduced-size (50%) Lewis rat livers are transplanted into green fluorescence protein transgenic Lewis recipients after they are fed alcohol or a control diet for 5 weeks. Here we show that normal small Lewis grafts transplanted to alcohol-fed Lewis hosts developed fibrosis, whereas no fibrosis was observed in control-fed recipients. Host-derived CD133 + 8-hydroxy-2'-deoxyguanosine (8-OHdG) cells were significantly increased in livers recovered from both alcohol-fed and control recipients, but only alcohol-fed recipients demonstrated co-staining (a marker of oxidative DNA damage). α smooth muscle actin (α-SMA) staining, a marker for myofibroblasts, also co-localized with CD133 + cells only in the livers of alcohol-fed recipients. Immunostaining and polymerase chain reaction analysis confirmed that chronic alcohol consumption decreased the proportion of bone marrow stem cells (BMSCs) expressing CD133, c-Kit, and chemokine (C-X-C motif) receptor 4 markers and caused oxidative mitochondria DNA (mtDNA) damage. Culture of CD133 + cells from normal rats with medium containing 3% ethanol for 48 hours resulted in elevated mitochondrial 8-OHdG and mtDNA deletion, and ethanol exposure diminished CD133 expression but dramatically increased α-SMA expression. In conclusion, oxidative mtDNA damage and deletions occur in BMSCs of chronic alcohol-fed recipients, and these damaged cells mobilize to the small liver grafts and become myofibroblasts where they play a key role in the subsequent development of fibrosis. Liver Transplantation 23 1564-1576 2017 AASLD.

Severe Aplastic Anemia and a Healthy Pregnancy Immediately Following Syngeneic Transplantation: An Extremely Rare Case Report.

Aplastic anemia is potentially fatal, particularly if the disease does not respond to immunotherapy and progresses to severe pancytopenia. Allogeneic hematopoietic stem cell transplant from an HLA-matched sibling donor, the first-line treatment in patients younger than 40 years, is used as a curative treatment option in severe aplastic anemia. The availability of an identical twin donor is infrequent, and there is limited experience in this context. Additionally, the choices for a conditioning regimen for a syngeneic transplant to prevent engraftment failure and the necessity of graft-vs-host disease prophylaxis are controversial. Although long-term survival gradually increases after an allogeneic hematopoietic stem cell transplant, hypogonadism and infertility are the main problems that significantly affect patients' quality of life. We present a patient diagnosed with severe aplastic anemia who has had a healthy pregnancy immediately after a syngeneic transplant.

Inhaled nitric oxide attenuates the adverse effects of transfusing stored syngeneic erythrocytes in mice with endothelial dysfunction after hemorrhagic shock.

BACKGROUND: The authors investigated whether transfusion with stored erythrocytes would increase tissue injury, inflammation, oxidative stress, and mortality (adverse effects of transfusing stored erythrocytes) in a murine model of hemorrhagic shock. They tested whether the adverse effects associated with transfusing stored erythrocytes were exacerbated by endothelial dysfunction and ameliorated by inhaling nitric oxide. METHODS: The authors studied mice fed a high-fat diet (HFD-fed; to induce endothelial dysfunction) or a standard diet for 4-6 weeks. Mice were subjected to 90 min of hemorrhagic shock, followed by resuscitation with leukoreduced syngeneic erythrocytes stored less than 24 h (fresh erythrocytes) or stored for 2 weeks (stored erythrocytes). RESULTS: In standard-diet-fed mice at 2 h after resuscitation, transfusion with stored erythrocytes increased tissue injury more than transfusion with fresh erythrocytes. The adverse effects of transfusing stored erythrocytes were more marked in HFD-fed mice and associated with increased lactate levels and short-term mortality. Compared with fresh erythrocytes, resuscitation with stored erythrocytes was associated with a reduction in P50, increased plasma hemoglobin levels, and increased indices of inflammation and oxidative stress, effects that were exacerbated in HFD-fed mice. Inhaled nitric oxide reduced tissue injury, lactate levels, and indices of inflammation and oxidative stress and improved short-term survival in HFD-fed mice resuscitated with stored erythrocytes. CONCLUSIONS: Resuscitation with stored erythrocytes adversely impacts outcome in mice with hemorrhagic shock, an effect that is exacerbated in mice with endothelial dysfunction. Inhaled nitric oxide reduces tissue injury and improves short-term survival in HFD-fed mice resuscitated with stored erythrocytes.

