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OBJECTIVE: Natural killer (NK) cells have been involved in the pathology of different inflammatory and autoimmune disorders. Inflammation is an important regulator of osteoarthritis (OA), but the molecular and cellular mechanisms regulating this process are not well defined. DESIGN: To understand the role of NK cells in OA, we have compared the phenotype (CD56 subsets and perforin and granzyme expression) and cytotoxic function of NK cells in peripheral blood and synovial fluid from patients with OA undergoing total knee arthroplasty. RESULTS: In contrast to peripheral blood lymphocytes (PBLs), the majority of NK cells from the synovial fluid were CD56brightCD16(-) cells. As expected the expression of the cytolytic mediators perforin and granzyme B in CD56brightCD16(-) cells was low and correlated with a poor cytotoxic potential against K562 sensitive target cells. Surprisingly, this low cytotoxic NK cell subset expressed high levels of granzyme A (a protease recently characterized as a key modulator of inflammation in mouse models) in synovial fluid but not in peripheral blood. The presence of the CD56(+)brightCD16(-) cells expressing granzyme A correlated with increased levels of pro-inflammatory cytokines in synovial fluid from OA patients. CONCLUSION: Our results indicate that NK cells from the synovium of patients with OA, which present an immunoregulatory non-cytotoxic phenotype, show different phenotype comparing with NK cells from peripheral blood, especially expressing granzyme A, a pro-inflammatory molecule which may contribute to the establishment of chronic articular inflammation in this type of patients.

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Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists.

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Purpose. Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. Methods/patients. Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. Results. Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. Conclusion. Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics (AU)

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PURPOSE: Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. METHODS/PATIENTS: Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. RESULTS: Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. CONCLUSION: Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics.

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Difuse proliferative lupus nephritis (DPLN) is the most common and severe form of lupus nephritis. A predominance of IFN-gamma-producing T cells in both peripheral and renal tissues of patients with DPLN has been identified which suggests an important role for cell-mediated immunity in the pathogenesis of this complication in SLE. The biological effects of IFN-gamma rely mainly on the activity of the transcription factor called signal transducer and activator of transcription (STAT)-1. To assess the IFN-gamma/STAT-1 pathway in DPLN, we examined the expression of STAT-1 in renal biopsies from 15 DPLN patients by immunohistochemical staining with an anti-STAT-1 antibody. The expression of STAT-1 in renal tissues was correlated with several clinical and laboratory findings in these DNPN patients.STAT-1 was activated in the tubular cells in all DPLN patients. Seven of 15 DPLN biopsies (46.7%) showed positive cells in glomeruli. Five of these seven DPLN biopsies (71.4%) with positive glomerular cells showed a serum creatinine >1.5 mg/mL at the time the biopsy was carried out whereas only one of eight DPLN biopsy specimens (12.5%) without positive glomerular cells, showed a serum creatinine >1.5 mg/mL (P = 0.041). Moreover, the percentage of DPLN patients with a worse renal outcome in those who showed expression of STAT-1 in glomerulari were higher in comparison to those without STAT-1 expression (P = 0.041). Our results show that STAT-1 is activated in DPLN suggesting that biological effects of IFN-gamma in renal tissues depend, at least in part, on the activation of STAT-1.

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A functional polymorphism in PTPN22, a gene encoding a phosphatase involved in T-cell signaling, has been associated with autoimmunity. We checked for the prevalence of the PTPN22 R620W polymorphism in multiplex families affected with systemic lupus erythematosus (SLE) and other autoimmune diseases. Its association with other polymorphisms in mannose binding lectin (MBL) and FcgammaRIIa (CD32A) genes was also studied. Deoxyribonucleic acid samples were obtained from 233 Spanish individuals who belonged to 21 families in which at least two members had been diagnosed with some autoimmune disease, mainly SLE. A healthy control population was also included (n= 129). Genotyping for the R620W single-nucleotide polymorphism (SNP) was performed by restriction fragment length polymorphism analysis of polymerase chain reaction products. Allele frequency for the T allele was slightly higher in the families with autoimmune disease, especially when considering the affected individuals (0.094 vs 0.062). Actually, 18.8% affected family members vs 11.6% controls had the polymorphism (P= 0.179). Nineteen percent of affected individuals had both the PTPN22 T and the CD32A R131 alleles, whereas only 8.5% unaffected relatives had both susceptibility alleles simultaneously [P= 0.031, odds ratios 2.508 (95% confidence interval 1.066-5.896)]. The tendency toward finding the T allele more frequently in members affected with some particular autoimmune disorder suggests that this SNP may confer susceptibility to autoimmunity. The fact that more affected than unaffected relatives carried both the T and the R131 alleles simultaneously leads us to think about the existence of a combinatorial effect between genes that could help define individuals prone to autoimmune diseases.

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OBJECTIVE: It has recently been reported that some autoimmune diseases seem to be associated with a functional polymorphism in PTPN22, a gene which encodes a phosphatase known to be important in T-cell signaling. The aim of our study was to check for the prevalence of the PTPN22 R620W polymorphism in patients with systemic sclerosis. METHODS: DNA samples from 54 systemic sclerosis patients and 55 healthy controls were obtained from peripheral blood and genotyping was performed by means of a restriction fragment length polymorphism analysis of PCR products (RFLP-PCR). RESULTS: Allele frequency for the T allele was slightly higher in the patients group (0.074 versus 0.055). Eight out of the 54 systemic sclerosis patients (14.8 %) were heterozygous for this single nucleotide polymorphism whereas the CT genotype was found in 6 out of the 55 controls (10.9%). Nevertheless, the difference did not reach statistical significance (p = 0.542). Neither certain antibodies linked to systemic sclerosis (anti-centromere and anti-topoisomerase I antibodies) nor any particular clinical involvement were associated with the polymorphism. CONCLUSION: This particular single nucleotide polymorphism of PTPN22 does not seem to be associated with systemic sclerosis.

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We report here a novel DRB1 allele identified during sequence-based HLA-DRB typing. This allele was detected during routine HLA typing of a patient and his family prior to bone marrow transplantation. The new allele, DRB1*0108, was found in the patient and in a brother. Molecular cloning and sequencing confirmed that the new DRB1 allele is identical to DRB1*0101 at exon 2 except for a single nucleotide substitution at codon 37 (TauCC-->TauAlphaC), changing the encoded serine to tyrosine. This position of the beta1 domain lies in the floor of the antigen-binding groove and shows the highest polymorphism among DRB1 alleles.

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