DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.

Detection and molecular typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and in different patient samples from southwest Germany.

The prevalence of different genospecies of Borrelia burgdorferi sensu lato in infected ticks could be a determinant for the risk of acquiring Lyme borreliosis (LB) and its clinical presentation. A total of 7373 ticks and 2761 samples from LB patients from the same area in southwest Germany were analyzed by PCR to assess the frequency of the occurrence of LB-associated genospecies. Fifteen percent of the tick samples and 19% of the human samples were found positive for the presence of B. burgdorferi sensu lato. Further identification of 1106 B. burgdorferi sensu lato positive tick samples by reverse line blotting and 125 positive patient samples by nested PCR using species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto, B. garinii and B. valaisiana. Both single-species and mixed infections were noted and a similar distribution of the different genospecies was found in ticks compared with human samples. It was also the purpose of this study to obtain more information about a possible correlation between the distribution of Borrelia species and clinical syndromes of LB. Skin biopsies of 59 patients with acrodermatitis chronica atrophicans and cerebrospinal fluid samples from 78 patients with possible neuroborreliosis were analyzed. In conclusion, the distribution of the different genospecies in ticks is the decisive factor for the occurrence of the different Borrelia genospecies in samples from LB patients. Borrelia afzelii is the predominant genospecies in all kind of samples from the observed area and there seems to be no association of particular Borrelia genospecies with distinct clinical manifestations of LB.

[Autonomy of the thyroid gland: is there an "organ hyperthyroidism" of "euthyroid" patients?]. / Schilddrüsenautonomie: Gibt es eine "Organhyperthyreose" bei "euthyreoten" Patienten?

BACKGROUND: Classic thyrotoxicosis is defined as the clinical syndrome of hypermetabolism that results when concentrations of serum free thyroxine (fT4), serum free triiodothyronine (fT3), or both are increased and serum level of TSH is suppressed. The term of subclinical thyrotoxicosis refers to a usually asymptomatic state associated with normal serum fT4 and fT3 and low serum TSH concentrations. AIM: We describe a new entity of thyrotoxicosis under the term of "organ-selective thyrotoxicosis". This refers to patients with mild clinical symptoms of thyrotoxicosis and with a non-suppressible thyroid toxic adenomas and normal serum concentrations of fT4, fT3 and TSH. PATIENTS AND METHODS: We compared symptoms and clinical signs of thyrotoxicosis with serum levels of fT4, fT3 and TSH in 33 patients with toxic adenomas. These patients were divided into 2 groups, 19 patients had normal concentrations of serum fT3, fT4 and TSH belonging to the group of "organ-selective thyrotoxicosis", 14 patients with subclinical thyrotoxicosis were in the control group. RESULTS: In both groups, mild symptoms of thyrotoxicosis were apparent but there was no significant difference between the 2 groups detectable. Therapeutic options were discussed with the patients referring to their symptoms. We describe 1 case of a female patient, in which we carried out an alcohol obliteration of a single toxic adenoma. CONCLUSION: Biological availability of thyroid hormones in patients with toxic adenomas might be elevated in selected organs although serum levels of fT4, fT3 and TSH are normal. This might be due to an increased production rate of T4 on the one hand and a specific peripheral T4/T3 conversion rate on the other hand. This might lead to an organ-selective thyrotoxicosis in the periphery without concerning the thyrotroph, so that TSH stays within the normal range. Necessity of therapeutic interventions depends on clinical signs and symptoms.

[QT syndrome: new diagnostic possibilities]. / QT-Syndrome: Neue diagnostische Möglichkeiten.

Electrocardiographic and clinical characteristics are currently used as diagnostic criteria for the long QT-syndrome. In borderline electrocardiographic findings associated with unclear syncope, it is often difficult to ensure or exclude long QT-syndrome. Schwartz and coworkers therefore created a point system as a guide in clinical decision making. In recent years genetic diagnostics have entered the arena of long-QT assessment. Aside from new insights into the pathophysiology of the long QT-disorder, it is expected that genetic diagnostics will offer substantial help to ascertain long QT-syndrome in patients with borderline electrocardiographic and clinical findings and improve risk stratification in long-QT family members. Performing linkage analysis, coupling of autosomal-dominant congenital long QT-syndrome (Romano-Ward Syndrome) to chromosomes 11 (LQT1/11p15.5), 3 (LQT3/3p21), 7 (LQT2/7q35), and 4 (LQT4/4q25-27) was demonstrated. More recently, the disease genes in long QT-syndrome 1, 2, and 3 could be identified. Analysis of the base-pair sequence allowed detection of several different mutations in different families illustrating genetic heterogeneity. Aside from diagnostic aspects, molecular genetics may also guide pharmacological therapy by identifying the specific ion-channel disorder leading to QT-prolongation and sudden death.

A role for helix 3 of the TRbeta ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone beta receptor (TRbeta) mutations which cluster in two regions (alphaalpha 310-353 and alphaalpha 429-461) of the hormone-binding domain and closely approximate the ligand-binding cavity. Here, we describe a third cluster of RTH mutations extending from alphaalpha 234-282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (Ka). T3-dependent recruitment of coactivators (SRC-1, ACTR) by mutant receptor-RXR heterodimers was reduced in comparison with wild-type. Cotransfection of SRC-1 restored transactivation by T277A. In the TRbeta crystal structure this helix 3 residue is surface-exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptor-coactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Delta276I, A279V, R282S) in this cluster correlated with their reduced Ka and they inhibited wild-type TRbeta action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions.

Time series prediction of plasma hormone concentration. Evidence for differences in predictability of parathyroid hormone secretion between osteoporotic patients and normal controls.

Recent evidence links osteoporosis, a disease of bone remodeling, to changes in the dynamics of parathyroid hormone secretion. We use nonlinear and linear time series prediction to characterize the secretory dynamics of parathyroid hormone in both healthy human subjects and patients with osteoporosis. Osteoporotic patients appear to lack the periods of high predictability found in normal humans. Our results may provide an explanation for why an intermittent administration of parathyroid hormone is effective in restoring bone mass in osteoporotic patients.

Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perifused human osteosarcoma cells (SaOS-2).

The pulsatile but not the continuous application of parathyroid hormone (PTH) increase bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1-34) (10(-7) M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC50 = 1 x 10(-10) M PTH(1-34)). Neither 8Br-cAMP (2 x 10(-4) M) nor PMA(1 x 10(-7) M) had an effect on the PTH(1-34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [125I]-[Tyr36]hPTHrP(1-36)amide binding to SaOS-2 cells was decreased by 60-70% by PTH(1-34) (1 x 10(-6) M), bPTH(1-84) (1.8 x 10(-6) M) and bPTH(3-34) (2 x 10(-6) M), whereas 8Br-cAMP (2 x 10(-4) M) had no effect on radioligand binding. PMA (1 x 10(-7) M) appeared to slightly increase [125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent EC50 = 3 x 10(-7) M PTH(1-34)). The maximal effect occurred after 10-30 min and was largely reversible within 2 h. Monensin (3 x 10(-5) M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1-34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.

Is there low-dimensional chaos in pulsatile secretion of parathyroid hormone in normal human subjects?

In many biological systems information is transferred by hormonal ligands, and it is assumed that these hormonal signals encode developmental and regulatory programs in mammalian organisms. The specificity of the biological response on activation by a hormone has so far been located within the interaction of a specific conformation of the ligand with the corresponding receptor structure. According to these classical explanations, the constant circulating hormonal pool described by the rate of its production and metabolic clearance is a major determinant of this interaction. Recently it has become apparent that hormone pulses contribute to this hormonal pool. Phase-space analysis of dynamic parathyroid hormone (PTH) secretion allowed the definition (in comparison to normal subjects) of a relatively quiet "low dynamic" secretory pattern in osteoporosis, and a "high dynamic" state in hyperparathyroidism. We now investigate whether this pulsatile secretion of PTH in healthy humans exhibits characteristics of low-dimensional deterministic chaos. Our findings suggest that this indeed appears to be the case. PTH secretion thus seems to be a first example of a chaotic hormonal rhythm in human physiology.

Pulse amplitude and frequency modulation of parathyroid hormone in early postmenopausal women before and on hormone replacement therapy.

Although the pathophysiology of postmenopausal osteoporosis has been investigated extensively, it is still not established in what respect PTH is related to the events. Recently, consistent data on the pulsatile secretion of PTH in man have been published. In this study intact PTH was measured in six early postmenopausal women before and after 6 months of hormone replacement therapy (HRT; 0.6 mg conjugated estrogens and 5 mg medrogestone). In addition to parameters of calcium metabolism and bone mass and to control HRT, intact PTH was measured in blood drawn over 6 h every 2 min. With HRT there was a 30% reduction in PTH secretion. Both the amount secreted per pulse (baseline, 26.8 +/- 6.9 ng/L; HRT, 21.4 +/- 7.6 ng/L; P < 0.05) as well as the basal secretion (baseline, 232.6 +/- 117.6 ng/L.h; HRT, 145.5 +/- 80.0 ng/L.h; P < 0.01) were reduced, whereas the pulse count per h remained constant (baseline, 5.1 +/- 2.2; HRT, 5.1 +/- 1.3). Power spectrum analysis showed a shift in spectral maxima consistent with these findings. Ionized and total calcium were slightly, but nonsignificantly, reduced with treatment. In summary we conclude that in early postmenopausal women, HRT reduces the secretion of PTH by reducing both the basal secretion and the amount secreted per pulse. It is conceivable that some of the known effects of HRT on bone metabolism might be mediated by the modulation of PTH secretion.

