Recruitment, genetic counseling, and BRCA testing for underserved women at a public hospital

Genetic counseling and testing for heritable susceptibility to breast cancer caused by mutations in BRCA genes are largely unavailable to underserved women in the United States. Starting in 2002 the UCSF Cancer Risk Program offered this service free of charge to poor and medically indigent women at San Francisco General Hospital (SFGH). One recruitment strategy was a single-page questionnaire in four languages administered to women waiting for mammograms at SFGH. This report analyzes our first 3 years of experience with the recruitment questionnaire and compares the patient demographics and BRCA test results at SFGH with a more typical population undergoing genetic counseling and testing at UCSF's Mt. Zion Hospital (MZH). To our knowledge this is the first comprehensive clinical service for hereditary breast cancer in a U.S. public hospital. The ethnic mix of all 350 patients counseled was Caucasian 49% (approximately 20% of Caucasians reported Ashkenazi Jewish ancestry), Latina, 26%; African American, 13%; and Asian/other, 12%. Compared to the MZH population, SFGH patients were more ethnically diverse, less educated and more likely to be unemployed. Of 72 patients tested for BRCA mutations, 51 (71%) were negative, 5 were BRCA1 positive, and 12 were BRCA2 positive. Four (1 Caucasian, 1 Latina, 2 African American) had a total of 13 BRCA variants of unknown significance (VUS). The ratio of BRCA1/BRCA2 positive SFGH patients (5/12) was reversed compared to MZH (119/91). We evaluated 4573 recruitment questionnaires and 280 (6%) were judged to represent a high risk of heritable cancer. After additional screening and referral negotiation, 74 were scheduled for counseling. We judged the recruitment questionnaire to be a feasible, efficient, and reasonably cost-effective way to identify women at high risk of hereditary cancer in a traditionally underserved population. Underserved populations present special challenges for genetic counselors because of large, geographically dispersed families, cultural taboos about cancer diagnoses, and social marginalization. Despite these complexities, the clinical service at SFGH has been well accepted by patients and staff. Our successful venture can serve as a model for other public hospitals contemplating this clinical service.     

C-MALISA (cellular magnetic-linked immunosorbent assay), a new application of cellular ELISA for MRI

A modified cellular ELISA (enzyme-linked immunosorbent assay), named cellular magnetic-linked immunosorbent assay (C-MALISA), has been developed as an application of magnetic resonance imaging (MRI) for in vitro clinical diagnosis. To validate the method, three contrast agents targeted to integrins were synthesized by grafting to USPIO (ultrasmall particles of iron oxide): (a) the CS1 (connecting segment-1) fragment of fibronectin (FN) (USPIO-g-FN); (b) the peptide GRGD (USPIO-g-GRGD); (c) a non-peptidic RGD mimetic (USPIO-g-mimRGD). Jurkat cells and rat mononuclear cells were stimulated to activate their integrins. After cell fixation on ELISA plates, incubation with the contrast agents, rinsing, and digestion in 5N HCl, the samples were analyzed by MRI. Paramagnetic relaxation rate enhancements (delta R2) were measured on images. Delta R2 was converted in values of iron concentration based on a calibration curve. The apparent dissociation constants (K(d)*) of the three contrast agents were estimated based on the MRI measurement of delta R2. K(d)* of 1.22 x 10(-7) M, of 7.00 x 10(-8) M, and of 1.13 x 10(-8) M were found respectively for USPIO-g-FN, USPIO-g-GRGD, and USPIO-g-mimGRG. The MRI confirmed a statistically significant difference (p < 0.01, p < 0.05) between the stimulated cells incubated with integrin-targeted compounds with respect to the controls (i.e., non-stimulated cells and stimulated cells incubated with non-specific USPIO). The integrin specificity of the three compounds was confirmed by the pre-incubation with GRGD (for USPIO-g-mimRGD and USPIO-g-GRGD) or FN (for USPIO-g-FN).     

