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Between April 1968 and February 1988, 311 patients with symptomatic and progressive Graves' ophthalmopathy were treated with megavoltage orbital radiotherapy. The patients were divided into three groups: I (156 patients) treated with 20 Gy/2 weeks; II (69 patients) treated with 30 Gy/3 weeks, and III (a most recent set of 86 patients) received 20 Gy/2 weeks. The degree of eye involvement was evaluated numerically before and after therapy for each of five parameters: soft tissue signs, proptosis, eye muscle impairment, corneal involvement, and sight loss. Pre-treatment and current thyroid diagnosis and status were also noted. To evaluate the effects of radiotherapy alone, follow-up was terminated at the time any eye surgery was done; for those not treated surgically the minimum follow-up was 12 months. Because there were significant demographic differences between the patient groups, the results of each group were analyzed separately. A stepwise linear regression analysis was performed to determine if there were any significant variables affecting outcome. Based on these data formulae were derived which enable outcome to be predicted in any patient. Before therapy more than 90% of patients in all groups had soft tissue and eye muscle involvement, whereas 65-75% had proptosis and about half 50% had some degree of sight loss. Radiotherapy arrested progression of ophthalmic parameters in all but 1-6% of the patients. Objective and symptomatic improvement was noted for all parameters assessed, but there was marked individual variability. The best responses were noted for soft tissue, corneal involvement, and sight loss; however over half the patients had some improvement in eye muscle function and proptosis. Factors which resulted in less favorable outcome included male gender, advanced age, need for concurrent therapy for hyperthyroidism, and no history of hyperthyroidism. No complications have been observed. No significant differences in outcome were observed between the two dosage schedules. Following radiotherapy 29% of patients subsequently underwent some form of eye surgery, mostly eye muscle surgery to correct diplopia. After radiotherapy corticosteroid therapy was stopped without relapse in 76%. Orbital radiotherapy can result in improvement in signs and symptoms of Graves' ophthalmopathy in the majority of patients. For the remainder of patients the disease manifestations can be stabilized to allow functional surgical correction.

Pretibial myxedema is considered an autoimmune complication or association of Graves' disease, Hashimoto's thyroiditis, and primary myxedema. The mechanism of lesion formation is unknown; the most plausible theory is that it arises as a result of a target cell in the skin, probably the fibroblast, being stimulated to produce abnormally high amounts of glycosoaminoglycans (especially hyaluronic acid) by autoantibodies directed against a thyroid antigen(s)--that is, by a cross reaction. One or more intermediary humoral agents may be involved in pathogenesis. The reason for the localization to the pretibial region is unknown; there is evidence that most patients with the disorder have similar abnormalities in the preradial skin. The condition may persist for months or years but often regresses spontaneously, accompanied by a parallel decline in, or disappearance of, serum anti-TSH-receptor autoantibody levels. Skin biopsies reveal evidence of increased amounts of hyaluronic acid and damage to collagen and elastic fibers. Local symptomatic treatment with corticosteroids is effective in most cases with slight to moderate severity of skin involvement. Repeated treatments are advised until such time that a spontaneous clinical remission occurs.

CT scans of 9 patients with orbital pseudotumor (bilateral in 6 and unilateral in 3) showed findings distinct from those observed in Graves' ophthalmopathy. In bilateral involvement, they ranged from localized mass lesions to complete obiliteration of normal orbital CT anatomical landmarks; diffuse or multifocal lesions involving the posterior globe and muscle insertions were most typical of the diagnosis. However, findings in unilateral psedotumor may be indistinguishable from orbital mass lesions other than Graves' ophthalmopathy. Serial CT scans were used to show progression of disease and response to treatment.

