Changes of peripheral mononuclear cell subpopulations in autoimmune thyroid diseases

We evaluated thyrotropin-binding inhibitor immunoglobulin (TBII) in sera from patients with Graves' disease (GD) by using the new human recombinant TSH receptor (TSH-R) assay to determine if it is useful for the diagnosis and follow-up of GD. Serum samples from 25 patients with untreated GD, 24 with relapsing GD, 15 with GD in remission and 197 treated GD patients were examined. TBII was also measured in sera from healthy individuals (n = 69). TBII was retrospectively measured by using the conventional porcine TBII assay (pTBII) and the human recombinant TBII assay (hTBII). Specificity was adjusted at 97.5% for both assays by receiver-operating characteristic (ROC) plot analysis, resulting in cut-offs at 10.5% inhibition (hTBII) and 9.9% (pTBII). Sensitivity was 87% for hTBII compared to 52% for pTBII (P < 0.0001). Even in pTBII-positive patients, hTBII values (57.6 +/- 26.1%) were significantly (P < 0.0001) higher than pTBII values (34.2 +/- 27.6%). When 139 pTBII-negative serum samples from patients with treated GD were examined, 97 samples (69.8%) were positive for hTBII. hTBII values (23.9 +/- 17.0%) were significantly (P < 0.0001) higher than pTBII values (4.1 +/- 3.3%). hTBII was positive in 13 of 13 patients who were taking antithyroid drugs but had normal serum TSH levels. When 19 serum samples from pTBII-negative untreated patients with GD were examined, 15 out of 19 (79%) were positive for hTBII. We also examined 24 patients with relapsing GD. hTBII was positive in 16 (64%) patients, whereas seven (16%) were positive for pTBII (P < 0.01). Positivity of hTBII in these untreated or relapsing GD patients was significantly higher than that of thyroid stimulating antibodies. We then examined TBII levels in 15 patients with GD in remission who had normal serum TSH values. hTBII was positive in seven patients at the time of blood withdrawal, and GD subsequently relapsed in three hTBII-positive patients in 6 months. Low TBII titres can now be followed more accurately during the course of treatment, which will also allow better longitudinal studies on the importance of TBII for the diagnosis as well as the prognosis of GD with respect to remission or relapse.

Introduction: This study aims to evaluate the correlation and agreement between 2 methodologies of CD4 lymphocyte enumeration. Materials and Methods: Fifty-two blood samples from patients with human immunodeficiency virus (HIV) infection were sent for CD4 lymphocyte enumeration at 2 major hospitals using dual-platform flow cytometry where the absolute lymphocyte counts were determined on separate haematology analysers. CD4 cell enumeration was accomplished using 3-colour flow cytometery on the FACSCalibur cytometer (Becton Dickinson) in Hospital A and 4-colour flow cytometery on the Coulter Epic XL cytometer (Beckman Coulter) in Hospital B. The percentages and absolute counts of CD4 lymphocytes obtained were analysed using both linear regression and Bland-Altman plots. Results: On linear regression plots, the total white cell counts, absolute lymphocyte counts, CD4 lymphocyte percentages and absolute CD4 counts from the 2 hospitals correlated well with correlation coefficients, r &gt;0.95. On the Bland-Altman plots, there was a mean difference of 0.13 x 109/L and 0.06 x 109/L in the total white cell and absolute lymphocyte counts from the 2 hospitals respectively. The CD4 lymphocyte percentages revealed a mean difference of only 0.05% (95% limits of agreement, -3.6 to 3.7%) but there was a mean difference of 14/µL in the absolute CD4 lymphocyte counts (95% limits of agreement, -113 to 142/µL). Conclusions: The CD4 lymphocyte percentages obtained using the 3-colour and 4-colour flow cytometry correlated and agreed well. However, the absolute CD4 lymphocyte counts obtained using the dual-platform technique in both the hospitals did not agree well. Hence, absolute CD4 lymphocyte counts from the 2 hospitals cannot be used interchangeably in clinical practice due to poor inter-laboratory comparability.

We have studied by flow cytometric analysis the antigen specific activation of CD4+ (helper/inducer) T lymphocytes by purified human thyroid peroxidase (TPO). Peripheral blood mononuclear cells were obtained from 26 patients with Graves' disease (GD), 16 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), and 14 normal subjects (N). Cells were cultured for 7 days in the presence or absence of TPO at final concentrations of 3, 30, and 300 ng/mL. When harvested, cells were reacted with an FITC-conjugated anti-CD4 and a PE-conjugated anti-HLA-DR murine monoclonal antibodies. The percentage of HLA-DR+ CD4+ cells (activated CD4+ cells) was determined by a flow cytometer. In the absence of TPO, CD4+ cells had been activated without any specific stimulant. This is known as the autologous mixed lymphocyte reaction (AMLR). In the AMLR, CD4+ cells from GD and HT were less activated compared to those from NG and N. Results of TPO-specific activation were expressed as an incremental increase of activated CD4+ cells (II) (percentage of activated CD4+ cells cultured with TPO minus percentage of activated CD4+ cells cultured without TPO). II of N, GD, HT, and NG were 0.37 +/- 0.21, 2.20 +/- 0.45,** 2.0 +/- 0.66,* and 0.35 +/- 0.27 (mean +/- SEM), respectively (**p less than 0.01; *p less than 0.05 vs N). When patients were further subdivided, the highest mean II was found in patients with hyperthyroid GD (p less than 0.01), followed by euthyroid HT (p less than 0.05) and euthyroid GD (p less than 0.05), however there was no significant difference between hypothyroid HT and N. In conclusion (1) AMLR reactivity of CD4+ cells from GD and HT was impaired, (2) however, CD4+ cells from both GD and HT were significantly more induced by TPO compared to N, and (3) this induction depends, in part, on the in vivo thyroid status.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Diversity of complement activation in different thyroid diseases

To evaluate the role of cell-mediated immunity in Graves's disease and Hashimoto's thyroiditis, we studied the percentage of erythrocyte (nonimmune) and erythrocyte-antibody-complement (immune) rosettes (i.e., thymus-dependent and bursa-equivalent lymphocytes) in the peripheral blood. The mean (± S.D.) percentage of nonimmune rosettes was 63.8 ± 5.8 in 24 control subjects, 93.1 ± 6.2 in 17 patients with untreated Graves's disease, 98.8 ± 8.7 in 16 patients with Hashimoto's thyroiditis and 64.5 ± 6.4 in eight "goiter controls," two of whom were hyperthyroid. Six patients with Graves's disease in remission after antithyroid drug therapy had normal counts of lymphocytes forming nonimmune rosettes. Conversely, in five patients maintained in a euthyroid state on antithyroid drugs, but not in remission, 92.4 ± 3.1 (mean ± S.D.) per cent of lymphocytes were of the type forming nonimmune rosettes.

Introduction. An absolute lymphocyte count is commonly used as an alternative to a CD4 count to determine initiation of antiretroviral therapy for HIV-infected individuals in Fiji when a CD4 count is unavailable. Methods. We conducted a retrospective analysis of laboratory results of HIV-infected individuals registered at all HIV clinics in Fiji. Results. Paired absolute lymphocyte and CD4 counts were available for 101 HIV-infected individuals, and 96% had a CD4 count of ≤500 cells/mm3. Correlation between the counts in individuals was poor (Spearman rank correlation r = 0.5). No absolute lymphocyte count could be determined in this population as a suitable surrogate for a CD4 count of either 350 cells/mm3 or 500 cells/mm3. The currently used absolute lymphocyte count of ≤2300 cells/μL had a positive predictive value of 87% but a negative predictive value of only 17% for a CD4 of ≤350 cells/mm3 and if used as a surrogate for a CD4 of ≤500 cells/mm3 it would result in all HIV-infected individuals receiving ART including those not yet eligible. Weight, CD4 count, and absolute lymphocyte count increased significantly at 3 months following ART initiation. Conclusions. Our findings do not support the use of absolute lymphocyte count to determine antiretroviral therapy initiation in Fiji.

