Segundo González

NKG2D ligands: key targets of the immune response.

NKG2D is an activating receptor expressed by NK and T cells. NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a key role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease. In this review, we analyse the recent advances in understanding the regulation of NKG2D ligand expression and their therapeutic implications.

The MICA‐A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to the development of psoriatic arthritis and is independent of the association of Cw*0602 in psoriasis

OBJECTIVE To investigate the relative contribution of HLA antigens in the susceptibility to psoriasis and to localize additional genetic factors involved in psoriatic arthritis (PsA). METHODS DNA from 45 patients with psoriasis, 65 with PsA, and 177 healthy control subjects was examined by polymerase chain reaction (PCR) using sequence-specific oligonucleotide probes to determine HLA-C. To examine whether MICA (class I major histocompatibility complex chain-related gene A) confers additional susceptibility, trinucleotide repeat polymorphism in the transmembrane region of the MICA gene was investigated by radioactive PCR. Further analysis of MICA was made by PCR-single-strand conformational polymorphism to determine the allelic variant corresponding to MICA transmembrane polymorphism. RESULTS Our results reveal new findings: 1) the frequency of the Cw*0602 allele was significantly increased in both patient groups: psoriasis (corrected P [Pcorr] < 10(-5), relative risk [RR] 6.2), PsA (Pcorr < 10(-6), RR 6.3), 2) the trinucleotide repeat polymorphism MICA-A9 was present at a significantly higher frequency in PsA patients (Pcorr < 0.00035, RR 3.2), whereas a similar distribution was found in both the control and psoriasis population, 3) this polymorphism corresponds to the MICA-002 allele and was found to be overrepresented in patients with the polyarticular form (Pcorr < 0.0008, RR 9.35), 4) the increase in MICA-A9 in PsA patients is independent of linkage disequilibrium with Cw*0602, 5) this allele confers additional relative risk (RR 3.27, etiologic fraction 0.44; etiologic fraction is the proportion of disease cases among the total population that are attributable to 1 allele when the relative risk is > 1) in PsA patients who carry Cw*0602. CONCLUSION The data obtained in this study are consistent with the polygenic inheritance of psoriasis. Cw*0602 appears to be the stronger genetic susceptibility factor for psoriasis. Independent of the HLA-C association, MICA-A9 polymorphism corresponding to the MICA-002 allele is a possible candidate gene for the development of PsA.

TNF-α− 308A promoter polymorphism is associated with enhanced TNF-α production and inflammatory activity in Crohn's patients with fistulizing disease

OBJECTIVE:Tumor necrosis factor-α (TNF-α) plays a key role in the inflammatory response and pathogenesis of Crohns disease (CD). TNF-α− 308A polymorphism within the TNF-α gene promoter has been associated with enhanced TNF-α production in vitro. The aim of this study was to investigate the effect of TNF-α promoter polymorphism at −308 on the susceptibility and phenotypic expression of fistulizing CD.METHODS:The distribution of −308 TNF-α genotypes was analyzed in 50 patients with fistulizing CD and 100 healthy matched controls. TNF-α, interleukin-1β, and interleukin-6 serum levels were measured by ELISA. Serum amyloid-A, C-reactive protein, α1-antitrypsin, α1-acid glycoprotein, and haptoglobin were measured by nephelometry.RESULTS:No significant differences were found in the allele frequencies of the polymorphism between patients and controls. However, compared with −308GG patients, those carrying −308AG had a significant increase of serum levels of TNF-α (58 ± 79 vs 8 ± 19 pg/ml, p < 0.001), interleukin-1β (36 ± 45 vs 16 ± 20 pg/ml, p = 0.048), and acute phase proteins (APPs). −308A carriers had also a higher frequency of arthritis (66% vs 26%, p = 0.039). The logistic regression model showed that the patients carrying −308A polymorphism had a relative risk for developing arthritis of 5.45 (95% CI = 1.1–25.6). No other clinical or analytical findings were predictive for the risk of development of arthritis.CONCLUSIONS:TNF-α− 308A polymorphism is associated with enhanced TNF-α production, more intense inflammatory activity, and an increased risk for arthritis susceptibility in CD patients with fistulizing disease.

