Synergistic suppression of pre-perfusion of donor livers with recipient serum and cobra venom factor treatment on hyperacute rejection following liver xenotransplantation.

PURPOSE: To investigate synergistic suppression of donor liver pre-perfusion with recipient serum (RS) and cobra venom factor (CVF) treatment on hyperacute rejection (HAR) following liver xenotransplantation. METHODS: Guinea-pigs (GP, n=24) and Sprague-Dawley rats (SD, n=24) were recruited. Before transplantation, serum was collected from SD rats and used for preparation of inactivated complements. GP and SD rats were randomly assigned into four groups (n=6), respectively: RS group, CVF group, RS+CVF group and control group. Orthotopic liver xenotransplantation was performed with modified two-cuff technique. The survival time and liver function of recipients, morphological and pathological changes in rat livers were investigated. RESULTS: There was no piebald like change in the recipient livers in all experiment groups. The survival time of recipients in all experiment groups was longer than that in control group (p<0.05). Moreover, the survival time in the RS+CVF group was markedly longer than that in the RS group (p<0.01) and CVF group (p<0.05). The serum ALT level in all experiment groups were lower than that in the control group (p<0.05). Furthermore, the ALT level in the RS+CVF group was significantly lower than that in the CVF group (p<0.05) and RS group (p<0.01). The histological damages were significantly improved when compared with the control group, and the histological damages in the RS+CVF group were milder than those in the remaining groups (p<0.05) CONCLUSION: Pre-perfusion of donor liver with recipient serum and cobra venom factor treatment can exert synergistic suppressive effects on the hyperacute rejection following liver xenotransplantation.

Synergistic suppression of pre-perfusion of donor livers with recipient serum and cobra venom factor treatment on hyperacute rejection following liver xenotransplantation / Supressão sinérgica da rejeição hiperaguda no xenotransplante hepático com uso da pre-perfusão dos fígados doadores tratados com soro do receptor e com fator veneno de cobra

PURPOSE: To investigate synergistic suppression of donor liver pre-perfusion with recipient serum (RS) and cobra venom factor (CVF) treatment on hyperacute rejection (HAR) following liver xenotransplantation. METHODS: Guinea-pigs (GP, n=24) and Sprague-Dawley rats (SD, n=24) were recruited. Before transplantation, serum was collected from SD rats and used for preparation of inactivated complements. GP and SD rats were randomly assigned into four groups (n=6), respectively: RS group, CVF group, RS+CVF group and control group. Orthotopic liver xenotransplantation was performed with modified two-cuff technique. The survival time and liver function of recipients, morphological and pathological changes in rat livers were investigated. RESULTS: There was no piebald like change in the recipient livers in all experiment groups. The survival time of recipients in all experiment groups was longer than that in control group (p<0.05). Moreover, the survival time in the RS+CVF group was markedly longer than that in the RS group (p<0.01) and CVF group (p<0.05). The serum ALT level in all experiment groups were lower than that in the control group (p<0.05). Furthermore, the ALT level in the RS+CVF group was significantly lower than that in the CVF group (p<0.05) and RS group (p<0.01). The histological damages were significantly improved when compared with the control group, and the histological damages in the RS+CVF group were milder than those in the remaining groups (p<0.05) CONCLUSION: Pre-perfusion of donor liver with recipient serum and cobra venom factor treatment can exert synergistic suppressive effects on the hyperacute rejection following liver xenotransplantation.

OBJETIVO: Investigar a supressão sinérgica da pré-perfusão do doador de fígado com soro do receptor (SR) e tratamento com fator veneno de cobra (FVC) na rejeição hiperaguda (RHA) após o xenotransplante de fígado. MÉTODOS: Foram utilizados Cobaias (GP, n=24) e ratos Sprague-Dawley (SD, n=24). Antes do transplante foram coletadas amostras de soro dos ratos SD e usados para a preparação dos complementos inativados. Cobaias GP e ratos SD foram randomicamente distribuídos em quatro grupos (n=6), respectivamente: grupo RS, grupo FVC, grupo SR+FVC e grupo controle. Xenotransplante ortotópico do fígado foi realizado com a técnica de dois cuffs modificados. Foram investigados o de tempo de sobrevida, a função hepática dos receptores e alterações morfopatológicas em fígados de ratos. RESULTADOS: Não houve alteração na coloração do parênquima dos fígados nos receptores. O tempo de sobrevida dos receptores em todos os grupos experimentais foi mais longo do que o grupo controle (p<0,05). Além disso, o tempo de sobrevida do grupo SR+ FVC foi marcadamente maior do que o grupo SR (p<0,01) e o grupo FVC (p<0,05). O nível sérico ALT foi menor em todos os grupos experimentais do que o grupo controle (p<0,05). O nível de ALT no grupo SR+ FVC foi significantemente menor do que no grupo FVC (p<0,05) e o grupo SR (p<0,01). As alterações histológicas foram significantemente melhoradas quando comparado com o grupo controle, e os danos histológicos no grupo SR+ FVC foram mais moderados do que nos grupos restantes (p<0,05). CONCLUSÃO: Pré-perfusão do fígado doador com soro do receptor e fator veneno de cobra pode exercer efeito supressor sinérgico da rejeição hiperaguda após xenotransplante de fígado.