The results of total skeletal scintigraphy with technetium 99m-labeled diphosphonate in 200 patients with biopsy-proven primary cancer demonstrated abnormalities in 114; 57 had normal roentgenograms. In 32 of the 57 patients, the abnormalities were ascribed to malignant lesions. This is a false-negative rate of 17% for the roentgenographic examinations. Only three patients had abnormal roentgenograms but normal scans, a yield of 1.6% false-negative scans. In a comparison of scan and roentgenographic findings with the skeleton divided into 12 regions, 119 of 160 abnormalities seen on scans but not on roentgenograms were attributed to tumor. Only five of 17 regions that were abnormal on roentgenogram but not on scans were unexplained false-negative findings. Thus, on a regional basis, the yields of false-negative roentgenograms and scans were 9.1% and 0.4%, respectively.

Gallium-67 citrate scintigraphy was performed in 33 patients with Hodgkin's disease and 25 patients with non-Hodgkin's lymphoma. Three hundred twenty-eight sites of potential involvement were investigated. Confirmation of involvement was made by physical examination, roentgenographic evaluation and histopathologic examination of tissue obtained at diagnostic laparotomy. The results of scintigraphy correlated with all other clinicopathologic data in only 35% of patients, the true-positive rate being significantly higher above the diaphragm (61%) than below (40%). The overall true-positive and true-negative rates were 53 and 90%, respectively. A significant correlation existed between tumor histology and scanning accuracy; the true-positive rate in Hodgkin's disease was 74% compared to only 13% in patients with lymphocytic lymphoma. A similar variation of 67Ga concentration with tumor histology was also noted in 70 tissue specimens obtained from 28 patients at the time of diagnostic laparotomy or biopsy. The routine use of 67Ga-citrate to detect splenic involvement with tumor appeared to be precluded by the low true-positive and high false-negative rates of 57 and 27%, respectively. Gallium-67-citrate scintigraphy may be useful as an adjunct to established clinical staging procedures in untreated patients and in the detection of recurrent disease in treated patients. Our data indicate it is not sifficiently reliable to replace established methods presently used for clinical staging.

Vesicles 500-600 A in diameter were formed by sonicating diluted samples of rat and mouse plasma. An average preparation of these vesicles formed from 1 ml of plasma contained 7.5 mg of plasma lipid and 7.3 mg of plasma protein. Plasma vesicles could be made to enclose such radiopharmaceuticals as 99mTcO4-, and the vesicles were found to be impermeable to this anion. We have studied the in vivo distribution patterns of 99mTc after intravenous injection into the rat or mouse of pertechnetate-plasma vesicles formed from rat or mouse plasma, and we find that the radioactivity remains primarily within the circulation even at 60 min after injection. In contrast, vesicles formed from artificial lipids are rapidly removed by the liver and spleen. Formation of vesicles from native plasma constituents offers a means of carrying drugs and radiopharmaceuticals in vivo in packages that have a low risk of being either toxic or antigenic.

A method for background subtraction is presented. The data are digitized scintigraphic images, stored in 64 X 64 frames. Within this array a rectangular area surrounding the target is defined. All data points outside of this area are set equal to zero. From each data point within the area, the computer subtracts a background value equal to the average of the two intersecting linear interpolations between the values at the edges of the rectangular area having the ordinate and abscissa values of the point of interest. The rationale for this approach is discussed and its application in myocardial perfusion studies is illustrated.

Artificial lipid vesicles interact with a variety of mammalian cells, including blood cells, spleen cells, and tumor cells, and during this interaction components can be transferred from the vesicle to the cell and from the cell to the vesicle. Transfer of intravesicular material is observed when artificial lipid vesicles carrying an intravesicular marker, 99mTc (as TcO4- ion), are incubated with mammalian cells. When vesicles prepared with [14C] phospholipid are incubated with mammalian cells, the labeled lipid is also transferable to mammalian cells. Conversely, if the mammalian cell surface is radiolabeled (with 125I), the cell marker is in part transferable to the vesicles. Thus, interaction of mammalian cells with vesicles alters the characteristics of the cell in several ways. There is a loss of some cell surface components, a gain of vesicle lipid components, and an acquisition of intravesicular contents. Such alteration may affect the biological behavior of the cell in vivo. Thus, the in vivo distribution in the mouse of isologous red blood cells is altered after interaction with vesicles; the liver and spleen remove large proportions of such cells in comparison to control erythrocytes labeled with 51Cr. The behavior of vesicle survivors from a cell interaction is also altered. Upon reexposure of such vesicles to a fresh population of cells, the intravesicular marker is no longer transferable to cells; and upon injection of such vesicles into mice, the liver accumulation of vesicular label is reduced as compared with that of nonincubated vesicles.