Protective Effect of the HLA-Bw4I80 Epitope and the Killer Cell Immunoglobulin-Like Receptor 3DS1 Gene against the Development of Hepatocellular Carcinoma in Patients with Hepatitis C Virus Infection

The aim of the present study was to investigate, in 152 Spanish patients infected with hepatitis C virus (HCV), the possibility that killer cell immunoglobulin-like receptors (KIRs) influence progression to hepatocellular carcinoma. KIRs are related to the activation and inhibition of natural killer cells and may play an important role in the innate response against infection with such viruses as HCV. We found that the human leukocyte antigen-Bw4I80 epitope and the KIR3DS1 gene were more frequent in HCV carriers than in patients with hepatocellular carcinoma. Moreover, these associations were not independent of each other--the KIR3DS1/Bw4I80 genotype clearly was also more frequent in HCV carriers (odds ratio, 24.22).

Polymorphism in MICA rather than HLA-B/C genes is associated with psoriatic arthritis in the Jewish population

The aim of this study was to examine whether the association of psoriatic arthritis (PsA) with human leukocyte antigen (HLA) class I genes is secondary to linkage disequilibrium with a nearby gene. We examined a sample of the Jewish population to investigate whether HLA-B/C and DR polymorphism is associated with susceptibility, or whether other closely related class I loci, such as the major histocompatibility complex class I chain-related gene A (MICA) and tumor necrosis factor (TNF), might play a role in disease development. Comparisons of different populations with different HLA profiles would be of value in identifying the candidate genes involved in PSA. Fifty-two patients with PsA and 73 random matched controls from a Jewish population were selected and DNA typed by polymerase chain reaction-single-strand oligonucleotide probe (PCR-SSOP) (HLA-C), PCR sequence-specific primers (PCR-SSP) (HLA-B, -DR), radioactive PCR (MICA-TM polymorphism in the transmembrane region), and PCR-RFLP (TNF). Some findings can be concluded from the study: (1) the frequency of HLA-B*5701, B*3801, B*39, B*27, Cw*0602, Cw*07, DRB1*0402, and DRB1*0701 were not found to be significantly increased in PsA; (2) no significant differences of TNFalpha promoter alleles at positions -308 and -238 were found between PsA and healthy controls; (3) the trinucleotide repeat polymorphism MICA-A9 was present at a higher frequency in PsA patients, (p(c) < 0.009, RR = 3.34, EF = 0.39); and (4) MICA-A9 polymorphism was found in linkage disequilibrium with HLA-B alleles (B*5701, B*3801) described to be associated with PsA in Caucasians. These results suggest that the MICA gene or other nearby gene(s) may be involved in the development of PsA, and it would thus appear that psoriasis vulgaris (PsV) and PsA are associated with different MHC susceptibility genes.

High serum tumor necrosis factor-α levels are associated with lack of response to infliximab in fistulizing Crohn’s disease

OBJECTIVES:Infliximab, a chimeric monoclonal antibody directed against tumor necrosis factor-α (anti-TNF-α), has been effective in the treatment of patients with active Crohns disease and with fistulas. We investigated the effect of infliximab on circulating cytokines and acute phase proteins in patients with fistulas to determine the clinical response to anti-TNF-α.METHODS:A total of 36 patients with fistulizing Crohns disease were selected for study. Serum from patients was drawn before the infusion on day 0 and at wk 2, 4, 6, 8, and 10 after completion of treatment. Circulating concentrations of TNF-α, interleukin-1β (IL-1β), and IL-6 were measured by ELISA. The functional activity of circulating TNF-α was assessed by the WEHI 164 TNF-α bioassay. Acute phase proteins were also determined.RESULTS:Elevated TNF-α, IL-1β, IL-6, and acute phase proteins were observed in patients with Crohns disease. Of the patients with fistulas, 22 (61.1%) responded to treatment. Before receiving infliximab, higher levels of serum TNF-α were found in patients who did not respond to infliximab compared with those who did (median interquartile range 26, 0–245 pg/ml; n = 14 vs 0, 0–22 pg/ml, n = 22). Patients showed no change in circulating levels of TNF-α during the course of the study.CONCLUSIONS:This treatment produces a clinical improvement in about two-thirds of CD patients with fistulas. The circulating levels of TNF-α are associated with the response to infliximab and could help to identify patients who would benefit from anti-TNF-α treatment.

Transcriptional regulation of ULBP1, a human ligand of the NKG2D receptor.

