Therapeutic Epigenetic Reprogramming of Trained Immunity in Myeloid Cells.

Infiltrating and tissue-resident myeloid cells are essential regulators of innate and adaptive immunity. During inflammation, and in response to microbial products, these cells can adapt to microenvironmental conditions and acquire specialized functions, including phagocytosis and the production of proinflammatory cytokines. Such myeloid plasticity is driven, in part, by epigenetic dynamics that can sustain stable phenotypes after activation, and which may lead to maladaptive cell polarization states associated with inflammation and autoimmunity. Here, we review recent reports describing epigenetic mechanisms linked to such polarization states and innate immune memory (tolerance and training) in monocyte and macrophage lineages. We discuss how these mechanisms might be targeted to develop putative immunomodulatory tools that might be used to treat a variety of immune-mediated diseases.

BET Proteins: An Approach to Future Therapies in Transplantation.

In order to develop new efficient therapies for organ transplantation, it is essential to acquire a comprehensive knowledge of the molecular mechanisms and processes, such as immune activation, chronic inflammation, and fibrosis, which lead to rejection and long-term graft loss. Recent efforts have shed some light on the epigenetic regulation associated with these processes. In this context, the bromo and extraterminal (BET) family of bromodomain proteins (BRD2, BRD3, BRD4, and BRDT) have emerged as major epigenetic players, connecting chromatin structure with gene expression changes. These proteins recognize acetylated lysines in histones and master transcription factors to recruit regulatory complex and, finally, modify the transcriptional program. Recent studies indicate that BET proteins are essential in the NF-kB-mediated inflammatory response, during the activation and differentiation of Th17-immune cells, and in profibrotic processes. Here, we review this new body of data and highlight the efficiency of BET inhibitors in several models of diseases. The promising results obtained from these preclinical models indicate that it may be time to translate these outcomes to the transplantation field, where epigenetics will be of increasing value in the coming years.

Methylation of NKG2D ligands contributes to immune system evasion in acute myeloid leukemia.

Engagement of the activating receptor NKG2D (natural killer group 2 member D) with its ligands (NKG2DL) major histocompatibility complex class I related-A and -B (MICA/B), UL-16 binding protein families (ULBPs 1-6) is important to ensure the innate immunity to tumor cells. However, these cells have developed strategies to downregulate NKG2DL expression and avoid immune recognition. We demonstrate that DNA methylation can contribute to the absence of NKG2DL expression during tumor progression. We analyzed the DNA methylation profiles for each NKG2DL by pyrosequencing in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), hepatocellular carcinoma (HC), breast cancer and colon cancer cell lines. High levels of DNA methylation for NKG2DL were found in some tumor cell lines, mainly in AML cells. This hypermethylation was correlated with the absence of transcription for NKG2DL. Higher DNA methylation levels for MICA, ULBP1 and ULBP2 were observed in AML patients (n=60) compared with healthy donors (n=25). However, no DNA methylation for NKG2DL was found in colon cancer patients (n=44). Treatment with demethylating agents (5-azacytidine and 5-aza-2'-deoxycytidine) restored the expression of NKG2DL on the cell surface of AML cells, leading to an enhanced recognition by NKG2D-expressing cells. Our data suggest that NKG2DL may be aberrantly silenced by DNA methylation as a consequence of tumor development in AML patients.

Activating killer immunoglobulin-like receptors genes are associated with increased susceptibility to ankylosing spondylitis.

