Limb lengthening in children with Russell-Silver syndrome: a comparison to other etiologies.

INTRODUCTION/BACKGROUND: Russell-Silver syndrome (RSS) is the combination of intrauterine growth retardation, difficulty feeding, and postnatal growth retardation. Leg length discrepancy (LLD) is one of four major diagnostic criteria of RSS and is present in most cases. We aimed to ascertain whether pediatric RSS patients will adequately consolidate bony regenerate following leg lengthening. MATERIALS AND METHODS: We retrospectively reviewed pediatric RSS patients who underwent limb lengthening and compared them to a similar group of patients with LLD resulting from tumor, trauma, or congenital etiology. The primary outcome measurement was the bone healing index (BHI). RESULTS: The RSS group included seven lengthened segments in five patients; the comparison group included 21 segments in 19 patients. The groups had similar lengthening amounts (3.3 vs. 3.9 cm, p = 0.507). The RSS group healed significantly faster (lower BHI) than the control group (BHI 29 vs. 43 days/cm, p = 0.028). Secondary analysis showed no difference between RSS and trauma patients in terms of the BHI (29 vs. 31); however, the BHI of the RSS group was significantly lower than both of the other congenital etiologies (29 vs. 41, p = 0.032) and tumor patients (29 vs. 66, p = 0.019). The RSS patients had fewer and less significant complications than the controls. DISCUSSION: The limb lengthening regenerate healing of RSS patients is faster than the healing of patients with other congenital etiologies and tumor patients, and is as fast as the regenerate healing of patients with posttraumatic LLD. Although all RSS patients were treated with human growth hormone (hGH), we are unable to isolate the hGH contribution to the regenerate bone healing. We conclude that RSS patients can have safe limb lengthening.

A randomized crossover study to evaluate Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist in women with stress urinary incontinence.

OBJECTIVES: To investigate the potential therapeutic benefits of the selective alpha1A/1l-adrenoceptor partial agonist Ro 115-1240 in women with mild-to-moderate stress urinary incontinence (SUI). PATIENTS AND METHODS: Thirty-seven women with mild-to-moderate SUI were enrolled in a randomized, placebo-controlled crossover study. Patients received 1.5 mg Ro 115-1240 twice daily or matching placebo for 2 or 4 weeks. Voiding diaries were used to record the number of SUI episodes, urge incontinence episodes and pads used. Sitting blood pressures and heart rate were recorded at each visit. RESULTS: Ro 115-1240 was associated with a significantly lower mean weekly number of SUI episodes than placebo (8.4 vs 6.0; P= 0.0079), a 28% relative improvement over placebo. There was also a significantly lower mean number of pads used and wet pads changed/week with Ro 115-1240 than with placebo (P = 0.0055 and 0.0066, respectively). The most frequently reported treatment-emergent adverse events were scalp tingling, headache, chills, piloerection, and pruritus. Generally these events were transient and mild to moderate. There was a slightly lower mean sitting heart rate with Ro 115-1240 than with placebo, but no difference in mean systolic or diastolic blood pressure between treatments. CONCLUSIONS: This study suggests that selective alpha1A/1l-adrenoceptor partial agonists have the potential to improve the symptoms of SUI with little or no cardiovascular effect. These results are encouraging and a randomized controlled trial of Ro 115-1240 in a larger population with SUI is warranted to substantiate these findings.

Meier-Gorlin syndrome: report of eight additional cases and review.

The Meier-Gorlin syndrome or ear, patella, short stature syndrome (MIM 224690) is a rare autosomal recessive disorder, characterized by the association of bilateral microtia, aplasia/hypoplasia of the patellae, and severe pre- and postnatal growth retardation. Twenty-one cases have been reported in literature thus far. Here we report on eight patients from seven families and compare them with previously described cases. One of the present cases had previously undescribed genital anomalies. There is a difference in facial characteristics between patients reported in early infancy and those described at older age; follow-up of patients is needed to substantiate this changing facial phenotype. We recommend radiographic survey of the patellae in patients at older age to investigate the weight of absent or hypoplastic patellae in the diagnosis of the syndrome.

Growth hormone therapy alone or in combination with gonadotropin-releasing hormone analog therapy to improve the height deficit in children with congenital adrenal hyperplasia.

