[Focal proliferation in reactive hyperplastic parathyroid tissue in end-stage renal failure--implication for autotransplantation after total parathyroidectomy]. / Fokale Proliferationen in reaktiv hyperplastischen Nebenschilddrüsen bei terminaler Niereninsuffizienz-Implikation für die Autotransplantation nach totaler Parathyreoidektomie.

Total parathyroidectomy with simultaneous autotransplantation may be associated with recurrence of graft-dependent hyperfunction due to excessive proliferation. We performed macroscopic tissue selection with a stereomicroscope prior to autotransplantation, which resulted in very low recurrence rates. As this technique greatly depends on experience, we investigated the possibility of additionally using proliferation staining (PCNA, MIB-1) for the detection of dysfunctional tissue. Selected tissue from 26 patients was investigated. Serial sections of freshly removed parathyroid tissue were correlated with their macroscopic appearance, HE and immunohistochemically stained paraffin sections, and with semithin Epon sections. The asymptotic growth mode of clonal proliferating regions was reflected by highest staining intensity (1-5%) in small to medium sized foci (diffuse, up to 3 mm in diameter) and very low staining in large areas (diffuse or nodular, 5-15 mm in diameter, from 0.03 to 0.003% positive cells). Thus, very large dysfunctional regions with (almost) no proliferation could not be detected by this method. However, they were very evident on macroscopic investigation. In conclusion, multiple fulminant recurrence after parathyroidectomy can be prevented by selecting tissue after proliferation staining. This may allow a delayed autotransplantation after total parathyoidectomy for those surgeons lacking experience in macroscopic tissue classification.

Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse.

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.

Effects of triiodothyronine on the morphology of cells and matrix, the localization of alkaline phosphatase, and the frequency of apoptosis in long-term cultures of MC3T3-E1 cells.

The effects of triiodothyronine (T3) on the localization and morphology of alkaline phosphatase (ALP)-positive cells, matrix formation, and apoptosis in MC3T3-E1 cells cultured up to 6 weeks were investigated by light and electron microscopy. Cell size, shape, and frequency of apoptosis were measured histomorphometrically. At all time points both ALP-positive and -negative cells were observed histochemically. Control cultures older than 3 weeks were characterized by colonies of small cuboidal ALP-positive cells. Cross sections revealed that these areas corresponded to unmineralized nodules. The thickening was caused by local accumulation of extracellular matrix. The internodular regions were characterized by ALP-positive spindle-shaped cells randomly distributed throughout all cell layers. Apoptotic nuclei were found within a frequency of 0.2%-1%. With increasing culture time the percentage of apoptotic cells became higher in the nodules. T3 treatment inhibited cell proliferation and stimulated ALP activity. After confluence, T3-treated cultures reached two to three cell layers at maximum and showed a different morphology and histochemical staining pattern. ALP-positive cells were stellar shaped and larger than unstained cells. Small ALP-positive colonies suggested nodule formation; however, the most striking differences between T3-treated and control cultures were a decrease in the amount of extracellular matrix with only few collagen fibers and the absence of local matrix accumulation. Furthermore, the number of apoptotic nuclei was increased. Our data extend beyond previous observations on the role of thyroid hormones in osteoblastic differentiation. Besides their effects on proliferation and cell morphology, they influence ALP activity, matrix composition, nodule formation, and apoptotic transformation.

Inhibitory effect of amylin on basal and parathyroid hormone-stimulated bone resorption in cultured neonatal mouse calvaria.

Amylin is a secretory product of pancreatic beta-cells which shares major sequence homology with calcitonin gene-related peptide. In neonatal mouse calvaria maintained in organ culture for 72 h, amylin inhibited basal (i.e., unstimulated) resorption in medium concentrations above 2.5 x 10(-9) M. In addition, amylin (> or = 1.0 x 10(-7) M) in a calcitonin-like fashion transiently inhibited bone resorption induced by 1.0 x 10(-8) M PTH ("escape phenomenon"). Pretreatment of calvarial bones with amylin (1.0 x 10(-8) - 1.0 x 10(-6) M) for 72 h attenuated the subsequent response to 1.0 x 10(-8) M PTH. Changes in location and appearance of osteoclasts in amylin-treated bones, as visualized by light microscopy, suggest that amylin inhibits bone resorption by causing a loss of specialized contact zones to the mineralized matrix in resorbing osteoclasts, and in addition, by preventing retraction of osteoblasts from the mineralized surface which impedes attachment of osteoclasts thereon.

[Experiences with total parathyroidectomy and autotransplantation of intraoperatively selected parathyroid tissue in reactive renal hyperparathyroidism]. / Erfahrungen mit der totalen Parathyreoidektomie und Autotransplantation intraoperativ ausgewählten Parathyreoidea-Gewebes beim reaktiven renalen Hyperparathyreoidismus.

Surgery is indicated in cases of therapy-resistant reactive renal hyperparathyroidism with clinical symptoms and signs. The method of choice is total parathyroidectomy, with autotransplantation of parathyroid tissue into the forearm musculature. Twenty-two patients were parathyroidectomized. In 19 cases the intraoperative selection of tissue for autotransplantation was made by means of a stereoscopic magnifying glass. With one exception, only fragments of type 1a glands and type 1a-like areas obtained from type 2 glands were transplanted. In 18 of the 22 cases parathyroid hormone levels decreased from 1131 +/- 657 preoperatively to 87 +/- 53 pg/ml postoperatively. In 4 patients with postoperative values over 200 pg/ml a transplant-related recurrence of hyperparathyroidism or a remaining 5th gland in the cervical region was suspected. In all 22 patients serum calcium, as well as alkaline phosphatase levels returned to normal and the clinical signs of hyperparathyroidism disappeared.

Morphologic and functional studies to prevent graft-dependent recurrence in renal osteodystrophy.

The cross sections of hyperplastic glands removed during total parathyroidectomy in patients with renal osteodystrophy were classified on the basis of macroscopic and functional criteria. Diffuse (type 1) and nodular (type 2) glands could be distinguished macroscopically. Functional in vitro studies showed no significant difference in suppressibility and basal parathyroid hormone (PTH) secretion (61% and 74%, respectively). In both types of glands A, B, C, and D regions could be distinguished with use of a stereo magnifier (magnification, x10 to x16). In diffuse A regions containing stroma fat cells, PTH secretion could be suppressed optimally (more than 50% of maximum secretion) in more than 90% of all specimens in vitro. In diffuse B regions containing no fat cells, hormonal secretion could be suppressed in only 17% and 25%, respectively. Confined (oxyphilic or chief cell) nodules (C regions), only found in type 2 glands, could be suppressed in 18%. Nodules of "degenerating" oxyphilic cells (D regions), only found in type 2 glands, were suppressible, but the low PTH secretion indicated degenerative processes. Thus only fragments from A regions should be selected for autotransplantation.

[Parathyroidectomy and autotransplantation in renal hyperparathyroidism. II. Morphologic and functional studies in transplant-dependent recurrence]. / Parathyreoidektomie und Autotransplantation beim renalen Hyperparathyreoidismus. II. Morphologische und funktionelle Untersuchung bei transplantatabhängigem Rezidiv.

Corresponding to predefined macroscopic criteria indicating morphological and functional characteristics, fragments of a type 1a-gland were used after total parathyroidectomy for auto-transplantation in 16 hemodialyzed patients with drug-resistant renal hyperparathyroidism (rHPT). Fragments of a type 1b-gland were used in two, fragments of a type 2-gland in 17 patients, respectively. The clinical and biochemical follow-up three to 84 months postoperatively showed one patient (3%) with graft-dependent hypocalcemia (type 1a-graft) and in two patients (6%) with type 2-grafts histological examinations confirmed graft-dependent recurrent hyperparathyroidism. Thus the estimated cumulative success (= normal parathyroid metabolism; Kaplan Meier) was 78 +/- 12% 5 years postoperatively. Comparing the estimated cumulative function rate of type 1a- and type 2-autotransplants, a better course was found in typ 1a-grafts three years postoperatively (Breslow: p = 0.0724; Mantel-Cox: p = 0.0677). The morphological examinations of the removed enlarged fragments of different size (between 0.2 cm and 0.9 cm) showed signs of an expansive, but never invasive growth. The chief and oxyphilic cells were intermixed and arranged in a follicular pattern as in the former glands selected for grafting. Mitoses could be found more often in larger fragments, thus correlating with a tendency to proliferation and a bad in vitro suppressibility. The results of the studies confirm the conclusions of an in vitro study, showing that the diffuse hyperplastic type 1a-parathyroids are most suited for autotransplantation. Glands or gland areas without fat-cells (type 1b) or nodules of type 2-glands which can be identified intraoperatively using a stereomagnifier should not be used.

