Daily Cyclosporine Research Articles

Comprehensive clinical imaging, histopathological analysis and liquid biopsy-based surveillance of human uveal melanoma in a prolonged rabbit xenograft model.

Uveal melanoma is the most common intraocular tumor in adults. Our group has previously developed a human uveal melanoma animal model; however, adverse effects caused by the immunosuppressive agent, cyclosporine A, prevented animals from surviving more than 12 weeks. In this study, we tested multiple cyclosporine A doses over an extended disease course up to 20 weeks, providing complete clinical imaging of intraocular tumors, histopathological analysis and liquid biopsy biomarker analysis. Twenty albino rabbits were divided into four groups with different daily cyclosporine A schedules (0-10 mg/kg) and inoculated with human uveal melanoma cell lines, 92.1 or MP41, into the suprachoroidal space. Rabbits were monitored with fundoscopy, ultrasound and optical coherence tomography. Intraocular tumors (macroscopic or microscopic) were detected in all study animals. Tumor size and growth were correlated to cyclosporine A dose, with tumors regressing when cyclosporine A was arrested. All tumors expressed HMB-45 and MelanA; however, tumor size, pigmentation and cell morphology differed in 92.1 vs. MP41 tumors. Finally, across all groups, circulating tumor DNA from plasma and aqueous humor was detected earlier than tumor detection by imaging and correlated to tumor growth. In conclusion, using three clinically relevant imaging modalities (fundoscopy, ultrasonography and optical coherence tomography) and liquid biopsy, we were successfully able to monitor tumor progression in our rabbit xenograft model of human uveal melanoma.

Rapid Efficacy of Ixekizumab for Pediatric Acute Generalized Pustular Psoriasis

Introduction: Generalized pustular psoriasis (GPP) is a rare, severe variant of psoriasis that is uncommon in pediatric patients and can be refractory to many therapies. Ixekizumab, a monoclonal antibody that selectively inhibits interleukin 17-A, has been reported as safe and efficacious in patients with GPP, though minimal data exists on its use in the pediatric population.
 Case presentation: A 17-year-old female with history of alopecia areata and pustular psoriasis, on ustekinumab, was admitted for a severe pustular psoriasis flare with systemic symptoms including fever and tachycardia which progressed to erythroderma and required vasopressor support. After minimal improvement with 24 hours on broad-spectrum antibiotics, she received cyclosporine 5mg/kg/day. On day three of admission, and after two days of cyclosporine with minimal improvement, she received a 160 mg loading dose of ixekizumab. She defervesced and transferred out of the ICU within 24 hours. She was successfully weaned off cyclosporine after her second dose of ixekizumab. She experienced no adverse reactions.
 Discussion: Generalized pustular psoriasis is less common and often more severe than plaque psoriasis, associated with increased morbidity and mortality in both pediatric and adult patients. Clinical trials and case series have reported rapid and sustained improvement in patients with pustular psoriasis refractory to other therapies, though there is little data on the pediatric population. This case demonstrates the rapid efficacy of ixekizumab for severe erythrodermic pustular psoriasis in a pediatric patient, highlighting its use not only for refractory disease, but also as a rescue therapy in an emergent setting.

Acquired Aplastic Anemia Patients Using Single Agent Cyclosporine a, in Resource Poor Setting