Short stature in the adult patient with congenital adrenal hyperplasia (CAH) is commonly seen, even among patients in excellent adrenal control during childhood and puberty. In this study we examine the effect of GH therapy on height prediction in children with both CAH and compromised height prediction. Leuprolide acetate, a GnRH analog (GnRHa), was given to patients with evidence of early puberty. GH (n = 12) or the combination of GH and GnRHa (n = 8) was administered to 20 patients with CAH while they continued therapy with glucocorticoids. Each patient in the treatment group was matched according to age, sex, bone age, puberty, and type of CAH with another CAH patient treated only with glucocorticoid replacement. The match was made at the start of GH treatment. Of the 20 patients, 12 have completed 2 yr of therapy. After 1 yr of GH or combination GH and GnRHa therapy, the mean growth rate increased from 5 +/- 1.9 to 7.8 +/- 1.6 cm/yr vs. 5.4 +/- 1.7 to 5 +/- 2 cm/yr in the group not receiving GH (P < 0.0001). During the second year of treatment, the mean growth rate was 6 +/- 1.6 vs. 4.2 +/- 2.1 cm/yr in the group not receiving GH (P < 0.001). The height SD score for chronological age in the treatment group at the end of 1 and 2 yr of treatment improved significantly more than the nontreatment group (P < 0.01). A similar improvement in the height SD score for bone age was found in the treatment group after 1 (-1.4 +/- 0.9 vs. -1.7 +/- 0.9; P < 0.0001) and 2 yr of therapy (-0.67 +/- 0.68 vs. -1.7 +/- 1.2; P < 0.0004). The mean predicted adult height improved from 159 +/- 11 (baseline) to 170 +/- 7.5 cm (after 2 yr of therapy) closely approximating target height (173 +/- 8 cm). All patients continued the hydrocortisone treatment. In patients with CAH and compromised height prediction, treatment with GH or the combination of GH and GnRHa results in an improvement of growth rate and height prediction and a reduction in height deficit for bone age.

Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma.

We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.

Osteoporosis: an unusual presentation of childhood Crohn's disease.

Osteoporosis is known to be associated with Crohn's disease. We report a 12-yr-old boy without a history of steroid use, in whom severe osteoporosis and multiple collapsed vertebrae were the presenting manifestations of Crohn's disease. After treatment of the Crohn's disease, he resumed normal growth and progressed through puberty. Concomitantly, he demonstrated a substantial recovery of vertebral bone mineral density and structure. Possible pathophysiological mechanisms underlying the osteoporosis and the subsequent improvement in bone density are discussed.

Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms.

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.

Therapy of human pancreatic carcinoma implants by irinotecan and the oral immunomodulator JBT 3002 is associated with enhanced expression of inducible nitric oxide synthase in tumor-infiltrating macrophages.

We determined the therapeutic effect of irinotecan (CPT-11) combined with the immunomodulator JBT 3002, a synthetic bacterial lipopeptide (N-acylated derivative of psi-amino-C1-C3-alkane-sulfonic acid), against highly metastatic human pancreatic carcinoma cells injected into the pancreas of athymic nude mice. Mice received four courses consisting of three daily oral doses of JBT 3002, followed by once weekly i.p. injection of CPT-11. Control mice were treated with CPT-11 alone, JBT 3002 alone, or saline. Tumor growth and metastasis were assessed by gross pathology and confirmed by histological examination. Treatment with CPT-11 alone significantly decreased the median volume of pancreatic tumors and the incidence of metastasis, whereas treatment with only JBT 3002 did not. The combination therapy of CPT-11 plus JBT 3002 decreased tumor volume and incidence of metastasis significantly more than CPT-11 alone. The number of apoptotic cells (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), the number of scavenger-receptor-positive macrophages, and expression level of inducible nitric oxide synthase (iNOS) within lesions directly correlated with therapeutic effects. Indeed, the in vitro incubation of tumor cells with macrophages activated by JBT 3002 plus IFN-gamma produced a significant lysis of tumor cells that could be blocked by a specific inhibitor of iNOS. Collectively, these data demonstrate that the oral administration of the immunomodulator JBT 3002 combined with i.p. injection of CPT-11 can decrease the growth of human pancreatic carcinoma and the incidence of metastasis in nude mice by both a direct antitumor effect and the activation of iNOS in infiltrating macrophages.

Thoracolumbar accessory penis: etiology, diagnosis, and management.

Human sacral appendages have rarely been reported. We present a neonate with a thoracolumbar appendage resembling a penis, and discuss the nature of the anomaly and its diagnosis and management.

In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice.

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.

