Clinical description of patients with cytoplasmic discrete dots pattern (lysosome) on indirect immunofluorescence on HEp-2 cells.

The cytoplasmic discrete dot (CDD) pattern is an unusual finding in indirect immunofluorescence, and its clinical value is unknown. To describe the clinical characteristics of patients with CDD pattern on indirect immunofluorescence (IIF) from our laboratory database and to evaluate possible associations with other autoantibodies and autoimmune diseases. This is a retrospective descriptive study. We included all patients with CDD pattern on IIF in HEp-2 cells with a titer equal or greater than 1/80, using a database of all IIF performed in a reference immunology and rheumatology laboratory between 2007 and 2015. Data on demographics, past medical history, and relevant laboratory findings were recorded and analyzed. We performed 13.056 IIF on HEp-2 cells tests between January 1, 2007 and December 31, 2015, with 6075 positive results. Among them, 5447 had nuclear pattern, 55 had both nuclear and cytoplasmic pattern, and 573 had cytoplasmic pattern. Only 21 showed a CDD pattern. Four patients were excluded since they did not have medical records at the institution. The prevalence of the CDD pattern in our laboratory was 0.35%. The median age was of 62.3 years (SD 9.16) and 100% were female. Fifty-three percent (9/17) had an autoimmune disease, Hashimoto's thyroiditis (4/9) being the most frequent one. In conclusion, cytoplasmic discrete dot pattern is an uncommon finding and its clinical value is uncertain. However, in our study, 53% of the patients had an autoimmune disease.

Parathyroid Diseases and T Cells.

PURPOSE OF REVIEW: This review summarizes studies into the permissive role of T cells in the bone catabolic effects of hyperparathyroidism and parathyroid hormone (PTH). RECENT FINDINGS: Work in animals combined with recent translational studies in humans now highlight the potent amplificatory action of T cells on PTH-induced bone resorption. Mechanistic animal studies reveal a complex pathway by which PTH exploits natural self-renewal functions of CD4+ T cells, to drive TNFα production that promotes formation of IL-17A secreting Th17 T cells. TNFα and IL-17 further amplify osteoblastic receptor activator of NF-κB ligand (RANKL) production and down-modulate osteoprotegerin (OPG), establishing conditions propitious for osteoclastic bone resorption. These findings are consistent with, and add to, the traditional view of PTH-induced bone loss involving only osteoblast-lineage cells. T cells potently amplify traditional pathways and provide permissive costimulatory signals to bone marrow stromal cells, facilitating the development of an increased RANKL/OPG ratio favourable to bone resorption and bone loss.

Advantage of tacrolimus/mycophenolate mofetil regimen for cytotoxic T cell-mediated defence and its inhibition by additive steroid administration in high-risk liver transplant recipients.

Our previous work revealed that the recipients with the highest pre-existing numbers of CD8(+) effector T cells (TE ) [hyperparathyroidism (HPT)E recipients] occupied approximately 30% of adult transplant recipients performed in our hospital. HPTE recipients demonstrated very poor clinical outcome compared with the remaining 70% of recipients with the lowest pre-existing TE (LPTE recipient). This study aimed to clarify the best combined immunosuppressive regimen related to function of cytotoxic T lymphocytes (CTLs) for HPTE recipients. Eighty-one HPTE recipients were classified into three types, according to the immunosuppressive regimens: type 1, tacrolimus (Tac)/glucocorticoid (GC); type 2, Tac/mycophenolate mofetil (MMF)/GC; and type 3, Tac/MMF. Frequencies of severe infection, rejection and hospital death were the highest in types 1 and 2, whereas the lowest occurred in type 3. The survival rate in type 3 was the highest (100%) during follow-up until post-operative day 2000. Regarding the immunological mechanism, in type 1 TE perforin and interferon (IFN)-γ were generated through the self-renewal of CD8(+) central memory T cells (TCM ), but decreased in the early post-transplant period due to marked down-regulation of interleukin (IL)-12 receptor beta-1 of TCM. In type 2, the self-renewal TCM did not develop, and the effector function could not be increased. In type 3, in contrast, the effectors and cytotoxicity were correlated inversely with IL-12Rß1(+) TCM levels, and increased at the highest level around the pre-transplant levels of IL-12Rß1(+) TCM . However, the immunological advantage of Tac/MMF therapy was inhibited strongly by additive steroid administration.

Dietary-induced hyperparathyroidism affects serum and gingival proinflammatory cytokine levels in rats.

BACKGROUND: Poor diet and inadequate nutrition are suggested to affect the periodontium as well as impair the systemic health. This study investigated the systemic and periodontal effects of dietary-induced hyperparathyroidism (dHPT) by evaluating serum and gingival proinflammatory cytokine levels. METHODS: Twenty-four Sprague-Dawley rats were used in the study. dHPT was induced in 12 rats by calcium/phosphorus imbalance, and 12 rats were fed a standard diet (SD). Afterward, endotoxin-induced periodontitis was induced on the right mandibular molar teeth (mmt). Four study groups were created: dHPT + mmt without periodontitis (group 1), dHPT + mmt with periodontitis (group 2), SD + mmt with periodontitis (group 3), and SD + mmt without periodontitis (group 4). Interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) levels were measured by enzyme-linked immunosorbent assay to evaluate the proinflammatory cytokine profiles. Serum cytokines were analyzed in the blood samples collected prior to periodontitis induction, whereas gingival cytokines were analyzed in the gingival supernatants of the four groups. RESULTS: Serum cytokines were higher in dHPT rats than in SD rats (P <0.001), with a positive correlation between parathormone and the cytokines (P <0.001). Gingival cytokines were highest in group 2 and lowest in group 4 (group 2 > group 3 > group 1) (P <0.001). There was a positive correlation between parathormone and the gingival cytokines in group 1 (P <0.001 for IL-1beta; P <0.01 for TNF-alpha). CONCLUSION: The results suggested that increased serum proinflammatory cytokine production may be a complication of dHPT, and this may affect healthy and diseased periodontia by increasing gingival proinflammatory cytokine levels.

T cells potentiate PTH-induced cortical bone loss through CD40L signaling.

Parathyroid hormone (PTH) promotes bone catabolism by targeting bone marrow (BM) stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption, and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell-expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, the receptor activator of nuclear factor-kappaB ligand (RANKL)/OSTEOPROTEGERN (OPG) ratio, and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, life span, and function through CD40L. T cell-SC crosstalk pathways may thus provide pharmacological targets for PTH-induced bone disease.

Evolution of PTH assays.

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function.

Evolution of PTH assays / Evolução dos ensaios para paratormônio

PTH metabolism is complex and the circulating forms include the intact 1-84 molecule as well as several carboxyl-terminal fragments. The first generation of PTH assays included several types of competitive assays, with specificities that spanned carboxyl, mid-region and amino-terminal portions of the molecule. The limitations of these assays and the methodological evolution led to the description of 2nd generation non-competitive immunometric assays for PTH in the late 80's, based on the recognition of the PTH molecule by two different antibodies, one directed against de amino-terminal and other against the carboxyl-terminal segments. The observation that in some circumstances "long" carboxyl-terminal segments were also measured by 2nd generation assays led to the development of 3rd generation assays based on amino-terminal specific antibodies that are specific for the first amino acids, measuring only the molecular forms that activate PTH1R. The practical and cost-benefit advantages of these assays are still debatable. The recent observation that carboxyl-terminal fragments of PTH have biological activity via a distinct receptor than PTH1R, points to the future need of more than one assay in order to evaluate parathyroid hormone function.

O metabolismo do PTH e complexo e as formas circulantes incluem o PTH 1-84, assim como fragmentos C-terminal. A primeira geração de ensaios para o PTH incluía vários ensaios competitivos com especificidades para as regiões carboxi, meio da molécula e amino-terminal. A limitação destes ensaios e a evolução metodológica, levaram ao desenvolvimento dos ensaios não competitivos de 2ª. geração no final dos anos 80, baseados no reconhecimento por dois anticorpos diferentes, contra a porção amino e carboxi-terminal respectivamente. A observação que em algumas circunstâncias segmentos carboxiterminais longos também eram detectados, levou ao desenvolvimento dos ensaios de 3ª. geração, baseados em anticorpos específicos para a porção aminoterminal com maior especificidade para os primeiros aminoácidos, e assim mensurando apenas a forma molecular que ativa o PTH1R. As vantagens práticas e o custo-benefício deste ensaio ainda e motivo de debate. A observação recente de que fragmentos carboxiterminais têm atividade biológica via receptor distinto, aponta para a necessidade futura de mais de um ensaio para avaliar a função do paratormônio.

Early stage of urolithiasis formation in experimental hyperparathyroidism.

PURPOSE: We have previously noted marked acceleration in the proliferative activity of parathyroid cells in rats with spontaneous hypercholesterolemia and secondary hyperparathyroidism. Using this proliferative potential we investigated whether transplantation of these enlarged parathyroids into normal rats would induce hyperparathyroidism and renal stones. MATERIALS AND METHODS: We used 26-week-old male rats with spontaneous hypercholesterolemia as donors, and 5-week-old normal male Sprague-Dawley rats and rats with spontaneous hypercholesterolemia as recipients. Enlarged parathyroid glands were transplanted into group 1--Sprague-Dawley rats with no treatment, group 2--Sprague-Dawley rats that received FK-506 as an immuno-suppressor, group 3--rats with spontaneous hypercholesterolemia rats that underwent parathyroidectomy plus FK-506 administration and group 4--Sprague-Dawley rats that underwent parathyroidectomy plus FK-506 administration. Parathyroidectomy was performed in recipients before transplantation to ensure a low calcium condition. RESULTS: Grafts were rejected within 11 and 15 weeks in groups 1 and 2, respectively. In group 3, 78% of the grafts were successful even after 19 weeks. In group 4 graft survival was 30% at 15 weeks with complete rejection at 19 weeks. In group 3 gradually elevated serum parathyroid hormone was observed as well as stone plaques containing calcium oxalate and calcium phosphate in renal tubules located mainly in the corticomedullary junction. An increased number of plaques was associated with higher parathyroid hormone. CONCLUSIONS: Our study shows that transplanted parathyroid glands function with an immunosuppressive agent and the maintenance of hypocalcemic conditions, and they secrete sufficient parathyroid hormone to demonstrate hyperparathyroidism. Plaque in these kidneys indicates an early stage of urolithiasis caused by hyperparathyroidism.

Restoration of impaired T-cell proliferation after parathyroidectomy in hemodialysis patients.

BACKGROUND/AIMS: Severe secondary hyperparathyroidism is not infrequent in hemodialysis patients and recent studies suggest that parathyroid hormone (PTH) may play a role in the genesis of cell immunity abnormalities in uremia. The aim of the present study is to describe the effect of parathyroidectomy on T- and B-cell functions in hemodialysis patients. METHODS: The study was performed on 6 patients with severe secondary hyperparathyroidism. iPTH, B, CD4(+), CD8(+), total number of lymphocytes, lymphoproliferative response to PHA, PWM and Candidin, and IgG, IgM, IL-2 production in vitro were determined 1 day before and 4 months after parathyroidectomy. RESULTS: The lymphoproliferative response to PHA increased significantly after parathyroidectomy. We also observed a trend to an increase in production of IgG and IgM after PWM stimulation before therapy. CONCLUSION: The present study suggests that patients with extremely high levels of PTH show a complete restoration of impaired T-cell proliferation after parathyroidectomy.

Major histocompatibility complex class II expression and parathyroid autoantibodies in primary hyperparathyroidism.