Abstract: Aplastic Anemia (AA) is a syndrome characterized by peripheral blood pancytopenia and bone marrow hypo-cellularity. Acquired AA is considered to have an underlying autoimmune etiology. Hematopoietic stem cell transplantation (HSCT) still remains the treatment of choice for AA patients. However, due to cost restrictions, cyclosporine A has been tried in resource poor setting as a single agent.Aim: The aim of this study is to observe the single agent Cyclosporine treatment response in AA patients.Materials and Methods: The following study is a retrospective study. Patients diagnosed for the first time between January 2010 and December 2020, aged 14 years old and up, treated with cyclosporine A and supportive therapy, were included in this study. Statistical analysis for this study was performed using SPSS.All subjects were started on 5mg/kg per day of cyclosporine. The patients were divided into three groups according to the IAASG classification criteria (Non severe AA, Severe AA and Very severe AA). Another subgroup analysis related to sex (females to males) and age was performed, classifying the patients into three subgroups (14-29; 30-59; 60+ years old).Overall Survival (OS) was evaluated using Kaplan-Meier method with Log-Rank test. Overall survival (OS) was defined as the duration from the first day of treatment to all-cause death. T-test and Chi-square test were used for categorical and continuous parameters analysis. P value lower than 0.05 was considered statistically significant.Results: A total of 35 patients were diagnosed as Acquired Aplastic Anemia between 2010-2020. The median age at diagnosis was 51 years old. The incidence of Aplastic Anemia in Albania is 1.35 per million inhabitants. 5 patients (19.2%) developed complete response. NSAA showed the best treatment response (38.5%). The relation between the treatment response at the end of the first year of cyclosporine treatment and the 5-year survival was statistically significant, Pearsons' correlation coefficient 0.441 (P=0.035).The estimated mean survival time of AA patients in our study is 68.6 months. When compared the survival to the severity, the SAA group showed a better OS (53 months) (P=0.081). The 5-year cumulative survival of the patients resulted 71.9%. VSAA group had the worst estimated survival (45.5%). The youngest age group showed the worst 5-year survival on cyclosporine alone (33.3%) (P=0.016). The subgroup analysis between males and females resulted in women having a slightly better survival than men, 59 and 58 months respectively (P=0.276). Those patients who failed the treatment with cyclosporine alone at the end of the first year, showed the worse 5-year survival (50%).Conclusions: This study emphasizes once again the importance of the IAASG classification on AA patients' prognosis and treatment outcome. The youngest patients should be considered for HSCT rather than be treated on cyclosporine alone. Treatment on cyclosporine alone and supportive therapy is a reasonable option for older patients in poor resource countries as Albania.Abbreviations: AA- Aplastic Anemia, NSAA- Non severe aplastic anemia, SAA- Severe aplastic anemia, VSAA- Very severe aplastic anemia, HSCT- Hematopoietic stem cell transplantation, IAASG-International Aplastic Anemia Study Group, SPSS-Statistical Package for the Social Sciences. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modeling chronic cervical spinal cord injury in aged rats for cell therapy studies

With an expanding elderly population, an increasing number of older adults will experience spinal cord injury (SCI) and might be candidates for cell-based therapies, yet there is a paucity of research in this age group. The objective of the present study was to analyze how aged rats tolerate behavioral testing, surgical procedures, post-operative complications, intra-spinal cell transplantation and immunosuppression, and to examine the effectiveness of human iPSC-derived Neural Progenitor Cells (IMR90-hiPSC-NPCs) in a model of SCI. We performed behavioral tests in rats before and after inducing cervical hemi-contusions at C4 level with a fourth-generation Ohio State University Injury Device. Four weeks later, we injected IMR90-hiPSC-NPCs in animals that were immunosuppressed by daily cyclosporine injection. Four weeks after injection we analyzed locomotor behavior and mortality, and histologically assessed the survival of transplanted human NPCs. As rats aged, their success at completing behavioral tests decreased. In addition, we observed high mortality rates during behavioral training (41.2%), after cervical injury (63.2%) and after cell injection (50%). Histological analysis revealed that injected cells survived and remained at and around the grafted site and did not cause tumors. No locomotor improvement was observed in animals four weeks after IMR90-hiPSC-NPC transplantation. Our results show that elderly rats are highly vulnerable to interventions, and thus large groups of animals must be initially established to study the potential efficacy of cell-based therapies in age-related chronic myelopathies.

