Association between human leukocyte antigens and graft-versus-host disease occurrence after allogenic hematopoietic stem cell transplantation / Associação entre antígenos leucocitários humanos e a ocorrência da doença do enxerto contra o hospedeiro após o transplante alogênico de células-tronco hematopoiéticas

CONTEXT AND OBJECTIVE: Graft-versus-host disease (GVHD) is one of the complications following allogenic stem cell transplantation. This study investigated an association between human leukocyte antigen (HLA) and the occurrence of acute and chronic GVHD in patients who had received stem cell transplantations from HLA-identical siblings. DESIGN AND SETTING: Retrospective study at Hematology and Hemotherapy Center, Universidade Estadual de Campinas (Unicamp). METHODS: The participants were 176 patients whose first transplant was between 1997 and 2009. HLA genotyping was performed serologically and using the polymerase chain reaction with specific primer sequence. RESULTS: Acute GVHD was positively associated with HLA-A10 (P = 0.0007), HLA-A26 (P = 0.002), B55 (P = 0.001), DRB1*15 (P = 0.0211) and DQB1*05 (P = 0.038), while HLA-B16 (P = 0.0333) was more frequent in patients without acute GVHD. Chronic GVHD was positively associated with HLA-A9 (P = 0.01) and A23 (P = 0.0292) and negatively with HLA-A2 (P = 0.0031) and B53 (P = 0.0116). HLA-B35 (P = 0.0373), B49 (P = 0.0155) and B55 (P = 0.0024) were higher in patients with acute GVHD grade 3 or above, than in other patients. In patients with extensive chronic GVHD, HLA-A9 (P = 0.0004), A24 (P = 0.0059) and A26 (P = 0.0411) were higher than in other patients, while HLA-A2 was lower (P = 0.0097). CONCLUSION: This study suggests that HLA can influence the incidence and severity of acute and chronic GVHD. However, a study with a better design and more patients will be needed to confirm these results.

CONTEXTO E OBJETIVO: A doença do enxerto contra o hospedeiro (DECH) é uma das complicações pós-transplante alogênico de células progenitoras hematopoéticas. Este estudo investigou uma associação entre o antígeno leucocitário humano (HLA) e a ocorrência de DECH aguda e crônica, em pacientes que receberam transplantes de irmãos HLA-idênticos. TIPO DE ESTUDO E LOCAL: Estudo retrospectivo no Centro de Hematologia e Hemoterapia da Universidade Estadual de Campinas (Unicamp). MÉTODOS: Os participantes foram 176 pacientes cujo primeiro transplante foi entre 1997 e 2009. A tipagem HLA foi realizada por sorologia e reação em cadeia da polimerase (PCR) com sequência específica de primers. RESULTADOS: A DECH aguda foi associada positivamente com HLA-A10 (P = 0,0007), HLA-A26 (P = 0,002), B55 (P = 0,001), DRB1*15 (P = 0,0211) e DQB1*05 (P = 0,038), enquanto HLA-B16 (P = 0,0333) foi mais frequente em pacientes sem DECH aguda. A DECH crônica foi associada positivamente com HLA-A9 (P = 0,01) e A23 (P = 0,0292) e, negativamente, com HLA-A2 (P = 0,0031) e B53 (P = 0,0116). HLA-B35 (P = 0,0373), B49 (P = 0,0155) e B55 (P = 0,0024) estavam aumentados em pacientes com DECH aguda grau 3 ou maior, em comparação aos outros pacientes. Em pacientes com DECH crônica extensa, HLA-A9 (P = 0,0004), A24 (P = 0,0059) e A26 (P = 0,0411) estavam aumentados em comparação aos outros pacientes, enquanto HLA-A2 estava diminuído (P = 0,0097). CONCLUSÕES: Este estudo sugere que o HLA pode influenciar a ocorrência de DECH aguda e crônica e a sua gravidade. No entanto, um estudo com melhor desenho e com mais pacientes será necessário para confirmar esses resultados.

Diagnosis of acute humoral rejection using immunofluorescence in renal allograft biopsies- one step towards better understanding!

