Search

Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would be mediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P). Male ZDF rats were randomized to RYGB (n = 7) or sham surgery (n = 7) and harvested after 28 days. Total tissue ceramide, ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), and S1P were quantified in the white gastrocnemius muscle using LC-ESI-MS/MS after separation with HPLC. Total SIRT1, AMPK, PGC-1α, and CS protein expression were measured by Western blot. Body weight, fasting glucose, insulin, and HOMA-IR decreased significantly after RYGB compared with sham control. These changes were paralleled by lower total ceramide (483.7 ± 32.3 vs. 280.1 ± 38.8 nmol/g wwt), C18:0 ceramide subspecies (P

Diabetes and obesity are associated with inflammasome-mediated low-grade, chronic inflammation that may induce pancreatic beta-cell dysfunction and apoptosis. We examined the effects of Roux-en-Y gastric bypass (RYGB) surgery on NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome-related genes from pancreatic islets of Zucker diabetic fatty rats. Islets were collected from Zucker diabetic fatty sham control and RYGB, 30 days after surgery. We assessed expression of genes that regulate glucose metabolism and the NLRP3 inflammasome (NLRP3, caspase-1, IL-1β, IL-18, apoptosis-associated speck-like protein), IL-6, and monocyte chemoattractant protein-1. Gene expression for NLRP3 (p

AIM High-fat diets are known to contribute to the development of obesity and related co-morbidities including non-alcoholic fatty liver disease (NAFLD). The accumulation of hepatic lipid may increase endoplasmic reticulum (ER) stress and contribute to non-alcoholic steatohepatitis and metabolic disease. We hypothesized that bariatric surgery would counter the effects of a high-fat diet (HFD) on obesity-associated NAFLD. METHODS Sixteen of 24 male Sprague Dawley rats were randomized to Sham (N = 8) or Roux-en-Y gastric bypass (RYGB) surgery (N = 8) and compared to Lean controls (N = 8). Obese rats were maintained on a HFD throughout the study. Insulin resistance (HOMA-IR), and hepatic steatosis, triglyceride accumulation, ER stress and apoptosis were assessed at 90 days post-surgery. RESULTS Despite eating a HFD for 90 days post-surgery, the RYGB group lost weight (-20.7 ± 6%, P

Objectives/Hypothesis: The increased risk of malignancy associated with post-transplant immunosuppression limits the potential of laryngeal transplantation as a reconstructive option. This risk may be mitigated by utilizing decoy nuclear factor kappa B (NF-κB) immature dendritic cells (iDC) to provide donor-specific tolerance. The purpose of this study was to explore whether tolerogenic properties of iDC can be applied to composite tissue transplantation. Study Design: Animal study. Methods: Five iDC-injected mice were euthanized at 15, 30, and 60 days post-laryngeal transplant. Control groups included five transplanted mice without immunosuppression, one iDC-injected mouse euthanized prior to transplantation, one mouse without injection or transplantation, and one mouse administered mature DC to serve as an accelerated rejection control. Larynges were graded for rejection severity according to a grading scale. Draining lymph nodes and spleens were evaluated by flow cytometry to determine immunogenic activity of iDC and T cells locally and peripherally. Results: Each time group demonstrated moderate allograft rejection (rejection severity scores: 4.38, 5.10, 5.29). NF-κB iDC-treated mice had significantly less rejection at all time points compared to nonimmunosuppressed mice. Flow cytometry showed inhibition of cytotoxic T cell infiltration and expansion of regulatory T cells at the allograft site. Conclusions: iDC immunosuppression maintains laryngeal allograft integrity up to 60 days post-transplantation. Regulatory T cells are enhanced at the allograft site, which serves to suppress immune cell activation and induce self-antigen tolerance. iDC injection may lessen post-transplant comorbidities by decreasing the systemic immune response and favorably affecting concurrent immunosuppressive dose sequencing for laryngeal allograft preservation. Laryngoscope, 2010

Objectives Laryngeal transplantation research has been studied in various animal models. For in-depth, immunology-based transplantation research, however, a thoroughly studied animal model must exist. The purpose of this study was to develop a reliable surgical technique in mice to serve as a model for further study of laryngeal transplantation. Methods Heterotopic laryngeal transplantation was attempted in 15 immunocompetent mice by use of modifications of previously described techniques established in rats. Results Various microvascular techniques were used that led to 8 successful transplants (of 15) with patent vascularity at the time of sacrifice. The first 7 attempts at transplantation were unsuccessful because of technical difficulties related to vessel size, soft tissue traumatic injury, and venous congestion. Subsequently, 8 transplantation procedures were successfully performed after modifications of the surgical technique. Conclusions This pilot study describes the reproducible surgical techniques performed in using mice for studying laryngeal transplantation. Mice are cost-effective and immunologically well studied, and are thus ideal for further laryngeal transplantation research.

