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2.
Transplant Proc ; 51(3): 987-992, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30979492

RESUMO

BACKGROUND: To explore the adsorption of heterologous antibodies in 6 xenotransplants of Landrace piglet kidneys into rhesus monkeys. METHODS: The Landrace piglets and rhesus monkeys were used as donors and recipients, respectively. The donor kidney was the left kidney excised from each Landrace piglet and lavaged with University of Wisconsin solution through the renal artery and vein ex vivo. The renal arteriovenous end of the recipient was preserved. After anastomosis of the renal artery and vein with the arteriovenous end of the recipient for reperfusion, a cross-lymphocyte cytotoxicity test of the heterogeneous kidney was performed. RESULTS: All 6 Landrace piglet kidneys absorbed heterologous antibodies that were pre-existing in the rhesus macaques' kidneys. The cross-lymphocyte toxicity test was performed after the kidney were completely blackened. The cross-lymphocyte toxicity in all each heterogeneous kidney changed from strong positive to weak positive. CONCLUSIONS: Heterologous antibodies were adsorbed in xenotransplants of Landrace piglet kidneys into rhesus monkeys. Xenotransplanted kidney can adsorb heterologous antibodies and consume relevant complements, which is a good model for research of hyperacute rejection in xenotransplantation.


Assuntos
Anticorpos Heterófilos/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim , Rim/imunologia , Adenosina , Adsorção , Alopurinol , Animais , Anticorpos Heterófilos/metabolismo , Modelos Animais de Doenças , Feminino , Glutationa , Insulina , Macaca mulatta , Soluções para Preservação de Órgãos , Rafinose , Suínos , Doadores de Tecidos , Transplante Heterólogo
3.
Clin Biochem ; 66: 103-105, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30738031

RESUMO

We report a case of a heterophile antibodies interference in a new high-sensitivity troponin commercial immunoassay (cTNIH Siemens), observed in a patient with possible acute coronary syndrome (ACS). The analytical interference was investigated with standard laboratories procedures. The false positive result was found with different troponin methods and kits. We also investigated the protein sequence of cTnl and no sequence variants were detected. The discordance between clinical pictures and high concentration of cTnl, together with the collaboration between clinicians and laboratory staff avoided possible erroneous diagnosis and further invasive investigations to the patient.


Assuntos
Dor no Peito/sangue , Troponina I/sangue , Animais , Anticorpos Heterófilos/imunologia , Anticorpos Monoclonais/imunologia , Bovinos , Reações Falso-Positivas , Cabras , Humanos , Imunoensaio/métodos , Masculino , Camundongos , Pessoa de Meia-Idade , Ovinos , Troponina I/imunologia
5.
Avian Pathol ; 48(2): 157-167, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30570345

RESUMO

Avian pathogenic E. coli (APEC) cause severe respiratory and systemic disease. To address the genetic and immunological basis of resistance, inbred chicken lines were used to establish a model of differential resistance to APEC, using strain O1 of serotype O1:K1:H7. Inbred lines 72, 15I and C.B12 and the outbred line Novogen Brown were inoculated via the airsac with a high dose (107 colony-forming units, CFU) or low dose (105 CFU) of APEC O1. Clinical signs, colibacillosis lesion score and bacterial colonization of tissues after high dose challenge were significantly higher in line 15I and C.B12 birds. The majority of the 15I and C.B12 birds succumbed to the infection by 14 h post-infection, whilst none of the line 72 and the Novogen Brown birds developed clinical signs. No difference was observed after low dose challenge. In a repeat study, inbred lines 72 and 15I were inoculated with low, intermediate or high doses of APEC O1 ranging from 105 to 107 CFU. The colonization of lung was highest in line 15I after high dose challenge and birds developed clinical signs; however, colonization of blood and spleen, clinical signs and lesion score were not different between lines. No difference was observed after intermediate or low dose challenge. Ex vivo, the phagocytic and bactericidal activity of lung leukocytes from line 72 and 15I birds did not differ. Our data suggest that although differential resistance of inbred lines 72, 15I and C.B12 to APEC O1 challenge is apparent, it is dependent on the infectious dose. Research Highlights Lines 15I and C.B12 are more susceptible than line 72 to a high dose of APEC O1. Differential resistance is dose-dependent in lines 15I and 72. Phagocytic and bactericidal activity is similar and dose independent.


