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Biomedical subjects

C R Newton

Publications and source records attributed to C R Newton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Development and validation of a screening test for 12 common mutations of the cystic fibrosis CFTR gene.

The results obtained using deoxyribonucleic acid (DNA) amplification-based tests must be accurate and reproducible. One such test that simultaneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in this report. An investigation was conducted into how changes of primer, DNA template and Taq DNA polymerase concentrations and of polymerase chain reaction annealing temperatures affect the test. A total of 383 DNA samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate results are obtained even if the test is performed under suboptimal conditions. Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remaining 1.6% of samples were not mistyped, rather they were not scored owing to failure to detect control DNA sequences. These were also archival DNA preparations rather than freshly prepared samples from venous blood. Careful primer design and optimization of reaction conditions are important in the development of multiplex deoxyribonucleic acid amplification-based diagnostic tests. Providing the recommended protocols are followed, the test described here is simple to carry out, gives accurate results and works well if performed within defined operational windows for each reaction variable.

Cystic Fibrosis↗
PubMed1998

Pathophysiology of fatal falciparum malaria in African children.

Children living in sub-Saharan Africa bear the brunt of the mortality from falciparum malaria, yet there is a dearth of relevant post-mortem data. Clinical studies from centers in Africa suggest that the pathophysiology of severe malaria is different in children and adults. Three overlapping clinical syndromes, metabolic acidosis manifesting as hyperpnea, cerebral malaria, and severe anemia, are responsible for nearly all the deaths in African children. Despite improvements in antimalarial treatment, there has not been a significant reduction in mortality. We review the pathology and pathophysiology of fatal falciparum malaria in African children. Many questions remain, the answers to which would facilitate the development and evaluation of new approaches to the management of this disease.

Acidosis↗
PubMed1998

A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping.

We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5'-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.

Alleles↗
PubMed1998

Nasal manipulation with intravenous sedation. Is it an acceptable and effective treatment?

Local anaesthesia is increasingly being used for nasal manipulation. Doubt remains over the discomfort associated with this procedure. We studied in a prospective manner, the acceptability and effectiveness of nasal manipulation with intravenous sedation and local anaesthesia, in a case series of thirty-five patients. Patients used linear analogue scales to assess outcome and pain associated with the procedure. More than half of the patients (60%) returned pain scores of only 1 out of 10. Thirty-two patients (90%) found the procedure less painful than receiving a tooth filling from the dentist, while thirty-four patients (97%) said they would undergo the same procedure again if they re-fractured their nose. The effectiveness of the treatment as assessed by an independent observer revealed that 90% of patients had achieved a significant reduction in their deformity (P = 1.27 x 10(-9).

Administration, Intranasal↗
PubMed1997

A colorimetric assay for phosphate to measure amplicon accumulation in polymerase chain reaction.

We describe a rapid colorimetric method for the detection of PCR products. Color generation is complete within 5 min of mixing reagents with a PCR. The method is simple and does not require affinity capture steps or special labeling to be carried out. The color development can be monitored by eye or a simple spectrophotometer can be used to read sample absorbance. We demonstrate the method by its use in the amplification refractory mutation system (ARMS) analysis [C. R. Newton, A. Graham, L. E. Heptinstall, S. J. Powell, C. Summers, N. Kalsheker, et al. (1989) Nucleic Acids Res. 17, 2503-2515] of cystic fibrosis [J. R. Riordan, J. M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, et al. (1989) Science 245, 1066-1073] and factor V [R. M. Bertina, B. P. C. Koeleman, T. Koster, F. R. Rosendaal, R. J. Dirven, H. de Ronde, P. A. van der Velden, and P. H. Reitsma (1994) Nature 369, 64-67] allelic variants.

Alleles↗
PubMed1997

Severe anaemia in children living in a malaria endemic area of Kenya.

