Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Mefenoxam and pyraclostrobin: toxicity and in vitro inhibitory activity against Pythium insidiosum.

The objective of this study is to verify in vitro susceptibility of Pythium insidiosum against the agricultural fungicides mefenoxam and pyraclostrobin and evaluate the toxicity of both compounds. Twenty-one P. insidiosum isolates were tested against mefenoxam and pyraclostrobin using the broth microdilution method. Minimum inhibitory and oomicidal concentrations for both compounds were established. Additionally, scanning electron microscopy was performed on P. insidiosum hyphae treated with the sublethal concentration of each fungicide. The toxicity of the compounds was evaluated in vivo Caenorhabditis elegans model. The concentration to inhibit 100% of P. insidiosum growth ranged from 0·625 to 10 µg ml-1 for mefenoxam and from 0·019 to 5 µg ml-1 for pyraclostrobin. The SEM analysis revealed changes on the surface of the hyphae treated with the fungicides, suggesting possible damage caused by these compounds. There was no evidence of toxicity in vivo models. Mefenoxam and pyraclostrobin did not show toxicity at the doses evaluated and have inhibitory effects on the pathogenic oomycete P. insidiosum. However, further evaluations of their pharmacokinetics and toxicity in different animal species and possible pharmacological interactions are necessary to infer a possible use in the clinical management of pythiosis.

Pneumotoxicosis in sheep caused by ingestion of trema micrantha.

Trema micrantha, a fast-growing tree distributed throughout the Americas, produces palatable leaves that have been associated with hepatic necrosis and acute death when consumed by livestock. This report describes fatal pulmonary disease of sheep triggered by consumption of Trema micrantha. Affected sheep had severe progressive dyspnea for a few days before death. Subcutaneous and mediastinal emphysema, reddened lungs, interalveolar septal thickening, and diffuse type II pneumocyte proliferation were the main pathological findings. After ingesting 77.5 and 102.5 g/kg (divided in 3 doses, at 30-day intervals) of T. micrantha leaves, 2 additional sheep developed the same condition. These findings indicate that T. micrantha toxicosis should be considered in the differential diagnosis of ovine respiratory disease.

Settleable atmospheric particulate matter affects the swimming performance and aerobic metabolic rate of Nile tilapia (Oreochromis niloticus).

Atmospheric particulate matter (APM) produced by the steel industry comprises a complex mixture of particles that includes a wide variety of metals and metallic nanoparticles. These particles settle out onto areas surrounding the industries. There is evidence that this 'settleable' APM (SePM) may cause air-to-water cross-contamination with significant effects on aquatic biota. Recent investigations have reported sublethal impacts on the gill structure and blood oxygen-carrying capacity of fishes, which raises the hypothesis that there will be consequences for gas exchange capacity and ability to support aerobic activities. Therefore, we investigated the effects of an environmentally relevant level of SePM contamination on swimming performance and associated aerobic metabolic rates in Nile tilapia, Oreochromis niloticus. Short-term exposure (96 h) to SePM reduced critical swimming speed, energetic efficiency of aerobic swimming, standard metabolic rate, maximum metabolic rate, and aerobic scope. The compromised swimming performance could have adverse ecological effects by limiting foraging ability, predator evasion, territorial protection, and migration. The impairments to aerobic capacity could also affect overall fish performance by influencing long-term energy balance and allocation to growth and reproduction. Thus, despite being sublethal, SePM contamination is considerably debilitating, and if its limiting effects are not compensated for in the longer term, this may reduce the survival and fitness of fish populations.

Settleable atmospheric particulate matter affects cardiorespiratory responses to hypoxia in Nile tilapia (Oreochromis niloticus).