Assuntos
Galinhas , Resistência à Doença , Infecções por Escherichia coli/veterinária , Escherichia coli/imunologia , Imunidade Inata , Doenças das Aves Domésticas/imunologia , Sacos Aéreos/microbiologia , Animais , Animais Endogâmicos , Anticorpos Heterófilos/imunologia , Carga Bacteriana , Relação Dose-Resposta Imunológica , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Macrófagos/imunologia , Masculino , Doenças das Aves Domésticas/microbiologia , Organismos Livres de Patógenos Específicos
6.
Exp Oncol ; 40(4): 275-281, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30593747

RESUMO

AIM: To investigate the effect of chicken embryo proteins (CEP) as a prototype of xenogeneic vaccine on immune reactions in mice immunized after Lewis lung carcinoma (LLC) surgical removal. MATERIALS AND METHODS: C57Bl male mice were immunized on days 1, 8, and 15 after surgical removal of LLC. The immune response was assessed on days 7, 14, 21 and 28 after tumor resection. Cytotoxic activity of natural killer cells (NK) and cytotoxic T-lymphocytes as well as antibody dependent cellular cytotoxicity was estimated in MTT-assay; specific antibodies were detected in ELISA; lymphocyte proliferation was tested in reaction of in vitro blast transformation. RESULTS: None of the immunized mice developed LLC metastases. Immunization with CEP seems to prevent the potential decrease in NK cell cytotoxic activity and spontaneous blast transformation activity of lymphocytes following the surgically induced stress. Further research on improving immunization schedule and elucidating the mechanisms of NK modulation with CEP is needed.


Assuntos
Proteínas Aviárias/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Animais , Anticorpos Heterófilos/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/secundário , Embrião de Galinha , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Imunização , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia
7.
J Thromb Haemost ; 16(9): 1779-1788, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29981270

RESUMO

Essentials Inhibitor formation remains a challenging complication of hemophilia A care. The Bethesda assay is the primary method used for determining bleeding risk and management. Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance. Antibodies that increase clearance contribute to antibody pathogenicity. SUMMARY: Background The development of neutralizing anti-factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low-titer inhibitors.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Fator VIII/imunologia , Animais , Anticorpos Heterófilos/administração & dosagem , Anticorpos Heterófilos/imunologia , Anticorpos Heterófilos/toxicidade , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/toxicidade , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/toxicidade , Epitopos/imunologia , Fator VIII/antagonistas & inibidores , Fator VIII/farmacocinética , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Hemorragia/etiologia , Concentração Inibidora 50 , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Modelos Animais , Fenótipo , Domínios Proteicos , Doenças de von Willebrand , Fator de von Willebrand/metabolismo
8.
Am J Transplant ; 18(11): 2763-2771, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29603642

RESUMO

Autoantibodies to the angiotensin II type 1 receptor (AT1R) are thought to be important in antibody-mediated rejection (AMR), especially in the absence of anti-HLA antibodies. We used a variety of methods to examine the specificity of a commercially available kit designed to quantitate anti-AT1R antibodies. We found that fibrin formation in serum samples from patients awaiting cardiac transplantation with ventricular assist devices (VADs) can produce falsely elevated anti-AT1R values. In addition, absorption studies with a variety of cell lines with or without expression of human AT1R, and those that express xenoantigens, suggest that many of the antibodies detected in the AT1R test system are heterophilic and have reactivity to xenoantigens. Furthermore, we provide data that show that reactivity to the sialic acid Neu5Gc is a common finding among samples that are highest in anti-AT1R levels. We conclude that a common laboratory method for quantitation of anti-AT1R antibodies is nonspecific and overestimates the frequency of true positives. A reevaluation of the role that anti-AT1R antibodies play in allograft function and patient outcomes is warranted.


