Heterotopic transplantation of glycerin-preserved trachea: effect of respiratory epithelium desquamation on acute rejection

An effective preservation method and decreased rejection are essential for tracheal transplantation in the reconstruction of large airway defects. Our objective in the present study was to evaluate the antigenic properties of glycerin-preserved tracheal segments. Sixty-one tracheal segments (2.4 to 3.1 cm) were divided into three groups: autograft (N = 21), fresh allograft (N = 18) and glycerin-preserved allograft (N = 22). Two segments from different groups were implanted into the greater omentum of dogs (N = 31). After 28 days, the segments were harvested and analyzed for mononuclear infiltration score and for the presence of respiratory epithelium. The fresh allograft group presented the highest score for mononuclear infiltration (1.78 ± 0.43, P <= 0.001) when compared to the autograft and glycerin-preserved allograft groups. In contrast to the regenerated epithelium observed in autograft segments, all fresh allografts and glycerin-preserved allografts had desquamation of the respiratory mucosa. The low antigenicity observed in glycerin segments was probably the result of denudation of the respiratory epithelium and perhaps due to the decrease of major histocompatibility complex class II antigens.

Relationship of cyclosporin and sirolimus blood concentrations regarding the incidence and severity of hyperlipidemia after kidney transplantation

The influence of drug concentrations on the development of persistent posttransplant hyperlipidemia was investigated in 82 patients who received cyclosporin A (CsA) and prednisone plus sirolimus (SRL) (52) or azathioprine (AZA) (30) during the first year after transplantation. Blood levels of CsA and SRL, daily doses of AZA and prednisone, and cholesterol, triglyceride, and glucose concentrations were determined during each visit (pretransplant and 30, 60, 90, 120, 180, and 360 days posttransplant). Persistent hyperlipidemia was defined as one-year average steady-state cholesterol (CavCHOL) or triglyceride (CavTG) concentrations above 240 and 200 mg/dL, respectively. Mean cholesterol and triglyceride concentrations increased after transplantation (P < 0.01) and were higher in patients receiving SRL compared to AZA (P < 0.001). Patients receiving SRL showed a significantly higher number of cholesterol (>229 or >274 mg/dL) and triglyceride (>198 or >282 mg/dL) determinations in the upper interquartile ranges. CsA and SRL interquartile ranges correlated with cholesterol concentrations (P = 0.001) whereas only SRL interquartile ranges correlated with triglyceride concentrations (P < 0.0001). Only pretransplant cholesterol concentration >205 mg/dL was independently associated with development of persistent hypercholesterolemia (CavCHOL >240 mg/dL, relative risk (RR) = 20, CI 3.8-104.6, P = 0.0004) whereas pretransplant triglyceride concentration >150 mg/dL (RR = 7.2, CI 1.6-32.4, P = 0.01) or >211 mg/dL (RR = 19.8, CI 3.6-107.9, P = 0.0006) and use of SRL (RR = 3, CI 1.0-8.8, P = 0.0049) were independently associated with development of persistent hypertriglyceridemia (CavTG >200 mg/dL). Persistent hypercholesterolemia was more frequent among patients with higher pretransplant cholesterol concentrations and was dependent on both CsA and SRL concentrations. Persistent hypertriglyceridemia was more frequent among patients with higher pretransplant triglyceride concentrations and was dependent on SRL concentrations.