Atmospheric particulate matter (APM) emitted by iron ore processing industries has a complex composition, including diverse metallic particles and nanoparticles. Settleable APM (SePM) causes air to water cross-contamination and has recently been demonstrated to have harmful sublethal impacts on fish, eliciting stress responses, affecting the immune system, and reducing blood oxygen-carrying capacity. These findings imply potential consequences for fish aerobic performance and energy allocation, particularly in their ability to tolerate respiratory challenges such as aquatic hypoxia. To assess that potential limitation, we analyzed metabolic, cardiorespiratory, and morphological alterations after exposing tilapia, Oreochromis niloticus, to an environmentally relevant concentration of SePM (96 h) and progressive hypoxia. The contamination initiated detectable gill damage, reducing respiratory efficiency, increasing ventilatory effort, and compromising fish capacity to deal with hypoxia. Even in normoxia, the resting respiratory frequency was elevated and limited respiratory adjustments during hypoxia. SePM increased O2crit from 26 to 34% of O2 (1.84 to 2.76 mg O2·L-1). Such ventilatory inefficacy implies higher ventilatory cost with relevant alterations in energy allocation. Progression in gill damage might be problematic and cause: infection, blood loss, ion imbalance, and limited cardiorespiratory performance. The contamination did not cause immediate lethality but may threaten fish populations due to limitations in physiological performance. This was the first investigation to evaluate the physiological responses of fish to hypoxia after SePM contamination. We suggest that the present level of environmental SePM deserves attention. The present results demonstrate the need for comprehensive studies on SePM effects in aquatic fauna.

Expression of heme oxygenase-1 can determine cardiac xenograft survival.

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

Relationship between the magnitude of the inbreeding coefficient and milk traits in Holstein and Jersey dairy bull semen used in Brazil.

Artificial insemination has been used to improve production in Brazilian dairy cattle; however, this can lead to problems due to increased inbreeding. To evaluate the effect of the magnitude of inbreeding coefficients on predicted transmitting abilities (PTAs) for milk traits of Holstein and Jersey breeds, data on 392 Holstein and 92 Jersey sires used in Brazil were tabulated. The second-degree polynomial equations and points of maximum or minimal response were estimated to establish the regression equation of the variables as a function of the inbreeding coefficients. The mean inbreeding coefficient of the Holstein bulls was 5.10%; this did not significantly affect the PTA for percent milk fat, protein percentage and protein (P = 0.479, 0.058 and 0.087, respectively). However, the PTAs for milk yield and fat decreased significantly after reaching inbreeding coefficients of 6.43 (P = 0.034) and 5.75 (P = 0.007), respectively. The mean inbreeding coefficient of Jersey bulls was 6.45%; the PTAs for milk yield, fat and protein, in pounds, decreased significantly after reaching inbreeding coefficients of 15.04, 9.83 and 12.82% (P < 0.001, P = 0.002, and P = 0.001, respectively). The linear regression was only significant for fat and protein percentages in the Jersey breed (P = 0.002 and P = 0.005, respectively). The PTAs of Holstein sires were more affected by smaller magnitudes of inbreeding coefficients than those of Jersey sires. It is necessary to monitor the inbreeding coefficients of sires used for artificial insemination in breeding schemes in Brazil, since the low genetic variability of the available sires may lead to reduced production.

Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis.

Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-alpha. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.

Abortion and neonatal mortality in sheep poisoned with Tetrapterys multiglandulosa.

Tetrapterys spp. have been reported as a cause of cardiac fibrosis, status spongiosus of the nervous system, and abortion. To study the intoxication by Tetrapterys multiglandulosa, 24 sheep were divided into 4 experimental groups of 4 sheep each and 1 control group. Groups 1 to 3, respectively, received 1, 1.5, and 3 g/kg body weight of the dry plant daily, starting on the 90th day of pregnancy. Group 4 received 1.5 g/kg from the 120th day of pregnancy to the end of gestation. All sheep from groups 2 and 3, except 1 that was submitted to cesarean delivery, aborted between 110 and 134 days of pregnancy. Seven fetuses had anasarca. Seven lambs from groups 1 and 4 were weak and died or were euthanatized. The other 2 were born weak with mild nervous signs but recovered. Cardiac fibrosis and status spongiosus of the nervous system were observed in newborn lambs and fetuses.

