Killer Cell Immunoglobulin-like Receptors (KIR) and Human Leucocyte Antigen C (HLA-C) Increase the Risk of Long-Term Chronic Liver Graft Rejection.

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2+ and rKIR2DS3+) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2+ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2-) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3+/dC1- (p = 0.015), rKIR2DS4/dC1- (p = 0.014) and rKIR2DL3+/rKIR2DS4+/dC1- (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1+rKIR2DS4+/dC1- at 5-10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1+/C1-ligands and the influence of KIR2DS4+/C1-ligands in long-term graft survival.

HLA-DRB1 and HLA-DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA-DRB1 alleles have previously been associated with ABPA-CF susceptibility; however, HLA-DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA-CF and determine their roles in susceptibility or protection. Patients with ABPA-CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA-DRB1 and -DQB1 genotyping was performed by the Luminex PCR-SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR-SSP was also performed by high-resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA-DRB1*5:01 and -DRB1*11:04 alleles occurred with greater frequency in patients with ABPA-CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA-CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA-DQB1 region, the HLA-DQB1*06:02 allele occurred more frequently in patients with ABPA-CF than in those with AST and CF and healthy controls, whereas HLA-DQB1*02:01 occurred less frequently in patients with ABPA-CF. These data confirm that there is a correlation between HLA-DRB1*15:01, -DRB1*11:04, DRB1*11:01, -DRB1*04 and -DRB1*07:01 alleles and ABPA-CF susceptibility and suggest that HLA-DQB1*02:01 is an ABPA-CF resistance allele.

Measurable residual disease study through three different methods can anticipate relapse and guide pre-emptive therapy in childhood acute myeloid leukemia.

PURPOSE: Although outcomes of children with acute myeloid leukemia (AML) have improved over the last decades, around one-third of patients relapse. Measurable (or minimal) residual disease (MRD) monitoring may guide therapy adjustments or pre-emptive treatments before overt hematological relapse. METHODS: In this study, we review 297 bone marrow samples from 20 real-life pediatric AML patients using three MRD monitoring methods: multiparametric flow cytometry (MFC), fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). RESULTS: Patients showed a 3-year overall survival of 73% and a 3-year event-free survival of 68%. Global relapse rate was of 25%. All relapses were preceded by the reappearance of MRD detection by: (1) MFC (p = 0.001), (2) PCR and/or FISH in patients with an identifiable chromosomal translocation (p = 0.03) and/or (3) one log increase of Wilms tumor gene 1 (WT1) expression in two consecutive samples (p = 0.02). The median times from MRD detection to relapse were 26, 111, and 140 days for MFC, specific PCR and FISH, and a one log increment of WT1, respectively. CONCLUSIONS: MFC, FISH and PCR are complementary methods that can anticipate relapse of childhood AML by weeks to several months. However, in our series, pre-emptive therapies were not able to prevent disease progression. Therefore, more sensitive MRD monitoring methods that further anticipate relapse and more effective pre-emptive therapies are needed.

Moderate to Intense Physical Activity Is Associated With Improved Clinical, CD4/CD8 Ratio, and Immune Activation Status in HIV-Infected Patients on ART.

BACKGROUND: Physical activity has anti-inflammatory effects and reduces morbidity and mortality in the general population, but its role in the clinical, CD4/CD8 ratio, and immune activation status of HIV-infected patients has been poorly studied. METHODS: A cross-sectional study was carried out in a cohort of 155 HIV-infected patients on stable antiretroviral therapy (ART) to compare clinical, biochemical, CD4/CD8 ratio, and immune activation status according to their physical activity in the last 2 years (sedentary/low vs moderate/intense) assessed by the iPAQ. A binary logistic regression and mixed analysis of variance were performed to evaluate the impact of levels of physical activity on CD4/CD8 ratio. RESULTS: In our series, 77 (49.7%) out of 155 patients were sedentary, and 78 (50.3%) practiced moderate/intense physical activity. Moderate/intense physical activity was associated with better metabolic control (lower body mass index, P = .024; glucose, P = .024; and triglyceride, P = .002) and CDC HIV stage (P = .046), lower CD8+ (P =  .018), CD4+CD8+ (P = .026), CD4+CD86+ (P = .045), CD4+HLA-DR+ (P = .011), CD8+HLA-DR+ (P = .048) T lymphocytes and CD16+HLA-DR+ natural killer cells (P = .026), and higher CD3+CD4+ T lymphocytes (P = .016) and CD4/CD8 ratio (P = .001). Sedentary lifestyle (odds ratio [OR], 2.12; P = .042), CD4 nadir (OR, 1.005; P < .001), and CD8+CD38+ T cells (OR, 1.27; P = .006) were independently associated with low CD4/CD8 ratio (<0.8). Earlier and more intense CD4/CD8 ratio recovery was observed in patients with higher physical activity in the 2-year follow-up with a significant interaction between these variables: F(2, 124) = 3.31; P = .049; partial η2 = 0.042. CONCLUSIONS: Moderate to high physical activity is associated with beneficial health effects, improvement in metabolic profile, and reduction of chronic inflammation in patients with HIV. Although more studies and clinical trials are needed to confirm these findings, a healthy lifestyle including at least moderate physical activity should be recommended to HIV patients on stable ART.