Severe anaemia is an important cause of morbidity and mortality in African children, but the causes, particularly falciparum malaria, are difficult to determine. We assessed the contribution of falciparum malaria to anaemia in Kenyan children by clinical examination and measurement of parasitaemia and haemoglobin (Hb) concentration in 559 children in the community and in 2412 children admitted to Kilifi district hospital during a 2-year period. We also attempted to characterize severe malarial anaemia by examining the causes and pathophysiology of anaemia in 101 children admitted with Hb concentration < or = 50 g/l during a 1-year period. Plasmodium falciparum infection was associated with reduced Hb concentration in children in the community and in those admitted to hospital irrespective of diagnosis. Falciparum malaria was the primary cause in 46 cases (46%) of severe anaemia admitted to hospital. There was no difference in the frequency of haemolysis or dyserythropoiesis in the children with malarial anaemia and those with anaemia from other causes, such as iron deficiency or sickle cell disease. The mortality rate in the children with severe malarial anaemia was 8.6% compared with 3.6% in children with severe anaemia due to other causes. Falciparum malaria does not present with a characteristic clinical or haematological picture, but is a major cause of the morbidity and mortality in children with severe anaemia who live on the Kenyan coast, a malaria endemic area.

Anemia↗
PubMed1997

Measurement of cerebral blood flow in dogs with near infrared spectroscopy in the reflectance mode is invalid.

Near infrared spectroscopy (NIRS) is used to measure CBF (CBFNIRS) in humans, based on Fick's principle, using oxygen as an intravascular tracer. We compared CBFNIRS with CBF measured by microspheres (CBF mu) and the venous outflow technique (CBFv) in 15 dogs, altering CBF with ventilation-induced changes in PaCO2. Five hundred forty-nine CBFNIRS measurements were attempted using an integration time of 2.5 s on the saturation signal from the tongue. One hundred ninety-eight (36.1%) of the measurements fulfilled predefined criteria. The coefficient of variation (CV) for six measurements under stable conditions was 29.1%. The CBFNIRS measurements correlated best with microsphere-measured blood flows in the cortical gray matter (median 0.43, range 0.16-0.93); the contributions of the skull and dura were variable. The CBFv varied by a médian of 12% (range 0-67%) during the CBFNIRS measurements. The percentage of acceptable CBFNIRS measurements, the CV, and the correlation coefficients of the CBFNIRS were improved by using saturation signal directly from the artery and varying the integration time with an estimate of the minimum transit time. The current method of measuring CBFNIRS in the reflectance mode is in-accurate when compared with other accepted techniques.

Animals↗
PubMed1997

Intracranial hypertension in Africans with cerebral malaria.

The causes of death and neurological sequelae in African children with cerebral malaria are obscure. Intracranial pressure (ICP) was monitored and cerebral perfusion pressure (CPP) calculated in 23 Kenyan children with cerebral malaria. Four children had severe intracranial hypertension (ICP > 40 mm Hg, CPP < 40 mm Hg): two died, one with an ICP of 158 mm Hg and signs of transtentorial herniation, the other one with an ICP of 42 mm Hg and cardiorespiratory arrest. The other two survived with severe neurological sequelae. Nine had intermediate intracranial hypertension (ICP > 20 mm Hg, CPP < 50 mm Hg) and 10 had mild intracranial hypertension (maximum ICP 10-20 mm Hg); all survived without severe sequelae. Mannitol controlled the ICP in children with intermediate intracranial hypertension, but it did not prevent the development of intractable intracranial hypertension in children with severe intracranial hypertension. Intracranial hypertension is a feature of Kenyan children with cerebral malaria and severe intracranial hypertension is associated with a poor outcome.

Child↗
PubMed1997

A homogeneous method for genotyping with fluorescence polarization.

We combined the amplification refractory mutation system (ARMS) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for delta F508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.

Apolipoproteins B↗
PubMed1996

Perturbations of cerebral hemodynamics in Kenyans with cerebral malaria.

The mechanisms of death and neurologic sequelae in African children with cerebral malaria are undetermined. Because pathologic features are confined to the cerebral vasculature, perturbations in cerebral hemodynamics may be responsible. We compared the transcranial Doppler findings in 50 children with cerebral malaria with those of 115 conscious Kenyan children. In addition, 10 children with cerebral malaria were studied during intracranial pressure monitoring and nine children were studied during the agonal stages. In the children with cerebral malaria, cerebral blood flow velocity was increased in 30%, usually associated with seizures. Of the 11 children who developed neurologic sequelae, six had sonographic abnormalities associated with lateralizing deficits, including four children with hemiparesis (in two children the contralateral middle cerebral artery could not be insonated and two had transient increases in blood flow velocity associated with seizures). In the children with severe intracranial hypertension, there was a significant linear relationship between the cerebral perfusion pressure and blood flow velocity, suggesting that autoregulation was impaired. Sonographic features of progressive intracranial hypertension, were observed in three children with cerebral malaria who died. Perturbations of cerebral hemodynamics are associated with a poor outcome in Kenyan children with cerebral malaria.

Blood Flow Velocity↗
PubMed1996

Falciparum malaria in children.

Recent reports have substantiated claims that the clinical spectrum of falciparum malaria is different in African children as compared with nonimmune adults. The SPf66 vaccine has been tried in African children with varying success, but only insecticide-treated bed nets have reduced the mortality. Developments in ancillary treatment may reduce the morbidity and mortality of severe falciparum malaria.

Animals↗
PubMed1995

Inter-observer agreement of the assessment of coma scales and brainstem signs in non-traumatic coma.

The authors evaluated the inter-observer agreement between two experienced clinicians examining 19 unconscious children who were not paralysed or ventilated. Inter-observer reliability was assessed by proportion of agreement, disagreement rate and kappa statistics. Corneal reflexes, pupillary responses to light and motor responses were the most reliably elicited. Reduction of the number of categories improved inter-observer agreement. Some of the disagreement may be attributed to changes in the child's condition during the period of assessment. There was more agreement about the five-category 0-IV scale than the summated Adelaide (10-category) and Jacobi (13-category) scales. The ability of these scales to follow changes in the patient's condition and to predict outcome needs to be evaluated in a prospective trial.

Brain Stem↗
PubMed1995

The management of severe malaria in children: a review.

We have attempted to summarise an approach to management of severe malaria from our experience and that of others from published data in this review. This represents our current state of knowledge and practices which may change as research continues in this field. It also represents what we feel should be the minimum aim in treating severe malaria even at district hospital level. It focuses on practical issues encountered when admitting such patients: initial assessment, immediate supportive management, use of transfusion, appropriate anti-malarial treatment and ongoing management.

Child↗
PubMed1994

Reagents for the preparation of two oligonucleotides per synthesis (TOPS).

In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.

Amides↗
PubMed1994

Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.

We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.

Affinity Labels↗
PubMed1994

Brain swelling and ischaemia in Kenyans with cerebral malaria.

Computed tomography was performed on 14 unconscious Kenyan children recovering from cerebral malaria (seven of whom had another scan 12-120 days later) to elucidate the cause of intracranial hypertension and neurological sequelae. Brain swelling, defined as a loss of cerebrospinal fluid spaces, was documented in six children, while a further two had conspicuously small ventricles only. There was severe intracranial hypertension in the two children with definite brain swelling in whom intracranial pressure was monitored. There was no evidence of acute hydrocephalus or vasogenic oedema. Four children with brain swelling also had widespread low density areas suggestive of ischaemic damage. The patterns of damage were not uniform but were consistent with a critical reduction in cerebral perfusion pressure (which was documented in the two in whom this was monitored), hypoglycaemia, or status epilepticus. All four had serious neurological sequelae. These data suggest that brain injury in cerebral malaria may be due in part to secondary systemic and intracranial factors as well as to the direct effect of intravascular sequestration.

Brain↗