Endocrine disorders among long-term survivors of childhood head and neck rhabdomyosarcoma.

PURPOSE: Head and neck rhabdomyosarcoma (HNRMS) survivors are at increased risk of developing pituitary dysfunction as an adverse event of radiotherapy. Our aim was to investigate the frequency and risk factors for pituitary dysfunction in these survivors. Secondly, we aimed to compare the prevalence of pituitary dysfunction between survivors treated with external beam radiation therapy (EBRT) and survivors treated with the ablative surgery, moulage technique after loading brachytherapy, and surgical reconstruction (AMORE) procedure. METHODS: Eighty HNRMS survivors treated in London (EBRT based) and Amsterdam (AMORE based: AMORE if feasible, otherwise EBRT) in the period 1990-2010 and alive ≥ 2 years post-treatment were evaluated. Survivors were evaluated in multidisciplinary late-effects clinics, with measurement of linear growth, determination of thyroid function, and growth hormone parameters. Additional data, such as baseline characteristics, anthropometrics, pubertal stage, and the results of additional laboratory investigations, were retrieved from patient charts. RESULTS: Pituitary dysfunction was diagnosed in 24 in 80 (30%) survivors, after a median follow-up time of 11 years. Median time to develop pituitary dysfunction after HNRMS diagnosis was 3.0 years. Risk factors were EBRT-based therapy (odds ratio [OR] 2.06; 95% confidence interval [CI] 1.79-2.46), parameningeal tumour site (OR 1.83; 95% CI 1.60-2.17) and embryonal RMS histology (OR 1.49; 95% CI 1.19-1.90). CONCLUSIONS: Radiotherapy used for the treatment of HNRMS confers a significant risk of the development of pituitary dysfunction. AMORE-based treatment in children with HNRMS resulted in less pituitary dysfunction than treatment with conventional EBRT. Our findings underscore the importance of routine early endocrine follow-up in this specific population.

Successful introduction and audit of a step-down oral antibiotic strategy for low risk paediatric febrile neutropaenia in a UK, multicentre, shared care setting.

PURPOSE: Patients with febrile neutropaenia (FN) can be stratified according to their risk of significant complications, allowing reduced intensity therapy for low risk (LR) episodes. Serious events are very rare in low risk episodes making randomised trials difficult. Introduction of new evidence-based guidelines followed by re-auditing of the outcome is an alternative strategy. METHODS: New guidelines for the management of LR FN were implemented in 4 specialist paediatric oncology centres (POCs) and in their associated shared care units (POSCUs). All patients commenced empirical intravenous antibiotic therapy and after 48h those with blood culture negative episodes designated LR were eligible for discharge on oral co-amoxiclav. Prospective data collection on FN episodes in all treatment centres was undertaken over a 1-year period. RESULTS: Seven hundred and sixty two eligible episodes of FN were recorded in 368 patients; 213 episodes were initiated in POCs and 549 episodes were initiated in POSCUs. In 40% of episodes no clinical or microbiological focus of infection was found. At 48h, 212 (27%) episodes were classified as LR and 143 of these (19%) were managed on the LR protocol. There was a low hospital readmission rate (8/143 episodes; 5.6%), no intensive care admissions and no deaths in LR episodes. Almost all LR episodes (209/212) occurred in the shared care setting. CONCLUSIONS: Rapid step-down to oral antibiotics was a feasible and safe management strategy for LR FN in the shared care setting in England.

Efficacy of carboplatin given in a phase II window study to children and adolescents with newly diagnosed metastatic soft tissue sarcoma.

AIM: The activity of carboplatin was evaluated in a phase II window study in previously untreated children with metastatic soft tissue sarcoma. METHODS: Children with poor-risk metastatic disease (over 10 years and/or with bone/bone marrow involvement) treated in the SIOP MMT 98 study were scheduled to receive two courses of intravenous carboplatin (area under curve [AUC] of 10), 21 days apart. RESULTS: Sixteen eligible patients were entered into the rhabdomyosarcoma (RMS) group. Response (complete remission or partial remission) was seen in five children (31%, 95% confidence interval (CI) 14-56%). Ten eligible patients with other soft tissue sarcomas were recruited into the non-RMS group. Two responses (20%, 95% CI 6-51%) were seen. Toxicity in both groups was predictable nausea, vomiting and marrow suppression and there were no toxic deaths. CONCLUSION: Single-agent carboplatin at AUC of 10 has an acceptable toxicity profile but only moderate efficacy in poor-risk metastatic soft tissue sarcoma.

A prospective study of admissions for febrile neutropenia in secondary paediatric units in South East England.