A group of Japanese were investigated for evidence of an association between Graves' disease and HL-A. Forty-four patients with the disease and 83 normal, unrelated random Japanese and Japanese-American controls were selected for study. The frequency of the W5 antigen among patients (57%) was significantly (P less than .0001) greater than among controls (20%). Of the 34 patients with abnormally elevated serum levels of anti-(thyroid) microsomal (anti-M) auto-antibodies, 56% had the W5 antigen. In contrast, of 48 control individuals tested for anti-M, only seven were seropositive and none (0%) of the seven had the W5 antigen. As expected the HL-A8 antigen was absent from this non-Caucasian population. These data demonstrate that the W5 antigen in Japanese, analogous to the HL-A8 antigen in Caucasians, is associated with Graves' disease but not with anti-M seropositivity in controls. The occurrence of different HL-A antigens in association with the same disease in different ethnic groups requires that the use of a major histocompatibility system antigen as a disease susceptibility marker must be confirmed for each ethnic group under study.

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular (99m)Tc marker. Injury to the vesicular membrane was assessed by measurement of (99m)Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in (99m)Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.

Thirty-two patients with orbital pseudotumor (18), reactive lymphoid hyperplasia (2), atypical lymphoid infiltrate (4) or malignant lymphoma (8) were treated in the Division of Radiation Therapy at Stanford University between January 1973 and May 1983. Of the 20 patients with pseudotumor or reactive lymphoid hyperplasia, 10 had unilateral lesions and 10 had bilateral lesions. Biopsy samples were obtained in 15 patients; in five patients with bilateral disease the diagnosis was made on the basis of computed tomography (CT) and clinical findings. The majority of patients were referred because of disease refractory to treatment with corticosteroids. The patients were given a mean dose of 2360 rad using complex, individualized megavoltage techniques including lens shielding. Radiotherapy was well tolerated with no significant acute or late complications. Fifteen patients had complete resolution of symptoms after treatment; five had continued symptoms. Of the 12 patients with malignant lymphoma or atypical lymphoid infiltrate, four had systemic lymphoma with orbital involvement and eight had orbital involvement only. The diagnosis was made by biopsy in all patients and immunophenotyping was done in six cases, of which 5 were monoclonal. Patients were evaluated with a chest radiograph, lymphogram or abdominal CT, bone marrow biopsy and orbital CT. A mean dose of 3625 rad was delivered to the orbit only. Most of the patients received complex megavoltage treatment using bolus. All patients in this group had a complete response and local control. There were no relapses in those with localized disease. Two patients developed cataracts. Carefully planned orbital radiotherapy provides local control without symptomatic sequelae for orbital masses ranging from pseudotumor to malignant lymphoma.

An improved assay for detecting blocking or inhibitory activity on TSH-binding (TBBA) in neat serum of patients with Graves' disease is described. The assay is sensitive, reproducible, simple, and quick. Crude membranes prepared from guinea pig fat (FCM) were used as the source of TSH receptors. One major feature of the assay is the use of small amounts of FCM to increase assay sensitivity. The sensitivity of the assay was found to be inversely proportional to the amount of FCM used. Using a quantity of 10-20 micrograms crude FCM achieved a satisfactory balance between assay sensitivity and accuracy. The assay employs two sequential one-hour incubations at room temperature, first incubating the FCM with the serum sample, followed by removal of excess sample and washing, and then incubating the exposed FCM with 125I-TSH for quantitative determination of specific TSH binding. The assay is recommended as a standard method for measurement of blocking activity in serum of specific TSH binding. Seventy-four percent of the patients with Graves' disease showed positive blocking activity while all normal individuals and patients with Hashimoto's thyroiditis and with nonautoimmune thyroid disorders were negative.

