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Biomedical subjects

N Yoshikawa

Publications and source records attributed to N Yoshikawa.

At least 127 records · Page 7Linked to original sources
PubMed1994

Studies of human thyroid xenografts from Hashimoto's thyroiditis in severe combined immunodeficient (SCID) mice: detection of thyroid stimulation-blocking antibody.

Human thyroid xenografts from 7 patients with Hashimoto's thyroiditis (HT) and 3 normal persons (N) were xenografted into severe combined immunodeficient (SCID) mice to study the intrathyroidal lymphocytes that were expected to survive in these animals. Human IgG was detected in all mice engrafted with HT thyroid tissue peaking at 6-10 weeks after xenografting. Thyroperoxidase-antibody (TPO-Ab) was also detected in all mice with HT thyroid grafts peaking at 4-6 weeks after xenografting, reaching up to 44% of donors' original concentrations. In contrast, maximal thyroglobulin (Tg)-Ab production in some SCID mice with HT thyroid grafts was higher than the donors' original level, and was detectable in mice with thyroid grafts from Tg-Ab-negative HT donors. Thyroid stimulation-blocking antibody (TSBAb) was found in 2 mice with thyroid xenografts from 1 HT patient whose original serum TSBAb and thyrotropin-binding inhibitor immunoglobulin (TBII) had been positive; the maximal TSBAb level in SCID mice exceeded the donor's original level. TSBAb production in SCID mice reached its peak at 10 weeks after xenografting, i.e., later than that of thyroid-stimulating antibody (TSAb) observed in our recent report, suggesting the existence of distinct intrathyroidal B cell autoreactive clones of different life span responsible for secreting TSAb or TSBAb. When autologous peripheral blood mononuclear cells (PBMC) were engrafted alone (without thyroid tissue), TSBAb was undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada.

It has been suggested elsewhere that the enteric pathogen Yersinia enterocolitica (Y.e.) might be implicated etiologically in autoimmune thyroid disease (AITD). To reevaluate this hypothesis in the Canadian population, where the prevalence of anti-Y.e. antibodies in the general population is very low (< 1%), we have studied the occurrence of antibacterial reactivity (against Y.e. 0:3 and 0:9, Escherichia coli and Staphylococcus aureus) in the sera of patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), nontoxic nodular goiter (NTG), and autoimmune rheumatic diseases (ARD) as well as normal controls (C). Using the tube agglutination method, no single positive sample was detected in these subjects. No differences in the mean levels of anti-Y.e. 0:3 or 0:9 by ELISA were observed between various groups of patients. Immunoreactivity in the course of medical therapy during 5-12 months did not show significant changes in any of 12 ARD and AITD patients. Some serological reactivity to the plasmid containing strain of Y.e. 0:3 was demonstrated in all subjects by the Western blotting technique. However, weaker signals and fewer bands were noticed in these sera compared to sera from patients with acute yersiniosis. Analysis of the pattern of reactivity did not show any difference in reactivity to any protein between the groups of subjects. The immunodominant antigen in Y.e. 0:3 to which IgG reacted in almost all subjects was the plasmid encoded 240-kDa protein. Our study favors the view that there is a merely coincidental incidence of seroreactivity to bacterial antigens, which appears to be irrespective of diagnosis.

Antibodies, Bacterial↗
PubMed1994

Effect of FK-506 on xenografted human Graves' thyroid tissue is severe combined immunodeficient mice.