KIR2DL2/S2 and KIR2DS5 in alcoholic cirrhotic patients undergoing liver transplantation.

INTRODUCTION: The molecular mechanisms underlying alcoholic liver fibrosis and cirrhosis are not completely understood. Hepatic fibrosis involves the interplay of diverse cells and factors, including hepatic stellate cells (HSCs), Kupffer, NK cells, and T-lymphocyte subsets. Killer-cell immunoglobulin-like receptors (KIR) are membrane receptors involved in mediation between NK and activated HSCs, regulating NK cell function through their interaction with HLA-I molecules. The aim of this study was to analyse the genetic association between KIR genes and the susceptibility to or protection from alcoholic cirrhosis (AC) in a cohort of male AC patients undergoing liver transplantation (LT) with and without concomitant viral infections. MATERIAL AND METHODS: KIR genotyping was performed in nuclear DNA extracted from 281 AC patients and compared with 319 male controls. RESULTS: Significant differences between total AC patients and healthy controls were only found in the case of KIR2DL2 and KIR2DS5. KIR2DL2 was significantly underrepresented in non-viral AC patients (52.6% vs. 63.3%; p = 0.015), while patients heterozygous for KIR2DL2 were also underrepresented in the non-viral AC group compared with controls (p = 0.034). KIR2DS5 was overrepresented in this group compared with healthy controls (p = 0.002). All these observations were only evident in AC patients older than 54 years old. CONCLUSIONS: Our data suggest a contrary effect of KIR2DL2 and KIR2DS5 in AC patients older than 54 years, in whom the presence of KIR2DL2 appears to be protective against AC, whereas the presence of KIR2DS5 seems to promote the fibrotic process, particularly in patients with no associated viral infection.

Expression of NK Cell Receptor Ligands on Leukemic Cells Is Associated with the Outcome of Childhood Acute Leukemia.

Acute leukemia is the most common malignancy in children. Most patients are cured, but refractory/relapsed AML and ALL are the first cause of death from malignancy in children. Maintenance chemotherapy in ALL has improved survival by inducing leukemic cell apoptosis, but immune surveillance effectors such as NK cells might also contribute. The outcome of B-ALL (n = 70), T-ALL (n = 16), and AML (n = 16) pediatric patients was evaluated according to leukemic cell expression of ligands for activating and inhibiting receptors that regulate NK cell functioning. Increased expression of ULBP-1, a ligand for NKG2D, but not that of CD112 or CD155, ligands for DNAM-1, was associated with poorer 5-year event-free survival (5y-EFS, 77.6% vs. 94.9%, p < 0.03). Reduced expression of HLA-C on leukemic cells in patients with the KIR2DL1/HLA-C*04 interaction was associated with a higher rate of relapse (17.6% vs. 4.4%, p = 0.035) and lower 5y-EFS (70.6% vs. 92.6%, p < 0.002). KIR2DL1/HLA-C*04 interaction was an independent predictive factor of events (HR = 4.795, p < 0.005) or death (HR = 6.731, p < 0.005) and might provide additional information to the current risk stratification. Children who carry the KIR2DL1/HLA-C*04 interaction were refractory to current chemotherapy treatments, including allogeneic stem cell transplantation; therefore, they should be considered as candidates for alternative biological therapies that might offer better results.

Monitoring of B Cell in Kidney Transplantation: Development of a Novel Clusters Analysis and Role of Transitional B Cells in Transplant Outcome.

