bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Solitary Pancreas Transplant Graft Survival in the Modern Immunosuppressive Era
Authors: Odorico Jon S. , Becker Yolanda T. , Groshek Marilyn , Werwinski Cathy , Becker Bryan N. , Pirsch John D. , Sollinger Hans W.
Published: June 2017

The results of solitary pancreas (SP) transplantation have traditionally lagged behind those of simultaneous pancreas–kidney (SPK) transplantation. This is one of the chief factors that has limited the wide-scale application of SP transplantation in nonuremic type I diabetic patients. The purpose of this study is to report our present experience with SP transplantation and compare it to a prior experience. Twenty-three SP transplants (14 PAK, 4 PTA, and 5 PASPK) performed since January 1997 were compared to 56 SP transplants (53 PAK, 1 PTA, and 2 PASPK) performed before 1994. Between 1993 and 1997, SP transplants were not performed because of high morbidity in the early experience. Early SP transplants were performed using bladder drainage of exocrine secretions, and enteric drainage without a Roux-en-Y was used in the recent series. In the early era, immunosuppressive therapy included cyclosporine (CsA), azathioprine (AZA), corticosteroids, and in half of the patients, ALG or OKT3. Recent SP transplants received tacrolimus (TAC), mycophenolate mofetil (MMF), corticosteroids, and induction with either anti-thymocyte globulin (n = 9), OKT3 (n = 1), daclizumab (n = 5), or basiliximab (n = 8). The 1-year Kaplan-Meier patient survival was 85% in the early era and 100% in the recent group of patients (p = 0.08). In the previous era, four patients suffered significant decrement in renal function, necessitating dialysis or kidney transplantation following pancreas transplantation. All patients transplanted since 1997 maintain near prepancreas transplant levels of renal function [mean pretransplant serum creatinine (Cr) 1.3 ± 0.3 mg/dl vs. mean current Cr 1.4 ± 0.4 mg/dl, p = NS]. The 1-year Kaplan-Meier graft survival (insulin independence) of recent SP transplants was 87%, whereas for prior SP transplants it was 19% (p = 0.0001). The rate of acute pancreas rejection was significantly different between the two groups. Of early SP transplants, 76% experienced at least one rejection episode within the first year. In contrast, 35% of recent SP transplants suffered acute rejection during the same time period (p = 0.04). Current experience with SP transplantation demonstrates improved graft survival and reduced rejection rates with the use of newer immunosuppressive agents.
Pancreas Preservation by the Two-Layer Method: Does it Have a Beneficial Effect Compared with Simple Preservation in University of Wisconsin Solution?
Authors: Noguchi Hirofumi , Levy Marlon F. , Kobayashi Naoya , Matsumoto Shinichi
Published: June 2017

A large number of reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW in islet transplantation. However, two recent large-scale studies showed no beneficial effect of TLM compared with UW storage in human islet transplantation. We reevaluated the effect of TLM by following three groups: group 1: UW simple storage; group 2: TLM performed by multiorgan procurement teams (not specialists of islet isolation); and group 3: TLM performed by specialists of islet isolation (Noguchi and Matsumoto). There were no significant differences between groups 1 and 2, whereas islet yields were significantly higher in group 3 compared with either group 1 or 2. Our data suggest that exact, complete performance of TLM could improve the outcome of islet isolation and transplantation. In this review, we describe the mechanisms of the TLM, the procedure of preoxygenated TLM, and the several possibilities for the reasons of the discrepancy.
Establishment of Mouse Pancreatic Stem Cell Line
Authors: Noguchi Hirofumi , Oishi Koichi , Ueda Michiko , Yukawa Hiroshi , Hayashi Shuji , Kobayashi Naoya , Levy Marlon F. , Matusmoto Shinichi
Published: June 2017

β-Cell replacement therapy via islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes. However, such an approach is severely limited by the shortage of donor organs. Pancreatic stem/progenitor cells could become a useful target for β-cell replacement therapy in diabetic patients because the cells are abundantly available in the pancreas of these patients and in donor organs. In this study, we established a mouse pancreatic stem cell line without genetic manipulation. The duct-rich population after islet isolation was inoculated into 96-well plates in limiting dilution. From over 200 clones, 15 clones were able to be cultured for over 3 months. The HN#13 cells, which had the highest expression of insulin mRNA after induction, expressed PDX-1 transcription factor, glucagon-like peptide-1 (GLP-1) receptor, and cytokeratin-19 (duct-like cells). These cells continue to divide actively beyond the population doubling level (PDL) of 300. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology in HN#13 cells induced insulin and pancreas-related gene expression. This cell line could be useful for analyzing pancreatic stem cell differentiation. Moreover, the isolation technique might be useful for identification and isolation of human pancreatic stem/progenitor cells.
Preconditioned Cell Array Optimized for a Three-Dimensional Culture of Hepatocytes
Authors: Miyamoto Yoshitaka , Ikeya Takeshi , Enosawa Shin
Published: June 2017