Interferon gamma and calcitonin induce differential changes in cellular kinetics and morphology of osteoclasts in cultured neonatal mouse calvaria.

The present study compares the effects of calcitonin (CT) and interferon gamma (IFN-gamma) on the size, distribution, and ultrastructure of osteoclasts in cultured neonatal mouse calvaria. The number and cross-sectional area of osteoclasts in cultured bones was increased by the addition of parathyroid hormone (PTH) to the culture medium for 24-48 h. Prolonged treatment (up to 72 h) with PTH led to extensive rarefication and formation of holes in the mineralized matrix. PTH-activated osteoclasts exhibited an elaborate ruffled border and showed a typical zonal arrangement of intracellular organelles with the outer cytoplasmic region containing numerous membrane-bound vesicles. CT (20 mU/ml) within 90 minutes caused a complete loss of the ruffled border in PTH-activated osteoclasts. The typical zonal architecture disappeared with intracellular vesicles spread through the entire cytoplasm. Prolongation of CT treatment to 24-48 h led to the appearance of vesicles with dark granular content in inactivated osteoclasts. This morphologically distinct vesicle population allowed us to identify the latter cells as "postosteoclasts" and thereby to distinguish them from osteoclasts precursors. Small active osteoclasts that had originated very likely from these precursors appeared in calvarial bones coincidentally with escape from CT inhibition of bone resorption and resulted in increased total number of osteoclasts. In contrast, IFN-gamma (500 U/ml) acting as a proliferation inhibitor, reduced the total number of osteoclasts. In the presence of PTH it caused no immediate (90 minutes) change in the ultrastructure of PTH-induced osteoclasts apart from an increase in the number of autophagic vacuoles. After prolonged exposure (24-48 h) in the presence of PTH, osteoclasts with low resorbing activity exhibited intermediate borders at their contact zone with the mineralized matrix. In the absence of PTH, the short-term effect of the immune interferon on osteoclast morphology was almost comparable to that of CT. After prolonged treatment in the absence of PTH, postosteoclasts comparable in vesicle population and size to those after CT treatment were found.

Effect of auranofin on resorption, prostaglandin synthesis and ultrastructure of bone cells in cultured mouse calvaria.

Auranofin (AF) in concentrations between 3 x 10(-7) and 3 x 10(-6) mol/l stimulated bone resorption in cultured neonatal mouse calvariae significantly with 1 x 10(-6) mol/l being most potent. Complete inhibition by 5 x 10(-7) mol/l indomethacin and increased medium concentrations of prostaglandin (PG) E2 and 6-keto-PGF1 alpha after 72 h indicate a PG mediated mechanism. Morphology revealed active osteoclasts. Cytotoxic effects were observed with 3 x 10(-6) and 1 x 10(-5) mol/l AF with osteocytes and osteoblasts being considerably more sensitive than osteoclasts. The latter concentrations inhibited bone resorption stimulated by parathyroid hormone (PTH) 1,25-dihydroxyvitamin D3, PGE2, thrombin and interleukin 1. The stimulatory effect of AF on PG production and subsequent bone resorption could limit its therapeutic usefulness.

Comparison between the effects of forskolin and calcitonin on bone resorption and osteoclast morphology in vitro.

