Risk of brainstem necrosis in pediatric patients with central nervous system malignancies after pencil beam scanning proton therapy.

Background: Radiation therapy (RT) plays an important role in management of pediatric central nervous system (CNS) malignancies. Centers are increasingly utilizing pencil beam scanning proton therapy (PBS-PT). However, the risk of brainstem necrosis has not yet been reported. In this study, we evaluate the rate of brainstem necrosis in pediatric patients with CNS malignancies treated with PBS-PT.Material and methods: Pediatric patients with non-hematologic CNS malignancies treated with PBS-PT who received dose to the brainstem were included. All procedures were approved by the institutional review board. Brainstem necrosis was defined as symptomatic toxicity. The actuarial rate was analyzed by the Kaplan Meier method.Results: One hundred and sixty-six consecutive patients were reviewed. Median age was 10 years (range 0.5-21 years). Four patients (2.4%) had prior radiation. Median maximum brainstem dose in the treated course was 55.4 Gy[RBE] (range 0.15-61.4 Gy[RBE]). In patients with prior RT, cumulative median maximum brainstem dose was 98.0 Gy [RBE] (range 17.0-111.0 Gy [RBE]). Median follow up was 19.6 months (range, 2.0-63.0). One patient who had previously been treated with twice-daily radiation therapy and intrathecal (IT) methotrexate experienced brainstem necrosis. The actuarial incidence of brainstem necrosis was 0.7% at 24 months (95% CI 0.1-5.1%).Conclusion: The rate of symptomatic brainstem necrosis was extremely low after treatment with PBS-PT in this study. Further work to clarify clinical and dosimetric parameters associated with risk of brainstem necrosis after PBS-PT is needed.

fdg-pet in two cases of neurofibromatosis type 1 and atypical malignancies.

Patients with neurofibromatosis type 1 (nf1) are at increased risk for both benign and malignant tumours, and distinguishing the malignant potential of an individual tumour is a common clinical problem in these patients. Here, we review two cases of uncommon malignancies (Hodgkin lymphoma and mediastinal germ-cell tumour) in patients with nf1. Although (18)F-fluorodeoxyglucose positron-emission tomography (fdg-pet) has been used to differentiate benign neurofibromas from malignant peripheral nerve sheath tumours, fdg-pet characteristics for more rare tumours have been poorly described in children with nf1. Here, we report the role of pet imaging in clinical decision-making in each case. In nf1, fdg-pet might be useful in the clinical management of unusual tumour presentations and might help to provide information about the malignant potential of uncommon tumours.

Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Defining optic nerve tortuosity.

BACKGROUND AND PURPOSE: Optic nerve tortuosity is one of several nonmalignant abnormalities documented on MR imaging in patients with neurofibromatosis type 1 and may be related to the development of optic pathway gliomas. This study seeks an operational definition for optic nerve tortuosity. MATERIALS AND METHODS: A focus group of 3 pediatric neuroradiologists reviewed 20 MR images of the brain and orbits of patients suspected to have optic nerve tortuosity in the absence of optic pathway glioma and found 6 radiographic factors that occurred frequently. Subsequently, 28 MR images were assessed for the presence of optic nerve tortuosity, using a global assessment question that reflects a neuroradiologist's confidence in the presence of optic nerve tortuosity, and for the presence of the 6 radiographic factors, to identify a combination of these factors that best predicted a diagnosis of optic nerve tortuosity. RESULTS: We found perfect inter-rater agreement between 3 readers on the presence/absence of tortuosity in 75% of cases. Lack of congruity of the optic nerves, in more than 1 coronal section and dilation of the subarachnoid space surrounding the optic nerves, when found together are sensitive (89%) and specific (93%) for a diagnosis of tortuosity on the global scale. The absence of these 2 factors, along with absence of deviation of the optic nerve within the axial plane, provides a reliable test to exclude tortuosity. CONCLUSION: Lack of congruity of the optic nerves in more than 1 coronal section and dilation of the subarachnoid space surrounding the optic nerves together provide an operational radiographic definition of optic nerve tortuosity.

Influences of diet and strain on the proliferative effect on the rat urinary bladder induced by sodium saccharin.

