Prevalence and impact on quality of life of peripheral neuropathy with or without neuropathic pain in type 1 and type 2 diabetic patients attending hospital outpatients clinics.

AIMS: Diabetic polyneuropathy (DPN) without or with neuropathic pain (DPN-P) is one of the most frequent complications of diabetes. To better delineate their respective prevalences, we conducted a cross-sectional study that included 1111 patients (767 type 2 and 344 type 1 diabetic patients) followed up in diabetic outpatients clinics. The association of DPN and DPN-P with other diabetic complications, the impact on quality of life (QoL) and pain management were also investigated. METHODS: Two validated tools (Neuropen) and the DN4 questionnaire) were used to diagnose the two conditions. Pain intensity was measured using a visual analogue scale, and participants completed the 12-item Short-Form Health Survey to evaluate the physical and mental components of QoL. Univariate and multivariate models were used for the statistical analyses. RESULTS: The prevalence of DPN was 43% (95% CI 40.1-45.9), and was higher in type 2 (50.8%) than in type 1 (25.6%) diabetic patients. The prevalence of DPN-P was 14% (95% CI 12.1-16.2) which, again, was higher in type 2 (17.9%) than in type 1 (5.8%) patients. These prevalences both increased with age and diabetes duration. Nephropathy, obesity, low HDL cholesterol and high triglyceride levels were independently associated with DPN and/or DPN-P. Physical and mental components of QoL were significantly altered by DPN-P, but not DPN. Only half of the DPN-P patients were using analgesic treatment, while 28% were using anticonvulsants or antidepressants. CONCLUSION: DPN and DPN-P are frequent complications of diabetes, especially in type 2, and can be identified with inexpensive and easy-to-use screening tools. Despite its profound impact on QoL, DPN-P remains undertreated.

Involvement of mTOR and Regulation by AMPK in Early Iodine Deficiency-Induced Thyroid Microvascular Activation.

Iodine deficiency (ID) induces TSH-independent microvascular activation in the thyroid via the reactive oxygen species/nitric oxide-hypoxia-inducible factor-1α/vascular endothelial growth factor (VEGF) pathway. We hypothesized the additional involvement of mammalian target of rapamycin (mTOR) as a positive regulator of this pathway and AMP-activated protein kinase (AMPK) as a negative feedback regulator to explain the transient nature of ID-induced microvascular changes under nonmalignant conditions. mTOR and AMPK involvement was investigated using an in vitro model (human thyrocytes in primary cultures) and 2 murine models of goitrogenesis (normal NMRI and RET-PTC mice [a papillary thyroid cancer model]). In NMRI mice, ID had no effect on the phosphorylation of ribosomal S6 kinase (p70S6K), a downstream target of mTOR. However, rapamycin inhibited ID-induced thyroid blood flow and VEGF protein expression. In the RET-PTC model, ID strongly increased the phosphorylation of p70S6K, whereas rapamycin completely inhibited the ID-induced increase in p70S6K phosphorylation, thyroid blood flow, and VEGF-A expression. In vitro, although ID increased p70S6K phosphorylation, the ID-stimulated hypoxia-inducible factor/VEGF pathway was inhibited by rapamycin. Activation of AMPK by metformin inhibited ID effects both in vivo and in vitro. In AMPK-α1 knockout mice, the ID-induced increase in thyroid blood flow and VEGF-A protein expression persisted throughout the treatment, whereas both parameters returned to control values in wild-type mice after 4 days of ID. In conclusion, mTOR is required for early ID-induced thyroid microvascular activation. AMPK negatively regulates this pathway, which may account for the transient nature of ID-induced TSH-independent vascular effects under benign conditions.

Peroxiredoxin 5 expression in the human thyroid gland.

Peroxiredoxin 5 (PRDX5) is a newly discovered thioredoxin peroxidase able to reduce peroxides that is implicated in antioxidant protective mechanisms. We report here its expression in the human thyroid gland. Twenty-seven human thyroid specimens were examined by immunohistochemistry. They included six normal thyroid tissues, five multinodular goiters, nine hot nodules, two Hürthle cell adenomas, and five thyroids from patients with Graves' disease. In the control tissue, PRDX5 expression was heterogeneous, being stronger in cubical functionally active follicular cells than in flat quiescent thyrocytes. It was diffuse in the cytoplasm, occasionally localized in inclusions that most likely corresponded to mitochondria. This feature was particularly marked in the Hürthle cell adenoma case. In multinodular goiters, hot nodules, and Graves' thyroids, the cytosolic labeling was enhanced compared to the control tissue and a signal was also detected in few nuclei. To determine whether the level of expression was different between multinodular goiters and hyperthyroid Graves' thyroids, PRDX5 immunoblotting was performed in these two respective tissues. We observed that PRDX5 expression was higher in the thyroid gland of patients with Graves' disease compared to multinodular goiters. In conclusion, our data show that PRDX5 is expressed in the thyroid gland where it could act as antioxidant. The level of expression is directly correlated with the functional status of epithelial cells, being higher in multinodular goiters, and even more pronounced in hyperthyroid tissues, such as Graves' disease.

Involvement of nitric oxide in iodine deficiency-induced microvascular remodeling in the thyroid gland: role of nitric oxide synthase 3 and ryanodine receptors.

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10µM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.

Estradiol sensitization of rat pituitary cells to gonadotropin-releasing hormone: involvement of protein kinase C- and calcium-dependent signaling pathways.

During the female reproductive cycle, estrogen enhances the actions of GnRH on the gonadotrope cell. Recently, we reported that in vivo exposure to estradiol causes a marked enhancement GnRH-induced transcription of the alpha gene promoter in primary cultures of pituitary cells. In the present study, we analyzed the GnRH signaling pathways that mediate the sensitizing effects of estradiol on the alpha promoter. Primary cultures of male and female rat pituitary cells were transfected with the -420alphaLUC reporter gene and treated with agonists or antagonists for 24 h. As found previously, the degree of GnRH (1 nM) stimulation was 15-fold greater in females (157-fold) than in males (9-fold). When cells were treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA); 10 nM], the level of stimulation was half that observed with GnRH, but the sexual dimorphism was preserved. When protein kinase C (PKC) activity was either depleted by long term treatment with phorbol esters (1 microM PMA for 24 h) or inhibited with staurosporine, the stimulatory effect of GnRH was minimally affected in males, but was markedly reduced in females. The reduced threshold of GnRH responsiveness after inhibition of PKC suggests that the actions of estrogen involve this pathway. Coexpression of c-jun and c-fos, which are increased by GnRH and PMA, suppressed basal alphaLUC activity, but did not alter the sensitivity to GnRH in a sexually dimorphic manner. Dominant negative mutants of the mitogen-activated protein kinase pathway, which is also activated by GnRH and PMA, failed to reveal sexually dimorphic alterations in GnRH responsiveness. These findings indicate that the mitogen-activated protein kinase pathway and activating protein-1 are probably not involved in estrogen sensitization of transcriptional responses to GnRH. The involvement of Ca2+-dependent pathways was analyzed either by chelating extracellular Ca2+ with EGTA (5 mM) or by using a Ca2+ channel blocker, methoxyverapamil (D600; 1 microM). Depletion of extracellular Ca2+ markedly reduced GnRH action in females, but not in males. Treatment with the Ca2+ channel blocker D600 did not alter GnRH-induced stimulation of -420alphaLUC in males, but in females, GnRH stimulation was significantly impaired (208- vs. 23-fold). Estrogen replacement in ovariectomized females reconstituted GnRH sensitivity and the inhibitory effect of methoxyverapamil (84- vs. 13-fold). We conclude that both PKC- and Ca2+-dependent signaling pathways are involved in estradiol-induced sensitization of female pituitary cells to GnRH.

Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C.