OBJECTIVE: We studied the macrolide antibiotic FK-506, an immunosuppressive agent, in an attempt to ameliorate the lesion of autoimmune thyroid disease in human thyroid tissue xenografted into severe combined immunodeficient (SCID) mice. It was not felt appropriate to employ this agent directly in patients with autoimmune thyroid disease because adequate therapeutic modalities are available and the introduction of new, experimental agents could not be justified. Moreover, the study of the tissue before and after treatment could not have been undertaken directly in patients. DESIGN: Human thyroid xenografts from four patients with Graves' disease and two normal persons were xenografted into SCID mice. Two weeks after xenografting, human immunoglobulin G (IgG) was detectable in all SCID mice xenografted with Graves' thyroid tissue. Mice were divided into two groups with human IgG levels similar to each other. Mice in the first group were treated with FK-506 daily for 6 weeks; mice in the second (similar) group were given phosphate-buffered saline (PBS) only (control group). MEASUREMENTS: Blood samples were taken every 2 weeks from the tail veins for human IgG, thyroid stimulating antibody, thyroperoxidase antibodies, thyroglobulin antibodies, and interferon-gamma (IFN-gamma). After 8 weeks treatment, animals were sacrificed; thyroid tissue was examined histologically and for thyrocyte HLA-DR expression. FK-506 was also added to thyrocytes in in-vitro tissue culture conditions. RESULTS: After 4-6 weeks of FK-506 therapy, human IgG, all thyroid antibodies and IFN-gamma were suppressed, while the levels remained elevated in the control group. Lymphocytic infiltration virtually disappeared in the human thyroid tissue of the FK-506-treated mice and thyrocyte HLA-DR expression markedly declined; in the control mice, lymphocytic infiltration remained heavy and HLA-DR expression remained high. On the other hand, FK-506 added directly to thyrocytes in vitro (without lymphocytes) did not reduce thyrocyte HLA-DR expression. CONCLUSIONS: FK-506 appears to suppress the activation of intrathyroidal lymphocytes, but not thyrocytes. From these observations, it is concluded that this agent, by its action on intrathyroidal lymphocytes, is able to ameliorate the immunologically mediated histological and serological disturbance in human autoimmune thyroid disease, at least under these circumstances.

Animals↗
PubMed1994

Regional analysis of ceramides within the stratum corneum in relation to seasonal changes.

We studied the regional variations and seasonal changes of ceramide quantities in the stratum corneum of human skin. In summer, the total lipid amounts extracted from the stripped stratum corneum were highest in the forehead, followed by the chest and upper back, with the lowest level in the sole of the foot. In winter, while the total lipid distribution followed the same trend as that in summer, the absolute amounts slightly decreased, especially in the forehead and chest, as compared with those found in summer. Ceramide analysis showed that while a higher level was seen in the cubital fossa in summer, the level was highest in the forehead during winter. A seasonal comparison of ceramide mass revealed a slightly increased level in winter compared with summer, at almost all sites tested, except in the cubital fossa and dorsum pedis, reflecting seemingly enhanced keratinization in winter. These findings indicate that in young adults, the mass of ceramide remains steady at similar levels at various skin sites, providing maintenance of a pertinent water reservoir and the barrier function of the stratum corneum, even under different seasonal conditions.

Adult↗
PubMed1994

Effect of removing human Graves' thyroid xenografts after eight weeks in nude mice and rexenografting them into SCID mice.

Human thyroid xenografts from four patients with Graves' disease (GD) and two normal persons were initially xenografted into nude mice. Eight weeks after xenografting, the thyroid tissue appeared normal; indeed, thyroid infiltrating lymphocytes in the GD xenograft could no longer be identified when analyzed histologically. Thus, human immunoglobulin G (IgG), thyroperoxidase (TPO)-antibodies (Abs), thyroglobulin (Tg)-Abs, thyroid-stimulating antibodies (TSAb), and thyrocyte histocompatibility leucocyte antigen (HLA)-DR expression were undetectable. These same tissues were retrieved from the nude mouse and rexenografted into severe combined immunodeficient (SCID) mice (with no prior xenograft); autologous peripheral blood mononuclear cells (PBMC) or CD8-depleted PBMC (non-CD8 cells) were simultaneously injected into some of these SCID mice. Engraftment of a GD thyroid rexenograft (TH) alone did not cause IgG, TSAb, TPO-Ab, or Tg-Ab production, thyrocyte HLA-DR expression, or lymphocytic infiltration in thyroid grafts. Engraftment of GD PBMC or non-CD8 cells alone (i.e. without a thyroid xenograft) caused human IgG to rise, but only minimal titers of thyroid antibodies appeared. When TSAb, TPO-Ab, and Tg-Ab were quantified, GD TH plus PBMC-engrafted SCID mice showed significantly higher production of each antibody than that of GD PBMC alone, and this phenomenon was further enhanced by the removal of CD8+ cells. GD thyrocytes showed marked HLA-DR expression at human surgery; however, after 8 weeks' sojourn in nude mice, DR expression disappeared. After a further 8 weeks following rexenografting into SCID mice, TH plus PBMC resulted in a reappearance of DR expression only in GD but not in grafts from normal persons, and this was enhanced by the depletion of CD8 cells. These results were also in parallel with histological findings inasmuch as the normal tissue remained normal with no thyroid antibodies appearing with PBMC or CD8-depleted cells. In experiments from two GD patients, autologous skeletal muscle as well as thyroid tissue were xenografted into nude mice. Eight weeks after xenografting, these were rexenografted into SCID mice that contained prior autologous primary GD thyroid xenografts. Histological findings showed new lymphocytic infiltration in rexenografted thyroid tissues in the SCID mice but not in autologous skeletal muscle. This signifies that the immune assault in GD is specifically targeted to the thyroid tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1994