Increased NPC1L1 and serum cholesterol in a chronic rejection rat.

PURPOSE: To measure serum cholesterol and triglyceride levels and NPC1L1 mRNA and protein as an index of cholesterol absorption during the development of chronic rejection (CR) in a rat model of intestinal transplantation. METHODS: Rats were randomly divided into two groups: Group 1 (n=20) underwent syngenic Lewis-to-Lewis transplantation and Group 2 (n=20) underwent allogenic F344-to-Lewis transplantation as well as treatment with FK506. Blood samples and intestinal tissue were procured on the 190th day after operation. Histological changes were analyzed and the semiquantitative scores of histological parameters were compared. The serum levels of cholesterol and triglyceride were determined. The expression of Niemann-Pick C1 Like 1(NPC1L1) mRNA and protein were analyzed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and immunohistochemistry, respectively. RESULTS: All the animals survived for the 190 days. The appearance and histology of isografts were almost normal whereas the allografts displayed thickened bowel wall and mesenteric fibrosis, concentric intimal thickening and interstitial fibrosis and inflammatory infiltration. The histology scores displayed a significant difference between the allografts and isografts (P < 0.001). No differences were observed for triglycerides for the two groups. The serum cholesterol levels increased significantly in the allogenic group in comparison with the syngenic group (P=0.034), while no difference was observed for triglyceride levels between groups. RT-PCR showed that the expression of NPC1L1 of allografts increased significantly (P=0.004). Immunohistochemistry confirmed RT-PCR findings. CONCLUSIONS: Neointima formation and mesenteric fibrosis were the dominant pathological features. The increased expression of NPC1L1 might contribute to hypercholesterolemia, which may be involved in the pathogenesis of transplant arteriosclerosis.

Survival and Functional Immune Reconstitution After Haploidentical Stem Cell Transplantation in Atm-Deficient Mice.

Hematopoietic stem cell transplantation (HSCT) has been proposed as a promising therapeutic opportunity to improve immunity and prevent hematologic malignancies in Ataxia-telangiectasia (A-T). However, experience in the transplantation strategy for A-T patients is still scarce. The aim of this study was to investigate whether different approaches of HSCT are feasible in regard to graft versus host response and sufficient concerning functional immune reconstitution. Atm-deficient mice were treated with a clinically relevant non-myeloablative host-conditioning regimen and transplanted with CD90.2-depleted, green fluorescent protein (GFP)-expressing, and ataxia telangiectasia mutated (ATM)-competent bone marrow donor cells in a syngeneic, haploidentical or allogeneic setting. Like syngeneic HSCT, haploidentical HSCT, but not allogeneic HSCT extended the lifespan of Atm-deficient mice through the reduction of thymic tumors and normalized T-cell numbers. Donor-derived splenocytes isolated from transplanted Atm-deficient mice filled the gap of cell loss in the naïve T-cell population and raised CD4 cell functionality up to wild-type level. Interestingly, HSCT using heterozygous donor cells let to a significantly improved survival of Atm-deficient mice and increased CD4 cell numbers as well as CD4 cell functionality equivalent to HSCT using with wild-type donor cells. Our data provided evidence that haploidentical HSCT could be a feasible strategy for A-T, possibly even if the donor is heterozygous for ATM. However, this basic research cannot substitute any research in humans.

Association between chronic liver and colon inflammation during the development of murine syngeneic graft-versus-host disease.

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4(+) T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals that developed colitis also develop chronic liver inflammation. Liver samples from control and SGVHD animals were monitored for tissue pathology, RNA for inflammatory mediators, and phenotypic analysis and in vitro reactivity of the inflammatory infiltrate. Diseased animals developed lesions of intrahepatic and extrahepatic bile ducts. Elevated levels of mRNA for molecules associated with chronic liver inflammation, including mucosal cellular adhesion molecule -1, the chemokines CCL25, CCL28, CCR9, and T(H)1- and T(H)17-associated cytokines were observed in livers of SGVHD mice. CD4(+) T cells were localized to the peribiliary region of the livers of diseased animals, and an enhanced proliferative response of liver-associated mononuclear cells against colonic bacterial antigens was observed. The murine model of SGVHD colitis may be a valuable tool to study the entero-hepatic linkage between chronic colon inflammation and inflammatory liver disease.