Cardiac Allograft Tolerance Induction with Ox40L Costimulatory Blockade

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism with allogeneic bone marrow and kidney co-transplantation. This model has failed without the regulatory mechanisms associated with the donor kidney. We hypothesized that rapamycin and anti-OX40L mAb (KY1005, Kymab Ltd) could substitute for kidney co-transplantation and induce cardiac allograft tolerance by providing a favorable immunologic milieu for regulatory T cells (Tregs). <h3>Methods</h3> Fourteen cynomolgus NHPs underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplant. Heart Alone recipients received a heart transplant (n=5), Heart/Kidney recipients received a heart and kidney co-transplant (n=7). Heart/OX40L recipients received a heart transplant plus 40 days of anti-OX40L mAb (n=2). All groups underwent a 12 day course of aCD154 mAb. The Heart Alone and Heart Kidney groups received 28 days of cyclosporine while the Heart aOX40L group received 100 days of rapamycin. <h3>Results</h3> All Heart Alone recipients had cellular rejection (ISHLT 3R) by 200 days post bone marrow transplant (pBMT). No Heart/Kidney recipients showed early rejection. Similarly, no Heart/OX40L recipients showed early rejection and one is currently 258 days pBMT without clinical or pathologic evidence of rejection (Figure 1). The other recipient died of viral complications. Mean peak lymphoid chimerism was 10% for the Heart Alone group, 26% for the Heart/Kidney group, and 29% for the Heart/OX40L group. The anti-OX40L protocol also showed an expansion of the Treg population with a peak of 35% Tregs in the CD4+ population. <h3>Conclusion</h3> The OX40L protocol achieved robust lymphoid chimerism with concomitant expansion of Tregs. The surviving Heart/OX40L recipient achieved longer freedom from allograft rejection when compared to the Heart Alone group. OX40L costimulatory blockade may allow for heart allograft tolerance without kidney co-transplantation.

Arabic Gum Role In Ameliorating Cyclosporine- A Initiated Nephrotoxicity In Albino Rats

Recently many researchers reported that treatment using cyclosporine A in extended period in order to avoide the rejection of allograft is connected with renal impairment progress. Arabic gum is a valuable antioxidants and anti-carcinogenic factor with a protective effect against renal intoxications. This research aims at assessing the improving function of Arabic gum in ameliorating nephrotoxicity of cyclosporine A in adult rats by measuring urea and creatinine as renal function tests and histopathological changes plus redox status parameters. 80 mature rats males and females were classified into four groups during the period of expirment (45 days) first one act as control given saline; the second one was given 7.5gm/kg/day of Arabic gum ,while third group was given cyclosporine A( 25 mg/kg /day ) finally the fourth one was given 7.5gm/kg/day of Arabic gum together with the 25 mg/kg /day of cyclosporine A. Cyclosporine A group three rats had renal impairment beside injury, when reported by elevated values of urea and creatinine and renal pathological changes, relative to the first group(control) with high level of cyclosporine A in blood and bad parameters of redox status. Receiving of (Arabic gum) accompanied with (cyclosporine A) ameliorating renal damage by decreasing the level of urea and creatinine in the blood, renal histo pathological amelioration, moreover the improved parametersof antioxidant. The levels of catalase, glutathione, peroxidase and superoxide dismutase were statistically reduced as regard group three G3 (cyclosporine) which was significant relative to the control & second groups G1,G2 although significantly elevated in the fourth group which given cyclosporine plus Arabic gum when compared with the third group (cyclosporine). Arabic gum has likely to behave like natural agent opposing cyclosporine A -provoked nephrotoxicity.

Limb Transplantation Following Ex Vivo Normothermic Perfusion in an Orthotopic Forelimb Swine Transplantation Model