Objective: Traditional immunosuppressive regimens make laryngeal transplantation in cancer patients prohibitive because of the increased risk of recurrence. Everolimus, a recently developed immunosuppressant, has demonstrated significant antitumor properties. The purpose of this study was to examine the effects of everolimus alone and in combination with other immunosuppressants on tumor growth in a combined laryngeal transplantation and tumor model. Study Design: Animal, prospective, randomized, controlled, and blinded. Methods: One million squamous cell carcinoma cells (SCC-158) were injected intravenously into a total of 40 rats 1 day before laryngeal transplantation. Rats were divided into four groups differing by immunosuppressive regimens. Lung surface metastases were counted 21 days after inoculation, and numerical transplantation rejection scores were recorded. A separate experiment for comparison was performed with no transplant on 24 rats, but with the same immunosuppressive treatment groups. Results: The median number of lung surface metastases were: a) control (i.e., no immunosuppression): 85; b) everolimus 1.0 mg/kg: 25; c) tacrolimus 1.2 mg/kg: 1650; d) everolimus 1.0 mg/kg + tacrolimus 0.05 mg/kg: 1300. Rats receiving everolimus alone showed a statistically significant decrease in pulmonary surface metastases compared with the other groups. Transplanted rats had no difference in their outcomes when compared with nontransplanted rats. Conclusion: Everolimus significantly decreases SCC-158 growth in our combined transplantation and tumor model compared with controls and other immunosuppressants.

Introduction The concept of donor bone marrow transplantation has been successfully used in human solid organ transplantation to increase recipient chimerism. The development of recipient chimerism is associated with a decreased need for immunosuppression and even donor-specific tolerance. In this study, we attempted to augment recipient chimerism by the transfer of donor bone marrow at the time of rat laryngeal transplant. Study design Experimental study in rats. Methods The study used a well-established semiallogeneic rat laryngeal transplant model with partial major histocompatibility complex (MHC)-mismatched Lewis-Brown-Norway donors and Lewis recipients. Donor bone marrow was introduced at transplantation via (1) intravascular injection and (2) transfer of a vascularized femoral bone graft. Recipients were treated with an established immunosuppressive regimen consisting of everolimus and anti- αβ TCR monoclonal antibody for a 7-day perioperative course. Animals received a 5-day boost of the same regimen at 90 days posttransplantation. Parathyroid hormone levels and histological examination were used for rejection surveillance and scoring. Results Animals treated with intravenous bone marrow injection followed by perioperative and pulsed immunosuppression commonly demonstrated early rejection (90%). Animals receiving transfer of vascularized donor femur had an average rejection score of 2.9 (scale, 1-6) at 180 days posttransplantation. Mixed-lymphocyte reaction did not demonstrate donor-specific tolerance in the latter group, and chimerism was less than 1%. Conclusions In the rat laryngeal transplant model, donor bone marrow does not consistently lead to augmentation of peripheral chimerism using our established pulsed immunosuppression protocol. In many cases, rejection occurred earlier than animals not receiving bone marrow. This may be due to several different factors including (1) an element of graft-vs-host disease, (2) inability to establish bone marrow engraftment due to our short-term perioperative immunosuppression regimen, or (3) preferential rejection of donor bone marrow cells.

Objectives/Hypothesis: Dendritic cells (DC) are potent antigen-presenting cells that instigate allograft rejection. Their migration kinetics vary depending on the type of organ transplanted. The timing of donor and recipient DC trafficking in laryngeal transplants is unknown. Study Design: Prospective animal model. Methods: Lewis to Brown Norway (BN) rat laryngeal allografts and BN to BN isografts were performed without immunosuppression. Recipient animals were sacrificed at seven posttransplant time points. Total DC, as well as recipient and donor DC (in allograft recipients), were enumerated in situ in the airway epithelium and subepithelium using monoclonal antibodies, immunofluorescence, confocal microscopy, and image analysis software. Results: Total DC densities in both laryngeal allografts and isografts decreased to approximately 10% of their initial values in the first 3 days and then rose beyond their starting values. In allografts, there was a net efflux of donor DC, reaching a nadir by 3 to 5 days; they were identified in recipient cervical lymph nodes from 12 hours to 5 days. Recipient DC infiltrated the laryngeal allograft, reaching a maximal density by day 7. Conclusion: The paradigm of donor DC efflux and recipient DC influx has been confirmed in a rat laryngeal transplant model, and the allograft-specific timing of these events has been elucidated. Similarities in total DC migration between allografts and isografts suggest that this phenomenon may not be driven entirely by major histocompatibility mismatch. Further understanding of trafficking may help with the goal of manipulating DC to induce allograft tolerance in the absence of generalized immunosuppression.

