Cancer genetics evaluation: barriers to and improvements for referral

Despite the availability of cancer susceptibility testing, little information exists regarding physicians' selection and referral of eligible patients. This study provides insight into whom, why, and when physicians refer for cancer genetics evaluation, as well as their comfort level within this role. Eighty-two physicians (51 primary care, 15 gynecology, 11 surgery and 5 oncology) completed a survey (response rate: 34%) regarding cancer genetics referral practices. Of these, 59% reported an awareness of the hospital's cancer genetics program. Program awareness was greater among oncologists, surgeons, and gynecologists than among primary care physicians (p < 0.0001). Patients were referred for enhanced risk assessment (88%), improved medical management (85%), and concern for family members (83%). Patient eligibility was based on family cancer history (96%), patient cancer history (83%), and patient request (73%). Patients were not referred mainly due to patient disinterest (54%) or physician concern about either insurance coverage (44%) or insurance discrimination (31%). Primary care physicians were less comfortable with identifying patients for referral (p < 0.001) and with discussing genetics (p < 0.002) than specialists. The largest barriers to referral were lack of program awareness and limited knowledge regarding patient eligibility, improved insurance coverage, and antidiscrimination legislation. Physician-targeted marketing and education may improve the referral process.     

Evidence that TGFA influences risk to cleft lip with/without cleft palate through unconventional genetic mechanisms

This study examined the association between markers in transforming growth factor alpha (TGFA) and isolated, non-syndromic cleft lip with/without palate (CL/P) using a case–parent trio design, considering parent-of-origin effects. We also tested for gene–environmental interaction with common maternal exposures, and for gene–gene interaction using markers in TGFA and another recognized causal gene, IRF6. CL/P case–parent trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 17 single nucleotide polymorphisms (SNPs) in TGFA. The transmission disequilibrium test was used to test individual SNPs, and the parent-of-origin likelihood ratio test (PO-LRT) was used to assess parent-of-origin effects. We also screened for possible gene–environment interaction using PBAT, and tested for gene–gene interaction using conditional logistic regression models. When all trios were combined, four SNPs showed significant excess maternal transmission, two of which gave significant PO-LRT values [rs3821261: P = 0.004 and OR(imprinting) = 4.17; and rs3771475: P = 0.027 and OR(imprinting) = 2.44]. Haplotype analysis of these two SNPS also supported excess maternal transmission. We saw intriguing but suggestive evidence of G × E interaction for several SNPs in TGFA when either individual SNPs or haplotypes of adjacent SNPs were considered. Thus, TGFA appears to influence risk of CL/P through unconventional means with an apparent parent-of-origin effect (excess maternal transmission) and possible interaction with maternal exposures.     

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.The development of an AIDS vaccine is an ongoing and urgent challenge. One of the major hurdles is that the specific correlates of protection against human immunodeficiency virus (HIV) are still largely unknown. Nonetheless, most agree that the full complement of cellular and humoral components of the immune system will be needed to combat this virus. This is especially true given that the virus resides permanently in its host, infects the very cells needed to direct effective immune responses, and evades the immune system, either by changing in appearance or hiding in subcellular compartments.A broadly reactive neutralizing antibody response is likely to be critical as a first line of defense upon initial HIV exposure by aiding in the clearance of cell-free virions, targeting infected cells for destruction, and preventing viral spread through cell-to-cell transmission. The presence of inhibitory antibodies in highly exposed persistently seronegative individuals testifies to the importance of the humoral response (9, 37). Additionally, broadly neutralizing serum has been associated with healthier prognoses for infected individuals (27, 65) and may be vital for protecting offspring from their infected mothers (7, 79) and preventing superinfection by heterologous HIV strains (23, 84). Even if complete protection cannot be achieved by vaccine-derived antibodies, an early, well-poised and effective neutralizing antibody repertoire may be able to lower the set point of the viral load following the initial burst of viremia, an outcome that has been reported to translate into improved disease outcomes and reduced transmission of HIV (66, 74). Further benefits of neutralizing antibodies have been seen with passive immunization studies in macaques, in which administration of broadly neutralizing monoclonal antibodies (MAbs) has demonstrated that it is possible to provide protection from—and even sterilizing immunity against—HIV infection (5, 51, 66). There is also evidence that such antibodies may provide therapeutic benefits for chronically infected individuals, analogous to benefits realized with anti-HIV drug treatment regimens (87).Despite the promising potential of broadly neutralizing MAbs, designing immunogens that can elicit such cross-reactive neutralizing responses against HIV has been a surprisingly difficult task. Since the majority of the host''s B-cell response is directed against the envelope (Env) glycoproteins, gp120 and gp41, vaccine efforts have concentrated on these proteins and derivatives thereof in approaches ranging from the use of Env-based peptide cocktails to recombinant proteins and DNAs made with varied or consensus sequences and diverse, heterologous prime/protein boost regimens (reviewed in references 36, 58, and 70). These iterative studies have shown notable improvements in the potency and breadth of neutralizing responses induced. However, concerns exist regarding immunogens containing extraneous epitopes, as is the case with intact subunits of Env, and the nature of the immune responses they may elicit. A polyclonal burst of antibodies against a multitude of nonfunctional epitopes may include a predominance of antibodies that are (i) low affinity and/or nonfunctional (reviewed in reference 72); (ii) isolate specific (25); (iii) able to interfere with the neutralizing capabilities of otherwise-effective antibodies (via steric hindrance or by inducing various forms of B-cell pathology) (67); or (iv) directed against irrelevant epitopes instead of more conserved (and sometimes concealed) epitopes that might be able to elicit more potent and cross-reactive neutralizing responses (28, 71, 91).We have developed a system that can be used to present essentially any chosen epitope in a stable, well-exposed manner on the surface of the cold-causing human rhinovirus (HRV). HRV is itself a powerful immunogen and is able to elicit T-cell as well as serum and mucosal B-cell responses (reviewed by Couch [22]) and has minimal immunologic similarity to HIV (data not shown). Chimeric viruses displaying optimal epitopes should be able to serve as valuable components in an effective vaccine cocktail or as part of a heterologous prime/boost protocol. We have shown previously that HRV chimeric viruses displaying HIV-1 gp120 V3 loop sequences are able to elicit neutralizing responses against HIV-1 (75, 82, 83).In this study, we focused our attention on presenting part of the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41, a region of approximately 30 amino acids adjacent to the transmembrane domain (reviewed in references 59 and 97). The MPER plays an important role in the process of HIV fusion to the host cell membrane (60, 78). This region is also involved in binding to galactosylceramide, an important component of cell membranes, thus permitting CD4-independent transcytosis of the virus across epithelial cells at mucosal surfaces (1, 2). These functions likely explain this region''s sequence conservation and the efficacy of antibodies directed against the MPER (97), particularly given that an estimated 80% of HIV-1 infections are sexually transmitted at mucosal membranes. In fact, potent responses against the MPER are associated with stronger and broader neutralizing capabilities in infected individuals (68). A conserved, contiguous sequence of the MPER, the ELDKWA epitope (HIV-1 HxB2 gp41 residues 662 to 668), is recognized by the particularly broadly neutralizing human MAb 2F5 (11, 62, 85) and is highly resistant to escape mutation in the presence of 2F5 (49). 2F5 was also used in the MAb cocktails reported to confer passive, protective immunity in macaques (5, 51). In addition, infected individuals producing neutralizing antibodies directed against the ELDKWA epitope have been seen to exhibit better health (16, 29), including persistent seronegativity (8), and reduced transmission of HIV to offspring (89). While none of the vaccine-induced immune responses generated against this region has been effective thus far (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), more appropriate presentations of MPER epitopes should produce valuable immunogens that can contribute to a successful vaccine.In this study, we have grafted the ELDKWA epitope onto a surface loop of HRV connected via linkers of variable lengths and sequences and selected for viruses well recognized and neutralized by MAb 2F5. In so doing, we have been able to create immunogens capable of eliciting antibodies whose activities mimic some of those of 2F5. The combinatorial libraries produced were designed to encode a large set of possible sequences and, hence, structures from which we could search for valuable conformations. This work illustrates that HRV chimeras have the potential to present selected HIV epitopes in a focused and immunogenic manner.     