PURPOSE: Ischemia-reperfusion injury remains a major limiting factor for limb replantation and transplantation. Ex-vivo normothermic limb perfusion has been proven to preserve viability and function of amputated limbs longer than cold storage. Our aim was to investigate the feastibility of limb transplantation following EVNLP and possible vascular, inflammatory and infectious complications in a mid-humerus porcine forelimb transplant model. METHODS: Eight Yucatan miniature pigs, 4 donors and 4 recipients, were used. Right forelimbs of donor animals were amputated at mid-humeral level, to maintain integrity of forearm fascial compartments, and preserved with EVNLP until replantation. The right forelimb of the recipient animal was amputated, and a central venous line was placed in the contralateral neck. Perfusate electrolytes, gases, O2 saturation, muscle contractility, surface temperature (infrared thermography), and peripheral perfusion (indocyanine green agiography) were assessed during EVNLP. The humerus was fixed with a single 3.5 mm LC-DCP plate; microsurgical anastomosis of the brachial artery, cephalic vein, and repair of radial, ulnar, and median nerves were performed under an operative microscope. Tendons of the biceps and triceps were repaired with pulvertaft weave technique. The first animal did not receive systemic immunosuppression. Systemic immunosuppression for the remaining 3 pigs included induction with antithymoglobulin, followed by daily cyclosporine (CSA), mycophenolate mofetil, and methylprednisolone. CSA trough levels were measured daily. Limbs were monitored clinically and histologically for signs of rejection. Bone healing was confirmed with CT scan at euthanasia. The endpoint of the study was 90 days. RESULTS: Warm ischemia time during limb procurement was 20.6 ± 9 minutes and 2.2 ± 0.25 hours during limb transplantation. EVNLP lasted an average of 4.3 ± 0.52 hours. Total time to revascularize was 6.8 ± 0.5 hours. PO2, pH, and lactate were 557 ± 72 mm Hg, 7.5 ± 0.1, 5.6 ± 0.9 mmol/L, respectively. Muscle contractions were 4/5 during EVNLP. All forelimbs were successfully transplanted with no vascular failure. CSA trough levels were on average 678 ± 450, 369 ± 445, and 336 ± 468 ng/ml. Animals 2, 3, and 4 developed septic thrombophlebitis of the central line, which was replaced on POD 14, 51, and 28. Animals 3 and 4 lost IV access on POD 54 and 64, respectively, and were transitioned to PO medications. The first transplanted limb (animal 1) showed evidence of acute rejection on POD 4 and the animal was euthanized on POD 6. In the remaining animals, the incisions healed and initial edema resolved by day 14.The third animal developed angioinvasive aspergillosis on POD 20 with 2 areas of full thickness skin necrosis of trunk. Voriconazol was started and the lesions were resected on POD 51. The animal lost the central line on POD 55, showed evidence of rejection on POD 57 and was euthanized on POD 60. Animals 2–4 showed bone healing and consolidation with the presence of bony callus on CT scans. At endpoint, animals 2 and 4 had reached complete weight bearing on the transplanted limb, at the hoof and wrist, respectively. CONCLUSION: Extremity transplantation can be successfully performed following EVNLP. EVNLP does not increase the risk of vascular or infectious complications. The mid-humerus porcine transplantation model is feasible and reproducible.

RAPID INTRAVENOUS DESENSITIZATION TO HORSE ANTITHYMOCYTE GLOBULIN IN A WOMAN WITH SEVERE APLASTIC ANEMIA

Introduction Antithymocyte globulin (ATG) and cyclosporine is a first line regimen for the treatment of severe aplastic anemia in patients without a matched sibling donor for transplant. Administration of ATG is associated with life threatening anaphylaxis in up to 5% of post-marketing cases. Accordingly, the manufacturer's insert recommends skin testing prior to first-time administration of ATG. We present a case of a 59 year-old woman with positive skin testing to horse ATG, who underwent successful desensitization to ATG for treatment of her severe aplastic anemia. Case Description A 59 year-old woman with severe aplastic anemia was admitted to our institution for induction therapy with ATG and cyclosporine. Pre-treatment skin testing to ATG was positive, with a 13 mm wheal after intradermal injection of 5 mcg of ATG. Epicutaneous skin testing was negative. She underwent successful rapid intravenous desensitization to ATG with no adverse reactions during the procedure. She completed a four-day cycle of ATG and was started on daily cyclosporine. She had good response to ATG treatment, and at six-month follow-up, she had continued clinical remission sustained with cyclosporine and eltrombopag. Discussion Our patient's positive intradermal skin testing was suggestive of a Type I hypersensitivity to ATG. Patients with positive skin testing should avoid ATG or undergo desensitization when medically necessary. Previous reports have described several cases of life threatening hypoxia and hypotension during ATG desensitization, but our patient successfully underwent a rapid intravenous desensitization and subsequently tolerated a four-day continuous infusion of ATG.

Effect of savory and yarrow on spermatogenesis in adult mice undergoing chemotherapy Cyclosporine

Spermatogenesis process during which the male germ cells are produced at each stage of the disorder, it can lead to infertility. The aim of this study was to evaluate the effect of fennel and yarrow on spermatogenesis in adult micereceiving chemotherapy is cyclosporine. For this study, 32 male Wistar rats with an average weight of 20 ± 200 g were randomly divided into four groups (control, cyclosporine A, cyclosporine and aqueous extracts of yarrow, cyclosporine and aqueous extract of fennel). Three groups of mice at a dose cyclosporine day / kg / mg40 dissolved in half ml sesame oil, received daily. The other two groups, water extracts of yarrow and fennel with a dose of day / kg / mg 150 received. After 21 and 45 days, all rats were euthanized, the testis and epididymis were separated in the next stage of sperm characteristics, morphological changes and morphological index were Trzya by spermatogenesis. Administration of cyclosporine A significant reduction in the number and motility of sperm, as well as a significant increase in the amount of dead and abnormal sperm has caused. If the plant extracts of yarrow and fennel is to improve these parameters. The results of this study show that cyclosporine A by oxidative stress can damage the testes provide, while the aqueous extract of yarrow and fennel with scavenging free radicals and oxidative processes can be reduced the adverse effects of this drug.

Human Endothelial Progenitor Cells as a Novel Therapeutic Option to Treat Severe Erectile Dysfunction in Obese Type 2 Diabetic Rats

Erectile dysfunction (ED) is a consequence of a number of medical conditions, with a high prevalence among men with type II diabetes. We investigated the effect of intracavernous injection of human umbilical cord blood derived endothelial progenitor cells (EPCs) on ED in a type II diabetic rat model. Erectile function was first assessed by cavernous nerve electrostimulation in Zucker Diabetic Fat (ZDF) rats aged 24 weeks to establish the baseline intracavernous pressure (ICP). At the age of 28 weeks, severe ED was confirmed. ZDF rats at age 29 weeks were randomised into 3 groups: treatment with 1million and 2million EPCs, and control, PBS. The injected animals were maintained with daily cyclosporine (10mg/kg) for 4 weeks, followed by the evaluation of erectile function and immunohistochemistry of penile tissue. The maximum (mean±SD) ICP observed for EPCs treated animals were significantly higher, 24.7±7.4 and 42.1±5.2 mmHg for 1million and 2million EPCs respectively in comparison to 4.4±1.6 mmHg in the age-matched PBS treated animals (p&lt;0.001). EPCs treatment also improved the smooth muscle content of the cavernosum (p&lt;0.001) and the expression of eNOS, RECA-1 and nNOS (p&lt;0.05) compared to the PBS control. In conclusion, we showed for the first time that human cord blood derived EPCs significantly improved severe ED in ZDF rats in a dose-dependent manner through improvement of the nerve and endothelium function and also through preservation of smooth muscle cells in the cavernosum.

Functional Repair of Rat Corticospinal Tract Lesions Does Not Require Permanent Survival of an Immunoincompatible Transplant.

Cell transplantation is one of the most promising strategies for repair of human spinal cord injuries. Animal studies from a number of laboratories have shown that transplantation of olfactory ensheathing cells cultured from biopsies of the olfactory bulb mediate axonal regeneration and remyelination and restore lost functions in spinal cord injuries. For translation from small laboratory experimental injuries to the large spinal cord injuries encountered in human patients the numbers of cells that can be obtained from a patient's own olfactory bulb becomes a serious limiting factor. Furthermore, removal of an olfactory bulb requires invasive surgery and risks unilateral anosmia. We here report that xenografted mouse bulbar olfactory ensheathing cells immunoprotected by daily cyclosporine restore directed forepaw reaching function in rats with chronic C1/2 unilateral corticospinal tract lesions. Once function had been established for 10 days, cyclosporine was withdrawn. Thirteen out of 13 rats continued to increase directed forepaw reaching. Immunohistochemistry shows that in all cases neurofilament-positive axons were present in the lesion, but that the grafted cells had been totally rejected. This implies that once grafted cells have acted as bridges for axon regeneration across the lesion site their continued presence is no longer necessary for maintaining the restored function. This raises the possibility that in the future a protocol of temporary immunoprotection might allow for the use of the larger available numbers of immunoincompatible allografted cells or cell lines, which would avoid the need for removing a patient's olfactory bulb.