Three-dimensional culture procedures have attracted attention in various fields of cell biology. A newly developed cell array assisted in the formation of hepatocyte spheroids by two innovations: 1) micropatterning by a hydrophilic polymer, and 2) the use of bovine carotid artery-derived HH cells as feeder cells. The former contributes to the standardization of the spheroid size and the latter to the maintenance of the spheroids. We created a way to provide a ready-to-use cell array by cryopreservation of an HH feeder cell cultured array. After inoculation of HH cells on the cell array, the culture medium was replaced by freezing medium containing dimethyl sulfoxide. Thereafter, the array was frozen and stored in a −80°C deep freezer. At the start of the hepatocyte culture, the cryopreserved HH cell array was thawed by adding warmed (37°C) culture medium. The morphology and biological activities of the cryopreserved HH cells were intact, as confirmed by phase contrast microscopy and functional staining with calcein and formazan. The rat hepatocytes formed perfect spheroids on the cryopreserved HH cell array without any differences from those on the freshly prepared HH cell array. The CYP3A drug metabolism activities of the hepatocytes were well maintained on the cryopreserved and fresh cell arrays. The present protocol greatly shortened the time and labor required to prepare a cell array for culturing hepatocytes.
Cell Transplantation for Myocardial Repair: An Experimental Approach
Authors: Marelli Daniel , Desrosiers Carolyne , El-Alfy Mohamed , Kao Race L. , Chiu Ray C.-J.
Published: June 2017

Myocardium lacks the ability to regenerate following injury. This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells. It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle. Fourteen dogs underwent explantation of the anterior tibialis muscle. Satellite cells were multiplied in vitro and their nuclei were labelled with tritiated thymidine 24 h prior to implantation. The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe. The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle. Medium without cells was injected into an adjacent site to serve as a control. Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography. In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory. In three implanted dogs, specimens were taken within 6-8 wk. There were persistence of the implantation channels in the experimental sites when compared to the controls. Macroscopically, muscle tissue completely surrounded by scar tissue could be seen. Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed. In two other dogs, specimens were taken at 14 wk postimplantation. Muscle tissue could not be found. These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment. Our specimens failed to demonstrate the presence of myocyte nuclei. It is therefore further hypothesized that in the late postoperative period, the muscle regenerate failed to survive.
Utilization of an Antibody Specific for Human Dystrophin to Follow Myoblast Transplantation in Nude Mice
Authors: Huard Johnny , Tremblay Geneviève , Verreault Steve , Labrecque Claude , Tremblay Jacques P.
Published: June 2017

Human myoblasts were transplanted in nude mice. The efficacy of these transplantations was analyzed using a monoclonal antibody (NCLDys3) specific for human dystrophin. This antibody did not reveal any dystrophin in nude mice that did not receive a human myoblast transplantation. However, about 30 days after a human myoblast transplantation, dystrophin-positive muscle fibers were observed. They were not abundant, and were present either in small clusters or isolated. This technique follows the fate of myoblast transplantation in animals that already have dystrophin, and distinguishes between new dystrophin-positive fibers due to the transplantation and the revertant fibers in mdx mice. Moreover, this technique does not require any labelling of the myoblasts before transplantation. It can also be used to detect dystrophin produced following the fusion of myoblasts transfected with the human dystrophin gene.
Alleviation of Pain in Cancer Patients by Adrenal Medullary Transplants in the Spinal Subarachnoid Space
Authors: Sagen Jacqueline , Pappas George D. , Winnie Alon P.
Published: June 2017

The treatment of intractable pain with currently available therapeutic regimens is often unsatisfactory due to tolerance and untoward complications. Studies in our laboratory have suggested that the transplantation of adrenal medullary tissue into the spinal subarachnoid space can significantly reduce pain in animal models, most likely via release of opioid peptides and catecholamines. The current study was an initial attempt to assess the potential for adrenal medullary transplants in the spinal subarachnoid space to alleviate pain in humans. Donor adrenal medullary tissue was prepared for transplantation in our laboratory. One cc of adrenal medullary tissue was transplanted via lumbar puncture in five patients suffering from terminal cancer pain. Pain levels were determined using a Visual Analog Pain Scale prior to and following the transplantation procedure. In addition, records of narcotic intake and activity were kept. When possible, CSF samples were collected via lumbar puncture for biochemical and cytological analysis. Four of the patients demonstrated progressive decreases in pain scores following the procedure, with concomitant reductions in narcotic intake. Three patients remained pain free, two for over 10 mo. One patient, who developed spinal cord compression secondary to metastasis, was initially pain free, but the pain returned after 10 wk. The fifth patient had no pain reduction by 1 mo following the procedure, but further information was unavailable due to poor patient compliance. In most cases, spinal cerebrospinal fluid (CSF) samples revealed increased levels of met-enkephalin and/or catecholamines following the transplants. The results of this study suggest that adrenal medullary transplants in the spinal subarachnoid space have potential as an alternative approach to the management of chronic pain in humans.
Effect of the YCD3-1 Anti-Mouse Monoclonal on Murine GvHD
Authors: Okunewick James P. , Kociban Deborah L. , Machen Laurie L. , Buffo Mary J.
Published: June 2017

