Thyroid organoid formation in simulated microgravity: influence of keratinocyte growth factor.

The generation of artificial human thyroid tissues in suspension (low-shear environment, present in simulated microgravity [MG] and generated by a rotary cell culture system [RCCS]), was enhanced by increasing medium kinematic viscosity with a (3% v/v) suspension of extracellular matrix (basement membrane extract [BME]) in serum-free medium to generate artificial human thyroid organoids. Recombinant human keratinocyte growth factor (KGF, 7 ng/mL) facilitated human thyrocyte aggregation and three-dimensional (3-D) differentiation. There was an MG-associated decrease in extractable DNA that was reversed after addition of keratinocyte growth factor (KGF). In simulated MG, the increase in extractable DNA after KGF addition was up to 170% over non-KGF control cultures. In contrast, monolayer cultures in unit gravity showed a maximum DNA increase of 39% after KGF addition. Morphologically, differentiated thyroid neofollicles displayed polarization and were located in close proximity after 2 weeks of culture. Immunogold labeling with antibody to human thyroglobulin (Tg) revealed staining of follicular lumina and secretory vesicles, and a time-dependent increase in human Tg was detected in the culture media. Culture under simulated MG thus allowed direct visualization of KGF-facilitated thyrocyte/extracellular matrix interaction. Such artificial human thyroid organoids-generated in MG and in the presence of KGF-structurally resembled natural thyroid tissue. The above findings may have implications for autoimmune thyroid disease where KGF (if, for example, secreted locally by intraepithelial gammadelta T cells among other cells) may contribute to thyroid cell growth.

Preservation of functioning human thyroid "organoids" in the scid mouse. IV. In vivo selection of an intrathyroidal T cell receptor repertoire.

To study the in vivo influence of thyroid cells on the T cell receptor repertoire in human autoimmune thyroid disease, we mixed lymphocyte-free thyrocytes (approximately 1.2 x 10[6]) from patients with Graves' disease with autologous peripheral blood mononuclear cells (PBMC; approximately 1.5 x 10[6]) and transplanted this mixture sc into scid mice while suspended in a basement membrane gel (approximately 0.4 ml). Controls included mice that received either thyrocytes only or PBMC only. The resulting artificial mixed cell thyroid organoids were explanted after 5 weeks, and their T cell receptor repertoire was examined. Of a total of 63 organoids constructed, 60 were recovered (95.2%). Total RNA was extracted and then analyzed by reverse transcription-PCR primarily for human T cell receptor (hTcR) Vbeta gene expression using 21 hTcR Vbeta amplimers. A restricted pattern of hTcR Vbeta gene expression was found, with 6 Vbeta genes (Vbeta5, 6, 7, 8, 13.1, and 18) predominantly expressed [P < 0.05, by ANOVA on ranks and Student-Newman-Keul's (SNK) test]. PBMC and control organoids showed no preferential selection of particular hTcR V gene-expressing T cells. This reductionist, mixed cell, thyroid model reflected earlier observations in human and murine autoimmune thyroid diseases in which a bias in hTcR V gene family expression had been observed. The model permitted in vivo T cell selection and/or enrichment of potentially disease relevant human T cells.

Preservation of functioning human thyroid organoids in the scid mouse: 1. System characterization.

We have characterized a system for preserving reconstituted human thyroid follicles in vivo by transplanting human thyrocytes into mice with severe combined immunodeficiency (scid mice). Human thyroid organoids were constructed from thyroid monolayer cells derived from both normal and abnormal thyroid tissue, and embedded within a basement membrane preparation which was then transferred sc to scid mice. As early as 4 weeks, and as late as 3 months post transplantation, histological examination of human thyroid organoids demonstrated widespread neofollicle formation and colloid accumulation which stained positive for human thyroglobulin (hTg). Although there were no changes in murine serum T4 levels; the transplanted thyroid epithelial cells secreted hTg into the scid mouse circulation (with an average level of 29 micrograms/L). In addition, hTg release was stimulated in vivo by ip administration of recombinant human TSH (0.1-1.0 IU/mouse) achieving greater than 20-fold increases in scid mouse serum hTg levels. In situ immunohistochemistry showed that thyroid organoids derived from patients with Graves' disease retained scattered lymphocytes in peripolesis with the thyroid epithelial cells; those lymphocytes were identified as human T cells of the memory (CD45RO +), rather than naive, type. These data demonstrate that functioning human thyroid organoids establish in scid mice and remain responsive to TSH stimulation. The system offers a unique opportunity to examine human thyroid-lymphocyte interaction within the confines of a predictable animal model.