IgG-associated primary glomerulonephritis in children.

Ten children with IgG-associated primary diffuse mesangial proliferative glomerulonephritis are presented. All cases showed diffuse global IgG deposits in the mesangium. IgG was the sole immunoglobulin deposited in the mesangium in 8 patients and was the predominant immunoglobulin in 2. C3 was also present in 8 patients. Light microscopy revealed variable diffuse and global mesangial proliferation in all cases, with additional focal segmental glomerular sclerosis in one. On electron microscopy, electron-dense deposits in the mesangium were identified in all patients. Clinically, 4 patients presented with the steroid-resistant nephrotic syndrome, and 6 had proteinuria and hematuria. At the latest follow-up, renal function was normal in all patients, 6 showed clinical remission and 4 had slight proteinuria and/or hematuria. These data suggest that there is a clinicopathologic entity, IgG-associated glomerulonephritis, characterized by diffuse mesangial proliferation, predominant mesangial IgG deposition, variable clinical presentation, and a relatively benign course.

Adolescent↗
PubMed1994

T-cell subpopulations in childhood nephrotic syndrome.

In order to clarify the T-cell abnormalities present in minimal-change nephrotic syndrome (MCNS), lymphocyte subsets were studied using two-color flow cytometry in children with MCNS and nephrotic syndrome caused by other types of glomerular disease, and normal healthy children. In MCNS, CD4+Leu8+ (suppressor-inducer) and CD3+CD25+ (IL-2 receptor positive T) cells were significantly increased, and CD4+Leu8- (helper-inducer), CD8+CD11+ (suppressor), CD3+HLA-DR+ (DR positive T) and CD4+HLA-DR+ (activated helper/inducer) cells were significantly decreased in comparison with normal healthy children. The same abnormalities were also found in nephrotic syndrome caused by other types of glomerular disease. These findings indicate that abnormalities of lymphocyte subsets are present in MCNS and nephrotic syndrome due to a variety of glomerular diseases, and suggest that such abnormalities are a consequence of nephrotic syndrome.

Adolescent↗
PubMed1993

The use of the severe combined immunodeficient mouse and the athymic "nude" mouse as models for the study of human autoimmune thyroid disease.

The athymic "nude" mouse and the severe combined immunodeficient (SCID) mouse have differing immunological properties which permit complementary studies of autoimmune thyroid disease (AITD). The nude mouse accepts human thyroid xenografts, but lyses the passenger lymphocytes, whereas in the SCID mouse both the xenograft and its lymphocytes survive. Human AITD thyroid xenografts manifest a worsening of the pathological picture in the SCID mouse, but show a return to normal morphology and function in the nude mouse at the time of sacrifice 6-8 weeks after engraftment. Such tissue which has been grafted into the nude mouse can be retrieved and then can be re-xenografted into the SCID mouse. Normalized AITD thyroid tissue (from the nude mouse xenograft) will remain normal in the SCID mouse, but if xenografted into a SCID mouse which already has a primary autologous AITD thyroid xenograft, or if autologous PBMC are added, the AITD lesion will be reproduced. This demonstrates the primacy of the immune system in AITD and constitutes a useful model for the study of this human disorder.

