In Vivo Detection of Amyloid-β Deposits Using Heavy Chain Antibody Fragments in a Transgenic Mouse Model for Alzheimer's Disease

This study investigated the in vivo properties of two heavy chain antibody fragments (VHH), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-β deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled VHH in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aβ was examined in more detail with fluorescently labeled VHH by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All VHH showed rapid renal clearance (10–20 min). Twenty-four hours post-injection 99mTc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for 99mTc-ni3A or DTPA(111In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aβ was confirmed for both fluorescently labeled VHH, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both VHH showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aβ. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that VHH detect Aβ deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aβ deposits

Progress Toward Long-Term Survivors of Glioblastoma

Objective: To identify the frequency and characteristics of long-term survivors of glioblastoma. Patients and Methods: Using all cases of glioblastoma with histopathological confirmation in the National Cancer Database from January 1, 2004, through December 31, 2009, clinical, institutional, and treatment-related factors were evaluated with multivariable logistic regression models so as to elucidate factors independently associated with higher than 5-year overall survival after diagnosis. Results: A total of 48,652 patients met the inclusion criteria, with 2249 (4.6%) achieving 5-year survival. Factors associated with odds of improved 5-year overall survival in multivariable analysis were younger age, female sex, less medical comorbidities, nonwhite race, highest median income quartile, left-sided tumors and tumors outside the brainstem, and treatment with radiotherapy (P\u3c.05 for all). The percentage of patients surviving 5 years remained relatively unchanged over the 6-year study period (P=.97). Conclusion: Despite improvements in median and short-term overall survival shown in recent large clinical trials for glioblastoma, the percentage of patients with glioblastoma achieving 5-year overall survival remains low. This observation calls for the development of practice-redefining therapies and justifies the increased application of radical novel and experimental treatment paradigms for all patients with glioblastoma

Blood clearance.

<p>These graphs represent the blood half lives of tagged ^99mTc-ni3A and -pa2H (<b>A</b>), and untagged DTPA(¹¹¹In)-pa2H (<b>B</b>) in APP/PS1 mice and wildtype littermates. Data is shown as percentage of injected dose per gram of blood (%ID/g) over time. Based upon this plot the clearance is suggested to respectively consist of a fast and a slow phase, or only a single phase.</p

Blood distribution of ^99mTc-pa2H.

<p>At different time point after bolus injection of ^99mTc-pa2H blood collected from the tail vein of 12–14 month old APP/PS1 mice or wildtype littermates. Separated into the cell pellet and plasma, samples were counted for radioactivity. Fractions are expressed in percentage of total activity at that time point. No significant differences were calculated using a student <i>t</i>-test (<i>p</i><0.05).</p

Blood half lives of radiolabeled V_HH.

<p>Half lives were determined by fitting a one or a two phase exponential decay model based on blood obtained from both tail vein and cardiac puncture at several time points after intravenous bolus injection of 2 µg radiolabeled V_HH in 12–14 month old APP/PS1 mice and wildtype littermates, as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038284#pone-0038284-g002" target="_blank">Figure 2</a>. Please note that DTPA(¹¹¹In)-pa2H was produced without any additional peptide tags.</p

Immunostaining on murine APP/PS1 sections using ni3A and pa2H.

<p>The upper panels (<b>A–D</b>) show 10× magnifications of the resulting staining with cryosections of aged APP/PS1 mouse brain tissue including negative controls, while the lower panels (<b>E–H</b>) show similar staining performed with wildtype littermates.</p

Biodistribution of radiolabeled pa2H in mice.

*<p> <i> = P<0.05 wildtype mice compared to APP/PS1 mice.</i></p><p>A bolus injection of 2 µg radiolabeled pa2H was administered intravenously into 12–14 month old APP/PS1 mice or their wildtype littermates. At three or one time points after injection of radiolabeled pa2H respectively with or without additional peptide tags, the animals were sacrificed and various tissues and entire organs were removed, weighed and counted for radioactivity. Values are expressed as a percentage of the injected dose per gram tissue (mean ± SD).</p

Specific <i>in vivo</i> Aβ binding after BBB disruption.

<p>After disruption of the BBB using a co-injection of 15% mannitol with pa2H-Alexa594 into the right carotid artery of an aged APP/PS1 mouse sacrificed 2 hrs post injection, amyloid plaques are clearly depicted in both hemispheres using a Thioflavin T (ThT) staining (<b>A</b>), while the pa2H-Alexa594 signal is only detected in the right hemisphere (<b>B</b>). More careful examination shows all Alexa594 signal colocalizes with ThT in the right hemisphere, while in the left only some autofluorescense can be detected. Furthermore, immunofluorescense anti-Aβ staining of the plaques using Alexa488 within the left hemisphere (<b>C</b>) results only in green signal, while within the right hemisphere (<b>D</b>) the red signal from pa2H-Alexa594 nicely colocalizes within the plaques. Experiments performed in a similar setting but sacrificed 24 hrs post-injection, showed similar results with pa2H-Alexa594 still nicely corresponding to the green labeling of the anti-Aβ staining within the right hemisphere (<b>E</b>).</p

Biodistribution of ^99mTc-ni3A in mice.

*<p> <i> = P<0.05 wildtype mice compared to APP/PS1 mice.</i></p><p>A bolus injection of 2 µg ^99mTc-ni3A was administered intravenously into 12–14 month old APP/PS1 mice or their wild type littermates. At three time points after injection the animals were sacrificed and various tissues and entire organs were removed, weighed and counted for radioactivity. Values are expressed as a percentage of the injected dose per gram tissue (mean ± SD).</p