Objectives: Dendritic cells (DCs) are key instigators of rejection after transplantation. Their distribution has not been systematically characterized in all locations of the larynx and its surrounding tissues. Methods: Rat larynges were stained with monoclonal antibodies identifying DCs. These cells were then enumerated by a new combination of techniques including immunofluorescence, confocal microscopy, and imaging software. Results: The vast majority of DCs were located in the epithelium and subepithelium of the airway; the mean DC density ranged from 9 cells per square millimeter (0.2% of cells) to 645 cells per square millimeter (10.3% of cells). Their density in the epithelium was 3 to 11 times higher than that in the subepithelium. Non-airway sites (thyroid, parathyroid, muscle, fat) had mean DC densities varying from 3 cells per square millimeter (0.2%) to 57 cells per square millimeter (0.8%). No DCs were detected in cartilage. Conclusions: Dendritic cells are concentrated in the laryngotracheal epithelium and subepithelium and represent a much smaller proportion in the other sites studied. A baseline for laryngeal DC population studies has been established, and a computerized model for consistent quantitation using confocal microscopy has been developed. This unique method will serve as a foundation for investigating DC trafficking after rat laryngeal transplantation.

Objectives: Laryngeal transplantation can restore the voice in patients who have undergone laryngectomy. However, the prospect of lifelong immunosuppression is a drawback to this procedure. We present data from a study aimed at minimizing the need for immunosuppression while maintaining graft viability through a novel pulsed-dosing protocol. Methods: Larynges were transplanted from Lewis–brown Norway (RT11+n, F1) rats to Lewis (RT11) recipients. All recipients received 7 days of treatment with everolimus and mouse anti-rat αβ T-cell receptor (anti-TCR) monoclonal antibodies beginning the day before transplantation. At 90 days after transplantation, all recipients received a pulse of the same treatment combination for 5 days. From 90 to 180 days after transplantation, the rats received no treatment (group 1, n = 5), 2.5 mg/kg everolimus per day (group 2, n = 5), or 1.0 mg/kg everolimus per day (group 3, n = 5). Results: Histologic analysis of rats that received everolimus as pulse therapy evidenced no signs of rejection, whereas animals that were untreated after 90 days had normal to mild chronic rejection. T-cell reconstitution occurred 65 days after perioperative immunosuppressive treatment, but less rapidly after pulse therapy. Also, peripheral chimerism was generated in all 3 groups. Conclusions: In the rat laryngeal transplantation model, short-term perioperative therapy with everolimus and anti-TCR followed by pulsing is a viable alternative to the concerns associated with continuous, lifelong immunosuppression.

BACKGROUND: The major limitation preventing expansion of laryngeal transplantation as a therapeutic modality is the necessity of lifelong immunosuppression. In this report, we describe an immunomodulatory strategy for tolerance induction in laryngeal allotransplantation that permits escape from chronic immunosuppression. MATERIALS AND METHODS: Larynges were transplanted from Lewis-Brown-Norway (RT1l/n, F1) donors to Lewis (RT1l) recipients. Recipients received 7 days of treatment with tacrolimus and mouse anti-rat alphabeta T-cell-receptor (TCR) monoclonal antibodies. Histology, mixed lymphocyte reaction (MLR), skin grafting, and flow cytometry assessed functional tolerance, efficacy of immunodepletion, and donor-specific chimerism. RESULTS: All 10 recipients survived until sacrifice at 100 days. Histology suggested functional allograft tolerance. Skin grafting, MLR, and flow cytometry revealed that tolerance is neither donor-specific nor related to systemic immunocompromise. CONCLUSIONS: In this rat laryngeal-transplantation model, functional tolerance was induced under combined tacrolimus and alphabeta TCR protocol. Mechanisms responsible for this tolerance induction require future elucidation.

Since the first successful human laryngeal transplantation in 1998, research continues toward developing less-morbid immunosuppressive protocols. Although irradiation of donor organs is known to decrease acute rejection, the most advantageous method of radiation delivery is still unknown. Using a rat laryngeal transplant model, we sought to determine the most beneficial timing and delivery method of irradiation. A prospective study was undertaken including 16 treatment arms of 10 to 30 animals each (189 transplantations). The animals received 800 cGy before transplantation to the donor larynx in vivo, the donor larynx in vitro, or the recipient animal's neck. The transplantation occurred at 24 hours, 5 days, or 10 days after irradiation. The transplanted larynges were harvested 15 days after transplantation and histologically scored for rejection. Irradiation of allogeneic transplantations demonstrated a strongly protective effect from rejection as compared to no irradiation (p < .001), regardless of the method of radiation delivery or the amount of time between irradiation and transplantation (p = .78). Irradiation of donor larynges between allogeneic rats has a protective effect, reducing the degree of acute rejection when irradiation is used as the single mode of immunosuppression, and is unrelated to either the timing or the mode of delivery of the irradiation.