Improvement of adynamic bone disease after renal transplantation

Low bone remodeling and relatively low serum parathyroid hormone (PTH) levels characterize adynamic bone disease (ABD). The impact of renal transplantation (RT) on the course of ABD is unknown. We studied prospectively 13 patients with biopsy-proven ABD after RT. Bone histomorphometry and bone mineral density (BMD) measurements were performed in the 1st and 12th months after RT. Serum PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and osteocalcin were measured regularly throughout the study. Serum PTH levels were slightly elevated at transplantation, normalized at the end of the third month and remained stable thereafter. Bone biopsies performed in the first month after RT revealed low bone turnover in all patients, with positive bone aluminum staining in 5. In the 12th month, second biopsies were performed on 12 patients. Bone histomorphometric dynamic parameters improved in 9 and were completely normalized in 6, whereas no bone mineralization was detected in 3 of these 12 patients. At 12 months post-RT, no bone aluminum was detected in any patient. We also found a decrease in lumbar BMD and an increase in femoral BMD. Patients suffering from ABD, even those with a reduction in PTH levels, may present partial or complete recovery of bone turnover after successful renal transplantation. However, it is not possible to positively identify the mechanisms responsible for the improvement. Identifying these mechanisms should lead to a better understanding of the physiopathology of ABD and to the development of more effective treatments.

Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution

We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group 2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51%) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23% of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38% at ~8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with <25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10(6) cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6) cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

A novel noninvasive method to detect rejection after heart transplantation

Prompt and accurate detection of rejection prior to pathological changes after organ transplantation is vital for monitoring rejections. Although biopsy remains the current gold standard for rejection diagnosis, it is an invasive method and cannot be repeated daily. Thus, noninvasive monitoring methods are needed. In this study, by introducing an IL-2 neutralizing monoclonal antibody (IL-2 N-mAb) and immunosuppressants into the culture with the presence of specific stimulators and activated lymphocytes, an activated lymphocyte-specific assay (ALSA) system was established to detect the specific activated lymphocytes. This assay demonstrated that the suppression in the ALSA test was closely related to the existence of specific activated lymphocytes. The ALSA test was applied to 47 heart graft recipients and the proliferation of activated lymphocytes from all rejection recipients proven by endomyocardial biopsies was found to be inhibited by spleen cells from the corresponding donors, suggesting that this suppression could reflect the existence of activated lymphocytes against donor antigens, and thus the rejection of a heart graft. The sensitivity of the ALSA test in these 47 heart graft recipients was 100%; however, the specificity was only 37.5%. It was also demonstrated that IL-2 N-mAb was indispensible, and the proper culture time courses and concentrations of stimulators were essential for the ALSA test. This preliminary study with 47 grafts revealed that the ALSA test was a promising noninvasive tool, which could be used in vitro to assist with the diagnosis of rejection post-heart transplantation.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Detection of human cytomegalovirus DNA in various blood components after liver transplantation

The quantification of human cytomegalovirus (HCMV DNA) by real-time PCR is currently a primary option for laboratory diagnosis of HCMV infection. However, the optimal sample material remains controversial due to the use of different PCR assays. To explore the best blood component for HCMV DNA surveillance after liver transplantation, whole blood (WB), serum (SE), and plasma (PL) specimens were collected simultaneously from targeted patients and examined for HCMV DNA using one commercially available assay. The HCMV DNA-positive rate with WB (16.67%) was higher than that with either SE or PL (8.33%, both P<0.01). Quantitative DNA levels in WB were of greater magnitude than those in SE (WB-SE mean log-transformed difference, 0.99; 95%CI=0.74-1.25; P<0.0001) and PL (WB-PL mean log-transformed difference, 1.37; 95%CI=1.07-1.66; P<0.0001). Dynamic monitoring revealed that HCMV DNA in WB was positive sooner and had higher values for a longer period of time during therapy. With earlier positive detection, higher sensitivity, and yield of greater viral loads, WB compared favorably to SE or PL and hence is recommended as the superior material for HCMV DNA surveillance after liver transplantation. In addition, infant recipients require more intensive monitoring and prophylactic care because of their higher susceptibility to primary HCMV infection.

Successful domino liver transplantation in maple syrup urine disease using a related living donor

Maple syrup urine disease (MSUD) is an autosomal recessive disease associated with high levels of branched-chain amino acids. Children with MSUD can present severe neurological damage, but liver transplantation (LT) allows the patient to resume a normal diet and avoid further neurological damage. The use of living related donors has been controversial because parents are obligatory heterozygotes. We report a case of a 2-year-old child with MSUD who underwent a living donor LT. The donor was the patient's mother, and his liver was then used as a domino graft. The postoperative course was uneventful in all three subjects. DNA analysis performed after the transplantation (sequencing of the coding regions of BCKDHA, BCKDHB, andDBT genes) showed that the MSUD patient was heterozygous for a pathogenic mutation in the BCKDHB gene. This mutation was not found in his mother, who is an obligatory carrier for MSUD according to the family history and, as expected, presented both normal clinical phenotype and levels of branched-chain amino acids. In conclusion, our data suggest that the use of a related donor in LT for MSUD was effective, and the liver of the MSUD patient was successfully used in domino transplantation. Routine donor genotyping may not be feasible, because the test is not widely available, and, most importantly, the disease is associated with both the presence of allelic and locus heterogeneity. Further studies with this population of patients are required to expand the use of related donors in MSUD.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Salvaged single-unit cord blood transplantation for 26 patients with hematologic malignancies not in remission

Treatments for patients with hematologic malignancies not in remission are limited, but a few clinical studies have investigated the effects of salvaged unrelated cord blood transplantation (CBT). We retrospectively studied 19 patients with acute leukemia, 5 with myelodysplastic syndrome (MDS with refractory anemia with excess blasts [RAEB]), and 2 with non-Hodgkin's lymphoma who received 1 CBT unit ≤2 loci human leukocyte antigen (HLA)-mismatched after undergoing myeloablative conditioning regimens between July 2005 and July 2014. All of them were in non-remission before transplantation. The infused total nucleated cell (TNC) dose was 4.07 (range 2.76-6.02)×107/kg and that of CD34+ stem cells was 2.08 (range 0.99-8.65)×105/kg. All patients were engrafted with neutrophils that exceeded 0.5×109/L on median day +17 (range 14-37 days) and had platelet counts of >20×109/L on median day +35 (range 17-70 days). Sixteen patients (61.5%) experienced pre-engraftment syndrome (PES), and six (23.1%) patients progressed to acute graft-versus-host disease (GVHD). The cumulative incidence rates of II-IV acute GVHD and chronic GVHD were 50% and 26.9%, respectively. After a median follow-up of 27 months (range 5-74), 14 patients survived and 3 relapsed. The estimated 2-year overall survival (OS), disease-free survival (DFS), and non-relapse mortality (NRM) rates were 50.5%, 40.3%, and 35.2%, respectively. Salvaged CBT might be a promising modality for treating hematologic malignancies, even in patients with a high leukemia burden.

Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

Histopathological analysis of pre-implantation donor kidney biopsies: association with graft survival and function in one year post-transplantation

Introduction: Pre-implantation kidney biopsy is a decision-making tool when considering the use of grafts from deceased donors with expanded criteria, implanting one or two kidneys and comparing this to post-transplantation biopsies. The role of histopathological alterations in kidney compartments as a prognostic factor in graft survival and function has had conflicting results. Objective: This study evaluated the prevalence of chronic alterations in pre-implant biopsies of kidney grafts and the association of findings with graft function and survival in one year post-transplant. Methods: 110 biopsies were analyzed between 2006 and 2009 at Santa Casa de Porto Alegre, including live donors, ideal deceased donors and those with expanded criteria. The score was computed according to criteria suggested by Remuzzi. The glomerular filtration rate (GFR) was calculated using the abbreviated MDRD formula. Results: No statistical difference was found in the survival of donors stratified according to Remuzzi criteria. The GFR was significantly associated with the total scores in the groups with mild and moderate alterations, and in the kidney compartments alone, by univariate analysis. The multivariate model found an association with the presence of arteriosclerosis, glomerulosclerosis, acute rejection and delayed graft function. Conclusion: Pre-transplant chronic kidney alterations did not influence the post-transplantation one-year graft survival, but arteriosclerosis and glomerulosclerosis is predictive of a worse GFR. Delayed graft function and acute rejection are independent prognostic factors.