Laser technique for the fabrication of blood vessels-like models for preclinical studies of pathologies under flow conditions.

In this work a method for fabricating functionalized preclinical devices is presented. The manufacturing process combines a laser indirect writing technique to fabricate a soda-lime glass master and soft-lithography methods to obtain the final structure in polydimethylsiloxane (PDMS). The roughness of the device is modified in a controlled manner by applying a post-thermal treatment to the master, and thus devices with different roughness values are created. The PDMS devices are fully covered with human umbilical vein cells in a two-step process. In order to determine the most suitable device to perform bioassays, the cell attachment to the channel is evaluated with regards to the walls roughness when flow experiments are carried out.

[Peroral endoscopic full and partial-thickness myotomy. A viability study in an animal model]. / Miotomía endoscópica por vía oral de espesor total y parcial. Estudio de factibilidad en un modelo animal.

BACKGROUND: Peroral endoscopic myotomy has recently been developed and performed on patients with good results. AIMS: To evaluate the technical feasibility of peroral endoscopic full-thickness and partial thickness myotomy in a porcine model. MATERIAL AND METHODS: Eighteen criollo pigs were randomly assigned to 2 groups: group A (partial-thickness myotomy) and group B (full-thickness myotomy). The mucosal defect proximal to the myotomy site was left open. On the seventh postoperative day the pig was euthanized and follow-up surgical exploration was performed. The duration of each procedure, postoperative progression of the animal, complications, and anatomopathologic findings were registered. RESULTS: The procedure was viable in all the pigs. The mean surgery duration was 81±35.3min (group A 51.11±11.12, group B 111±22.61; P<.05). The main complication during myotomy was subcutaneous emphysema (16%). The histopathologic study of the group A surgical specimens reported complete circular myotomy in all cases, and complete circular and longitudinal myotomy was reported in 100% of the group B sample. CONCLUSIONS: The endoscopic myotomy technique is feasible. Endoscopic partial-thickness myotomy was associated with shorter surgery duration and better results during the intraoperative period and the 7-day follow-up.

Analysis of different routes of administration of heterologous 5-azacytidine-treated mesenchymal stem cells in a porcine model of myocardial infarction.

Stem cell therapy constitutes an exciting, powerful therapy to repair the heart. Nevertheless, there are numerous doubts about the best route of stem cell administration to achieve implantation into the injured myocardium. Development of a preclinical, large animal model may be useful to obtain a better approach to clinical situations. The aim of this work was to study the effectiveness of various routes of heterologous bone marrow mesenchymal stem cell (MSCs) administration in a porcine model of myocardial infarction. MSC treated with 5-azacytidine were stained with a fluorescent compound (DiO) before their administration to previously infarcted pigs via 3 routes: intracoronary (IC), intramyocardial (IM), or endocardial (EC; n = 5 each group). Healthy, noninfarcted animals were used as a control group. At 30 days after delivery, hearts were divided into 12 parts: infarcted zone (1-6), right-left atria, interatrial and interventricular septa, and right-left ventricles. In each zone we looked for and quantified, injected fluorescence-stained cells. In the animals in which presence of DiO-stained cells was detected, cells were located preferentially in the infarcted zone and not in the atria, ventricles, or septa. Comparing various administration routes, the mean number of engrafted cells within the infarct zone was significantly greater after IC infusion than either IM or EC injection. Fluorescent cells were not observed in healthy zones of the myocardium or in healthy animals.

Gene expression profiles in a porcine model of infarction: differential expression after intracoronary injection of heterologous bone marrow mesenchymal cells.

Myocardial infarction is one of the main causes of mortality in developed countries. Injection of bone marrow mesenchymal stem cells (BMMSC) with the ability to regenerate lost cardiomyocytes is a promising therapy for heart failure. To evaluate this strategy, an in vivo porcine model of infarction was used. Gene expression profiles of 3 groups of pigs (n = 5 each) were analyzed and compared by real-time reverse transcription-polymerase chain reaction (RT-PCR). One of the groups underwent anterior descending coronary occlusion followed by BMMSC injection; a placebo group was injected with culture medium without cells after infarction; and a third group was formed by healthy pigs. Four weeks later, cells or medium was administered by intracoronary injection and, a month later, animals were sacrificed and samples collected. Genes related to cardiomyogenesis (Mef2C, Gata4, Nkx2.5), mobilization and homing of resident or circulating stem cells (Sdf1, Cxcr4, c-Kit), contractibility (Serca2a), and fibrosis (CollA1) were analyzed. Gene expression profiles changed in various heart areas in the 3 groups. Expression of genes related to cardiomyogenesis decreased in infarcted zones compared with homologous regions of healthy hearts. Sdf1 expression increased in the apex of infarcted hearts. Serca2a expression was reduced in the ventricles and atria of infarcted hearts. Also, increases in Cxcr4 and CollA1 expression were observed in infarcted hearts of cell-treated pigs compared with the placebo group. In conclusion, infarction induced changes in genes involved in various biological processes. Intracoronary injection of heterologous BMMSC resulted in localized changes in the expression of Cxcr4 and Col1A1.