Objective Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control. Methods Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase–1, apoptosis-associated speck-like protein (ASC), IL–1β, IL–18, IL–6 and MCP–1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT). Results Compared to Sham surgery controls, RYGB surgery decreased IL–6, MCP–1, NLRP3, IL–18, caspase–1 and ASC in omental fat, and decreased IL–6, MCP1, IL–1β, IL–18, caspase–1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL–6 and IL–1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL–18, caspase–1 and IL–1β protein expression in omental tissue. We found a positive correlation between caspase–1, ASC, MCP–1, IL–18 and IL–6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase–1 gene expression in subcutaneous fat. Conclusion This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.

The rat laryngeal transplant model, introduced in 1992, laid the basic science foundation that contributed to the first successful human larynx transplant in 1998. Over 1,500 rat transplants later, numerous modifications have improved the model, increasing the initially reported evaluability rate of 50% to almost 100%. The observed histologic rejection process has been altered, as well. We report the technique modifications, as well as the results of a new study using nonimmunosuppressed, allogenic transplantations, in order to define a new rejection grading system. Using the updated model, we performed 50 transplantations between LBN(fl) donor rats and Lewis recipients. Larynges from 8 groups of 5 to 10 animals were harvested at intervals between 1 and 20 days after transplantation. The larynges were examined grossly and microscopically in a blinded manner for evidence of rejection. A multivariable linear regression model was used to define a new rejection grading scale. All 50 animals survived their assigned posttransplantation period. No animals exhibited vascular thromboses, for an evaluability rate of 100%. Histologic criteria in 7 categories and gross criteria in 5 categories demonstrated increased rejection proportional to the amount of time after transplantation. The equation [Group = −2.209 + 0.465 × (Size) + 0.901 × (VAFlow) + 0.613 × (Muscle) + 1.040 × (Thyroid)] reproducibly grades rejection on the basis of gross and histologic findings. New modifications to the rat laryngeal transplant model have conferred greatly improved animal survival rates and anastomotic patency rates. Additionally, the observed rates of rejection have been reduced in comparison to those of initial studies. This updated rejection staging system will be used to compare immunosuppressive regimens in future rat laryngeal transplant studies.

Adding oral prednisone or in vitro organ irradiation to an immunosuppressive regimen for laryngeal transplantation may allow cyclosporine dosage to be decreased without compromising organ rejection rates. Using an established rat laryngeal transplant model, we sought to determine whether combined immunosuppressive regimens decreased rejection rates. Twenty-two treatment groups of 10 animals each were studied. Cyclosporine was administered at 2 different historically low dosages (1.5 and 2 mg/kg per day) alone, in combination with prednisone, and with in vitro irradiation of the harvested larynx. At 15 days after transplantation, cyclosporine combined with prednisone did not significantly alter the mean severity of rejection. At 30 days after transplantation, rats treated with a combination of cyclosporine 2 mg/kg and prednisone 1 mg/kg showed the least amount of rejection. Rejection rates were significantly higher with 2 mg/kg cyclosporine alone (p < .00024). Irradiation did not significantly decrease the severity of rejection. These results demonstrate that at 1 month after transplantation, a combined steroid and cyclosporine regimen reduces the severity of rejection after laryngeal transplantation in an animal model. Our findings suggest that laryngeal transplant patients may be treated with low cyclosporine doses without experiencing organ rejection if steroids are added to the immunosuppressive regimen.

Background Roux-en- Y gastric bypass (RYGB) surgery achieves high remission rates of type 2 diabetes mellitus in obese diabetic patients. It has been hypothesized that the changes in bowel nutrient exposure after RYGB results in altered release of gut hormones and improved glucose homeostasis. Our objective was to assess the feasibility of, and report on, our technique and initial experience with selective gut stimulation in a gastric bypass rat model at an academic medical center in the United States. Methods We performed RYGB with simultaneous placement of a gastrostomy tube in the excluded gastric remnant in 8 obese Sprague-Dawley rats. A second group of 8 obese Sprague-Dawley rats underwent gastrostomy tube placement without gastric bypass and served as the controls. Each rat was tested for oral glucose tolerance preoperatively. On postoperative days 14 and 28, glucose tolerance was re-evaluated using the oral and gastrostomy tube routes. Results The gastrostomy tubes were successfully inserted in all the rats with no tube-related complications. The area under the curve after oral glucose gavage decreased significantly after gastric bypass (P = .01 at 14 d and P = .003 at 28 d). The gastric remnant glucose gavage after RYGB essentially reversed the effects of surgery on glucose metabolism. The areas under the curve showed no significant differences in the control group between the preoperative and postoperative oral or tube results. Conclusion Placing a gastrostomy tube into the gastric remnant at RYGB in a rat model is technically feasible. Our initial findings support the role of duodenal exclusion in improving glucose metabolism after RYGB.