Activated CD4 T cells' express CD40 ligand (CD154) interacting with CD40 on the B cells surface, protecting them from Fas-mediated apoptosis and in this study, influence humoral response. The aim of the study was to assess soluble CD40 and CD154 in Graves' disease (GD) and Hashimoto's thyroiditis (HT) in relation to Fas and FasL and to the markers of humoral response: aTPO, aTG and aTSHR. The study was carried out in 5 groups of subjects: 1/14 patients with GD in euthyreosis on methimazol (euGD), 2/20 patients with hyperthyroid GD (hrGD), 3/15 patients with HT in euthyreosis on levothyroxine (euHT), 4/16 patients with hypothyroid Ht (hoHT), 5/12 healthy volunteers, age and sex-matched to groups 1–4. The serum levels of CD40, CD154, Fas and FasL, aTPO and aTG were determined by ELISA and aTSHR was determined by the RIA method. CD40 serum concentration was significantly higher in hoHT individuals: 55.8 (24.0–83.2) pg/ml (p<0.01) and euHT patients: 51.2 (20.0–80.1) (p<0.05) as compared to the controls. Also sCD40L values were significantly increased in euHT individuals: 5.1 (1.0–11.8) (p<0.05) and hoHT patients: 3.9 (0.7–11.2) ng/ml (p<0.05) as compared to the controls. There was a positive correlation between sCD40 and sCD154 in the patients studied (r = 0.36, p<0.001). In HT patients we found positive correlations between sCD40 and aTPO (r = 0.45, p<0.001) and sFas (r = 0.36, p<0.05) as well as a negative correlation between sCD40 and FasL (r = –0.24, p<0.05). In GD patients there was a positive correlation between sCD40 and aTSHR (r = 0.28, p<0.05). In summary, our results suggest that CD40/CD154 interaction plays an important role in the regulation of autoimmune humoral response, both in Hashimoto's thyroiditis and Graves' disease. Fas-mediated apoptosis seems to be involved in this process especially in Hashimoto thyroiditis. Soluble CD40 may serve as a marker of the active stage of autoimmune thyroid disease.

Introduction: Hashimoto's Thyroiditis (HT) and Graves' Disease (GD) reflect two extremes in the spectrum of autoimmune thyroid diseases. In HT, there are anti-thyroid peroxidase antibodies thought, but not proven, to be destructive to thyroid follicles as well as thyroid stimulating hormone (TSH) stimulation-blocking antibodies which block the action of TSH and cause damage and atrophy of the thyroid gland leading to hypothyroidism. On the other hand, GD is characterized by primary hyperthyroidism due to stimulating autoantibodies against TSH receptors. While some of the patients with GD can develop spontaneous hypothyroidism due to HT, development of GD with hyperthyroidism following HT is unusual (1). We report a case of development of GD in a case of long standing HT. Case Report: A 55-year-old woman with a 20-year history of HT on stable doses of levothyroxine was found to be hyperthyroid on routine follow up. She presented with clinical signs of hyperthyroidism including weight loss of 40 pounds over 1 year and periorbital edema. The dose of levothyroxine was tapered and ultimately stopped over six months. Despite discontinuation of thyroid hormone, she continued to remain hyperthyroid. Labs were significant for elevated free T4 2.28 (0.82-1.77 ng/dL), elevated free T3 9.2, (2.0-4.4 pg/mL) and suppressed TSH, 0.006 (0.0-0.55 IU/L). Further testing revealed elevated thyroid antibodies including TSI of 10.4 (0.0-0.55 IU/L), Thyroglobulin Ab of 50.6 (0.0-0.9 IU/mL), TSH receptor Ab of 6.1 (>1.5 U/L positive) and TPO Ab of > 600 (0.0-34 IU/mL). Thyroid ultrasound disclosed an enlarged, heterogenous and hyperemic thyroid parenchyma. The 24-hour radioactive iodine uptake was elevated at 52% (8-35%) indicative of hyperthyroidism, most likely from GD. She was treated with methimazole and atenolol, followed by total thyroidectomy. Surgical pathologyrevealed diffuse chronic lymphocytic thyroiditis with lymphoid aggregates, fibrosis, adenomatous nodules with oncocytic changes and reactive nuclear atypia. Conclusion: Although development of GD following HT is rare, it can occur suggesting that HT does not always result in irreversible destruction of the thyroid gland. Patients with thyroid autoimmune disease have both thyroid hormone receptor stimulating and thyroid hormone receptor blocking antibodies and clinical manifestations depend upon the level of these antibodies and the state of the thyroid gland. Because patients with HT can transform to GD at any point due to a change of blocking to stimulating antibodies, clinicians should be aware of the potential for this transformation.

Autoimmune thyroid diseases (AITDs) are chronic organ-specific autoimmune disorders, predominantly including Graves' disease (GD), and Hashimoto's thyroiditis (HT). This study aimed to investigate whether single-nucleotide polymorphisms (SNPs) in MAGI2 and MAGI3 gene contributed to the etiology of AITDs. We conducted a case-control study including 1001 patients with AITDs (625 GD, 376 HT) and 846 healthy controls. Subgroup analyses in GD and HT were also performed. The genotypes of rs2160322 in MAGI2 showed a borderline association with AITDs (P = 0.048), and they had a strong correlation with GD (P = 0.012). The frequency of the minor allele G of rs2160322 was significantly higher in the GD patients than in the controls (P = 0.027; OR 1.91; 95% CI 1.020-1.391), especially for GD females (P = 0.008; OR 1.304; 95% CI 1.072-1.587), and those who had positive family history (P = 0.011; OR 1.412; 95% CI 1.083-1.843). For genetic model analysis, the recessive model and homozygous model of rs2160322 showed significant associations with AITDs (P = 0.009; P = 0.019) and GD (P = 0.004; P = 0.005). Nevertheless, our study could not identify any relationship between these SNPs and HT. Due to the low mutation rate of rs1343126 in MAGI3, we were unable to obtain a credible conclusion on its association with AITDs. Our study identified that MAGI2 rs2160322 was strongly associated with GD susceptibility. The potential dysfunction of tight junction proteins and aberrant epithelial barrier caused by abnormal MAGI2 expression may be a novel mechanism of GD.

Osteopontin (OPN) is recognized as a potent immunoregulator of autoimmune disease. In the study, we tried to explore the association of serum OPN levels with autoimmune thyroid disease, including Graves' disease (GD) and Hashimoto's thyroiditis (HT), in an ethnic Chinese population. We enrolled 131 patients with GD, 33 patients with HT and 123 healthy controls. Serum OPN, B cell-activating factor (BAFF) and interferon (IFN)-α levels were quantified. Graves' disease patients with high thyroid function at the time of sample collection were defined as having active GD, while the other patients were defined as having inactive GD. Serum OPN levels were higher in active GD than in inactive GD and the control groups (P=0.001 and P=0.018, respectively). In GD, significant associations of OPN levels with thyroid-stimulating hormone receptor antibody (TSHRAb) levels were observed in women (r=-0.344, P=0.002, and r=0.440, P=0.004, respectively) but not in men. Osteopontin levels were associated with BAFF levels only in women with GD or HT (r=0.506, P<0.001 and r=0.430, P=0.025, respectively), but not in men with GD or HT. Serum OPN levels were upregulated in active GD, and serum OPN levels were associated with thyroid function and TSHRAb levels in GD. Additionally, OPN levels were correlated with BAFF levels in GD and HT. The associations of OPN levels with clinical phenotypes of GD and BAFF levels showed a dimorphic pattern.

To characterize and compare autoimmune comorbidity patterns and temporal dynamics in patients with Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) within an autoimmune polyglandular syndrome (APS) framework. We retrospectively analysed patients with an autoimmune thyroid disease (AITD) and at least one additional autoimmune disease fulfilling APS criteria, followed at a tertiary APS referral centre between 2000 and 2025. The prevalence of associated autoimmune diseases was compared between HT and GD. Time to development of subsequent autoimmune diseases was assessed using Kaplan–Meier analysis and Cox proportional hazards models, with adjustment for sex and age. A total of 1,057 patients (76% women; mean age 53.7 ± 16.4 years) were included, of whom 964 had HT and 93 GD. Type 1 diabetes was the most frequent autoimmune comorbidity, but was significantly less prevalent in GD than in HT (OR 0.48, 95% CI 0.31–0.75; FDR-adjusted p = 0.008). GD was selectively enriched for systemic sclerosis (OR 51.54, 95% CI 10.96–242.41; FDR-adjusted p < 0.001) and vitiligo (OR 3.46, 95% CI 1.82–6.55; FDR-adjusted p < 0.001). In patients in whom AITD was the first autoimmune manifestation (n = 333), GD was associated with a longer latency to additional autoimmune diseases and a lower hazard of subsequent autoimmunity compared with HT (HR 0.52, 95% CI 0.37–0.73; p < 0.001). Within APS-related AITD, HT and GD anchor distinct autoimmune phenotypes with different clustering patterns and temporal trajectories. These differences have relevant clinical implications for risk stratification and personalized surveillance of patients with autoimmune thyroid disease.

In the present study we have measured in parallel thyrotrophin binding inhibiting immunoglobulins (TBII) and thyroid adenylate cylase stimulating immunoglobulins (TACSI) in patients with Graves' disease (GD) both before and during long-term antithyroid treatment. A statistical model based on the calculation of the differences (TACSI-TBII) is presented, comparing the changes in this parameter to the analytical variation. The correlation between TBII and TACSI in 52 patients with GD before treatment was: r = 0.58, P less than 0.0001. During long-term antithyroid treatment of GD the 2 activities changed in parallel in 39 of 45 patients followed. In a few patients discrepancies were observed, and 1 patient, initially TACSI positive, developed adenylate cyclase inhibiting IgG during treatment but without detectable TBII. In conclusion, 1) TBII and TACSI are significantly correlated in patients with GD both before and during long-term antithyroid treatment, 2) in some patients discrepancies between TBII and TACSI suggest, that these IgGs are heterogeneous with varying capacity for stimulation of the adenylate cyclase and receptor binding, and 3) adenylate cyclase inhibitory IgG without TBII activity was demonstrated in GD.

Interim Absolute Lymphocyte Count (ALC) As a Predictor of Survival in Hodgkin Lymphoma

Polymorphic variants of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and forkhead box protein P3 (FOXP3) genes are implicated in dysregulated immune homeostasis and autoimmune disorders. We analyzed the association between CTLA-4 rs231775 and FOXP3 rs3761548, rs3761549 polymorphisms and predisposition to autoimmune thyroid disease (AITD), inclusive of Hashimoto's thyroiditis (HT) and Graves' disease (GD) in South-Indian population. A total of 355 AITD subjects (comprising 275 HT and 80 GD) and 285 randomly selected age- and sex-matched control subjects were genotyped for the aforementioned polymorphisms by PCR-RFLP method. The rs231775 "G" allele was preponderant in HT and GD subjects when compared with controls and exerted a dominant influence on the susceptibility to HT (p = 0.009) and GD (p = 0.02), respectively. There was no allelic association of rs3761548 and rs3761549 polymorphisms with AITD susceptibility, albeit a significant difference in genotype distribution with respect to rs3761549. Haplotype analysis revealed an increased frequency of rs3761548 "C"-rs3761549 "T" in HT and GD subjects, thereby associating it with disease predisposition (p = 0.03). Epistatic interaction analysis by multifactor dimensionality reduction approach revealed redundancy between CTLA-4 and FOXP3 genes in influencing the susceptibility to AITD. The genetic variation in CTLA-4 gene with reference to rs231775 polymorphism contributes to an increased predisposition to HT and GD. Also, in conjunction with FOXP3 gene variants it seems to influence the susceptibility to HT and GD respectively. The significance of these findings in combination with antithyroid antibody screening could plausibly contribute towards meticulous case-finding for effective treatment of HT and GD.