Biomedical subjects
Publications and source records attributed to H Phillips.
Capnometer measurements during 46 evaluable treatments in which patients were administered carbogen (95% O2, 5% CO2) have demonstrated that the mean inspired carbon dioxide level was 3.1% (range 0-4.7) and the mean inspired oxygen concentration was 70.6% (range 26.4-94). The explanation for this observation is leakage of air into the breathing system during radiotherapy.
This pilot study investigated the feasibility, toxicity and effect of continuous, hyperfractionated, accelerated radiotherapy (CHART) in 19 patients with adenocarcinoma of the rectum who were treated at Mount Vernon Hospital between April 1992 and April 1994. A total dose of 54 Gy was given in 36 fractions over 12 consecutive days; three fractions of 1.5 Gy were employed each day with an interfraction interval of 6 hours. Of these 19 patients, 13 had local pelvic recurrence from rectal carcinoma, four had a primary rectal carcinoma (which was unsuitable for abdominoperineal resection because of patient frailty), and two had large, fixed, unresectable tumours and were given radiotherapy preoperatively to a lower dose of 42 Gy in 28 fractions over 10 days to facilitate surgery. However, none of the 19 patients subsequently underwent surgical resection. Treatment compliance with radiotherapy was 95%. Acute bowel toxicity was assessed by means of a daily bowel chart for 6 weeks and then at 8, 12 and 26 weeks after the completion of radiotherapy. Patients were subsequently scored using the World Health Organization (WHO) grading system. No toxic deaths were observed in this study. There were five grade 3 and one grade 4 toxicities (WHO scale) observed in six patients. There has been minimal acute bladder toxicity in two patients and skin reactions have been surprisingly minimal in all but three patients. Small bowel obstruction and late bowel damage, possibly attributable to radiotherapy, occurred in three patients. Complete pain relief was achieved in recurrent rectal cancer in 100% of patients, at a median of 15 days since commencing radiotherapy (range 7-63 days), and has been maintained for mean and median durations of 19 and 18 months respectively (range 1.5-45 months). Five patients have had a recurrence of pain at 3, 4, 8, 9 and 18 months respectively. All four patients with primary rectal adenocarcinoma achieved a complete response, which has been sustained for 4 months (until death), 54 and 57 months, although one patient relapsed locally at 8 months. In summary, CHART appears to be a feasible treatment for pelvic tumours with regard to toxicity. Patients with pain from a locoregional recurrence achieved rapid complete pain relief, which was sustained for many months.
Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.
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Ethylmalonic encephalopathy (EE), an organic aciduria of unknown etiology characterized by developmental delay, hypotonia, and vascular instability associated with lactic acidemia and urinary excretion of ethylmalonic acid (EMA) and methylsuccinic acid (MSA), has been described in 11 patients. To test the possibility that the underlying biochemical defect involves isoleucine catabolism, we determined the response to oral L-isoleucine (IIe) load (150 mg/kg) in a 5-year-old girl with EE and in three healthy, age- and sex-matched controls. Following IIe load in the patient, there was accumulation of 2-methylbutyrylglycine (2-MBG) and a delayed and lower peak urinary excretion of tiglylglycine (TGL), suggesting a partial defect in 2-methyl-branched chain acylcoenzyme A dehydrogenase (2M-BCAD). In vitro measurements 2M-BCAD activity in cultured skin fibroblasts from patients with EE have been reported to be normal. Our results show that isoleucine is a source for the elevated EMA and MSA in patients with EE, and suggest a functional, possibly secondary, deficiency of activity of 2M-BCAD in vivo.
Glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGFbeta protein family that is essential for neuronal survival and renal morphogenesis. We show that mice who are deficient in the glycosyl-phosphatidyl inositol (GPI) -linked protein GFRalpha1 (GDNFRalpha) display deficits in the kidneys, the enteric nervous system, and spinal motor and sensory neurons that are strikingly similar to those of the GDNF- and Ret-deficient mice. GFRalpha1-deficient dopaminergic and nodose sensory ganglia neurons no longer respond to GDNF or to the structurally related protein neurturin (NTN) but can be rescued when exposed to GDNF or neurturin in the presence of soluble GFRalpha1. In contrast, GFRalpha1-deficient submandibular parasympathetic neurons retain normal response to these two factors. Taken together with the available genetic and biochemical data, these findings support the idea that GFRalpha1 and the transmembrane tyrosine kinase Ret are both necessary receptor components for GDNF in the developing kidney and nervous system, and that GDNF and neurturin can mediate some of their activities through a second receptor.
Results from the multicentre randomized trial of CHART (continuous, hyperfractionated, accelerated radiotherapy) in non-small-cell lung cancer (NSCLC) showed a significant increase in survival (P=0.004) compared with conventional radiotherapy and a therapeutic benefit relative to late radiation-induced morbidity. However, 60% of patients died because of failure to control locoregional disease. These findings have stimulated interest in assessing the feasibility of dose escalation using a modified CHART schedule. Acute and late morbidity with a CHARTWEL (CHART WeekEnd Less) schedule of 54 Gy in 16 days was compared with that observed with 60 Gy in 18 days in patients with locally advanced NSCLC. The incidence and severity of dysphagia and of analgesia were scored using a semiquantitative clinical scale. Late radiation-induced morbidity, namely pulmonary, spinal cord and oesophageal strictures, were monitored using clinical and/or radiological criteria. Acute dysphagia and the analgesia required to control the symptoms were more severe and lasted longer in patients treated with CHARTWEL 60 Gy (P< or = 0.02). However, at 12 weeks, oesophagitis was similar to that seen with 54 Gy and did not lead to consequential damage. Early radiation pneumonitis was not increased but, after 6 months, there was a higher incidence of mild pulmonary toxicity compared with CHARTWEL 54 Gy. No cases of radiation myelitis, oesophageal strictures or of grade 2 or 3 lung morbidity have been encountered. CHARTWEL 60 Gy resulted in an enhancement of oesophagitis and grade 1 lung toxicity compared with CHARTWEL 54 Gy. These were of no clinical significance, but may be important if CHARTWEL is used with concomitant chemotherapy. These results provide a basis for further dose escalation or the introduction of concurrent chemotherapy.
We report gene localization in a family with a benign autosomal dominant familial periodic fever (FPF) syndrome characterized by recurrent fever associated with abdominal pain. The clinical features are similar to the disorder previously described as familial Hibernian fever, and they differ from familial Mediterranean fever (FMF) in that FPF episodes usually do not respond to colchicine and FPF is not associated with amyloidosis. Frequent recombination with the marker D16S2622, <1 Mb from FMF, at 16p13.3, excluded allelism between these clinically similar conditions. Subsequently, a semiautomated genome search detected linkage of FMF to a cluster of markers at 12p13, with a multipoint LOD score of 6.14 at D12S356. If penetrance of 90% is assumed, the FPF gene maps to a 19-cM interval between D12S314 and D12S364; however, if complete penetrance is assumed, then FPF maps to a 9-cM region between D12S314 and D12S1695. This interval includes the dentatorubropallidoluysian atrophy locus, which, with FPF, gave a maximum two-point LOD score of 3.7 at a recombination fraction of 0. This is the first of the periodic-fever genes, other than FMF, to be mapped. Positional candidate genes may now be selected for mutation analysis to determine the molecular basis for FPF. Together with the recent identification of the defective gene in FMF, identification of a gene for FPF might provide new insights into the regulation of inflammatory responses.
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Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two structurally related, potent survival factors for sympathetic, sensory and central nervous system neurons. GDNF mediates its actions through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) and the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by which the NTN signal is transmitted is not well understood. Here we describe the identification and tissue distribution of a GPI-linked protein (designated NTNR-alpha) that is structurally related to GDNFR-alpha. We further demonstrate that NTNR-alpha binds NTN (K[d] approximately 10 pM) but not GDNF with high affinity; that GDNFR-alpha binds to GDNF but not NTN with high affinity; and that cellular responses to NTN require the presence of NTNR-alpha. Finally, we show that NTN, in the presence of NTNR-alpha, induces tyrosine-phosphorylation of Ret, and that NTN, NTNR-alpha and Ret form a physical complex on the cell surface. These findings identify Ret and NTNR-alpha as signalling and ligand-binding components, respectively, of a receptor for NTN and define a novel family of receptors for neurotrophic and differentiation factors composed of a shared transmembrane protein tyrosine kinase and a ligand-specific GPI-linked protein.
Carbogen and nicotinamide have been evaluated in a phase II study as hypoxia-modifying agents during radical radiotherapy for bladder cancer using a standard daily 20-fraction schedule. Three groups of patients have received (a) nicotinamide alone, given orally in a dose of 80 mg kg(-1) daily with 52.5 Gy in 20 fractions over 4 weeks, (b) carbogen alone, with 50 Gy in 20 fractions over 4 weeks, and (c) carbogen and nicotinamide, with 50-52.5 Gy in 20 fractions over 4 weeks. Ten patients were treated in each group. All patients completed carbogen and radiotherapy as prescribed, but only 45% completed daily nicotinamide over the 4-week treatment period. The end points of this study were acute bowel and bladder morbidity and local control at cystoscopy 6 months after treatment. An expected level of acute bowel and bladder morbidity was seen that reverted to normal in most patients by 12 weeks with no difference between the three treatment groups. Complete response rates at 6 months were seven out of ten (100%) in the nicotinamide alone group, nine out of ten (90%) in the carbogen alone group and seven out of ten (70%) in the carbogen and nicotinamide group. It is concluded that carbogen and nicotinamide may improve the results of daily fractionated radiotherapy in bladder cancer and that further evaluation is required.
The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.
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Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in that NT-3(-/-) mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss between NT-3(-/-) and trkC(-/-) mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.
Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic midbrain dopaminergic, spinal motor, cranial sensory, sympathetic, and hindbrain noradrenergic neurons, and is available to these cells in vivo. It is therefore considered a physiological trophic factor and a potential therapeutic agent for Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here we show that at postnatal day 0 (P0), GDNF-deficient mice have deficits in dorsal root ganglion, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons. These mice completely lack the enteric nervous system (ENS), ureters and kidneys. Thus GDNF is important for the development and/or survival of enteric, sympathetic and sensory neurons and the renal system, but is not essential for catecholaminergic neurons in the central nervous system (CNS).
Schwann cells are one of the principal components of the peripheral nervous system. They play a crucial role in nerve regeneration and can be used clinically in the repair of injured nerves. We have established serum-free, defined culture conditions that rapidly expand adult human Schwann cells without fibroblast growth. We find that Gas6, a ligand for the Axl and Rse/Tyro3 receptor protein tyrosine kinase family, stimulates human Schwann cell growth, increasing both cell number and thymidine incorporation. Gas6 has synergistic effects with the other known human Schwann cell mitogens, heregulin/glial growth factor and forskolin. Addition of Gas6 causes phosphorylation of Axl and Rse/Tyro3 simultaneously and results in ERK-2 activation. A combination of Gas6 with heregulin and forskolin, on a defined background, supports maximal Schwann cell proliferation, while preserving the typical Schwann cell morphology and expression of the Schwann cell markers S-100, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor. Gas6 mRNA is present in both spinal motor neurons and large neurons of the dorsal root ganglia, and neural injury has been reported to upregulate Rse/Axl in the schwann cell. This is the first demonstration of a potentially important biological role for the human Gas6/Rse-Axl system.
The presenilin 1 gene has recently been identified as the locus on chromosome 14 which is responsible for a large proportion of early onset, autosomal dominantly inherited Alzheimer's disease (AD). We have elucidated the intron/exon structure of the gene and designed intronic primers to enable direct sequencing of the entire coding region (10 exons) of the presenilin gene in a large number of families. This strategy has enabled us to find a further two novel mutations in the gene. We discuss the distribution of mutations and the proportions of autosomal dominant AD with a mean age of onset below 60 years caused by mutations in this gene.