Objectives: The risks of daily immunosuppression limit the use of laryngeal transplantation as a reconstructive option. Pulsed immunosuppressive dosing can lessen these risks. The study objective was to develop a long-term pulsing regimen that minimizes exposure to immunosuppressive agents. Methods: Rat laryngeal transplantation was performed. Everolimus (1 mg/kg per day) and anti–αβ T-cell receptor (TCR) antibodies (250 μg) were given for 7 days beginning 1 day before transplantation and for 5 days beginning on day 90 after transplantation. On day 180, group 1 (n = 5) received the initial regimen for 3 days, and group 2 (n = 5) received everolimus (1 mg/kg per day) until euthanization, which occurred when parathyroid hormone (PTH) levels dropped to less than 11 pg/mL or at 300 days. Results: Four of the 5 rats in group 1 had normal PTH levels at 300 days. The PTH level for 1 rat was less than 11 pg/mL at 270 days. In group 2, none of the 5 rats had normal PTH levels at 300 days. Two had PTH levels below 11 pg/mL at 270 days, and 3 had PTH levels below 11 pg/mL at 300 days. The allografts that survived beyond 300 days had an essentially normal histologic appearance. Conclusions: Pulsed immunosuppression prevented allograft rejection for 10 months and was more effective than daily everolimus. Short-term perioperative therapy followed by pulsed, tapered dosing is a viable alternative to traditional regimens and may decrease associated risks.

The increased risk of malignancy associated with post-transplant immunosuppression limits the potential of laryngeal transplantation as a reconstructive option. This risk may be mitigated by utilizing decoy nuclear factor kappa B (NF-kappaB) immature dendritic cells (iDC) to provide donor-specific tolerance. The purpose of this study was to explore whether tolerogenic properties of iDC can be applied to composite tissue transplantation.Animal study.Five iDC-injected mice were euthanized at 15, 30, and 60 days post-laryngeal transplant. Control groups included five transplanted mice without immunosuppression, one iDC-injected mouse euthanized prior to transplantation, one mouse without injection or transplantation, and one mouse administered mature DC to serve as an accelerated rejection control. Larynges were graded for rejection severity according to a grading scale. Draining lymph nodes and spleens were evaluated by flow cytometry to determine immunogenic activity of iDC and T cells locally and peripherally.Each time group demonstrated moderate allograft rejection (rejection severity scores: 4.38, 5.10, 5.29). NF-kappaB iDC-treated mice had significantly less rejection at all time points compared to nonimmunosuppressed mice. Flow cytometry showed inhibition of cytotoxic T cell infiltration and expansion of regulatory T cells at the allograft site.iDC immunosuppression maintains laryngeal allograft integrity up to 60 days post-transplantation. Regulatory T cells are enhanced at the allograft site, which serves to suppress immune cell activation and induce self-antigen tolerance. iDC injection may lessen post-transplant comorbidities by decreasing the systemic immune response and favorably affecting concurrent immunosuppressive dose sequencing for laryngeal allograft preservation.

Development of a rat laryngeal transplantation model allowed for the first total human laryngeal transplantation by the senior author in 1998. In an effort to further our knowledge of the immune system's role in laryngeal rejection, a change to the mouse model was required. Prior to initiating immunosuppressive research protocols, a reliable mouse larynx rejection classification had to be established.Animal study.Thirty-one mouse laryngeal transplants (C57 BL/6 donors to C3H recipients) were performed and allowed to reject. Six time points were evaluated histologically: 1, 3, 5, 7, 9, and 15 days post-transplant. Eight anatomic sites were evaluated and assigned a point value. A linear regression model was constructed using the group number as the response and the scores from the eight histological criteria as predictors. Severity classifications were determined by observing patterns in the sum of scores of variables found to be significant contributors. Group 1 was normal; group 2, minimal rejection; groups 3, 4, and 5, moderate rejection; and group 6, severe rejection.All mice survived the transplants. Of the observed histological changes, cartilage, fat, muscle, and magnitude of lymphocytic infiltration significantly correlated with rejection severity. The rejection model created demonstrated 100% accuracy in predicting the severity classification for the 31 specimens in the study.The model established provides an accurate and reliable way to classify rejection severity in mice receiving laryngeal allografts. This sets the stage for future advanced study of manipulating the immune system as a mechanism for establishing allograft tolerance.

Objective The purpose of this study was to explore the mechanism and utility of everolimus as a single-agent therapy in preventing mouse laryngeal allograft rejection. Study Design Prospective animal study. Setting Academic research at a tertiary medical center. Subjects and Methods Fifteen recipient mice (five per group) were injected with everolimus (1 mg/kg/d) until euthanized at 15, 30, and 60 days posttransplantation. Five mice received transplants without immunosuppression and were euthanized at day 15. Larynges were graded for rejection severity. Draining lymph nodes and spleens were evaluated by flow cytometry to assess the systemic immunological environment. Results Each time group demonstrated minor allograft rejection (rejection severity scores: 2.51, 2.46, 2.78; no rejection, 1; severe, 6). This was not significantly different between groups. Everolimus-treated mice had significantly less rejection at all time points compared with non-immunosuppressed mice. Flow cytometry showed a blunted cytotoxic T-cell response, differentiation favoring regulatory T-cells, and decreased number and function of dendritic cells. Conclusions Everolimus successfully prevents laryngeal allograft rejection up to 60 days posttransplantation. It appears to increase the production of regulatory T-cells while decreasing cytotoxic T-cell and dendritic cell response. Everolimus alone or in combination with other immunosuppressants may enable laryngeal transplantation to become a viable reconstructive option following laryngectomy for malignancy.