Interface-driven Sr-morin complexation at Langmuir monolayers for bioactive coating design.

Flavonoid-metal complexes are widely studied because of their interesting luminescent behavior and biological activity. Despite the extensive exploration of flavonoid-metal coordination processes in solution, the formation of complexes using the flavonoid molecule inserted in a lipid membrane has been little investigated. This effect could provide important insight into the biological activity of flavonoids at lipid membranes and could represent an attractive strategy to design supramolecular structures. Here, we studied the complexation between Sr2+ and morin inserted in an octadecylphosphonic acid (OPA) Langmuir monolayer. This is a relevant system due to the synergism imposed by the association of the Sr2+ ability to control bone formation/resorption with the morin antioxidative effect. Morin incorporation into the OPA monolayers and further Sr2+ complexation were monitored by surface pressure isotherms. Electronic absorption spectroscopy and fluorescence techniques showed Sr-morin complexation both in solution and at the air-liquid interface. Although morin complexation has been described to occur only at basic pH, the specific thermodynamic properties at the air-liquid interface drove metal complexation. LB films were deposited on Ti surfaces, and the resulting OPA/Sr-morin coatings exhibited high surface free energy and increase on its polar component. This optimized surface feature supported further serum protein adsorption and osteoblast growth and differentiation, indicating that these lipid-based coatings are promising for bioactive coating design. This study paves the way for the use of this lipid-based coating in the design of implants for faster osteointegration. Moreover, flavonoid-metal complexation at membranes could also help to shed light on the biological role played by flavonoids.

Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism.

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (<27 repeats) and long repeats (L) (>/=27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.

Inhibition of bovine endothelial cell activation in vitro by regulated expression of a transdominant inhibitor of NF-kappa B.

The activation of endothelial cells is a recurrent phenomenon linked to pathologic conditions such as inflammation, chronic arthritis, allo- and xenograft rejection. To inhibit endothelial cell activation we have constructed a transactivation-deficient derivative of the p65/RelA subunit of NF-kappa B, a transcription factor known to be crucial for the induction of adhesion molecules, cytokines and procoagulants in activated endothelial cells. This protein (p65RHD) comprises the Rel homology domain of the RelA subunit, retaining dimerization, DNA binding, and nuclear localization functions, but is deficient in transcriptional activation, and acts as a competitive inhibitor of NF-kappa B. Our data demonstrate that p65RHD is a potent and specific inhibitor of NF-kappa B-mediated induction of a number of genes, such as I kappa B alpha, IL-8, E-selectin, P-selectin, and tissue factor in endothelial cells. Furthermore, tetracycline-inducible expression of p65RHD in stably transfected primary endothelial cells inhibits the induction of gene expression equally well. This regulated system of gene expression provides the basis for a novel therapeutic approach to the pathologic effects of endothelial cell activation, especially in delayed xenograft rejection, by using transgenic animals as organ donors.

Statin-mediated cytoprotection of human vascular endothelial cells: a role for Kruppel-like factor 2-dependent induction of heme oxygenase-1.

BACKGROUND: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. METHODS/RESULTS: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. CONCLUSIONS: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.

New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain.

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.

Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury.

Heme oxygenase-1 (HO-1) is induced under a variety of pro-oxidant conditions such as those associated with ischemia-reperfusion injury (IRI) of transplanted organs. HO-1 cleaves the heme porphyrin ring releasing Fe2+, which induces the expression of the Fe2+ sequestering protein ferritin. By limiting the ability of Fe2+ to participate in the generation of free radicals through the Fenton reaction, ferritin acts as an anti-oxidant. We have previously shown that HO-1 protects transplanted organs from IRI. We have linked this protective effect with the anti-apoptotic action of HO-1. Whether the iron-binding properties of ferritin contributed to the protective effect of HO-1 was not clear. We now report that recombinant adenovirus mediated overexpression of the ferritin heavy chain (H-ferritin) gene protects rat livers from IRI and prevents hepatocellular damage upon transplantation into syngeneic recipients. The protective effect of H-ferritin is associated with the inhibition of endothelial cell and hepatocyte apoptosis in vivo. H-ferritin protects cultured endothelial cells from apoptosis induced by a variety of stimuli. These findings unveil the anti-apoptotic function of H-ferritin and suggest that H-ferritin can be used in a therapeutic manner to prevent liver IRI and thus maximize the organ donor pool used for transplantation.