Endoscopic features in a model of multivisceral xenotransplantation.

INTRODUCTION: Xenotransplantation and multivisceral transplantation are advanced therapeutic methods that still require a scientific basis. There are no experimental models of multivisceral transplantation available, particularly not the monitoring by endoscopy. Here, we describe the endoscopic features in a model of multivisceral xenotransplantation. METHODS: The distal esophagus, stomach, intestine, colon, liver, pancreas, and the kidneys with a common vascular pedicle were harvested from rabbits and implanted in swine (group I, n = 9) or in rabbits (group II, n = 4). Endoscopy was performed in the stomach, jejunum, and ascending colon at four consecutive time points (immediate after surgery and 10, 90, and 180 min after reperfusion). Lesions were macroscopically graded as mild, moderate, and severe. Biopsies were taken following sacrifice at 180 min after reperfusion. RESULTS: In group I, the stomach, jejunum, and colon manifested a progression of lesions with predominance of mild lesions after 10 min, mild to moderate lesions after 90 min, and moderate to severe lesions after 180 min. In animals from group II, endoscopy showed normal features at all time points after reperfusion. Histopathologic analysis confirmed the diagnosis of hyperacute rejection in group I. Grafts from group II animals presented normal or mild ischemic/reperfusion injury. CONCLUSION: All animals subjected to multivisceral xenotransplantation showed a progression of endoscopic lesions with time after transplantation, while animals subjected to allotransplantation showed no aberrations in endoscopy. We conclude that endoscopy is a useful tool in the assessment of hyperacute rejection of a xenograft.

Unexpected immunoresponse to Gal and APA antigens in diabetic type 1 patients receiving neonatal pig islets after 6 years.

Cotransplantation of porcine islets and Sertoli cells into preimplanted subcutaneous devices improve metabolic control in type 1 diabetic patients, and survive grafted for more than 4 years. We report here, further assessment of the endocrine and porcine nature of the surviving cells and the immune responses elicited toward Gal alpha(1,3)-Gal beta(1,4)-GlcNAc (Gal) antigen in patients who received a second and third transplants. No immunosuppressive drugs were administered. We were able to immunostain insulin- and glucagon-positive cells in all biopsies of patients and Sertoli cell markers in 60.9% of biopsies. Additionally, all biopsies tested, amplified the porcine COII gene. Patients demonstrated an increase in antipig antibodies in response to the first transplant with a decreasing response toward the second and third transplants. In all transplants, the IgG levels promptly returned to basal values after 3-4 months. The long-term survival of porcine cells and the reduced humoral immune response to multiple transplants indicate a form of tolerance. We have not been able to find CD25-positive cells, indicating that it is probably an immune accommodation of the graft.

Keloid heterograft in the hamster (Mesocricetus auratus) cheek pouch, Brazil.

PURPOSE: To study the integration of keloid heterograft in hamster (Mesocricetus auratus) cheek pouch. METHODS: The sample is formed by 18 male hamsters, heterogenic ones, aged between 10 and 14 weeks. Keloid fragments were obtained from keloid scars of the breast region of adult female mulatto patient. Each hamster received keloid fragments into both of its pouches, in a total of 36 grafted fragments. Animals were distributed into 6 groups for having their grafts assessed in the days 5, 12, 21,42, 84, and 168. A macroscopic assessment is performed by comparing the pouch containing the grafted fragment, at each time point, with the same pouch in the immediate post surgical moment through a comparison of standardized photographs. Under microscope, the presence of blood vases is considered within the conjunctive tissue of the grafted fragment, as a criterion of its integration. Other events, as keratin secretion, the presence of cellular infiltrated, epithelium and keloid collagen fibers aspects are also analyzed. RESULTS: Macroscopy reveals intensive vascularization of the pouch up to 12 days from the transplantation and the presence of constant dark brown pigmentation on the grafted keloid fragments. In microscopy, the integration of keloid fragments is considered by the presence of blood capillary vases within conjunctive tissue. The presence of intensive cellular inflammatory type infiltrated up to 12 days is also observed, as well as the remaining of keloid epithelium up to 21 days, and the appearing of melanocytes from the day 42. CONCLUSION: Hamster cheek pouch represents, a priori, an experimental model for the investigation of keloid.