Autoantibodies to the self-antigen thyroglobulin (Tg) are usually not found in sera of normal individuals, but are often present in sera of patients with autoimmune thyroid diseases. To determine if the presence of such autoantibodies could be due to the abnormal appearance of self-reactive B cells, which are otherwise absent in normal subjects, or to an alteration in the mechanisms regulating such B cells, peripheral blood lymphocytes (PBL) from normal individuals and patients with autoimmune thyroid diseases were cultured and stimulated in vitro with the polyclonal stimulant pokeweed mitogen (PWM). A modified plaque assay was used to enumerate cells secreting protein A-binding immunoglobulins (Igs) and specific antibodies against Tg. PBL from all individuals tested, including normal subjects (n = 26), could be induced by PWM to produce antibodies against Tg in vitro and these antibodies were of IgG isotypes. PBL from patients with detectable serum anti-Tg had more inducible cells secreting anti-Tg [27,000 +/- 10,700 (+/- SD)/10(6) PBL] than those from patients with autoimmune thyroid diseases, who had no detectable serum anti-Tg (8,000 +/- 5,000), and those from normal individuals (7,200 +/- 4,200). The demonstration of inducible mature (IgG) anti-Tg-producing cells in normal individuals suggests that subclinical autoimmunity against certain self-antigens may be a normal phenomenon in man and that its escalation into clinical autoimmune conditions is prevented through regulation of the specific self-reactive cells.

A radiometric two-site assay for soluble thyroglobulin-antithyroglobulin immune complex (TgA) applicable to human serum has been developed. Rabbit anti-(human)IgG globulin and anti-(human)thyroglobulin (anti-Tg) were purified by affinity chromatography. When these antibodies were labeled with 125I, 45% and 62% could be bound to their corresponding antigens, IgG globulin and thyroglobulin, respectively. TgA was prepared by dissolving the immune precipitate (formed with human thyroglobulin [Tg] and human anti-Tg) in excess Tg and chromatographing the mixture on Sepharose 4B. On immunoelectrophoresis TgA migrated between IgG and Tg and formed precipitin lines against anti-IgG and anti-Tg. Serum unknowns were chromatographed on Sepharose 4B to separate TgA from free IgG; the heavy TgA eluated in the first fraction. Standard TgA or prepared unknown was first incubated in plastic cups precoated with anti-IgG. After washing cups (free Tg was removed at this step), bound TgA was identified by binding to cups of added 125I-anti-Tg, the bound radioactivity being directly proportional to the amount of TgA present. Minimal detectable TgA was 0.4 ng/cup corresponding to a serum concentration of 16 ng/ml. The response curves of serial dilutions of serum eluate paralleled the standard curve. Coefficients of within assay variation ranged from 3.7 to 4.9%; coefficients for between assay variation ranged from 20 to 28%. Preliminary data indicated that TgA was not detected in the sera of 10 normal subjects, but was detectable in 7 of 29 subjects (24%) with Graves' disease. The clinical significance of serum TgA levels remains to be determined by more extensive testing. The results indicate that a soluble immune complex, TgA, can be detected in serum with a high degree of sensitivity and reliability.

Extraocular muscle enlargement was symmetrical in 70% and asymmetrical in 30% of patients with Graves' ophthalmopathy. True unilateral muscle involvement occurred in 6%. Computed tomography (CT) showed bilateral orbital involvement in 50% of patients presenting clinically with unilateral eye signs. In patients without a clinically apparent ophthalmopathy, CT demonstrated muscle enlargement in 40%. The medial and inferior rectus muscles were the most frequently and most severely involved. Orbital radiation therapy can result in a decrease in size of involved muscles.