Using stressor gradients to determine reference expectations for great river fish assemblages

We describe a simple empirical modeling approach for determining least-disturbed conditions for the great rivers of the Upper Mississippi River basin: Missouri, Upper Mississippi, and Ohio Rivers. We used multivariate analysis to identify reference strata (reaches for which a single reference expectation was appropriate) on each river. Strata included the Upper Missouri, Lower Missouri, impounded Upper Mississippi, unimpounded Upper Mississippi, and the Ohio River. We created a multimetric stressor gradient for each stratum using a suite of site- and landscape-scale metrics. Site-scale metrics included water chemistry, aquatic and riparian habitat, and human disturbance metrics. Landscape-scale metrics included land use, land cover, and proximity to human disturbance. The gradient was scaled from 0 (least stressed) to 1 (most stressed). Multimetric indices of condition based on fish assemblages for the Lower Missouri and Upper Mississippi River were responsive to stressor gradients based on 18–24 abiotic stressor metrics. Ohio River fish assemblages were responsive to a hand-picked three-metric gradient. We used the y-intercept of quantile regression to predict the fish index value for a stressor gradient value of 0 (the fish index value at a site with the lowest mean stressor gradient score in the reference stratum) which we designated as least-disturbed condition for the fish index for that stratum. We trisected the difference between predicted least-disturbed condition (ceiling value) and a floor value set at the 5th percentile of the sample to create thresholds for three condition classes: least-disturbed, intermediate, and most-disturbed. Based on the derived condition class thresholds for the fish index, 10% (by length) of the Lower Missouri was in least-disturbed condition, compared to 14% of the Ohio River and 19% of the impounded Upper Mississippi River. The index of condition exhibited longitudinal variation that was associated with the location of major urban areas along each river. We conclude that empirical modeling based on an abiotic stressor gradient can provide an alternative approach for deriving internal reference expectations for great rivers with few, if any, minimally disturbed sites.     

Comparative Immunogenicity of HIV-1 Clade C Envelope Proteins for Prime/Boost Studies

Background

Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses.

Methodology/Principal Findings

A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested.

Conclusions/Significance

While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.     

Deletion of the TPM1 and MDM20 Genes Affect the Mechanical and Structural Properties of Yeast Cells

Many diseases including cancer are associated with a disorganised cytoskeleton. The process of characterising how cytoskeletal disorganisation affects the mechanical properties of cells offers the potential to develop new drugs and treatment regimes that may exploit mechanical weakness in cells and tissues. This work investigated the role of actin associated proteins, namely tropomyosin 1 (tpm1p) and mitochondrial distribution and morphology protein 20 (mdm20p), on the mechanical and morphological properties of yeast cells. For the first time it was shown that deletion of both the TPM1 and MDM20 genes resulted in a decrease in Young’s modulus when compared to the wild-type cells. The deletion strains appeared to have aberrant cell walls when compared to the wild-type strain and also appeared to have lost the characteristic elliptical morphology that is normally exhibited by yeast. Deletion of the TPM1 gene resulted in a significant increase in mean conjugate cell diameter when compared to the wild-type cells, however deletion of the MDM20 gene did not have any significant effect upon the mean conjugate diameter of the yeast cells.