Tumor cells expressing ligands of the NKG2D receptor stimulate anti-tumor immunity mediated by natural killer and T cells. In humans, NKG2D ligands (NKG2DL) are encoded by MIC and ULBP proteins. NKG2DL exhibit highly restricted expression in healthy tissues but are widely expressed in tumors. However, regulation of each NKG2DL differs substantially in different cancer cells. In this study, we characterized the mechanisms that regulate the expression of ULBP1. We show that the transcription of ULBP1 strictly depends on the binding of Sp1 and Sp3 to a CRE(1) site located in the ULBP1 minimal promoter. The mutation or deletion of this Sp1/Sp3 binding site abolished the transcription of ULBP1. It also diminished the transactivation of ULBP1 promoter by Sp3 overexpression, but not by Sp1, indicating that Sp3 is the main transcription factor that regulates ULBP1 through the CRE(1) site. Experiments in SL2 cells showed that the ULBP1 promoter was inactive in the absence of the Sp proteins and indicate that Sp3 is the essential activator of ULBP1 transcription, because the overexpression of Sp3 up-regulated its promoter activity >500-fold. Additionally, we demonstrated that AP-2α repressed the expression of ULBP1 in HeLa cells by interfering with the binding of Sp3 and Sp1 to the ULBP1 promoter. These data indicate that Sp1, Sp3, and AP-2α may play an important role in the immunosurveillance against cancer. Finally, the definition of ULBP1 regulation may have implications for development of new therapeutic strategies against cancer cells.

Contribution of KIR3DL1/3DS1 to ankylosing spondylitis in human leukocyte antigen-B27 Caucasian populations

Killer cell immunoglobulin-like receptors (KIRs) and humaAn leukocyte antigen (HLA) loci are both highly polymorphic, and some HLA class I molecules bind and trigger cell-surface receptors specified by KIR genes. We examined whether the combination of KIR3DS1/3DL1 genes in concert with HLA-B27 genotypes is associated with susceptibility to ankylosing spondylitis (AS). Two HLA-B27-positive Caucasian populations were selected, one from Spain (71 patients and 105 controls) and another from the Azores (Portugal) (55 patients and 75 controls). All were typed for HLA-B and KIR (3DS1 and 3DL1) genes. Our results show that in addition to B27, the allele 3DS1 is associated with AS compared with B27 controls (p < 0.0001 and p < 0.003 in the Spanish population and Azoreans, respectively). We also observed that the association of KIR3DS1 to AS was found in combination with HLA-B alleles carrying Bw4-I80 in trans position in the Spanish population (30.9% in AS versus 15.2% in B27 controls, p = 0.02, odds ratio (OR) = 2.49) and in Azoreans (27.2% in AS versus 8.7% in B27 controls, p = 0.01, OR = 4.4 in Azoreans). On the other hand, 3DL1 was decreased in patients compared with B27 controls (p < 0.0001 in the Spanish population and p < 0.003 in Azoreans). The presence of this allele in combination with Bw4-I80 had a protective effect against the development of AS in the Spanish population (19.7% in AS, 35.2% in B27 controls; p = 0.03, OR = 0.45). The presence of KIR3DS1 or KIR3DL1 in combination with HLA-B*27s/HLA-B Bw4-I80 genotypes may modulate the development of AS. The susceptibility to AS could be determined by the overall balance of activating and inhibitory composite KIR-HLA genotypes.

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.

Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

MHC class I‐related genes A/B (MICA/B) are ligands of the NKG2D receptor expressed on T and NK cells. Their expression is highly restricted in normal tissues, but is up‐regulated in tumoral and infected cells. We show that the minimal promoter of both genes contains a CCAAT box, which binds to NF‐Y, and a GC box, which binds to Sp1, Sp3 and Sp4. We also demonstrate that MICB promoter is polymorphic, showing three single nucleotide polymorphisms (C>G at +16, –341, –408) and a deletion of two base pairs at –66 (AG>‐‐) that is located close to the CCAAT box (‐70) and the GC box (‐86). Transcriptional activity associated with MICB promoter variants carrying this deletion, present in the 45.3% of Spanish population, showed a remarkable decrease (18‐fold, p <0.01). By functional analysis, we show that the deletion plays a critical role in MICB promoter activity by diminishing Sp1 transcriptional activation. These important variations in MICB expression among normal individuals could imply a significant difference in the natural immune response against infections or tumor transformation, and might thereby contribute to an increased aberrant immune response against self cells, providing the molecular basis for the associations of the MICB gene to different autoimmune diseases.