Comparison of gene expression profiles in a porcine infarct model after intracoronary, transthoracic, or transendocardiac injection of heterologous bone marrow mesenchymal cells.

An in vivo porcine model of myocardial infarction was developed with the aim of comparing the effectiveness for cardiac repair of intracoronary, transthoracic, or transendocardial delivery strategies for bone marrow mesenchymal stem cells (BMMSC) using an analysis of expression levels of transcripts related to various cellular processes at 8 heart regions using quantitative reverse transcriptase polymerase chain reaction. We observed significant rises in cardiomyogenic markers Mef2C, Gata4 and Nkx2.5, and contractibility marker Serca2A at infarcted regions for cell-treated pigs. We also observed differences in Sdf1 expression related to the organ stress response between delivery strategies. Unexpectedly, increased expression of Col1A1 was detected in 2 cell-treated groups at various heart regions. Our results suggest improvements in both contractility and cardiomyogenic capability of damaged tissue after BMMSC injection, but also warned us about the relevance of the chosen delivery strategy and potential undesired effects like increasing fibrosis after treatment.

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

The serum level of xenoantibodies, and hDAF or alphaGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage.

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and alphaGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-alphaGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and alphaGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of alphaGal on pig cells.

Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay.

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-alphaGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-alphaGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.

Neither acute rejection nor immunosuppressant drug therapy (cyclosporine or tacrolimus) correlates with expression of either CD40 or CD154 on peripheral blood cells among human cardiac transplant patients.

Acute allograft rejection (AAR) is an important cause of graft loss following heart transplantation (HT). Increasing evidence shows that CD40-CD154 interactions play a central role in the immune processes leading to AAR. In this study we investigated the expression of CD40 and CD154 on peripheral blood cells from HT patients so as to determine possible association with AAR. Using two-color flow cytometry, we determined the expression of CD40 and CD154 in 102 samples of peripheral blood taken from 53 adult HT patients and in 17 samples from healthy adult volunteers. Samples from patients were obtained at the same time as endomyocardial biopsy was performed. We analyzed the relationships between the expression of these molecules and the following parameters: immunosuppressive treatment (cyclosporine vs tacrolimus), gender, age, time post-HT, and AAR (indicated by an ISHLT rating > or =3A). The percentages of HT patients' blood samples showing above-normal CD40 or CD154 expression did not differ significantly from those of controls. The percentage of patients' samples showing above-normal CD40 expression decreased with time after HT. Expression of these molecules was not above normal during rejection episodes, and for neither was there any statistically significant correlation between expression level and the immunosuppressor drug.

Porcine endothelial cell activation in hDAF pig hearts transplanted in baboons with prolonged survival and lack of rejection.

Depletion of anti-alphaGal antibodies before and after transplantation with GAS 914, a polylysine containing alphaGal epitopes, together with immunosuppression, has been shown to prevent acute humoral xenograft rejection (AHXR) in hDAF pig-to-baboon xenotransplantation. This therapy was associated with low levels of serum anti-alphaGal antibodies and lack of antipig hemolytic antibodies (APA) during the entire transplant course. In the present study we investigated the condition of xenograft endothelial cells and the presence of other antipig antibodies. No xenograft failed because of AHXR. However, endothelial cell markers of activation, such as CD62, CD106, ET-1, and particularly 5A6/8, were detected at necropsy, along with a lack or scarce deposits of IgM and total absence of complement and fibrin. The endothelial cell markers were negative or slightly positive at biopsy obtained 30 minutes after transplantation. At the time of animal death serum xenoantibodies against pig aortic cells were also detected by immunochemistry whereas anti-alphaGal and APHA were almost absent, suggesting that the presence of non-anti-alphaGal and noncytotoxic xenoantibodies may cause endothelial activation.

Monitoring cytotoxicity against pig cells after transplantation using two-color fluorescence viability assay.