Highly Angiogenic, Nonthrombogenic Bone Marrow Mononuclear Cell-Derived Spheroids in Intraportal Islet Transplantation.

Highly angiogenic bone marrow mononuclear cell-derived spheroids (BM-spheroids), formed by selective proliferation of the CD31+CD14+CD34+ monocyte subset via three-dimensional (3D) culture, have had robust angiogenetic capacity in rodent syngeneic renal subcapsular islet transplantation. We wondered whether the efficacy of BM-spheroids could be demonstrated in clinically relevant intraportal islet transplantation models without increasing the risk of portal thrombosis. The thrombogenic potential of intraportally infused BM-spheroids was compared with that of mesenchymal stem cells (MSCs) and MSC-derived spheroids (MSC-spheroids). The angiogenic efficacy and persistence in portal sinusoids of BM-spheroids were examined in rodent syngeneic and primate allogeneic intraportal islet transplantation models. In contrast to MSCs and MSC-spheroids, intraportal infusion of BM-spheroids did not evoke portal thrombosis. BM-spheroids had robust angiogenetic capacity in both the rodent and primate intraportal islet transplantation models and improved posttransplant glycemic outcomes. MRI and intravital microscopy findings revealed the persistence of intraportally infused BM-spheroids in portal sinusoids. Intraportal cotransplantation of allogeneic islets with autologous BM-spheroids in nonhuman primates further confirmed the clinical feasibility of this approach. In conclusion, cotransplantation of BM-spheroids enhances intraportal islet transplantation outcome without portal thrombosis in mice and nonhuman primates. Generating BM-spheroids by 3D culture prevented the rapid migration and disappearance of intraportally infused therapeutic cells.

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Hemodynamic regulation of myosin heavy chain gene expression. Studies in the transplanted rat heart.

Cardiac work is a major determinant of heart size and growth. Heterotopic cardiac isografts are hemodynamically unloaded and undergo atrophy. To determine the molecular changes that occur as a result of hemodynamic unloading, we have studied the rate of synthesis of total cardiac proteins and myosin heavy chain (MHC) and the expression of the myosin heavy chain gene as reflected in the messenger RNA levels for alpha- and beta-MHC isoforms. 72 h after transplantation there is a significant decrease in left ventricular size accompanied by a 27% decrease in the rate of total cardiac protein synthesis and a 53% decrease in the rate of myosin heavy chain synthesis. In contrast to isografts 14 d after transplantation which have a decrease in protein synthetic capacity, simultaneous measurements of 18S ribosomal RNA and myosin messenger RNA suggest that after 3 d the decrease in synthesis is due to a change in the efficiency of protein translation. While the working in situ heart expresses primarily alpha-MHC mRNA (97%) hemodynamic unloading leads to a 43% decrease in alpha-MHC mRNA concentration and the de novo expression of the beta-MHC mRNA. Total MHC mRNA (alpha plus beta) concentration analyzed by a quantitative S1 nuclease protection assay was similar in the two groups of hearts. Thus, in association with hemodynamic unloading there are changes in cardiac myosin heavy chain content as a result of both gene transcription and protein translation mechanisms.

Effect of liver histopathology on islet cell engraftment in the model mimicking autologous islet cell transplantation.

