Faculty Bibliography

Our original findings, showing the effectiveness of active and passive tau immunizations in mouse models, have now been confirmed and extended by many groups, with several clinical trials underway in Alzheimer's disease and progressive supranuclear palsy. Here, we report on a unique and sensitive two-photon imaging approach to concurrently study the dynamics of brain and neuronal uptake and clearance of tau antibodies as well as the acute removal of their pathological target in live animals. This in vivo technique is more sensitive to detect clearance of pathological tau protein than western blot tau analysis of brain tissue. In addition to providing an insight into the mechanisms involved, it allows for an efficient in vivo assessment of the therapeutic potential of tau antibodies, and may be applied to related protein misfolding diseases.

Objective-To study transfection efficiency of folate-modified chitosan (FA-CS) nanoparticles as a non-viral vector delivering pEGFP-C3plasmid (FA-CS/P) to 293T cells with or without the combination of ultrasound and microbubble. Method-pEGFP-C3 was used as reporter gene and FA-CS nanoparticles, which prepared by complex coagulation method were used as biological carriers. Transfection efficiency to 293T cells mediated by FA-CS/P nanoparticles, ultrasound (US) and microbubble (MB) was assessed by fluorescence microscopy and flow cytometry. Result-FA-CS/P nanoparticles have a particle size of 355.1 nm and zeta potential of 10.4 mV. Significant green fluorescence could be observed in CS/P group, FA-CS/P group, US+MB/P group, US+FA-CS/P group, Liposome 2000 (L) group under inverted fluorescence microscope, while US+MB+FA-CS/P group only scattered fluorescence observed. Result of flow cytometry showed that transfection rate of US+MB+FA-CS/P group was (2.0 ± 0.2)%, which was significantly lower than other groups (P＜0.05). CCK-8 experiments showed that cell vitality of US+MB+FA-CS/P was (64.1±4.6)%, which was also lower than other groups (P＜0.05). Conclusion-In this study, FA-CS was successfully synthesized. FA-CS could combine with pEGFP-C3 effectively forming nanoparticles with nanoparticle size, well dispersion, high encapsulation efficiency and no significant toxicity to cells. The application of ultrasound increased the transfection rate of FA-CS/P. However, while exposed to ultrasound and microbubble, the transfection rate of FA-CS/P decreased obviously, may indicating that there was no synergistic effect for gene transfection by the combination of ultrasound, folate modified chitosan and microbubbles.

Alzheimer's disease is characterized by amyloid-β plaques and neurofibrillary tangles composed of tau aggregates. Several β-sheet dyes are already in clinical use to detect amyloid-β plaques by in vivo positron emission tomography (PET), and related dye compounds are being developed for targeting pathological tau aggregates. In contrast to β-sheet binders, antibody-derived ligands should provide greater specificity for detecting tau lesions, and can be tailored to detect various pathological tau epitopes.For preclinical in vivo evaluation of these ligands prior to PET development, we have established an in vivo imaging system (IVIS) protocol to detect tauopathy in live mice. Antibodies and their derivatives are conjugated with a near infrared fluorescent dye and injected intravenously into anesthetized mice, which subsequently are imaged at various intervals to assess their pathological tau burden, and clearance of the ligand from the brain. The in vivo signal obtained through the skull correlates well with the degree of tau pathology in the mice, and the injected ligand can be found intraneuronally within the brain bound to tau aggregates. Control IgG and injections of the tau antibodies/fragments into wild-type mice or mice with amyloid-β plaques lead to minimal or no signal, confirming the specificity of the approach.

Amyloid-β (Aβ) and tau pathologies are intertwined in Alzheimer's disease, and various immunotherapies targeting these hallmarks are in clinical trials. To determine if tau pathology influences Aβ burden and to assess prophylactic benefits, 3xTg and wild-type mice received tau immunization from 2-6 months of age. The mice developed a high IgG titer that was maintained at 22 months of age. Pronounced tau and Aβ pathologies were primarily detected in the subiculum/CA1 region, which was therefore the focus of analysis. The therapy reduced histopathological tau aggregates by 70-74% overall (68% in males and 78-86% in females), compared to 3xTg controls. Likewise, western blot analysis revealed a 41% clearance of soluble tau (38-76% in males and 48% in females) and 42-47% clearance of insoluble tau (47-58% in males and 49% in females) in the immunized mice. Furthermore, Aβ burden was reduced by 84% overall (61% in males and 97% in females). These benefits were associated with reductions in microgliosis and microhemorrhages. In summary, prophylactic tau immunization not only prevents tau pathology but also Aβ deposition and related pathologies in a sustained manner, indicating that tau pathology can promote Aβ deposition, and that a short immunization regimen can have a long-lasting beneficial effect.