Temperature-sensitive paramagnetic liposomes for image-guided drug delivery: Mn versus [Gd(HPDO3A)(H2O)]

Temperature-sensitive liposomes (TSLs) loaded with doxorubicin (Dox), and Magnetic Resonance Imaging contrast agents (CAs), either manganese (Mn^2 +) or [Gd(HPDO3A)(H₂O)], provide the advantage of drug delivery under MR image guidance. Encapsulated MRI CAs have low longitudinal relaxivity (r₁) due to limited transmembrane water exchange. Upon triggered release at hyperthermic temperature, the r₁ will increase and hence, provides a means to monitor drug distribution in situ. Here, the effects of encapsulated CAs on the phospholipid bilayer and the resulting change in r₁ were investigated using MR titration studies and ¹H Nuclear Magnetic Relaxation Dispersion (NMRD) profiles. Our results show that Mn^2 + interacted with the phospholipid bilayer of TSLs and consequently, reduced doxorubicin retention capability at 37 °C within the interior of the liposomes over time. Despite that, Mn^2 +-phospholipid interaction resulted in higher r₁ increase, from 5.1 ± 1.3 mM^− 1 s^− 1 before heating to 32.2 ± 3 mM^− 1 s^− 1 after heating at 60 MHz and 37 °C as compared to TSL(Gd,Dox) where the longitudinal relaxivities before and after heating were 1.2 ± 0.3 mM^− 1 s^− 1 and 4.4 ± 0.3 mM^− 1 s^− 1, respectively. Upon heating, Dox was released from TSL(Mn,Dox) and complexation of Mn^2 + to Dox resulted in a similar Mn^2 + release profile. From 25 to 38 °C, r₁ of [Gd(HPDO3A)(H₂O)] gradually increased due to increase transmembrane water exchange, while no Dox release was observed. From 38 °C, the release of [Gd(HPDO3A)(H₂O)] and Dox was irreversible and the release profiles coincided. By understanding the non-covalent interactions between the MRI CAs and phospholipid bilayer, the properties of the paramagnetic TSLs can be tailored for MR guided drug delivery.     

Altered glucose homeostasis in mice lacking the receptor protein tyrosine phosphatase sigma

Several protein tyrosine phosphatases (PTPs) expressed in insulin sensitive-tissues are proposed to attenuate insulin action and could act as key regulators of the insulin receptor (IR) signaling pathway. Among these PTPs, RPTPsigma is expressed in relatively high levels in insulin-target tissues. We show that RPTPsigma-/- knockout mice have reduced plasma glucose and insulin concentrations in the fasted state compared with their wild-type siblings. The knockout animals were also more sensitive to exogenous insulin as assayed by insulin-tolerance tests. Despite increased whole-body insulin sensitivity, tyrosine phosphorylation of the IR was not increased in muscle of RPTPsigma-/- animals, as would be expected in insulin-sensitive animals. Instead, the levels of IR tyrosine phosphorylation and PI3-kinase activity were reduced in the muscle of knockout animals stimulated with insulin in vivo. However, insulin-stimulated Akt serine phosphorylation was essentially identical between both groups of mice. Accordingly, muscles isolated from RPTPsigma-/- mice did not have a significant increase in glucose uptake in response to insulin, suggesting that RPTPsigma did not play a direct role in this process. Taken together, our results suggest an indirect modulation of the IR signaling pathways by RPTPsigma. Since low dose injection of growth hormone (GH) normalized the response to exogenous insulin in RPTPsigma-/- mice, we propose that the insulin hypersensitivity observed in RPTPsigma-/- mice is secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency.     

Employing a Recombinant HLA-DR3 Expression System to Dissect Major Histocompatibility Complex II-Thyroglobulin Peptide Dynamism: A GENETIC, BIOCHEMICAL, AND REVERSE IMMUNOLOGICAL PERSPECTIVE*

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.     

Ultrasmall particle of iron oxide—RGD peptidomimetic conjugate: synthesis and characterisation

Ultrasmall particles of iron oxide (USPIOs) coated with 3,3′-bis(phosphonate)propionic acid were covalently coupled to a home-made Arg-Gly-Asp (RGD) peptidomimetic molecule via a short oligoethyleneglycol (OEG) spacer. The conjugation rate was measured by X-ray photoelectron spectroscopy (XPS). The particle size and magnetic characteristics were kept. Our novel conjugate targeted efficiently Jurkat cells (increase of 229% vs the control).