BACKGROUND: The inflammatory milieu in the liver as determined by histopathology is different in individual patients undergoing autologous islet cell transplantation. We hypothesized that inflammation related to fatty-liver adversely impacts islet survival. To test this hypothesis, we used a mouse model of fatty-liver to determine the outcome of syngeneic islet transplantation after chemical pancreatectomy. METHODS: Mice (C57BL/6) were fed a high-fat-diet from 6 weeks of age until attaining a weight of ≥28 grams (6-8 weeks) to produce a fatty liver (histologically > 30% fat);steatosis was confirmed with lipidomic profile of liver tissue. Islets were infused via the intra-portal route in fatty-liver and control mice after streptozotocin induction of diabetes. Outcomes were assessed by the rate of euglycemia, liver histopathology, evaluation of liver inflammation by measuring tissue cytokines IL-1ß and TNF-α by RT-PCR and CD31 expression by immunohistochemistry. RESULTS: The difference in the euglycemic fraction between the normal liver group (90%, 9/10) and the fatty-liver group (37.5%, 3/8) was statistically significant at the 18th day post- transplant and was maintained to the end of the study (day 28) (p = 0.019, X2 = 5.51). Levels of TNF-α and IL-1ß were elevated in fatty-liver mice (p = 0.042, p = 0.037). Compared to controls cytokine levels were elevated after islet cell transplantation and in transplanted fatty-liver mice as compared to either fatty- or islet transplant group alone (p = NS). A difference in the histochemical pattern of CD31 could not be determined. CONCLUSION: Fatty-liver creates an inflammatory state which adversely affects the outcome of autologous islet cell transplantation.

Transplantation of preconditioned intestinal grafts is associated with lower inflammatory activation and remote organ injury in rats.

Reperfused grafts--particularly the intestine--release free radicals and cytokines into the systemic circulation. The type of discharge, which is greatly dependent on the local injury, may also induce inflammatory activation in distant organs and leading to multiple system and organ failure. It has been suggested that intestinal grafts from tacrolimus (TRL)-pretreated donors show improved morphology and microcirculation. We studied whether transplantation of intestines from TRL-pretreated donors influenced inflammatory response and remote organ injury posttransplantation. Donor Sprague Dawley rats received TRL or saline (controls) intravenously at 6 hours prior to graft harvest. The intestinal grafts were preserved in saline for 3 hours before transplantation. At 6 and 12 hours postreperfusion hepatic and renal cortical microcirculation were assessed using laser-Doppler flowmetry (n = 8-12 per group). Blood pressure was measured; liver, kidney, and serum samples were obtained. We analyzed hepatic and renal ICAM-1 expression and caspase-3-like activity as well as plasma content of tumor necrosis factor-alpha and interleukin-6. Pretreated graft recipients had higher mean arterial pressure (82 +/- 10 vs 51 +/- 17 mm Hg, P < .05) and renal perfusion at 6 hours whereas liver perfusion was similar at both 6 and 12 hours. Liver and renal functions were also superior among recipients of pretreated grafts. Both caspase-3-like activity and ICAM-1 expression in liver and kidney were lower in pretreated graft recipients. Plasma IL-6 levels were lower in animals receiving pretreated grafts. Transplantation of intestines from TRL-pretreated donors was followed by a lower systemic inflammatory response, improved organ function and decreased remote injury early posttransplantation compared with animals receiving grafts from untreated donors.

Bioengineering the Endocrine Pancreas: Intraomental Islet Transplantation Within a Biologic Resorbable Scaffold.

Transplantation of pancreatic islets is a therapeutic option to preserve or restore ß-cell function. Our study was aimed at developing a clinically applicable protocol for extrahepatic transplantation of pancreatic islets. The potency of islets implanted onto the omentum, using an in situ-generated adherent, resorbable plasma-thrombin biologic scaffold, was evaluated in diabetic rat and nonhuman primate (NHP) models. Intraomental islet engraftment in the biologic scaffold was confirmed by achievement of improved metabolic function and preservation of islet cytoarchitecture, with reconstitution of rich intrainsular vascular networks in both species. Long-term nonfasting normoglycemia and adequate glucose clearance (tolerance tests) were achieved in both intrahepatic and intraomental sites in rats. Intraomental graft recipients displayed lower levels of serum biomarkers of islet distress (e.g., acute serum insulin) and inflammation (e.g., leptin and α2-macroglobulin). Importantly, low-purity (30:70% endocrine:exocrine) syngeneic rat islet preparations displayed function equivalent to that of pure (>95% endocrine) preparations after intraomental biologic scaffold implantation. Moreover, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients. Collectively, our feasibility/efficacy data, along with the simplicity of the procedure and the safety of the biologic scaffold components, represented sufficient preclinical testing to proceed to a pilot phase I/II clinical trial.