The aim of this study was to analyse the association of specific killer cell immunoglobulin-like receptors (KIR) genes and haplotypes with susceptibility to ankylosing spondylitis (AS) and its different clinical manifestations in a Spanish population. The presence or absence of all KIR genes was studied for their association with AS. A total of 176 patients with AS and 435 healthy control subjects were selected for this study based on clinical criteria. The commercial KIR-sequence-specific oligonucleotides (SSO) typing kit was used to investigate KIR typing. Frequencies of KIR2DS1 and KIR3DS1 genes were increased significantly in patients compared with healthy controls [52·8 versus 38·2%, PBonf < 0·01, odds ratio (OR) = 1·81 (1·28-2·59); 51·7 versus 37·5%, PBonf < 0·01, OR = 1·79 (1·25-2·54)]. Moreover, the frequency of activating genotypes in the AS patient group was significantly higher than in the healthy control group (P < 0·05). KIR2DS1 and KIR3DS1, in addition to human leucocyte antigen (HLA)-B27, may play an important role in the pathogenesis of AS. However, we show that the contribution of the KIR genes to AS susceptibility extends beyond the association with individual KIRs, with an imbalance between activating and inhibitory KIR genes seeming to influence the susceptibility to AS.

Intensity of the humoral response to cytomegalovirus is associated with the phenotypic and functional status of the immune system.

Cytomegalovirus (CMV) infection exerts an enormous effect on human immunity, as it is associated with an immune-impaired response, a variety of chronic diseases, and overall survival in elderly individuals. Levels of anti-CMV antibodies may be associated with the differentiation degree of T cell subsets. Titers are significantly higher in the elderly and positively correlated with specific CD4(+) T cell responses to CMV. In the elderly, antibody titers are associated with the degree of differentiation and the T cell receptor excision circle (TREC) content in CD4(+) T cells, with other features of the immune risk profile, and with a reduced ability to respond to immunization in vivo. Associations may be absent in young subjects because their anti-CMV antibody titers are lower than those of the elderly. However, comparing young and elderly individuals with similar antibody levels reveals differences in their highly differentiated and naïve T cells. These are more marked in individuals with high titers. In parallel with the increase in anti-CMV antibodies, the elderly experience a significant reduction in absolute counts of naïve CD4(+) T cells, which may be a strategy to compensate for the expansion of differentiated cells and to avoid an increase in total T cells. In summary, our results show that titers of anti-CMV antibodies, and not only CMV seropositivity, are related to differentiation status and immunocompetence in the elderly, making this as an important prognostic marker of the status of immune system function.

Old and new HLA associations with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease that primarily involves the axial skeleton and the sacroiliac joint, but may also affect peripheral joints and entheses. AS susceptibility is clearly attributable to genetic factors and the link between human leukocyte antigen (HLA)-B27 and AS is the strongest association between an HLA class I molecule and a disease. However, there is evidence for the involvement of other, non-B27 factors within the major histocompatibility complex (MHC) in AS susceptibility. MHC class I is clearly the most significant genetic region for the disease, although most of the genetic association of this region is driven by HLA-B27. Moreover, several studies have investigated the MHC class II region and its association with AS. This review summarizes the current findings concerning the MHC genetics of the disease, focusing in particular on the associations of HLA with AS found in different ethnic populations throughout the world, and the possible mechanisms underlying them.

Effect of killer immunoglobulin-like receptors in the response to combined treatment in patients with chronic hepatitis C virus infection.

Killer immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells and may play an important role in the innate response against infection with viruses such as hepatitis C virus (HCV). We examined whether the different combinations of KIRs with their HLA class I ligands influenced the response to combined treatment (pegylated alpha interferon and ribavirin) of patients infected by HCV. A total of 186 consecutive patients diagnosed with chronic HCV infection were analyzed. Seventy-seven patients exhibited HCV RNA levels at 6 months posttreatment and were called nonresponders (NR), while 109 cleared viral RNA and were named sustained viral responders (SVR). Patients were typed for HLA-B, HLA-Cw, KIR genes, and HCV genotype. In our study, the frequency of the KIR2DL2 allele was significantly increased in NR (P < 0.001; odds ratio [OR] = 1.95), as was the frequency of the KIR2DL2/KIR2DL2 genotype (P < 0.005; OR = 2.52). In contrast, the frequencies of the KIR2DL3 genotype (P < 0.001) and KIR2DL3/KIR2DL3 genotype (P < 0.05; OR = 0.54) were significantly increased in the SVR. Different combinations of KIR2DL2 and KIR2DL3 alleles with their ligands were analyzed. The frequency of the KIR2DL2/KIR2DL2-HLA-C1C2 genotype was significantly increased in the NR (P < 0.01; OR = 3.15). Additionally, we found a higher frequency of the KIR2DL3/KIR2DL3-HLA-C1C1 genotype in the SVR group (P < 0.05; OR = 0.33). These results were not affected by the HCV genotype. In conclusion, patients who carried the KIR2DL2/KIR2DL2-HLA-C1C2 genotype were less prone to respond to treatment. However, the KIR2DL3/KIR2DL3-HLA-C1C1 genotype clearly correlated with a satisfactory response to treatment, defined by the clearance of HCV RNA.