Rats were fed sodium saccharin as 5 or 7.5% of the diet by weight, and proliferation of the bladder epithelium was assessed by autoradiography, histology, and scanning electron microscopy. In Experiment 1, male F344 rats, 5 weeks old, were placed on a diet of 0, 5, or 7.5% NaS mixed in Prolab 3200, NIH-07, or AIN-76A diet for 4 or 10 weeks. In Experiment 2, 5-week-old F344 rats or 4-week-old Sprague-Dawley rats were fed 0, 5, or 7.5% NaS in Prolab 3200 or Purina 5002 diet for 10 weeks. In Experiment 1, at both the 4- and 10-week intervals, NaS had a greater effect on the urothelium when administered in the Prolab diet compared to the NIH diet, and there was little response with the AIN diet. Eight of 10 rats fed 7.5% NaS in Prolab 3200 for 4 or 10 weeks had bladders with simple or nodular hyperplasia, and eight of nine bladders contained abnormal surface features visible by scanning electron microscopy. At 10 weeks for control animals, the average labeling index following [3H]thymidine incorporation into bladder epithelium was approximately 0.05%. For rats fed 7.5% NaS diets, the labeling index was 0.43% for Prolab, 0.14% for NIH-07, and 0.04% for AIN-76A. In Experiment 2, the response to NaS was considerably greater in F344 rats than in Sprague-Dawley rats fed the same diet, and for both strains, the response to NaS was greater in Prolab than in Purina diets. In conclusion, the proliferative effect of NaS on male rat urinary bladder depended on rat strain as well as on type of diet.

Isoprenylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of isoprenyl groups, is a widespread phenomenon. Here we provide direct evidence for this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of [3H]mevalonolactone, specific C. elegans polypeptides became labelled in both aqueous and detergent (Triton X-114)-enriched extracts. Chemical and GC-MS analysis of modifying groups, cleaved from C. elegans polypeptides, revealed that geranylgeranylation and, to a lesser extent, farnesylation of target polypeptides occurred. Immunoblot analysis provided preliminary evidence that the ras-like let-60 polypeptide was a target for isoprenylation in C. elegans.

The effect of vanadate upon the expression of phenylalanine hydroxylase in streptozotocin-diabetic rat liver.

Induction of diabetes in rats is associated with a significant elevation in the phenylalanine hydroxylating capacity of the liver. This phenomenon reflects an increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine hydroxylase-specific mRNA. These changes can be abolished by insulin-dependent control of diabetes. We show here that the control of diabetes by oral administration of sodium orthovanadate will also nullify the diabetes-related alterations in phenylalanine hydroxylase expression. In addition, diabetes-induced changes in the extent of phosphorylation of phenylalanine hydroxylase are reversed by either insulin or vanadate treatment in vivo. These treatments also abolished the diabetes-related, approx. 30-fold, decrease in glucagon sensitivity of phenylalanine hydroxylation in isolated liver cells.

Fatty acylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of fatty acyl groups, is a widespread phenomenon. Here we describe the occurrence of this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of either [3H]-myristic acid or [3H]-palmitic acid, specific C. elegans polypeptides became labelled. Chemical analysis revealed that following incubation of C. elegans with [3H]-myristic acid, polypeptides became labelled with myristoyl, palmitoyl or stearoyl moieties; after incubation with [3H]-palmitic acid, palmitoyl or stearoyl moieties were incorporated into polypeptides. Fatty acyl groups were linked to target polypeptides, predominantly through alkali-labile thioester or ester linkages and acid-labile amide linkages. Where myristoylation involved an amide linkage, the modified amino acid was usually glycine. Preliminary immunological evidence indicated that heterotrimeric GTP-binding protein alpha subunit(s) are possible target(s) for acylation in C. elegans.

Diadenosine polyphosphate-mediated activation of phospholipase D in isolated rat liver cells.

Diadenosine polyphosphates (ApnAs) can, through interaction with appropriate purinoceptors, affect a range of cellular activities. Ap4A, the most prominent naturally occurring diadenosine polyphosphate, stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that Ap4A, and other naturally occurring diadenosine polyphosphates, also stimulates phospholipase D (PLD) activity in isolated rat liver cells. The characteristics of Ap4A-mediated activation of PLD are similar to those for the activation of PLD by extracellular ATP. These results are discussed in the context of the relation between diadenosine polyphosphate- and adenine mononucleotide-mediated cellular signalling processes.

Adenine dinucleotide-mediated activation of glycogen phosphorylase in isolated liver cells.