BACKGROUND: B lymphocytes (BL) seem to play an important role in transplantation, although the and role of different subpopulations in monitoring and outcome is not clear. Our aim was to monitoring immunological profiles based on BL subpopulations in kidney recipients (KR) with the risk of acute rejection (AR). METHODS: Monitoring of BL subpopulations was performed by flow cytometry in PBLs before transplantation and three and six months after transplantation (PTX). We used two methodological approaches, a traditional analysis, and a novel cluster analysis, to determine the association between BL subpopulations, AR incidence, and graft function. RESULTS: After three months of PTX, KRs with a B phenotype enriched in transitional BL and plasmablasts had better kidney function and lower AR incidence. KRs with decreased transitional BL and plasmablasts were associated with lower kidney function and higher AR PTX. KRs that had an increase in transitional BL PTX had a better clinical outcome. The increase in transitory BL during PTX was also associated with an increase in Tregs. Indeed, KRs receiving thymoglobulin as induction therapy showed a slight decrease in the relative frequency of naive BLs after three months of PTX. CONCLUSION: The monitoring of BL subpopulations may serve as a non-invasive tool to improve immunological follow-up of patients after kidney transplantation. However, further studies are needed to confirm the obtained results, define cut-off values, and standardize more optimal and even custom/customized protocols.

Patient Sex in the Setting of Liver Transplant in Alcoholic Liver Disease.

OBJECTIVES: The aim of this study was to analyze alcoholic cirrhosis in women who were to undergo liver transplant, including their biochemical and clinical characteristics, main complications, survival rates, and main causes of death compared with men with alcoholic cirrhosis. MATERIALS AND METHODS: Our study included 400 patients with alcoholic cirrhosis, which we divided according to sex and viral infections. Biochemical parameters and the presence and degree of ascites and encephalopathy, liver function status, and liver rejection and survival rates were analyzed from 1 to 10 years and the main cause of death at 10 years. RESULTS: Patients with nonviral alcoholic cirrhosis and liver transplant had significantly better survival rates (84.1%) at 1 year versus those with viral alcoholic cirrhosis (74.5%; P = .036). Men with nonviral alcoholic cirrhosis (14%) and women with hepatitis C virus (29%) had the lowest short-term survival rates. In long-term survival analysis, the lowest rate was observed in women with nonviral alcoholic cirrhosis (26.1%), and the highest rate was observed in women with hepatitis C virus (42.9%). Liver graft failure was one of the main causes of death in male patients (19.5%). CONCLUSIONS: Women with alcoholic cirrhosis showed a higher rate of ascites and encephalopathy but lower liver graft rejection than men with alcoholic cirrhosis. Survival rates were similar between men and women, although slightly lower in women who had hepatitis C virus.

NKG2D Polymorphism in Melanoma Patients from Southeastern Spain.

BACKGROUND: Natural killer (NK) and CD8+ T cells are involved in the immune response against melanoma. C-Type lectin-like NK cell receptors are located in the Natural Killer Complex (NKC) region 12p13.2-p12.3 and play a critical role in regulating the activity of NK and CD8+ T cells. An association between polymorphisms in the NKC region, including the NKG2D gene and NKG2A promoter, and the risk of cancer has been previously described. The aim of this study was to analyze the association of polymorphisms in the NKC region with cutaneous melanoma in patients from southeastern Spain. METHODS: Seven single-nucleotide polymorphisms (SNPs) in the NKG2D gene (NKC3,4,7,9,10,11,12), and one SNP in the NKG2A promoter (NKC17) were genotyped by a TaqMan 5' Nuclease Assay in 233 melanoma patients and 200 matched healthy controls. RESULTS: A linkage disequilibrium analysis of the SNPs performed in the NKC region revealed two blocks of haplotypes (Hb-1 and Hb-2) with 14 and seven different haplotype subtypes, respectively. The third most frequent haplotype from the block Hb-2-NK3 (CAT haplotype)-was significantly more frequent on melanoma patients than on healthy controls (p = 0.00009, Pc = 0.0006). No further associations were found when NKC SNPs were considered independently. CONCLUSIONS: Our results suggest an association between NKG2D polymorphisms and the risk of cutaneous malignant melanoma.

Allelic diversity of MICA gene and MICA/HLA-B haplotypic variation in a population of the Murcia region in southeastern Spain.