Liquid hybridization analysis of TSH receptor mRNA in normal and abnormal human thyroid tissues.

Radiolabeled human TSH receptor (hTSHR) cRNA probes encoding nucleotides 37-2298 and 37-209, with unlabeled sense RNA control segments, were used in a liquid hybridization assay and found to be highly specific and sensitive enabling detection of 0.5 fmol of hTSHR mRNA. Using normal human thyroid monolayer cell cultures we calculated that the average number of TSHR mRNA transcripts was 95 +/- 5 per cell under in vitro basal conditions. We found no significant difference between the hTSHR mRNA concentrations of intact normal human thyroid tissue (n = 4) and specimens from patients with multinodular goiter (n = 5) and Graves' disease thyroid tissues (n = 5) (23.0, 25.2, and 27.6 fmol of hTSHR and mRNA/mg total cellular RNA, respectively). However, there was a relative deficiency of hTSHR mRNA in some samples of thyroid papillary carcinoma tissue (n = 5) (12 fmol of hTSHR mRNA/mg total RNA, p < 0.05). The hTSHR 37-2298 probe was fully protected in normal and abnormal thyroid tissues, consistent with the absence of large deletions or insertions in the hTSHR mRNA transcripts but additional bands were present, consistent with the production of splicing variants.

Intrathyroidal accumulation of T cell phenotypes in autoimmune thyroid disease.

In this study we have correlated peripheral T cell subset phenotypes with intrathyroidal lymphocyte accumulation in patients with autoimmune thyroid disease (Graves' and Hashimoto's disease). Our study utilized euthyroid family members for one of our control groups (n = 48) thus significantly limiting familial, but not disease-specific, influences on these T cell phenotypes. Our principal new observations were found only in patients with Graves' disease. As previously reported, there was a decrease in CD8+ (suppressor/cytotoxic) T cells in the peripheral blood of patients with untreated hyperthyroid Graves' disease (n = 27) (mean +/- SEM, 19 +/- 1.1% in patients compared with 25 +/- 1.2% in controls, p = 0.03), a finding not observed in treated, euthyroid Graves' disease patients or their relatives. However, the relative number of CD8+ T cells, assessed by CD4:CD8 ratios, was increased in the intrathyroidal T cell populations (n = 10), when compared to normal and patient peripheral blood. There were no consistent changes in total CD4+ (helper) T cells in the peripheral blood of patients with treated and untreated Graves' disease but a reduction in CD4+2H4+ (suppressor-inducer) T cells was seen in patients undergoing surgery for Graves' disease (13 +/- 6.9% compared with 39 +/- 3.4%). Again, however, this T cell subset was increased within the target organ of the same patients (41 +/- 5.9%). These data point to either a selective accumulation, or a specific "homing", of certain T cell subsets within the thyroid gland of patients with Graves' disease where T cell differentiation may be strongly influenced by antithyroid drug treatment and the local immune environment.

Successful production of intrathyroidal human T cell hybridomas: evidence for intact helper T cell function in Graves' disease.

We have developed a series of human intrathyroidal T-T cell hybridomas and evaluated their phenotypic characteristics and lymphokine secretions in order to further understand the role of the T cell in Graves' disease. Mitogen-stimulated T cell blasts were generated from intrathyroidal lymphocyte preparations and fused with a hypoxanthine-, aminopterin-, and thymidine-sensitive variant of the Molt 4 human leukemia T cell line. The resulting intrathyroidal T-T cell hybridomas and T-T cell hybridomas obtained from normal peripheral blood mitogen-stimulated T cell blasts were expanded and tested for their biological function. None of the generated T cell hybridomas exhibited antigen-specific IL-2 secretion when stimulated with autologous thyrocytes, although 60% of the hybridomas expressed CD3 antigen and the T cell receptor alpha/beta heterodimer. However, 9 intrathyroidal and 11 peripheral blood T cell hybridomas secreted a factor(s) that significantly enhanced immunoglobulin G secretion in vitro (P less than 0.005, by Student-Newman-Keuls test; mean +/- SEM, 338 +/- 60% increase). In summary, we have successfully used a technique that allows the construction of T-T cell hybridomas derived from intrathyroidal T cell cultures. The data demonstrated that a predominance of helper factor-secreting T cells were available for fusion within the Graves' thyroid gland. Such observations are further evidence for intact T cell help within the thyroid gland of patients with Graves' disease.

