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Biomedical subjects

A Centeno

Publications and source records attributed to A Centeno.

15 recordsLinked to original sources
PubMed2004

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.

Animals↗
PubMed2004

[Experimental model of auxiliary heterotopic liver-intestinal transplantation].

In pigs, orthotopic liver-intestine transplantation (LITX) has high per operative morbidity and mortality. It is due to hemodynamic, coagulation and metabolism disorders during native liver hepatectomy (total hepatic vascular exclusion) and the postoperative diarrhea secondary to initial dysfunction of the graft and enterectomy of native intestine. To avoid those disturbances and to increase the survival, we have developed a porcine model of auxiliary heterotopic LITX. The allograft was harvested in-bloc, containing the liver, duodenum, pancreas, and jejunum. In the recipient, the liver and intestine were left intact. The allograft was implanted heterotopically, caudal to the native liver. Venous drainage was achieved with anastomosis of donor (D) to recipient (R) infrahepatic cava; and arterialization with anastomosis of D aortic conduit containing the celiac axis and SMA to infra-renal R aorta. The D jejunum was hooked-up to R jejunum. The experiment was performed in 16 animals without intraoperative deaths, hemodynamic stability and no blood requirements. Four animals were left alive 7 days with functioning grafts, suggesting the model viability.

Animals↗
PubMed2003

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.

Acute Disease↗
PubMed2003

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.

Acute Disease↗
PubMed2003

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.

Animals↗
PubMed2001

Removal of bowel aerobic gram-negative bacteria is more effective than immunosuppression with cyclophosphamide and steroids to decrease natural alpha-galactosyl IgG antibodies.

Natural alpha-Galactosyl (Gal) antibodies play an important role in the rejection of pig xenografts by humans and Old World monkeys. In this study we investigate the efficacy of two different strategies to reduce the serum level of natural anti-Gal antibodies. On the one hand, removal of aerobic gram-negative bacteria from the intestinal flora, because anti-Gal antibodies appear to be produced as a result of the continuous sensitization by these microorganisms. On the other hand, we studied the effect on these antibodies of an immunosuppressive regimen of cyclophosphamide and steroids. Ten baboons were treated for three months with norfloxacin (Nor Group; n=6) or cyclophosphamide and steroids (CyP Group; n=4). A further four baboons did not receive any treatment (Control Group). Aerobic gram-negative bacteria became negative in stools of the Nor Group after two weeks of treatment, and remained undetectable until week 7. Thereafter, a gradual increase on the fecal concentration of aerobic gram-negative bacteria was observed despite the norfloxacin treatment. The mean anti-Gal IgG in the Nor Group gradually declined from week 4 to 9 to a mean of 62.7 +/- 18% of the baseline level, and during this period were significantly lower than in the CyP (P<0.02) and the Control (P<0.05) groups. No differences were observed between the three groups during the 16 weeks of follow-up in serum levels of anti-Gal IgM, hemolytic anti-pig antibodies, total IgG, IgM and IgA. In conclusion, removal of normal aerobic gram-negative bacteria from the intestinal flora is more effective than immunosuppression with CyP and steroids in reducing the level of natural anti-Gal antibodies, although there is no discernible effect on IgM antibodies.

Animals↗
PubMed1995

[Videothoracoscopy in staging of lung cancer].

We present a review of 108 cases of lung cancer in patients who, for a variety of reasons outlined in this report, underwent video assisted thoracoscopy prior to staging. Overall results of the procedure were positive in 94%. We report the usefulness of this tool for identifying adenopathic locations, for effecting biopsies of tumors that could not be typed by other means, for resecting peripheral nodules for screening purposes, and for making etiological diagnoses of accompanying pleural effusions. For diagnostic purposes, this procedure provides a better image than does conventional thoracoscopy, involves the participation of an endoscopy team and even calls for the presence of a pathologist. We consider video assisted thoracoscopy to be a highly effective tool for avoiding so-called pointless thoracotomies and for completing alternative treatments. Its cost effectiveness is indisputable, as are its low rates of morbidity and mortality.

Adult↗
PubMed1983

Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells.

Adhesiveness↗
PubMedDate not supplied

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.

Animals↗
PubMedDate not supplied

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.

Animals↗