Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara.

Immunization with irradiated sporozoites can protect against malaria infection and intensive efforts are aimed at reproducing this effect with subunit vaccines. A particular sequence of subunit immunization with pre-erythrocytic antigens of Plasmodium berghei, consisting of single dose priming with plasmid DNA followed by a single boost with a recombinant modified vaccinia virus Ankara (MVA) expressing the same antigen, induced unprecedented complete protection against P. berghei sporozoite challenge in two strains of mice. Protection was associated with very high levels of splenic peptide-specific interferon-gamma-secreting CD8+ T cells and was abrogated when the order of immunization was reversed. DNA priming followed by MVA boosting may provide a general immunization regime for induction of high levels of CD8+ T cells.

Factors influencing disease phenotype and penetrance in HFE haemochromatosis.

Haemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is present in approximately 1 in 200 people of Northern European origin. However, not all p.C282Y homozygotes develop clinical features of haemochromatosis, and not all p.C282Y homozygotes even present abnormal iron parameters justifying venesection therapy. This situation was not apparent from initial genotype/phenotype correlation studies as there was a selection bias of patients. Only those patients with a significant iron burden were included in these early studies. It is now largely accepted that the p.C282Y/p.C282Y genotype is necessary for the development of HFE haemochromatosis. However, this genotype provides few clues as to why certain symptoms are associated with the disease. Expression of iron overload in people with this genotype depends on the complex interplay of environmental factors and modifier genes. In this review, we restrict our discussion to work done in humans giving examples of animal models where this has helped clarify our understanding. We discuss penetrance, explaining that this concept normally does not apply to autosomal recessive disorders, and discuss the usefulness of different biochemical markers in ascertaining iron burden. Hepcidin, a peptide synthesized primarily by the liver, has been identified as the central regulator in iron homeostasis. Consequently, understanding its regulation is the key. We conclude that the main goal now is to identify important modifiers that have a significant and unambiguous effect on iron storage.

A family of cation ATPase-like molecules from Plasmodium falciparum.

We report the nucleotide and derived amino acid sequence of the ATPase 1 gene from Plasmodium falciparum. The amino acid sequence shares homology with the family of "P"-type cation translocating ATPases in conserved regions important for nucleotide binding, conformational change, or phosphorylation. The gene, which is present on chromosome 5, has a product longer than any other reported for a P-type ATPase. Interstrain analysis from 12 parasite isolates by the polymerase chain reaction reveals that a 330-bp nucleotide sequence encoding three cytoplasmic regions conserved in cation ATPases (regions a-c) is of constant length. By contrast, another 360-bp sequence which is one of four regions we refer to as "inserts" contains arrays of tandem repeats which show length variation between different parasite isolates. Polymorphism results from differences in the number and types of repeat motif contained in this insert. Inserts are divergent in sequence from other P-type ATPases and share features in common with many malarial antigens. Studies using RNA from the erythrocytic stages of the malarial life cycle suggest that ATPase 1 (including the sequence which encodes tandem repeats) is expressed at the large ring stage of development. Immunolocalization has identified ATPase 1 to be in the region of the parasite plasma membrane and pigment body. These findings suggest a possible model for the genesis of malarial antigens.

Iron genes, iron load and risk of Alzheimer's disease.

BACKGROUND: Compound heterozygotes of the haemochromatosis gene (HFE) variants, H63D and C282Y, have raised transferrin saturation compared with that in the wild type. In the cohort of the Oxford Project To Investigate Memory and Ageing (OPTIMA), bicarriers of the HFE C282Y and the transferrin C2 gene variants are at five times greater risk of developing Alzheimer's disease; the addition of HFE H63D may raise the risk still further. OBJECTIVE: To investigate transferrin saturation by HFE and transferrin genotype among people without dementia-that is, controls and those with mild cognitive impairment (MCI)-and also among those with Alzheimer's disease. METHODS: Serum iron status and genotype were examined of 177 patients with Alzheimer's disease, 69 patients with MCI and 197 controls from the OPTIMA cohort. RESULTS: Although each of these variants alone had relatively little effect on iron status, the combination of either HFE C282Y and HFE H63D or of HFE C282Y and transferrin C2 markedly raised transferrin saturation in those without dementia, but had little effect in those with mature Alzheimer's disease. CONCLUSIONS: These combinations may raise the risk for Alzheimer's disease, owing to higher iron loads and therefore oxidative stress in the preclinical phase. If replicated, these findings will have implications for the prevention of Alzheimer's disease.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