Purpose The use of induction therapy with αβ T-cell receptor ( αβ TCR) monoclonal antibody in association with tacrolimus in an allogeneic rat laryngeal transplant model permits rigorous long-term evaluation of potential short-term synergism offered by these agents. Materials and methods The allogeneic model consisted of 19 Brown Norway larynges transplanted to Lewis recipients. Treatment consisted of tacrolimus (1.2 mg/kg per day) alone and in combination with 7 days of αβ TCR (0.5 mL/d). Control groups consisted of untreated animals, as well as a semiallogeneic model with Lewis-Brown Norway larynges transplanted to Lewis recipients and treated with tacrolimus monotherapy. Each group consisted of 6 to 11 rats. The median duration of engraftment was 44 days (range, 14–106 days). Graft histopathology was graded according to an established 31-point scale in a blinded fashion. Long-term grafts (>75 days) were followed with serum parathyroid hormone levels. Results Untreated controls experienced almost complete rejection (mean score, 27; SD, 0). Allogeneic transplants treated with tacrolimus monotherapy experienced near-complete rejection (mean score, 25.2; SD, 2.1). Allogeneic transplants treated with combination therapy and followed for a median duration of 100 days demonstrated significantly better rejection scores than controls (mean score, 11.1; SD, 1.7; P = .003). Combination therapy was also significantly more effective in preventing acute rejection than monotherapy with tacrolimus in a semiallogeneic model (mean score, 22.1; SD, 4.6; P Conclusions Induction therapy with tacrolimus and αβ TCR prevents rejection in an allogeneic model for up to 100 days. This regimen was associated with significantly better histopathologic rejection scores than untreated controls, or monotherapy with tacrolimus in allogeneic or semiallogeneic models.

Lifelong immunosuppression carries significant morbidity, and techniques to reduce or eliminate such immunosuppression might expand laryngeal transplantation. This study investigates the ability of donor-specific transfusion to establish tolerance in a rat model of laryngeal transplantation. A total of 289 transplants were performed from Lewis-Brown-Norway donors to Lewis recipients. Donor-specific transfusion was provided as single intravenous injections of donor splenocytes 1 hour before transplantation. Different combinations of in vitro irradiation of donor larynges and postoperative cyclosporine doses provided additional immunomodulation. Allograft rejection was scored histologically at sacrifice 15 or 30 days posttransplant. Multivariate analysis was performed across all groups, and paired analyses compared groups with and without donor-specific transfusion. Donor-specific transfusion did not improve rejection score, although increased cyclosporine dose did (P < .001). Donor splenocyte injection on the day of transplantation does not establish tolerance to laryngeal allografts or permit reduction of other immunosuppression. Other immunomodulatory strategies to establish tolerance in rat laryngeal transplantation require further investigation.

Parathyroid gland allotransplantation has been a challenging goal for decades. Our objective was to optimize a parathyroid allotransplantation model for analysis of short-term or low-dose immunosuppressive regimens. Rats that had undergone parathyroidectomy received parathyroid allografts either by direct microvascular anastomoses as part of a composite laryngotracheal graft or by direct implantation into hind limb muscle. All rats were maintained on daily low-dose FK506 (tacrolimus). Intact serum parathyroid hormone (PTH) levels for both groups were recorded over a period of at least 45 days and compared with a repeated-measures mixed model. We found that microvascular anastomosis was superior to implantation for parathyroid gland survival, as all revascularized grafts immediately produced normal levels of PTH, whereas implanted grafts had a significantly slower recovery of function (p < .001). Four of the 11 implanted grafts (36%) never produced detectable PTH levels. Using this model, we are developing innovative strategies that will lead to successful parathyroid allotransplantation without the need for chronic immunosuppression.

Problem: Transplant research continues to seek immunosuppressive regimens that minimize recipient morbidity and do not promote tumor growth. Several animal studies have demonstrated long-term donor-specific allograft tolerance after posttransplant total lymphoid irradiation (TLI) when used in conjunction with anti-lymphocyte globulin (ALG) and donor-specific transfusion (DST). The goals of the current study were as follows: (1) to assess the ability of posttransplant TLI to induce allograft tolerance in an established murine laryngeal transplant model, (2) to compare postoperative TLI regimens with varying doses of radiation and anti-lymphocyte globulin, and (3) to evaluate the ability of sirolimus to augment tolerance when used in combination with a posttransplant TLI regimen. Methods: Using 8 treatment arms containing 10 animals each, recipient rats received 1 of 4 postoperative regimens: (1) 1,000 rads of TLI in 5 fractions, ALG and DST, (2) 2,400 rads of TLI in 10 fractions, ALG and DST, (3) 2,400 rads of TLI in 10 fractions, ALG, DST, and intraperitoneal sirolimus, and (4) ALG only. Recipient groups were assessed at 15 or 30 days after the completion of the assigned treatment regimen. Laryngeal allografts were graded for histologic rejection level using an established scale. Results: Both 1,000 and 2,400 rads of TLI were well tolerated by recipient animals. Laryngeal allograft preservation was significantly better using 2,400 rads of TLI when compared to 1,000-rad regimens. The addition of sirolimus did not augment graft preservation. All 2,400-rad TLI regimens were superior to ALG alone, with only mild to moderate rejection observed on average at 30 days after treatment. No treatment regimen yielded consistent allograft preservation on histologic analysis. Conclusion: Posttransplant total lymphoid irradiation regimens significantly reduce the severity of allograft rejection at 15 and 30 days. However, consistent histologic evidence of allograft tolerance was not observed. Significance: At this point, TLI has limited applicability to human laryngeal transplantation. Support: This study was funded by the Cleveland Clinic Department of Otolaryngology and Communicative Disorders and a matching Cleveland Clinic Research Programs Council grant.

Problem: A mouse anti-rat T-cell receptor monoclonal antibody (TCR) has great potential in the development of long-term immunologic tolerance. Combination therapy with FK-506 and TCR may enable the establishment of long-term immunotolerance while reducing dose-related toxicities. The use of combined TCR and FK-506 in a completely allogeneic model permits a rigorous evaluation of potential synergism. Methods: Nineteen Brown Norway larynges were transplanted to Lewis recipients. Treatment consisted of 7 days of FK-506 (1.2 mg/kg/day) alone and in combination with TCR (0.5 mL/day). Control groups consisted of untreated animals, as well as 11 Lewis-Brown Norway larynges transplanted to Lewis recipients and treated with FK-506 monotherapy. Each group consisted of 6–11 rats. Median duration of engraftment was 44 days (range, 14–106 days). Graft histopathology was graded according to an established 31-point scale in a blinded fashion. Long-term grafts (>75 days) were followed with serum PTH levels. Results: Untreated controls experienced almost complete rejection (mean score, 27, SD 0). Allogeneic transplants treated with FK-506 monotherapy experienced near-complete rejection (mean score, 25.2, SD 2.1). Allogeneic transplants treated with combination therapy and followed for a median duration of 100 days demonstrated significantly better rejection scores than controls (mean score, 11.1, SD 1.7; P = 0.003). Combination therapy was also significantly more effective in preventing acute rejection than monotherapy with FK-506 in a semi-allogeneic model (mean score, 22.1, SD 4.6; P < 0.04). Conclusion: Short-term combination therapy with FK-506 and TCR prevents rejection in an allogeneic model for up to 100 days. This regimen was associated with significantly better histopathologic rejection scores than monotherapy with either agent, or monotherapy with FK-506 in a semi-allogeneic model. Significance: The blockade of primary antigen recognition with TCR may prevent the need for daily immunosuppressive therapy, thereby limiting treatment-associated toxicities. Pulsing protocols may also provide a mechanism for intermittent immunomodulation with this regimen. Support: None reported.

Objectives/Hypothesis Short-term immunosuppressive protocols in a preclinical rat model that have led to prolonged survival of the graft in preliminary trials were investigated. Rat allografts, by their heterotopic nature, do not allow direct examination or functional interpretation. The primary objective of the study was to identify a reliable functional biological marker allowing monitoring of graft status. A secondary goal was to use this marker to indicate onset of rejection. Study Design The authors elected to study rat parathyroid hormone as a functional marker of laryngotracheal complex rejection. Theoretically, a pulsed immunosuppressive regimen that is administered when parathyroid hormone levels begin to fall could prevent rejection. Methods Eleven Lewis recipient rats underwent parathyroidectomy one week before laryngeal transplantation. Parathyroid hormone levels were obtained on various days following transplantation, then analyzed with an intact rat parathyroid hormone assay. Animals were killed on different days to allow for comparison of parathyroid hormone levels with histopathological signs of rejection. Results All animals had undetectable levels of parathyroid hormone before transplantation. By 24 hours after transplantation, parathyroid hormone levels were within normal range. At 72 hours, the parathyroid hormone levels dropped precipitously. Rejection of parathyroid glands paralleled the early histological changes of rejection seen in the other anatomical areas of interest in the laryngotracheal graft. Conclusion The study represents the first time a hematological marker has been identified that has predictive value for rejection of transplanted rat larynges. As such, it not only enables a 3-month experimental window with reasonable numbers of animals, but also opens the door for novel sequencing of immunosuppressive modalities.

Objectives/Hypothesis Tacrolimus is efficacious in several transplantation settings. Some studies have demonstrated improved results using combination therapy with mycophenolate mofetil. Our primary objective was to evaluate the efficacy and optimal dosing of tacrolimus in preventing rejection, using an established rat model of laryngeal transplantation. Further, the ability of mycophenolate to allow lower dosing of tacrolimus while achieving equivalent immunosuppression was investigated. Study Design A dosage efficacy study with 10 experimental arms was conducted. Methods Dosage groups were 0.1, 0.2, 0.3, and 0.6 mg/kg tacrolimus alone and 0.1 mg/kg tacrolimus combined with 15 mg/kg mycophenolate mofetil, 0.1 mg/kg tacrolimus combined with 30 mg/kg mycophenolate mofetil, 0.1 mg/kg tacrolimus combined with 40 mg/kg mycophenolate mofetil, 0.2 mg/kg tacrolimus combined with 15 mg/kg mycophenolate mofetil, 0.2 mg/kg tacrolimus combined with 30 mg/kg mycophenolate mofetil (30 d only), and 0.2 mg/kg tacrolimus combined with 40 mg/kg mycophenolate mofetil. Each group contained 8 to 10 rats. Grafts were harvested for histopathological analysis on day 15 or 30 after transplantation. Histopathological appearance of the graft was blindly graded according to an established scale. Dosage groups were compared on rejection score using Wilcoxon's rank sum test and the Jonckheere-Terpstra test for trend. Results There was a significant association between increasing dose of tacrolimus and decreasing rejection score at both 15 and 30 days (P

Using a rat model of laryngeal transplantation, we sought to define the relationships between acute laryngeal rejection grade (RG) and cyclosporin A (CSA) concentration and CSA dosage. Five recipient Lewis rat groups (N = 10 per group) were administered intramuscular CSA doses of 1.0 (group 1), 2.5 (group 2), 5.0 (group 3), 7.5 (group 4), and 10 mg/kg per day (group 5) for 14 days. Immediately before sacrifice, 5 mL of whole blood was obtained to assay CSA trough levels by high-performance liquid chromatography. The specimens were graded microscopically by blinded reviewers by day of RG, 0 to 14 days after transplantation, as described in earlier reports. Despite high intragroup variability in CSA levels, significantly different mean CSA concentrations were achieved among all CSA dosage groups: 1,2,3,4, and 5 (.0001 < p < .02). The mean laryngeal RGs did not test significantly different from each other with groups 3, 4, and 5 (RG, 2.3 ± 1.3 versus 1.9 ± 1.1 versus 1.7 ±0.3, respectively, .2 < p < .6). The RG for group 1 was significantly greater than those for groups 2 through 5 (p < .001), and the group 2 RG was greater (p < .02) than those for groups 3, 4, and 5. Polynomial fitting was used to determine the continuous relationship between each individual specimen's CSA concentration and the RG. Significant pathological allograft rejection correlated with CSA concentrations below 250 ng/mL.

Total lymphoid irradiation is an acknowledged adjunctive immunosuppressant in whole organ transplantation in humans and animals. Local irradiation administered for a similar purpose is at best controversial. We evaluated in vitro donor larynx irradiation immediately preceding laryngeal transplantation as an immunomodulator. Each donor larynx was pretreated with 7.34 Gy of radiation in vitro. After transplantation, cyclosporine was administered in doses of 5 mg/kg per day, 2.5 mg/kg per day, and 1 mg/kg per day for trial lengths of 15 days and 30 days. Each of these 6 groups consisted of 10 rats per group. Earlier data have shown cyclosporine dosed at 5 mg/kg per day, without irradiation, administered for 1 month to have varied efficacy. Established histologic criteria were used to determine rejection patterns. All recipient rats survived the 15-day and 30-day trials. In all 10 rats receiving 5 mg/kg per day of cyclosporine for 15 days, the harvested transplanted larynges were viable without evidence of meaningful rejection (mild rejection). In 9 of the 10 rats receiving 5 mg/kg per day of cyclosporine for 30 days, the transplanted larynges displayed no meaningful rejection (mild rejection). In 9 of the 10 rats receiving 2.5 mg/kg per day of cyclosporine for 15 days, the transplanted larynges displayed no meaningful rejection (mild rejection). One rat receiving 2.5 mg/kg per day of cyclosporine for 15 days had a transplanted larynx that displayed moderate rejection. In all 10 rats receiving 2.5 mg/kg per day of cyclosporine for 30 days, the transplanted larynges displayed no meaningful rejection (mild rejection). At 15 days, 5 rats treated with 1 mg/kg per day of cyclosporine displayed mild rejection, 2 displayed moderate rejection, 2 displayed advanced to moderate rejection, and 1 displayed severe rejection. At 30 days, 4 rats treated with 1 mg/kg per day of cyclosporine displayed moderate rejection, 2 displayed advanced to moderate rejection, and 4 displayed severe rejection. We conclude that pretransplantation in vitro irradiation of donor larynges has immunomodulatory effects, allowing reduced cyclosporine immunosuppression with less rejection.