The in vivo distribution of vesicles containing radiopharmaceuticals in their cavities has been studied using three routes of administration: intravenous, subcutaneous, and intraperitoneal. The in vivo distribution in mice was determined by dissection of the animals and calculation of radioactivity in the organs. In rats the in vivo distribution was assessed by scintigraphy using a scintillation camera-digital computer unit. After intravenous injection of vesicles, radioactivity is concentrated to some extent in the liver and spleen but the pattern of distribution is different from that of the corresponding free radiopharmaceutical or radiocolloid made of the corresponding radionuclide. The permeability of the vesicular membrane to contained radiopharmaceutical has been shown to vary according to the chemical composition of the vesicles. Vesicles can be used to introduce materials in vivo and the potential exists for their specific targeting by coupling other molecules to their surfaces.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

The number of normal cellular elements present in bone marrow biopsies was compared to the extent of uptake at corresponding sites on 111InCl3 bone marrow scans in 87 patients with malignant lymphoma. Of the biopsies free of tumor, 49/54 interpreted as normocellular and 16/17 hypocellular or aplastic confirmed the scan interpretation. Of the biopsies demonstrating tumor, 10/11 of those interpreted as normocellular and 14/15 hypocellular or aplastic confirmed the scan interpretation. The 111InCl3 bone marrow scan is accurate in depicting the presence or absence of normal marrow elements, but is of value in detecting tumor only when the normal elements are extensively replaced.

The ability of various thyroid subcellular fractions to bind [125I]iodo TSH and to absorb long-acting thyroid stimulator (LATS) and thyroid stimulating immunoglobulins (TSI) activities was examined. Membranes purified from thyroid homogenates or isolated thyroid cells absorbed LATS/TSI activities and specifically bound [125I]iodo TSH. Purified thyroglobulin, nuclei, mitochondria and ribosomes did not bind [125I]iodo TSH nor did they absorb LATS/TSI activities. Cell sap obtained by gentle lysis of isolated thyroid cells failed to absorb LATS/TSI activities and to bind labeled hormone. However, freeze-thawing of the cells fragmented the membranes, releasing [125I]iodo TSH binding as well as LATS/TSI absorbing activities into the soluble (cell sap) fraction. The results suggest that the LATS/TSI antigen is of cell surface origin, includes the TSH receptor or larger membrane fragments containing the receptor, and that its release into the soluble fraction is due to the fragmentation of the thyroid membrane during homogenization and preparative procedures.

Artificial spherules or vesicles of 900 A in diameter formed from phosphatidylcholine and gangliosides and enclosing 99mTcO4 - (standard preparation) survive intact in the circulation of the mouse. Polyamino acids and protein have been incorporated into and onto the vesicles; such vesicles remain intact as determined by diffusion dialysis studies and by electron paramagnetic resonance studies of vesicles enclosing spin label. In studying the distribution of polyamino acid-vesicles and protein vesicles in vivo, it was found that the latter distribute differently from standard vesicles or free protein alone whereas aromatic polyamino acid-vesicles concentrate in the liver and spleen to a greater extent than standard vesicles. We conclude that the permeability and stability characteristics of vesicles may be preserved when they are modified by the addition of protein or polyamino acids and that such modification of vesicles may be associated with an alteration of their fate in vivo. The potential exists to use vesicles as carriers of radiopharmaceuticals and other drugs and to direct the vesicles preferentially to tissue targets in vivo.

Thyroid disorders can be associated with elevated concentrations of serum antithyroglobulin antibodies (anti-Tg) and/or thyroglobulin (Tg), but none of the available anti-Tg assays deals with anti-Tg measurements in the presence of abnormally high Tg levels. The competitive binding radioassay produces falsely elevated values for anti-Tg if serum Tg is elevated and either falsely elevated or depressed values if both Tg and anti-Tg are abnormally high. The falsely elevated anti-Tg values can be identified by measuring the formation of Tg[125I]anti-Tg complexes in the supernatant of the anti-Tg assay (supernatant assay). For screening purposes, we modified the original anti-Tg RIA into a solid phase, sandwich-type RIA. Anti-Tg in serum or standard is first bound to plastic cups coated with Tg and then quantitated by binding of [125I]Tg. This assay has a detection limit of 2 U/ml serum, intra- and interassay coefficients of variation of 9--15%, and a normal range of less than 2 U/ml. When sera with normal Tg concentrations were analyzed, the results for anti-Tg obtained by the competitive binding RIA and the new sandwich RIA were comparable as far as positives and negatives were concerned, and the numerical values for positive sera correlated moderately well (r = 0.79); the sandwich assay, in general, gave lower values for anti-Tg. The major advantages of the sandwich anti-Tg RIA are the elimination of false positive results and its applicability to sera containing high levels of both Tg and anti-Tg. In the latter case, the results indicate the level of free anti-Tg present, as opposed to antibody present in the form of Tg-anti-Tg complexes.