CREB and its associated proteins act as survival factors for human melanoma cells.

cAMP response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), members of the CREB/ATF family, have been implicated in cAMP- and calcium-induced transcriptional activation. We have previously demonstrated that quenching of CREB-associated proteins in metastatic melanoma cells by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreased their radiation resistance, and their tumorigenic and metastatic potential in nude mice. As the induction of apoptosis by diverse exogenous signals is dependent on the elevation of intracellular Ca2+, the purpose of this study was to determine the role of CREB and its associated proteins in apoptosis using KCREB. We used thapsigargin (Tg), which inhibits endoplasmic reticulum-dependent Ca2+-ATPase and thereby increases cytosolic Ca2+, to induce apoptosis. MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analyzed for their susceptibility to Tg-induced apoptosis. Here we demonstrate that expression of KCREB in MeWo cells rendered them susceptible to Tg-induced apoptosis. Tg treatment induced phosphorylation of CREB and possibly ATF-1 transcription factors. Treatment with Tg induced CRE-dependent transcription in parental cells, whereas this activation was reduced in the KCREB-transfected cells. In addition, CAT activity driven by the CRE-dependent promoter was inhibited in parental MeWo cells cotransfected with increasing concentrations of KCREB in a dose-dependent manner. We did not observe any changes in Bcl-2 or Bcl-2-related proteins (Bcl-x, Bax, and Bad) in control or KCREB-transfected cells before or after treatment with Tg. Collectively, these data indicate that CREB and its associated proteins act as survival factors for human melanoma cells, and hence contribute to the acquisition of the malignant phenotype.

Examination of genotype and phenotype relationships in 14 patients with apparent mineralocorticoid excess.

Apparent mineralocorticoid excess (AME) is a genetic disorder causing pre- and postnatal growth failure, juvenile hypertension, hypokalemic metabolic alkalosis, and hyporeninemic hypoaldosteronism due to a deficiency of 11 beta-hydroxysteroid dehydrogenase type 2 enzyme activity (11 beta HSD2). The 11 beta HSD2 enzyme is responsible for the conversion of cortisol to the inactive metabolite cortisone and therefore protects the mineralocorticoid receptors from cortisol intoxication. Several homozygous mutations are associated with this potentially fatal disease. We have examined the phenotype, biochemical features, and genotype of 14 patients with AME. All of the patients had characteristic signs of a severe 11 beta HSD2 defect. Birth weights were significantly lower than those of their unaffected sibs. The patients were short, underweight, and hypertensive for age. Variable damage of one or more organs (kidneys, retina, heart, and central nervous system) was found in all of the patients except one. The follow-up studies of end-organ damage after 2-13 yr of treatment in six patients demonstrated significant improvement in all patients. The urinary metabolites of cortisol demonstrated an abnormal ratio with predominance of cortisol metabolites, i.e. tetrahydrocortisol plus 5 alpha-tetrahydrocortisol/tetrahydrocortisone was 6.7-33, whereas the normal ratio is 1.0. Infusion of [11-3H]cortisol resulted in little release of tritiated water, indicating the failure of the conversion of cortisol to cortisone. Thirteen mutations in the HSD11B2 gene have been previously published, and we report three new genetic mutations in two patients, one of whom was previously unreported. All of the patients had homozygous defects except one, who was a compound heterozygote. Our first case had one of the most severe mutations, resulting in the truncation of the enzyme 11 beta HSD2, and died at the age of 16 yr while receiving treatment. Three patients with identical homozygous mutations from different families had varying degrees of severity of clinical and biochemical features. Due to the small number of patients with identical mutations, it is difficult to correlate genotype with phenotype. In some cases, early and vigilant treatment of AME patients may prevent or improve the morbidity and mortality of end-organ damage such as renal or cardiovascular damage and retinopathy. The outcome of treatment in more patients may establish the efficacy of treatment.

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.

Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration.