The adenylate cyclase activator forskolin (1-10 mumol/L) inhibited 45Ca release from parathyroid hormone (PTH; 10 nmol/L) stimulated prelabeled neonatal mouse calvaria in short term culture (24 h). This effect of forskolin was potentiated by rolipram, Ro 20-1724, and isobutyl-methylxanthine, three structurally different inhibitors of cyclic AMP phosphodiesterase. Forskolin (10 mumol/L) and calcitonin (30 mU/mL) inhibited the mobilization of stable calcium and inorganic phosphate as well as the release of the lysomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase from PTH-stimulated unlabeled bones. Osteoclasts in PTH-stimulated calvaria showed active ruffled borders with numerous membrane infoldings. Treatment of PTH-stimulated bones with forskolin and calcitonin resulted in a rapid (2 h) loss of the active ruffled border. In addition, forskolin and calcitonin induced similar changes with respect to the number and size distribution of cytoplasmic vesicles in PTH-activated osteoclasts. After 24 h, all signs of osteoclast inactivation were still prominent, whereas after 48 h of treatment with forskolin or calcitonin, the reappearance of a ruffled border on a number of osteoclasts signaled an escape from the inhibitory action of both calcitonin or forskolin. These data indicate that forskolin inhibits bone resorption by a cyclic AMP dependent mechanism and that the effect of forskolin and calcitonin on bone resorption and osteoclast morphology are comparable. These observations lend further support to the view that cyclic AMP may be an intracellular mediator of the inhibitory action of calcitonin on multinucleated osteoclasts.

[Recurrent renal hyperparathyroidism: reoperation on the autograft]. / Rezidivierender renaler Hyperparathyreoidismus: Reoperationen am Autotransplantat.

Graft-dependent recurrence was observed 24 and 27 months after total parathyroidectomy and immediate autotransplantation in 2 out of 35 haemodialized patients (6%) with reactive (renal) hyperparathyroidism. In order to normalize the altered parathyroid metabolism in these 2 patients 7 reductions of the graft have been required so far in the patients and cervico-mediastinal reexploration was necessary as an additional procedure in one of the patients. Histological examination of the more or less enlarged fragments showed nearly the same architecture as the original glands used for grafting. Proliferating chief cell nodules with mitoses and signs of expansive, but never invasive growth were seen. Reviewing the literature, 38 authors describe 61 graft-dependent recurrences in 783 patients (7.8%) since 1975. Our own experiences and those in literature are discussed with respect to diagnosis, differential diagnosis, localization and histology of the graft-dependent recurrence.

[Parathyroidectomy and autotransplantation in renal hyperparathyroidism. I. Morphologic studies for tissue selection]. / Parathyreoidektomie und Autotransplantation beim renalen Hyperparathyreoidismus. I. Morphologische Untersuchungen zur Gewebeauswahl.

During total parathyroidectomy and autotransplantation 140 enlarged glands were removed in 35 hemodialyzed patients (normocalcemic: n = 14; hypercalcemic: n = 21). The cross-sections of all glands were classified intraoperatively. Diffuse hyperplastic (type 1) and nodular hyperplastic (type 2) glands could be distinguished. Using a stereo-magnifier (magnification: x 10 -x 16), type 1a- (stromal fat cells!) and type 1b- glands (without stromal fat cells!) could be differentiated. Those areas were also found between the nodules of type 2-glands. Significantly, nodular hyperplastic glands predominated in hypercalcemic patients (chi 2-Test: p less than 0.001). The colour of the nodules on the cross-sections of type 2-glands correlated with the predominating cell type ("dark": nodule of oxyphile cells; "medium": nodule of chief cells; "light": nodule of 'degenerating' oxyphile cells). As sign of proliferation the mitotic index was elevated (greater than 1:10,000) in type 1b-glands, in type 1b-like areas and in nodules of type 2-glands. These areas should not be used for autotransplantation.

[Parathyroidectomy and autotransplantation in renal hyperparathyroidism. II. Functional in vitro studies for tissue selection]. / Parathyreoidektomie und Autotransplantation beim renalen Hyperparathyreoidismus. II. Funktionelle in vitro Untersuchungen zur Gewebeauswahl.

The PTH secretion was studied in fragments of 83 (reactive) hyperplastic parathyroids (type 1 a: n = 24; type 1 b: n = 20, type 2: n = 39) simulating hypo- (0.3 mmol Ca++ = maximal PTH secretion), normo- (0.9 and 1.2 mmol Ca++) and hypercalcemia (3.0 mmol Ca++ = basic PTH secretion, maximal suppressibility) in vitro. 56 out of 83 glands (67%) were suppressible (PTH secretion decreased under 50% of maximal secretion) with no significant difference in suppressibility of diffuse and nodular hyperplasia (50% and 74%, respectively). Differentiating diffuse hyperplastic glands in those with (type 1 a) and without (type 1 b) stroma fat cells, 92% of type 1 a glands and only 25% of type 1 b glands were suppressible (Fischer's test: p less than 0.001). Separated (oxyphilic and/or chief cell) nodules, type 1 b- and type 1 a-like areas (both localized between nodules) of types 2 glands were suppressible in 18%, 17% and 91%, respectively (Fischer's test: p less than 0.001). Thus fragments of type 1 a regions should be autotransplanted for preference. Type 1 b regions or nodules should not be used for grafting.

The structure of tight junctions in human thyroid tumors. A systematic freeze-fracture study.

Tight junctions of normal human epithelium comprise 6--8 intramembranous ridges. Tight junctions of adenomas and one case of follicular carcinoma were morphologically identical with normal controls. Another follicular carcinoma (clinically less malignant than the case mentioned above), however, showed loss of junctional polarity with spreading of ridges on lateral cell membranes. One case of papillary carcinoma (clinically low malignancy) displayed only focal attenuations of the tight junction belt. Another more malignant tumor of the same histologic type showed breaks of ridges and focal interruptions of the tight junction meshwork. Cell membranes of two anaplastic carcinomas of high malignancy were completely devoid of tight junction ridges. We conclude that tight junction alterations are not necessarily implicated in malignant transformation and that they correspond with tumor differentiation rather than directly with a single parameter of thyroid tumor malignancy.

The proliferation of adrenal medullary cells in newborn and adult mice. A light and electron microscopic autoradiographic study.

The proliferative activity of newborn and adult mouse adrenal medullary cells was determined with light and electron microscopic autoradiography. The H3 thymidine labelling index of 2 weeks old mice adrenal medullary cells was about 9.4% and declined to less than 1% in adult mice. In electron microscopic autoradiography labelled norepinephrine as well as epinephrine cells could be seen. Only in 1 and 2 weeks old mice some morphologically undifferentiated cells were visible. In formaldehyde induced fluorescence combined with light microscopic autoradiography the fluorescence intensities of labelled and unlabelled medullary cells were measured. On average the fluorescence intensity of labelled cells was lower than that of unlabelled cells. The differences could be explained by a higher number of autoradiographic silver grains laying on the cytoplasm of labelled cells. These results give evidence that fully differentiated adrenal medullary cells are capable of division.

'VA', a new type of erythrocyte polyagglutination characterized by depressed H receptors and associated with hemolytic anemia. II. Observations by immunofluorescence, electron microscopy, cell electrophoresis and Biochemistry.

With help of immunoflorescence, best with anti-AHP from Helix pomatia, a stippled structure could be demonstrated on the patient"s red blood cells. Thus an "A-like" receptor could be detected on the erythrocyte membrane of this group O patient. The reactive antigen was proved not to be a crypt antigen exposed by the action of neuraminidase. The same stippled fluorescence with antiAhp was observed on the red blood cells of a patient suffering from hemolytic anemia induced by influnza A2 virus. In this case this virus was shown not to be responsible for polyagglutination. No virus or microorganism could be isolated from the patient"s blood. Also by immunofluorescence the weak expression of the H antigen could be demonstrated with an extract of Evonymus europaeus. Electron microscopy of erythrocytes was normal. The neuraminic acid content and the electrophoretic mobility were found to be decreased to a minor degree. No distinct cell populations could be observed.