Potential role of NKG2D and its ligands in organ transplantation: new target for immunointervention.

NKG2D is one of the best characterized activating receptors on Natural Killer (NK) and CD8+ T cells. This receptor recognizes several different ligands (MICA/MICB and ULBPs) induced by cellular stress and infection. In addition to the role described in cancer surveillance, recent data highlight the importance of NKG2D and its ligands in organ transplantation. Allografts show evidence of MICA and MICB expression in both acute and chronic rejection. The presence of anti-MICA antibodies has been correlated with incidence of graft rejection. Furthermore, NKG2D-ligand engagement activates NK cells, which provides T-cell costimulation, and enhances antigen specific CTL-mediated cytotoxicity. Activated NK cells may function as a bridge between innate and adaptive immunity associated with transplantation. Activated NK cells in response to IL-15 can also trigger organ rejection through NKG2D and affect the maturation of both donor and recipient antigen presenting cells (APCs) and ultimately the T-cell allogeneic response. Regulatory T cells, which modulate T-cell responses in organ transplantation and infections, were reduced in numbers by NK cells exposed to intracellular pathogens, possibly via interaction with one NK2GD receptor. Blockage of NKG2D-NKG2D-L interactions provides a novel pathway for development of inhibitors. These studies have important clinical and therapeutic implications in solid organ transplantation.

A search for cyclophilin-A gene (PPIA) variation and its contribution to the risk of atherosclerosis and myocardial infarction.

Cyclophilin A is secreted by vascular smooth muscle cells in response to inflammatory stimuli, and could thus contribute to atherosclerosis. We hypothesized that the genetic variation at the cyclophilin A gene (PPIA) could affect the risk for developing atherosclerosis and myocardial infarction. This study included 250 myocardial infarction patients (all male and < 60 years; 95% are smokers). All these cases had at least one atherosclerotic diseased coronary vessel. DNA was obtained from patients and from 250 healthy controls. The variation at the PPIA gene was determined in the patients through single-strand conformation analysis and direct sequencing of seven polymerase chain reaction fragments. Allele and genotype frequencies were compared between patients and controls. The effect of a promoter polymorphism (-11 G/C) on gene expression was in vitro analysed with luciferase-reporter assays. We found two common polymorphisms in the PPIA promoter (-11 G/C) and the 5' non-translated (+36 G/A) regions. Cells transfected with luciferase-plasmids containing the -11 G had significantly higher luciferase activity. Genotype frequencies for these polymorphisms did not differ between patients and controls. In conclusion, we reported a functional variant in the PPIA promoter. However, the PPIA variation did not significantly contribute to the risk of suffering from myocardial infarction among patients with atherosclerotic diseased vessels.

[Relapsing acute pancreatitis associated with gluten enteropathy. Clinical, laboratory, and evolutive characteristics in thirty-four patients]. / Pancreatitis aguda recidivante con enteropatía por gluten asociada, Características clínico-analíticas y evolutivas en 34 pacientes.