Our laboratory is testing the hypothesis that hypomethylation of DNA [a decreased content of 5-methylcytosine (5MeC) compared with cytosine] facilitates aberrant oncogene expression involved in tumorigenesis, using a model system of mouse strains with differing susceptibilities to liver tumorigenesis. The B6C3F1 (C57BL/6 x C3H/He) mouse serves as the relatively susceptible strain and C57BL/6 serves as the relatively resistant strain. Phenobarbital (PB) and/or administration of a choline-devoid, methionine-deficient diet (CMD) were employed as non-genotoxic hepatocarcinogens. We have examined hepatocyte and nonhepatocyte proliferation in conjunction with an assessment of global methylation changes in liver DNA of B6C3F1 and C57BL/6 mice following these promoter treatments. Bromodeoxyuridine incorporation into DNA, used to measure cell proliferation indirectly, was visualized by immunohistochemistry and quantified by a Macintosh-based image analysis system. Increased hepatocyte proliferation was demonstrated following all three treatments. This increase was larger in C57BL/6 (the relatively resistant strain) as compared with B6C3F1. In contrast, global hypomethylation was evident to a larger extent in the B6C3F1 mouse, as compared with C57BL/6. PB led to hypomethylation (>20% decrease as compared with controls) at weeks 1, 2 and 4 in B6C3F1, but not in C57BL/6 at the same time points. CMD diet administration led to hypomethylation in both strains. At week 1, 21 and 9% decreases in global methylation status were observed in B6C3F1 and C57BL/6 respectively. Evaluation of these data suggests that the heightened sensitivity of the B6C3F1 mouse compared with the C57BL/6 is due, in part, to a decreased capacity for, or fidelity of, maintaining normal methylation status. The relatively resistant strain is better able to maintain the normal methylation status of DNA in the face of a higher level of cell proliferation.

Congenital fusion of the fourth and fifth metacarpals associated with primary gonadal failure.

Congenital fusion of the fourth and fifth metacarpals is described in a male infant and his maternal grandfather. Primary gonadal failure, which is present in the infant, has not been noted in previously, reported cases. The pedigree in this family is compatible with X-linked recessive or autosomal dominant inheritance with incomplete penetrance.

Prostate, adrenocortical, and brown adipose tumors in fetal globin/T antigen transgenic mice.

Targeted oncogenesis in transgenic mice has unexpectedly produced predictable tissue-specific tumors. We previously showed that hybrid gene constructs of the human fetal G gamma- or mouse embryonic beta h1-globin promoter linked to the viral simian virus 40 T antigen (G gamma/T and beta h1/T) expressed appropriately in embryonic erythroid tissue, with some unexpected expression elsewhere. Tumors arising in the G gamma/T and beta h1/T transgenic mice were identified by histology, electron microscopy, cell culture, and RNase protection analyses. In one G gamma/T transgenic line, males developed prostate tumors that showed mixed neuroendocrine and epithelial cell features, whereas females developed adrenocortical tumors. In several other G gamma/T lines, brown adipose tumors, or hibernomas, developed in the subcutaneous interscapular neck and shoulder area, as well as internally in the periadrenal and pericardial areas. Little or no expression of T antigen was detected in adult animals before visible tumor formation. In contrast, beta h1/T transgenic mice developed only choroid plexus tumors. Transient transfection assays in prostate and adrenocortical tumor-derived cell lines showed that the G gamma-globin promoter is 7-to 10-fold more active than the beta h1-globin promoter. Activity of 5' G gamma-globin promoter-deletion DNA plasmids was analyzed by transient transfection in a variety of human prostate cancer cell lines. The G gamma-globin promoter region between -140 and -201 also showed high activity in the androgen-independent human prostate cancer cell lines DU-145 and PPC-1, but low activity in the androgen-responsive human prostate cell line LNCaP. We conclude that tumor formation in the G gamma/T transgenic lines apparently results from cryptic positive DNA cis elements active in prostate and adrenocortical cells. Because G gamma-globin promoter activity is highest in embryonic tissue, tumors in adult transgenic mice may result from expression of T antigen in embryonic prostate, adrenal glands, and brown adipose tissue.

Several homozygous mutations in the gene for 11 beta-hydroxysteroid dehydrogenase type 2 in patients with apparent mineralocorticoid excess.

Four deleterious mutations are described in the gene for HSD11B2, which encodes the type 2 isoenzyme of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD2). In seven families with one or more members affected by apparent mineralocorticoid excess, this disorder is shown to be the result of a deficiency in 11 beta HSD2. Surprisingly, the patients are all homozygous for their mutation. This results from consanguinity in two families and possibly from endogamy or a founder effect in four of the other five families. The absence of compound heterozygotes remains to be investigated.

Protein characterization and targeted disruption of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex.

The Grg gene encodes a 197 amino acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. Analysis with a polyclonal antisera specific for the Grg protein revealed that Grg is a 25 kd nuclear protein that can participate in specific protein-protein interactions. A null mutation of the Grg gene was constructed by gene targeting. Mice homozygous for this mutation completed embryogenesis and were born, but exhibited varying degrees of post-natal growth deficiency. No dosage-sensitive genetic interaction was detected between the Notch1 and Grg genes in mice heterozygous for a Notch1 mutant allele and homozygous for the Grg null mutation.