Pre- and posttransplant monitoring of alloantibodies by complement-dependent cytotoxicity and luminex methodologies in liver transplantation.

BACKGROUND: This study evaluated the influence of circulating anti-HLA antibodies on outcomes of 97 liver allografts from deceased donors. METHODS: Human leukocyte antigen (HLA) antibody screening was performed by both complement-dependent cytotoxicity (CDC) and multiparameter Luminex microsphere-based assays (Luminex assay). RESULTS: The agreements between T- and B- cell CDC and Luminex assays were 67% and 77% for pre- and posttransplant specimens, respectively. Graft dysfunction was not associated with either positive pretransplant CDC or Luminex panel-reactive antibody (PRA) values. Likewise, positive posttransplant T- or B- cell CDC PRA values were not associated with graft dysfunction. In contrast, posttransplant Luminex PRA values were significantly higher among patients with graft dysfunction compared with subjects with good outcomes (P = .017). CONCLUSION: Posttransplant monitoring of HLA antibodies with Luminex methodology allowed identification of patients at high-risk for poor graft outcomes.

Non-human leukocyte antigen antibodies reactive with endothelial cells could be involved in early loss of renal allografts.

Preformed donor-specific human leukocyte antigen (HLA) antibodies have been associated with allograft dysfunction and failure. However, recipients of HLA-identical kidneys can develop acute humoral rejection, implicating putative pathogenic antibodies that are directed against non-HLA antigens. We investigated the presence of endothelial cell-reactive antibodies in 11 patients who experienced early loss of their transplanted kidneys owing to humoral rejection and 1 loss from renal venal thrombosis. We examined the potential efficacy of intravenous immunoglobulin to block the binding of these antibodies, as previously suggested for anti-HLA antibodies.

Microchimerism does not correlate with survival of murine cardiac allografts.

The development of microchimerism was evaluated at different time points after infusion of a mixed population of bone marrow and spleen cells from (BALB/c x C57Bl/6)F1 mice in the presence or absence of a cardiac transplant. Microchimerism was observed in the spleen, bone marrow and thymus of transplanted BALB/c mice even after graft rejection. In the absence of transplantation, donor cells persisted especially in the thymus. The results show that despite augmentation of graft survival after donor cell infusion compared to nontreated controls, the development of microchimerism did not sustain cardiac semihistocompatible grafts. Moreover, the persistence of donor cells in the thymus in both situations suggests a role for this organ in the increased graft survival in our model.

Antibodies eluted from acutely rejected renal allografts bind to and activate human endothelial cells.

This study was designed to investigate how antiendothelial antibodies (EAbs) are involved in acute irreversible renal graft rejection. Eluates from 25 renal allografts, lost by irreversible rejection (n = 22) and by renal vein thrombosis (controls n = 3), were tested against a panel of cultured human umbilical vein endothelial cells (HUVEC). All patients were under immunosuppression at the time of nephrectomy. EAbs binding and membrane expression of adhesion molecules ELAM-1 and VCAM-1 were analyzed by flow cytometry (FACS) and by semiquantitative RT-PCR for mRNAs coding for those molecules. The absence of anti-HLA antibodies against the donor was ascertained at transplant, and before and after nephrectomy by the negativity of specific crossmatches performed using the most sensitive techniques. EAbs eluted from eight rejected kidneys bound to HUVEC. They did not induce any cytotoxicity, but their incubation with HUVEC (4 h at 37 degrees C; 2.5 mg/ml) led to upregulation of mRNAs coding for VCAM-1 (35- to 60-fold increases) and ICAM-1 (8- to 12-fold increases) as compared with control EAbs. Membrane expression of adhesion molecules was also strikingly increased, with 80% of the cells expressing VCAM-1 and 65% expressing ELAM-1 upon incubation. EAbs were detected in eight out of nine (88.8%) eluates from kidneys lost from acute vascular rejection, but in none of the 13 (0.0%) kidneys lost from other types of rejection (p < 0.0001). We conclude that EAbs, capable of activating human endothelial cells, can be recovered from acutely rejected kidneys and may play a direct role in the pathogenesis of acute rejection.

Cytomegalovirus infection amplifies class I major histocompatibility complex expression on cultured human endothelial cells.

Cytomegalovirus infection, a common complication in immunosuppressed graft recipients, bears an adverse impact on graft survival. Cytomegalovirus enhances the expression of the monotypic determinants of the class I major histocompatibility complex molecule by the endothelium, possibly rendering the endothelial cells more immunogenic and prone to attack by the allogeneic lymphocytes. In the present study, we focused on the effect of cytomegalovirus on the endothelial cell expression of different class I genes, on the relation between the extent of endothelial cell infection and the class I effect, and on the time course of the class I changes induced by the cytomegalovirus infection. Cytomegalovirus infection of primary cultures of human umbilical vein endothelial cells augmented the expression of the A2, A3, and B7 class I major histocompatibility complex genes when compared with uninfected cells. beta 2 microglobulin upregulation by the infected cells paralleled the changes in specific class I expression; this effect was significant only after 7 days after infection. Double immunocytochemical staining and fluorescence-activated cell sorter analysis revealed that the class I enhancement was uniform throughout the umbilical vein endothelial cell monolayer and not restricted to the cells that expressed cytomegalovirus early or late antigens. Ultraviolet-inactivated supernatants from infected umbilical vein endothelial cell did not increase class I expression on uninfected cells. In conclusion, cytomegalovirus might affect graft survival by amplifying the changes in class I expression beyond the sites of viral replication.

Rejeiçäo humoral com prova cruzada negativa: análise de 31 casos / Humoral rejection with negative crossmatch: analysis of 31 cases

Analisamos os dados clínicos, anatomopatológicos e imunológicos de 31 casos de rejeiçäo humoral (RH) com prova cruzada (PC) negativa e perda do enxerto, observados nos últimos 487 transplantes renais. Diferenciamos três tipos de rejeiçäo humoral: imediata - RHI - (1,84%), tardia - RHT (2,25%) e vascular aguda - RVA - (2,25%). Em relaçäo ao tipo de doador, a RH ocorreu em 3,96% dos casos com doador vivo parente, 7,9% com doador cadáver e em 10,4% com doador vivo näo partente e foi mais freqüente no retransplante. A prova cruzada pré-transplante foi negativa contra linfócitos T em todos os casos. Em 12 casos, foi feita utilizando-se antiglobulina humana (AGH) e manteve-se negativa. Em 11 casos, os soros foram testados contra monócitos do doador, com resultados também negativos. Apenas três casos em 12 tiveram PC positiva contra linfócitos B do doador. Doze casos de RHI e RHT tiveram as provas cruzadas repetidas pós-perda do enxerto. Um caso apresentou resultados positivos para células T, B e monócitos e outro só para monócitos. Nos seis casos de RVA que tiveram a PC repetida no pós-transplante, três apresentaram positividade para T, B e monócito e os três restantes apenas para B. Esses resultados sugerem que, apesar do PCs altamente sofisticadas, continuamos impossibilitados de prever todas as perdas por RH. Além disso, cremos que os alvos estudados parecem näo apresentar diversidade suficiente, sugerindo a participaçäo de antígenos ainda näo pesquisados na rotina pré-transplante

Immunological tolerance in human transplantation. The possible existence of a maternal effect.

We analyzed data from 93 patients who received a kidney graft from their parents; 55 were transplanted with a kidney from their mothers (Mat.) and thirty-eight from their fathers (Pat.) The Pat. group has shown a better graft and patient survival as well as long-term renal function when compared with the Mat. group. This pattern was more evident with time. Long-term graft function assessed by creatinine levels also showed differences between the groups, favoring the Pat. group. The results are interesting since recent reports have claimed a tolerance developed by the individual to noninherited HLA antigens of their mother.

HLA typing in 67 Italian patients with idiopathic hemochromatosis and their relatives.