Cryopreservation of rat aortic valves results in increased structural failure.

BACKGROUND: The cause of valve allograft failure is most likely multifactorial and may include mechanical, immunological, and other factors. Cryopreservation of these valves is often used to extend storage times. However, there has been considerable confusion as to the effects of cryopreservation on valve durability. Our objective was to determine the effects of cryopreservation on histopathological changes in rat aortic valve grafts. METHODS AND RESULTS: Syngeneic rat aortic valve grafts (Lewis to Lewis; n=24) and allogeneic rat aortic valve grafts (Brown Norway to Lewis; n=24) were implanted infrarenally, either fresh or after cryopreservation. At 7, 14, and 28 days, the valves were explanted, and histological and immunohistochemical examinations were performed in a blinded fashion. Fresh syngeneic graft leaflets retained their normal structure for the 28-day period of observation. Cryopreserved syngeneic grafts showed retrovalvar thrombus formation, with leaflet destruction at 7, 14, and 28 days. Fresh allogeneic graft leaflets showed significant leaflet thickening and progressive destruction at 14 and 28 days. Cryopreserved allogeneic grafts had evidence of retrovalvar thrombus formation with leaflet destruction at 7, 14, and 28 days. Cryopreserved syngeneic grafts resulted in significant infiltration of mononuclear (ED1(+)) cells not seen with fresh syngeneic grafts but similar to fresh allogeneic grafts. All allogeneic grafts resulted in significant infiltration of T-lymphocytes (CD3(+), CD8(+), CD43(+)). CONCLUSIONS: Cryopreservation appears to predispose syngeneic and allogeneic rat aortic valve leaflets to accelerated injury and destruction. This mode of failure resembles that of fresh allogeneic valve grafts.

Secondary malignancies after marrow transplantation.

Secondary malignancies after marrow transplantation have been observed in 20 patients: 19 patients underwent marrow transplantation for the treatment of a hemopoietic malignancy and one for aplastic anemia. All but three were given total body irradiation at doses of 8.0-15.75 Gy as part of the conditioning regimen. Secondary malignancies were composed of three groups: (a) Six patients had recurrence of leukemia (three acute lymphoblastic, two acute myeloblastic, and one chronic myelocytic) in cells of donor origin 62-1074 days after grafting. (b) Eight patients developed lymphoproliferative disorders (four of immunoblastic sarcoma type, one lymphoblastic, one follicular center cell, and one Hodgkin's lymphoma and one acute lymphoblastic leukemia) 54-730 days after grafting. In four of seven patients with appropriate studies these tumors were of donor-cell origin and in three of four tested the cells contained Epstein-Barr virus genome or expressed viral antigens. (c) Six patients developed solid tumors (two glioblastoma multiforme, two adenocarcinomas, one squamous cell carcinoma, and one sarcoma) 347-1875 days after grafting. All but two patients (one with glioblastoma and one with squamous cell carcinoma) have died. These data suggest that patients undergoing marrow transplantation for a hemopoietic malignancy may be at risk of developing secondary malignancies. The etiology appears to be multifactorial, including irradiation, immunosuppression, Epstein-Barr virus infections, and other factors.

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic, but Not Syngeneic Recipient Mice.

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.

Infectious complications of human bone marrow transplantation.