Immunofluorescence (IF) studies are important diagnostic tool in understanding pathogenesis involved in graft injury. Acute humoral rejection (AHR) associated with circulating donor-specific cytotoxic antibodies, is a poor prognosticator for graft survival. It can be diagnosed by staining for C4d antibody using indirect IF technique. C4d staining required to diagnose AHR was made mandatory for reporting renal allograft biopsies in 7th Banff conference. We present 2 years experience of IF studies using C4d polyclonal antibody on 546 renal allograft biopsies belonging to two groups of patients; 464 from group A (tolerance induction protocol) and 82 from group B (controls). We observed C4d focal positivity in 4 (0.9%) biopsies from group A and 4 (4.9%) from group B. We conclude that it is advisable to collect simultaneous core biopsy samples for IF studies and light microscopy to give better definition of allograft injury and thereby support in clinical management.

Role of Complement Regulatory Proteins in the Survival of Murine Allo-transplanted Sertoli Cells

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.

Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution

We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group 2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51 percent) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23 percent of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38 percent at ~8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with <25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.

4-1BB (CD137) signals depend upon CD28 signals in alloimmune responses

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.

Pruebas de Histocompatibilidad en el Programa de Trasplantes / Histocompatibility tasting provides data to evaluate the immunological risk for transplantation

The importance of the role of the histocompatibility laboratory in solid organ transplantation is to perform HLA typing and determine the degree of HLA matching between recipient/donor. It is a useful tool to increase graft survival and decrease chronic rejection. HLA matching has a positive effect on kidney transplants and it has variable impact on other organ transplants. The crossmatch procedure is the most important test in a solid organ transplantation to evaluate the presence of recipient antibodies to antigens expressed on donor white cells. This test decreases the risk of hyperacute humoral rejection or early graft loss. Positive crossmatch is a contraindication for transplantation because it represents the existence of IgG recipient antibodies that will reath againts donor antigens. Antibody evaluation is important in donor-recipient selection and the responsability of the histocompatibility laboratory is to identify clinically relevant anti-donor HLA antibodies. This detection is useful to determine the degree of humoral alloimmunization, expressed as a percent panel reactive antibody (96PRA). This test also provides information about the antibody specificity and can be used for evaluate a patient's immune status providing a significant correlation in selecting donors.

La importancia del laboratorio de histocompatibilidad en los programas de trasplante de órganos sólidos es llevar a cabo la tipificación HLA para determinar el grado de compatibilidad que exhibe la pareja receptor/donador para el trasplante. Se tiene conocimiento que el grado de compatibilidad HLA representa un efecto positivo en el trasplante renal y en la disminución de los episodios de rechazo. Su impacto en la sobrevida de los injertos es variable en otros órganos. El procedimiento más importante para evaluar la presencia de anticuerpos preformados presentes en el suero del receptor en contra de los antígenos expresados en los linfocitos del donador es la prueba cruzada. Esta prueba permite disminuir el riesgo de un rechazo hiperagudo o la pérdida temprana del injerto. Una prueba cruzada positiva se considera como contraindicación para el trasplante por presencia de anticuerpos preformados detectables en el suero del receptor del tipo IgG en contra de los antígenos del donador en estudio. Parte de la responsabilidad del laboratorio de histocompatibilidad es la detección en el receptor de anticuerpos anti-HLA clínicamente relevantes dirigidos en contra de las especificidades antigénicas de su potencial donador. Esta evaluación es útil para conocer el grado de aloinmunización humoral del paciente (sensibilización) y se expresa como un porcentaje de reactividad de anticuerpos (%PRA). Esta prueba también permite conocer la especificidad del anticuerpo anti HLA presente, y así evaluar el estatus inmunológico del paciente y la selección del donador.

HLA expression in aortic and pulmonary homografts: effects of cryopreservation.

