Nitrogen can substitute helium as a mobile phase in the analysis of wastewater treatment matrices for persistent organic pollutants by gas chromatography - electron capture detection system.

Municipal wastewater treatment plants are required to monitor persistent organic pollutants (POPs) in their wastewater treatment related discharges and to assess the impact of the discharges on the environment and public health. One tool for monitoring chlorinated organic pollutants particularly is a gas chromatographic (GC) system coupled to a pair of halogen-specific electron capture detectors (ECDs) with helium (He) as the mobile phase. He supplies, however, has become inconsistent and unreliable lately. In its place, N2 gas is evaluated in this study as a potential substitute for He in quantifying organochlorine pesticides, polychlorinated biphenyls, chlordane congeners and toxaphene in wastewater treatment related matrices (influent, effluent, benthic sediment, mussel tissue, and biosolids/sludge). N2 is inert, inexpensive and requires no additional hardware to incorporate into the basic functions of a GC-ECD. Our results show that, with the usual data quality controls (blank, laboratory control, matrix spike/duplicate and proficiency testing samples, and the fact that certified reference materials data met requirements), N2 can replace He for regulatory purposes. And when necessary, the N2-based retention times (tN) can be predicted reliably from He-based retention times (tHe), irrespective of column chemistry or POPs (here: tN = 1.90tHe + 0.04, R2 = 0.996).

Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17.

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rß. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rß mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.

Rituximab treatment of patients with severe, corticosteroid-resistant thyroid-associated ophthalmopathy.

PURPOSE: To study the effectiveness of anti-CD20 (rituximab [RTX]; Rituxan; Genentech, Inc., South San Francisco, CA) therapy in patients with severe, corticosteroid (CS)-resistant thyroid-associated ophthalmopathy (TAO). DESIGN: Retrospective, interventional case series. PARTICIPANTS: Six consecutive subjects with severe, progressive TAO unresponsive to CS. METHODS: Electronic medical record review of consecutive patients receiving RTX during the previous 18 months. Responses to therapy were graded using standard clinical assessment and flow cytometric analysis of peripheral lymphocytes. MAIN OUTCOME MEASURES: Clinical activity score (CAS), proptosis, strabismus, treatment side effects, and quantification of regulatory T cells. RESULTS: Six patients were studied. Systemic CS failed to alter clinical activity in all patients (mean CAS+/-standard deviation, 5.3+/-1.0 before vs. 5.5+/-0.8 during therapy for 7.5+/-6.4 months; P = 1.0). However, after RTX treatment, CAS improved from 5.5+/-0.8 to 1.3+/-0.5 at 2 months after treatment (P<0.03) and remained quiescent in all patients (CAS, 0.7+/-0.8; P<0.0001) at a mean follow-up of 6.2+/-4.5 months. Vision improved bilaterally in all 4 patients with dysthyroid optic neuropathy (DON). None of the 6 patients experienced disease relapse after RTX infusion, and proptosis remained stable (Hertel measurement, 24+/-3.7 mm before therapy and 23.6+/-3.7 mm after therapy; P = 0.17). The abundance of T regulatory cells, assessed in 1 patient, increased within 1 week of RTX and remained elevated at 18 months of follow-up. CONCLUSIONS: In progressive, CS-resistant TAO, rapid and sustained resolution of orbital inflammation and DON followed treatment with RTX. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Increased generation of fibrocytes in thyroid-associated ophthalmopathy.

CONTEXT: The pathogenic basis for Graves' disease (GD) continues to elude our understanding. Specifically why activating antibodies are generated against self-antigens remains uncertain as does the identity of the antigen(s) that provokes orbital involvement in GD, a process known as thyroid-associated ophthalmopathy (TAO). OBJECTIVE: The aim of the study was to determine whether CD34(+) fibrocytes are generated more frequently in GD, whether they infiltrate orbital connective tissues in TAO, and whether they express the thyrotropin receptor (TSHR). DESIGN/SETTING/PARTICIPANTS: Generation of fibrocytes from peripheral blood mononuclear cells was examined in samples from 70 patients with GD and 25 healthy control subjects. Fibrocytes were characterized by flow cytometry. Orbital tissues and fibroblast culture strains were examined for their presence. MAIN OUTCOME MEASURES: The frequency of CD34(+) fibrocyte generation from peripheral blood cells, characterization of their phenotype, cytokine production, and their presence in affected orbital tissues were analyzed. RESULTS: CD34(+)CXCR4(+)Col I(+) fibrocytes expressing IGF-I receptor are far more frequently generated from cultured peripheral blood mononuclear cells of donors with GD compared with healthy subjects. They express TSHR at high levels and TSH induces fibrocytes to produce IL-6 and TNF-alpha. Numerous CD34(+) fibrocytes were detected in orbital tissues in TAO but were absent in healthy orbits. Tissue-infiltrating fibrocytes express TSHR in situ and comprise a subpopulation of TAO-derived orbital fibroblasts. CONCLUSIONS: Our findings suggest that fibrocytes may participate in the pathogenesis of TAO because they express relevant autoantigens such as IGF-I receptor and functional TSHR and differentially accumulate in orbital tissue in TAO.

Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1.

PURPOSE: Fibroblast diversity represents an emerging concept critical to our understanding of tissue inflammation, repair, and remodeling. Orbital fibroblasts heterogeneously display Thy-1 and exhibit unique phenotypic attributes that may explain the susceptibility of the human orbit to thyroid-associated ophthalmopathy (TAO). In the present study the authors investigated the role of CD40 ligation on macrophage chemoattractant protein-1 (MCP-1), IL-6, and IL-8 expression in fibroblasts from patients with TAO. METHODS: Human orbital fibroblasts were cultured from tissues obtained with informed consent from patients with TAO and from patients undergoing surgery for other noninflammatory conditions. The fibroblasts were then examined by flow cytometry, microscopy, and cytokine assays. RESULTS: The authors report that orbital fibroblasts from patients with TAO expressed elevated levels of CD40. Surface CD40 could be further upregulated by IFN-gamma in TAO and control fibroblasts. This upregulation was mediated through Jak2 and could be blocked by dexamethasone and AG490, a powerful and specific inhibitor of tyrosine kinase. Treatment with CD154, the ligand for CD40, upregulated the expression of IL-6, IL-8, and MCP-1 in TAO fibroblasts but failed to do so in control cultures. Thy-1(+) fibroblasts displayed higher CD40 levels than did their Thy-1(-) counterparts and were largely responsible for this cytokine production. IL-1beta also induced MCP-1, IL-6, and IL-8 more vigorously in TAO-derived fibroblasts. CONCLUSIONS: Characterization of orbital fibroblasts and their differential expression of cytokines and receptors should prove invaluable in understanding the site-specific nature of TAO and the development of specific therapies.

B cells from patients with Graves' disease aberrantly express the IGF-1 receptor: implications for disease pathogenesis.

Graves' disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R(+) memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R(+) phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R(+) B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease.

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.