The potential for applying the YCD3-1 rat-anti-mouse IgG2b anti-CD3-ϵ monoclonal antibody (MAB) to the study of graft-versus-host disease (GvHD) in mouse models has been examined. This MAB, unlike the previously developed hamster-anti-mouse-CD3 MABs, had been reported to exhibit strong cytolytic properties when applied in vitro in the presence of complement. Therefore, it was of interest to determine whether it could be effectively used for T-cell depletion in mice to suppress GvHD in the same manner as the anti-human-CD3 MABs have been applied in clinical transplantation. Evaluation of the effectiveness of this antibody was carried out both under fully allogeneic MHC-mismatched and under unrelated-donor MHC-matched marrow transplant conditions. For both types of transplantation, depletion of the donor cells with YCD3-1 plus complement prior to their injection into lethally irradiated recipients significantly suppressed GvHD, resulting in survivals of 75-79%, as compared to 8-13% in the controls that received undepleted cells from the same donors (p < 0.0001). These results suggest that the YCD3-1 MAB may have a potential for use as a negative selection agent in the further definition of the roles of the various T-cell subtypes, as well as the possible roles of natural-killer cells, in future studies into the mechanisms of GvHD, and of the graft-versus-leukemia effect.
Bone Marrow Transplantation for Aplastic Anemia
Authors: Storb Rainer
Published: June 2017

The increased survival of aplastic anemia patients treated by human leukocyte antigens (HLA)-identical marrow transplants is due in part to a decrease in the incidence of graft rejection. The decrease in rejection, in turn, results from the more judicious use of transfusions before transplant, the removal of sensitizing white blood cells from transfusion products, and improvements in the immunosuppressive conditioning programs used to prepare patients for transplant. In regards to the latter, radiation-based programs have been effective, although a cydophosphamide/antithymocyte globulin program begins to look impressive with only one rejection among 33 patients transplanted. In regards to transfusions before transplant, in vitro radiation of all blood products may further reduce the risk of sensitization to minor histocompatibility antigens in the future. The incidence and severity of acute graft-versus-host disease (GvHD) have declined with the use of the methotrexate/cyclosporine regimen, and this has also contributed to improved survival. Chronic GvHD is difficult to treat and future emphasis should be on prevention rather than on treatment. Whether an extended course of cyclosporine beyond 6 mo after transplant will reduce the risk of chronic GvHD is under study. As more and more patients become long-term survivors, problems from long-term sequelae from the conditioning programs and from postgrafting immunosuppression must be considered, in particular secondary malignancies. Perhaps less toxic conditioning programs can be designed. Because of the higher likelihood of causing secondary cancer, possible deleterious effects on growth and development for pediatric patients, and the problem of sterility, radiation-based regimens should not be used in HLA-identical recipients.
Extracorporeal Application of a Gel-Entrapment, Bioartificial Liver: Demonstration of Drug Metabolism and Other Biochemical Functions
Authors: Nyberg Scott L. , Shirabe Ken , Peshwa Madhusudan V. , Sielaff Timothy D. , Crotty Paul L. , Mann Henry J. , Remmel Rory P. , Payne William D. , Hu Wei-Shou , Cerra Frank B.
Published: June 2017

Metabolic activity of a gel-entrapment, hollow fiber, bioartificial liver was evaluated in vitro and during extracorporeal hemoperfusion in an anhepatic rabbit model. The bioartificial liver contained either 100 million rat hepatocytes (n = 12), fibroblasts (n = 3), or no cells (n = 7) during hemoperfusion of anhepatic rabbits. Eight other anhepatic rabbits were studied without hemoperfusion as anhepatic controls, and three sham rabbits served as normal controls. Albumin production rates (mean ± SEM) were similar during in vitro (17.0 ± 2.8 μg/h) and extracorporeal (18.0 ± 4.0 μg/h) application of the hepatocyte bioartificial liver. Exogenous glucose requirements were reduced (p < 0.01) and euglycemia was prolonged (p < 0.001) in anhepatic rabbits treated with the hepatocyte bioartificial liver. The maximum rate of glucose production by the hepatocyte bioartificial liver ranged from 50-80 μg/h. Plasma concentrations of aromatic amino acids, proline, alanine, and ammonia were normalized in anhepatic rabbits during hepatocyte hemoperfusion. Gel-entrapped hepatocytes in the bioartificial liver performed sulfation and glucuronidation of 4-methylumbelliferone. P450 activity was demonstrated during both in vitro and extracorporeal application of the BAL device by the formation of 3-hydroxy-lidocaine, the major metabolite of lidocaine biotransformation by gel-entrapped rat hepatocytes. In summary, a gel-entrapment, bioartificial liver performed multiple hepatocyte-specific functions without adverse side effects during extracorporeal application in an anhepatic, small animal model. With its potential for short term support of acute liver failure, scale-up of the current bioartificial liver device is indicated for further investigations in large animal, preclinical trials.
Ultrastructural Characterization of a Regular Schwann-Axon-Islet Complex after the Autograft of Pancreatic Fragments into the Spleen of the Adult Dog
Authors: Madureira Mário L. C.
Published: June 2017

After autotransplantation of pancreatic fragments into the dog's spleen, the morphogenesis of the reinnervating process has evolved as an highly differentiated model, reproducing the most peculiar and systematic relationships between Schwann cells, axons, and islet cells reported in the dog's islet, despite it's modulation by the restrictive conditions derived from the intrasplenic location of the dispersed pancreatic tissue. The reinnervating process is described, emphasizing the peculiar ultrastructural features and topography of the Schwann cells and of the axonal network that impose the concept of a true anatomical reinnervation, which make previsible the possibility of a very selective and direct neurochemical and/or electrotonic control of the engrafted islet cells. The schwann-axon-islet complexes are a very peculiar and regular arrangement between islet cells and nervous elements and are reproduced after the autotransplant without the engrafted ganglia, whose potential but aleatory contribution could not be unequivocally characterized. Axonal profiles or Schwann cells on the abundant regenerated ductal-acinar structures were not identified.
Staining and in Vitro Toxicity of Dithizone with Canine, Porcine, and Bovine Islets
Authors: Clark Samuel A. , Borland Kermit M. , Sherman Sandra D. , Rusack Thelma C. , Chick William L.
Published: June 2017

Dithizone (DTZ) is a recognized diabetogenic agent in vivo, and a supravital stain commonly used for identification of islets to be used for transplantation. In the present studies, we compared DTZ staining of freshly isolated and cultured canine, bovine, and porcine islets, and the effect of DTZ on the function and viability of islets. Incubation with DTZ resulted in staining of canine and porcine islets, but no discernible staining with bovine islets. Insulin content of porcine, canine, and bovine islet was 2.0 ± 0.2, 2.2 ± 0.3, and 1.9 ± 0.2 mU/EIN, indicating a lack of correspondence of DTZ staining and insulin content. Seven days of culture with canine islets resulted in ≥50% reduction of DTZ stained cells. Exposure to DTZ at 50 μg/mL resulted in a maximal number of stained cells in preparations of purified islets (80-85%; counted after dispersion), a lower percentage of cells stained faintly at 20 μg/mL (50-55%), with no discernible staining at 10 μg/mL.

Prolonged exposure of islets (4-48 h) to 20 μg/mL DTZ led to reduced insulin secretion and islet cell death. Incubation of canine or porcine islets with 100 μg/mL of DTZ for 0.5 h resulted in a dramatic loss of viability and diminished insulin secretory function, which was not reversed with continued culture. The concentration dependence of toxic effects paralleled the concentration dependence of cellular staining. The minimally effective staining concentration (20 μg/mL) also resulted in a loss of viability. An additional assessment of DTZ toxicity was made using the RIN-38 β-cell line, which shows no discernible staining with DTZ. A 1 h exposure to dithizone resulted in a dose-dependent loss of viable RIN-38 cells.

We conclude first, that DTZ is cytotoxic to islet cells in vitro, at concentrations used for islet staining. Although the toxicity of DTZ appears to be related to its staining properties, high concentrations have toxic effects that are unrelated to staining properties. We propose that cellular accumulation of DTZ (staining), produces toxicity by concentrating DTZ to toxic levels. Secondly, we conclude that DTZ does not stain islets of all species, despite the equivalent insulin content.
Immunocytochemical Detection and Characterization of Intrahepatic Human Pancreatic Islets after Combined Liver-Islet Allotransplantation
Authors: Scopsi Lucio , Andreola Salvatore , Socci Carlo , Bertuzzi Federico , Di Carlo Valerio , Pozza Guido , Rilke Franco , Gennari Leandro , Coleela Giovanni , Regalia Enrico , Mazzaferro Vincenzo
Published: June 2017

The unique availability of an explanted liverislet allograft, removed for primary nonfunction of the liver, led us to evaluate distribution and phenotype of exocrine and endocrine components of the pancreatic graft. Immunocytochemistry was used to map patterns of gene products for islet hormones, proprotein processing enzymes, panneuroendocrine markers, and pancreatic exocrine markers. When compared with age-matched control pancreases, insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells were similarly represented and distributed within the grafted islet. We also demonstrate that the intrahepatic transplanted islets retained the enzyme machinery able to process the hormone precursors into bioactive fragments. In the clinical setting, this resulted in an immediate functioning of the graft and insulin-independence of the patient one month after transplantation. The purity in islets, as assessed by immunocytochemistry with antibodies to tissue constituents of endocrine and exocrine lineages, was around 40%. Despite the massive intraportal presence of pancreatic acinar tissue, no signs or symptoms attributable to ectopic hypersecretion of exocrine enzymes occurred. In fact, when tested with antibodies to such enzymes, low levels of immunoreactivity were observed in the grafted acinar cells.
CD36+-Dendritic Epidermal Cells: A Putative Actor in the Cutaneous Immune System
Authors: Rouabhia Mahmoud , Jobin Nicolas , Doucet Robert , Bergeron Julie , Auger François A.
Published: June 2017

In the present study we have investigated by indirect immunofluorescence staining and mixed lymphocyte reaction methods, the localization, distribution, percentage, and the immunological involvement of CD36+- dendritic epidermal cells (CD36+-DECs) in normal human skin. Human epidermal cell suspensions were obtained from skin specimen of healthy persons. First, an indirect immunofluorescent staining method was performed on frozen skin sections, freshly isolated cells, nonadherent and adherent cells and second, the allogeneic mixed epidermal cell-lymphocyte reaction (ELR) method was performed with human peripheral blood mononuclear cells and irradiated CD36+-DECs plus CD-1a+ (Langerhans cells) and/ConA (at 10 μg/mL). We found that CD36+-DECs were localized in the epidermis mainly in the basal layer. They were non adherent cells. The percentage of these CD36+-DECs was of about 2%. These CD36+-DECs were AE3(which recognizes keratin normally expressed by keratinocytes) positive cells. Our immunoreactivity study using allogeneic mixed ELR, showed that CD36+-DECs stimulated allogeneic lymphocyte proliferation. Their stimulatory effects were important when Langerhans cells and ConA were added separately or together to the PBMCs culture. The above results suggest that CD36+-DECs may contribute to the immunological role of skin and could be involved in cutaneous allograft recognition and rejection. Abbreviations: DECs: dendritic epidermal cells; ConA: concanavalin A; DPM: disintegrations per minute; ELR: epidermal cell-lymphocyte reaction; LC: Langerhans cells; PBMCs: peripheral blood mononuclear cells.
Formation of Porcine Hepatocyte Spheroids for use in a Bioartificial Liver
Authors: Lazar Arye , Peshwa Madhusudan V. , Wu Florence J. , Chi Chung-Ming , Cerra Frank B. , Hu Wei-Shou
Published: June 2017

Xenogeneic hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, form multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 μm. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Ureagenesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.
Immunosuppressive Effect of 2′-Deoxycoformycin (Pentostatin®) for Rat Islet Allotransplantation
Authors: Ar'rajab Aamer , Harris Richard B. , Khair-El-Din Tarik A. , Sentementes James T. , Lu Christopher , Dawidson Ingemar J.A.
Published: June 2017

Adenosine deaminase (ADA) is an important enzyme for proper function of lymphocytes and congenital absence of ADA results in a form of severe combined immunodeficiency syndrome. 2′-Deoxycoformycin (Pentostatin®, DCF) irreversibly inhibits ADA and therefore has been suggested as an immunosuppressive drug. The present study evaluated the immunosuppressive effect of DCF for islet allotransplantation in rats. Isolated islets (1,500 islets) from Lewis rats were transplanted into the kidney subcapsular space of streptozotocin-induced diabetic Wistar-Furth rats. DCF was administered IP either as a single injection at 1 mg/kg/wk, 1 mg/kg twice weekly, 5 mg/kg/twice weekly or 1 mg/kg/day, or as a continuous infusion at 0.8 or 1 mg/kg/day. Daily administration of DCF at 0.8 mg/kg in both methods, single daily injection or continuous infusion, resulted in a lymphopenia and a decrease in concanavalin A stimulation of splenic lymphocytes. However, DCF (in all doses) was not effective in preventing islet allograft rejection as evaluated by measuring the duration of normoglycemia following islet transplantation and by microscopic examination of the islet grafts. In fact, the duration of normoglycemia following islet transplantation was 7.5 ± 0.9 and 9.0 ± 1.0 days in rats receiving DCF in single daily injection or continuous infusion, respectively. This was not significantly different from control nontreated transplanted rats (8.5 ± 0.7 days). Increasing the dose of DCF to 1 mg/kg, administered by continuous infusion, resulted in 100% mortality. For comparison, cyclosporine A (20 mg/kg, IP daily injection for 14 days) prolonged islet allograft survival to 27.3 ± 1.5 days (p < 0.001). It is concluded that inhibition of ADA by DCF, despite its marked lymphotoxic effect, does not prevent rejection of islet allograft.
Article Commentary: Development of the Human Striatum: Implications for Fetal Striatal Transplantation in the Treatment of Huntington's Disease
Authors: Freeman Thomas B. , Sanberg Paul R. , Isacson Ole
Published: June 2017

Fetal neural transplantation has recently been demonstrated to ameliorate motor and other behavioral deficits in animal models of Huntington's disease, and reconstruct many of the damaged striatal circuits. However, there has been significant variability in the histological appearance of these grafts, most likely related to differences of the regions of dissection of the donor tissue. Selective dissection and transplantation of the lateral ventricular eminence in rodents has resulted in grafts consisting of primarily striatal-like tissue. This data, combined with data from our own and other laboratories has led to a description of the development of the human striatum, with a particular emphasis on the relevance of human striatal development to the field of fetal tissue transplantation for the treatment of Huntington's disease. If the goal of transplantation is to graft GABAergic striatal projection neurons, it is our impression that optimal grafting results will occur when transplants are derived from the lateral ventricular eminence and the lateral aspect of the body of the ventricular eminence anterior to the foramen of Monro. Optimal results are likely to occur when donor ages range from Stage 19 to 23, with possible graft success when donor age extends to as late as postovulatory week 22.
A Method for Mass Harvesting Islets (Brockmann Bodies) from Teleost Fish
Authors: Yang Hua , Wright James R.
Published: June 2017

In certain species of fish, the insulin-producing tissue is uniquely located in separate structures called Brockmann bodies (BBs). Tilapia BBs have been shown to be a simple and inexpensive source of islet cells for xenotransplantation research. Each donor tilapia contains roughly 12-15 BBs, measuring from 0.3 to 5.0 mm in maximum dimension, in a triangular region of adipose tissue bounded by the liver, stomach, and spleen/gallbladder. At present, the larger BBs (usually 2-4) are harvested by microdissecting these “BB regions” using jeweler's forceps and microvascular scissors while being visualized with the aid of a dissecting microscope. It is a simple but time-consuming task that would not be applicable for harvesting massive amounts of BB tissue for large animal studies. Therefore, we have developed an easier and more efficient method of harvesting BBs based on a standard enzymatic method for isolating human adipocytes. BB regions are harvested from donor fish and pooled into a 50 mL plastic tube containing collagenase Type II (3 mg/mL) in Hank's balanced salt solution (HBSS); the tube is then placed in a 37°C waterbath/shaker for roughly 15 min. The exact length of the digestion interval is determined by visual inspection of the tube to determine whether the BBs have been liberated. The digestion is then stopped by adding excess cold HBSS. The adipocytes float while the BBs and residual connective tissue (i.e., a few blood vessels, nerves, and bile ducts) form a pellet. The pellet is washed several times in HBSS and then placed in a culture dish. The BBs are easily handpicked with a siliconized pipette. Based on functional data and DNA content, this new method roughly doubles or triples our yield of BB tissue per donor fish. To determine whether BBs harvested in this manner functioned in a manner similar to those harvested by microdissection, we performed a series of transplants using mass-harvested BBs. Long-term normoglycemia was achieved in streptozotocin-diabetic nude mice and mean graft survival time was not altered in streptozotocin-diabetic euthymic balb/c mice. However, the total weight of donor fish required per recipient was decreased by 50% in both strains.
Arterial Delivery of Genetically Labelled Skeletal Myoblasts to the Murine Heart: Long-Term Survival and Phenotypic Modification of Implanted Myoblasts
Authors: Robinson Shawn W. , Cho Peter W. , Levitsky Hyam I. , Olson Jean L. , Hruban Ralph H. , Acker Michael A. , Kessler Paul D.
Published: June 2017

The ability to replace damaged myocardial tissue with new striated muscle would constitute a major advance in the treatment of diseases that irreversibly injure cardiac muscle cells. The creation of focal grafts of skeletal muscle has been reported following the intramural injection of skeletal myoblasts into both normal and injured myocardium. The goals of this study were to determine whether skeletal myoblast-derived cells can be engrafted into the murine heart following arterial delivery. The murine heart was seeded with genetically labeled C2C12 myoblasts introduced into the arterial circulation of the heart via a transventricular injection. A transventricular injection provided access to the coronary and systemic circulations. Implanted cells were characterized using histochemical staining for β-galactosidase, immunofluorescent staining for muscle-specific antigens, and electron microscopy. Initially the injected cells were observed entrapped in myocardial capillaries. One week after injection myoblasts were present in the myocardial interstitium and were largely absent from the myocardial capillary bed. Implanted cells underwent myogenic development, characterized by the expression of a fast-twitch skeletal muscle sarco-endoplasmic reticulum calcium ATPase (SERCA1) and formation of myofilaments. Four months following injection myoblast-derived cells began to express a slow-twitch/cardiac protein, phospholamban, that is normally not expressed by C2C12 cells in vitro. Most surprisingly, regions of close apposition between LacZ labeled cells and native cardiomyocytes contained structures that resembled desmosomes, fascia adherens junctions, and gap junctions. The cardiac gap junction protein, connexin43, was localized to some of the interfaces between implanted cells and cardiomyocytes. Collectively, these findings suggest that arterially delivered myoblasts can be engrafted into the heart, and that prolonged residence in the myocardium may alter the phenotype of these skeletal muscle-derived cells. Further studies are necessary to determine whether arterial delivery of skeletal myoblasts can be developed as treatment for myocardial dysfunction.
Expression of Tyrosine Hydroxylase in an Immortalized Human Fetal Astrocyte Cell Line; in Vitro Characterization and Engraftment into the Rodent Striatum
Authors: Tornatore Carlo , Baker-Cairns Belinda , Yadid Gal , Hamilton Rebecca , Meyers Karen , Atwood Walter , Cummins Alex , Tanner Virginia , Major Eugene
Published: June 2017

The use of primary human fetal tissue in the treatment of neurodegenerative disorders, while promising, faces several difficult technical and ethical issues. An alternative approach that would obviate these problems would be to use immortalized cell lines of human fetal central nervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro and in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is vimentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neurofilament and neuron specific enolase, consistent with its glial origin. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constructed (phTH/Neo) and stably expressed in the SVG cells for over 18 months as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cells, termed SVG-TH, consistently found 4-6 pmol/ml/min of 1-dopa produced with the addition of BH4to the media. Furthermore, in cocultivation experiments with hNT neurons, PC-12 cells and primary rat fetal mesencephalic tissue, both the SVG and SVG-TH cells demonstrated neurotrophic potential, suggesting that they constituitively express factors with neuroregenerative potential. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the striatum of Sprague-Dawley rats and followed over time. A panel of antibodies was used to unequivocally differentiate the engrafted cells from the host parenchyma, including antibodies to: SV40 large T antigen (expressed in the SVG-TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomittantly, rat CD4 and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxydopamine lesioned rats, rotational behavior of the rat decreased as much as 80% initially, then slowly returned to baseline over the next four weeks, parallelling graft rejection. Thus, the SVG-TH cells can induce a functional recovery in an animal model of Parkinson's disease, however as a xenograft, the SVG cells are recognized by the immune system.
The Effect of Fetal Neocortical Transplants on Lesion-Induced Cerebral Cortex Plasticity
Authors: Schulz M.K. , Sørensen J.C. , Tillotson G.L. , Castro A.J. , Zimmer J.
Published: June 2017

Sensorimotor cortical lesions in newborn rats lead to the formation of abnormal projections from the opposite intact sensorimotor cortex. In the present study the influence of fetal neocortical transplants on this lesion-induced plasticity was examined. Newborn rats received unilateral frontal neocortical lesions. One experimental group received grafts of fetal neocortical tissue (E14–E16) into the lesion cavities. Another group served as lesion-only animals, while a third group was left unlesioned and without grafts as normal controls. At 3 mo of age, the animals received injections of the anterograde tracer biotinylated dextran amine (BDA) into the sensorimotor cortex contralateral to the lesion/transplantation area. After sacrifice 2 wk later, the brains were processed histochemically for detection of BDA-labeled cells and fibers. As a measure of the lesion-induced axonal sprouting response, corticothalamic and corticopontine fibers crossing the midline were counted. Significantly fewer cortical efferent fibers crossed the thalamic midline in the transplanted rats compared to the lesion-only controls. In contrast, the presence of transplants did not reduce the corticopontine sprouting response. These results therefore indicate that fetal neocortical grafts have a modulatory, yet variable effect on the lesion-induced axonal sprouting of contralateral sensorimotor cortical neurons.
Neural Transplantation for Huntington's Disease: Experimental Rationale and Recommendations for Clinical Trials
Authors: Shannon Kathleen M. , Kordower Jeffrey H.
Published: June 2017

Huntington's disease (HD) is a neurodegenerative disorder affecting motor function, personality, and cognition. This paper reviews the experimental data that demonstrate the potential for transplantation of fetal striatum and trophic factor secreting cells to serve as innovative treatment strategies for HD. Transplantation strategies have been effective in replacing lost neurons or preventing the degeneration of neurons destined to die in both rodent and nonhuman primate models of HD. In this regard, a logical series of investigations has proven that grafts of fetal striatum survive, reinnervate the host, and restore function impaired following excitotoxic lesions of the striatum. Furthermore, transplants of cells genetically modified to secrete trophic factors such as nerve growth factor protect striatal neurons from degeneration due to excitotoxicity or mitochondrial dysfunction. Given the disabling and progressive nature of HD, coupled with the absence of any meaningful medical therapy, it is reasonable to consider clinical trials of neural transplantation for this disease. Fetal striatal implants will most likely be the first transplant strategy attempted for HD. This paper describes the variable parameters we believe to be critical for consideration for the design of clinical trials using fetal striatal implants for the treatment of HD.
Significant Improvement of Survival by Intrasplenic Hepatocyte Transplantation in Totally Hepatectomized Rats
Authors: Vogels Birgit A.P.M. , Maas Martinus A.W. , Bosma Anne , Chamuleau Robert A.F.M.
Published: June 2017