In Vitro, In Vivo Comparison of Cyclosporin A Induced Hepatic Protein Expression Profiles

To reduce the amount of laboratory animals which are used to analyze hepatotoxic properties of chemicals and drugs, the development of alternative in vitro models is necessary. Ideally these in vitro models reflect the in vivo toxicological response and cholestasis. In this study the protein expression in livers from C57BL/6 mice after cyclosporin A-induced cholestasis was analyzed. After 25 days of a daily cyclosporine A treatment the cholestatic phenotype was established. An in vitro to this in vivo study comparison was made by using the results of our previous studies with HepG2 and primary mouse hepatocytes. The in vivo proteomics data show cyclosporin Ainduced oxidative stress and mitochondrial dysfunction was actually induced, leading to a decreased mitochondrial ATP production and an altered urea cycle. These processes were also altered by cyclosporin A in the in vitro models HepG2 and primary mouse hepatocytes. In addition, detoxification enzymes like methyl- and glutathione-Stransferases were differentially expressed after cyclosporin A treatment. Changes in these detoxification enzymes were mainly detected in vivo, though primary mouse hepatocytes show a differential expression of some of these enzymes. By means of a functional classification of differentially expressed proteins we demonstrated similarities and differences between in vitro and in vivo models in the proteome response of cyclosporin A-induced hepatotoxicity.

Evaluation of cyclosporine-sparing effects of polyunsaturated fatty acids in the treatment of canine atopic dermatitis

A randomised, double-blinded, placebo-controlled multicentre trial was conducted in 36 dogs with atopic dermatitis to evaluate the cyclosporine-sparing effect of polyunsaturated fatty acids. Dogs were stable on their individual cyclosporine dosage and received either a mainly omega-3 fatty acid product with a minor omega-6 fatty acid fraction or placebo, orally for 12 weeks. Dogs were examined every 4 weeks and the Canine Atopic Dermatitis Extent and Severity Index (CADESI-03) was determined by a clinician. Pruritus, quality of life, global condition and coat quality were scored by the owner. If the dog's CADESI-03 and/or pruritus score improved by at least 25% compared with the previous visit, the cyclosporine dosage was decreased by approximately 25%. If the scores deteriorated by at least 25%, the cyclosporine dosage was increased by the same percentage.The median daily cyclosporine dosage/kg bodyweight decreased in the active group from 4.1 mg to 2.6 mg and in the placebo group from 3.5 mg to 3.3 mg over the study period. The difference between the two groups was significant (P = 0.009). The improvement in median pruritus score from inclusion to completion was significantly greater in the active group than in the placebo group (P = 0.04). There was no significant difference in CADESI-03 changes between groups (P = 0.38). The results of this study indicate a cyclosporine-sparing effect of a mainly omega-3 fatty acid supplement in dogs with atopic dermatitis.

In vivo high-frequency contrast-enhanced ultrasonography of choroidal melanoma in rabbits: imaging features and histopathologic correlations

PurposeTo evaluate the use of in vivo imaging of rabbit model of choroidal melanoma using high-frequency contrast-enhanced ultrasound (HF-CE-US) with two-dimensional (2D) or three-dimensional (3D) modes and to correlate the...

A reproducible mouse model of chronic allograft nephropathy with vasculopathy

While short-term outcomes in kidney transplantation have improved dramatically, long-term survival remains a major challenge. A key component of long-term, chronic allograft injury in solid organ transplants is arteriosclerosis characterized by vascular neointimal hyperplasia and inflammation. Establishing a model of this disorder would provide a unique tool, not only to identify mechanisms of disease, but also test potential therapeutics for late graft injury. To this end, we utilized a mouse orthotopic renal transplant model in which C57BL/6J (H-2b) recipients were given either a kidney allograft from a completely mismatched Balb/cJ mouse (H-2d), or an isograft from a littermate. A unilateral nephrectomy was performed at the time of transplant followed by a contralateral nephrectomy on post-transplant day seven. Recipients were treated with daily cyclosporine subcutaneously for 14 days and then studied 8 and 12 weeks post transplantation. Renal function was significantly worse in allograft compared to isograft recipients. Moreover, the allografts had significantly more advanced tubulointerstitial fibrosis and profound vascular disease characterized by perivascular leukocytic infiltration and neointimal hyperplasia affecting the intrarenal blood vessels. Thus, we describe a feasible and reproducible murine model of intrarenal transplant arteriosclerosis useful to study allograft vasculopathy.