BACKGROUND: The Paediatric Oncology Centres (POCs) treating childhood cancer in South East England produce unified supportive care guidelines for use in the secondary pediatric (shared care) units. This study evaluated the adherence to current guidelines for febrile neutropenia (FN) and documented outcome in terms of bacterial isolates, antibiotic resistance patterns, length of hospital stay, and mortality. PROCEDURE: Prospective study of pediatric FN admissions between July 2001 and December 2002. RESULTS: Data were received on 433 eligible FN episodes in 212 patients. The recommended empirical antibiotics (piptazobactam + gentamicin) were used in 354 (82%) admissions. Blood cultures were positive in 129 episodes (30%). Gram-positive organisms predominated (120/149 organisms isolated) and the majority were coagulase-negative Staphylococci (95/120). There were 27 Gram-negative isolates and 1 fungal isolate. No Gram-negative isolate was resistant to both first-line antibiotics. Only one death was recorded in the study group. The median length of hospital stay was 5 days. CONCLUSIONS: We obtained data on a large number of shared care episodes of FN. The antibiotic guidelines were followed in most episodes. Bacteremia was common, but little resistance to first-line antibiotics was documented among Gram-negative isolates, confirming the safety of the strategy in our population.

Response to influenza immunisation during treatment for cancer.

AIMS: To assess the annual risk of influenza infection in children with cancer and the immunogenicity of a trivalent split virus influenza vaccine in these children. METHODS: Eighty four children with cancer were tested for susceptibility to the circulating strains of influenza virus in autumn 1995 and 1996. Non-immunised children were reassessed the following spring for serological evidence of natural infection. Forty two patients received two doses of influenza vaccine. These children were receiving continuing chemotherapy for acute lymphoblastic leukaemia or were within six months of completing chemotherapy. RESULTS: Among the 84 children tested for influenza virus susceptibility only 8% of patients were fully protected (antibody titres >/= 40) against all three of the prevalent influenza virus strains; 33% were susceptible to all three viruses. Evidence of acquired natural infection was seen in 30% of unimmunised patients. Among immunised susceptible patients, 66% made some protective response to the vaccine and 55% showed protective antibody titres to all three viral strains following vaccination. Older age was associated with increased response to the H1N1 and H3N2 vaccine components, but total white cell count or neutrophil count at immunisation, type of cancer, or length of time on treatment for acute lymphoblastic leukaemia did not affect response. CONCLUSIONS: Most children with cancer studied were at risk of influenza infection. A significant response to immunisation was seen, supporting annual influenza vaccination for children being treated for cancer.

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.

A mitochondrial bottleneck hypothesis of Alzheimer's disease.

Alzheimer's disease, in its early onset familial form, is known to be a heterogeneous disorder. This suggests that the different degenerative mechanisms, initiated by different genetic causes and ending in the shared phenotype of the disease, should intersect at some point in the degenerative cascade to form a 'bottleneck' from which the pathological features that are common to each of the genetic forms emerge. A growing body of evidence suggests that disturbances of energy metabolism may play a fundamental role in the onset and progression of Alzheimer's disease. In light of this, we propose a 'mitochondrial bottleneck hypothesis', which unifies the various forms of the disease in which different causes lead to the disorder via disturbances of mitochondrial function. The characterization of such a bottleneck would present a unique target for therapeutic intervention because it would be the earliest point in a neurodegenerative cascade shared by all forms of Alzheimer's disease, independent of cause.

Persistent hemolysis after transcatheter occlusion of a patent ductus arteriosus: surgical ligation of the duct over the occlusion device.

A 14-month-old child underwent routine transcatheter closure of her patent ductus arteriosus (PDA) using the Rashkind double-umbrella device. She developed severe persistent hemolysis in association with residual ductal flow. The problem was managed by the previously unreported approach of standard surgical ligation of the duct via a left thoracotomy, leaving the occlusion device in situ.

Dysgerminoma in mother and daughter: use of lactate dehydrogenase as a tumor marker in the child.

A 7-year-old girl presented with an extragonadal dysgerminoma arising from the pelvis. Her mother had been treated for a histologically identical pituitary tumor 3 years previously. The child's serum lactate dehydrogenase (LDH) level was markedly elevated at presentation and fell as the tumor responded to treatment. The potential use of LDH as a marker for gonadal dysgerminoma is well documented, but raised LDH in association with primary extragonadal dysgerminoma has not been described previously. In addition, this is the first report of extragonadal dysgerminoma occurring in female relatives.

The Ca2+ influx induced by beta-amyloid peptide 25-35 in cultured hippocampal neurons results from network excitation.

Although a neurotoxic role has been postulated for the beta-amyloid protein (beta AP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (beta AP25-35), in particular, has been reported to be toxic in cultured neurons. We report that beta AP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, beta AP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The beta AP25-35-induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D-aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by beta AP1-40. Responses to beta AP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of beta AP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by beta AP25-35 in voltage-clamp studies. We conclude that beta AP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of beta AP25-35.

A new glibenclamide-insensitive neuroselective hyperpolarizing agent.

We describe a novel compound, (-)-N-(2-ethoxyphenyl)-N'-(1,2,3-trimethylpropyl)-2-nitroethene-1, 1-diamine), Bay x9227, that demonstrates dose-dependent hyperpolarizing activity of remarkable potency (EC50 3 picomolar) and selectivity for CNS neurons and clonal neurotypic cells compared to smooth muscle cells. Single cell membrane potential measurements were obtained in physiologic buffer using the fluorescent probe, bisoxonol. Unlike K+ATP-channel activators including its (+)-enantiomer (Hoffman et al., 1993, Biochem. Biophys. Res. Commun. 190(2), 551), this activity was insensitive to glibenclamide antagonism. These data suggest a novel pharmacologic site for effecting neuroselective hyperpolarization.

Analysis of the fifth cell cycle of mouse development.

The 5th cell cycle of mouse development was analyzed to determine the lengths of each cell cycle phase. The DNA content of Feulgen-stained blastomere nuclei was measured at various times throughout the cell cycle by microdensitometry. To achieve precise timing of the start of the 5th cell cycle, experiments utilized isolated 16-cell blastomeres and cell pairs obtained by in-vitro division of isolated 8-cell blastomeres. The following estimates were made for a mixed population of polar and apolar 16-cell blastomeres: G1, less than or equal to 2 h; S, 8-9 h; G2 + M, 2 h. No significant difference was found in the timing of DNA synthesis between polar and apolar cells or between cell pairs and whole embryos.

Modulation by 1,25-dihydroxycholecalciferol of the acute change in cytosolic free calcium induced by thyrotropin-releasing hormone in GH4C1 pituitary cells.

Receptor-mediated regulation of prolactin synthesis by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the pituitary cell strain GH4C1 is dependent on the concentration of extracellular calcium. We have now investigated the actions of 1,25(OH)2D3 on cytosolic free calcium concentrations [( Ca2+]i) in these cells using the fluorescent indicator quin2. Basal resting [Ca2+]i was unchanged in cells treated with 1 nM 1,25(OH)2D3 either acutely (from 0 to 15 min) or for periods of up to 48 h. However, the initial peak of the biphasic change in [Ca2+]i induced by thyrotropin-releasing hormone (TRH) was enhanced more than twofold in cells pretreated for 24 or 48 h with 1,25(OH)2D3. This 1,25(OH)2D3-enhanced calcium response was restricted to the initial phase of TRH action; the secondary plateau phase was unaffected. Neither the affinity nor number of TRH receptors nor the early time course of [3H]MeTRH binding to GH4C1 cells were affected by pretreatment with 1,25(OH)2D3. Because TRH binding was not altered, four sites along the intracellular signal transduction pathway of TRH action were examined. Neither protein kinase C activation nor inositol polyphosphate accumulation were enhanced in response to TRH, in 1,25(OH)2D3 pretreated cells, indicating that phosphatidylinositol hydrolysis was unchanged by pretreatment. A low concentration of ionomycin was used to probe the size of the nonmitochondrial intracellular calcium pool that is sensitive to TRH. Ionomycin was not able to mobilize more calcium from 1,25(OH)2D3 pretreated cells, indicating that TRH-responsive intracellular calcium stores were probably not enhanced by pretreatment. Chelation of extracellular calcium, however, did eliminate enhancement of the TRH response in 1,25(OH)2D3-pretreated cells. We conclude that 1,25(OH)2D3 modulates acute dynamic changes in [Ca2+]i induced by TRH without affecting basal [Ca2+]i. The mechanism of the enhanced response of 1,25(OH)2D3-pretreated cells to TRH appears to depend upon a postreceptor event independent of phosphatidylinositol hydrolysis that involves increased calcium conductance at the level of the plasma membrane. A less likely explanation involves enhancement of intracellular calcium stores in an ionomycin-resistant, EGTA-sensitive, TRH-mobilizable reservoir.

Cytokeratin filament assembly in the preimplantation mouse embryo.

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.

The establishment of the embryonic-abembryonic axis in the mouse embryo.

The influence of cell division order on the establishment of the embryonic-abembryonic axis (EA axis) of the mouse embryo was investigated. Aggregate embryos were constructed in which a labelled cell (or pair of cells) was combined with a group of unlabelled cells all of which were up to one cell cycle earlier or later in their progress through development to the blastocyst stage. The aggregates were cultured first to the nascent blastocyst stage and then to the expanded blastocyst stage. The positions of the progeny of the labelled cells in relation to the nascent blastocoel and to the orientation of the embryonic-abembryonic axis were recorded. It was concluded that cell division order does influence the establishment of the EA axis, early dividing cells tending to be associated with the nascent blastocoel and the site of the nascent blastocoel tending to mark the site of the abembryonic pole. However, the influence of division order was diminished by a requirement for intercellular cooperation during blastocoel formation and by a counteracting influence of division order arising from its effects on the allocation of cells to the inner cell mass.

Transient, benign, serum alkaline hyperphosphatasemia in an adult.

A rare occurrence of transient, benign, serum alkaline hyperphosphatasemia (TBSAH) in an adult is described. This patient's serum alkaline phosphatase rose transiently and asymptomatically to 29 times the upper limit of normal. Although TBSAH has been described in the pediatric literature, no cases of this entity have been reported in adults. Awareness of TBSAH occurring in adults is equally important to physicians who care for adult patients.