Animals↗
PubMed1993

Long-term ciclosporin treatment in children with steroid-dependent nephrotic syndrome.

We report the effect of 18-month ciclosporin (CS) treatment in 19 children with steroid-dependent frequently relapsing nephrotic syndrome. CS was started at 3-5 mg/kg per day after remission with steroid therapy, was adjusted to maintain a trough blood level of between 200 and 600 ng/ml and was administered for 6 months (high-dose CS). Then, the dosage of CS was decreased and 2.5 mg/kg per day was administered for the subsequent 12 months (low-dose CS). Only 2 patients had relapses during the initial 6 months of CS therapy. Eight patients had infrequent relapses, 4 had frequent relapses and 7 had no relapses during the 12 months of low-dose CS. Not only did continuation of CS at a lower dosage decrease the relapse rate, it also reduced steroid toxicity, allowed increased growth in 16 of the 19 patients and also decreased obesity scores in 12 of these patients. All of the side-effects that occurred during the 18-month period of CS treatment were reversible and none was serious enough to necessitate discontinuation of therapy. Our 18-month CS treatment was helpful in preventing relapses and reducing steroid toxicity in children with steroid-dependent frequently relapsing nephrotic syndrome.

Adolescent↗
PubMed1993

Reduced activation of suppressor T lymphocytes by specific antigens in autoimmune thyroid disease.

In order to study the activation of suppressor T lymphocytes by thyroid-specific antigens in autoimmune thyroid disease (AITD), we have investigated the effects of the organ-specific antigens, thyroperoxidase (TPO), thyroglobulin (Tg), and thyroid microsomal antigen (TMc), as well as renal microsomes (RMc) as a control antigen, on the activation of suppressor T lymphocytes; this was accomplished by measuring major histocompatibility complex class II (HLA-DR) expression on their surfaces by flow cytometric analysis. Peripheral blood mononuclear cells (PBMC), obtained from 33 patients with Graves' disease (GD), 26 with Hashimoto's thyroiditis (HT), 5 with nontoxic nodular goiter (NTG), and 30 normal persons (N), were cultured for 7 days in the presence or absence of TPO, Tg, or RMc at final concentration of 10, 100, and 1000 ng/ml. Cultured cells were stained with fluorescent-conjugated monoclonal antibodies (anti-CD8, anti-CD11b, and anti-HLA-DR), and the activation of CD8+ and CD8+CD11b+ (pure suppressor) T cells by the antigens was analyzed on a flow cytometer. In the absence of antigen, i.e., the autologous mixed lymphocyte reaction (AMLR), CD8+ and CD8+CD11b+ T lymphocytes from patients with GD and HT showed significantly lower activation as compared to N. We measured the Stimulation Index (Sl) of activated T lymphocytes to compare antigen-specific activation between CD8+ and CD8+CD11b+ cells from normal persons and patients. With stimulation of 100 and/or 1000 ng/mL of TPO or Tg, Sl of activated CD8+ cells was significantly (p < 0.05 to 0.01) lower in GD and HT as compared with N.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

The effect of interferon alpha and prednisolone on the serum concentrations of the aminoterminal domain of procollagen type III(PIIIP) and type IV (7-S collagen) in patients with chronic hepatitis B.