Histological Assessment of Cutaneous Acute Graft-Versus-Host Disease in a Preclinical Swine Model of Hematopoietic Cell Transplantation and Vascularized Skin Flap Tolerance

Abstract 1894Graft-versus-host disease (GVHD) remains a frequent complication of hematopoietic cell transplantation with skin being a principal target organ. Murine models have provided some insight into the mechanisms of this complex disease process. However, mouse skin differs from human skin, and results of studies in rodents may not translate well to the clinic. Miniature swine is a well-recognized animal model for preclinical studies of skin including dermal toxicology, transdermal drug delivery and wound healing. Unlike skin of rodents, dogs or non-human primates, porcine skin is similar to human skin in terms of structure of epidermal rete ridges (Figure 1A), hair follicle structure and density, and presence of sweat glands and subcutaneous fat.We have developed a miniature swine model of haploidentical hematopoietic cell transplantation that results in stable stem cell engraftment, hematopoietic chimerism and donor skin flap tolerance without GVHD. Swine leukocyte antigen (SLA)ad animals receive 100cGy total body irradiation and T cell depletion using porcine CD3 immunotoxin prior to cytokine mobilized peripheral blood hematopoietic cell transplantation from a haploidentical SLAac donor. Daily cyclosporine is administered at doses adjusted to maintain serum levels between 400–800 ng/ml starting one day prior to transplantation and continued for 30 days with taper until day 45.Mixed hematopoietic cell chimerism persists in these animals and donor vascularized skin flaps are accepted without the need for maintenance immunosuppression regardless of whether they are placed at the time of hematopoietic cell transplantation or greater than 100 days later. Despite the initial resistance to GVHD in this model, acute cutaneous GVHD could be induced through infusion of a high dose of donor leukocytes (DLI) including 15 million donor T cells/kg recipient body weight administered 463 days post hematopoietic cell transplantation and 314 days post vascularized donor skin flap transplantation. Conversion to full donor hematopoiesis was observed between 3–4 weeks post DLI. At this time a mild skin rash was evident in both donor and host skin which appeared to progress specifically within host skin. The presence of both donor and host skin on a tolerant animal whose peripheral blood consists of a mixture of both donor and host derived hematopoietic cells provides a novel model to evaluate specific targets of acute GVHD.In this study we assess histologically the manifestations of acute GVHD in both donor and host skin and compare it to typical histological manifestations of acute GVHD observed in patients. Specifically, recipient skin developed superficial lymphoid infiltrates that progressed to confluence along the dermal-epidermal junction by day 21. These infiltrates were associated with intraepidermal migration, prominent basal cell layer keratinocyte apoptosis, and satellitosis virtually indistinguishable from grade 3–4 human cutaneous acute GVHD (Figure 1B). Superficial portions of hair follicles were involved, as also seen in human disease. Of interest, mild disease was detected in the donor skin grafts by day 62 post-DLI, where selective lymphoid infiltration in the tips of the epidermal rete ridges and in association with target cell apoptosis/satellitosis was documented, a finding remarkably similar to early targeting events in humans.These findings emphasize the value of the porcine model for translational studies of cutaneous GVHD. Preliminary data suggest differences in cellular targeting between recipient and donor skin whereby the latter may be triggered by the presence of chimeric recipient-derived hematopoietic elements that have populated donor grafts. Further studies are now possible to explore the effector-target cell interactions in this clinically-relevant model of human cutaneous GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased Cyclosporine Concentrations in the Absence of Cyclosporine Administration

A 9-year-old girl was admitted to our hospital with juvenile metachromatic leukodystrophy (arylsulfatase A deficiency). Symptoms of this lysosomal storage disease, such as decreased school performance and compromised motor skills, had started 1 year earlier. She underwent bone marrow transplantation from a matched unrelated donor after receiving conditioning with fludarabine, treosulfan, and thiotepa, together with thymoglobulin, a rabbit antithymocyte globulin. Conditioning was well tolerated, and the posttransplantation period was uneventful except for 1 febrile episode. Graft-vs-host disease (GvHD)4 prophylaxis consisted of 3 methotrexate doses in combination with a starting daily cyclosporin A (CsA) dosage of 3 mg · kg−1 · day−1. Trough CsA concentrations were monitored with the antibody-conjugated magnetic immunoassay (ACMIA) for CsA (RxL Dimension; Siemens). CsA concentrations entered the therapeutic interval after 3 days, and the dosage was adjusted to achieve trough concentrations of 120–150 μg/L (Fig. 1). The patient received CsA for 16 weeks. During this period, the CsA concentration was measured 31 times, with results from 98 μg/L to 219 μg/L (mean, 156 μg/L). Four weeks after transplantation, the patient developed a mild GvHD of the skin, which disappeared immediately after commencement of prednisolone treatment. Because B lymphocytes were absent or low early after transplantation, the patient received immunoglobulins for 5 months (Fig. 1). Endogenous immunoglobulin production started only in the later phase of immune reconstitution. Six weeks after discontinuation of CsA therapy, a concentration of 147 μg/L was still detected in whole blood. Although the patient was not supposed to have received CsA and the parents had denied the administration of CsA or drugs other than those prescribed, CsA concentrations between 116 μg/L and 174 μg/L were obtained over the next 4 months. Fig. 1. Time courses of the whole-blood CsA concentration measured with the ACMIA CsA assay, the administered daily CsA dose, and the B-lymphocyte count. ### QUESTIONS TO CONSIDER 1. Has the CsA therapy really been discontinued? 2. Can delayed drug elimination explain the continued increased CsA concentrations? 3. What …

