T and B cell reactivity to extraocular and skeletal muscle in Graves'' ophthalmopathy.

We have sought human eye muscle membrane binding antibodies in patients with Graves' ophthalmopathy using an enzyme-linked immunoassay. Antibodies were found in patients with thyroid autoimmunity irrespective of eye signs, and binding correlated closely (r = 0.94) with binding to skeletal muscle, showing that these antibodies are not site-specific. T cells from patients with thyroid autoimmunity proliferated in response to eye muscle, but again this was not specific for eye muscle or the presence of ophthalmopathy. No single antigen was responsible for inducing proliferation. These results fail to confirm a recent report of eye muscle membrane binding antibodies in a high proportion of patients with ophthalmopathy, and suggest instead that T and B cell autoreactivity to striated muscle antigens is a frequent feature of autoimmune thyroid disease, unlikely to be directly related to eye disease.     

Advances in nutrition care of children with neoplastic diseases: a review of treatment, research, and application

Within the last decade, significant advances have been made both in treating children with cancer and in providing proper nutrition support. Oncologic treatment and nutrition research and their application to the nutrition care of children with cancer are reviewed. Quality nutrition care is now possible because of an improved understanding of (a) the prevalence and significance of protein-energy malnutrition (PEM) in high-risk groups, (b) the staging and assessment of nutritional status, and (c) the efficacy and limitations of nutrition support options. Nutrition staging, assessment, and support should be integrated into treatment protocols for children with neoplastic diseases. Common risk factors for the development of PEM have been identified from serial monitoring of newly diagnosed children with a variety of tumors. Certain tumor types and their treatment can be classified within either low or high nutritional risk groups. A comprehensive nutrition program (intense nutrition counseling, favorite nutritious foods) is preferred for low nutritional risk groups but is ineffective in preventing or reversing PEM in high-risk groups. For high-risk patients, central parenteral nutrition (CPN) is the method of choice as a relatively short-term but important support measure that allows children to withstand long intervals of intense treatment during periods of growth and development. Current data suggest that bone marrow suppression may be attenuated and treatment tolerance improved with the use of CPN in selected children with advanced cancer (e.g., acute nonlymphocytic leukemia or advanced neuroblastoma).     

Persistent thyroid autoimmunity after subacute thyroiditis

Subacute (de Quervain's) thyroiditis (SAT) is accompanied by temporary abnormalities in thyroid function and, in a minority of patients, by transient thyroglobulin and thyroid microsome autoantibody formation. In this report we have examined the sera of patients with SAT for the presence of multiple thyroid autoantibodies using qualitative immunoblotting (Western blotting). For this purpose we used a crude thyroid extract (2,000 g supernatant) as a source of antigen, in order to identify antibodies reacting with a wide range of potential autoantibodies. Eight of the 9 patients tested had autoantibodies which reacted with between 2-18 antigenic determinants (molecular weights 177-10 kd) present in the thyroid antigen preparation. None of the sera reacted with thyroglobulin or thyroid microsomes. The majority of these autoantibodies were directed against antigens which were absent from liver membrane preparations. Furthermore there was no diminution in the levels of these autoantibodies over a period of up to 39 months after the onset of SAT. The prolonged presence of these novel thyroid autoantibodies may explain the recent findings of subtle thyroid defects, such as altered gland iodine content, long after apparent clinical resolution of SAT. The accumulated evidence for a viral aetiology in this condition suggests that these sequelae may be due to a virally-induced autoimmune response.     

The tissue distribution of thyroglobulin-responsive B and T cells in experimental autoimmune thyroiditis (EAT)

The tissue distribution of thyroglobulin antibody producing lymphocytes has been studied during the course of immunisation-induced experimental autoimmune thyroiditis in the rat. The iliac lymph nodes were initially the most important source of autoantibody but later in the disease the bone marrow and thyroid-draining lymph nodes were also involved. In addition small amounts of thyroglobulin antibody were made by thyroid-derived lymphocytes. A blastogenic response to thyroglobulin was found using lymphocytes from the iliac nodes initially and later from the spleen. These results demonstrate the widespread and sequential involvement of the lymphoid system in the autoimmune response to thyroglobulin.     

Enzyme-linked immunoassay of monoclonal and serum microsomal autoantibodies

An automated enzyme-linked immunoassay for the detection of antibodies to human thyroid microsomes has been assessed. This assay correlated closely with the established commercial passive haemagglutination method. Variations in the purity of crude microsome preparations and the degree of thyroglobulin contamination make careful comparison of different preparations essential for meaningful interpretation of results, and attempts to circumvent these problems by further purification of microsome preparations using gel filtration are discussed. The application of this method for routine screening of serum samples is demonstrated using populations of normal subjects and patients with rheumatoid arthritis and anti-glomerular basement membrane disease. This assay has also permitted the establishment of murine hybrid myelomas secreting monoclonal antibodies to human thyroid microsomes.     

Graves′病患者经~(131)碘治疗后循环活性T细胞亚群的动态观察

本文应用直接、间接双色免疫荧光染色,流体细胞测定仪技术,观察了经~(131)碘治疗的23例Graves′病患者的循环活性T细胞亚群的动态变化。~(131)碘治疗后的第一个月至第三个月,HLA-DR、Ta_1和UCHL_1活性T细胞亚群数目明显增加,以Vicia-villosa为标志的抗抑制细胞亚群较治疗前明显增加。实验结果提示:Graves′病患者经~(131)碘治疗后的抗甲状腺自身抗体浓度增加可能是由于T细胞的激活和抗抑制细胞亚群增加的共同结果。     

Antibodies to porcine eye muscle in patients with Graves' ophthalmopathy: identification of serum immunoglobulins directed against unique determinants by immunoblotting and enzyme-linked immunosorbent assay

The immunological mechanisms involved in Graves' ophthalmopathy are not known. To explore the pathophysiology of this disorder, we used an enzyme-linked immunosorbent assay to detect antibodies that bound to the 100,000 X g sediment fraction of porcine eye muscle. Serum from patients with Graves' ophthalmopathy had enhanced immunoglobulin binding to porcine eye muscle compared to serum from control subjects (P less than 0.0001); however, several individual normal serum samples also had elevated binding activity. Incubation of serum from some Graves' patients with skeletal muscle or liver tissue resulted in reduction in immunoglobulin binding to porcine eye muscle. Thyroglobulin and TSH reduced binding only at high concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some obvious differences in protein bands between porcine eye muscle and skeletal muscle. Serum from many normal subjects and patients with autoimmune thyroid disease formed bands with skeletal muscle protein fractions on immunoblotting, and no specific bands were found using serum from Graves' ophthalmopathy patients. Immunoblots of Graves' patients' serum after reaction with either porcine skeletal or eye muscle showed no reactivity with thyroglobulin (200K and 320K) or thyroid microsomal antigen (105K). Instead, immunoblots of eye muscle and serum from Graves' patients, with or without eye disease, showed two bands at 64K and 73K, which were shared by serum from normal subjects. In addition, several unique eye muscle determinants were detected in serum from 6 of 13 Graves' disease patients analyzed. These unique bands usually had mol wt of less than 50K, and 5 of the 6 patients whose serum reacted with these determinants had significant ophthalmopathy. Our findings support the presence of potential antigenic differences between eye muscle and skeletal muscle accounting for the immunological specificity of eye muscle as a target in Graves' ophthalmopathy.     

Immunoglobulin class and subclass distribution of eye muscle and fibroblast antibodies in patients with thyroid-associated ophthalmopathy

OBJECTIVES The investigation of the antibody response in thyroid-associated ophthalmopathy (TAO) using different antigens and assays has given inconsistent results. We have analysed antibodies against eye muscle and control antigens in a large group of TAO patients to assess whether specific eye muscle antibodies exist in TAO. We have also evaluated the presence of IgA and IgM class antibodies and examined IgG subclass distribution. DESIGN Sera were obtained from all patients (TAO, Graves' disease without ophthalmopathy and Hashimoto's thyroiditis) within one year of diagnosis. Sera were also collected from healthy controls, with no family history of autoimmune thyroid disease. PATIENTS Thirty-eight patients had Graves' disease with Grade III or greater TAO; 15 patients had Graves' disease without ophthalmopathy and nine had Hashimoto's thyroiditis without any eye signs. The control group consisted of 14 subjects. MEASUREMENTS Antibodies against porcine eye and skeletal muscle, human eye (membrane and soluble antigen) and skeletal muscle, human thyroid microsomal and thyroglobulin antigens and dermal and orbital fibroblast antigens were assessed using ELISA. Antibody isotypes and IgG subclasses were studied for porcine and human eye muscle antibodies. Eye muscle (porcine and human) and orbital fibroblast antibodies were further analysed by immunoblotting. RESULTS There were no significant differences in the ability of either IgG or IgA in sera from the different groups to bind porcine and human eye muscle antigens. There was a significant correlation (P < 0·0001) between the binding to porcine eye muscle and skeletal muscle antigens (for both IgG and IgA). There was no difference between sera from TAO patients and control subjects in their binding to eye muscle fibroblasts for both IgG and IgA antibodies. However, IgA antibody activity against dermal fibroblasts differed significantly between TAO patients and controls (P <0·05). By immunoblotting, the frequency of IgA antibodies recognizing 21 kDa (40% of patients) and 62 kDa (52%) bands in porcine eye muscle blots and 20, 24 and 38 kDa bands in blots of human eye muscle (soluble) antigen differed significantly between patients with TAO and controls (P <0·05 in all cases). IgG antibodies recognizing 80 and 92 kDa bands in blots of the subcellular membrane antigen prepared from orbital fibroblasts were found more frequently in patients with TAO compared with controls (P <0·05 in both cases). CONCLUSIONS We found no evidence that eye muscle membrane or fibroblast antibodies are present in a significant proportion of TAO patients, using ELISAs based on antigens prepared from several sources. We have also failed to demonstrate the presence of previously described specific, TAO-associated antibodies, including those directed against a 64 kDa protein in eye muscle and a 23 kDa protein in fibroblasts. IgA class antibodies reactive with orbital components appeared to be more strongly associated with TAO than those of the IgG class, though even this relationship is weak. These results suggest that antibodies are of secondary importance in the pathogenesis of TAO, which is most likely a T cell-mediated disorder.     

Potentiation by 2,2'-pyridylisatogen tosylate of ATP-responses at a recombinant P2Y1 purinoceptor.

1. 2,2''-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5''-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (ICl,Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2. PIT (0.1-100 microM) failed to stimulate P2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 microM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 microM), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 microM (pKB = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3. Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1-3 microM) potentiated ATP-responses in three experiments, while higher concentrations (3-100 microM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4. In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: alpha 1, alpha 2-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D1, D2, muscarinic, central benzodiazepine, H1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5. In guinea-pig isolated taenia caeci, PIT (12.5-50 microM) irreversibly antagonized relaxations to ATP (3-1000 microM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 microM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 microM) and guanethidine (17 microM), were not affected by PIT (50 microM, for 25-60 min). 6. These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.