Safety of invasive procedures in patients with the coagulopathy of liver disease.

To assess the need for prophylactic fresh frozen plasma in patients with chronic liver disease before procedures, we followed 39 consecutive patients with prothrombin times (PT) of 15-29 s who had 71 invasive procedures. A total of 57 procedures was done in 30 patients receiving no blood product support, while 12 patients received blood products within 12 h of 14 procedures. Only three of the latter group received fresh frozen plasma prophylactically to improve the PT. The two groups were similar in the severity of their liver disease. There were nine surgical procedures as well as paracenteses, thoracenteses, lumbar punctures, and central venous line placement. Three bleeding episodes occurred. Two of the bleeding episodes required no further treatment. Because of the low incidence of bleeding and the ease in controlling the bleeding once it occurred, fresh frozen plasma is not recommended for prophylaxis of an elevated prothrombin time.

Hereditary spherocytosis in the elderly.

Five patients with hereditary spherocytosis diagnosed in their seventh to ninth decades of life are presented. These patients are remarkable for absent or mild clinical manifestations of disease. Splenectomy is the recommended treatment for hereditary spherocytosis to avoid the complications of aplastic or hemolytic crisis. When the diagnosis is made in the elderly, the treatment of choice may be careful observation with folic acid supplementation rather than splenectomy. This recommendation is based on the incidence of complications of splenectomy in the elderly in comparison to the severity and incidence of complications from the disease itself.

Variability in production and immunoreactivity of in-vitro secreted human thyroglobulin.

We examined basal and bTSH-stimulated human thyroglobulin (hTg) secretion by autologous normal and abnormal (benign and malignant) human thyroid cell monolayers. Basal and bTSH-stimulated hTg secretion was highly variable and ranged from 50-700 ng/ml/10(5) cells over a 6 day period. All normal and benign 'non-functioning' adenoma cells demonstrated a dose and time related stimulation of hTg secretion in response to bTSH. Comparison of hTG secretion by autologous normal and abnormal cells showed that in six of eight pairs, the normal thyroid cells had a greater output of hTg than the benign adenoma cells in contrast to our previous studies using non-autologous cells. Malignant thyroid cell hTg production was less predictable than that obtained with normal and benign thyroid cells varying from absent to normal responses to bTSH. Characterization studies of the secreted hTg showed no difference between normal, benign and malignant thyroid cell hTg with reference to molecular weight. However, hTg secreted in vitro was non-iodinated and had a marked reduction (up to 200-fold) in immunoreactivity assessed by both polyclonal and monoclonal antibodies to hTg when compared to hTg standard prepared from intact thyroid tissue (which had 4.58 micrograms iodine/mg). This reduction in hTg immunoreactivity was greatest for hTg secreted by malignant thyroid cells. These data demonstrate the wide variability in the hTg secretory capacity of human thyroid cell monolayers and indicate, when compared to autologous normal cells, that abnormal human thyroid epithelial cells may be relatively deficient in their ability to secrete hTg in vitro. There were also qualitative differences in the immunoreactivity, and iodine content, of in-vitro secreted hTg. These observations suggest that there may be much greater heterogeneity in hTg secreted in vitro than previously realized, perhaps secondary to differences in iodine content and/or degree of glycosylation. Human thyroglobulin (hTg) is the major secretory protein of the thyroid cell (Van Herle et al., 1979). Intracellular hTg is the site of thyroid hormone iodination yet its extrathyroidal role, if any, remains unclear. While hTg is usually present in the peripheral circulation of normal individuals, in species of differing molecular weight (Feldt-Rasmussen et al., 1978), there have been few studies of in-vitro production of hTg by isolated human thyroid cells. Our interest in hTg is in its role as an antigen in thyroid autoimmune disease (De Bernardo et al., 1983; 1986).(ABSTRACT TRUNCATED AT 400 WORDS)

Preservation of the parathyroid glands in total thyroidectomy.