Assuntos
Anticorpos Heterófilos/sangue , Anticorpos Heterófilos/imunologia , Fibrina/metabolismo , Transplante de Coração , Coração Auxiliar , Ácidos Neuramínicos/imunologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células CHO , Bovinos , Galinhas , Cricetulus , Feminino , Fibrina/imunologia , Humanos , Masculino , Receptor Tipo 1 de Angiotensina/imunologia , Transplantados
11.
Hinyokika Kiyo ; 63(10): 435-437, 2017 Oct.
Artigo em Japonês | MEDLINE | ID: mdl-29103259

RESUMO

We described a 63-year-old man who was diagnosed with clinical T1c prostate cancer, with a Gleason score of 6 (3+3), and a preoperative prostate-specific antigen (PSA) level of 5. 27 ng/ml. Radical prostatectomy(RP) was performed and final pathologyshowed Gleason score 3+4, pT2c with negative surgical margin. In spite of suggested surgical radicality, PSA was 3.32, 4.78, 5.93 ng/ml, at 1, 2, and 3 months after RP, respectively. However, radiological investigation revealed no metastasis. Because of this clinical discrepancy, we checked the PSA-α1-antichemotrypsin level and found it to be ≦0.1 ng/ml. From these results, false PSA elevation caused byinterference of positive heterophilic antibodies was suggested and demonstrated byseveral immunoassays.


Assuntos
Anticorpos Heterófilos/sangue , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Anticorpos Heterófilos/imunologia , Reações Falso-Positivas , Humanos , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/imunologia , Prostatectomia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia
12.
PLoS One ; 12(2): e0172188, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28222144

RESUMO

The use of heterologous immunoassays containing antibodies raised against a different biological species for quantification of serum proteins is studied and discussed, taking as example the case of the use of a commercially available heterologous assay containing antibodies against human C-reactive protein (hCRP) for quantification of CRP in serum of dogs. This assay was adapted and validated for measurements of canine CRP (cCRP) and compared with three different homologous assays containing species-specific canine antibodies, which are currently commercially available for cCRP determination. Serum samples from healthy and diseased dogs (n = 44) were used. Analytical evaluation included precision, accuracy, limit of detection and lower limit of quantification for all assays. In the case of the heterologous assay also cross-reactivity of the antibody of the heterologous assay with cCRP was evaluated by a Western-Blot analysis giving a positive result. The heterologous assay showed similar results than the homologous assays in all the tests of the analytical evaluation that indicated that the assay was precise and accurate. Method comparison showed a high correlation between all assays (r≥0.9). The Bland-Altman test revealed that the heterologous assay showed a proportional error when compared with the homologous automated assays and a random error when compared with the point-of-care assay. All four CRP assays were able to detect higher CRP values in dogs with inflammatory conditions compared with healthy dogs. It is concluded that heterologous immunoassays could be used for quantification of serum proteins in different species, provided that the antibody has cross-reactivity with the protein to be measured and the assay give satisfactory results in the analytical validation tests. In addition, use of species-specific calibrators and an appropriate batch validation are recommended in these cases.


Assuntos
Anticorpos Heterófilos/imunologia , Proteína C-Reativa/análise , Imunoensaio/métodos , Animais , Western Blotting , Proteína C-Reativa/imunologia , Reações Cruzadas/imunologia , Doenças do Cão/imunologia , Cães , Feminino , Humanos , Imunoensaio/veterinária , Inflamação/imunologia , Inflamação/veterinária , Masculino , Reprodutibilidade dos Testes
13.
BMJ Case Rep ; 20162016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27908913

RESUMO

A previously well woman aged 63 years presents to the emergency department with vomiting, palpitations and 3 presyncopal episodes. She had no previous medical or cardiac history, with the patient stating that she tried a herbal remedy of boiled comfrey leaves for insomnia 18 hours before arrival to the department. Her ECG showed multiple abnormalities, including bradycardia, second-degree atrioventricular node block, Mobitz Type 2, a shortened QT interval, downsloping ST depression and presence of U waves. After viewing the images of comfrey and foxglove, it highlighted the possibility of mistaken ingestion of Digitalis, containing the organic forms of cardiac glycosides, such as digoxin and digitoxin. Raised serum digoxin levels confirmed this. The patient was haemodynamically stable, and given digoxin-binding antibodies. After 5 days of cardiac monitoring, her ECG returned to normal rhythm, and she was discharged home.


Assuntos
Acidentes , Anticorpos Heterófilos/uso terapêutico , Bloqueio Atrioventricular/induzido quimicamente , Confrei , Digitalis/envenenamento , Digoxina/envenenamento , Intoxicação por Plantas/diagnóstico , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Anticorpos Heterófilos/imunologia , Bradicardia/etiologia , Digitalis/imunologia , Digoxina/imunologia , Eletrocardiografia , Feminino , Humanos , Pessoa de Meia-Idade , Folhas de Planta/envenenamento , Intoxicação por Plantas/complicações , Intoxicação por Plantas/tratamento farmacológico , Plantas Medicinais , Resultado do Tratamento , Vômito/etiologia
14.
Biochem Biophys Res Commun ; 480(3): 474-478, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27773813

RESUMO

The number of patients in need of organ transplantation is continuously on the rise. However, because of organ donor shortage, xenotransplantation has been highlighted as an alternative. Among the various porcine organs and tissues, porcine islets are considered to be the best-matching implantable candidates for clinical application based on recent progress in nonhuman primate pre-clinical studies. Nevertheless, before initiation of clinical trials, it should be confirmed whether the requisite xeno-antigen sensitization would have a deleterious effect on subsequent allo-transplantation or vice versa. Therefore, in the present study, the survival rate of islets grafted in naïve recipients was compared with that in cross-sensitized recipients. Enzyme-linked immunosorbent spot, fluorescence-activated cell sorting, and immunohistochemistry were conducted to assess the cellular and humoral immune responses. The survival days of Balb/c mouse islets transplanted into B6 mice that had been previously sensitized with porcine cells (i.e., xeno-sensitized) showed no significant difference from that of naïve B6 mice. Moreover, the survival days of porcine islets transplanted into allo-antigen (Balb/c)-sensitized B6 recipients was not significantly different from that in naïve B6 mice. Furthermore, our data provide the first demonstration that the cellular xenogeneic immune response (against porcine antigen) measured by an enzyme-linked immunosorbent spot assay is not cross-reactive to the allogeneic immune responses in a murine islet transplantation model. These results suggest that clinical application of islet xenotransplantation is not likely to have a deleterious effect on subsequent allogeneic islet transplantation.


Assuntos
Antígenos Heterófilos/imunologia , Reações Cruzadas/imunologia , Sobrevivência de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/imunologia , Isoantígenos/imunologia , Animais , Anticorpos Heterófilos/imunologia , Imunização/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Suínos
15.
J Small Anim Pract ; 57(11): 626-630, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27726133

RESUMO

OBJECTIVE: The clinical feasibility of passive immunotherapy has not been demonstrated in dogs naturally infected with canine distemper. In this study, porcine anti-canine distemper virus IgG and F(ab')2 antibody fragments were used to treat infected puppies. METHODS: A total of 41 naturally infected puppies (age Äsix months) exhibiting severe respiratory signs, but lacking neurological signs, were enrolled in the study. Twenty-five puppies were treated with a combination of IgG or F(ab')2 antibody fragments (Group 1) and supportive therapy and 16 puppies received routine supportive care only (Group 2). RESULTS: The survival rate of dogs in Group 1 (19/25; 76%) was significantly higher than that in Group 2 (5/16; 31·3%) (P<0·05). During the therapy, 8 of the 25 dogs (32%) in Group 1 developed neurological signs versus 12 of the 16 dogs (75%) in Group 2 (P<0·05). Adverse reactions were limited to elevated body temperature in dogs that received IgG antibodies. CLINICAL SIGNIFICANCE: Porcine anti-canine distemper virus antibodies improved survival in puppies affected with canine distemper with minimal adverse effects. Therefore, this therapy could be considered for treatment of endangered animal species infected with canine distemper virus.