The frequency of HLA A3 and B7 antigens was significantly higher in 67 unrelated patients with idiopathic hemochromatosis (IH) than in 700 controls (62.7% vs 22.5%, p less than 10(-8) and 26.9% vs 9.3%, p less than 10(-3), respectively). A3 B7, A3 B35 and A3 B5 were significantly more frequent in 72 haplotypes linked to IH gene than in 278 control haplotypes. The prevalence of B35 and A3 B35 was significantly higher in IH patients from North-Eastern Italy than from other regions (60% vs 21%, p less than .05 and 54.5% vs 8.2%, p less than 0.0001, respectively). All 15 siblings HLA identical to the respective proband were homozygous for IH with variable expression of the disease, whereas minor abnormalities of iron-related indexes were present in 23% of heterozygous relatives. Homozygous-heterozygous mating probably occurred in three of 40 families, accounting for the overt disease in three offspring and in one HLA semi-identical sibling; however, in this last case the possibility of a recombination event cannot be excluded.

Association of hereditary spherocytosis and idiopathic hemochromatosis. A synergistic effect in determining iron overload.

Two siblings, both splenectomized at an early age for hereditary spherocytosis, had a severe hemochromatosis develop. The human leukocyte antigen (HLA) system typing showed that they were half HLA identical. All the other members of the family who did not have evidence of hereditary spherocytosis, including those who displayed identical HLA haplotypes with the two patients, did not have any increase in iron stores. These results suggest that the two siblings are heterozygous for idiopathic hemochromatosis and that the coexistence of this condition with hereditary spherocytosis can cause a severe iron overload.

Impaired natural killing activity in patients with rheumatoid arthritis. Clinical characteristics and a study of defective mechanisms.

We have studied NK activity against K562 cells of peripheral blood mononuclear cells (PBMC) from 83 patients affected with RA and searched for correlations with some clinical and laboratory parameters. In 65 patients T lymphocyte subsets were investigated by laser flow cytometry using monoclonal antibodies against OKT3, OKT4 and OKT8 antigens and in 25 patients also HNK-1+ cells were enumerated. NK activity in patients with RA resulted significantly decreased compared with controls (relative cytotoxic index = 0.68 +/- 0.74 versus 1.00 +/- 0.60, p less than 0.01). Decreased NK activity was not correlated with sex, age, duration of disease, ESR, haemoglobin, serum alpha-2-globulin, serum gamma-globulin, rheumatoid factor titre. The only clinical parameter correlated with decreased NK activity was the anatomical stage of the disease. NK activity depression resulted to be significantly correlated with OKT3+ cell percentage and at a lesser extent with OKT4+ and OKT8+ cell percentages. HNK-1+ cell percentage resulted only slightly reduced in patients with RA (13.1 +/- 8.7 versus 15.0 +/- 7.0) and there was only a modest correlation (p approximately equal to 0.10) between NK activity and HNK-1+ cell percentage. In order to elucidate the mechanisms of impaired NK activity in RA, experiments in vitro were carried out on PBMC of 23 patients to investigate the effects of the depletion of cells adherent to plastic, incubation with beta-interferon (1000 IU/ml) and incubation with indomethacin (10 -6M). Our data suggest that decreased NK activity in RA is mainly due to functional immaturity of NK cells and sometimes to inhibition by monocytes in some cases probably through prostaglandin release.

Low-dose heparin and deep-vein thrombosis after total hip replacement.

The effect of subcutaneous low-dose heparin on postoperative deep-vein thrombosis (D.V.T.) (diagnosed by the 125I-labelled fibrinogen test) has been investigated in a trial of 143 patients undergoing the operation of total hip replacement. Two randomized studies were carried out: in one the scanning for D.V.T. was carried out daily for 7 days postoperatively andin the other for 15 days. In both, the incidence of D.V.T. was significantly lower in the heparin-treated patients (P less than 0.005). Bilateral D.V.T. was also prevented (P less than 0.05), through the extension of D.V.T. to the distal veins of the thigh was not significantly reduced. Heparin treatment was, however, followed by a higher incidence of severe postoperative bleeding (P less than 0.02) and wound haematoma formation (P less than 0.005), and the postoperative haemoglobin was significantly lower than in the control group (P less than 0.005). A higher number of transfused blood units was also needed by the heparin treated patients (P less than 0.001).