We studied the regulatory activities of T cells on specific antithyroglobulin (anti-Tg) and nonspecific immunoglobulin secretion in cultures of peripheral blood lymphocytes (PBL) of five patients with autoimmune thyroid diseases and high levels of serum anti-Tg. PBL were separated into a non-T population, including B-cells and monocytes, and a T-cell population by rosetting with sheep red cells. T-Cells were further separated into T helper (Th) and T suppressor (Ts) subsets by a panning technique using the monoclonal antibodies anti-Leu 3a and anti-Leu 2a, respectively. The three sets of cells, i.e. B, Th, and Ts, from patients and from normal individuals were cocultured in various combinations and stimulated with the polyclonal stimulant pokeweed mitogen. A sensitive plaque assay was used to enumerate cells producing anti-Tg and protein A-binding immunoglobulins. The PBL of both patients and normal individuals had Tg-specific suppressor cells. Ts-cells from patients in syngeneic or allogeneic combinations with B- and Th-cells at a ratio of 1:1:1 suppressed the pokeweed mitogen-induced anti-Tg response to 41 +/- 8% (+/-SE) and 50 +/- 20% of the control value, respectively, while Ts from normal individuals suppressed the response to 7 +/- 3% of the control value. The suppressive effect of the Ts-cells from patients and normal individuals on nonspecific immunoglobulin secretion was similar (reduced to 10-15% of control). Thus, there appeared to be a deficiency in Tg-specific suppressor activity in PBL of patients. On the other hand, Th-cells from patients (syngeneic or allogeneic) cocultured with patient B-cells produced a greater anti-Tg response than Th-cells from normal individuals. The helper activities of Th-cells of patients and normal individuals on nonspecific immunoglobulin secretion were similar. Thus, there appeared to be an increase in Tg-specific helper activity in PBL of patients.

The CT scan with the 160 x 160 matrix demonstrated both the normal orbital anatomy and the abnormal orbital anatomy of Graves' ophthalmopathy in great detail. In Graves' ophthalmopathy, the cardinal pathologic feature of extraocular muscle enlargement was accurately reflected on the CT scan and was a distinctive, diagnostically reliable finding. Enlargement of the medial and lateral rectus muscles and of the apex of the muscle cone were the most consistent findings. The severity of the CT scan abnormalities correlated well with clinical severity. Because muscle cone abnormality was observed characteristically in those patients with sight loss, we suggest that pressure by the extraocular muscles on the optic nerve may contribute to visual acuity loss in this disease.

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

To determine the reliability of creatinine as a measure of the glomerular filtration rate (GFR), we compared the simultaneous clearance of creatinine to that of three true filtration markers of graded size in 171 patients with various glomerular diseases. Using inulin (radius [rs] = 15 A) as a reference marker, we found that the fractional clearance of 99mTc-DTPA (rs = 4 A) was 1.02 +/- 0.14, while that of a 19 A rs dextran was 0.98 +/- 0.13, with neither value differing from unity. In contrast, the fractional clearance (relative to inulin) of creatinine (rs = 3 A) exceeded unity, averaging 1.64 +/- 0.05 (P less than 0.001), but could be lowered towards unity by acute blockade of tubular creatinine secretion by IV cimetidine. Cross-sectional analysis of all 171 patients revealed fractional creatinine secretion to vary inversely with GFR. This inverse relationship was confirmed also among individual patients with either deteriorating (N = 28) or remitting (N = 26) glomerular disease, who were studied longitudinally. As a result, changes in creatinine relative to inulin clearance were blunted considerably or even imperceptible. We conclude that true filtration markers with rs less than 20 A, including inulin, are unrestricted in glomerular disease, and that creatinine is hypersecreted progressively by remnant renal tubules as the disease worsens. Accordingly, attempts to use creatinine as a marker with which to evaluate or monitor glomerulopathic patients will result in gross and unpredictable overestimates of the GFR.

One hundred and two previously treated lymphoma patients were studied with 111Indium bone marrow scans and bone marrow biopsies. The biopsies were considered to represent sampling errors when the cellularity of the biopsy did not reflect the general state of the marrow organ cellularity as demonstrated by the scan. In each instance the accuracy of the scan was confirmed by either another biopsy or the subsequent clinical course of the patient. Sampling errors were infrequent (1/51) in patients with normal peripheral blood counts and whose marrow had never been involved with tumor. Errors were especially likely (17/51) in patients who had had marrow involvement or those who had anemia, leukopenia, or thrombocytopenia. The 111Indium bone marrow scan allows the clinician to avoid selecting a biopsy site with a high risk for sampling error.

Artificial lipid vesicles (artificial membranes) were shown to bind human 125I-antithyroglobulin (anti-Tg) and human 125I-thyrotropin. Vesicles made with gangliosides bound more antibody and hormone than vesicles lacking them. These gangliosides contained a variety of carbohydrates including glucose, galactose, N-acetyl-galactosamine, and sialic acid. The in vivo stability of antibody-vesicle complexes was a function of vesicle composition: vesicles were most stable when formed from phosphatidylcholine, cholesterol, and gangliosides. Anti-Tg-vesicle complexes bind to thyroglobulin, indicating that at least some of the antibody associated with the vesicle still retains ability to bind to its specific antigen. The addition of a specific antibody or hormone to artificial lipid vesicles may serve as a mechanism to confer specificity to the vesicle in vivo.

Single compartmental spherules of various lipid constituents (vesicles), enclosing (99m)TcO(4) (-) as a radioactive marker, were injected intravenously into C(3)H mice, and the distribution of radioactivity was studied. About 25% of the administered radioactivity was present in the liver 5 min and 30 min after the injection of vesicles composed of phosphatidylcholine and gangliosides, which were sonicated for 5 min (standard preparation). About 10-20% of the radioactivity remained in the circulation. By use of a nonradioactive spin label (tempocholine) enclosed within vesicles, intact vesicles were demonstrated in the circulation for 46 min after intravenous injection. The distribution of radioactivity from (99m)TcO(4) (-) inside vesicles is very different from that of free (99m)TcO(4) (-) or of (99m)Tc sulfur colloid. Increase in the length of sonication or incorporation of cholesterol into the wall of the vesicles enhanced hepatic levels and reduced blood levels of radioactivity. These same manipulations also slowed the rate of transfer of (99m)TcO(4) (-) out of vesicles in dialysis experiments in vitro. Addition of phosphatidic acid, phosphatidylethanolamine, or phosphatidylserine to the standard constituents did not greatly alter the distribution of radioactivity in vivo but did increase the number and type of active coupling sites on the outside of the vesicle. The results indicate that vesicles might be valuable as carriers of diagnostic or therapeutic agents.

