Macrophages promote tumour growth and liver metastasis in an orthotopic syngeneic mouse model of colon cancer.

PURPOSE: Tumour-associated macrophages have been shown to promote proliferation, angiogenesis and metastasis in several carcinomas. The effect on colon cancer has not yet been clarified. Furthermore, Kupffer cells in the liver might initiate the formation of metastases by directly binding tumour cells. METHODS: An orthotopic syngeneic mouse model of colon cancer as well as a liver metastases model has been studied, using murine CT-26 colon cancer cells in Balb/c-mice. Macrophages were depleted in both models by clodronate liposomes. Tumour sizes and metastases were determined using 7-Tesla MRI. The macrophage and vascular density in the orthotopic tumours as well as the Kupffer cell density in the livers were evaluated using immunohistochemistry. RESULTS: Animals in the macrophage-depleted group displayed significantly smaller primary tumours (37 ± 20 mm(3)) compared to the control group (683 ± 389 mm(3), p = 0.0072). None of the mice in the depleted group showed liver or peritoneal metastases, whereas four of six control mice displayed liver and five out of six mice peritoneal metastases. The vascular density was significantly lower in the macrophage-depleted group (p = 0.0043). In the liver metastases model, animals of the Kupffer cell-depleted group (14.3 ± 7.7) showed significantly less liver metastases than mice of the two control groups (PBS liposomes, 118.5 ± 28.2, p = 0.0117; NaCl, 81.7 ± 23.2, p = 0.0266). The number of liver metastases correlated directly with the Kupffer cell density (p = 0.0221). CONCLUSION: Macrophages promote tumour growth, angiogenesis and metastases in this orthotopic syngeneic mouse model. Kupffer cells enhance the formation of metastases in the liver.

A syngeneic orthotopic murine model of pancreatic adenocarcinoma in the C57/BL6 mouse using the Panc02 and 6606PDA cell lines.

BACKGROUND/AIMS: To develop a clinically relevant immunocompetent murine model to study pancreatic cancer using two different syngeneic pancreatic cancer cell lines and to assess MRI for its applicability in this model. METHODS: Two cell lines, 6606PDA and Panc02, were employed for the experiments. Cell proliferation and migration were monitored in vitro. Matrigel™ was tested for its role in tumor induction. Tumor cell growth was assessed after orthotopic injection of tumor cells into the pancreatic head of C57/BL6 mice by MRI and histology. RESULTS: Proliferation and migration of Panc02 were significantly faster than those of 6606PDA. Matrigel did not affect tumor growth/migration but prevented tumor cell spread after injection thus avoiding undesired peritoneal tumor growth. MRI could reliably monitor longitudinal tumor growth in both cell lines: Panc02 had a more irregular finger-like growth, and 6606PDA grew more spherically. Both tumors showed local invasiveness. Histologically, Panc02 showed a sarcoma-like undifferentiated growth pattern, whereas 6606PDA displayed a moderately differentiated glandular tumor growth. Panc02 mice had a significantly shorter (28 days) survival than 6606PDA mice (50 days). CONCLUSION: This model closely mimics human pancreatic cancer. MRI was invaluable for longitudinal monitoring of tumor growth thus reducing the number of mice required. Employing two different cell lines, this model can be used for various treatment and imaging studies.

Genetic and molecular aspects of hypospadias.

Hypospadias, a midline fusion defect of the male ventral urethra, is a relatively common genital anomaly occurring in 0.3 - 7 of 1000 live male births. The anatomical location of the misplaced urethral meatus determines the severity of this anomaly with the severity increasing from distal to proximal. Glandular and penile hypospadias, the most common forms, often appear as an isolated anomaly and account for the majority of hypospadias, whereas about 20 % are classified as scrotal and perineal types. These latter forms frequently occur in association with other genital anomalies such as microphallus, bifid scrotum, penoscrotal transposition, and cryptorchidism, and may represent an intersex phenotype. Besides a higher incidence in consanguineous families and a suggested recessive inheritance, in other families a dominant transmission is likely. The recurrence risk in the next generation seems to be correlated with the severity of hypospadias. Only 30 % of severe hypospadias can be attributed to defects in the synthesis of testosterone or adrenal steroid hormones, receptor defects, syndrome-associated hypospadias, chromosomal anomalies, defects in other genetic factors, or exogenous forms. To identify the underlying causes of the remaining 70 % "idiopathic" hypospadias, familial and twin studies were performed. Familial studies can help identify gene loci and, subsequently, candidate genes by mutational analysis. Either linkage analysis in large families with many affected individuals suspicious for a monogenic trait or association studies in cases of a complex inheritance in many families with a few affected individuals can be performed. Microarrays and proteomics can help detect gene expression or protein differences. Furthermore, genetically modified animal models can be used to detect phylogenetically homologous genes in man. In addition to an optimal documentation and acquisition of blood and tissue samples this requires a close cooperation between clinicians in the operative and non-operative specialties as well as geneticists.

Longitudinal data for intrauterine levels of fetal IGF-I and IGF-II.

OBJECTIVE: An increasing body of evidence supports a major role for the insulin-like growth factors (IGFs) in the control of human fetal growth. Individual data at various times of pregnancy suggest that IGF-I and IGF-II levels remain stable up to the 33rd week of pregnancy. Thereafter, both increase to reach values 2-3 times higher at term. In order to provide an accurate reflection of fetal IGFs in utero, we sampled fetal blood from the umbilical cord by cordocentesis. METHODS: We measured IGF-I and IGF-II in 12 fetuses longitudinally for up to 5 times between the 21st week of gestation and delivery. RESULTS: All patients showed a progressive increase in IGF-I and IGF-II levels. Data determined during different time intervals (before 29th, 29th to 32nd, after 32nd week) were compared and the main increase was found after the 32nd week. The median for IGF-I before the 29th week was 33.5 ng/ml (range 19-40.5) and increased to 41 ng/ml (32-59) between the 29th to 32nd and further to 54.1 ng/ml (range 17-70) thereafter. During the same time interval, the median for IGF-II increased from 217 ng/ml (86-326) to 349 ng/ml (227-467). In 7 patients, cord blood after delivery was available. For IGF-II a further increase was consistently found after birth (from 282 ng/ml (175-511) to 393 ng/ml (297-513)), whereas only 2 fetuses showed an increase in IGF-I. CONCLUSION: We conclude that in human fetuses, IGF-I and IGF-II levels increase longitudinally throughout pregnancy. Therefore, they may become important markers of healthy fetal development.

McCune-Albright syndrome--the German experience.

The classical McCune-Albright syndrome (MAS) consists of peripheral precocious puberty (PPP), fibrous bone dysplasia and café-au-lait spots. We conducted a survey among pediatric endocrinologists in Germany, Austria and Switzerland, most of them participating in the German Working Group for Pediatric Endocrinology (APE). Thirty-three physicians reported 58 patients. A detailed questionnaire yielded extensive data from 41 patients. Patients (36 females, 5 males) were diagnosed between the 4th week of life and 8 years. Symptoms included precocious puberty (37 patients), café-au-lait spots (35), fibrous bone dysplasia (32), hyperthyroidism (5), liver disease (5), phosphate wasting (4), GH hypersecretion (3), anemia in infancy (2), hyperprolactinemia (1), and Cushing's disease (1). Therapy of PPP included testolactone (15), tamoxifen (7), cyproterone acetate (CPA) (5), anastrozole (1) and exemestane (1). Testolactone, tamoxifen and CPA showed varying degrees of clinical remission; none was unanimously effective, but side effects were very rare. New aromatase inhibitors were not well validated in MAS. Eleven children received bisphosphonate therapy (pamidronate i.v. q 3 months) for fibrous bone dysplasia. Pamidronate was well tolerated, and pain improved when present, but data on preventive effects are not yet available. In conclusion, this survey describes a large cohort of patients with MAS, many with severe clinical problems, including major physical handicaps. Our results show that there is no 'gold standard' for the therapy of PPP; tested treatment regimens are not ideal, and new aromatase inhibitors need to be evaluated in controlled studies. Pamidronate was well tolerated, but preventive effects on bone dysplasia have not yet been proven.

Effective aromatase inhibition by anastrozole in a patient with gonadotropin-independent precocious puberty in McCune-Albright syndrome.

Testolactone is used to treat conditions with excessive estrogen synthesis, e.g. gonadotropin-independent precocious puberty in McCune-Albright syndrome (MAS). Unfortunately, daily treatment with testolactone requires 3 to 4 doses (10-20 tablets) and even at these doses it is sometimes ineffective. We treated a patient with MAS (café-au-lait spots; thelarche at age 2- 6/12 yr; menarche at 5- 5/12 yr; accelerated bone age [BA 10 yr]) with the highly selective aromatase inhibitor anastrozole (1 mg once per day). Tamoxifen 1 mg/kg per day was added for 1 year but was discontinued when an ovarian cyst developed with markedly elevated estradiol levels. Estradiol levels returned to normal after resuming anastrozole-only treatment and accelerated BA progressed only 6 months during 2 1/2 years of treatment. The potent estrogen suppressive action and simple dosage regimen of anastrozole suggest it may be advantageous compared to other aromatase inhibitors such as testolactone or anti-estrogens.

Complete virilization in congenital adrenal hyperplasia: clinical course, medical management and disease-related complications.

OBJECTIVE: In girls with congenital adrenal hyperplasia (CAH), genital ambiguity usually leads to a rapid neonatal diagnosis. Rarely, CAH causes complete virilization and male sex assignment with a delayed diagnosis. After being confronted with very specific problems in two of such patients, we collected data of patients with CAH and complete virilization in a nationwide study to delineate specific problems of these rare patients in order to improve their management. DESIGN AND PATIENTS: Through the German Working Group of Paediatric Endocrinology (Arbeitsgemeinschaft Pädiatrische Endokrinologie, APE), questionnaires were sent to all members caring for patients with CAH and complete virilization in their endocrine clinics. Data from 16 patients from 10 paediatric endocrine centres were assessed by questionnaire. RESULTS: The following problems have been encountered. (1) Sex assignment/gender identity: initially all patients had a male sex assignment. Six patients were diagnosed during the first month of life. Five were reassigned to female sex immediately, one at the age of 19 months. Except in one girl demonstrating some tomboyish behaviour, gender role behaviour in these patients did not differ from unaffected girls. Ten patients were diagnosed late at 3.4--7 years of age. In seven patients with a late diagnosis, male sex assignment was maintained; one of them expressed some concerns about living as a male. In three patients late sex reversal was performed, gender identity is very poor in one and new sex assignment is currently under consideration. (2) SURGERY: irrespective of the sex assigned, all patients had between one and three surgical procedures, including clitoris reduction and (repeated) vaginoplasties in patients with female sex assignment. Hysterectomy and ovarectomy were performed in patients with male sex assignment. (3) Short stature: patients with a late diagnosis of CAH had extremely advanced bone ages of +6.3 to +9.5 years, leading to severely reduced final height of 137 to 150 cm in adult patients. Patients tended to follow height percentiles of genetic females. One pubertal patient was suicidal due to short stature. (4) Central precocious puberty (CPP): prolonged exposition to adrenal androgens led to CPP in one patient. He was treated with GnRH analogues until gonadectomy. CONCLUSIONS: Patients with CAH and complete virilization have a high risk of being diagnosed late. There are major problems and uncertainties of the patients' families and the treating physicians concerning gender assignment. Gender identity is disturbed in some patients. In addition, multiple surgical procedures are necessary and short stature as well as central precocious puberty might be important to avoid late sequelae. While some surgical interventions are probably unavoidable, most of these issues could be resolved with an early diagnosis. Thus, especially for these patients, a neonatal screening programme for CAH would be of paramount importance.

Overview of pulse actions in the human.

Many hormones are secreted in a pulsatile fashion. The knowledge of this pulsatility has brought about detailed descriptions of hormone fluctuations employing sophisticated methods, but only a few advantages in patient care. Two areas of research comprise the analysis of the effects of single pulses on target cells and the development of circadian rhythms in newborn humans. This article gives an overview of these aspects of hormone physiology.

Diagnosis and management of juvenile hyperthyroidism in Germany: a retrospective multicenter study.

This retrospective multicenter study was designed to survey the management of childhood and adolescent hyperthyroidism in six pediatric endocrinological units in Germany. Fifty-six patients aged between 1.1 and 17.0 yr (median 10.5 yr) were enrolled. Data were collected retrospectively from the patients' records by a trained pediatric endocrinologist using standardized questionnaires. After the diagnosis of hyperthyroidism was established on the basis of clinical and biological findings, treatment with antithyroid drugs (carbimazole, methimazole, thiamazole, propylthiouracil) was started in all patients. In 55/56 of the patients treated with antithyroid drugs, euthyroidism was achieved (98%). However, 26 patients (47%) were still hyperthyroid after discontinuation of the medication. Eight children with continued hyperthyroidism ultimately underwent subtotal thyroidectomy 13-136 (median 28) months after the initial diagnosis. Management principles of the participating centers were heterogeneous. As a consequence, prospective multicenter studies are urgently needed to establish clear standards for the diagnosis and therapy of childhood hyperthyroidism.

Longitudinal changes of circadian leptin, insulin and cortisol plasma levels and their correlation during refeeding in patients with anorexia nervosa.

OBJECTIVE: To study the longitudinal changes in plasma levels of leptin, insulin and cortisol during the transition from the state of starvation to the state of refeeding focussing on diurnal secretion characteristics and their temporal relationships. DESIGN: Leptin, insulin and cortisol were measured every 2h for 24h during acute starvation (T1). Sampling was repeated after reaching half the target-body mass index (BMI) (T2) and again at target-BMI (17. 5kg/m(2); T3). The temporal relationships between the diurnal secretion patterns were assessed by cross-correlation analysis. RESULTS: Although BMIs at T1 were uniformly low, leptin levels varied widely within a range clearly below normal levels (0.03-1. 7microg/l). With increasing body fat during the course of refeeding, mean leptin levels increased from 0.64microg/l (range: 0.27-1. 73microg/l) (T1) to 1.61microg/l (range: 0.36-4.2microg/l) (T2) and to 3.67microg/l (range: 0.7-9.8microg/l) (T3). Circadian leptin secretion patterns showed maximal values uniformly around 0200h and minimal values around 0800h at all stages of the study. At all three weight levels, plasma leptin levels were highest between midnight and the early morning hours and lowest around the late morning hours. Refeeding neither profoundly changed secretion patterns of leptin nor did it change the positive, time-delayed relationship between leptin and insulin with increments in insulin secretion preceding those of leptin by 6h. A temporal relationship between leptin and cortisol could not be demonstrated in the state of semistarvation but emerged after a substantial weight gain; at that time, leptin increases preceded cortisol increases by 8h. CONCLUSIONS: Absolute leptin, insulin and cortisol levels are profoundly changed during starvation in anorectic patients, while refeeding, paralleled by a BMI gain, reverses these changes. During refeeding the relationship between leptin and cortisol changed profoundly, showing no significant correlation in the state of starvation, whereas at T3 after refeeding a strong inverse relationship could be observed. Leptin and insulin did not correlate significantly at any of the three stages studied.

Basal inhibin B and the testosterone response to human chorionic gonadotropin correlate in prepubertal boys.

During childhood, the quiescent phase of testicular activity, the hCG stimulation test is widely used to evaluate testicular function. Inhibin B, a gonadal peptide regulating FSH secretion, is an established marker of Sertoli cell function and spermatogenesis in adults. In contrast to the other hormones of the hypothalamo-pituitary-gonadal axis, inhibin B is also secreted in detectable amounts during childhood. The aim of this study was to determine whether basal inhibin B levels are able to predict prepubertal testicular function, so as to avoid a stimulation test. Inhibin B and testosterone before and after hCG stimulation were measured in 54 male children with various testicular disorders by an immunoassay specific for inhibin B. Basal inhibin B was compared to the testosterone increase after hCG. Inhibin B and the hCG-induced testosterone increment correlated strongly (r = 0.84; P<0.0001). Patients with anorchia were clearly distinguishable from those with abdominal testes, having undetectable (inhibin B, <15 pg/mL) respective normal inhibin B levels for age. Inhibin B and the testosterone response to hCG were low in boys with testicular damage (delayed diagnosis of cryptorchidism; after testicular torsion) and in patients with gonadal dysgenesis, but were normal or increased in children with androgen insensitivity syndrome. We conclude that basal inhibin B predicts the testosterone response to hCG in boys and therefore gives reliable information about both the presence and function of the testes. The diagnostic procedure in cryptorchidism may be reduced to a single inhibin B measurement. Furthermore, inhibin B levels show specific alterations in patients with sexual ambiguity, adding a valuable diagnostic tool to the complex differential diagnosis of male pseudohermaphroditism.

Time relationship between circadian variation of serum levels of leptin, insulin and cortisol in healthy subjects.

BACKGROUND: Leptin is involved in the regulation of eating behavior. Its serum levels are determined by fat mass but a diurnal rhythm is also described. It is not clear whether leptin levels are also controlled in vivo by hormonal stimuli, like insulin or cortisol. METHODS AND RESULTS: This possible temporal relation was investigated by serial measurements during 24 h (group A) and 46 h (group B) in 15 healthy volunteers and another 10 subjects (group C) while fasting for 72 h. Maximal leptin levels were observed at 4:00 a.m. and 4:00 p.m. in subjects on a normal diet. During 24 h starvation (group B), there was a 40% decrease of mean leptin concentration when compared to baseline values. In group C, the leptin concentration under starvation dropped to 25% of basal levels after 72 h. Pooled data from group A and the nonfasting data from group B showed an insulin increase preceding leptin increase by 6 h (r = 0.405, p < 0.0001), while cortisol decreased 4 h (r = 0.361, p < 0.001) after leptin decrease. CONCLUSION: Starvation results in a fall of circulating leptin, ending leptin rhythmicity. Food intake is causally involved in the fluctuation of leptin levels in serum. Presumably this effect is mediated by insulin, while cortisol does not seem to affect leptin release directly in vivo.

Homozygous alpha-thalassaemia and hypospadias--common aetiology or incidental association? Long-term survival of Hb Bart's hydrops syndrome leads to new aspects for counselling of alpha-thalassaemic traits.

UNLABELLED: Fetuses with homozygous alpha-thalassaemia develop Hb Bart's hydrops fetalis syndrome, which usually leads either to abortion or fetal/neonatal death. We report diagnosis, intrauterine transfusion therapy, neonatal intensive care management and long-term follow-up of a Vietnamese infant who survived Hb Bart's hydrops fetalis syndrome. During the first 2 years the child had normal development. In addition, the patient exhibited penoscrotal hypospadias. Despite a thorough endocrinological work-up the aetiology of genital ambiguity could not be elucidated. A review of the literature showed an association of homozygous alpha-thalassaemia and hypospadias in all surviving male children, suggesting a common aetiology for both entities. CONCLUSION: On the basis of our findings, we speculate that an unknown gene on chromosome 16 responsible for genital formation is altered in homozygous alpha-thalassaemia.

Acute N-methyl-D,L-aspartate administration stimulates the luteinizing hormone releasing hormone pulse generator in the ovine fetus.

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130-142 days of gestation; term 147 +/- 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 +/- 0.36 ng/ml) and oFSH (1.21 +/- 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 microg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 microg/day i.v. x 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D, L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10(-10) to 10(-6) M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10(-8) M; p < 0.0004) and oFSH (10(-8) M; p < 0. 0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

Circadian plasma leptin levels in patients with anorexia nervosa: relation to insulin and cortisol.

In anorexia nervosa, underweight results from a loss of body mass due to a restricted energy intake. Circulating leptin levels have been shown to be low in the acute stage of the disorder. We studied diurnal secretion characteristics of leptin, insulin and cortisol in a study group of anorectic patients prior to refeeding, a second study group of anorectic patients after initiation of refeeding and study groups of healthy underweight and normal-weight controls. Spontaneous secretion of leptin, insulin and cortisol was measured by drawing blood samples every 2 h for 24 h. The temporal relationships between the diurnal secretion patterns of the three hormones were assessed by cross-correlation analysis in every study group. Plasma levels of leptin and cortisol were secreted with a specific circadian rhythmicity and displayed an intricate temporal relationship in anorectic patients. Semistarvation in the non-refed patients was associated with (1) exceedingly low plasma leptin levels, (2) a qualitative alteration in the circadian rhythm of leptin and cortisol levels and (3) an alteration in the temporal coupling between cortisol and leptin. In contrast, in the patients who had gained weight, leptin levels were higher; furthermore, the diurnal pattern of leptin and the temporal relationship between leptin and cortisol were similar to controls. Increments in insulin secretion preceded those of leptin by 4-6 h in both anorectic patients and in controls. Leptin levels increased 4 h prior to those of cortisol in controls and in refed patients, whereas in the non-refed patients cortisol increased prior to leptin. Thus, anorexia nervosa leads to pronounced, albeit reversible changes in the secretion dynamics of leptin and cortisol.

Management of recombinant human growth hormone therapy at puberty.

Treatment modalities for growth hormone (GH) substitution in GH-deficient children at puberty is still a matter of debate. Although circulating GH levels increase during puberty, it has not been proven that the increase of exogenous GH is necessary for a normal pubertal growth spurt. Increasing GH levels may thus well be the consequence and not the cause of the pubertal growth spurt. A permissive role of GH for pubertal growth has been hypothesized. Some effects of exogenous GH may even be detrimental to pubertal growth; specifically, puberty seems to be shortened by GH administration. Further aspects of the physiology of GH secretion and the poor imitation by current replacement schemes are discussed. Only randomized, prospective studies will allow to define the optimal dose of GH at puberty. As long as these studies are missing, a pragmatic approach is an individualized, minimalistic GH substitution scheme under close surveillance. GH dose at puberty should be kept at prepubertal levels, but must be increased once height velocity drops below the 50th percentile. This widely used approach ensures unnecessary, costly and potentially harmful overdosages of GH.