Major histocompatibility complex class I-related chain A (MICA) is located at 46 kb centromeric of HLA-B. It is highly polymorphic and interacts with NKG2D, its receptor on the surface of NK, Tgammadelta and T CD8 lymphocytes. Data on MICA polymorphism in different populations are still limited. Our aim was to establish allelic diversity of MICA gene and linkage disequilibrium with HLA-B in our population. DNA was obtained from 154 unrelated healthy individuals from the Murcia region in southeastern Spain. HLA-B genotyping was performed using polymerase chain reaction (PCR)-sequence-specific oligonucleotide probes and allele-specific PCR-sequence-specific primers, and MICA genotyping by using PCR-sequence-specific oligonucleotide probes. A total of 19 MICA alleles were detected on this study. MICA*008 was the most frequent allele (25.3%), followed by MICA*002 (16.1%), MICA*004 (14.9%), MICA*001 (7.8%), MICA*009 and MICA*016 (7.1%), and MICA*010 (4.6%). Eleven alleles had frequencies of <1%. In the haplotype analysis, MICA*008-B*0702 was found to be the most common, followed by MICA*004-B*4403 and MICA*001-B*1801, MICA*002-B*3501, MICA*008-B*4402, MICA*004-B*4901, MICA*008-B*0801, and MICA*002-B*3801. The frequency of MICA*010-B*1501, MICA*008-B*1302, MICA*015-B*4501, and MICA*008-B*4001 was remarkable inasmuch as these two last haplotypes have not been reported in Spanish population. Indeed, MICA*016 linked to B*1402 has also not been reported in the literature. In conclusion, the allelic diversity in our population is similar to other Caucasian populations; however we found a series of less frequent alleles, in addition to as-yet-undescribed haplotypic associations in other populations of Caucasian origin.

MHC class I chain-related gene a diversity in patients with cutaneous malignant melanoma from southeastern Spain.

A limited number of studies have been performed so far on the polymorphism in the transmembrane region (exon 5) of the major histocompatibility complex class I chain-related gene A (MICA) in patients with melanoma. However, the influence of MICA polymorphism in extracellular domains (exons 2, 3, and 4) has not been investigated on melanoma disease. This study aims to characterize the influence of extracellular MICA polymorphism, and its previously described linkage disequilibrium with HLA-B locus, on patients with cutaneous melanoma from southeastern Spain. For this purpose, MICA and HLA-B genotyping was performed in 233 patients and 200 ethnically matched controls by luminex technology. Patients were classified according to the presence of methionine or valine at codon 129 of MICA gene. We found a high frequency of MICA(*)009 in melanoma patients compared with controls (P = 0.002, Pc = 0.03). Our results also showed an association between MICA(*)009 and HLA-B(*)51 alleles in both patients and controls. This association was stronger in patients than controls (P = 0.015). However, a multivariate logistic regression model showed that neither MICA(*)009 nor the combination MICA(*)009/HLA-B(*)51 was associated with melanoma susceptibility. No relationship was observed between MICA-129 dimorphism and melanoma nor when MICA polymorphism was evaluated according to clinical findings at diagnosis.

Association analysis of MICA gene polymorphism and MICA-129 dimorphism with inflammatory bowel disease susceptibility in a Spanish population.

MICA is located at 46 kb centromeric of HLA-B, is highly polymorphic and interactions with NKG2D, its receptor on the surface of NK, Tgammadelta, and T CD8 lymphocytes. A variation at amino acid position 129 of the alpha2-heavy chain domain seems to categorize MICA alleles into strong and weak binder of NKG2D receptor, and thereby to influence effector cell function. Our aim was to study allele polymorphism of MICA and the functionally relevant dimorphism (129val/met) of MICA gene in inflammatory bowel disease (IBD) patients in our population. DNA was obtained from IBD patients (n = 88) and unrelated healthy Murcians (n = 154) and used to MICA genotyping using polymerase chain reaction-sequence-specific oligonucleotides. We did not find statistical differences in the distribution of MICA alleles between the IBD and control groups. However, we found a higher frequency of MICA-129met/met and a lower frequency of MICA-129val/met genotypes in IBD patients (mainly in ulcerative colitis) than in controls (pc = 0.02). These preliminary data could suggest a relevant role of MICA-129-val/met SNP (weak/strong binders of NKG2D receptor) in the pathogenesis of IBD.

Identification of apoptosis-related PLZF target genes.

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.