The interpretations of 156 99mtechnetium pyrophosphate myocardial scintigrams by four observers were analyzed in order to determine the reliability and reproducibility of the subjective process of reading scintigrams. The scintigrams were scored on an integral scale from 0 to 4, depending upon the degree of myocardial radionuclide accumulation, and the site and nature of uptake were specified. Exact agreement upon score was generally poor but approximate concurrence of interpretation was good (90.4 and 92.5% inter- and intra-observer agreement, respectively). There was somewhat less agreement on scintigrams with the higher scores of 3 and 4 (83.3 and 78.0%, respectively). A high level of concurrence upon the differentiation between diffuse and localized uptake, and upon the site of uptake, was found. We conclude that only approximate rather than exact agreement of individual readers' interpretations can be expected in this subjective technique, that scintigrams with higher degrees of radionuclide accumulation produce slightly greater observer disagreement, and that variability of interpretation could account for some of the diagnostic inaccuracy of 99mtechnetium pyrophosphate myocardial scintigraphy.

111In has been complexed to a series of metal-complexing phthaleins and sulfonphthaleins, and the hepatobiliary excretion of the compounds were compared in rats. The highest biliary excretion was obtained with 111In-o-cresolphthalexon. In a normal human subject, sequential imaging with 111In-o-cresolphthalexon visualized his gallbladder, common bile duct, and the bowel from jejunum to rectum. The potential utility and limitation of this biliary scanning agent is discussed.

Seventy-six thallium-201 myocardial perfusion studies were performed on twenty-five patients to assess their reproducibility and the effect ofvarying the level of exercise on the results of imaging. Each patient had a thallium-201 study at rest. Fourteen patients had studies on two occasions at maximum exercise, and twelve patients had studies both at light and at maximum exercise. Of 70 segments in the 14 patients assessed on each of two maximum exercise tests, 64 (91%) were reproducible. Only 53% (16/30) of the ischemic defects present at maximum exercise were seen in the light exercise study in the 12 patients assessed at two levels of exercise. Correlation of perfusion defects with arteriographically proven significant coronary stenosis was good for the left anterior descending and right coronary arteries, but not as good for circumflex artery disease. Thallium-201 myocardial imaging at maximum exercise is reproducible within acceptable limits, but careful attention to exercise technique is essential for valid comparative studies.

To test the hypothesis that Graves' dermopathy is due to cross-reactivity of thyroid autoantibody(ies) with a cellular target in pretibial skin, we tested the serum and serum immunoglobulin fraction of 20 such patients for their effects on the metabolic activities of cultured thyrocytes (rat FRTL cells), human pretibial skin fibroblasts, and human fibroblasts of other origins. The incorporation of 3H-labeled thymidine, amino acids, and glucosamine into DNA, protein, and glycosaminoglycans, respectively, was measured. TSH and the serum of each of the 20 patients with Graves' dermopathy markedly stimulated the synthesis of DNA, protein, and glycosaminoglycans by FRTL cells, but not by fibroblasts, whereas assays of serum from 38 of 40 patients with Graves' disease without dermopathy did not stimulate these processes in FRTL cells more than normal serum. Stimulatory activity was associated with immunoglobulins. Serum dermopathy-associated antibodies disappeared with the disappearance of the skin lesions. These results suggest that the serum of patients with dermopathy contains antibodies that recognize a component of the TSH receptor different from that recognized by serum of Graves' patients without dermopathy, the former acting in some manner to induce lesions in pretibial skin. The skin target remains unidentified.

99m-technetium (Tc) pyrophosphate myocardial scintigrams of 55 patients with stable angina pectoris were compared with those of 13 normal subjects. The mean scintigraphic score, obtained by averaging the blinded interpretations of four readers scoring on an integral scale from 0 to 4, was significantly higher for the patients with angina than for the control subjects (1.36 compared with 0.48, P less than 0.001). Among the patients with angina, those who had a prior myocardial infarction had a higher mean scintigraphic grade than those without a previous infarction (1.73 versus 1.15, P less than 0.005), and the mean grade in both groups was higher than that of control subjects (P less than 0.001). Radionuclide uptake was predominantly diffuse in the patients with angina pectoris (70%), although in those with greater uptake accumulation tended to be localized. Three of the 68 subjects had high levels of radionuclide uptake but no clinical evidence of acute myocardial injury. This study demonstrates that excess myocardial accumulation of 99m-Tc pyrophosphate can occur in patients with stable angina pectoris.