Importance: The lack of prospective randomized clinical trials demonstrating that full-body skin examination (FBSE) reduces melanoma morbidity or mortality has prompted an "I" rating from the United States Preventive Services Task Force for population-based skin cancer screening. More data on these screening programs are needed. Objectives: To describe a skin cancer screening quality initiative in a large health care system and to determine if the intervention was associated with screening of a demographically higher-risk population than previous screening programs and if melanoma incidence and thickness differed in screened vs unscreened patients. Design, Setting, and Participants: This observational evaluation of a prospectively implemented quality initiative was conducted in a large health care system in western Pennsylvania (University of Pittsburgh Medical Center, UPMC) among adults seen in an office visit by a UPMC-employed primary care physician (PCP) in 2014. Interventions: Implementation of a campaign promoting annual skin cancer screening by FBSE, including training of PCPs, promotion of the initiative to physicians and patients, and modification of the electronic health record (EHR) to include FBSE as a recommended preventive service for patients 35 years or older. Main Outcomes and Measures: Characteristics of screened and unscreened patients and melanomas detected among them. Results: Of 333735 adult patients seen in an office visit by PCPs in 2014, 53196 patients (15.9% of the screen-eligible population) received an FBSE, and 280539 did not. Screened patients were slightly older (median age, 60 vs 57 years; P < .001) but did not differ significantly by sex (43.2% vs 43.1% men; P = .49) from the unscreened population. Fifty melanomas were diagnosed in screened patients and 104 melanomas were diagnosed in unscreened patients. Screened patients were more likely than unscreened patients to be diagnosed with melanoma (adjusted risk ratio [RR], 2.4; 95% CI, 1.7-3.4; P < .001) and to have a thinner invasive melanoma (median thickness, 0.37 mm vs 0.65 mm; P < .001). The incidence of melanoma lesions 1 mm or thicker was similar in screened vs unscreened patients (adjusted RR, 0.7; 95% CI, 02.-2.2; P = .52). Conclusions and Relevance: Large-scale screening for melanoma within a United States health care system is feasible and can result in increased detection of thinner melanomas. This intervention also resulted in screening of a higher proportion of men and an older patient population than previous screening interventions in which younger individuals and women predominated.

BACKGROUND: A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. RESULTS: Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. CONCLUSIONS: Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

BACKGROUND: Peripheral surgical trauma may incite neuroinflammation that leads to neuronal dysfunction associated with both depression and cognitive deficits. In a previous study, we found that adult mice developed neuroinflammation and short-term working memory dysfunction in a delayed, transient manner after splenectomy that was ameliorated by the cyclooxygenase-2 inhibitor meloxicam. We tested the hypothesis that splenectomy in mice would also cause anhedonia, the diminished response to pleasure or rewarding stimuli that is a hallmark of depression, and that treatment with meloxicam would be ameliorative. METHODS: After Institutional Animal Care and Use Committee approval, Swiss-Webster mice underwent sucrose preference training before being randomized into groups on day 0, when they had either splenectomy and anesthesia or anesthesia alone. Within each group, half were randomized to receive intraperitoneal saline at 24 hours, while the other half received intraperitoneal meloxicam at 24 hours. Sucrose preference ratios were determined on days 1, 5, 9, and 14. Additional mice were randomized into groups for brain histochemistry. Specimens were stained for glial fibrillary acidic protein (GFAP), a marker of astrocytes, and CD45, a protein tyrosine phosphatase that identifies microglial activation. RESULTS: On day 5, mice receiving splenectomy and saline demonstrated diminished sucrose preference, which was not seen in mice receiving splenectomy and meloxicam. Semiquantitative analysis of histological slides taken from splenectomized mice treated with meloxicam revealed reduced microglial-based neuroinflammation and reactive astrocytosis compared to mice receiving saline. CONCLUSION: Splenectomy in mice is associated with neuroinflammation and anhedonia, as evidenced by reactive microgliosis, astrocytosis, and behavioral changes. Postsurgical treatment with meloxicam attenuates both neuroinflammation and anhedonia. These findings suggest that cyclooxygenase-2-dependent mechanisms may play a role in the development of postoperative mood disorders, possibly via modulation of peripheral effects on neuroinflammation.

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid beta (Abeta)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Abeta C terminus noncovalently and hydrolyzed Abeta rapidly, with no reactivity to the Abeta precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Abeta dipeptide unit. The catabody dissolved preformed Abeta aggregates and inhibited Abeta aggregation more potently than an Abeta-binding IgG. Intravenous catabody treatment reduced brain Abeta deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Abeta hydrolysis appears to be an innate immune function that could be applied for therapeutic Abeta removal.

Antibodies or their derivatives as imaging probes for pathological tau protein have great potential, but have not been well studied. In particular, smaller, single-chain-variable antibody fragments (scFv's) are attractive for detecting tau lesions in live subjects. Here, we generated libraries of scFv's and identified numerous phospho-tau-selective scFv's. Peripheral injection of one of these scFv's consistently resulted in a strong in vivo brain signal in transgenic tauopathy mice, but not in wild-type or amyloid-beta plaque mice. The parent tau antibody provided similar results, albeit with a weaker signal intensity. The imaging signal correlated very well with colocalization of the probe with intraneuronal tau aggregates. Both were associated with markers of endosomes, autophagosomes, and lysosomes, suggesting their interaction in these degradation pathways. Such specific antibody-derived imaging probes have great potential as diagnostic markers for Alzheimer's disease and related tauopathies.