Assuntos
Anticorpos Heterófilos/imunologia , Anticorpos Antivirais/imunologia , Vírus da Cinomose Canina/imunologia , Cinomose/prevenção & controle , Vacinas Virais/administração & dosagem , Animais , Animais Recém-Nascidos , Cães , Feminino , Masculino , Resultado do Tratamento , Vacinação/veterinária
16.
Biomed Pharmacother ; 83: 1247-1252, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27565847

RESUMO

An accumulating body of evidence suggests that xenogeneic vaccines can be very effective in breaking the immune tolerance to human tumor-associated antigens (TAAs). We assessed adverse effects, as well as clinical and immune responses induced by a lyophilized xenogeneic polyantigenic vaccine (XPV) prepared from murine melanoma B16 and carcinoma LLC cells in 60 stage IV colorectal cancer patients. Neither grade III/IV toxicities, nor laboratory and clinical signs of systemic severe autoimmune disorders were documented in any XPV-treated patient. Clinical effects of various grades (complete response, partial response and disease stabilization) with duration of no shorter than 6 months was observed in 25 (41.67%) vaccinated patients. The average survival time of the XPV-treated patients was markedly longer than that of the clinically matched control patients (20 vs. 7 months). The overall 3-year survival rate in the XPV-treated and control group was 16.7% (10 patients) and 0%, respectively. Following a course of ten XPV vaccinations, peripheral blood mononuclear cell (PBMC) proliferation assays revealed increased T-cell immune responses to human Caco-2 colon adenocarcinoma-associated antigens. In addition, relative contents of CD25+ FoxP3+regulatory T-cells in patients with proven immunotherapy-mediated clinical effects (responders) were significantly decreased in the blood, which was paralleled by marked increases in serum levels of proinflammatory cytokines, such as interferon-alpha (IFN-α), IFN-É£, and interleukin-8 (IL-8). Serum levels of tumor necrosis factor-alpha (TNF-α), IL-1, IL-4, and IL-6 were not affected in both responder and non-responder patients. In conclusion, this study provides evidence for the safety, clinical feasibility and immunogenicity of xenogeneic composite cell vaccine administration in colorectal cancer patients. This is the first demonstration that clinical effects of such a vaccine are associated with vaccine-induced, proinflammatory immune responses.


Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Imunidade Celular/imunologia , Imunoterapia Ativa/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Heterófilos/imunologia , Feminino , Seguimentos , Humanos , Masculino , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Resultado do Tratamento
17.
Transfusion ; 56(10): 2495-2501, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27383738

RESUMO

BACKGROUND: Complement has significant status in the field of transfusion medicine. The accepted stability profile of complement is based on historical studies of diluted human serum hemolyzing rabbit heterophile antibody-sensitized sheep red blood cells (RBCs). Contemporary tools are available to reevaluate these historical observations using human heterophile antibodies, undiluted serum, and antigen-modified human RBCs. STUDY DESIGN AND METHODS: Human RBCs were made into "animal-like" kodecytes with heterophile Galα3Galß4GlcNAcß function-spacer-lipid constructs. These α-Gal-kodecytes were prepared with an antigen dilution capable of consistently producing 50% antibody-mediated hemolysis against human α1-3galactose heterophile antibodies and undiluted standardized serum. Standardized human serum aliquots from a two-donor pool stored at -85, -20, 4, 22, and 37°C for durations of up to 150 days were evaluated for loss of hemolytic activity. Where practical methodologic procedures were aligned with historical studies. RESULTS: Comparison of the historical assay with the α-Gal-kodecyte assay against complement activity standards showed concordance. However, in most scenarios complement was found to be more than twice as stable as generally accepted. At least 60% of complement hemolytic activity was observed in serum stored at 22°C for 1 week or 2 months at 4°C. No loss of hemolytic activity was observed after 5 months' storage at temperatures below -20°C. CONCLUSIONS: An alternative method using undiluted serum and modified human RBCs observed that classical-pathway complement hemolytic activity in stored human serum is at least twice as stable as previously accepted.


Assuntos
Anticorpos Heterófilos/imunologia , Via Clássica do Complemento , Proteínas do Sistema Complemento/química , Hemólise/imunologia , Animais , Antígenos Heterófilos/imunologia , Ativação do Complemento , Proteínas do Sistema Complemento/imunologia , Galactose/imunologia , Humanos , Estabilidade Proteica
18.
Basic Res Cardiol ; 111(4): 39, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27154491

