Risk assessment for inherited susceptibility to cancer: a review of the psychosocial and ethical dimensions

The objective of this study was to conduct a broad-based systematic review of social, ethical, and legal considerations associated with genetic cancer risk assessment technologies (CaRATs). This paper focuses on psychosocial and ethical issues. Search results were limited to papers published in English, French, or German from January, 1990, to May, 2003. A quality assessment tool was developed and applied to retrieved papers. Application of the quality assessment tool resulted in 77 of 247 qualitative and quantitative primary research papers being reviewed and synthesized. A broad range of issues were addressed and grouped into content areas. Despite a large literature addressing psychosocial and ethical issues associated with CaRATs, many existing studies are not adequate to inform decision-makers and stakeholders. Careful policy analysis, as in some of the economic analyses reviewed here, is important to bridge this gap.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.     

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.     

NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation

The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.     

Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the beta-amyloid peptide (Abeta) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Abeta, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Abeta and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Abeta-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Abeta-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.     

A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage

Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation.     

Tensile yield in compact bone is determined by strain, post-yield behaviour by mineral content

Compact bone specimens from many species were examined to determine the relationships, in tension, between mineral content, Young's modulus, yield stress, yield strain, post-yield stress, post-yield strain, ultimate stress, ultimate strain and work under the stress-strain curve. Yield strain varied much less than the post-yield strain, and yield stress was strongly dependent on Young's modulus. Mineral content was a rather poor predictor of yield stress. However, post-yield events were predicted better by mineral (calcium) content than by Young's modulus. The greater the mineral content the less the post-yield work under the curve and the less the increase in post-yield stress and strain. The findings are compared with those of Les et al. who compressed specimens from equine metacarpals. Where they can be compared, the results are consistent with each other.     

Novel Approach to the Formulation of an Epstein-Barr Virus Antigen-Based Nasopharyngeal Carcinoma Vaccine

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.Epstein-Barr virus (EBV) is a member of the herpesvirus family and is one of the most common human viruses. It occurs worldwide, and most people become infected with the virus sometime during their lives. EBV is associated with a range of neoplasms. These include various B- and T-cell non-Hodgkin''s lymphomas such as posttransplant lymphoproliferative disease (PTLD), Hodgkin''s lymphoma (HL), and several lymphoepithelioma-like carcinomas, of which nasopharyngeal carcinoma (NPC) is the archetype (1). The association of the virus with these malignancies and its oncogenic potential have been well established (19).Worldwide, NPC is characterized epidemiologically by foci of relatively high endemicity in certain geographic regions including southern China, Hong Kong, Taiwan, the Philippines, Singapore, Vietnam, Kenya, Tunisia, Sudan, and Uganda. The reason for the focal distribution of NPC is uncertain, although genetics and environmental factors have been suggested to be causes (14, 49).Currently, the mainstay for the treatment of NPC is radiation and chemotherapy. Indeed, this treatment is frequently successful when the extent of the tumor is small and confined. However, when disease is advanced at diagnosis and where metastatic spread has become apparent, more radical treatments may need to be adopted, including surgery. In either case, all these treatments are associated with severe short- and long-term side effects including secondary malignancies (16). Hence, there is a need for specific therapies that target the tumor itself rather than therapies that are associated with the destruction of normal tissue.Virus-associated malignancies offer a distinct advantage in this regard since therapy can be directed specifically toward viral proteins expressed in the tumor, thus avoiding collateral damage to normal tissue. This has been dramatically demonstrated in the case of PTLD, where the adoptive transfer of EBV-specific cytotoxic T lymphocytes (CTLs) activated in vitro by using autologous lymphoblastoid cell lines (LCLs) has resulted in a resolution of disease with a very low frequency of side effects (9, 18, 40). In this case, it is likely that the effector cells infused into these patients are directed mainly toward the dominant EBV nuclear antigen 3A (EBNA3A), EBNA3B, and EBNA3C. The concept of immunological intervention as a treatment option for NPC is greatly enhanced by a range of previously reported studies that indicated the presence of transport-associated proteins (TAP1 and TAP2) and major histocompatibility complex class I and class II in NPC (23, 37, 42, 48), all of which are required for efficient CTL recognition. In NPC, EBNA1, latent membrane protein 1 (LMP1), and LMP2 offer the best opportunity for specific targeting since these are the only EBV proteins expressed in this malignancy. This is particularly so in the case of immunotherapy since defined CD4⁺ and CD8⁺ T-cell determinants in each of these proteins have been defined (12, 15, 20, 31). However, the CTL response in the case of the LMPs is relatively weak (particularly LMP1), and the glycine-alanine repeat sequence within EBNA1 may affect immunological processing (29), although this may not be the absolute barrier that was first hypothesized (46). Recent studies have provided some encouragement that immunotherapeutic intervention may be a realistic treatment option for NPC (4, 5, 7, 8, 10, 30, 43, 45). For example, Straathof et al. (43) and Comoli et al. (10) adoptively transferred effector cells expanded in vitro by using LCLs to activate CTLs in patients with advanced NPC, resulting in some cases in the resolution of disease, although in other cases, efficacy was limited and transient (3). Those studies, however, have provided a promising hint that the immunotherapeutic control of NPC might be feasible.Indeed, recent studies have shown that multiple human leukocyte antigen (HLA) A2-restricted LMP1 CTL epitopes, when used as a polyepitope vaccine in a poxvirus vector, efficiently induced a strong CTL response, and this response could reverse the outgrowth of LMP1-expressing tumors in HLA-A2/Kb mice (13). The poxvirus-based LMP1-polyepitope vaccine tested in these studies contained only HLA A2-restricted epitopes, and targeting just one HLA allele will not be suitable for all ethnic groups. If a CTL-based therapy for NPC is to be universally applicable, the target epitopes must bind to a range of HLA alleles preferably present at a high frequency in patient populations and include determinants irrespective of whether they have previously been defined.It is likely that the essential difference between the very successful treatment of patients with PTLD and the partial success in the case of NPC is that in the former case, immunodominant targets are available, while in the latter case, only relatively weak responses are seen even for healthy individuals. The present communication has arisen in an attempt to maximize the possibility of activating a response toward the three proteins present in NPC rather than skewing the effector population toward the immunodominant EBNA3A, -B, and -C proteins. We have achieved this by generating a “scrambled-antigen vaccine” (referred to as SAVINE) incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2. This SAVINE has been incorporated into a replication-deficient adenovirus (Ad5/F35) as a 6.9-kb insert (Ad-SAVINE). An important feature of the Ad-SAVINE strategy is that it provides a platform for the activation of all possible immunological determinants (including helper cells and CTLs) within EBNA1, LMP1, and LMP2 and should be applicable to all populations for which NPC is endemic. This report describes the construction of this Ad-SAVINE construct and its utility in generating LMP1 and LMP2 responses from peripheral blood mononuclear cells (PBMCs) from healthy individuals and NPC patients.