The use of CRP within a clinical prediction algorithm for the differentiation of septic arthritis and transient synovitis in children.

Clinical prediction algorithms are used to differentiate transient synovitis from septic arthritis. These algorithms typically include the erythrocyte sedimentation rate (ESR), although in clinical practice measurement of the C-reactive protein (CRP) has largely replaced the ESR. We evaluated the use of CRP in a predictive algorithm. The records of 311 children with an effusion of the hip, which was confirmed on ultrasound, were reviewed (mean age 5.3 years (0.2 to 15.1)). Of these, 269 resolved without intervention and without long-term sequelae and were considered to have had transient synovitis. The remaining 42 underwent arthrotomy because of suspicion of septic arthritis. Infection was confirmed in 29 (18 had micro-organisms isolated and 11 had a high synovial fluid white cell count). In the remaining 13 no evidence of infection was found and they were also considered to have had transient synovitis. In total 29 hips were categorised as septic arthritis and 282 as transient synovitis. The temperature, weight-bearing status, peripheral white blood cell count and CRP was reviewed in each patient. A CRP > 20 mg/l was the strongest independent risk factor for septic arthritis (odds ratio 81.9, p < 0.001). A multivariable prediction model revealed that only two determinants (weight-bearing status and CRP > 20 mg/l) were independent in differentiating septic arthritis from transient synovitis. Individuals with neither predictor had a < 1% probability of septic arthritis, but those with both had a 74% probability of septic arthritis. A two-variable algorithm can therefore quantify the risk of septic arthritis, and is an excellent negative predictor.

The association between clubfoot and developmental dysplasia of the hip.

The association between idiopathic congenital talipes equinovarus (CTEV) and developmental dysplasia of the hip is uncertain. We present an observational cohort study spanning 6.5 years of selective ultrasound screening of hips in clubfoot. From 119 babies with CTEV there were nine cases of hip dysplasia, in seven individuals. This suggests that 1 in 17 babies with CTEV will have underlying hip dysplasia. This study supports selective ultrasound screening of hips in infants with CTEV.

Microbiological culture results for the femoral head. Are they important to the donor?

We determined the rate of contamination of donated femoral heads at primary hip arthroplasty within a single region between July 1992 and July 2001. We established the null hypothesis that culture results played no role in predicting early failure of the joint because of infection. The rate of contamination was 9%. A positive culture, at the time of retrieval, was found in 367 of 4045 femoral heads. Coagulase-negative staphylococcus was isolated in 77% of the positive cases. At a minimum follow-up of one year, there was no statistically significant difference in the rate of complications or of revision of age-matched patients whose femoral heads had a positive culture compared with those whose femoral heads were sterile. Our findings confirm that culture of the femoral head plays no part in determining future failure of joint replacement in the donor.

Identification of recurrent regions of chromosome loss and gain in vestibular schwannomas using comparative genomic hybridisation.

BACKGROUND: Schwannomas are benign tumours of the nervous system that are usually sporadic but also occur in the inherited disorder neurofibromatosis type 2 (NF2). The NF2 gene is a tumour suppressor on chromosome 22. Loss of expression of the NF2 protein product, merlin, is universal in both sporadic and NF2 related schwannomas. The GTPase signalling molecules RhoA and Rac1 regulate merlin function, but to date only mutation in the NF2 gene has been identified as a causal event in schwannoma formation. METHODS: Comparative genomic hybridisation (CGH) was used to screen 76 vestibular schwannomas from 76 patients (66 sporadic and 10 NF2 related) to identify other chromosome regions that may harbour genes involved in the tumorigenesis. RESULTS: The most common change was loss on chromosome 22, which was more frequent in sporadic than in NF2 related tumours. Importantly, eight tumours (10%) showed gain of copy number on chromosome 9q34. Each of the two NF2 patients who had received stereotactic radiotherapy had non-chromosome 22 changes, whereas only one of eight non-irradiated NF2 patients had any chromosome changes. Three tumours had gain on 17q, which has also been reported in malignant peripheral nerve sheath tumours that are associated with neurofibromatosis type 1. Other sites that were identified in three or fewer tumours were regions on chromosomes 10, 11, 13, 16, 19, 20, X, and Y. CONCLUSIONS: These findings should be verified using techniques that can detect smaller genetic changes, such as microarray-CGH.

Effects of maternal iron restriction on placental vascularization in the rat.