BACKGROUND: Autoimmune diseases are characterized by induced parenchymal expression of major histocompatibility complex (MHC) class II antigens and circulating autoantibodies directed toward surface structures on the target cells. MHC class II expressions can be modified by viral infections of potential pathogenic importance in autoimmune reactions. Primary hyperparathyroidism exhibits incompletely clarified cause. METHODS: With cryosections, human parathyroid glands were stained with monoclonal antibodies to MHC class II antigens according to a peroxidase-antiperoxidase technique. Human parathyroid adenoma tissue transplanted to nude mice and rat parathyroid glands was tested with serum from patients with hyperparathyroidism and control subjects. RESULTS: Induced MHC class II expression was demonstrated on parathyroid parenchymal cells in 13 of 54 adenomatous and eight of 23 hyperplastic glands of patients with primary hyperparathyroidism. This reactivity was absent in 12 normal glands, nine normal-sized glands associated with the adenomas, and 17 enlarged glands of patients with hyperparathyroidism caused by uremia. Staining of parathyroid tissue was found with serum from 27 of 38 patients with primary hyperparathyroidism, whereas this reactivity was absent on rat thyroid and pancreatic tissue, as well as with control sera. CONCLUSION: The concurrent induction of MHC class II antigen expression and c circulating antiparathyroid autoantibodies in 16 or 38 patients with primary hyperparathyroidism suggests hitherto unrecognized immunologic involvement in this disease.

Effects of parathyroid hormone and serum calcium on the phenotype and function of mononuclear cells in patients with primary hyperparathyroidism.

BACKGROUND: Several studies have demonstrated specific influence of parathyroid hormone (PTH) on immune parameters, especially on T- and B-cell function, migration of polymorphonuclear leucocytes (PMNLs) and antibody synthesis, in patients with secondary hyperparathyroidism and chronic renal failure and recently also in patients with primary hyperparathyroidism (pHPT). METHODS: We therefore examined 12 patients with pHPT before and 6 months after parathyroidectomy (PTX) and nine sex- and age-matched control subjects to determine the impact of PTH and serum calcium concentrations on several immune parameters, including (a) serum concentrations of immunoglobulins, (b) immunophenotype of peripheral blood lymphocytes, (c) phytohaemagglutinin (PHA)-induced lymphocyte proliferation and (d) monocytic surface marker expression. RESULTS: Serum concentrations of immunoglobulins (IgG, IgA, IgM) were unaffected by elevated serum PTH and calcium levels. T lymphocytes (CD3), B lymphocytes (CD19), NK cells (CD16/56) and monocytes (CD16) revealed a normal distribution and were not different before and after PTX in patients with pHPT when compared with the control group. CD4+ T-helper lymphocytes were significantly elevated pre- and post-operatively in patients with pHPT. The lymphocyte proliferation response to PHA in the highest concentration (12.5 micrograms L-1) tested was significantly suppressed in patients with pHPT preoperatively when compared with the patients post-operatively and the control group. In addition, both CD4+ and CD8+ lymphocytes showed a lower expression of activation markers, interleukin 2 (IL-2) receptor (CD25) and transferrin receptor (CD71), which could be partially restored 6 months after PTX, but did not reach normal values. CONCLUSION: In summary, in contrast to the findings in patients with secondary HPT, pHPT appears to be associated with less alterations of immune functions. Chronically elevated serum PTH and calcium concentrations in patients with pHPT induce a higher percentage of CD4+ helper T lymphocytes and a suppressed lymphocyte response to PHA as well as a reduced expression of activation markers on peripheral blood lymphocytes.

Parathyroid hormone-related protein: hormone and cytokine.

While PTHrP acts as a hormone when it is produced in excess by certain cancers, and perhaps also in lactating women, the foetus, and lower vertebrates, it seems clear that PTHrP is an important paracrine regulator of several tissue-specific functions. Its roles in smooth muscle relaxation, placental calcium transport, and bone development are becoming firmly established. However, it is likely to be an important player in the control of cellular growth and differentiation, although much of our understanding of this role to date comes from indirect evidence. The next decade will be an exciting time in defining further both the hormonal and paracrine actions of PTHrP.

Expression and function of a CD3-like molecule on normal and abnormal human parathyroid cells.

BACKGROUND: Normal and abnormal human parathyroid tissue express the T-lymphocyte protein CD4, and parathyroid and lymphocyte cells show similarities with respect to mechanisms of calcium permeability and regulation of the cytoplasmic calcium concentration. METHODS: Anti-Leu4, a monoclonal antibody recognizing the T-lymphocyte glycoprotein complex CD3, is used to immunohistochemically stain normal and abnormal human parathyroid cells and to explore influences on the parathyroid hormone (PTH) secretion of enzymatically dispersed parathyroid cells. RESULTS: Parathyroid glands of patients with different forms of hyperparathyroidism displayed variable expression of the anti-CD3 reactive complex. The stainings correlated both positively and inversely to immunoreactivity for a previously defined calcium sensor, the decreased expression of which may constitute a molecular basis for hyperparathyroidism. Incubation of parathyroid cells with the anti-Leu4 antibody inhibited PTH secretion and reduced its sensitivity to external calcium without influence on parathyroid cytoplasmic calcium concentration. CONCLUSIONS: The results suggest that the human parathyroid cells express a CD3-like molecule with the ability to interact in PTH release.

Immunohistochemical studies on specificity and affinity of the BB5-antibody against human parathyroid tissue.

Localisation of parathyroid tissue in hyperparathyroidism may be difficult with standard methods. Immunoscintigraphy, using radiolabeled antibodies against epitopes of human parathyroid cells, could be a promising alternative. Therefore, we studied the necessary preconditions, whether the so-called BB5-antibody, directed against parathyroid cell membranes possesses sufficient specificity and affinity to be employed in immunoscintigraphy. Specificity was tested immunohistochemically with APAAP-staining of 39 different human tissues. Additionally, an immunoscore-based quantitative comparison was performed to test the affinity of BB5-antibody for normal and pathologic parathyroid tissue. Specificity was proven by the fact that of all 39 tested tissue-types only the parathyroid tissue was BB5-positive. Normal parathyroid tissue showed a significantly higher affinity to the BB5-antibody than pathologic parathyroid tissue (p < 0.03). However, all tissue samples from primary or secondary hyperparathyroidism exhibited sufficient staining. We conclude that the BB5-antibody fulfills the necessary conditions to be tried for immunoscintigraphical localisation of the parathyroid glands.

Neutrophil leucocyte function in primary hyperparathyroidism.

Neutrophil leucocyte chemotaxis, phagocytosis and oxidative metabolism were measured in six patients with primary hyperparathyroidism (HPT) who underwent parathyroidectomy. The preoperative neutrophil chemotaxis value was 6.2 +/- 0.3 arb.U. and this decreased to 5.8 +/- 0.5 arb.U. (p less than 0.05) on the third postoperative day and to 5.4 +/- 0.3 arb.U. three weeks later. Serum calcium levels were 2.95 +/- 0.06 mmol/l preoperatively and decreased to 2.20 +/- 0.05 (p less than 0.05) and 2.34 +/- 0.05 mmol/l (p less than 0.05) on the third and 21st day after parathyroidectomy. Phagocytosis measured as the intracellular uptake of complement-opsonized yeast particles by neutrophils was not influenced by operation. In a control group, six patients undergoing hemithyroid ectomy due to microfollicular tumours were studied. In this group leucocyte chemotaxis, phagocytosis and serum calcium levels were not significantly different before and after surgery. Neutrophil oxidative metabolism, measured by the ability of the cells to reduce nitroblue tetrazolium (NBT), was similar in the HPT and control groups both before and after operation. The results indicate that HPT is associated with an abnormal leucocyte migration which is reversed after successful parathyroidectomy.

Effect of parathyroidectomy on T cell functions in patients with primary hyperparathyroidism.

The notion that parathyroid hormone (PTH) can serve as an immunomodulator was examined. T cell function tests were performed in 3 patients with primary hyperparathyroidism before and 1 month after parathyroidectomy (PTX). Three normal volunteers, age and sex matched, were used as controls. One patient with lipoma of the neck was also examined before and after surgical removal of the lesion. In the primary hyperparathyroidism patients the total T cells were lower, the suppressors were higher and the helper to suppressor ratio was significantly lower than in control subjects. The lectin-stimulated lymphocyte transformation was significantly inhibited. All these abnormalities were restored to normal after PTX. Depressed lymphocyte activity was found also in the patient with lipoma. However, no change occurred after surgery. These results support the assumption that excess blood levels of PTH may have an immunosuppressive effect.