Acute humoral xenograft rejection (AHXR) is now the major hurdle for the long-term survival of pig organs transplanted into nonhuman primates. Mechanisms involved in this rejection are not well understood, albeit that it has been proposed to require the participation of antibodies with specificities other than alphaGal. In this study, we evaluated a two-color fluorescence method, fluorescein dicetate (FAD)/propodium iodide (PI), to stain live versus dead cells, respectively, to monitor complement-mediated antibody cytotoxicity in hDAF pig-to-baboon xenotrasplants. FDA/PI flow cytometry assays showed a high correlation (rho Spearman=.736; P=.003) with the cytotoxic activities of baboon serum antibodies against PK15 cells, using either endogenous or exogenous complement. Average serum cytotoxicity against AOC40 was higher (59.82+/-17.90) compared with PK15 (33.69+/-13.05) and L35 (37.64+/-12.77) cells, albeit the difference did not reach statistical significance. Incubation of serum samples with low-molecular weight heparin reduced serum cytotoxicity against PK15 cells in dose-dependent fashion. Therefore, FDA-PI two-color fluorescence is a suitable method to study antibody-mediated cytotoxicity by endogenous or exogenous complement for various pig cells.

Elicited non-anti-alphaGAL antibodies may cause acute humoral rejection of hDAF pig organs transplanted in baboons.

The combination of immunosuppression and GAS 914, a polylysine containing alphaGal trisaccharide type 2 (TRI 2), has been associated with the prevention of acute humoral xenograft rejection (AHXR) in human decay accelerating factor (hDAF) pig-to-baboon xenotransplants. The aim of this study was to investigate the role of immunosuppression and GAS 914 to neutralize xenoantibodies before and after xenotransplantation. Eight baboons underwent heteropic heart xenotransplantation with hDAF transgenic pig organs, receiving GAS 914 before and after transplantation. Six baboons (Group A) were treated with an immunosuppression protocol that included cyclophosphamide (CyP), Neoral, ERL, and steroids. The other 2 baboons (Group B) were treated with the same immunosuppression but with a 50% reduction in the doses of CyP. No xenograft from Group A underwent acute humoral xenograft (median survival, 27 days), whereas the 2 in Group B experienced rejection (median survival, 6 days). GAS 914 depleted both immunoglobulin (Ig)M and IgG anti-alphaGAL disaccharide (DI), trisaccharide type 2 (TRI 2), and trisaccharide type 6 (TRI 6), before and after transplantation in Groups A and B. However, cytotoxic antibodies with other anti-pig specificities were elicited by the xenografts in Group B leading to AHXR.

Coronary stent implantation in diabetic versus nondiabetic patients. Early and late outcomes.

OBJECTIVE: To assess whether coronary stenting in diabetic patients provides in-hospital results and clinical evolution similar to those in nondiabetic patients. METHODS: From July '97 to April '99 we performed coronary stent implantation in 386 patients with coronary heart disease, who were divided into two groups: diabetic patients and nondiabetic patients. The in-hospital results and the clinical evolution of each group were retrospectively analyzed. RESULTS: The nondiabetic group comprised 305 (79%) patients and the diabetic group 81 (21%) patients. Basic clinical and angiographic characteristics were similar. Angiographic success was in diabetics = 96.6% vs in nondiabetics = 97.9% (p=ns). Among the major complications in the in-hospital phase, the rate of myocardial infarction was higher in the diabetic group (7.4% vs 1.9%) (p=0.022). In the follow-up, a favorable and homogeneous evolution occurred in regard to asymptomatic patients, myocardial infarction, and death in the groups. A greater need for revascularization, however, existed in the diabetic patients (15% vs 2.4%, p<0.001). CONCLUSION: Coronary stenting in diabetic patients is an efficient procedure, with a high angiographic and clinical success rate similar to that in nondiabetic patients. Diabetic patients, however, had a higher incidence of in-hospital myocardial infarction and a greater need for additional myocardial revascularization.

[Vulvar angiomyofibroblastoma. Report of a case and review of the literature]. / Angiomiofibroblastoma vulvar. Informe de un caso y revisión de la literatura.

Angiomyofibroblastoma is a rare mesenchymal tumor predominantly of vulvar location. It is characterized being of superficial and slow-growth, low propensity for local recurrence and often misdiagnosed as a Bartholin's gland cyst, hydrocele of the canal of Nuck and aggressive angiomyxoma. A case of vulvar angiomyofibroblastoma in a 56 year-old woman is presented. The specimen was processed by structural and immunohistochemical analysis. It's important to suspect the diagnosis and to distinguish it of aggressive angiomyxoma.