Since interferons are reported to inhibit collagen synthesis in vivo and in vitro, they are possible anti-fibrogenic drugs. However, little is known about the effect of interferons on connective tissue metabolism in liver disease. To investigate the effect, serum concentrations of the aminoterminal domain of procollagen type III and type IV (7-S collagen) were measured in 16 patients with chronic hepatitis B during alpha interferon treatment. The levels were also determined during prednisolone withdrawal treatment to investigate the effect on hepatic collagen turnover. In patients with chronic hepatitis B, the serum levels of both markers were significantly elevated before any treatment (procollagen type III: 1.10 +/- 0.39 U/ml, 7-S collagen: 6.69 +/- 3.71 ng/ml) compared with levels in 41 healthy volunteers (pro-collagen type III: 0.53 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.72 +/- 0.57 ng/ml, P < 0.001). Both levels were even more elevated during interferon therapy (procollagen type III: 1.36 +/- 0.58 U/ml, P < 0.05; 7-S collagen: 8.77 +/- 4.68 ng/ml, P < 0.01). On the other hand, serum procollagen type III and 7-S collagen levels were both decreased during prednisolone treatment (procollagen type III: 0.62 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.66 +/- 1.69 ng/ml, P < 0.01). These preliminary data suggest that interferon alpha enhanced, but prednisolone inhibited the turnover of procollagen type III and IV in the liver. Further study is required to interpret this observation in relation to anti-fibrotic therapy.

Adolescent↗
PubMed1993

Human mesangial cells and peripheral blood mononuclear cells produce vascular permeability factor.

Vascular permeability factor, or vascular endothelial growth factor (VPF/VEGF) is a disulfide-linked dimeric glycoprotein of about 40 kD that promotes fluid and protein leakage from blood vessels. Various human tumor cell lines and cells including fetal vascular smooth muscle cells produce VPF/VEGF. Since glomerular mesangial cells (MC) are closely related to vascular smooth muscle cells, we examined whether cultured human MC produce VPF/VEGF. Northern blotting analysis revealed that cultured human MC expressed a 3.7 kilobases (kb) VPF/VEGF mRNA. Human peripheral blood mononuclear cells (PBMC) also expressed VPF/VEGF transcripts of 8.6 and 3.8 kb. Although the sizes of the transcripts suggested the existence of unique molecular species of VPF/VEGF mRNA in PBMC, RT-PCR analysis revealed that PBMC as well as human MC expressed 121, 165, and 189 amino acid-containing isoforms of VPF/VEGF, implying that there are no unique alternative splicing products of VPF/VEGF mRNA in PBMC. Fetal calf serum and 12-o-tetradecanoyl- phorbol-13-acetate (TPA) transiently enhanced VPF/VEGF mRNA expression in cultured human MC. Transforming growth factor-beta 1 enhanced VPF/VEGF mRNA expression in cultured human MC at least within 24 hours. Dexamethasone (DEX) inhibited the TPA-induced increase in VPF/VEGF mRNA expression, whereas DEX did not change the basal level. The DEX depressed the TPA-induced increase in VPF/VEGF mRNA expression is therefore probably a result of transcriptional control. VPF/VEGF protein was detected in cultured human MC with immunoperoxidase staining using anti-VPF/VEGF antibody. TPA increased VPF/VEGF protein levels as well as those of VPF/VEGF mRNA in cultured human MC.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

New animal models for human autoimmune thyroid disease. Xenografts of human thyroid tissue in severe combined immunodeficient (SCID) and nude mice.

We have been employing two mouse models for the study of human thyroid xenografts from patients with autoimmune thyroid disease (AITD). The first mouse strain is that of the athymic "nude" mouse which accepts human thyroid xenografts, but the passenger lymphocytes are lysed in this model over several weeks; in the second model, the severe combined immunodeficient (SCID) mouse, both the xenograft and its lymphocytes survive. The AITD thyroid xenograft returns to normal function and morphology in the nude mouse over several weeks whereas the same AITD thyroid xenografts undergo aggravation of their lesions in the SCID mouse. Normalized ("cleansed") thyroid tissue in the nude mouse, now bereft of the passenger lymphocytes, can be removed and then re-xenografted into SCID mice. There it will remain normal unless autologous peripheral blood mononuclear cells are added, whereupon the AITD lesion will be reproduced. Autologous "irrelevant" muscle tissue having undergone the same process will not show such lesions. There is thus no evidence for a primary thyroid cell disturbance in AITD, the abnormality appearing to be only in the immune system. Moreover peripheral blood mononuclear cells appear to contain sufficient memory cells to be able to mount an immune assault on the autologous normalized thyroid tissue (to which they had been previously sensitized) but not on irrelevant autologous (muscle) tissue.

