A multicenter evaluation of dysthyroxinemia in a defined patient cohort.

BACKGROUND: In the region Limburg (The Netherlands) almost all of the five participating laboratories use a different immunoassay platform to determine thyroid stimulating hormone (TSH) and free thryoxine (FT4). With the frequent transfer of patients within the region, harmonization of test result interpretation is necessary. In this study, we investigated dysthyroxinemia classification between participating laboratories and developed procedures for improvement. METHODS: Two ring surveys with an interval of 2 years were performed. Four patient groups (n=100) with different dysthyroxinemia classification were based on biochemical results of the Autodelphia analyzer. Samples were tested in five participating laboratories. In each group the percentage of patients classified with dysthyroxinemia was calculated and differences were analyzed by the Fisher's exact test. RESULTS: After the first survey, the percentage of patients with hyperthyroxinemia was more than 20% lower in three laboratories compared to the other two. Bhattacharya analysis revealed that the upper reference limit of FT4 was 20%-30% too high in two laboratories. Adjustments of reference ranges appeared to be effective in the second survey. The third laboratory reported significantly lower percentages of patients with hyperthyroxinemia in the second survey. New FT4 reference ranges were determined for this laboratory, resulting in adequate classification of hyperthyroxinemia. CONCLUSIONS: This study illustrates the potential of a multicenter evaluation of dysthyroxinemia in a biochemical-defined patient cohort. In particular, classification of hyperthyroxinemia differed between laboratories. Adjustments of reference ranges resulted in better agreement of dysthyroxinemia classification. Even using internal and external quality assurance programs, application of multicenter ring surveys is advised to prevent inadequate reference ranges.

The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

Increased osteopontin plasma levels in multiple sclerosis patients correlate with bone-specific markers.

The pro-inflammatory cytokine osteopontin has been found to be highly expressed in multiple sclerosis lesions and plasma levels are increased during relapses in relapse-onset multiple sclerosis patients. The objective was to determine the relationship between osteopontin plasma and cerebrospinal fluid levels in relation to the immunoglobulin G index. In addition, osteopontin plasma levels were compared with osteopontin mRNA levels in peripheral blood mononuclear cells and bone-specific markers to analyse whether osteopontin may be peripherally produced. Osteopontin and bone-specific markers were determined in paired plasma-cerebrospinal fluid samples and serum samples of relapse-onset multiple sclerosis patients (n = 36), respectively. Osteopontin mRNA levels were determined by quantitative polymerase chain reaction analysis. Compared to healthy controls (n = 20), plasma osteopontin levels were significantly increased in relapsing-remitting multiple sclerosis patients and correlated (r = 0.43, p = 0.013) with the immunoglobulin G index. In contrast, cerebrospinal fluid osteopontin levels correlated neither with plasma osteopontin in paired samples nor with the immunoglobulin G index. Since osteopontin mRNA levels in peripheral blood mononuclear cells of relapsing-remitting multiple sclerosis patients did not correlate with osteopontin plasma levels, peripheral blood mononuclear cells might not be the major source for the increased osteopontin plasma levels. Osteopontin plasma levels correlated (r = 0.42, p = 0.035) with the bone-specific degradation product C-telopeptide of type-1 collagen. In addition, another immunomodulatory molecule involved in bone metabolism, 25-OH vitamin D, correlated negatively (r = -0.359, p = 0.048) with the immunoglobulin G index. This study suggests that bone-related molecules like osteopontin and vitamin D with important immunomodulatory functions are related to the immunoglobulin G index in relapsing-remitting multiple sclerosis patients.

Identification of soluble CD14 as an endogenous agonist for Toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins.

Human astrocytes express a limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Also, TLR3 but not TLR4 activation on astrocytes induces expression of several factors involved in neuroprotection and down-regulation of inflammation rather than in the onset of traditional pro-inflammatory reactions. The notion that astrocyte TLR may thus play a role not only in host defense but also in tissue repair responses prompted us to examine the possibility that endogenous TLR agonists could be expressed in the human central nervous system to regulate the apparently dual astrocyte functions during trauma or inflammation. As a potential source of endogenous agonists, a cDNA library derived from several human brain tumor cell lines was used. Gene pools of this library were transfected into COS-7 cells and the expression products were screened for their ability to induce TLR activation in human primary astrocytes. The screening resulted in the identification of soluble CD14. By using a panel of TLR-transfected HEK293 cells, we found that signaling by soluble CD14 was TLR2 dependent. Moreover, the CD14-triggered TLR2-mediated response in astrocytes lead to the production of CXCL8, IL-6, and IL12p40, whereas typical TLR-induced pro-inflammatory cytokines, like TNF-alpha and IL-1beta, were not produced at detectable levels. In conclusion, our data indicate that apart from its well-known ability to act as a co-receptor for TLR-dependent signaling by peptidoglycans or LPS, soluble CD14 can also act as a direct agonist for TLR2.

Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation.

BACKGROUND: Stem-cell grafts between HLA-identical siblings are less likely to succeed when there is a sex mismatch. This lack of success can be interpreted as enhanced activity directed against minor histocompatibility antigens encoded by the Y chromosome (H-Y). So far, in man, only cytotoxic T lymphocytes (CTLs) specific for several minor histocompatibility antigens have been reported. We aimed to identify and clarify the role of MHC class II-restricted H-Y-specific T-helper cells in these transplant settings. METHODS: H-Y-specific MHC class II-restricted CD4+ T cells were isolated from blood of a female patient who rejected an HLA-identical male stem-cell transplant. By molecular cloning of H-Y genes and functional T-helper experiments, we elucidated antigen specificity and the functional properties of these H-Y-specific T-helper cells. FINDINGS: CD4+ T-helper cells recognise the Y gene-encoded peptide VIKVNDTVQI presented by HLA-DRbeta3*0301. These T-helper cells mature dendritic cells and enhance expansion of minor histocompatibility antigen-specific MHC class I-restricted CD8+ CTLs. INTERPRETATION: Characterisation of an MHC class II-restricted H-Y epitope that evoked CD4+ T-helper responses adds a novel cellular component to the alloimmune response against Y chromosome-encoded minor histocompatibility antigens. This component completes the H-Y-directed alloimmune response and aids understanding of the poorer outcome of sex-mismatched transplants.

Elevated osteopontin levels in active relapsing-remitting multiple sclerosis.

In the search for proteins that might play a role in the pathogenesis of multiple sclerosis (MS), osteopontin (OPN) has been identified as the most prominent cytokine-encoding gene expressed within MS lesions. Here, we report significantly increased OPN protein levels in plasma of relapsing-remitting MS patients. In contrast, OPN protein levels in primary progressive and secondary progressive MS patients were similar to healthy control levels. Interestingly, active relapsing-remitting patients had higher OPN protein levels than patients without relapses.

The DBY gene codes for an HLA-DQ5-restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease.

Graft rejection or graft-versus-host (GVH) disease after HLA-identical stem cell transplantation is the result of recognition of minor histocompatibility antigens (mHags) by immunocompetent T lymphocytes from recipient or donor origin, respectively. Cytolytic T lymphocyte (CTL) clones can be isolated during graft rejection and GVH disease to identify mHags and their corresponding genes. Thus far, all human mHags identified appeared to be HLA class I-restricted. Here, we report the characterization of the first human HLA class II-restricted sex-linked mHag involved in GVH disease. Previously, we isolated an HLA-DQ5-restricted CD4(+) CTL clone from a male patient with chronic myeloid leukemia who developed acute GVH disease grade III-IV after transplantation of HLA genotypically identical female stem cells. Using a panel of female HLA-DQ5(+) EBV cells that we stably transfected with Y chromosome-specific genes, we determined that the HLA class II male-specific mHag (H-Y) was encoded by the Y chromosome-specific gene DBY. The H-Y epitope was localized in the DBY protein using female HLA-DQ5(+) peripheral blood mononuclear cells loaded with DBY protein fragments. The minimal peptide sequence leading to maximal recognition by the specific HLA-DQ5-restricted CTL clone was characterized as the 12-amino acid sequence HIENFSDIDMGE. Although the epitope differed by 3 amino acids from its X-homolog DBX, only 2 polymorphisms were shown to be essential for recognition by the CTL clone.