The effect of intrasplenic hepatocyte transplantation (HTX) was studied in an experimental model of acute liver failure in rats with chronic liver atrophy. Rats underwent a portacaval shunt operation on Day -14 to induce liver atrophy, and underwent total hepatectomy on Day 0 as a start of acute liver failure. Intrasplenic hepatocyte or sham transplantation was performed on Day -7, -3, or -1 (n = 4 to 6 per group). During the period following hepatectomy, mean arterial blood pressure was maintained above 80 mm Hg and hypoglycaemia was prevented. Severity of hepatic encephalopathy was assessed by clinical grading and EEG spectral analysis, together with determination of blood ammonia and plasma amino acid concentrations, and “survival” time. Histological examination of the spleen and lungs was performed after sacrifice. Intrasplenic hepatocyte transplantation resulted in a significant improvement in clinical grading in all transplanted groups (p < 0.05), whereas a significant improvement in EEG left index was seen only in the group with transplantation on Day -1 (p < 0.05). In contrast to hepatocyte transplantation 1 day before total hepatectomy, rats with hepatocyte transplantation 3 and 7 days before total hepatectomy showed a significant 3- and 2-fold increase in “survival” time compared to sham transplanted controls: HTX at Day -1: 7.5 ± 0.3 h vs. 5.9 ± 0.6 h (p > 0.05), HTX at Day -3:19.7 ± 3.7 h vs. 6.5 ± 0.3 h (p < 0.05), and HTX at Day -7: 13.8 ± 3.2 h vs. 6.3 ± 0.3 h (p < 0.05). Furthermore, rats with hepatocyte transplantation on Day -3 and -7 showed significantly lower blood ammonia concentrations after total hepatectomy (p < 0.0001). Histological examination of the spleens after sacrifice showed clusters of hepatocytes in the red pulp. Hepatocytes present in the spleen for 3 and 7 days showed bile accumulation and spots of beginning necrosis. The present data show that in a hard model of complete liver failure in portacaval shunted rats, intrasplenic hepatocyte transplantation is able to prolong “survival” time significantly 2- to 3-fold. The relevance of this observation for human application is discussed.
Correction of Fanconi Anemia Type C Phenotypic Abnormalities Using a Clinically Suitable Retroviral Vector Infection Protocol
Authors: Freie Brian W. , Dutt Parmesh , Clapp D. Wade
Published: June 2017

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.
Differences in the Contribution of CD4+ T Cells to Proislet and Islet Allograft Rejection Correlate with Constitutive Class II MHC Alloantigen Expression
Authors: Simeonovic Charmaine J. , Brown Debra J. , Townsend Michelle J. , Wilson J. Dennis
Published: June 2017

Allografts of BALB/c (H-2d) fetal proislets facilitated long-term (>100 days) reversal of streptozotocin-induced diabetes in CBA/H (H-2k) mice treated with a combination of anti-CD4 and anti-CD8 mAbs. Anti-CD8 monotherapy was partially effective in restoring normoglycemia but anti-CD4 mAb treatment of host animals failed to promote allograft function. In contrast, allografts of BALB/c adult islets demonstrated indefinite reversal of diabetes in recipient mice treated only with anti-CD8 mAb. Anti-CD4 monotherapy resulted in only transient restoration of normoglycemia. These findings clearly demonstrate (1) a critical role for CD8 T cells in the acute rejection of pancreatic islet tissue allografts and (2) tissue-specific differences in the participation of CD4 T cells as primary effectors in the rejection reaction. Immunohistochemical studies showed that the capacity for CD4 T cells to initiate the rejection of proislet but not adult islet allografts correlates with the presence/absence, respectively, of graft parenchymal cells that constitutively express Class II MHC alloantigens. Proislet grafts, unlike transplants of purified adult islets, contain heterogeneous tissue components including Class II MHC+ve duct epithelium. Thus, the participation of CD8 and CD4 T cells as primary effectors of graft rejection depends on which class or classes of MHC antigens are constitutively expressed on graft parenchymal cells and are available for recognition. Islet tissue in both rejecting proislet and islet allografts showed de novo induction of Class II MHC alloantigens only after severe disruption to islet architecture had been achieved by infiltrating mononuclear cells. Thus, at this stage of advanced allograft injury, CD4 T cells have the potential to act as secondary effectors, possibly by amplifying the inflammatory reaction and thus accelerating graft destruction. The capacity for antirejection mAb therapy to establish transplant tolerance was facilitated in the islet allograft model where it was necessary to target only the CD8 T cell subpopulation.