The ability of purine nucleotides (e.g. ATP) to cause a dose-dependent activation of glycogen phosphorylase in isolated liver cells is well known. These agents mediate their effects through interaction with specific P2-purinoceptors in the plasma membrane. We have investigated the ability of a range of synthetic and naturally occurring adenine dinucleotides to cause a similar stimulation of glycogen phosphorylase activity in isolated rat liver cells. Our results indicate that Ap3A and Ap4A, the most abundant naturally occurring adenine dinucleotides, cause a dose-dependent activation of glycogen phosphorylase similar to that observed with ATP. Similar responses were seen with Ap5A, Ap6A and a series of phosphorothioate analogues. In contrast, the response to phosphonate analogues depended on the position of the P-C-P bridge. The dinucleotides appear to exert their effects directly, rather than through hydrolytic products such as adenosine and/or ATP. The possibility that adenine dinucleotides are physiologically significant extracellular purinergic effectors is discussed in the light of these observations.

Molecular determinants of response to TRAIL in killing of normal and cancer cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a potent inducer of death of cancer but not normal cells, which suggests its potential use as a tumor-specific antineoplastic agent. TRAIL binds to the proapoptotic death receptors DR4 and the p53-regulated proapoptotic KILLER/DR5 as well as to the decoy receptors TRID and TRUNDD. In the present studies, we identified a subgroup of TRAIL-resistant cancer cell lines characterized by low or absent basal DR4 or high expression of the caspase activation inhibitor FLIP. Four of five TRAIL-sensitive cell lines expressed high levels of DR4 mRNA and protein, whereas six of six TRAIL-resistant cell lines expressed low or undetectable levels of DR4 (chi 2; P < 0.01). FLIP expression appeared elevated in five of six (83%) TRAIL-resistant cell lines and only one of five (20%) TRAIL-sensitive cells (chi 2; P < 0.05). Two TRAIL-resistant lines that expressed DR4 contained an A-to-G alteration in the death domain encoding arginine instead of lysine at codon 441. The K441R polymorphism is present in 20% of the normal population and can inhibit DR4-mediated cell killing in a dominant-negative fashion. The expression level of KILLER/DR5, TRID, TRUNDD or TRID, and TRUNDD did not correlate with TRAIL sensitivity (P > 0.05). These results suggest that the major determinants for TRAIL sensitivity may be the expression level of DR4 and FLIP. TRAIL-resistant cells became susceptible to TRAIL-mediated apoptosis in the presence of doxorubicin. In TRAIL-sensitive cells, caspases 8, 9, and 3 were activated after TRAIL treatment, but in TRAIL-resistant cells, they were activated only by the combination of TRAIL and doxorubicin. Our results suggest: (a) evaluation of tumor DR4 and FLIP expression and host DR4 codon 441 status could be potentially useful predictors of TRAIL sensitivity, and (b) doxorubicin, in combination with TRAIL, may effectively promote caspase activation in TRAIL-resistant tumors.

Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer.

Allelic loss of chromosome 8p21-22 occurs frequently in cancer, including lung and head and neck squamous cell cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, including proapoptotic DR4 and KILLER/DR5, are located on 8p21-22. TRAIL receptors are candidate tumor suppressor genes, because their inactivation would be expected to result in deficient apoptotic signaling. To investigate the involvement of DR4 in human cancer, we have determined the genomic structure of DR4 and screened 31 lung cancer cell lines [14 small cell lung cancer and 17 non-small cell lung cancer (NSCLC)], many with deletions at 8p21-22, and 21 primary NSCLC samples for mutations in DR4. We found two missense alterations in the ectodomain of DR4. One, at nucleotide 626, changes a cytosine to a guanine (C626G) and results in a substitution of an arginine for threonine. The other, at nucleotide 422, changes a guanine to adenine (G422A) and results in a substitution of a histidine for arginine. Using genomic DNA sequencing and RFLP analysis, we show that these two alterations cosegregated in 96% of all of the samples (n = 243) evaluated (tumor and normal). The frequency of being homozygous for both altered alleles was 35% in the lung cancer cell lines but only 13% in age- and race-matched controls, which was a significant increase (chi(2) = 5.2, P = 0.023). The frequency of homozygosity for both alleles was also significantly increased in the primary NSCLC samples (chi(2) = 9.2, P = 0.002) as compared with the age- and race-matched controls. To determine whether the altered alleles are specific for lung cancer, we evaluated 19 head and neck squamous cell cancer and 25 gastric adenocarcinoma samples. Forty-seven % of the former and 44% of the latter were homozygous for both the C626G and G422A alterations, and this was significantly elevated relative to age- and race-matched controls (chi(2) = 8.6, P = 0.003 and chi(2) = 8.2, P = 0.004). These alterations result in amino acid changes in or near the ligand-binding domain of DR4 and, based on the crystal structure of DR5 and its homology with DR4, have the potential to affect TRAIL binding to DR4. Our results suggest that the altered DR4 alleles may be associated with, and should be investigated additionally as potential markers for, predisposition to common malignancies.