BACKGROUND: Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. METHODS: We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. FINDINGS: In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

Synergy between the C2 allele of transferrin and the C282Y allele of the haemochromatosis gene (HFE) as risk factors for developing Alzheimer's disease.

BACKGROUND: There is evidence that iron may play a role in the pathology of Alzheimer's disease (AD). There may be genetic factors that contribute to iron deposition resulting in tissue damage thus exacerbating AD. METHODS: We have genotyped 269 healthy elderly controls, 191 cases with definite or probable AD, and 69 with mild cognitive impairment (MCI) from the OPTIMA cohort. RESULTS: We have examined the interaction between the C2 variant of the transferrin (TF) gene and the C282Y allele of the haemochromatosis (HFE) gene as risk factors for developing AD. Our results showed that each of the two variants was associated with an increased risk of AD only in the presence of the other. Neither allele alone had any effect. Carriers of both variants were at 5 times greater risk of AD compared with all others. The interaction was significant by logistic regression (p = 0.014) and by synergy factor analysis (p = 0.015, synergy factor = 5.1). Further, carriers of these two alleles plus apolipoprotein E epsilon4 (APOE4) were at still higher risk of AD: of the 14 tri-carriers of the three variants, identified in this study, 12 had AD and two MCI. CONCLUSION: We suggest that the combination of TF C2 and HFE C282Y may lead to an excess of redox-active iron and the induction of oxidative stress in neurones, which is exacerbated in carriers of APOE4. Since 4% of Northern Europeans carry the two iron-related variants and since iron overload is a treatable condition, these results merit replication.

Recent advances in understanding haemochromatosis: a transition state.

Mutations in the hepcidin gene HAMP and the hemojuvelin gene HJV have recently been shown to result in juvenile haemochromatosis (JH). Hepcidin is an antimicrobial peptide that plays a key role in regulating intestinal iron absorption. Hepcidin levels are reduced in patients with haemochromatosis due to mutations in the HFE and HJV genes. Digenic inheritance of mutations in HFE and HAMP can result in either JH or hereditary haemochromatosis (HH) depending upon the severity of the mutation in HAMP. Here we review these findings and discuss how understanding the different types of haemochromatosis and our increasing knowledge of iron metabolism may help to elucidate the host's response to infection.

The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading.

BACKGROUND: Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene. METHODS: Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts. RESULTS: The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively). CONCLUSIONS: Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

Pilot study into optimisation of viewing conditions for electronically displayed images.

The increased use of soft-copy reporting introduces new concerns over the effect of viewing conditions on the observer's ability to report images. Owing to their lower luminance, electronic display screens may be more susceptible to poor viewing conditions than conventional viewing boxes and there is the potential for images to be displayed in locations not optimised for viewing radiographs. In the present work, the effects of sub-optimal viewing conditions on the observer's performance for images on an electronic display device are investigated. A test object was used to produce a computed radiography image containing a wide range of grey levels. The image was scored under quasi-ideal and sub-optimal conditions and the effect of changing the viewing conditions on the observer's performance determined. Basic photometric quantities were used to characterise the viewing conditions and the degradation in observer performance related to these quantities. The presence of structured reflection had a significant effect on the observer's ability to discern low-contrast objects. The study demonstrates the need for adequate viewing conditions especially when images are displayed on low luminance devices in sub-optimal conditions.

Sequence diversity in the intron of the calmodulin gene from Plasmodium falciparum.