BACKGROUND: The present study was undertaken to find out the HLA allo-antigens on cardiac homografts. METHODS AND RESULTS: One pulmonary and eight aortic homografts were studied for the presence of major HLA class I and class II antigen expression. Cadaveric hearts were procured from the mortuary and kept in Hank's balanced salt solution with antibiotics at 4 degrees C. Bits were taken from the conduits and valves every 24 hours for 14 days during storage and snap-frozen using liquid nitrogen. A total of 1368 sections were made using a cryostat. These sections were stained using 4 monoclonal antibodies: BLA class I (MO736), class II HLA-DR (MO746), CD45 (MO701), and endothelial stain (MO616). All monoclonals were procured from DAKO. Class I antigen molecules could be demonstrated on the endothelial surface of the vessel wall from day 1 to day 4 to 5 of storage. They stained weaker and could not be demonstrated after day 10 of storage. Class I antigen molecules were positive in very fresh valves and by day 5-6 could not be seen on the valve surface. Class II (HLA-DR) antigen expression was present in the subendothelial layer from day 1 to day 12-14 of storage. They could also be demonstrated in valves and conduits released after cryopreservation. These class II staining cells were also stained by CD45 monoclonal antibody and hence could be macrophages, histiocytes or leucocytes. The endothelium was very well demonstrated in the vessel walls from day 1 to day 12-14 of storage; it could only be seen in very fresh valves. Storage in the liquid medium and sterilization procedures led to loss of endothelial lining of the valves. After cryopreservation and thawing, class I antigen molecules could not be demonstrated on the valves and conduits. Class II antigen molecules and CD45-stained cells continued to be demonstrated in the subendothelial layer and the valve matrix. The endothelium was intact in the vessel wall after cryopreservation and thawing, but could not be seen in the released valves. CONCLUSIONS: Allograft aortic and pulmonary conduits and valves are immunogenic, and HLA-ABC and HLA-DR antigen molecules can be demonstrated on different components of the vessel wall and valve leafets.

Effect of angiopeptin and aspirin on accelerated graft atherosclerosis in transplanted mouse heart

In this study of the inhibitory effects of angiopeptin and aspirin on the development of accelerated graft atherosclerosis (AGAS), 22 B10.BR mice received intra-abdominal heterotopic heart transplants from B10.A mice, without immunosuppression. Group 1 (n = 5) received no pharmacological intervention, Group 2 (n = 6) was treated with angiopeptin, Group 3 (n = 5) with aspirin, and Group 4 (n = 6) with both. There was no significant difference in the incidence of AGAS among these groups. The magnitude of intimal lesion development showed less narrowing of large vessels (> 100 microns in diameter) in groups 2 and 4--i.e. the groups received angiopeptin (Group 1 = 46.9 +/- 9.3%, Group 2 = 28.5 +/- 9.2%, Group 3 = 44.1 +/- 10.9%, Group 4 = 24.2 +/- 5.9%; p < 0.01). Comparison of the fraction of tropomyosin-positive staining cells in the intima revealed a lesser degree of staining in Group 2 (p < 0.01). No intervention was effective in preventing smooth muscle cell proliferation in the media or inflammatory cell infiltration in the adventitia. In conclusion, our data suggest that angiopeptin is effective in the direct inhibition of intimal smooth muscle cell proliferation in relatively large vessels, whereas aspirin exhibits no inhibitory role in the progression of AGAS. Angiopeptin appears to be a potential therapeutic agent for inhibiting the progression of postoperative AGAS in clinical heart transplantation.

Donor valves as substitutes for heart valve replacement.

The main problems of heart valve replacement in Sri Lanka are the cost of prosthetic valves and anticoagulant related complications. The use of human donor heart valves (homografts; allogeneic heart valves [AHV]) will alleviate these shortcomings. Recipients of AHV do not require anticoagulant therapy. Moreover, cryopreservation of AHV offers the opportunity for the storage of valves for an extended length of time with the preservation of valve integrity which is essential for their function after implantation. A donor valve bank can potentially provide diameter matched valves for recipients. Current research suggests that the adverse immunological reactions initiated by AHV cause tissue degeneration in a proportion of these implants. However, the grafts may be improved before implantation during the disinfection and storage of the valves. In this essay an overview on the advantages of using AHV, current concepts of valve banking, recent advances in the understanding of AHV immunogenicity, emerging techniques for immunomodulation of AHV and the possibility of setting up a donor heart valve bank in Sri Lanka are discussed.

Rat limb allotransplantation: efficacy of subtherapeutic dose combination immunotherapy with cyclosporine (CsA) and RS-61443

The synergic effect of subtherapeutic doses of cyclosporine and RS-61443 was demonstrated in a vascularized rat hindlimb allotransplantation across a strong histocompatibility barrier (Brown-Norway as donors and Fischer 344 as recipients). Low doses of agents in combination minimized the toxicity while increasing the therapeutic efficacy. All animals showed weight loss during the first 15 days posttransplantation and they regained protective sensation within 45 postoperative days. Only 15.38 per cent of the animals presented complications: thrombosis, enteritis, autophagia and disorders of unknown etiology.