Keloid heterograft in the hamster (mesocricetus auratus) cheek pouch

OBJETIVO: Investigar a integração do transplante heterólogo de quelóide na bolsa jugal do hamster (Mesocricetus auratus). MÉTODOS: A amostragem consiste de 18 hamsters machos, heterogênicos, com 10 a 14 semanas de idade. Fragmentos de quelóide foram obtidos de cicatrizes queloidianas da região mamária de paciente adulta parda. Cada hamster foi enxertado em ambas as bolsas com fragmentos de quelóide, totalizando 36 fragmentos enxertados. Os animais foram distribuídos em 6 grupos para exame dos fragmentos enxertados, com 5, 12, 21, 42, 84 e 168 dias. Uma avaliação macroscópica é realizada comparando a bolsa contendo o fragmento enxertado em cada período com a mesma bolsa no pós-operatório imediato, mediante a comparação de fotografias padronizadas. À microscopia, considera-se a presença de vasos sangüíneos no tecido conjuntivo do fragmento enxertado como critério de integração do mesmo. Outros eventos, como secreção de queratina, presença de infiltrados celulares e aspecto do epitélio e das fibras colágenas do quelóide, também são observados. RESULTADOS: A macroscopia revela intensa vascularização na bolsa até 12 dias de enxertia, e a presença constante de pigmentação castanho-escura nos fragmentos de quelóide enxertados. Na microscopia constata-se a integração dos fragmentos de quelóide pela presença de capilares sangüíneos no tecido conjuntivo. Observa-se, também, a presença de intenso infiltrado celular do tipo inflamatório até 12 dias, a permanência do epitélio do quelóide até 21 dias, e o aparecimento de melanócitos a partir de 42 dias. CONCLUSÃO: A bolsa jugal do hamster representa, a priori, modelo experimental para investigação do quelóide.

Immunologic and osteogeneic properties of xenogeneic and allogeneic demineralized bone transplants.

Xenografting is increasingly being developed as a response to the shortage of human tissues. However, antigenic components of bone material eliciting immune responses--particularly of cellular nature--are blamed for the reduction of the osteoinductive properties of bone and bone-derived implants. The aim of our study was to compare the immunologic response and osteogenesis induced by antigen-depleted allogeneic and xenogeneic bone-derived implants to that induced by partially antigen-depleted material heterotopically placed (muscular pouch) in rats. Wistar rats received bone-derived implants of different antigeneic condition, from both xenogeneic (rabbit) and allogeneic (rat) origin. After sacrifice, animals were evaluated for osteogenesis and immune response. New bone formation was observed around all bone-derived implants, whether fully or partially antigen-extracted, and from both xenogeneic and allogeneic origin. No significant humoral response resulted following bone implantation. Cellular response showed a similar pattern in partial and fully antigen-extracted bone of both allogeneic and xenogeneic origin. Xenogeneic antigen-extracted bone from safe donor sources could be a suitable solution to human tissue shortage in a near future.

Pig peripheral blood mononuclear cells are directly associated with the thrombotic microangiopathy that complicates the induction of chimerism in pig-to-baboon xenotransplantation.

The infusion of large numbers of porcine cells into primates in order to induce specific immunologic tolerance by mixed hematopoietic chimerism, results in thrombotic microangiopathy that can be fatal. For this reason, it is important to study in vitro the interaction of primate endothelial cells with pig cells. We show that pig peripheral blood mononuclear cells (p-PBMC) activate human endothelial cells (hECs) through direct contact. Thus, when endothelial cells are cultured in the presence of p-PBMC, overexpression of VCAM-1 and E-selectin adhesion molecules occurs within 3 h of culture and continues for at least 9 h. The co-culture of p-PBMC and hECs also results in an important adhesion of human platelets to both types of cell. Thus, viewed with the microscope, platelets aggregate above the endothelial cells and also around the pig cells. We present data that suggest that the presence of p-PBMC may be more important with regard to the increase of platelet adhesion to the endothelial cells than the activation alone of the cells. Our results also show that p-PBMC, and not the activated endothelia or the culture supernatant of activated hECs, are able to activate the coagulation cascade because they are able to generate thrombin when added to defibrinated human plasma. Overall, these findings suggest that p-PBMC are of primary importance for the development of the thrombotic disorders that occur in primates transplanted with pig progenitor cells.

Apoptosis and suppression of tumor growth by artepillin C extracted from Brazilian propolis.

Artepillin C was extracted from Brazilian propolis. Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) has a molecular weight of 300.40 and possesses antibacterial activity. When artepillin C was applied to human and murine malignant tumor cells in vitro and in vivo, artepillin C exhibited a cytotoxic effect and the growth of tumor cells was clearly inhibited. The artepillin C was found to cause significant damage to solid tumor and leukemic cells by the MTT assay, DNA synthesis assay, and morphological observation in vitro. When xenografts of human tumor cells were transplanted into nude mice, the cytotoxic effects of artepillin C were most noticeable in carcinoma and malignant melanoma. Apoptosis, abortive mitosis, and massive necrosis combined were identified by histological observation after intratumor injection of 500 microg of artepillin C three times a week. In addition to suppression of tumor growth, there was an increase in the ratio of CD4/CD8 T cells, and in the total number of helper T cells. These findings indicate that artepillin C activates the immune system, and possesses direct antitumor activity.