In vivo depletion of xenoreactive natural antibodies with an anti-mu monoclonal antibody.

Hyperacute rejection of vascularized discordant xenografts, such as pig-to-primate kidney or heart xenotransplants, is thought to be mediated by xenoreactive natural antibodies (XNA) of the IgM isotype and the activation of the classic pathway of complement. Using the guinea pig-to-rat discordant xenograft model, we have developed a potential therapeutic protocol leading to long-term depletion of circulating IgM in adult animals. This protocol consists of the injection into adult LOU/C rats of an antirat IgM MAb (MARM-7) after splenectomy, plasma exchange, and the administration of an anti-B cell immunosuppressant, mycophenylate mofetil (RS61443). Splectomized plasma exchanged adult rats receiving RS61443 showed strongly decreased IgG and IgM serum concentrations for a relatively short period during which these isotypes remained nevertheless detectable by a sensitive ELISA technique. In contrast to IgM, IgG in serum returned, shortly after the end of this treatment, to normal concentrations. Splenectomy alone was able to significantly decrease, for a long period (more than 70 days), IgM but not IgG serum concentrations in these rats. During this treatment, IgM XNA concentration mirrored total IgM. The injection of MARM-7 MAb to adult LOU/C rats was able to deplete circulating IgM and IgM XNA for a period of several weeks during which IgM was undetectable by a sensitive ELISA technique. Depletion time was dose-dependent--the higher the dose of injected MARM-7, the longer the period for which IgM and IgM XNA remained undetectable. Moreover depletion of circulating IgM was correlated with the detection in the serum of these rats of noncomplexed, free MARM-7. Finally, MARM-7 administration was significantly more efficacious in rats that had decreased levels of circulating IgM after splenectomy, plasma exchange, and administration of RS61443. These experiments suggest that the anti-mu approach may allow depletion of IgM XNA for a sufficiently long period to test the hypothesis of "accommodation" in other xenograft models such as the pig-to-primate xenograft or even in ABO-incompatible allografts.

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts.

BACKGROUND: The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. METHODS: Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. RESULTS: Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. CONCLUSION: Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.

Survival of accommodated cardiac xenografts upon retransplantation into cyclosporine-treated recipients.

BACKGROUND: Accommodation designates the survival of vascularized grafts in the presence of circulating antigraft antibodies and complement. In the hamster-to-rat model, accommodation is associated with an ongoing T helper (Th)2 cytokine response and the expression of "protective genes" by the graft endothelial cells and smooth muscle cells. In this report, we tested whether accommodated xenografts would be protected from rejection upon retransplantation into second recipients treated with cyclosporine (CsA), a treatment that does not prolong survival of a fresh hamster heart. METHODS: Long-term survival of hamster-to-rat cardiac xenografts was achieved using either CsA plus cobra venom factor (CVF) or CsA plus rapamycin. Xenografts that survived long term in their first recipients were retransplanted into second recipients treated with CsA. RESULTS: Long-term xenograft survival in CsA/CVF-treated recipients was associated with an ongoing Th2 response, expression of protective genes, and deposition of elicited xenoreactive antibodies and complement on the graft endothelium. In CsA/rapamycin-treated recipients, long-term xenograft survival occurred in the presence of basal levels of antigraft antibodies and was not associated with a Th2 cytokine response or the expression of protective genes. Xenografts from CsA/CVF-treated rats survived significantly longer upon retransplantation into second recipients treated with CsA (77.3% >10 days) as compared with xenografts from CsA/rapamycin-treated rats (4-11 days) or naive hearts (3-4 days). Moreover, 30-35% of xenografts from CsA/CVF rats survived long term and accommodated in the second recipient. CONCLUSIONS: Accommodated xenografts can have significantly prolonged acceptance when retransplanted into second recipients treated with CsA alone; in contrast, naive hearts or hearts that survived long term in first recipients, but did not accommodate, did not survive long term in the second recipients. We suggest that prolonged survival of accommodated xenografts is due to the expression of the protective genes A20, bcl-2 bcl-xL, and heme oxygenase-1 in the xenograft endothelium and possibly smooth muscle cells.

