Testing for BRCA1 mutations: a cost-effectiveness analysis

Breast carcinoma is the most common type of cancer affecting women in the Western world. The hereditary forms, which amount from 5 to 10% of all the cases of breast cancer, mainly involve BRCA1 or BRCA2 mutations. Due to the diagnostic strategy used by the patent owner, Direct DNA sequencing (DS) may become the only BRCA1/2 test procedure available, although there exist several alternative strategies. A cost-effectiveness study was carried out using BRCA1 testing as a model. The main techniques available for performing mutation searches were assessed: DS, denaturing high performance liquid chromatography (DHPLC), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), fluorescent assisted mismatch analysis (FAMA) and the protein truncation test (PTT). Twenty strategies involving the use of one or more techniques were then devised for performing the complete genetic analysis. DS was adopted as the 'gold standard' for effectiveness. All the strategies except for DS involved a two-step procedure. The first step consisted of pre-screening the 22 coding exons of BRCA1. The second step consisted of performing DS only on the variations detected in the coding sequence. The cost of the strategies tested, including a pre-screening stage, turned out to be 30 to 90% lower than that of DS, whatever annual use was made of the equipment. The most cost-effective strategy, ie, that corresponding to the lowest cost per mutation detected, was found to be a combination between PTT on exon 11 (60% of the coding sequence) and HA on the remaining 21 exons (PTT(11)+ HA(21)). However, since a high false negative rate is associated with this strategy, at least four other strategies are worth mentioning: PTT(11)+ DHPLC(21), DHPLC alone, FAMA(11)+ DHPLC(21) and FAMA alone. Our results on genetic testing for breast cancer show that DS is not the most cost-effective method available. The monopolist approach of the firm which owns the patents on the BRCA1/2 genes, may, therefore limit the use of the most cost-effective strategies.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Modulation of the transcripts for tumor necrosis factor-α and its receptors in vivo

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor-α (TNF) and its receptors: TNF receptor type I (TNF-R 55 kDa or TNF-R1) and TNF receptor type II (TNF-R 75 kDa or TNF-R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF-R1 and TNF-R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF-R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF-R of both types are capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.     

Reinfektionsversuche an Lymnaea stagnalis mit Isthmiophora melis (Trematoda,Echinostomatidae) durch Miracidienexposition nach vorausgegangener Redienimplantation

Zusammenfassung Die natürliche intramolluskäre Entwicklung des Darmegels Isthmiophora melis verläuft über zwei morphologisch distinkte Redienformen: Auf zwei Generationen makropharyngeater Redien (Mutterredien) folgen nur noch Generationen mikropharyngeater Redien (Tochterredien) sowie Cercarien. Die Bildung von Mutterredien unterbleibt in Schnecken, die nur durch Implantation von Tochterredien experimentell infiziert wurden. Werden Schnecken nacheinander beiden Infektionsmodi unterworfen, der Tochterredienimplantation und der Miracidienexposition, so läßt sich aus dem Vorhandensein oder Fehlen von Mutterredien ablesen, welche der Infektionen von Erfolg war.Mittels dieser Methode wurde geprüft, ob Isthmiophora-parasitierte Lymnaea-Individuen gegen homologen Miracidienbefall resistent sind. Sie erwiesen sich als nicht resistent gegen Reinfektion, und zwar auch dann, wenn sie bereits in die Phase des Cercarienausstoßes eingetreten waren.

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Experimental re-infection of Lymnaea stagnalis with Isthmiophora melis (trematoda, echinostomatidae) by exposition to miracidia after implantation of rediae

Summary The natural intramolluscular development of the intestinal fluke Isthmiophora melis takes place in two morphologically distinct forms of rediae: Two generations of macropharyngeate rediae (mother rediae) are followed only by generations of micropharyngeate rediae (daughter rediae) and cercariae. Mother rediae are not produced in snails infected by implantation of daughter rediae only. If snails are subjected to the implantation of daughter rediae and exposed to miracidiae successsively, the presence, or absence, of mother rediae indicates which of the infections has been successful.This method of investigation was used in order to ascertain whether Lymnaea individuals parasitized by Isthmiophora are resistant against invading miracidia of homologous species. They proved not resistant against re-infection, even if they had already entered the stage of shedding cercariae.

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Ermöglicht durch Beihilfen der Deutschen Forschungsgemeinschaft.     

Fluorescence in situ hybridization reveals closely correlated results in cytological and histological specimens of hematological neoplasias compared to conventional cytogenetics.

OBJECTIVES: Fluorescence in situ hybridization (FISH) has become a useful tool to identify chromosomal aberrations in non-dividing cells. Numerous studies have compared chromosomal banding analysis (CBA) and FISH on fixed cultured bone marrow cells. However, up to now, there has been no study comparing two main sources of diagnostic material, i.e. bone marrow aspirates and trephine biopsies. We therefore analyzed these materials by FISH in comparison with CBA. METHODS: CBA revealed chromosomal aberrations in 18 patients suffering from myelodysplastic syndrome (n = 13), acute myeloid leukemia (n = 3), or chronic myeloproliferative syndrome (n = 2). FISH was performed on fixed cultured bone marrow cells, aspirates and trephine biopsies from each patient. RESULTS: Percentages of aberrant cells in the different materials correlated highly with Pearson values of 0.909 for biopsy/fixed cultured cells (p < 0.001), 0.830 for biopsy/aspirate (p < 0.001) and 0.768 for aspirate/fixed cultured cells (p < 0.001). Moreover, in bone marrow biopsies peritrabecular and central intertrabecular areas yielded very similar FISH results with a high correlation (r = 0.968, p < 0.001). FISH revealed a lower proportion of aberrant cells than CBA in 90% of the specimens. CONCLUSIONS: In summary, the different materials available for the FISH examination are comparable in sensitivity and show similar quantitative results. Therefore, the use of biopsy sections for the routine FISH examination of chromosomal abnormalities is a valid method.     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.     

NPHS2 gene, nephrotic syndrome and focal segmental glomerulosclerosis: a HuGE review.

Nephrotic syndrome, characterized by edema, proteinuria, hyperlipidemia and low serum albumin, is a manifestation of kidney disease involving the glomeruli. Nephrotic syndrome may be caused by primary kidney disease such as focal segmental glomerulosclerosis. Mutations in the podocin gene, NPHS2, have been shown in familial and sporadic forms of steroid-resistant nephrotic syndrome, including focal segmental glomerulosclerosis. Podocin is an integral membrane protein located at the slit diaphragm of the glomerular permeability barrier. Complete information is lacking for the population frequency of some NPHS2 variants for all racial and ethnic groups. The most frequently reported variant, R229Q, is more common among European-derived populations than African-derived populations. We calculated crude odds ratios and 95% confidence intervals of childhood nephrotic syndrome and focal segmental glomerulosclerosis associated with R229Q heterozygosity using data from five studies. The R229Q variant is not associated with focal segmental glomerulosclerosis in the US population of African descent. In contrast, the R229Q variant is associated with a trend toward increased focal segmental glomerulosclerosis risk in European-derived populations, with an estimated increased risk of 20-40%. Our insight into the association between NPHS2 variants and nephrotic disease is hampered by the limitations of the existing studies, including small numbers of affected individuals and suboptimal control groups. Nevertheless, the available data suggest that large epidemiological case-control studies to examine the association between NPHS2 variants and nephrotic syndrome are warranted.     

A gamma-herpesvirus immune evasion gene allows tumor cells in vivo to escape attack by cytotoxic T cells specific for a tumor epitope

DNA vaccines induce CTL attack on target tumor epitopes, but tumor elimination in vivo also requires sufficient effector CTL to enter the site, guided by inflammatory chemokines. Many herpesviruses contain genes for chemokine and chemokine receptor-like proteins to protect infected cells from immune attack. To assess if this evasion strategy could protect tumor cells, we used a model where CTL specific for a single epitope were the only effectors. Following DNA vaccination, CTL eliminated tumor cells from a subcutaneous site. However, introducing a viral gene encoding a secreted broad-spectrum chemokine-binding protein (M3) into tumor cells completely blocked CTL attack. Transduced tumor cells also protected neighboring non-transduced tumor. These findings confirm the importance of chemokines for migration of CTL to a non-lymphoid site. They may have relevance for escape of human virus-associated malignancies, and raise the question of whether analogous molecules might contribute to the failure of CTL to eliminate tumors.     

Issues to debate on the Women's Health Initiative: estrogen: an instrument or the conductor of the orchestra?

Although it is well known that cyclic production of sex hormones is essential to establish reproductive function and female characteristics, distant impacts of the activity of the female endocrine system result from a concert of delicate mechanisms. Estrogen is rather an instrument than a conductor in this physiological orchestra of the female. Thus, controversies in the explanation of results from studies on hormone replacement therapy (HRT) and cardiovascular disease (CVD) prevention might be eliminated, if we analyse not only the role of estrogen but a broader spectrum of factors leading to CVD. Authors would like to hypothesize that haemorheological changes in women around menopause, such as increased blood and plasma viscosity, haematocrit and fibrinogen, are largely responsible for the increased mortality in the post-menopausal life period. We believe that a cyclic withdrawal bleeding establishes a more favourable haemorheological condition, thus, sequentially administered estrogen might be protective in post-menopausal women. Nevertheless, other factors, that decrease blood viscosity, such as daily exercise, intake of ample amount of fluids as well as ideal nutrition, are equally important. We are confident that sequential HRT, as well as healthy life style and risk prevention programmes have their proper place in the management of this issue.     

Standard skin prick testing and sensitization to inhalant allergens across Europe--a survey from the GALEN network

Skin prick testing (SPT) is the standard method for diagnosing allergic sensitization but is to some extent performed differently in clinical centres across Europe. There would be advantages in harmonizing the standard panels of allergens used in different European countries, both for clinical purposes and for research, especially with increasing mobility within Europe and current trends in botany and agriculture. As well as improving diagnostic accuracy, this would allow better comparison of research findings in European allergy centres. We have compared the different SPT procedures operating in 29 allergy centres within the Global Allergy and Asthma European Network (GA(2)LEN). Standard SPT is performed similarly in all centres, e.g. using commercial extracts, evaluation after 15-20 min exposure with positive results defined as a wheal >3 mm diameter. The perennial allergens included in the standard SPT panel of inhalant allergens are largely similar (e.g. cat: pricked in all centres; dog: 26 of 29 centres and Dermatophagoides pteronyssinus: 28 of 29 centres) but the choice of pollen allergens vary considerably, reflecting different exposure and sensitization rates for regional inhalant allergens. This overview may serve as reference for the practising doctor and suggests a GA(2)LEN Pan-European core SPT panel.