Sequence variation in the single intron of the calmodulin gene of Plasmodium falciparum has been examined following amplification using the polymerase chain reaction (PCR). The intron has 4 repeating motifs varying in length: 3 of these contain a dinunucleotide repeat, dA-dT, the fourth is a pentameric repeating unit, dA-dT-dA-dT-dT. These DNA polymorphisms can be applied to the study of parasite populations in mixed infections and in strain identification. The function of these repeating motifs is unknown. Computer modelling of the possible intron structures demonstrates that each of these repeating motifs forms individual stem loops such that any changes in repeat number are self-compensatory and do not change the overall intron structure. The implications of this sequence variation are discussed.

The structure of the calmodulin gene of Plasmodium falciparum.

The calmodulin gene and its flanking sequences from the malaria parasite, Plasmodium falciparum, have been analysed. The structure of this gene is unique amongst other known calmodulin genes. It exists as a single copy on chromosome 14 and has a single intron. The nucleotide sequence of this 4-kb region suggests the existence of three transcriptional units, each separated by a highly A+T-rich sequence. Sequences controlling gene expression might be expected to occur in these intergenic regions. The predicted protein sequences suggest that these other genes are transcribed in different orientations. Primer extension studies suggest that calmodulin mRNA has a major start site 62 bases upstream of the initial ATG. The calmodulin gene possesses consensus eukaryotic TATA, CAAT box, polyadenylation and splice junction sequences. This is the first detailed report of the DNA sequence surrounding a housekeeping gene in P. falciparum.

Cloning and expression of the thrombospondin related adhesive protein gene of Plasmodium berghei.

Sporozoite recognition of host cells is a key step in the life-cycle of malaria parasites. Two sporozoite proteins have so far been characterized in some detail, the circumsporozoite protein (CS) and thrombospondin related adhesive protein (TRAP). We report here the cloning and expression of the TRAP gene homologue from Plasmodium berghei, PbTRAP. The PbTRAP gene encodes a protein of 606 amino acids having a deduced molecular mass of 66 kDa. The overall structure is clearly that of the TRAP family having a signal sequence followed by an integrin A domain, a sulphatide binding motif, followed by a proline based repeat before a transmembrane domain and helical cytoplasmic tail. The observed molecular mass is almost 50% larger than expected, this can be explained almost entirely by the abnormal behaviour in SDS-PAGE of the proline based repeat. As would be expected PbTRAP shows greatest similarity with the P. yoelli TRAP homologue sporozoite surface protein 2 (SSP2) than with PfTRAP, the TRAP gene from P. falciparum. The pattern of expression is similar to that of SSP2.

Polymorphism of the TRAP gene of Plasmodium falciparum.

Natural sequence variation of the thrombospondin related anonymous protein (TRAP) gene of Plasmodium falciparum has been investigated by DNA analysis following the polymerase chain reaction amplification, and this shows the gene to be highly polymorphic. The region containing the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), common to TRAP, the circumsporozoite protein, properdin, and thrombospondin, was invariant. Elsewhere in the molecule, over 50 amino acid substitutions are described including the insertion of an in-frame, small-variable tandemly repeating motif between amino acid residues 352 and 353. Only one silent mutation was observed. Most nucleotide changes that occur in the first two codon positions result in conservative amino acid changes. Restriction fragment length polymorphism (RFLP) analysis was used to examine inheritance of TRAP in a cross between the HB3 and 3D7 clones of P. falciparum. Out of nine progeny examined, four possessed the HB3 gene and five the 3D7 gene. The TRAP gene hybridized to chromosome 13. Previous work has shown that a subtelomeric region of chromosome 13 from the 3D7 parent (marked by the HRP-III gene) was favoured strongly in this cross. The TRAP gene, however, is over 1 Mb away from this subtelomeric region and exhibits no such linkage because of chromosome crossovers. Five geographically separate isolates shared the same TRAP sequence as well as the same variant of the Th2R/Th3R region from the circumsporozoite protein. The correlation between independent markers in these isolates suggests that they have a common provenance.