To investigate the effects of maternal iron deficiency and anaemia on the placenta the composition and vascularization of the placental labyrinth was investigated in iron-restricted rats. Rats in the experimental groups were placed on iron-restricted diets either 1 or 2 weeks before mating and continued on these diets throughout gestation. Placentae were studied at day 21 of gestation. Tissue sections were stained with lectin to allow identification of fetal capillaries and analyzed using stereological techniques. Capillary surface area density and total capillary surface area were decreased in both iron-restricted groups compared with the control group. Capillary length density was decreased in both iron-restricted groups compared with the control group. Total capillary length was significantly reduced in the 1-week, but not in the 2-week, iron-restricted group compared with the control group. Endothelial cell volume was increased in both iron-restricted groups compared to the controls. There were no significant differences in the volume of fetal capillaries, the volume of the maternal blood spaces or the surface area of the maternal-fetal interface between the control and iron-restricted groups. Labyrinthine volume, labyrinthine tissue volume and the surface area of the maternal fetal interface were increased in the 2-week group when compared with the 1-week group. These changes in placental vascularization may contribute to the fetal growth retardation observed in iron-restricted litters.

Effects of maternal iron restriction in the rat on hypoxia-induced gene expression and fetal metabolite levels.

The mechanism by which maternal Fe deficiency in the rat causes fetal growth retardation has not been clearly established. This study compared the effects on the fetuses from dams fed a control diet with two groups of dams fed Fe-restricted diets. One Fe-restricted group was fed the Fe-restricted diet for 1 week prior to mating and throughout gestation and the second Fe-restricted group was fed the Fe-restricted diet for 2 weeks prior to mating and throughout gestation. On day 21 of gestation Fe-restricted dams, and their fetuses, were anaemic. Fetal weight was reduced in both Fe-restricted groups compared with controls. Expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) are induced by hypoxia. The levels of HIF-1alpha mRNA were highest in placenta, then in kidney, heart and liver but were not different between the groups. Levels of plasma VEGF were not different between the groups. Maternal plasma triacylglycerol was decreased in the 1-week Fe-restricted dams compared with controls. Maternal plasma cholesterol and free fatty acid levels were not different between the groups. In fetal plasma, levels of triacylglycerol and cholesterol were decreased in both Fe-restricted groups. In maternal plasma, levels of a number of amino acids were elevated in both Fe-restricted groups. In contrast, levels of a number of amino acids in fetal plasma were lower in both Fe-restricted groups. Fetal plasma lactate was increased in Fe-restricted fetuses but fetal plasma glucose and beta-hydroxybutyrate were not affected. These changes in fetal metabolism may contribute to fetal growth retardation in this model. This study does not support the hypothesis that the Fe-restricted fetus is hypoxic.

Genomic alterations associated with loss of heterozygosity for TP53 in Li-Fraumeni syndrome fibroblasts.

Studies of Li-Fraumeni syndrome fibroblasts heterozygous for germline TP53 mutations have shown that loss of heterozygosity (LOH) occurs during passaging and is associated with genomic instability, such as chromosomal aberrations and aneuploidy to investigate the genomic changes associated with LOH in Li-Fraumeni (LF) fibroblasts, we have analysed cell strains at increasing population doublings (PD) using Comparative Genomic Hybridization (CGH). We have looked at three groups of cell strains: LF mutation-carrying strains which showed LOH for TP53, LF mutation-carrying strains which did not show LOH, and strains from normal individuals. Using CGH, we have detected loss of distinct chromosomal regions associated with LOH in 4 out of 5 mutation-carrying strains. In particular we have found loss of chromosomal regions containing genes involved in cell cycle control or senescence, including loss of 9p and 17p in these strains. Other recurrent changes included loss of chromosomes 4q and 6q, regions shown to contain one or more tumour suppressor genes. No genomic alterations were detected at cumulative PD in the normal strains or in the LF mutation-carrying strains which did not show LOH for TP53. We have also analysed the three groups of strains for microsatellite instability and somatic TP53 mutations, and have found genetic alterations in only one strain.

Multiple endocrine neoplasia type 1: clinical and genetic features of the hereditary endocrine neoplasias.

MEN1 is a syndrome of parathyroid adenomas, gastrinomas, prolactinomas, and other endocrine tumors. Collagenomas and facial angiofibromas are newly recognized but common skin expressions. Many tumors in MEN1 are benign; however, many entero-pancreatic neuroendocrine tumors and foregut carcinoid tumors are malignant. MEN1 is thus the expression of a cancer gene but without available prevention or cure for malignancy. Hereditary (as compared to sporadic) endocrine tumors show early onset age and multiplicity, because each cell of the body has "one hit" by inheritance. Multiple neoplasia syndromes with endocrine tumor(s) all include nonendocrine components; their known defective genes seem mainly to disturb cell accumulation. Hereditary neoplasia/hyperplasia of one endocrine tissue reflects a defect that is tissue selective and directed at cell secretion. Though the hereditary endocrine neoplasias are rare, most of their identified genes also contribute to common sporadic endocrine neoplasms. Hereditary tumors may be caused by activation of an oncogene (e.g., RET) or, more often, by inactivation of a tumor suppressor gene (e.g., P53, MEN1). Recently, MEN1 was identified by positional cloning. This strategy included narrowing the gene candidate interval, identifying many or all genes in that interval, and testing the newly identified candidate genes for mutation in MEN1 cases. MEN1 was identified because it showed mutation in 14 of 15 MEN1 cases. NIH testing showed germline MEN1 mutations in 47 of 50 MEN1 index cases and in seven of eight cases with sporadic MEN1. Despite proven capacity to find germline MEN1 mutation, NIH testing found no MEN1 mutation among five families with isolated hyperparathyroidism, suggesting that this often arises from mutation of other gene(s). Analogous studies in Japan found that familial isolated pituitary tumors also did not show MEN1 germline mutation. MEN1 mutation testing can now be considered for cases of MEN1 and its phenocopies and for asymptomatic members of families with known MEN1 mutation. Germline MEN1 testing does not have the urgency of RET testing in MEN2a and 2b, as MEN1 testing does not commonly lead to an important intervention. Somatic MEN1 mutation was found in sporadic tumors: parathyroid adenoma (21%), gastrinoma (33%), insulinoma (17%), and bronchial carcinoid (36%). For each of these, MEN1 was the known gene most frequently mutated. MEN1 has a widely expressed mRNA that encodes a protein (menin) of 610 amino acids. The protein sequence is not informative about domains or functions. The protein was mainly nuclear. Menin binds to JunD, an AP-1 transcription factor, inhibiting JunD's activation of transcription. Most of the germline and somatic MEN1 mutations predict truncation of menin, a likely destructive change. Inactivating MEN1 mutations in germline and in sporadic neoplasms support prior predictions that MEN1 is a tumor suppressor gene. Germline MEN1 mutation underlies all or most cases of MEN1 (familial or sporadic). Somatic MEN1 mutation is the most common gene mutation in many sporadic endocrine tumor types.

Highly consistent genetic alterations in childhood adrenocortical tumours detected by comparative genomic hybridization.

We have examined 11 cases of childhood adrenocortical tumours for copy number changes using comparative genomic hybridization (CGH). The changes seen are highly consistent between cases, and are independent of tumour type (carcinoma versus adenoma) or the presence of a germline TP53 mutation. The regions of chromosomal gain and loss identified in this study indicate the location of genes that are potentially important in the development and progression of childhood adrenocortical tumours. Finally, the copy number changes identified in childhood tumours are distinctly different to those seen in adult cases (Kjellman et al (1996) Cancer Res 56: 4219-4223), and we propose that this indicates that childhood tumours are of embryonal origin.

Are there low-penetrance TP53 Alleles? evidence from childhood adrenocortical tumors.

We have analyzed a panel of 14 cases of childhood adrenocortical tumors unselected for family history and have identified germline TP53 mutations in >80%, making this the highest known incidence of a germline mutation in a tumor-suppressor gene in any cancer. The spectrum of germline TP53 mutations detected is remarkably limited. Analysis of tumor tissue for loss of constitutional heterozygosity, with respect to the germline mutant allele and the occurrence of other somatic TP53 mutations, indicates complex sequences of genetic events in a number of tumors. None of the families had cancer histories that conformed to the criteria for Li-Fraumeni syndrome, but, in some families, we were able to demonstrate that the mutation had been inherited. In these families there were gene carriers unaffected in their 40s and 50s, and there were others with relatively late-onset cancers. These data provide evidence that certain TP53 alleles confer relatively low penetrance for predisposition to the development of cancer, and they imply that deleterious TP53 mutations may be more frequent in the population than has been estimated previously. Our findings have considerable implications for the clinical management of children with andrenocortical tumors and their parents, in terms of both genetic testing and the early detection and treatment of tumors.

Exclusion of the genes CDKN2 and PTEN as causative gene defects in Li-Fraumeni syndrome.

We have analysed Li-Fraumeni syndrome families, previously shown to be negative for mutations in TP53, for mutations to the tumour suppressor genes PTEN and CDKN2. These genes function in cell cycle progression or are mutated in a variety of tumours. We have detected no mutations in the family members tested.

Comparative genomic hybridization as a tool in tumour cytogenetics.

The quality of cytogenetic analysis of solid tumours has greatly improved in the past decade, but a number of technical difficulties remain which limit the characterization of solid tumour chromosomes by conventional cytogenetics alone. The identification of regions of chromosomal abnormality has been aided by the introduction of molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Of these, a recently developed approach, comparative genomic hybridization (CGH), has had a particular impact on the cytogenetic analysis of solid tumours. It incorporates the sensitivity of in situ techniques and overcomes many of the drawbacks of conventional cytogenetic analysis. This review first outlines the CGH method, giving details for the preparation of DNA probes and target human metaphase chromosomes together with information on the in situ technique and data handling criteria used in our laboratory. It then presents an overview of some of the current applications of CGH, together with a discussion of future directions in the field.

Glutamine oxidation and utilization by rat and human oesophagus and duodenum.

The rates of utilization and oxidation of glutamine and glucose by oesophageal and duodenal tissues have been investigated in both rats and human subjects. In the rat, glutamine utilization by oesophageal tissue was 2-3-fold lower than that in the duodenum, and this substrate contributed less than 10% to the total oxidative metabolism of the tissue, even when glutamine was the only substrate provided. In contrast, rat duodenal tissue derived about 34% of the total CO2 production from glutamine-C, and this contribution was not suppressed by the addition of either glucose or a mixture of the other substrates. Rates of glucose utilization and oxidation by the duodenum were lower than those for glutamine, and were significantly (P < 0.001) suppressed by addition of glutamine. In both oesophageal and duodenal tissues, less than 10% of the glutamine-C utilized was fully oxidized, approximately 60-70% was converted to glutamate, and 30-40% to alanine. Results obtained using human biopsy tissue samples were similar to those observed in the rat. Glutamine oxidation contributed 34 (SD 4)% of the total CO2 production by the duodenal tissue, but only 8 (SD 4)% to oesophageal tissue oxidation. The findings suggest that glutamine is not an important or preferred fuel for oesophageal tissue, whereas it is for duodenal tissue. Thus, these tissues can be expected to respond differently to glutamine administration.

Comparative genomic hybridisation of ductal carcinoma in situ of the breast: identification of regions of DNA amplification and deletion in common with invasive breast carcinoma.

Comparative genomic hybridisation has been used to map copy number changes in nine cases of ductal carcinoma in situ of the breast obtained from wax-embedded archive material. A wide variety of abnormalities were detected including gain of regions of 1q, 17q, 19q, 20p and 20q and loss on 13q, 14q, 17p, 16q and 22q. Amplification of areas on 10p, 8q and 20q were also observed. Chromosomal alterations were more frequent in higher grade DCIS and closely resemble those previously detected in invasive breast cancer using the same technique. These data provide strong molecular support for the view that DCIS is a precursor lesion of invasive breast carcinoma.

Walking, cloning, and mapping with YACs in 3q27: localization of five ESTs including three members of the cystatin gene family and identification of CpG islands.

Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescence in sity hybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there.

Primary structure around the lipoate-attachment site on the E2 component of bovine heart pyruvate dehydrogenase complex.

Bovine heart pyruvate dehydrogenase complex was acetylated by using [3-14C]pyruvate in the presence of N-ethylmaleimide, with approx. 1 mol of acetyl groups being incorporated per mol of E2 polypeptide. After peptic digestion, lipoate-containing peptides were purified by high-voltage electrophoresis and ion-exchange and reverse-phase h.p.l.c. The amino acid sequence around the lipoic acid-attachment site of E2 was determined by automated Edman degradation. Acetylation of a lipoate cofactor bound to a lysine residue was verified by fast-atom-bombardment m.s.

Protein phosphorylations in poliovirus infected cells.

In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis.