A model of sequential heart and composite tissue allotransplant in rats.

BACKGROUND: Some of the 600,000 patients with solid organ allotransplants need reconstruction with a composite tissue allotransplant, such as the hand, abdominal wall, or face. The aim of this study was to develop a rat model for assessing the effects of a secondary composite tissue allotransplant on a primary heart allotransplant. METHODS: Hearts of Wistar Kyoto rats were harvested and transplanted heterotopically to the neck of recipient Fisher 344 rats. The anastomoses were performed between the donor brachiocephalic artery and the recipient left common carotid artery, and between the donor pulmonary artery and the recipient external jugular vein. Recipients received cyclosporine A for 10 days only. Heart rate was assessed noninvasively. The sequential composite tissue allotransplant consisted of a 3 x 3-cm abdominal musculocutaneous flap harvested from Lewis rats and transplanted to the abdomen of the heart allotransplant recipients. The abdominal flap vessels were connected to the femoral vessels. No further immunosuppression was administered following the composite tissue allotransplant. Ten days after composite tissue allotransplantation, rejection of the heart and abdominal flap was assessed histologically. RESULTS: The rat survival rate of the two-stage transplant surgery was 80 percent. The transplanted heart rate decreased from 150 +/- 22 beats per minute immediately after transplant to 83 +/- 12 beats per minute on day 20 (10 days after stopping immunosuppression). CONCLUSIONS: This sequential allotransplant model is technically demanding. It will facilitate investigation of the effects of a secondary composite tissue allotransplant following primary solid organ transplantation and could be useful in developing future immunotherapeutic strategies.

Efeito da ciclofosfamida sobre o desenvolvimento de germes dentais de molares transplantados para a câmara anterior do olho de camundongos.

Mice foetus were killed between 13 and 16 days of the gestational period and their molar tooth germs grafted into the anterior chamber of the eye. The hosts were injected either with 6.25 mg/Kg or with 125 mg/Kg of cyclophosphamide and killed 15 days later. The cyclophosphamide irreversibly, interferes on tooth germ development causing either its total degeneration or an alteration on its morphology forming hypodeveloped tooth germs; its action is directly proportional to the foetus age and to the dose used; it does not induce the formation of osteodentin while some predentin partial cell inclusion may occur.

Electroporation-mediated interleukin-10 overexpression in skeletal muscle reduces acute rejection in rat cardiac allografts

OBJECTIVES: Human interleukin 10 (hIL-10) may reduce acute rejection after organ transplantation. Our previous data shows that electroporation-mediated transfer of plasmid DNA to peripheral muscle enhances gene transduction dramatically. This study was designed to investigate the effect of electroporation-mediated overexpression of hIL-10 on acute rejection of cardiac allografts in the rat. METHODS: The study was designed to evaluate the effect of hIL-10 gene transfer on (a) early rejection pattern and (b) graft survival. Gene transfer was achieved by intramuscular (i.m.) injection into the tibialis anterior muscle of Fischer (F344) male recipients followed by electroporation 24 h prior to transplantation. Heterotopic cardiac transplantation was performed from male Brown Norway rat to F344. Four groups were studied (n = 6). Treated animals in groups B1 and B2 received 2.5 microg of pCIK hIL-10 and control animals in groups A1 and A2 distilled water. Graft function was assessed by daily palpation. Animals from group A1 were sacrificed at the cessation of the heart beat of the graft and those in group B1 were sacrificed at day 7; blood was taken for ELISA measurement of hIL-10 and tissue for myeloperoxidase (MPO) measurement and histological assessment. To evaluate graft survival, groups A2 and B2 were sacrificed at cessation of the heart beat of the graft. RESULTS: Histological examination revealed severe rejection (IIIB-IV) in group A1 in contrast to low to moderate rejection (IA-IIIA) in group B1 (p = 0.02). MPO activity was significantly lower in group B1 compared to group A1 (18 +/- 7 vs. 32 +/- 14 mU/mg protein, p = 0.05). Serum hIL-10 levels were 46 +/- 13 pg/ml in group B1 vs. 0 pg/ml in group A1. At day 7 all heart allografts in the treated groups B1 and B2 were beating, whereas they stopped beating at 5 +/- 2 days in groups A1 and A2 vs. 14 +/- 2 days in group B2 (p = 0.0012). CONCLUSIONS: Electroporation-mediated intramuscular overexpression of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in rats. This study demonstrates that peripheral overexpression of specific genes in skeletal muscle may reduce acute rejection after whole organ transplantation.

Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report

Cryopreservation of ovarian tissue has been proposed for storing gametes of young patients at high risk of premature ovarian failure. Autotransplantation has recently provided some promising results and is still the unique option to restore ovarian function from cryopreserved ovarian tissue in humans. In this article, we analyse data from the combined orthotopic and heterotopic transplantation of cryopreserved ovarian tissue that restored the ovarian function and fertility. Orthotopic transplantation of cryopreserved ovarian tissue at ovarian and peritoneal sites, together with a heterotopic transplantation at the abdominal subcutaneous site, was performed to restore the ovarian function of a 29-year-old woman previously treated with bone marrow transplantation (BMT) for Hodgkin's disease. Ovarian reserve markers progressively suppress within values 5 months after the transplantation (basal FSH 5 mUI/ml and inhibin B 119 ng/ml). Follicular development was observed at all transplantation sites but was predominant at the ovarian site. Six natural cycles were fully documented and analysed. The patient became spontaneously pregnant following the sixth cycle, but unfortunately she later miscarried. Combined orthotopic and heterotopic transplantations succeeded in the restoration of normal spontaneous cycles. Furthermore, this spontaneous pregnancy confirmed the efficiency of this procedure for restoring human fertility.

Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation

BACKGROUND Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. METHODS Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry. RESULTS In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days. CONCLUSIONS We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Fertility preservation: Successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease

Cryopreservation of ovarian tissue is now offered as an experimental procedure to preserve the fertility of young patients with a high risk for premature ovarian failure resulting from cancer therapy. This is the only available option to preserve the fertility of prepubertal patients treated with gonadotoxic chemotherapy. At present, thousands of patients all over the world have undergone this procedure with the hope of later restoring their fertility. Although the efficiency of the transplantation of cryopreserved ovarian tissue to restore ovarian function has been established, reports of pregnancy are still very scarce. Here, we describe the second published full-term spontaneous pregnancy after an orthotopic and heterotopic transplantation of cryopreserved ovarian tissue in a 31-year-old woman previously treated by conditioning therapy for bone marrow transplantation for Hodgkin's disease. This birth gives compelling evidence for the graft origin of the gamete and confirms the efficacy of ovarian tissue transplantation in restoring human natural fertility after oncological treatment. This case report stresses the importance of proposing the ovarian tissue cryopreservation procedure to all young patients who require potentially sterilizing treatment, with all alternative options to preserve fertility being duly taken into consideration.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

Em estudo recente demonstramos que a infusão de células da medula óssea do doador após o transplante intestinal não aumentou a sobrevida do enxerto quando se utilizou series curtas de drogas imunossupressoras. OBJETIVO: Neste estudo avaliamos se a utilização de diferentes regimes de irradiação em associação com a infusão de medula óssea altera a sobrevida do enxerto e a morbidade sobre receptor. MÉTODOS: Realizou-se o transplante heterotópico de intestino delgado, utilizando-se ratos Lewis como receptores e da como doadores, imunossuprimidos com FK 506 na dose de 1mg/kg/dia por 5 dias e distribuídos em 4 grupos: G1 (n= 4), não irradiado e sem infusão de medula óssea; G2 (n= 6), G3 (n= 9) e G4 (n= 6) foram infundidos com 100 x 10(6) células de medula após o transplante. Grupos 3 e 4 foram irradiados com 250 e 400 rd respectivamente. Os animais foram examinados diariamente para a detecção de rejeição e reação do enxerto versus hospedeiro, tendo sido colhidas amostras semanais de sangue para estudos de quimerismose biopsias quinzenais da estomia. RESULTADOS: Animais nos G1 e G2 apresentaram rejeição mínima no 15º pós-operatório, enquanto a reação do enxerto versus hospedeiro foi caracterizada nos G3 e G4. Os níveis de quimerismo total e de células T foram maiores nos grupos irradiados em comparação aos não irradiados. A causa mortis nos G1 e G2 foi a rejeição enquanto que nos G3 e G4 foi a reação do enxerto versus hospedeiro. CONCLUSÃO: Concluímos que a utilização de baixas doses de irradiações retardam o aparecimento da rejeição, mas não previne a ocorrência da reação do enxerto versus hospedeiro.

Experimental pancreas transplantation: the consequences of the portocaval shunt on the blood glucose, plasma insulin, and glucagon

A proposição foi realizada, no período pós-operatório imediato, em 40 ratos Wistar, distribuídos por sorteio em doi grupos: grupo NC, vinte ratos correspondentes ao grupo controle, não diabético, submetidos a operação simulada e o grupo PT, 20 ratos correspondentes ao grupo diabético que recebeu transplante de pâncreas heterotópico de ratos Wistar normais. Durante sete dias, antes do transplante, e 1, 3, 6, 12, 24, 48, 72, 96 horas após, determinava-se a glicose sanguínea, a insulina plasnática e o glucagon. Estes parâmetros eram obtidos também do grupo NC. Diabetes mellitus experimental era induzida pela administração intravenosa de aloxana. O grupo PT era imunosuprimido com ciclosporina A. O grupo NC apresentou níveis normais de glicose sanguínea, de insulina plásmica e de glucagon, durante todo o experimento. Foi encontrada nítida hiperinsulinemia no sangue venoso periférico do grupo PT. A insulina plasmática era significantemente maior no grupo PT comparada ao grupo NC começando 72 horas após o transplante. O glucagon plasmático, elevado no período pré-transplante, não se alterou após o transplante. Apesar de hiperinsulinemia e hiperglucagonemia, os níveis de glicose sanguínea eram elevados 6 horas após o transplante e mantiveram-se normais após este período. Considerando-se os níveis de glicose sanguínea 12 horas pós-transplante, não houve diferença estatisticamente significante entre os grupos PT e NC, até o sacrifício.

Evaluation of a porcine whole-limb heterotopic autotransplantation model

Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field.