Nancy Pham

Extracranial Sources of S100B Do Not Affect Serum Levels

S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrPC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.

Complications of Aortic Valve Surgery: Manifestations at CT and MR Imaging

Aortic valve replacement accounts for a significant portion of cardiac surgeries in the United States. Despite advances in prosthetic heart valve design, surgical technique, and postoperative care, complications after aortic valve replacement remain a leading cause of morbidity and mortality. Routine surveillance of prosthetic heart valves with transthoracic echocardiography (TTE), transesophageal echocardiography (TEE), and fluoroscopy is important, as these techniques allow accurate detection of prosthetic valve dysfunction. However, echocardiography and fluoroscopy may not allow identification of the specific underlying cause, including paravalvular leak, dehiscence, endocarditis, obstruction, structural failure, pseudoaneurysm formation, aortic dissection, and hemolysis. Magnetic resonance (MR) imaging and computed tomography (CT) have an emerging role as diagnostic tools complementary to conventional imaging for detection and monitoring of complications after aortic valve replacement. The choice between CT and MR imaging depends on individual patient characteristics, the type of prosthetic valve, and the acuity of the clinical situation. In general, screening with TTE followed by TEE is recommended. When results of TTE and TEE are inconclusive, cardiac CT and MR imaging should be considered. The choice between these imaging techniques depends on the presence of patient-specific contraindications to CT or MR imaging.

Ligand binding promotes prion protein aggregation – role of the octapeptide repeats

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu2+ and Zn2+. Here, we report our findings that GAG and Cu2+ promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu2+, mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild‐type PrP. When the GAG‐binding motif, KKRPK, is deleted the effect of GAG but not that of Cu2+ is abolished. By contrast, when the Cu2+‐binding motif, the octapeptide‐repeat region, is deleted, neither GAG nor Cu2+ is able to promote aggregation. Therefore, the octapeptide‐repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti‐PrP mAbs, whereas none of the tested anti‐PrP mAbs block Cu2+‐promoted aggregation. However, a mAb that is specific for an epitope at the N‐terminus enhances aggregation in the presence of either GAG or Cu2+. Therefore, although binding of either GAG or Cu2+ promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.

Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization

The thyrotropin receptor (TSHR), the major autoantigen in Graves’ disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves’ hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, “non-self” regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

Particle embolization of the bilateral superior and inferior alveolar arteries for life threatening dental socket hemorrhage

Particle embolization for epistaxis and intra-oral hemorrhage are performed on an as needed basis by neurointerventionalists. A case is presented of massive oral hemorrhage associated with end stage liver disease coagulopathy after tooth extraction of bilateral maxillary and mandibular molars. A man in his fifties with end stage liver disease who presented for evaluation of a syncopal episode was determined to be in hemorrhagic shock from 2 days of persistent oral bleeding after elective tooth extractions. Conservative management with multiple blood transfusion products, packing and vasoconstrictive spray was ineffective. Microcatheter angiograms of the alveolar arteries demonstrated blood and contrast pooling within the sockets of the extracted teeth. Selection of the bilateral supplying alveolar and inferior alveolar artery branches was achieved followed by polyvinyl alcohol particle embolization (250–355 μm). Polyvinyl alcohol particle embolization of dental socket hemorrhages is technically feasible and effective.