RESUMO

Pre-clinical and clinical data have unequivocally demonstrated the usefulness of decellularized heart valve (HV) matrices implanted for HV replacement therapy. However, human donor valves applicable for decellularization are in short supply, which prompts the search for suitable alternatives, such as porcine grafts. Since decellularization might be insufficient to remove all xenoantigens, we analysed the interaction of human preformed antibodies with decellularized porcine HV in vitro to assess potential immune reactions upon implantation. Detergent-decellularized pulmonary HV from German Landrace wild-type (wt) or α1,3-galactosyltransferase knockout (GGTA1-KO) pigs were investigated by inhibition ELISA and GSL I-B4 staining to localize and quantify matrix-bound αGal epitopes, which represent the most prominent xenoantigen. Additionally, preformed human xenoantibodies were affinity purified by perfusing porcine kidneys. Binding of purified human antibodies to decellularized HV was investigated by inhibition ELISA. Furthermore, binding of human plasma proteins to decellularized matrices was determined by western blot. Decellularized human pulmonary artery served as controls. Decellularization of wt HV led to a reduction of αGal epitopes by 70 %. Residual epitopes were associated with the subendothelial extracellular matrix. As expected, no αGal epitopes were found on decellularized GGTA1-KO matrix. The strongest binding of preformed human anti-pig antibodies was found on wt matrices, whereas GGTA1-KO matrices bound similar or even fewer xenoantibodies than human controls. These results demonstrate the suitability of GGTA1-KO pigs as donors for decellularized heart valves for human patients. Besides the presence of αGal antibodies on decellularized heart valves, no further preformed xenoantibodies against porcine matrix were detected in tested human sera.


Assuntos
Anticorpos Heterófilos/imunologia , Galactosiltransferases/deficiência , Próteses Valvulares Cardíacas , Valvas Cardíacas/imunologia , Xenoenxertos/imunologia , Animais , Antígenos Heterófilos/imunologia , Bioprótese , Western Blotting , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Suínos
19.
Clin Biochem ; 49(9): 729-731, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26968106

RESUMO

OBJECTIVES: This case report investigates the origin of a false positive result on a serum qualitative human chorionic gonadotropin (hCG) device. PATIENT AND METHODS: A 46-year-old woman diagnosed with chronic myeloid leukemia presented with nausea and vomiting. A qualitative serum hCG test was interpreted as positive; however, a quantitative serum hCG test was negative (<5IU/L). To further investigate this discrepancy, the sample was pretreated with heterophilic blocking reagent (HBR). Additionally, the sample was tested on other qualitative hCG devices composed of antibodies from different animal sources. Blocking reagent from an automated quantitative immunoassay was also tested for its ability to inhibit the heterophile antibody interference. RESULTS: The qualitative test result was negative after pretreatment with heterophilic blocking reagent. Other devices composed of antibodies from different animal sources also demonstrated mixed results with the patient's sample. Blocking reagent obtained from the automated quantitative assay inhibited the heterophile antibody interference in the patient's sample. CONCLUSION: This case demonstrates that positive serum point-of-care hCG results should be interpreted with caution and confirmed with a quantitative serum hCG immunoassay when clinical suspicion is raised.


Assuntos
Anticorpos Heterófilos/imunologia , Biomarcadores/análise , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/urina , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Urinálise/instrumentação , Anticorpos Heterófilos/sangue , Reações Falso-Positivas , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/urina , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/normas , Prognóstico , Urinálise/métodos
20.
Transplantation ; 100(3): 571-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26906939

RESUMO

BACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.


Assuntos
Plaquetas/metabolismo , Galactosiltransferases/genética , Fígado/metabolismo , Oxigenases de Função Mista/genética , Fagocitose , Trombocitopenia/prevenção & controle , Animais , Animais Geneticamente Modificados , Anticorpos Heterófilos/imunologia , Anticorpos Heterófilos/metabolismo , Antígenos Heterófilos/imunologia , Antígenos Heterófilos/metabolismo , Receptor de Asialoglicoproteína/deficiência , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/imunologia , Plaquetas/imunologia , Células Cultivadas , Galactosiltransferases/deficiência , Galactosiltransferases/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Fígado/imunologia , Oxigenases de Função Mista/deficiência , Oxigenases de Função Mista/imunologia , Adesividade Plaquetária , Suínos , Trombocitopenia/sangue , Trombocitopenia/imunologia , Trombocitopenia/metabolismo , Fatores de Tempo
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