Apoliprotein B polymorphism: Application to monitoring early bone marrow transplantation in Saudi population

Seventy-three unrelated Saudi individuals and seven consecutive bone marrow transplant donor-recipient pairs were studied to characterize and determine the frequency of 3 apolipoprotein B alleles by polymerase chain reaction. The samples analyzed were either peripheral blood or bone marrow aspirates. Eleven different alleles were detected. The index of heterozygosity was 0.66. Apolipoprotein B analysis was informative in 57% of the studied donor-recipient pairs. Engraftment was detected as early as day three post-transplantation. The threshold of detection of this allele was up to 0.01xl0[6]/ml. Mixed chimerism of the order of 1% could be detected. We concluded that apolipoprotein B is a highly polymorphic allele among the Saudi population and this makes the region a useful marker for monitoring engraftment following allogeneic bone marrow transplantation

Inhibitory effects of cimetidine on graft survival of allotransplanted rats submitted to an active-passive enhancement protocol

The objetive of the presented study was to determine wheter cimetidine, a type-2 histamine receptor antagonist, inhibits the immunological enhancement of allografted rats achieved by treatment with donor antigen plus anti-donor antibody. Groups of rats submitted to this active-passive enhancement protocol and treated ip with 30 (APEC30; Group I; N = 4) or 60 (APEC 60; Group II; N = 8) mg/day cimetidine for 14 days had a significantly shorter graft survival (20.2 ñ 5.1 and 11.1 ñ 2.6 days, respectively) than the control group (animals submitted to the enhancement protocol and killed on day 72 after transplant when the graft was beating normally; APE; Group III; N = 6; P<0.05). On the other hand, these animals had a significantly longer graft survival than rats allotransplanted but not treated for enhancement (ALLO; Group V; N = 5; 8.2 ñ 0.8 days). The surgical control, consisting of isotransplanted animals, had a long-term survival (ISO; Group V; N = 6; rats killed 120 days after transplant with the graft beating normally). Animals treated with cimetidine, but not submitted to the enhancement protocol (AC 60; Group IV, N = 4) had a significantly shorter graft survival (6.25 ñ 0.5) than the allotransplanted animals (Group V). These results indicate inhibition of the suppressor mechanisms which participate in this type of immunological enhancement

Linfolisis mediada por células en el transplante renal / Cell-mediated lympholysis in kidney transplantation

La sobrevida de los transplantes renales mejoró con la utilización con la utilización de ciclosporina A (CyA) y globulina antilinfocitaria (GAL). Pero la necesidad de una inmunosupresión continua produce morbilidad y mortalidad significativas y la dosificación necesaria para evitar rechazo es generalmente sólo estimada por sus efectos sobre la función renal. Es importante entonces aplicar técnicas para el seguimiento inmunológico, a fin de diagnosticar precozmente una sobreinmunosupresión, e incluso para poder suspender totalmente la inmunosupresión. S e estudiaron 17 receptores de riñón (3 cadavéricos y 14 con dador vivo relacionado), que fueron tratados inicialmente con CyA, 3-5 mg/kg/d, GAL y prednisolona, 0,5 mg/kg/d (tratamiento actual) o azatioprina, 2 mg/kg/d y prednisolona 1 mg/kg/d (tratameiento convencional). La técnica de la citotoxicidad mediada por células (cell mediated lympholysis = CML) fue realizada, de acuerdo a métodos descriptos (15), entre el receptor y, alternativamente, su dador específico y un control no relacionado. La respuesta fue mayor con medicación convencional (14,73 ñ 1,69), que con medicación actual (3,14 ñ 0,8) (P < 0,02). (Tabla 2). La respuesta entre receptores y controles no relacionados ascendió significativamente con medicación convencional a 77,67 ñ 8,17 (P < 0,05), valores similares a los encontrados entre controles no relacionados (72,7 ñ 3,9). La respuesta con medicación actual, si bien aumentó a 34,25 ñ 2,54 (P < 0,05), permaneció ...

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity.

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.

Autolysed antigen-extracted allogeneic bone for repair of diaphyseal bone defects in rabbits

Autolysed antigen-extracted allogeneic bone (AAA bone) was used to bridge a large osteoperiosteal gap in the diaphysis of the radius of 50 rabbits. Periodic observations of the graft were made clinically, radiologically and histologically every week up to fourteen weeks. The continuity of the radius was evaluated macroscopically and histologically. The AAA bones were progressively resorbed and replaced by the new bone. The bone remodelled to the mature tubular bone and did not undergo absorption during the experimental period. The AAA bone proceeded to be an osteoinductive and osteoconductive material. There were no appreciable histologic signs of immune or foreign body reaction.