OBJECTIVES: To describe the frequency and the clinical and laboratory characteristics of relapsing acute pancreatitis (AP) associated with gluten enteropathy (GE). PATIENTS AND METHODS: We prospectively examined all acute pancreatitis cases admitted to our Department in 2006. We recorded a total of 185 patients. With recurring forms, 40 (22%) in all, we used a clinical-lab protocol including serologic and genetic markers, and duodenal biopsy to rule out GE. RESULTS: A total of 34 patients (18%) met clinical-biological criteria for GE (group1), and were compared to the remaining non-GE AP cases (n=161) (group2). Mean age in the GE group was 54 +/- 25 years, slightly younger than group 2 (61 +/- 14) (NS). There was a mild predominance of women (50%) in group 1 versus group 2 (38.5%) (NS). Seven patients in group 1 (20%) had severe AP, as compared to 27 (17%) in group 2 (NS). The presence of cholelithiasis in group 1 involved 6 cases (18%), which was significantly lower than in group 2--72 cases (45%) (p < 0.05). Four patients with GE developed pseudocysts (12%) versus 13 (8%) in group 2 (NS). Tissue transglutaminase (tTG) was elevated only in 3 patients (9%). Nine patients (34%) were DQ2 (+) and 4 (12%) DQ8 (+); the rest (54%) were all negative for both markers. From an endoscopic perspective there was diffuse duodenitis in 32 patients (95%). Duodenal biopsies revealed villous atrophy (Marsh 3) in 2 patients (6%); submucosal inflammatory infiltration (Marsh 2) in 10 (29.4%); increased intraepithelial lymphocytes (Marsh 1) in 8 cases (23.5%), and normal mucosa (Marsh 0) in 14 patients (41.2%). Response to GFD after 1 year was excellent in 30 patients (88%). CONCLUSIONS: Relapsing AP with GE represents a relatively common association that is indistinguishable from other APs from a clinical-evolutive standpoint, except for a lower presence of cholelithiasis (p < 0.05). A specific diagnostic protocol is much needed in the identification of these patients since GFD is the only effective therapy to prevent new AP events from developing.

Single dose of Rituximab plus plasmapheresis in an HIV patient with acute humoral kidney transplant rejection: a case report.

Before the highly active antiretroviral therapy (HAART) era, kidney transplantation was not considered an option for patients infected with human immunodeficiency virus (HIV) because of its poor outcome. However, recent studies have demonstrated results comparable to those of recipients without HIV infections. They have shown that HIV-positive patients maintained on HAART mount an immune response. Immunosuppressive agents are chosen to minimize aggravation of HIV infection, bearing in mind the potential side effects of the combination of HAART and immunosuppressive drugs. Herein we have reported the case of a 43-year-old HIV- and hepatitis C virus-infected woman with preserved immune function who received a cadaveric kidney transplant and developed an acute humoral rejection, which was successfully treated with Rituximab.

[Increased prevalence of celiac disease in first and second-grade relatives. A report of a family with 19 studied members]. / Prevalencia aumentada de enfermedad celiaca en familiares de primer y segundo grado: descripción de una familia con 19 miembros estudiados.

INTRODUCTION: celiac disease (CD) is an autoimmune condition that is triggered by the ingestion of gluten, a substance present in most cereals, and that affects genetically predisposed individuals. As a result, this condition is clearly familial, and mainly associated with HLA class II markers. OBJECTIVES: in this work we set out to analyze the prevalence of CD in an extensive family based on an index subject who had already died from this disease a few years ago, where CD had been complicated by the development of a small-bowel malignancy, namely an adenocarcinoma. METHODS: nineteen members were studied. They all were subjected to a diagnostic protocol including a detailed medical history, hemogram, coagulation tests, and blood biochemistry (including liver function tests, serum iron metabolism, circulating folic acid and vitamin B12 levels, thyroid function tests, tissue transglutaminase measurement, and genetic markers (DQ2 and DQ8). Suspect cases underwent gastroscopy plus multiple duodenal biopsy for confirmation. RESULTS: overall we encountered CD in 9/19 studied members, which represents 47.4% with the following distribution according to degree of kinship -four of seven siblings (57%); one of three children (33.3%); three of eight nephews and nieces (37.5%), and the only grandnephew, who was 9 years old. CONCLUSIONS: from all this it may be seen that family studies are needed every time a patient is diagnosed with celiac disease; these studies should include both first- and second-degree relatives, given the high prevalence encountered and the fact that these tests are relatively straighforward to perform.

Interaction between KIR3DL1 and HLA-B*57 supertype alleles influences the progression of HIV-1 infection in a Zambian population.

KIR and HLA loci are both highly polymorphic, and some HLA class 1 products bind and trigger cell-surface receptors specified by KIR genes. We examined whether KIR genes act in concert with HLA-B locus to control HIV-1 infection in a sample of Zambian patients. DNA samples from 88 Zambian patients with HIV-1 were examined. Patients were classified as either slow progressors (SP; n = 54) or rapid progressors (RP; n = 34) to AIDS. All were typed for HLA-B and KIR genes. Our results reveal an association between B*57 supertype (B*57s, which includes B*57 and B*58 alleles) and delayed progression to AIDS (p = 0.0007 by pc = 0.015; OR = 5.25). We also observed an increase incidence of Bw4-I80 in patients with slow progression (p = 0.001 by pc = 0.003, OR = 5). This increase was found to be secondary to B*57s. The presence of both KIR3DL1 and B*57S has a significant effect on progression to AIDS (p = 0.0008; OR = 5.61). B*57s genotypes with another HLA-B allele different from those in the trans position, which also had a specificity different to Bw4-I80 (Bw4-T80 or Bw6), was also greater in the SP than in the RP group (p = 0.00003; OR = 10.11). The presence of the inhibitory allele KIR3DL1 in combination with the HLA-B*57s alleles that contain the Bw4-I80 epitope, has a highly protective effect against progression to AIDS in Zambian patients.

Loss of heterozygosity and mutation analysis of the p16 (9p21) and p53 (17p13) genes in squamous cell carcinoma of the head and neck.

We analyzed allelic loss at the p53 gene (17p13) and at chromosome region 9p21 in 35 primary head and neck squamous cell carcinomas. Loss of heterozygosity (LOH) at p53 and 9p21 was found in 50 and 75% of informative cases, respectively. LOH at the p53 gene did not increase significantly with tumor stage, but was more frequent in moderately and poorly differentiated tumors than in well-differentiated tumors. LOH plus mutation or homozygous deletion of p53 was limited to advanced stage and poorly differentiated tumors. Allelic loss at 9p21 is frequent in early stage head and neck squamous cell carcinoma and is not significantly associated with LOH at p53. The second exon of the p16/MTS1/CDKN2 gene was found to be homozygously deleted in 1 of 19 cases showing LOH at 9p21, but direct sequencing did not show mutations in the remaining 18 cases. This suggests that p16 plays a limited role in the development of head and neck squamous cell carcinoma.

Major histocompatibility complex associations of ankylosing spondylitis are complex and involve further epistasis with ERAP1.

Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,647 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype.

The OTF3 gene polymorphism confers susceptibility to psoriasis independent of the association of HLA-Cw*0602.

Psoriasis has been strongly associated to HLA-Cw6, but it remains unclear whether Cw6 itself or a closely linked gene is associated with the disease. The aim of this study was to clarify whether the HLA-C itself determines disease susceptibility or whether it acts only as a marker for the susceptibility allele. We examined a sample of 95 type I psoriasis patients and 104 Spanish matched controls to investigate whether HLA-Cw*0602 or other closely related class I loci, such as HLA-B and MICA (which are centromeric to HLA-C), or corneodesmosin gene and octamer transcription factor-3 genes (which are telomeric to HLA-C), might play a part in disease development. DNA samples were genotyped by polymerase chain reaction/sequence-specific primers (HLA-C), polymerase chain reaction/sequence-specific primers (HLA-B), radioactive polymerase chain reaction (MICA-TM polymorphism in the transmembrane region), and polymerase chain reaction/restriction fragment length polymorphism (protein S and octamer transcription factor-3). Our results show a significant increase of Cw*0602 in psoriasis patients (odds ratio = 3.64; pc < 0.0006). A significant association between the beta allele of octamer transcription factor-3 (HindIII) and psoriasis was also detected (odds ratio = 3.76; pc < 0.0003). The allele octamer transcription factor-3B (etiologic fraction = 0.62) was found to be more strongly associated to psoriasis vulgaris than Cw*0602 (etiologic fraction = 0.35) and the increase of octamer transcription factor-3 B allele is independent of the linkage disequilibrium with Cw*0602 as this was also found in Cw*0602 negative patients (odds ratio = 3.63; pc < 0.015, etiologic fraction = 0.55). We did not detect an association between the corneodesmosin gene and psoriasis. This fact suggests that the psoriasis susceptibility gene is located within a critical region of 147 kb, telomeric to HLA-C and centromeric to the corneodesmosin gene, and the association of Cw6 to psoriasis may be secondary to linkage disequilibrium.

The germline repertoire of T cell receptor beta-chain genes in multiple sclerosis patients from Spain.

Recently several reports have described contradictory results after studying the association between restriction fragments length polymorphisms (RFLP) of T cell receptor (TcR) beta-chain genes and multiple sclerosis (MS). We studied the allelic, genotypic and haplotypic distribution of RFLPs of TcR beta chain gene segments C beta, V beta 8 and V beta 11 in 97 unrelated multiple sclerosis (MS) patients, 11 with chronic progressive MS and 86 with relapsing/remitting (R/R) MS. We found the distribution of the TcR haplotypes defined by the alleles of the three loci studied in the MS patients was significantly different from that found in control individuals. The distribution of TcR haplotypes in R/R MS patients was also different from that observed in controls. Our data suggest that the TcR beta chain gene complex contains one or more genes involved in genetic susceptibility to develop MS.

DNA analysis of HLA-DR4B1 subtypes in multiple sclerosis by specific oligonucleotide probes.

Conversely to the well-established association of DR2/Dw2 with multiple sclerosis (MS) susceptibility in Caucasoids, several studies have found an association of DR4 in populations from Mediterranean origin. We have studied the distribution of the different DR4B1 subtypes in Spanish MS patients. Oligonucleotide probes were selected in order to type samples amplified by polymerase chain reaction (PCR) from Spanish DR4+ MS patients (25) and controls (28). No DR4B1 subtypes were found to be increased in MS. MS susceptibility linked to DR4 may be due to the presence of shared functional epitopes common to the different HLA-DR4B1 subtypes.

HLA-B27 alone rather than B27-related class I haplotypes contributes to ankylosing spondylitis susceptibility.

Characterization of non-B27 susceptibility genes will be required to know the pathogenesis of AS. The aim of this study was to examine whether ankylosing spondylitis (AS) susceptibility is controlled by B27 alone rather than B27 haplotypes and, whether other closely related class I loci, such as MICA and TNFA genes might play a role in AS. Three hundred eleven B27-positive samples from Caucasoid, Asian, and African populations were selected and genotypes were carried out by PCR/SSOP (HLA-B27 and HLA-C), PCR/SSP (MICA-TM polymorphism in the transmembrane region), PCR/SSCP (MICA alleles), and PCR-RFLP (TNF-alpha). Of these, 161 were AS patients, chosen in order to investigate the contribution of TNFA and MICA loci to AS in HLA-B27 positive individuals. Some findings can be concluded from the study: (a) No significant differences of TNF-alpha promoter alleles at position -308 and -238 (A/G) were found between AS patients and B27 matched alleles from healthy controls; (b) strong linkage disequilibrium was found between the B27 and the MICA alleles. The MICA-A4 was found to be in association with B*2705,02,03 and 08; MICA-A5 with B*2704 and B*2707 and MICA-A.5.1 with B*2706; (c) no significant differences of MICA alleles were found between AS and controls carrying the B27-associated alleles, and therefore no evidence is provided for an additional role of MICA gene in AS susceptibility; (d) there are a striking correlations between the structure of B27 extended haplotypes (from MICA region to HLA-C) and the ethnic distribution of these subtypes. The results of differential linkage disequilibrium with HLA-B27 subtypes suggest that B27 itself remains the primary gene for AS susceptibility, and TNFA and MICA are not involved in the pathogenesis of the disease.