A series of 183 patients who underwent total thyroidectomy is presented. The operative method, which emphasizes visualization and preservation of the parathyroid glands and their blood supply, as well as exposure of the recurrent laryngeal nerves, is discussed in detail. Permanent hypoparathyroidism was a complication in six patients (3.3 per cent). Permanent nerve injury occurred in one patient (0.55 per cent). Postoperatively, 78 patients had radioactive iodine uptake studies to evaluate the amount of residual thyroid tissue in the neck. Sixty-four (82 per cent) had 24 hour uptake studies of less than 2 per cent and 52 had an uptake of less than 1 per cent. Although a careful and consistent technique of total thyroidectomy substantially reduces the danger of permanent hypocalcemia, we experienced this complication sporadically. Since no operative technique completely removes the danger of permanent hypoparathyroidism, it is our opinion that total thyroidectomy should not be the standard management for carcinoma of the thyroid gland. It is best reserved for those patients in whom the extent of disease or the aggressiveness of the histologic cell type warrants the increased risk.

Intrathyroidal T cell clones from patients with autoimmune thyroid disease.

We cloned activated T cells from thyroid tissue of patients with autoimmune thyroid disease. After separation on 40% Percoll gradients, T cells were cultured for 2-7 days with T cell growth factor (interleukin 2; 20 U/mL) and cloned by limiting dilution (0.3 cells/well) in the presence of irradiated autologous peripheral blood mononuclear cells (PMC; 10,000/well) as feeder cells. Fifty-seven clones were successfully expanded and tested for reactivity, cytotoxicity, helper/suppressor function, and phenotype. In the reactivity assays clones were tested for responses to autologous and allogeneic PMC, thyroid cells, human thyroglobulin (hTg), and microsomal antigen. Two distinct patterns of functional T cell clones emerged from these characterization studies. Seventy-five percent of T cell clones recovered from Graves' disease thyroid tissue (n = 21) were of helper-induced (CD4+/4B4+) phenotype, and most were effective immunoglobulin helper clones. Fifty percent of Graves' T cell clones responded to autologous PMC, and 33% had a proliferative response to autologous thyroid cells. No cytotoxic clones were derived from Graves' thyroid tissue. By contrast, intrathyroidal T cell clones from patients with autoimmune thyroiditis (n = 36) were 59% suppressor/cytotoxic (CD8+) phenotype, 17% suppressed immunoglobulin secretion, and 55% were cytotoxic to allogeneic blast cells. Fifty-five percent of clones also responded to autologous PMC, and one clone was nonspecifically autocytotoxic. In the thyroid antigen proliferation assays 11% of thyroiditis clones reacted to human thyroglobulin, but none responded to microsomal antigen. Two clones were cytotoxic to autologous but not allogeneic thyroid cells. These data demonstrate that the majority of intrathyroidal T cells in autoimmune thyroid disease are autoreactive. However, small numbers of thyroid-specific T cell clones are present within the thyroid of such patients; they are principally helper-inducer T cells in Graves' disease thyroid and cytotoxic T cells in autoimmune thyroiditis.

Oral analgesia for treatment of painful crisis in sickle cell anemia.

A therapeutic plan that emphasized oral narcotic analgesia was instituted for the treatment of painful crisis of sickle cell anemia. Of the 100 adult sickle cell syndrome patients registered at North Central Bronx Hospital, 15 were identified as using the emergency department facilities three or more times per year. This "frequent user" patient population was tracked in their hospital and drug usage patterns during the first full year of the oral protocol and compared to their own patterns during the year prior to the protocol. The patients used the ED at the same rate but their frequency of admissions to the hospital dropped by 75%. The oral program produced a significant fall in the amount of narcotics dispensed in the ED (P less than .01).

TSH receptor antibody induction of thyroglobulin release from human thyroid cell monolayers.

We investigated the influence of TSH receptor antibody (TRA), as detected by inhibition of 125I-bTSH binding to detergent solubilized porcine TSH receptors, on in-vitro thyroglobulin (hTg) production using normal thyroid cells in monolayer. Secretion of hTg into the culture medium was analysed by a non-competitive enzyme immunosorbent (ELISA) technique utilizing two murine monoclonal antibodies. Basal hTg release (mean +/- SD 124 +/- 27 ng/10(5) cells/6 d, n = 5) was stimulated by bTSH (10, 10(2), 10(3) microU/ml) in a dose related manner (mean +/- SD 191 +/- 24, 587 +/- 80, 695 +/- 66 ng/10(5) cells/6 d, respectively). IgG (2 mg/ml) from seven patients with hyperthyroid Graves' disease, and known titres of TRA, similarly enhanced production of hTg, in a dose and time-dependent manner, when compared to control IgG. The degree of induction varied from a 140-230% increase in total hTg release over a 6-day incubation period. There was a direct correlation between the degree of 125I-bTSH binding inhibitory activity and the hTg response (r = 0.9, P less than 0.01). These data demonstrate that TSH receptor antibodies enhance hTg release from human thyroid cell monolayers and allow an assessment to be made of antibody-activated post receptor mechanisms.

Short- and long-term evaluation of normal and abnormal human thyroid cells in monolayer culture.

We have investigated the TSH responsiveness of normal and abnormal human thyroid cells cultured in the short term with high serum concentrations and for up to 6 months in a low serum, chemically-defined, medium. Cells from normal human thyroid tissue (n = 9), multinodular goitre (n = 6), benign follicular adenomata (n = 6), and differentiated thyroid carcinoma (n = 3) formed confluent monolayers which were sensitive to bovine TSH (bTSH) in concentrations greater than 25 microU/ml when assessed by the intracellular response of cyclic AMP at 7 d of culture. Such sensitivity was less than that observed with a continuously proliferating thyroid cell line (FRTL-5) derived from Fisher rat thyroid and which responded to concentrations of bTSH as low as 5-10 microU/ml. Human cells derived from iodine/antithyroid drug treated Graves' thyroid tissue (n = 6) were less sensitive than normal cells requiring up to 500 microU/ml bTSH to increase intracellular cyclic AMP and poorly differentiated thyroid cancer cells (n = 3) failed to respond to bTSH. Long-term human thyroid cultures of normal and follicular adenoma cells in the chemically-defined medium used for the FRTL-5 cells had absent fibroblast growth and continued in monolayer form without significant follicle formation. These cells remained highly sensitive to bTSH stimulation when tested after 4, 13, and 26 weeks of continuous culture. All such cell preparations failed to proliferate under conditions which favoured the rapid growth of the rat thyroid cells. These data demonstrated that while thyroid cell culture conditions described in the literature do not permit proliferation of human thyroid cells, they do allow an assessment of their functional state in vitro which may lead to a further understanding of thyroid cell pathophysiology.

Pseudothrombocytopenia masking true thrombocytopenia.

Pseudothrombocytopenia owing to platelet clumping is usually associated with blood specimens anticoagulated with EDTA. It may also be seen if specimens possessing IgM cold agglutinins are processed at room temperature. A patient with a temperature-independent, EDTA-independent agglutinin is reported whose pseudothrombocytopenia was masking true thrombocytopenia. A technique for blood collection when evaluating similar cases is described.

Polytetrafluoroethylene grafts in the peripheral venous circulation of rabbits.

We demonstrated by venography that the patency of 3 mm PTFE grafts in the jugular veins of rabbits could be maintained by pretreating the animals with either an anticoagulant (warfarin sodium) or an antiplatelet agent (aspirin, dipyridamole, or both). Examination of the lining of the grafts up to 4 months after grafting by scanning electron microscopy or light microscopy showed that endothelial cells extended across the anastomosis for a short distance and that a neointima lined the remainder of the graft. This lining could hypertrophy to the point of almost occluding the graft unless the drugs were continued.

The place of fine needle biopsy in the diagnosis of nodules of the thyroid.

Fine needle aspiration cytology with an accuracy rate of 90.2 per cent confirmed by surgical specimens was found to be a useful adjunct in the diagnosis of tumors of the thyroid but did not replace large needle core biopsy. Both methods offered information complementary to each other, and our data indicated that, when possible, they should be used together. Although both false-positive and false-negative results were reported with fine needle cytology aspiration biopsy, it was the false-negative reports which limited its use as a definitive diagnostic tool. Fine needle aspiration biopsy, however, did constitute a safe and useful method of increasing diagnostic ability in the evaluation of thyroid nodules.

Patency of venous grafts in small veins: influence of aspirin and warfarin sodium.

Venous autografts were prepared in jugular veins of rabbits as a test model to determine the usefulness of antiplatelet and anticoagulant agents (aspirin and warfarin sodium) alone or in combination for maintaining patency in the reconstruction of small veins. Graft patency was determined by periodic venography and postmortem examination. The grafts clotted in all of the untreated controls, five of six animals receiving aspirin, and three of four receiving warfarin, but in none of the animals receiving the combination of aspirin and warfarin.