NLRP3 E311K mutation in a large family with Muckle-Wells syndrome--description of a heterogeneous phenotype and response to treatment.

INTRODUCTION: Muckle-Wells syndrome (MWS) is an inherited autoinflammatory disease characterized by fever, rash, arthralgia, conjunctivitis, sensorineural deafness and potentially life-threatening amyloidosis. The NLRP3/CIAS1 E311K mutation caused a heterogeneous phenotype of MWS in a large family. This study analyzes the clinical spectrum, patterns of inflammatory parameters and reports on response to treatment. METHODS: A total of 42 patients and family members were screened for the presence of the NLRP3 mutation. Clinical symptoms were reviewed in all family members. Classical (erythrocyte sedimentation rate (ESR, C-reactive protein (CRP)) and novel MWS inflammatory markers (serum amyloid A (SAA), cytokines, cytokine receptor levels) were determined. Patients were treated with the IL-1 inhibitors Anakinra or Canakinumab. RESULTS: All 13 clinically affected patients were heterozygous carriers of the amino acid substitution p.Glu311Lys/E311K encoded by exon 3 of the NLRP3 gene, but none of the healthy family members. Disease manifestations varied widely. Except for one child, all carriers suffered from hearing loss and severe fatigue. TNF-α, IL-6, TNF-RI, and TNF-RII levels as well as SAA were elevated in three, two, one, six and ten patients, respectively. Both clinical and laboratory parameters responded quickly and sustainedly to treatment with Anakinra or Canakinumab. CONCLUSION: The NLRP3 E311K mutation is associated with a heterogeneous clinical spectrum, which may expand the view on MWS presentation. The leading symptom was hearing loss. Pericarditis, a rare but severe clinical feature of MWS, was diagnosed in three patients. One patient had a severe course, which led to renal failure secondary to amyloidosis. IL-1 inhibition leads to rapid and sustained improvement of symptoms.

Impaired suppression of synovial fluid CD4+CD25- T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells.

OBJECTIVE: Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25- effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells. METHODS: The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in (3) H-thymidine proliferation assays. Cytokine levels were analyzed using Bio-Plex Pro assay. RESULTS: Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA-DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells. CONCLUSION: Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

Spinal involvement in chronic recurrent multifocal osteomyelitis (CRMO) in childhood and effect of pamidronate.

There are only a few studies that address the frequency and type of spinal involvement in patients with chronic recurrent multifocal osteomyelitis (CRMO) as well as the outcome of these patients treated with pamidronate (PAM). We performed a retrospective study on patients with CRMO and analyzed clinical and pain assessments as well as regional and whole body MRI findings and compared with posttreatment findings. Of 102 children and adolescents with CRMO, 27 (26%) had involvement of the spine. Vertebral deformities were seen in 14 of these 27 patients, scoliosis or kyphosis in 6. After routine whole body MRI, 19 complained of back pain, whereas eight were asymptomatic with spinal lesions detected incidentally. A total of 72 spinal lesions were detected, thoracic vertebrae being the most commonly affected. Seven patients were treated with PAM; all of whom had vertebral deformities and ongoing back pain. Pain resolution was achieved within 3 months of PAM treatment in every case. One patient subsequently developed a pain amplification syndrome. Repeat MRI performed at a mean interval of 13 months revealed partial or complete resolution of vertebral hyperintensities in every patient. Improvement of vertebral height was seen in a total of three vertebrae in two patients. Severe side effects were not observed. In conclusion, we demonstrated that spinal involvement and associated vertebral deformities with or without kyphoscoliosis are not rare in CRMO, and PAM appears to be an effective and safe treatment for this condition. Although controlled studies are urgently needed, the use of PAM for refractory CRMO with extended spinal involvement (vertebral deformities, kyphosis, and scoliosis) should be considered, especially after failing of conventional therapy.

Involvement of CD91 and scavenger receptors in Hsp70-facilitated activation of human antigen-specific CD4+ memory T cells.

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress-inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen-specific CD4(+) T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen-specific cells were detected combining HLA-DRB1*0401 or HLA-DRB1*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen-specific CD4(+) T cell responses.

70-kDa heat shock proteins: specific interactions with HLA-DR molecules and their peptide fragments.

Heat shock protein 70 (HSP70):peptide complexes are involved in MHC class I and class II-restricted antigen presentation enabling enhanced activation of antigen-specific T cells. Here, we investigated the potential of bacterial and mammalian HSP70 molecules to interact with peptide fragments from HLA-DR and the corresponding complete HLA-DR molecules. Peptide fragments were found to interact with DnaK, the HSP70 homologue from E. coli, but less with stress-inducible human Hsp70. Only a peptide sequence exclusively found in rheumatoid arthritis-protective HLA-DR molecules did not interact with DnaK. Subsequently, we investigated the interaction of complete HLA-DR molecules with HSP70 and detected a specific HSP70:HLA-DR interaction, with highest affinity for human stress-inducible Hsp70. In contrast to the peptide fragments, no allele-specific differences in Hsp70 affinity were detected with complete HLA-DR molecules. Interaction with HLA-DR molecules was increased at lowered pH values, whereas HSP70-chaperoned peptides were released at acidic pH, thus HSP70 could serve as scanner and carrier for antigenic peptides of self or foreign origin and transfer chaperoned peptides onto MHC class II molecules in acidic late endosomal compartments. Our findings indicate that direct interaction between mammalian HSP70 and HLA-DR molecules could be involved in the HSP70-mediated enhancement of MHC class II-restricted peptide presentation and CD4(+) T cell activation.

Pseudodominant inheritance of the hyperimmunoglobulinemia D with periodic fever syndrome in a mother and her two monozygotic twins.

Hyperimmunoglobulinemia D with periodic fever syndrome (HIDS) is a recessively inherited recurrent fever syndrome. We describe a family of 2 monozygotic twins and their mother with characteristic symptoms of HIDS, but normal levels of IgD and IgA, and with a dominant inheritance pattern. Mevalonate kinase (MK) activity was deficient in both children, and analysis of the MVK gene revealed compound heterozygosity for 2 new mutations, G25G and R277H. Being positioned adjacent to a donor splice site, the G25G mutation was shown by reverse transcription-polymerase chain reaction analyses to cause aberrant splicing of the MVK messenger RNA, thus being disease-relevant. The mother, who was also symptomatic during her childhood and adolescence, was a compound heterozygote for I268T and R277H. Our findings expand the genetic and ethnic spectrum of HIDS and show that the possible presence of this disease cannot be excluded based solely on inheritance patterns. In each case in which HIDS is clinically suspected, analysis of MK activity and/or the MVK gene (especially exons 9 and 11) should be performed.

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells.

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.

Effects of insulin-like growth factors and insulin-like growth factor binding protein-2 on the in vitro proliferation of peripheral blood mononuclear cells.

Increasing evidence has implicated that insulin-like growth factors (IGFs), polypeptides structurally related to proinsulin, are involved in the function and development of the immune system. To probe the relevance of IGF binding protein 2 (IGFBP-2) in T-cell activation and proliferation, we studied the role of IGFBP-2 in anti-CD3 monoclonal antibody (mAb)-activated peripheral blood mononuclear cells (PBMCs). Secretion of IGF-I, IGF-II, and IGFBP-2 by PBMCs from healthy adult donors was determined by radioimmunoassays (RIAs). The PBMC proliferative response after stimulation with anti-CD3 mAb and exposure to increasing concentrations of IGF-I, IGF-II, IGFBP-2, and anti-IGFBP-2 were determined by bromodeoxyuridine enzyme-linked immunosorbent assay. Observations were tested for significance by paired t-tests. We demonstrate an increase in IGFBP-2 secretion associated with both activation of PBMC by anti-CD3 mAb and increasing cell density. Incubation with exogenous IGFBP-2 increased the proliferation of PBMCs, whereas anti-IGFBP-2 had an antiproliferative effect on PBMCs that was reversed by simultaneous exposure to IGFBP-2. The stimulatory activity of IGFBP-2 (1-10 ng/ml) on anti-CD3 mAb-activated PBMCs was similar to that of IGF-I and IGF-II (1-100 ng/ml), with the mean increase in PBMC proliferative response ranging between 150% and 160% for IGFBP-2 (p = 0.03), 150% and 170% for IGF-I (p < 0.01), 133%-161% for IGF-II (p < 0.01), and 157% and 175% for IGF-I + IGF-II (p < 0.01). Thus, our data strongly suggest a role for IGFBP-2 as a local growth factor contributing to the proliferation and activation of mononuclear cells.

Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance.

Agonistic antibodies against CD137 act as costimulators in the activation of CD8 T cells. They enhance the immune response against syngeneic tumour grafts and suppress T cell-dependent humoral immune responses in vivo. The present study was undertaken to determine whether suppression of antibody production by anti-CD137 mAb affects the development of collagen-induced arthritis (CIA). Male DBA/1J mice were immunized with bovine collagen II (CII) and treated with an agonistic anti-CD137 mAb or an isotype-matched control mAb. Mice were assessed regularly for macro- and microscopic signs of arthritis and for the appearance of collagen-specific antibody production. Interferon (IFN)-gamma determination, FACS analysis of splenocytes and histopathological joint examinations were performed after the animals were killed. Administration of anti-CD137 mAb at the time of collagen immunization blocked the development of disease and inhibited the humoral immune response against CII. Agonistic anti-CD137 mAb exhibited therapeutic efficacy even after the immune response to CII had succeeded and the disease became apparent. Furthermore, it induced a protective memory in the animals, enabling resistance to subsequent challenges with the pathogenic antigen. Our results suggest a key role for CD137 in the pathogenesis of CIA. This model provides insights into immunoregulatory conditions that control the pathogenesis of autoimmune diseases.

Autoantibodies in juvenile idiopathic arthritis: glucose-6-phosphate isomerase is not a specific target.

OBJECTIVE: Antibodies recognizing the ubiquitous cytosolic enzyme glucose-6-phosphate isomerase (GPI) cause arthritis in the K/BxN mouse model. Studies have shown that these antibodies are not specific for rheumatoid arthritis (RA) in humans. We evaluated GPI as a target of autoantibodies in juvenile idiopathic arthritis (JIA). METHODS: We studied 324 serum and 48 synovial fluid (SF) samples from 103 patients with JIA, 36 with RA, and 8 with arthralgia and 11 controls. Anti-GPI antibodies were assessed by densitometrically evaluating immunoblots and ELISA using native and recombinant GPI. We determined the GPI activity of the soluble antigen in serum and SF. RESULTS: Although several samples contained anti-GPI-IgG antibodies, this was not specific for JIA or its subgroups, or for RA. Other proteins in the GPI preparation were also frequently recognized by antibodies. Additionally, we observed increased GPI activity in patients with the systemic manifestation of JIA, but not in other patients. Neither anti-GPI concentrations nor GPI activity were associated with disease activity. CONCLUSION: In addition to the findings in RA, our results indicate that GPI is not a general target of autoantibodies in JIA.

Musculoskeletal abnormalities of the forearm in patients with juvenile idiopathic arthritis relate mainly to bone geometry.

OBJECTIVE: Alterations of the skeletal system, including reduced bone mineral density, increased frequency of fractures, and changes in markers of bone metabolism, have been described in juvenile idiopathic arthritis (JIA), but measurement of volumetric bone density and bone geometry is not possible with the techniques used in most studies. This study was undertaken to obtain a more detailed understanding of the musculoskeletal system in JIA by analyzing bone density, bone geometry, and muscle force as the most important factor in bone development. METHODS: The technique of peripheral quantitative computed tomography was used in a cross-sectional study of 57 children with the oligoarticular, polyarticular, or systemic form of JIA. Density of trabecular and cortical bone as well as geometric parameters of bone and muscle were measured at the forearm. RESULTS: Children in all subgroups had significantly reduced muscle cross-sectional area, which was strongly correlated with muscle force and abnormalities in geometric parameters of bone, including a significant reduction in cortical thickness. Trabecular density was affected only in the polyarticular JIA group, and cortical density was normal in all subgroups. CONCLUSION: Our results suggest that an important problem in JIA is the lack of muscle mass and force, together with abnormal bone geometry. The thinned bony cortices might predispose to fractures even though cortical bone density itself is normal. These results might have implications with regard to therapeutic approaches to preserve musculoskeletal integrity in JIA.