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.

BACKGROUND: Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. OBJECTIVE: To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. METHODS: Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. RESULTS: One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. CONCLUSION: The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

Differential effects of streptozotocin-induced diabetes on phenylalanine hydroxylase protein and mRNA abundance in isolated rat liver cells.

Phenylalanine hydroxylase catalyzes the major regulatory step of the phenylalanine degradation pathway. In view of the glucogenic nature of phenylalanine breakdown, and hence its potential contribution to glucose homeostasis, we have investigated the impact of streptozotocin-induced diabetes upon the expression of rat phenylalanine hydroxylase. Northern blot analysis revealed that induction of diabetes was associated with an increase in the in vivo abundance of hepatic phenylalanine hydroxylase-specific mRNA. This increase in mRNA abundance was maintained for at least 8 hr in liver cells isolated from diabetic animals. In contrast, phenylalanine hydroxylase immunoreactivity and enzymic activity decreased, over the 8 hr incubation period, to levels similar to those observed in liver cells from normal animals. These changes were retarded, but not prevented, by the presence of dexamethasone in incubation media. In liver cells from normal animals the abundance of phenylalanine hydroxylase-specific mRNA, immunoreactivity and enzymic activity, were largely insensitive to treatment with dexamethasone and/or glucagon over an 8 hr incubation period. It is concluded that, whereas diabetes-related alterations in phenylalanine hydroxylase-specific mRNA abundance persist after isolation of liver cells, changes in phenylalanine hydroxylase protein abundance do not. Additionally, in contrast to certain other enzymes (e.g. phosphoenolpyruvate carboxykinase) it is not possible to mimic diabetes-related alterations in the expression of phenylalanine hydroxylase, in liver cells from normal animals, by simple hormonal manipulation of incubation media. This implies that other additional factors must also contribute to diabetes-related alterations in hepatic enzyme expression.

The immediate 5'-flanking region of the rat phenylalanine hydroxylase-encoding gene.

We have characterized the immediate (465 bp) 5'-flanking region of the rat phenylalanine hydroxylase (PAH)-encoding gene. This sequence shows considerable similarity to the 5'-flanking region of the human PAH gene [Konecki et al., Biochemistry 31 (1992) 8363-8368]. Both sequences lack obvious TATA elements; however, putative regulatory sites, including a potential cyclic AMP-response element and glucocorticoid response elements, are present.

Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells.

Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.

Adenine dinucleotide-mediated cytosolic free Ca2+ oscillations in single hepatocytes.

Single rat hepatocytes microinjected with aequorin respond to Ca(2+)-mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP.

Non-peptide RGD surrogates which mimic a Gly-Asp beta-turn: potent antagonists of platelet glycoprotein IIb-IIIa.

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Fused bicyclic Gly-Asp beta-turn mimics with specific affinity for GPIIb-IIIa.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

Diamino benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors. 5. Potency, efficacy, and pharmacokinetic properties of modified C-3 side chain derivatives.

A systematic investigation of the structure-activity relationships of the C-3 side chain of the screening hit 1a led to the identification of the potent thrombin inhibitors 23c, 28c, and 31c. Their activities (1240, 903, and 1271 x 10(6) L/mol, respectively) represent 2200- and 2900-fold increases in potency over the starting lead 1a. This activity enhancement was accomplished with an increase of thrombin selectivity. The in vitro anticoagulant profiles of derivatives 28c and 31c were determined, and they compare favorably with the clinical agent H-R-1-[4aS, 8aS]perhydroisoquinolyl-prolyl-arginyl aldehyde (D-Piq-Pro-Arg-H; 32). The more potent members of this series have been studied in an arterial/venous shunt (AV shunt) model of thrombosis and were found to be efficacious in reducing clot formation. However, their efficacy is currently limited by their rapid and extensive distribution following administration.