Animals↗
PubMed1993

Striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capilloviruses.

The sequence of the 3'-terminal 2956 nucleotides, excluding the poly(A) tail, of the citrus tatter leaf virus (CTLV) genome was determined and compared with that of the apple stem grooving virus (ASGV) genome. The sequence of the 3'-terminal region of CTLV contains two overlapping open reading frames (ORFs) and a 3'-terminal non-coding region of 142 nucleotides. The long, incomplete ORF1 ends at UAG (position 2812) and encodes a protein with at least 938 amino acids (M(r) > 108,703). This protein contains the GDD motif associated with the RNA polymerase. ORF2, in a different frame within ORF1, starts at AUG (position 1248) and stops at UGA (position 2208) encoding a protein with an M(r) of 36,179 (36K). Partial homologies were found among the 36K protein of CTLV, the 50K protein of apple chlorotic leaf spot closterovirus, the 40K protein of potato virus T and the gene 1 products of caulimoviruses. The arrangement of ORFs in the 3'-terminal region of the CTLV genome is in perfect agreement with that of the ASGV genome. The sequence of the 3'-terminal 2956 nucleotides, excluding the poly(A) tail, of the CTLV genome shows 86.1% identity to that of the ASGV genome. Similarities of amino acid sequences encoded by ORF1 and ORF2 of CTLV with the corresponding regions of ASGV are 86.1% and 97.3%, respectively. These results indicate that CTLV is a capillovirus closely related to ASGV.

Amino Acid Sequence↗
PubMed1993

Complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus.

The complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus (ACLSVA) was determined. The genome is 7552 nucleotides excluding the poly(A) tail and contains three open reading frames (ORFs 1, 2 and 3), encoding proteins with M(r) values of 216,503 (216.5K), 50,453 (50.4K) and 21,394 (21.4K), respectively. Nucleotide sequence comparisons between ACLSV-A and the previously sequenced ACLSV from plum (ACLSV-P) showed that the sequence identity at the nucleotide level was 79.8%. Amino acid sequence identities of ORFs 1 and 2 between both isolates were 88.4% and 79.9%, respectively. The 21.4K protein encoded by ORF 3 of ACLSV-A had an amino acid sequence identity of 88.6% with the 28.3K protein encoded by ORF 3 of ACLSV-P. Immunoblot analysis of the 21.4K protein expressed in Escherichia coli showed that this protein is the coat protein of ACLSV-A.

Amino Acid Sequence↗
PubMed1993

Quantitative analysis of stratum corneum lipids in xerosis and asteatotic eczema.

Sphingolipids, a major constituent of intercellular lipids, are an important determinant for both water-holding and permeability barrier function in the stratum corneum. To assess the pathogenic role of sphingolipids in the stratum corneum of dry skin disorders such as xerosis and asteatotic eczema in leg skin, ceramides were quantified by thin layer chromatography after n-hexane/ethanol extraction of resin-stripped stratum corneum and evaluated as micrograms/mg stratum corneum. In healthy leg skin (n = 49), there was age-related decline in the total ceramide, whereas xerosis (n = 25) and asteatotic eczema (n = 16) suffering significantly reduced water-holding properties, exhibited no definite decrease, rather slight increase in ceramide quantity with the same composition of each individual ceramide as compared to healthy age-matched controls. These data indicate that the seemingly elevated level of ceramide is an artificial effect due to inflammatory processes which result from susceptibility to dryness. Analysis of sebum-derived lipids present in the stratum corneum revealed that there was a significant decline in free fatty acids in xerosis and asteatotic eczema as compared to age-matched healthy controls, and a similar decline in triglycerides in the above three groups when compared to younger controls. Although the observed decrease in the stratum corneum lipids may well explain the high incidence of winter dry skin in older people, the progression toward asteatotic eczema can not be accompanied solely by a decrease in ceramide quantity, suggesting that the evolution of xerotic skin is associated with other moisturizing factors and/or environmental stimuli.

Adult↗
PubMed1993

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.

Adolescent↗