Natural polymorphism in the thrombospondin-related adhesive protein of Plasmodium falciparum.

We have developed a typing system using natural sequence variation in the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum. This method permits a haplotype to be assigned to any particular TRAP gene. We have applied this method to a hospital-based, case control-study in Mali. Previous sequence variation and conservation in TRAP has been confirmed. Particular TRAP haplotypes can be used as geographic hallmarks. Because of the high level of conflict between characters, we have examined the phylogenetic relationships between parasites using a network approach. Having received patient samples from urban and periurban areas of Bamako, the majority of haplotypes were closely related and distinct from TRAP sequences present in other continents. This suggests that the structure of TRAP can only tolerate a limited number of sequence variations to preserve its function but that this is sufficient to allow the parasite to evade the host's immune system until a long-lived immune response can be maintained. It may also reflect host genetics in that certain variants may escape the host immune response more efficiently than others. For vaccine design, sequences from the major regional variants may need to be considered in the production of effective subunit vaccines.

Cutaneous manifestations of systemic diseases.

Many aspects of dermatologic diagnosis are either of importance or interest to the nondermatologist, and there are many excellent textbooks available for guidance. This article focuses on four categories of conditions that are the source of frequent queries from the primary care setting: (1) common skin diseases that frequently mimic systemic illness, (2) common skin diseases that have important systemic associations, (3) common systemic diseases that have prominent cutaneous findings, and (4) the seldom seen but frequently raised concerns regarding cutaneous signs of internal malignancy.

Mammographic image restoration using maximum entropy deconvolution.

An image restoration approach based on a Bayesian maximum entropy method (MEM) has been applied to a radiological image deconvolution problem, that of reduction of geometric blurring in magnification mammography. The aim of the work is to demonstrate an improvement in image spatial resolution in realistic noisy radiological images with no associated penalty in terms of reduction in the signal-to-noise ratio perceived by the observer. Images of the TORMAM mammographic image quality phantom were recorded using the standard magnification settings of 1.8 magnification/fine focus and also at 1.8 magnification/broad focus and 3.0 magnification/fine focus; the latter two arrangements would normally give rise to unacceptable geometric blurring. Measured point-spread functions were used in conjunction with the MEM image processing to de-blur these images. The results are presented as comparative images of phantom test features and as observer scores for the raw and processed images. Visualization of high resolution features and the total image scores for the test phantom were improved by the application of the MEM processing. It is argued that this successful demonstration of image de-blurring in noisy radiological images offers the possibility of weakening the link between focal spot size and geometric blurring in radiology, thus opening up new approaches to system optimization.

Receptor dose in digital fluorography: a comparison between theory and practice.

A method of identifying the dose per image when quantum mottle no longer dominates the image statistics is presented as a first step towards quantitative optimization in native and subtracted digital fluorography. The method is based on measurements of threshold contrast over a range of receptor doses and the application of a simple model of the threshold contrast detection task to estimate the magnitude of system noise sources. The point at which system and quantum noise sources are equal in magnitude is proposed as the practical upper limit for dose per image. The method is applied to a typical digital fluorography system and the results are placed into the context of the range of dose per image values found from a regional survey of digital fluorography units. While there is broad agreement between the dose per image values in the survey with values predicted from the experimental method, the considerable spread in survey doses suggests there are instances where the use of a high dose per image is unjustified.

Detection of C282Y and H63D in the HFE gene.

The gene for hemochromatosis was identified in 1996 and two mutations were found. Homozygosity for one of these, C282Y, is associated with hemochromatosis in a high percentage of patients. Genetic analysis of patient DNA is, therefore, a very useful tool to aid and confirm diagnosis and to screen asymptomatic relatives of patients to identify those at risk of developing this common, easily treated disease.

Uncommon mutations and polymorphisms in the hemochromatosis gene.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns.