Identification of peripheral CD154+ T cells and HLA-DRB1 as biomarkers of acute cellular rejection in adult liver transplant recipients.

Decreasing graft rejection and increasing graft and patient survival are great challenges facing liver transplantation (LT). Different T cell subsets participate in the acute cellular rejection (ACR) of the allograft. Cell-mediated immunity markers of the recipient could help to understand the mechanisms underlying acute rejection. This study aimed to analyse different surface antigens on T cells in a cohort of adult liver patients undergoing LT to determine the influence on ACR using multi-parametric flow cytometry functional assay. Thirty patients were monitored at baseline and during 1 year post-transplant. Two groups were established, with (ACR) and without (NACR) acute cellular rejection. Leukocyte, total lymphocyte, percentages of CD4+ CD154+ and CD8+ CD154+ T cells, human leukocyte antigen (HLA) mismatch between recipient-donor and their relation with ACR as well as the acute rejection frequencies were analysed. T cells were stimulated with concanavalin A (Con-A) and surface antigens were analysed by fluorescence activated cell sorter (FACS) analysis. A high percentage of CD4+ CD154+ T cells (P = 0·001) and a low percentage of CD8+ CD154+ T cells (P = 0·002) at baseline were statistically significant in ACR. A receiver operating characteristic analysis determined the cut-off values capable to stratify patients at high risk of ACR with high sensitivity and specificity for CD4+ CD154+ (P = 0·001) and CD8+ CD154+ T cells (P = 0·002). In logistic regression analysis, CD4+ CD154+ , CD8+ CD154+ and HLA mismatch were confirmed as independent risk factors to ACR. Post-transplant percentages of both T cell subsets were significantly higher in ACR, despite variations compared to pretransplant. These findings support the selection of candidates for LT based on the pretransplant percentages of CD4+ CD154+ and CD8+ CD154+ T cells in parallel with other transplant factors.

Three new HLA class I alleles with synonymous mutations: HLA-A*03:01:62, -C*07:02:82 and -C*12:03:42.

Three new HLA class I alleles with synonymous mutations were identified.

Pretransplant CD28 Biomarker (Levels of Expression and Quantification of Molecules per Cell) in Peripheral CD4+ T Cells Predicts Acute Rejection Episodes in Liver and Kidney Recipients.

BACKGROUND: Acute rejection (AR) remains a significant cause of graft loss. Better approaches to predict AR are being investigated. Surface CD28 protein is essential for T-cell proliferation and survival as well as cytokine production. PATIENTS AND METHODS: Pretransplant CD4+CD28+ peripheral T cells were examined in 30 liver recipients (LRs) and 31 kidney recipients (KRs) by flow cytometry. RESULTS: Pretransplant CD4+CD28+ T cells in LRs were significantly lower in rejectors than nonrejectors (P = .002). Furthermore, the total number of CD28 molecules per cell in LRs (P = .02) as well as KRs (P = .047) was significantly lower in rejectors than nonrejectors. The healthy group did not display differences when compared with patients with end-stage liver disease or renal failure; however, stratification analysis displayed higher levels of CD4+CD28+ when compared with rejected LRs (P = .04) but not KRs. CD28 levels <41.94% were able to discriminate LRs at high risk of AR (P = .003). Similarly, a total number of CD28 molecules ≤8359 (P = .031) in LRs and ≤7669 (P = .046) in KRs correlated with high risk of AR. CONCLUSION: The preliminary results presented herein exhibit a fast and noninvasive method that assists clinicians to prevent AR by monitoring CD4+CD28+ peripheral T cells.

Pro- and anti-inflammatory cytokine gene single-nucleotide polymorphisms in inflammatory bowel disease.

Anti-inflammatory cytokines have an important role in disease, tumour and transplant processes. Alterations in the regulation of several cytokines have been implicated in a variety of inflammatory disorders, including IBD (inflammatory bowel disease) [Crohn's disease (CD) and ulcerative colitis (UC)]. Cytokine polymorphisms are also known to affect the level of gene expression. Thus, the aim of this study was to determine the relationship between cytokine polymorphisms and the IBD pathologies in a Spanish population. Polymorphisms analysis was performed using PCR-SSOP using a microbeads luminex assay. The following polymorphisms were determined: TNFα [-238G/A (rs361525) and -308G/A (rs1800629)], IFNγ [+874A/T (rs62559044)], TGFß [+869C/T (rs1982073) and +915G/C (rs1800471)], IL10 [-1082A/A (rs1800896), -592A/C (rs1800872), -819C/T (rs1800871)], IL6 [-174C/G (rs1800795)], IL12p40 [3'UTR -1188A/C (rs3212227)], IL1α [-889C/T (rs1800587)], IL1ß [-511C/T (rs1143634) and +3962C/T (rs1143633)], IL1R [Pst-1 1970C/T] and IL1RA [Mspa-1 11100C/T]. No statistical differences in TNFα, IFNγ, TGFß, IL10, IL6, IL1α, IL1ß, IL1R and IL1Ra genotypes and allele distributions between the IBD groups and healthy controls were found. However, we observed significant differences in the 3'UTR -1188A/C polymorphism of IL12p40. So -1188A allele was increased in patients with UC and the -1188C allele (high IL12p40 production) was increased in patients with CD with respect to controls. These data are in concordance with the fact that CD has been shown to be associated with a Th1 T-cell-mediated inflammation model and high IL12/IFNγ production at histological affected sites. These data suggest that cytokine polymorphisms in TNFα, IFNγ, TGFß, IL10, IL6 and IL1α, IL1ß, IL1R and IL1Ra cytokine gene do not seem to be relevant in IBD susceptibility and IL12p40 3'UTR -1188A/C polymorphism seems to be associated with a differential IBD development.

Genetic polymorphisms of tumour necrosis factor alpha (TNF-α) promoter gene and response to TNF-α inhibitors in Spanish patients with inflammatory bowel disease.

Tumour necrosis factor alpha (TNF-α) has an important role in inflammatory response. Alterations in the regulation of TNF-α have been implicated in a variety of inflammatory disorders, including Inflammatory bowel disease (IBD). Indeed, a common treatment for IBD is the use of TNF-α inhibitors. Polymorphisms in the TNF-α promoter region are known to affect the level of gene expression. Our aim was to investigate the influence of these single nucleotide polymorphisms (SNPs) in TNF-α promoter gene play in the risk of IBD in a Spanish population and their individual response to anti-TNF-α treatment. DNA samples from patients with IBD and controls were screened for TNF-α -238G/A (rs361525) and -308G/A (rs1800629) SNPs by PCR-SSOP using a microbeads luminex assay and compared with response to TNF-α inhibitors. There were not statistical differences in -238G/A and -308G/A allele and genotype frequencies between patients. However, we found an increased frequency of -308A allele and -308GA genotype in these nonresponders patients to TNF-α inhibitors with respect to responders patients (Pc < 0.05). This -308GA genotype has been classified as high producer of this cytokine. This fact could actually be interesting to explain the different response of patients with IBD with respect to TNF-α inhibitors. TNF-α promoter gene polymorphism does not seem to play a role in IBD susceptibility, but particular TNF-α genotypes may be involved in the different responses to TNF-α inhibitor treatment in Spanish patients with IBD.

Complement component C6 deficiency in a Spanish family: implications for clinical and molecular diagnosis.

Complement component C6 deficiency is a genetic disease presenting as increased susceptibility to invasive Neisseria meningitidis infections. This disorder has rarely been diagnosed in the Spanish population. In this work we report the immunochemical and molecular characterization of complement C6 deficiency in a Spanish patient showing no detectable functional activity of either the classical or alternative complement pathways and reporting a history of several episodes of meningococcal meningitis. The levels of individual complement components C3, C4, C5, C7, C8 and C9 were within the normal range. However, C6 level was low in the patient's serum as measured by radial immunodiffusion. Exon-specific polymerase chain reaction and sequencing of the C6 gene revealed a previously described homozygous single base deletion in exon 6 (c.821delA), leading to a shift in the reading frame that caused the generation of a downstream stop codon, which, in turn, provoked the truncation of the C6 protein (p.Gln274fs). To our knowledge, this is the first report on the c.821delA mutation in the Spanish population, which has previously only been identified in individuals of African ancestry. Characterization of this mutation was thought interesting in order to elucidate its source and help understand the molecular basis of this uncommon deficiency in our population. Moreover, this report highlights the importance of complement screening in cases of repeated meningococcal infections in order to establish its involvement and to consider adequate clinical recommendations such as prophylactic antibiotics or meningococcal vaccines and, subsequently, for genetic counselling.

