Germline polymorphisms of the NOD2 pathway may predict the effectiveness of radioiodine in differentiated thyroid cancer treatment.

PURPOSE: Differentiated thyroid cancer (DTC) presents a complex clinical challenge, especially in patients with distant metastases and resistance to standard treatments. This study aimed to investigate the influence of specific genes and their germline single nucleotide polymorphisms (SNPs) linked to both inflammatory processes and other neoplasms on the clinical and pathological characteristics of DTC, particularly their potential impact on radioiodine (RAI) treatment efficacy. METHODS: This retrospective analysis involved a cohort of 646 patients diagnosed with DTC after thyroidectomy. Study covering 1998-2014, updated in 2023, included 567 women and 79 men (median age: 49; range: 7-83). SNP selection targeted functional significance, while mutational status was assessed by pyrosequencing for comprehensive characterization. Patient genetic profiles were assessed for associations with disease characteristics, RAI response, and cancer pathology. RESULTS: Significant correlations emerged between certain SNPs and DTC features. Notably, the NOD2 c.802 T > C variant (rs2066842) was identified as a marker distinguishing between papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Moreover, the c.802 T allele was associated with an enhanced response to RAI treatment, indicating a more substantial decrease in posttreatment stimulated thyroglobulin (sTg) concentrations. The NFKB1A allele c.126A (rs696) exhibited connections with lower FTC stages and a reduced probability of multifocality. CONCLUSION: This study explored the molecular mechanisms of particular SNPs, highlighting the role of NOD2 in innate immunity and the stress response, and its potential impact on RAI efficacy. This research underscores the clinical promise of SNP analysis and contributes to personalized treatment strategies for DTC, emphasizing the relevance of genetic factors in cancer progression and treatment outcomes.

Decreased staging of differentiated thyroid cancer in patients with chronic lymphocytic thyroiditis.

PURPOSE: The biological association between chronic lymphocytic thyroiditis (CLT) and differentiated thyroid cancer (DTC) has not been elucidated yet. The aim of the study was to assess whether the presence of CLT exerts any influence on clinical or histological presentation of DTC. METHODS: Nine hundred and seven consecutive patients with DTC treated in the years 1998-2016 were divided into two groups according to the presence or absence of concomitant CLT. The statistical differences were analysed. RESULTS: Out of 907 patients included in the study, 331 were diagnosed with DTC and CLT (studied group), while 576 patients with DTC but without CLT constituted a control group. The distribution of papillary and follicular thyroid cancer did not differ. In CLT group, the prevalence of pT1 was greater than for pT2-pT4 DTC (P = 0.0003; OR = 1.69, 95% CI 1.27-2.24) compared to controls (68.3 vs. 56.1%, respectively). The presence of multifocal lesions was similar. The thyroid capsule infiltration without extrathyroidal invasion (P < 0.0001; OR = 0.21, 95% CI 0.14-0.31) was more frequent in the studied group, unlike extracapsular invasion, which was significantly more often present in patients with DTC but without CLT (P = 0.004; OR = 1.66; 95% CI 1.17-2.34) as well as nodal involvement (P = 0.048; OR = 0.65, 95% CI 0.42-0.99). CONCLUSIONS: The collected data indicate a protective role of CLT in preventing the spread of the DTC. The presence of CLT might limit tumour growth to the primary site.

Two coexisting heterozygous frameshift mutations in PROP1 are responsible for a different phenotype of combined pituitary hormone deficiency.

The role of genetic background in childhood-onset combined pituitary hormone deficiency (CPHD) has been extensively studied. The major contributors are the PROP1, POU1F1, LHX3, LHX4 and HESX1 genes coding transcription factors implicated in pituitary organogenesis. The clinical consequences of mutations encompass impaired synthesis of a growth hormone (GH) and one or more concurrent pituitary hormones (i.e. LH, FSH, TSH, PRL). Manifestation of the disorder may vary due to various mutation impacts on the final gene products or an influence of environmental factors during pituitary organogenesis. We describe the clinical and molecular characteristics of two brothers aged 47 and 39 years presenting an uncommon manifestation of congenital hypopituitarism. Sequencing of the PROP1, POU1F1, LHX3, LHX4 and HESX1 genes was performed to confirm the genetic origin of the disorder. A compound heterozygosity in the PROP1 gene has been identified for both probands. The first change represents a mutational hot spot (c.150delA, p.R53fsX164), whereas the second is a novel alteration (p.R112X) that leads to protein disruption. Based on precise genetic diagnosis, an in silico prediction of a p.R112X mutation on protein architecture was performed. The resulting clinical phenotype was surprisingly distinct compared to most patients with genetic alterations in PROP1 reported in the current literature. This may be caused by a residual activity of a newly identified p.R112X protein that preserves over 70 % of the homeodomain structure. This examination may confirm a key role of a DNA-binding homeodomain in maintaining PROP1 functionality and suggests a conceivable explanation of an unusual phenotype.

Low free triiodothyronine levels are related to poor prognosis in acute ischemic stroke.

BACKGROUND: Neuroendocrine changes are important processes which accompany critical illness, however, the number of clinical studies concentrating on the role of thyroid gland hormones in stroke pathogenesis is relatively small. The aim of this prospective study was to investigate the relation between free triiodothyronine (fT3) levels and the prognosis of patients with stroke. METHODS: The prospective study included 387 patients with acute (<24 h of symptoms onset) ischemic stroke consecutively admitted to Stroke Units. The subjects with known conditions that could interfere with thyroid gland metabolism were excluded. We analyzed: the routine blood tests, fT3, free thyroxine (fT4), thyroid-stimulating hormone (TSH) levels, unenhanced CT scans, initial clinical status (NIH Stroke Scale, NIHSS), 30- and 360- days outcome (modified Rankin Scale-mRS) and calculated the survival rate. RESULTS: A higher NIHSS score was in the 1 (st) fT3 levels tertile, whereas a lower in the 3 (rd) fT3 levels tertile (p=0.006). The 30- and 360-days mRS scores showed that patients in the lowest fT3 tertile had more severe neurological impairment than those in the highest tertile (p=0.001 and p=0.03, respectively). A 1-year mortality of the patients with the first tertile fT3 levels was significantly higher than that of the patients with the third tertile hormone levels (p=0.008). Additionally, subjects with fT3 level in the lowest tertile demonstrated higher WBC counts and the ventricular system on Computed Tomography of head performed on admission to hospital was statistically more frequent compressed than that in the patients with fT3 level in the highest tertile (p=0.02 and p=0.03, respectively). CONCLUSION: In acute stroke patients lower free T3 levels are an important factor related to unfavorable outcome, i. e., severe disability and death.

The influence of growth hormone (GH) therapy on cardiac performance in patients with childhood onset GH deficiency.

OBJECTIVE: There is accumulating evidence that growth hormone (GH) plays an important role in the maintenance of normal cardiac growth and function. Abnormalities in left ventricular diastolic function and impairment of systolic function have also been reported in patients with GHD. In this study, we investigated the effects of 12 months GH replacement therapy on cardiac functional indices measured by echocardiography, the ECG stress test and SPECT imaging. DESIGN: Sixteen patients with childhood onset GHD (age 42.3+/-13.1 years, 10 males) were investigated before, and after, 12 months of GH treatment at a dosage of 0.02 IU/kg/day (7 microg/kg/day). The GH administration resulted in serum IGF-I levels within the normal range in all the patients. The following investigations were performed initially and after 12 months: electrocardiography, systolic and diastolic blood pressure, heart rate measurement, a complete Doppler-echocardiographic examination, treadmill exercise test and Technetium-99m sestamibi single-photon emission computer tomography (SPECT) imaging at rest and after exercise. RESULTS: Echocardiography showed improvement in left ventricular systolic function after GH treatment. End-systolic volume fell from 29.9+/-12.4 to 24.4+/-6.9 ml (p<0.05) and the ejection fraction increased from 56.2+/-7.2% to 63.2+/-6,1% (p<0.01). Left ventricular diameter and wall thickness did not change after GH treatment, although systolic increase in interventricular septum thickness (IVS%) and systolic increase in posterior wall thickness (PWT%) increased significantly (IVS% 52.2+/-31.9% vs. 67.3+/-30.4% and PWT% 48.7+/-20.2% vs. 58.0+/-17.7%, p<0.01, p<0.01, respectively). Contractile function, measured at midwall level, improved as left ventricular midwall fractional shortening (MWS) increased (16.11+/-6.55 vs. 23.30+/-5.89 %, p<0.01) and stress-corrected MWS increased between the examinations performed before and after 12 months of GH treatment (90.97+/-36.66 vs. 133.10+/-32.84 %, p<0.01). Diastolic function did not change, as assessed by early diastolic flow (E), diastolic flow secondary to atrial contraction (A), or the E/A ratio. The LV-mass index did not change significantly after GH treatment (78.4+/-22.1 vs. 81.9+/-21.1g/m(2)). After 12 months of GH treatment the myocardial performance index (MPI) decreased significantly from 0.483+/-0.146 at baseline to 0.410+/-0.086 at the end of the study (p<0.05). There was a trend towards an increase in exercise duration and capacity after GH treatment but the differences did not reach levels of statistical significance. SPECT imaging basally and after 12 months showed normal myocardial perfusion at rest and after exercise in all the patients. In conclusion, GH replacement therapy in adults with GHD demonstrated the beneficial effects on cardiac functions.

Iodine prophylaxis intensification. Influence on radioiodine uptake and activity of 131I used in the treatment of hyperthyroid patients with Graves' disease.

UNLABELLED: Poland, a country with mild/moderate iodine deficiency introduced an obligatory iodination salt system in 1996. AIM: To compare the results of radioiodine (131I) uptake after 5 h and 24 h with the activity of radioiodine used in the treatment of hyperthyroid patients with Graves' disease in the years 1995 and 2003. PATIENTS, METHODS: The marker of iodine content in the diet was urinary iodine excretion. 1000 randomly chosen patients (average age: 46 +/- 12 years) were included in the study. Every patient had routinely estimated radioiodine uptake after 5 h and 24 h and the activity of 131I was calculated using scintigraphy and ultrasonography of the thyroid gland. Urinary iodine excretion in samples from year 1995 and 2003 was also determined in some patients and healthy volunteers. RESULTS: The iodine load in the diet increased from 66 microg (average) in the year 1995 to 115 microg in the year 2003. Thyroid radioiodine uptake was 40% lower in comparison with the results from 1995. The average activity of 131I given in the year 2003 (10 mCi) was about 40% higher than in the year 1995 (7 mCi). CONCLUSION: There was significant negative correlation between higher iodine content in the diet and lower values of radioiodine uptake, which led to the application of the higher activity of 131I during treatment.

Recombinant human thyroid peroxidase produced in insect cells has similar properties to native human thyroid peroxidase.

Purified native human thyroid peroxidase (nTPO) isolated from thyroid tissue and recombinant (r)TPO produced in High Five insect cells have been compared. nTPO and rTPO were purified to about 95% homogeneity and showed similar UV and visual spectra and similar 412 nm per 280 nm absorbance ratios (0.4 for nTPO and 0.4 for rTPO). The nTPO and rTPO guaiacol oxidation enzyme activities were about 1,000 guaiacol units per milligram of protein. TPO autoantibody binding characteristics of nTPO and rTPO were analyzed in an assay based on 125I-labeled nTPO and precipitation with protein A. In the assay, the effect of unlabeled nTPO or rTPO on TPO autoantibody binding from 25 patients sera was studied. Unlabeled nTPO or rTPO (from 0 to 160 ng/mL) inhibited the binding of TPO autoantibodies in a dose-dependent manner in the case of each serum studied (from 100% in the absence of unlabeled TPO to 5%-10% in the presence of 160 ng/mL of TPO). The inhibition profile for each serum was essentially identical in the case of both TPO preparations. The effect of TPO autoantibodies on enzyme activity of rTPO was analyzed after incubation of rTPO with TPO autoantibody-positive serum immunoglobulin G (IgG) (n = 12), TPO monoclonal antibodies reactive with two different epitopes on the TPO, IgG (n = 3) from glutamic acid decarboxylase autoantibody positive patient sera, and IgG (n = 3) from healthy blood donors. Effective complexing of TPO by TPO autoantibodies was tested by precipitating the complexes with solid phase protein A and measuring the TPO enzyme activity in the resulting supernatants. These studies showed that the TPO enzyme activity was not affected by incubation with TPO autoantibody-positive IgG or monoclonal antibodies despite effective complexing of the autoantibodies with TPO. Overall, our studies demonstrate that nTPO and rTPO produced in insect cells are very similar in terms of enzyme activity, UV and visible spectra, and reactivity with autoantibodies. Furthermore, in our study, TPO autoantibodies did not appear to inhibit TPO enzyme activity.

Analysis of autoantibody epitopes on human thyroid peroxidase.

A number of studies have indicated that the major autoantibody epitopes on human thyroid peroxidase (TPO) are conformational and are formed by two overlapping immunodominant regions on the TPO molecule. In order to investigate further autoantibody reactivity with TPO, we have studied the TPO binding characteristics of sera from patients with autoimmune thyroid disease (n = 20), autoimmune adrenal disease (Addison's disease; n = 8) and apparently healthy blood donors (n = 9) using recombinant TPO expressed with a series of truncations and internal deletions. This material was obtained using an in vitro transcription/translation system in the presence of 35S-methionine and the reactivity of TPO autoantibodies tested in an immunoprecipitation assay. In addition, we have studied the effects of denaturing purified recombinant TPO by reduction and/or sodium dodecyl sulphate on its reactivity with TPO autoantibodies by Western blotting analysis. These studies show that TPO autoantibodies can recognise TPO in Western blotting analysis when large amounts of purified TPO are run on the gels and the blotted proteins renatured prior to addition of antibody. Under these conditions TPO autoantibodies in all 20 Graves' or Hashimoto's sera tested reacted strongly with blots of non-reduced TPO but reduction of TPO had a marked effect on the ability of autoantibodies to recognise it in Western blotting analysis. Analysis of TPO autoantibody binding to 35S-labelled TPO proteins containing N-terminal, central or C-terminal deletions indicated that all modifications studied caused a statistically significant lowering of binding. In the case of some modifications, there were differences in the reactivity of TPO autoantibodies in sera from patients with Addison's disease compared to TPO autoantibodies in autoimmune thyroid disease and/or healthy blood donor sera. Overall, our results of analysis of T PO autoantibody binding in Western blotting and with modified TPO proteins in immunoprecipitation assays suggest that the main autoantibody binding sites on the TPO molecule involve extensive amino acid sequences. Our studies also suggest that TPO autoantibodies from patients with autoimmune thyroid disease, Addison's disease and apparently healthy blood donors show some differences in epitope recognition on TPO and this approach may allow differentiation between disease related and unrelated TPO autoantibodies.

Recombinant human thyroid peroxidase expressed in insect cells is soluble at high concentrations and forms diffracting crystals.

Human thyroid peroxidase (TPO), the key enzyme in thyroid hormone synthesis, can be produced in active form in the High Five insect cell line and when purified from the cell culture medium is soluble at concentrations of up to 18 mg/ml. This contrasts to a recent report in which human TPO produced in insect cells was found to be insoluble at high concentrations. Our concentrated TPO grows trigonal trapezohedral crystals of up to 0.5 mm in length in a vapour diffusion apparatus using polyethelene glycol as a precipitant. The crystals diffract X-rays to a 6 A resolution and the diffraction data from the crystals have been analysed giving unit cell dimensions. A potential molecular replacement solution has been identified using myeloperoxidase (MPO) as a phasing model.

High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line.

Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotinlabelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r = 0.96, P < 0.001, n = 50 and r = 0.99, P < 0.001, n = 80 respectively). In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor delta-aminolevulinic acid was included in the culture medium. Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO-TPO autoantibody complexes.