GnRH regulates gonadotropin biosynthesis and secretion. Multiple intracellular signaling pathways are activated by GnRH, including phosphoinositol turnover, release of intracellular calcium and influx of extracellular calcium, and activation of protein kinase C (PKC), among others. In this study, we investigated whether GnRH stimulates mitogen-activated protein kinase (MAPKs), and whether this pathway plays a role in the transcriptional activation of the gonadotropin alpha-gene. In alpha T3-1 gonadotrope cells, treatment with GnRH caused 4- to 5-fold induction of MAPK activity. Stimulation of MAPK activity was detected within 5 min of GnRH treatment and persisted for 60 min. MAPK activation by GnRH was also seen in primary cultures of rat pituitary cells. Treatment with phorbol 12-myristate 13-acetate (TPA) caused 4- to 5-fold induction of MAPK activity in alpha T3-1 cells. Pretreatment with TPA, however, decreased both GnRH- and TPA-induced MAPK activation, suggesting that PKC is involved in GnRH-mediated activation of MAPK. Western blot analyses of PKC isoforms alpha and epsilon confirmed that they were depleted by chronic treatment with TPA, whereas MAPK protein levels were unaffected. Because transcriptional stimulation of the glycoprotein hormone alpha-gene by GnRH is also inhibited by PKC depletion, additional experiments were performed to explore a potential role for MAPK in alpha-gene expression. Cotransfection of a dominant negative inhibitors of MAPK isoforms (ERK1 and ERK2) suppressed basal expression of the alpha-promoter by 60%, but had less effect on the extent of GnRH stimulation in alpha T3-1 cells. These experiments indicate that GnRH stimulates MAPK activity, probably through a pathway involving PKC. Although PKC depletion inhibits both MAPK- and GnRH-stimulated alpha-gene transcription, pathways other than MAPK are also likely to be involved in mediating the transcriptional effects of GnRH.

Sexually dimorphic transcriptional responses to gonadotropin-releasing hormone require chronic in vivo exposure to estradiol.

GnRH regulates secretion of the gonadotropins, LH and FSH, in a sexually dimorphic manner. In the present study, we examined GnRH regulation of the gonadotropin alpha-subunit promoter to assess whether sex-dependent hormonal effects are manifest at the transcriptional level. Primary cultures of male or female rat pituitary cells were transfected with a reporter gene containing the alpha-promoter linked to luciferase (-420 alpha-LUC) and then subjected to treatment with GnRH for 24 h. Basal alpha-LUC expression was 4.2-fold greater in pituitary cells from males than in those from females. alpha-LUC activity was stimulated 5.3-fold by GnRH in males, whereas GnRH induced a 148-fold increase in alpha-promoter activity in females. The GnRH responsiveness of the transfected alpha-promoter did not vary in pituitary cells isolated at different stages of the female reproductive cycle, suggesting that acute changes in the hormonal milieu are not sufficient to alter transcriptional responses to GnRH. In males, orchidectomy minimally influenced alpha-LUC activity, indicating that testosterone does not exert a suppressive effect on GnRH responsiveness. In ovariectomized females, basal expression of alpha-LUC increased 3.7-fold, and GnRH stimulation was reduced from 165- to 11-fold, suggesting that an ovarian factor suppresses basal activity and enhances GnRH stimulation. Treatment of ovariectomized females with estrogen suppressed basal activity and restored GnRH stimulation of alpha-LUC, but the estrogen effects required long term treatment (10 days). Addition of progesterone to estrogen or treatment with the progesterone antagonist, RU486, had little effect on GnRH responsiveness. We conclude that estrogen exerts dual effects to suppress basal expression and to dramatically enhance GnRH responsiveness of the alpha-promoter. This model reveals potent actions of estrogen at the level of transcription and should provide new insights into the mechanisms that control estrogen priming of gonadotrope cells.

Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation.

The thyroid gland is a highly vascular tissue, and its blood flow changes dramatically in various pathological conditions. Although the mechanisms regulating these changes in vascularity and blood flow are not well understood, candidate mediators include endothelin-1 (ET-1) and nitric oxide (NO). In the present study, we used a reverse transcriptase-polymerase chain reaction assay to determine which components of these vasoregulatory pathways are present in the thyroid and to analyze changes in gene expression in an experimental model of goiter formation and involution. Expression of messenger RNAs (mRNAs) encoding ET-1, ET receptors (ETA and ETB), ET-converting enzyme, and the three nitric oxide synthase (NOS) isoforms (NOS I, NOS II, and NOS III) was readily detected in the rat thyroid. After goiter formation was induced by thiouracil and a low iodine diet, there was increased expression of the genes encoding ET-related proteins (ET-1, 3.2-fold; ETA, 2.9-fold; ETB, 3.5-fold) as well as two of the three NOS isoforms (NOS I, 2.7-fold; NOS III, 4.9-fold). During iodide-induced involution, the ET-related mRNA levels remained elevated, whereas those of the two NOS isoforms returned to basal values. ET-converting enzyme, NOS II, and thyroglobulin mRNAs were minimally affected in this model, providing evidence for selective regulation of these genes. To assess whether NO plays a role in vascular changes during goiter formation, animals were treated with a NOS inhibitor, N-nitro-L-arginine methyl ester (NAME). NOS activity in the thyroid was inhibited by more than 75% after treatment with NAME. Thyroid hormone and TSH levels were unchanged. Although NAME had little effect on overall thyroid size, vascular expansion during goiter formation was decreased by 36%. We conclude that the thyroid gland expresses a complex network of vasoactive genes whose expression is regulated dynamically during thyroid goiter formation and involution. NO production and probably other locally produced vasoactive substances are involved in changes in thyroid vascularization.

Structural changes in the angiofollicular units between active and hypofunctioning follicles align with differences in the epithelial expression of newly discovered proteins involved in iodine transport and organification.

In animals, as well as in humans, the thyroid gland is made of active follicles, with cuboidal cells and hypofunctioning follicles, with flattened cells. In this study, the functional status of human follicles was dissected out, based on immunohistochemical detection of TSH receptor, Na(+)/I(-) symporter, pendrin, thyroperoxidase (TPO), thyroid oxidases (ThOXs), and T(4)-containing iodinated Tg (Tg-I). To ascertain that angiofollicular units exist in the human, we studied the microvascular bed of each follicle, in correlation with detection of vascular endothelial growth factor (VEGF), of nitric oxide synthase III, and of endothelin in normal and goitrous thyroids. In hypofunctioning follicles, pendrin, TPO, and ThOXs were not detected, and there was no Tg-I in the colloid. At the opposite, in active follicles, pendrin, TPO, and ThOXs were detected in thyrocytes, and Tg-I was present in the colloid. In normal and goitrous thyroids, the capillary networks surrounding active follicles were larger than those surrounding hypofunctioning follicles. Immunoreactivity for nitric oxide synthase III and endothelin was solely detected in active follicles. Only a few follicles in normal thyroids were immunostained for VEGF, regardless of their functional status. In multinodular goiters, VEGF was detected in contact with the extracellular matrix at the basal pole of the cells. In conclusion, the present study endorses the likelihood of angiofollicular units in the human thyroids. Vascular changes are related to the functional status of thyrocytes.

Correlation between the loss of thyroglobulin iodination and the expression of thyroid-specific proteins involved in iodine metabolism in thyroid carcinomas.

Progress in biotechnology has provided useful tools for tracing proteins involved in thyroid hormone synthesis in vivo. Mono- or polyclonal antibodies are now available to detect on histological sections the Na(+)/I(-) symporter (NIS) at the basolateral pole of the cell, the putative iodide channel (pendrin) at the apical plasma membrane, thyroperoxidase (TPO), and members of the NADPH-oxidase family, thyroid oxidase 1 and 2 (ThOXs), part of the H(2)O(2)-generating system. The aim of this study was to correlate thyroglobulin (Tg) iodination with the presence of these proteins. Tg, T(4)-containing Tg, NIS, pendrin, TPO, ThOXs, and TSH receptor (TSHr) were detected by immunohistochemistry on tissue sections of normal thyroids and various benign and malignant thyroid disorders. Tg was present in all cases. T(4)-containing Tg was found in the adenomas, except in Hurthle cell adenomas. It was never detected in carcinomas. NIS was reduced in all types of carcinomas, whereas it was detected in noncancerous tissues. Pendrin was not expressed in carcinomas, except in follicular carcinomas, where weak staining persisted. TPO expression was present in insular, follicular carcinomas and in follicular variants of papillary carcinomas, but in a reduced percentage of cells. It was below the level of detection in papillary carcinomas. The H(2)O(2)-generating system, ThOXs, was found in all carcinomas and was even increased in papillary carcinomas. Its staining was apical in normal thyroids, whereas it was cytoplasmic in carcinomas. The TSHr was expressed in all cases, but the intensity of the staining was decreased in insular carcinomas. In conclusion, our work shows that all types of carcinomas lose the capacity to synthesize T(4)-rich, iodinated Tg. In follicular carcinomas, this might be due to a defect in iodide transport at the basolateral pole of the cell. In papillary carcinomas, this defect seems to be coupled to an altered apical transport of iodide and probably TPO activity. The TSHr persists in virtually all cases.

Expression of the endothelin-1 gene in the rat thyroid gland and changes in its peptide and mRNA levels in goiter formation and iodide-induced involution.

Endothelin-1 (ET-1) is a major vasoconstrictor peptide, first found in endothelial cells, and later in many other tissues, including the thyroid gland. We analysed the expression of the ET-1 gene in the rat thyroid gland and changes in ET-1 mRNA and peptide levels in goiter development and involution, two circumstances characterised by vascular changes. Thyroid hyperplasia was induced in adult Wistar rats by feeding a low iodine diet (LID) supplemented with 0.25% thiouracil for 10 days, and LID alone for 2 further days (H.12 group). Involution was induced by injecting 100 micrograms iodide and refeeding a normal diet during 6 h, 12 h, and 24 h (I.6h, I.12h, I.24h groups). Rats fed a normal iodine diet were used as controls. A specific 488 bp cDNA corresponding to the known sequence of pre-pro ET-1 was found by RT-PCR from RNA extracts in all thyroid experimental groups, as well as in lung and kidney which were used as positive controls. RP-HPLC analysis showed that ET-1 immunoreactivity eluted similarly as mature ET-1. During hyperplasia, ET-1 mRNA and peptide levels were increased 3.5- and 5-fold respectively. The relative volume of the vascular bed was more than doubled. During iodide-induced involution, the glandular ET-1 mRNA level remained elevated. The concentration of ET-1 peptide increased and was significantly greater at 12 h involution than in the H.12 group. At this time, the capillary reticulum reverted to individual capillaries and the vascular bed was significantly reduced. These data demonstrate that the ET-1 gene is expressed in the rat thyroid gland and that the ET-1 mRNA and peptide levels are increased during thyroid hyperplasia and remain elevated during a phase of rapid iodide-induced involution. These data suggest that changes in ET-1 production may play a role in control of thyroid gland trophic regulation and vascularity.

Nitric oxide is involved in interleukin-1alpha-induced cytotoxicity in polarised human thyrocytes.

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1alpha (100 U/ml), alone or in combination with interferon-gamma (IFN-gamma; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1alpha with IFN-gamma, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-gamma was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1alpha effect on LDH release. The NOS inhibitor, L-nitro-arginine-methyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1alpha+IFN-gamma and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier.

Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease.

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.

Evidence for processing of compact insoluble thyroglobulin globules in relation with follicular cell functional activity in the human and the mouse thyroid.

OBJECTIVE: Thyroglobulin (Tg) is stored within the follicular lumen mainly in a soluble form, but globules made of insoluble multimers are also present and considered to be a mechanism to store prohormone at high concentration. We investigated the immunohistochemical properties of these intrafollicular globules and their possible processing by thyroid cells upon stimulation in the human and in the mouse. DESIGN: Human thyroids (normal, Graves' disease and hot adenomas) and thyroids from old ICR mice without or with goitrogenic treatment were processed for light microscopy. METHODS: Immunohistochemistry for Tg with a polyclonal antibody and two monoclonal antibodies, one specific for thyroxine-rich-iodinated Tg and the other recognizing Tg independently of its iodine level, staining with periodic-acid-schiff, and binding of lectins specific for mannose and sialic acid were performed on all tIssue sections. Intrafollicular globules were quantified, with distinction between 'active' or 'hot' and 'hypofunctioning' or 'cold' follicles. RESULTS: In normal human and old mouse thyroids, the intrafollicular globules were strongly stained with PAS, but negative for the three anti-Tg antibodies and the two lectin-binding assays, while the surrounding soluble Tg was positive. In normal human tIssue, globules were more frequent in 'hypofunctioning' than in 'active' follicles. They were exceptional in Graves' disease and hot adenomas. In old mice, Tg globules were more frequent in 'cold' than in 'hot' follicles. Along with the goitrogen treatment, they became fewer, fragmented and more often present in follicles with a 'hot' aspect. CONCLUSIONS: Upon TSH stimulation, thyrocytes become able to process colloid globules suggesting that this stock of Tg can be used in vivo for thyroid hormone synthesis.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism.

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Evidence for co-ordinated changes between vascular endothelial growth factor and nitric oxide synthase III immunoreactivity, the functional status of the thyroid follicles, and the microvascular bed during chronic stimulation by low iodine and propylthiouracyl in old mice.

Vasoactive and angiogenic factors are involved in the autocrine/paracrine thyroid regulation of microvascular bed during goiter development. In the thyroid of old mice, the presence of slowly functioning ('cold') follicles allowed us to study the microvascular regulation of each follicle in correlation with the immunohistochemical expression of vascular endothelial growth factor (VEGF) and nitric oxide synthase III (NOSIII). Mice aged 20 months did or did not receive a goitrogenic treatment (low iodine diet and propylthiouracyl), and their thyroids were processed for light and electron microscopy, and for autoradiography. The relative volumes (Vv) of the capillaries, the number of vessels per follicular area, the mean capillary area and the number of [(3)H]thymidine labeled nuclei were measured separately for 'hot' and 'cold' follicles. Already in control mice, the capillary bed surrounding 'hot' follicles was significantly larger than that seen around 'cold' follicles, because of larger diameters and twice the number of capillaries. This difference persisted whatever the length of the stimulatory treatment. During this treatment, the Vv of the capillaries increased to a larger extent around 'hot' follicles than around 'cold' ones. All vascular changes around 'cold' follicles were less extended and the increase in the capillary diameter was delayed. In control mice, the 'cold' follicles were negative for NOSIII and positive for VEGF while 'hot' follicles were positive for both. During stimulation, all follicles became progressively NOSIII positive. These data support the concept of 'angio-follicular units' in the thyroid and demonstrate their differential regulation in chronic stimulation during which local secretion of VEGF and NO is clearly involved.

An unusual neuropathy in a diabetic patient: evidence for intravenous immunoglobin-induced effective therapy.

We report the case of a 68-year old type-2 diabetic male patient who was admitted to hospital for progressive weakness in the right lower limb. Although his metabolic control was good, he lost more than 20 kg of weight. Despite intensive physio- and vitaminotherapy, his neurological condition kept on degrading with a severe amyotrophy and pain of the right thigh. He was unable to walk and to stand alone. Besides a yet known sensitive polyneuropathy, the electrophysiological study revealed an obvious motor involvement with signs of demyelination and axonal degeneration. Combined with the albuminocytologic dissociation observed in the cerebrospinal fluid, these specific clinical and electrophysiological features led us to postulate a diagnosis of inflammatory neuropathy. The patient underwent a treatment by methylprednisolone and immunoglobins that rapidly induced a striking improvement of his neurological condition. This case report illustrates that rare forms of neuropathy such as inflammatory neuropathies close to chronic inflammatory demyelinating polyneuropathy (CIDP) can occur in diabetic patients and superimpose on the more commonly described forms of neuropathies. It recalls the importance of recognizing CIDP-like neuropathies because unlike other forms of neuropathy, inflammatory neuropathies are perfectly curable.

Functional lymphocyte subset assessment of the Th1/Th2 profile in patients with autoimmune thyroiditis by flowcytometric analysis of peripheral lymphocytes.

BACKGROUND: Hashimoto's thyroiditis (HT) results from a parenchymal infiltration by Th1 T cell clones that ultimately may cause tissue destruction. We analysed here whether the quantitative assessment of the cytokine profile in peripheral lymphocytes could help for the evaluation of patients with HT. METHODS: We added to a flow cytometric evaluation of lymphocyte subpopulations, an assay to identify specific Th1 and Th2 functional subsets. Whole blood diluted in RPMI was activated with PMA and ionomycin for 4h at 37 degrees C in the presence of brefeldin. Immunophenotyping of samples was performed with PerCP-Cy5.5-conjugated CD3, and APC-conjugated CD4 antibodies. After staining for surface antigens, red cells were lysed and white cells were permeabilized. Intracellular cytokines were detected with FITC-conjugated INFgamma (Th1) and PE-conjugated IL-4 (Th2) monoclonal antibodies (mabs). RESULTS: Twenty-three consecutive patients were selected based on the detection of anti TPO ab concentration equal or higher than 600 UI/L. They were compared to 17 healthy control subjects (with undetectable TPO abs). The lymphocyte count and the proportion of the different lymphocyte subsets (T cells, B cells, NK cells, T-CD4+, T-CD8+, Th1 CD3, Th2 CD3, Th1 CD4, Th2 CD4 cells) were alike when both groups were compared. A significant difference appeared when the Th1/Th2 ratio measured on CD3 T cells was considered. This ratio was significantly increased in HT patients when compared to controls (24.91+/-2.9 vs. 15.5+/-1.4 (mean +/- SEM); p <0.05). When the same analysis was performed on CD4 T cells, the Th1/Th2 ratio was again higher in HT patients, although without reaching significance (13.5+/-1.9 vs. 8.8+/-0.6; p >0.05). CONCLUSIONS: Our data indicate that the Th1 context analysed in peripheral lymphocytes is dominant in HT patients. Flowcytometry could be used as a diagnostic tool to better understand the pathogeny and the outcome of destructive autoimmune thyroiditis.

Central and extrapontine myelinolysis in a patient in spite of a careful correction of hyponatremia.

We report the case of a 54-year-old alcoholic female patient who was hospitalized for neurologic alterations along with a severe hyponatremia (plasma Na+: 97 mEq/l). She suffered from potomania and was given, a few days before admission, a thiazide diuretic for hypertension. A careful correction of plasma Na+ levels was initiated over a 48-hour period (rate of correction < 10 mEq/l/24h) in order to avoid brain demyelination. After a 2-day period of clinical improvement, her neurologic condition started to deteriorate. By the 5th day of admission, she became tetraplegic, presented pseudobulbar palsy, ataxia, strabism, extrapyramidal stiffness and clouding of consciousness. Scintigraphic and MRI investigations demonstrated pontine and extrapontine lesions associated with Gayet-Wernicke encephalopathy. After correction of ionic disorders (hyponatremia, hypokaliemia) and vitamin B (thiamine) deficiency, the patient almost completely recovered without notable disabilities. This case illustrates that profound hyponatremia, in a paradigm of slow onset, can be compatible with life. It also demonstrates that demyelinating lesions, usually considered as a consequence of a too fast correction of hyponatremia, may occur despite the strict observance of recent guidelines. There is increasing evidence to suggest that pontine swelling and dysfunction may sometimes occur in alcoholic patients even in absence of disturbance in plasma Na+ levels. It is therefore of importance, while managing a hyponatremic alcoholic patient, to identify additional risk factors (hypokaliemia, hypophosphoremia, seizure-induced hypoxemia, malnutrition with vitamin B deficiency) for brain demyelination and to correct them appropriately.

Switching from premixed insulin to basal-bolus insulin glargine plus rapid-acting insulin: the ATLANTIC study.

INTRODUCTION: Data on switching from premixed insulin to a basal-bolus regimen in routine clinical practice are sparse. The aim was to evaluate the efficacy and safety of switching from twice-daily premixed insulin to basal glargine plus rapid-acting insulin in a "real-world" clinical practice setting in Belgium and The Netherlands. METHODS: This prospective, 6-month, noninterventional, observational study was conducted in 37 centres in Belgium and 19 centres in The Netherlands. Adults (> or =18 years of age) with type 2 diabetes were eligible if they were not taking oral antihyperglycaemic drugs or only taking metformin. The primary objective was the proportion of patients attaining glycated haemoglobin (HbA1c) <7% at months 3 and 6. Secondary objectives included changes in HbA1c, weight, body mass index (BMI), insulin doses, hypoglycaemic events, and treatment satisfaction. RESULTS: There were 214 patients from Belgium and The Netherlands enrolled. Mean age was 64.6 years, weight was 89.5 kg, BMI was 31.4 kg/m2, and duration of diabetes was 12.1 years. At month 6, the percentage of patients with HbA1c <7% increased from 3.3% to 24.9% (p<0.001). Mean HbA1c at baseline was 8.9%; mean change from baseline was -1.5% (p<0.001). Glargine and prandial insulin doses increased (p<0.001, each), while body weight and BMI were unchanged. Hypoglycaemic events did not increase. Overall treatment satisfaction improved significantly (p<0.001). CONCLUSIONS: In a Belgian and Dutch clinical practice setting, patients with type 2 diabetes that is poorly controlled on premixed insulin experienced significant improvements in glycaemic control, without a concomitant increase in hypoglycaemic events or weight, when switched from premixed insulin to basal-bolus glargine plus rapid-acting insulin. As a result, treatment satisfaction significantly improved.