C1q-fixing human leukocyte antigen assay in immunized renal patients: correlation between Luminex SAB-C1q and SAB-IgG.

BACKGROUND: There is no consensus about the impact of thresholds of complement-fixing antibody assays. Recently, a C1q-SAB assay has been developed to identify complement-fixing HLA antibodies with high sensitivity and specificity. Our aim was to determine the correlation between IgG single antigens beads (SAB) and C1q-SAB assay results among patients on the renal waiting list. PATIENTS AND METHODS: Serum samples from immunized renal waiting list patients as well as negative and positive controls were valided by Luminex (LMX). These sera, which were positive for 166 antibody specificities, were tested for HLA class I in parallel by LMX-IgG and LMX-C1q. RESULTS: Comparison of antibody detection revealed no correlation based on median fluorescent intensity (MFI), levels between the IgG SAB and the C1qSAB assay (P > .05). IgG-positive sera with MFIs as low as 700 were able to fix C1q, whereas other sera with MFIs as high 14,500 did not. Furthermore, there appeared to be disparities in the profiles of class I antigens able to fix C1q-SAB. In our series, only 34% class I IgG SAB antibodies were also C1qSAB+. In several patients, we detected C1qSAB+ against IgGSAB- that was surely due to IgM antibodies. So, the C1qSAB assay detected IgM antibodies that fix complement. CONCLUSION: These data suggested that the C1q-SAB assay could be an important method to evaluate pretransplant virtual crossmatch and to define nonpermitted specificities (C1q-fixing) in kidney transplantation.

Evolution of soluble forms of CD86, CD95 and CD95L molecules in liver transplant recipients.

Co-stimulatory factors such as CD86 and apoptotic molecules such as CD95 and CD95L required to start and to turn off the allogenic immune response may also be present as soluble proteins. To determine the role of the soluble forms of CD86 (sCD86), CD95 (sCD95) and CD95L (sCD95L) in the outcome of liver transplants, we analyzed the circulating levels of these molecules in patients subjected to liver transplantation in the pre-operative period and during the first month post-transplantation. Serum samples were obtained from sixty-nine first orthotopic liver transplants (OLT). The patients were classified into acute rejection (AR=24) and not acute rejection (NAR=45), or considering the presence of chronic active hepatitis B or C (VP=30) or other primary liver diseases (VN=39). The levels of sCD86, sCD95 and sCD95L were analyzed by solid phase sandwich enzyme-linked immunoabsorbent assays. Our results first showed that the pre-transplantation serum levels of sCD86 in the AR group were significantly higher than in the NAR group (1007±82U/mL vs. 739±46U/mL, p=0.006), and in the post-transplantation period these levels decreased sharply. Second, the levels of sCD95L and sCD95 in the pre-transplantation period did not point to statistically significant differences between the AR and NAR groups. Considering primary liver disease, the pre-transplantation levels of sCD86 and sCD95L in the VP group were significantly higher than those of the VN group (VP, 977±69U/mL vs. VN, 722±51U/mL, p<0.002, and VP, 482±78pg/mL vs. VN, 221±31pg/mL, p=0.002, respectively). Multivariate analysis revealed that only the pre-transplantation levels of sCD86 were independently associated with the development of episodes of acute rejection (p=0.005, OR=2.1, IC 95%=1.27-3.47). In conclusion, the present work shows that primary liver disease could influence the pre-transplantation levels of sCD86 and sCD95L. High pre-transplantation serum levels of sCD86 could favor the development of episodes of acute rejection.

A compound heterozygous mutation in the EDAR gene in a Spanish family with autosomal recessive hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder characterised by sparse hair, lack of sweat glands and malformation of teeth. The X-linked form of the disease, caused by mutations in the EDA gene, represents the majority of HED cases. Autosomal forms result from mutations in either the EDAR or the EDARADD gene. The X-linked and autosomal forms are phenotypically indistinguishable. For the purpose of genetic counselling, it is, therefore, important to know which gene is involved. In this study, we ascertained a Spanish family demonstrating the autosomal recessive form of HED. Affected individuals in the family showed the characteristic features of HED, including fine and sparse scalp hair, sparse eyebrows and eyelashes, periorbital hyperpigmentation, prominent lips, hypodontia and conical teeth, reduced sweating, and dry and thin skin. Sequence analysis of the EDAR gene revealed a novel compound heterozygous mutation [c.52-2A>G; c.212G>A (p.Cys71Tyr)]. Our finding extends the body of evidence that supports the significance of the EDAR signalling pathway in the ectodermal morphogenesis.

Estudio descriptivo de actividad del banco y registro de donantes de médula ósea de la región de Murcia (sureste de España) (1994-2004) / Descriptive study of activity of the bone marrow donor registry and bank from the Murcia region (Southeast of Spain) (1994-2004)

Objetivo: Realizar un estudio descriptivo de la actividad del banco y registro de donantes de médula ósea de la región de Murcia. Sujetos y método: Constituido por los donantes disponibles en el Banco de médula desde 1994 hasta 2004 (n = 3137). El estudio consiste en un análisis del número de donantes, su procedencia y, las búsquedas realizadas. Los donantes se tiparon por técnicas serológicas de microlinfocitoxicidad, y moleculares de PCR-SSO y -SSP. Resultados: El banco dispone de 3.137 donantes voluntarios tipados en baja y alta resolución, realizando un total de 680 búsquedas de donante. La procedencia de los donantes según las áreas de salud en que esta dividida la Comunidad de Murcia fue: área I (28%), área II (18%), área III (23%), área IV (6%), área VI (10%) y otras provincias (12%). Conclusiones: Observamos un aumento exponencial del número de donantes anuales, así como un aumento muy notable de las búsquedas por año, especialmente el 2004

Objective: To perform a descriptive study of the activity of the Bank of marrow donors from Murcia Region. Subjects and methods: All donors in the Bank of bone marrow from 1994 until 2004 (n=3137). This study analysed the number of donors, their origin and, performed donor searches activity. Donors were typed by serological microlymphocytotoxicity and molecular PCR-SSO and PCR-SSP techniques. Results: The Bank of bone marrow has 3,137 voluntary donors typed in low- and high-resolution. A total of 680 donor searches have been realized. The origin of the donors according to several Areas of Health in which the Autonomous Community of Murcia is divided, is the following one: Area I (28%), Area II (18 %), Area III (23 %), Area IV (6 %), Area VI (10 %) and other provinces (12%). Conclusions: An increase is observed in the number of annual donors as well as an increase very marked of donor searches that are realized every year, especially in 2004

Study of Fas (CD95) and FasL (CD178) polymorphisms in liver transplant recipients.

The Fas receptor is capable of transducing apoptotic cell death upon interaction with their ligand (FasL). Recent studies suggest that the Fas/FasL system is involved both in graft rejection and in transplantation tolerance. In this study, we analyzed the effect of Fas and FasL polymorphisms in liver allograft outcome. Fas and FasL polymorphisms were analyzed in 151 primary liver graft recipients. The Fas (-670 A/G) and the FasL (IVS2nt -124 A/G and IVS3nt 169 T/delT) polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Fas -1377 G/A polymorphism was determined by allele-specific amplification. Fas and FasL polymorphisms were not associated with acute and chronic rejection in liver transplant. In contrast, those recipients bearing the AA -670 Fas genotype showed significantly lower graft survival rate (S = 40%) than those bearing the GA genotype (S = 63.1%). These differences were detected from the first year post-transplant. Multivariate analysis confirmed that the AA genotype increased the risk of liver graft loss. This work suggests for the first time a possible harmful effect of Fas -670 AA genotype on liver graft survival, whereas the Fas and FasL polymorphisms are not associated with acute or chronic rejection in liver graft recipients.

[Descriptive study of activity of the bone marrow donor registry and bank from the Murcia Region (southeast of Spain) (1994-2004)]. / Estudio descriptivo de actividad del banco y registro de donantes de médula ósea de la región de Murcia (sureste de España) (1994-2004).

OBJECTIVE: To perform a descriptive study of the activity of the Bank of marrow donors from Murcia Region. SUBJECTS AND METHODS: All donors in the Bank of bone marrow from 1994 until 2004 (n=3137). This study analysed the number of donors, their origin and, performed donor searches activity. Donors were typed by serological microlymphocytotoxicity and molecular PCR-SSO and PCR-SSP techniques. RESULTS: The Bank of bone marrow has 3,137 voluntary donors typed in low- and high-resolution. A total of 680 donor searches have been realized. The origin of the donors according to several Areas of Health in which the Autonomous Community of Murcia is divided, is the following one: Area I (28%), Area II (18 %), Area III (23 %), Area IV (6 %), Area VI (10 %) and other provinces (12%). CONCLUSIONS: An increase is observed in the number of annual donors as well as an increase very marked of donor searches that are realized every year, especially in 2004.

Evaluation of CD86 gene polymorphism at +1057 position in liver transplant recipients.

BACKGROUND: Efficient T cell-APC interaction requires the participation of primary and co-stimulatory signals. The main co-stimulatory pathway involves the interaction of CD80 and CD86, expressed on the APCs, with their T cell counter-receptor, CD28 and CTLA-4. Recently, a G to A transition has been described at position +1057 of the CD86 gene, located in their cytoplasmic tail. METHODS: CD86 polymorphism was analyzed by sequence based typing in DNA samples obtained from 205 liver transplant recipients. Acute rejection and chronic rejection were diagnosed based upon conventional clinical, biochemical and histological criteria. RESULTS: The study of CD86 +1057 (G/A) polymorphism revealed that recipients bearing the A allele or the AA genotype have a reduced risk of acute rejection. In fact, the AA genotype was absent in the group of patients showing acute rejection episodes, whereas its frequency in those patients without acute rejection episodes was 8.8% (P=0.009, OR=0.07). This polymorphism did not reveal any association with the incidence of chronic rejection, but patients bearing the AA genotype showed a higher graft survival rate (83.3%) than those bearing the GA genotype (49.3%) or GG genotype (56.5%). CONCLUSIONS: The results of the present report suggest that the CD86 AA genotype at +1057 position could be involved in liver transplant acceptance, given that its presence is related to a decrease of acute rejection frequency and to a graft survival increase.

Relationship between CDC cross-match in liver recipients and antibody screening by flow cytometry.

Several authors have shown that anti-donor antibodies before liver transplantation are associated with decreased graft survival. The aim of this study was to investigate the relationship between anti-donor antibodies detected by the CDC technique or by FlowPRA, and acute or chronic rejection as well as graft survival. Furthermore, we sought to determine whether anti-donor antibodies, detected by the CDC technique, correlated with those discovered by cytometric screening. The acute rejection incidence among patients with complement-dependent cytotoxicity positive CDC cross-match was similar to that for patients with a negative cross-match. None of the patients with a positive cross-match developed chronic rejection. Allograft survival was significantly lower among recipients with a positive T-lymphocyte cross-match. Indeed, the majority of recipients with positive CDC cross-matches displayed graft failures before first posttransplant year. The results of a positive FlowPRA determination were concordant with a positive CDC cross-match in 85.71% of cases. Our data demonstrate that pretransplant FlowPRA correlates with the final CDC cross-match results. This finding suggests that in the future prospective pretransplant antibody screening with FlowPRA or CDC techniques may be useful to identify high-risk recipients.

Impact of HLA-C on acute rejection in liver transplantation.

Although liver transplants show a special tolerogenic behaviour, rejection remains an important problem that involves several immunological mechanisms, some of which are unknown. Our study sought to analyze the influence of HLA-C polymorphism on short-term liver graft acceptance by HLA-C genotyping of 100 orthotopic liver transplant recipient-donor pairs. Recipients were statified according to the occurrence of acute rejection. HLA-Cw*06 allele appeared to be underrepresented among recipients without versus those with acute rejection or those in control groups. With regard to HLA-C allelic compatibility, the frequency of acute rejection or those in episodes decreased with fewer HLA-C mismatches. These findings suggest the participation of HLA-C molecules in liver graft alloresponses, involving HLA-C genotyping, as well as compatibility.