Pulse amplitude and frequency modulation of parathyroid hormone in primary hyperparathyroidism.

Pulsatile secretion of PTH in human subjects has been described recently. However, the pattern of PTH secretion in primary hyperparathyroidism (pHPT) remains to be characterized. In this study intact PTH was measured in 9 female patients with pHPT. As a control group we present data from 10 postmenopausal women. In addition to parameters of calcium metabolism and bone mass, PTH was measured in samples drawn over 4 or 6 h every 2 min by central venous blood sampling. The mean intact PTH concentration was 39.0 +/- 20.3 ng/L in healthy women and 193.2 +/- 127.9 ng/L in female patients with pHPT (P < 0.01). Pulse rhythm analysis showed significant differences between both groups for total PTH secretion per h (patients, 1196.4 +/- 485.3 ng/L; control group, 271.7 +/- 132.2 ng/L), basal PTH secretion per h (patients, 852.4 +/- 459.1 ng/L; control group, 185.6 +/- 126.1 ng/L), and average PTH secretion per pulse (patients, 112.6 +/- 54.8 ng/L; control group, 23.2 +/- 7.1 ng/L). Both patients and control subjects had, on an average, five pulses per h, and the pulsatile secretion accounted for about 50% of the total secretion. Differences in power spectrum analysis were consistent with these findings. The cross-correlation of PTH and calcium indicates an impaired feedback regulation in pHPT. PTH secretion in female patients with pHPT results from both an increased basal secretion and an increased amplitude of PTH pulses. Other features of secretion are the same as those in normal women. Feedback regulation of PTH and calcium is impaired in pHPT.

Temporal patterns of alpha 1-receptor stimulation regulate amplitude and frequency of calcium transients.

The pulsatile release of neurotransmitters and many hormones might encode specific biological information according to temporal pattern. We tested this hypothesis by applying pulsed alpha 1-adrenoceptor stimulation to single aequorin-injected hepatocytes. The amplitude of free Ca2+ transients induced by rapid phenylephrine pulses (20-s interpulse interval) and continuous stimulation was similar (approximately 640 nM) but increased to approximately 1,000 nM as the interpulse interval was increased to 120 s. The same overall response was maintained despite a 13-fold reduction in average phenylephrine concentration. Some regimes of pulsed phenylephrine stimulation could give a higher frequency of pulsed phenylephrine stimulation could give a higher frequency of free calcium oscillations than continuous stimulation, or more rapid stimulation when some agonist pulses failed to elicit a free Ca2+ transient. For the same average phenylephrine concentration (0.3-0.6 microM), pulsed regimes could result in significantly higher frequencies and integrated responses than constant application. The lags between phenylephrine pulses and free Ca2+ transients reduced as the period between pulses increased. The amplitude and lag data are consistent with a refractory period of 18 s and a recovery phase with a time constant of approximately 100 s, perhaps corresponding to dephosphorylation of alpha 1-adrenoceptors phosphorylated by protein kinase C during each free Ca2+ transient.

[A structural parameter for characterizing early trabecular changes in the spine in osteoporosis]. / Ein Strukturparameter zur Beschreibung früher trabekulärer Veränderungen der Wirbelsäule bei Osteoporose.

The pattern of the spongiosa in lumbar vertebrae demonstrated by ultra high resolution CT was treated statistically. Comparison of normals with osteoporotic vertebrae showed no significant difference in the large dominant trabeculae. However, there is a significant difference when quantifying the fine structures which are not to resolve lying between the larger trabeculae.

Early adaptation of thyrotropin and thyroglobulin secretion to experimentally decreased iodine supply in man.

Five healthy male volunteers (aged 25 to 28 years) were studied both after 4 weeks of treatment with 200 micrograms iodine/d orally (PO) and following experimental iodine depletion by treatment with 3 x 300 mg perchlorate/d PO over a 4-week period, in an attempt to better define the early adaptive responses to an alteration in iodine supply in thyroid function. Intrathyroidal iodine, serum triiodothyronine (T3), free T3 (FT3), thyroxine (T4), free T4 (FT4), reverse T3 (rT3), thyroxine-binding globulin (TBG), thyroglobulin (Tg), and thyrotropin (TSH) levels (10-minute sampling over 24 hours) were measured at the end of iodine administration and at the end of perchlorate treatment. Thyroid volume was determined by sonography, and iodine content was determined by fluorescence scintigraphy. TSH pulses were analyzed by computer-assisted programs. Comparing both experimental situations, perchlorate treatment significantly reduced intrathyroidal iodine concentration (4.0 +/- 1.3 to 3.0 +/- 1.2 nmol/mL, P less than .05), but thyroid volume and total serum T4, T3, FT3, and TBG levels were not altered. Mean 24-hour serum TSH levels (1.8 +/- 0.3 to 1.0 +/- 0.3 mU/L, P less than .001), amount of TSH secreted/pulse (0.5 +/- 0.1 to 0.3 +/- 0.1 mU/L, P less than .001), and FT4 levels (15.7 +/- 1.7 to 14.3 +/- 1.4 pmol/L, P less than .005) were significantly diminished, whereas Tg levels (18.6 +/- 10.0 to 35.1 +/- 14.0 ng/mL, P less than .01) were significantly increased. Thyroid-specific antibodies were normal and were not altered by treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Classification of dynamical diseases by new mathematical tools: application of multi-dimensional phase space analyses to the pulsatile secretion of parathyroid hormone.

The biological importance of dynamic hormonal secretion has been demonstrated. There is good evidence from recent studies that parathyroid hormone (PTH) which plays an important role in bone physiology is secreted in a pulsatile manner. In this study we performed a classification of two 'dynamical diseases' namely osteoporosis and hyperparathyroidism by the visualization of dynamic PTH-secretion in multidimensional phase spaces.

Evidence for P2-purinoceptors on human osteoblast-like cells.

ATP released from damaged cells or by controlled secretion could be an important factor in the formation or remodeling of bone. In a variety of other tissues ATP has been shown to control cellular processes by acting on P2-purinoceptors and activating the calcium signaling pathway. Here we demonstrate for the first time that extracellular ATP increases the intracellular free calcium [Ca2+]i concentration in normal human osteoblasts and in SaOS-2 cells, a human osteosarcoma-derived cell line, but not in ROS 17/2.8 cells. The ATP-induced increase in [Ca2+]i was dose dependent, and the concentrations of ATP required were similar to those reported to regulate cellular functions in other cell types. Although ATP is metabolized rapidly by bone cells, the effects on [Ca2+]i appeared to be mediated directly by ATP rather than one of its metabolites. Adenosine 3-thiotriphosphate, a nonhydrolyzable analog of ATP, induced similar changes in [Ca2+]i. This indicates that P2-purinoceptors are present on osteoblast-like cells and that extracellular ATP from various sources might be an important factor in the regulation of osteoblast functions.

The nude rat is established as a model for endocrine studies: regulation of thyroid function and xenotransplantation of a thyroid tumor cell line.

The thyroid physiology of athymic nude rats, rnu/rnu, is characterized and established here as an animal model to study transplanted thyroid tumors. Male rats were catheterized 5 days before experiments were started. The mean thyroid-stimulating-hormone (TSH) plasma concentrations were 2.9 +/- 0.6 ng/ml during infusion of 0.25 ml/h of 0.9% NaCl (n = 12). T3 plasma concentrations were 2.6 +/- 0.4 ng/ml. T4 plasma levels were 22.0 +/- 5.6 micrograms/dl. A bolus of 0.1 mg thyrotropin-releasing hormone (TRH) significantly increased TSH plasma concentrations (P less than or equal to 0.001; from 2.9 +/- 0.6 to 7.8 +/- 1.1 ng/ml, n = 12). No pulsatile TSH secretion was observed in a 2-hour period with blood samples taken every 10 minutes (n = 12) and hourly sampling disclosed no circadian variation of TSH during a 24-hour period (n = 4). Successful xenografting was possible in 12 of 15 cases using a follicular thyroid carcinoma cell line (FTC 133). Measurement of human thyroglobulin (hTg) by a hTg IRMA revealed high levels in rats with functional FTC tumors, whereas no hTg was detected in untransplanted rats or animals with nonfunctional transplants.

[The osteoprotective effect of ovarian hormones]. / Die osteoprotektive Wirkung der Ovarialhormone.

Osteoporosis is regarded as a disease of the systems controlling bone evolution. Estrogen modulates such a control system; without estrogen 50% of bone is not formed to the peak bone mass or may be lost lateron. Substitution with estrogen can be interpreted as a natural right of the women in our society. Estrogen modulates by dynamic hormonal system the genexpression in bone and determines its three-dimensional architecture. Substitution with estrogen has to consider the individual genetic situation of each women and its biographic and health situation. Individual estrogen substitution is not fully realized yet but estrogen substitution prevents osteoporosis effectively. We question if senile osteoporosis exists at all if there were no estrogen deficiency.