Risks of cyclosporine therapy in paediatric age

Cyclosporine has been found to be effective and safe in many inflammatory skin disorders such as psoriasis and atopic dermatitis (AD), in adults and in children. Its use in paediatrics is still under scope. We present three patients who started cyclosporine but stopped due to complications. It is our aim to warn about potential side effects of cyclosporine and recommend cautious utilization. Two children, aged 4 and 13 years, with AD and one child, aged 2 years, with erythrodermic psoriasis, were treated with oral cyclosporine. developed secondary impetigo on the 6th day of treatment. Started topical corticosteroids and topical calcineurin inhibitors afterwards, with no relapses. developed herpetic infection, hepatic and renal impairment (eventual drug interaction) on the 4th day of treatment. THIRD CASE: Psoriasis and impetigo, treated with flucloxacillin, gentamicin. Generalized angioedema and urticariform lesions after 6 days of cyclosporine. Beta lactam hypersensitivity reaction under study. Eventual cyclosporine toxicity to consider. The data on cyclosporine use in children is still scarce. Use should be limited to cases with precise indication, after considering risks and benefits.

Tolerance and Long-Lasting Peripheral Chimerism After Allogeneic Intestinal Transplantation in MGH Miniature Swine

Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)-inbred miniature swine. Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4/CD25(HIGH+)/Foxp3(+) cells. To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

T-helper 2 cells are essential for modulation of vascular repair by allogeneic endothelial cells

Endothelial cells (ECs) embedded within 3-dimensional matrices (MEEC) control lumenal inflammation and intimal hyperplasia when placed in the vascular adventitia. Matrix embedding alters endothelial immunogenicity in vitro. T-helper (Th) cell-driven host immunity is an impediment of allogeneic grafts. We aimed to identify if modulation of Th balance would affect immune compatibility and endothelial regulation of vascular repair in vivo. Pigs (n = 4/group) underwent carotid artery balloon injury and were left untreated (Group 1) or received perivascular porcine MEEC implants (Group 2), 12 days of cyclosporine A (CsA; Group 3), or MEEC and CsA (Group 4). Host immune reactivity was analyzed after 28 and 90 days. MEEC treatment induced formation of EC-specific immunoglobulin (Ig) G(1) antibodies (41 +/- 6 mean fluorescence intensity [MFI]) and differentiation of host splenocytes into Th2, but not Th1, cytokine-producing cells (interleukin [IL]-4, 242 +/- 102; IL-10, 273 +/- 114 number of spots). Concomitant CsA therapy reduced IgG(1) antibody frequency (25 +/- 2 MFI; p < 0.02) and Th2-cytokine producing splenocytes upon MEEC treatment (IL-4, 157 +/- 19; IL-10, 124 +/- 26 number of spots; p < 0.05). MEECs inhibited luminal occlusion 28 and 90 days after balloon injury (12 +/- 7%) vs untreated controls (68 +/- 14%; p < 0.001) but to a lesser extent with concomitant CsA treatment (34 +/- 13%; p < 0.02 vs Group 2). MEECs do not induce a significant Th1-driven immune response but do enhance differentiation of splenocytes into cells producing Th2 cytokine. Reduction in this Th2 response reduces the vasoregulatory effects of allogeneic ECs after injury.