Suppression of delayed xenograft rejection by specific depletion of elicited antibodies of the IgM isotype.

BACKGROUND: Hamster hearts transplanted into untreated rats undergo delayed xenograft rejection (DXR). This acute inflammatory response is associated with the deposition of anti-graft antibodies of the immunoglobulin (Ig)M isotype in the vasculature. We have previously shown that these antibodies are generated in a T cell-independent manner. In this study, we tested whether the generation of anti-graft IgM antibodies is involved in the pathogenesis of DXR. In addition, we tested whether the suppression of this antibody response would overcome DXR. METHODS: Hamster hearts were transplanted into rats treated with an anti-mu monoclonal antibodies (mAb) to deplete circulating IgM or with an isotype-matched control mAb recognizing the dinitrophenyl epitope. T cell immunosuppression was achieved with cyclosporin A (CsA). RESULTS: Depletion of circulating IgM by anti-mu mAb inhibited DXR, whereas the control mAb had no effect on DXR. In anti-mu-treated rats, xenografts were rejected 5-7 days after transplantation through a T cell-dependent mechanism associated with the generation of antibodies of the IgG isotype. Combination of anti-mu with CsA suppressed the anti-graft IgM and IgG response and resulted in long-term xenograft survival (> 50 days). Xenograft long term survival occurred despite the return of anti-graft IgM antibodies to the circulation, a phenomenon referred to as accommodation. CONCLUSION: This study demonstrates that the pathogenesis of DXR can be initiated by anti-graft antibodies of the IgM isotype, which are generated in a T-cell independent manner. In addition, we show that under T cell immunosuppression, specific depletion of this IgM response by anti-mu mAb administration results in xenograft long-term survival and accommodation.

Transient complement inhibition plus T-cell immunosuppression induces long-term survival of mouse-to-rat cardiac xenografts.

BACKGROUND: The use of anti-B-cell and T-cell immunosuppressive agents leads to only a few weeks' survival of mouse-to-rat cardiac xenografts. METHODS: BALB/c cardiac xenografts were transplanted to Lewis rats treated with cyclosporine (CsA) and/or cobra venom factor (CVF). RESULTS: CsA alone did not prolong xenograft survival (2.2+/-0.4 days), whereas CVF alone led to minimal prolongation of survival (5.6+/-0.8 days) as compared with nontreated recipients (2.4+/-0.5 days). The combination of CsA plus CVF, the latter given for either 2 days or 11 days, resulted in long-term survival of 14/16 hearts (> 100 days). Production of IgM elicited xenoreactive antibodies (EXA) peaked on day 4 after transplantation and decreased thereafter. Production of IgG EXA occurred only in the control group, whereas, in the CsA/CVF-treated group, IgG EXA were totally suppressed. Long-term surviving grafts showed (i) excellent preservation of morphology and minimal leukocyte infiltration, (ii) deposition of IgM, IgG and weak C3 deposition on the graft endothelium, (iii) low level infiltration by rat macrophages, (iv) replacement of mouse dendritic cells by class II+ rat macrophages, and (v) expression within endothelial and smooth muscle cells, macrophages, and myocytes of HO-1, a "protective gene" not seen in the rejected hearts. CONCLUSIONS: Our present findings suggest that long-term mouse-to-rat cardiac xenograft survival is induced by temporary suppression of C activation and sustained T-cell suppression leading to inhibition of IgG EXA production. Florid expression of a protective gene (HO-1) may contribute to survival.