Infections are an almost inevitable complication of human bone marrow transplantation and account for the majority of deaths in transplant recipients. Even prior to the initiation of the transplantation procedure, patients may present with infections complicating previously unsuccessful chemotherapy for hematological malignancy or aplastic anemia. Nevertheless, these pre-transplantation infections should not exclude the possibility of bone marrow transplantation if they can be successfully controlled with specific antimicrobial therapy and necessary adjunctive measures. The immediate post-transplantation period prior to engraftment is characterized by severe marrow aplasia that results from high-dose chemotherapy and total-body irradiation. Infections are primarily septicemias and localized processes caused by bacteria and fungi and their incidence increases as the intensity of immunosuppression is escalated. The high mortality associated with bacterial septicemia makes early, empirical antibacterial therapy mandatory. However, the reduction in mortality from bacterial infection resulting from such an aggressive approach may be offset by a higher mortality from invasive fungal infection, especially in patients with prior fungal colonization and undergoing prolonged conditioning therapy. Thus, until more specific and sensitive tests for the diagnosis of invasive fungal infection become available, empirical intravenous amphotericin should be considered in patients who are persistently febrile and deteriorate clinically in the face of appropriate antibacterial therapy. Interstitial pneumonia associated with severe GVHD is the major infectious complication after successful marrow engraftment and is the most significant barrier to long-term survival. Trimethoprim-sulfamethoxazole is effective prophylaxis against interstitial pneumonia due to Pneumocystis carinii, but one half of the patients still develop a pneumonitis either associated with CMV or of unknown etiology. Mortality from interstitial pneumonia is related to prior radiation therapy while survival is associated with a four-fold rise in CMV CF antibody titer. The latter observation supports the need to investigate passive immunization with CMV antibody as a means of preventing some interstitial pneumonias. Despite the progress made in many areas of human bone marrow transplantation, the majority of graft recipients still die of infectious complications. Thus, new approaches to the management of infections in transplant recipients are urgently needed. Better-tolerated oral nonabsorbable antibiotics, laminar-air-flow rooms, granulocyte transfusions, and chemotherapy and immunotherapy for CMV are among the prophylactic and therapeutic measures that must be critically evaluated in well-controlled, prospective studies. Continued assessment of the infectious complications of bone marrow transplantation is a critical aspect of any ongoing transplant program, not just a research goal...

Simultaneous transplantation of kidney and duct-ligated segmental pancreas in the rat. En bloc technique using two vascular anastomoses.

A new surgical technique of simultaneous kidney and pancreas transplantation is described here. It was used to perform 28 isografts in inbred male Wistar ( RT1y ) rats: 3 animals died within 3 days from early complications and 4 had long-term complications (2 severe pancreatitis and 2 urinary tract complications); 1 animal was killed at 3 months, and its organs were examined histologically. At greater than 4 months after transplantation 2 animals died normoglycemic from undetermined causes. In 3 animals the isografted pancreas was removed at 6 months to assess the effectiveness of the transplant. Currently, 15 animals are alive and normoglycemic greater than 6 months after transplantation. Morphological aspects of simultaneously isografted organs are unchanged when compared with separately transplanted organs. In our opinion this technique provides a useful experimental model to study several technical and immunological problems still present in kidney and pancreas transplantation in diabetic patients.

Unexpected T-cell diversity in syngeneic graft-versus-host disease revealed by interaction with peptide-loaded soluble MHC class II molecules.

BACKGROUND: Administration of cyclosporine after syngeneic bone marrow transplantation elicits a systemic autoaggression syndrome termed syngeneic graft-versus-host disease (SGVHD). The effector T cells recognize a peptide from the invariant chain termed CLIP (MHC class II invariant chain peptide) presented on MHC class II molecules. Moreover, the N-terminal flanking region of CLIP interacts with the T-cell receptor (TcR) beta chain. METHODS: The current study uses a novel approach to isolate and examine the responding T cells ex vivo. A soluble MHC class II molecule using immunoglobulin (Ig)G (MHC class II-Ig) as a scaffold was constructed. The MHC class II-Ig molecule loaded with different peptide variants of CLIP (lacking the N-terminal or C-terminal flanking regions of CLIP) was used to isolate antigen-specific T cells from animals with SGVHD by panning or by flow cytometric sorting. RESULTS: Two subsets of antigen specific T cells restricted by the N- and C-terminal flanking regions of CLIP can be isolated during acute SGVHD that express the Vbeta8.5 TcR determinant but secrete different cytokines (interferon [IFN]-gamma, interleukin [IL]-10) as detected by real-time quantitative polymerase chain reaction (PCR). Spectratyping of the complementarity-determining region 3 regions reveals that the N-terminal CLIP reactive population has greater diversity in both the CDR3 and J regions than the C-terminal CLIP reactive subset. Interestingly, the N-terminal CLIP reactive cells can be preferentially detected in the target tissues during acute SGVHD. However, only IFN-gamma messenger (m)RNA was detected in the tissues. CONCLUSION: The ability to isolate and examine antigen specific T cells ex vivo has revealed unexpected differences in TcR diversity and cytokine production in SGVHD.