These studies focused on a new radiolabeling technique with copper ((64)Cu) and zirconium ((89)Zr) for positron emission tomography (PET) imaging using a CD45 antibody. Synthesis of (64)Cu-CD45 and (89)Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC) was assessed in vitro (viability, population doubling times, colony forming units). hPBSC viability was maintained as the dose of (64)Cu-TETA-CD45 increased from 0 (92%) to 160 µCi/mL (76%, p>0.05). Radiolabeling efficiency was not significantly increased with concentrations of (64)Cu-TETA-CD45 >20 µCi/mL (p>0.50). Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p<0.05). For (89)Zr, there were no significant differences in viability (p>0.05), and a trend towards increased radiolabeling efficiency was noted as the dose of (89)Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0-40 µCi/mL (p<0.05). A greater than 2,000 fold-increase in the level of (89)Zr-Df-CD45 labeling efficiency was observed when compared to (64)Cu-TETA-CD45. Similar to (64)Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05) (growth, colony formation). Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×10(6) hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI), then 0.3 mCi of (89)Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that (89)Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.

Lymphatic dysfunction is associated with the progression of many cardiovascular disorders due to their role in maintaining tissue fluid homeostasis. Promoting new lymphatic vessels (lymphangiogenesis) is a promising strategy to reverse these cardiovascular disorders via restoring lymphatic function. Vascular endothelial growth factor (VEGF) members VEGF-C and VEGF-D are both potent candidates for stimulating lymphangiogenesis, though maintaining spatial and temporal control of these factors represents a challenge to developing efficient therapeutic lymphangiogenic applications. Injectable alginate hydrogels have been useful for the controlled delivery of many angiogenic factors, including VEGF-A, to stimulate new blood vasculature. However, the utility of these tunable hydrogels for delivering lymphangiogenic factors has never been closely examined. Thus, the objective of this study was to utilize ionically cross-linked alginate hydrogels to deliver VEGF-C and VEGF-D for potential lymphangiogenic applications. We demonstrated that lymphatic endothelial cells (LECs) are sensitive to temporal presentation of VEGF-C and VEGF-D but with different responses between the factors. The greatest LEC mitogenic and sprouting response was observed for constant concentrations of VEGF-C and a high initial concentration that gradually decreased over time for VEGF-D. Additionally, alginate hydrogels provided sustained release of radiolabeled VEGF-C and VEGF-D. Finally, VEGF-C and VEGF-D released from these hydrogels promoted a similar number of LEC sprouts as exogenously added growth factors and new vasculature in vivo via a chick chorioallantoic membrane (CAM) assay. Overall, these findings demonstrate that alginate hydrogels can provide sustained and bioactive release of VEGF-C and VEGF-D which could have applications for therapeutic lymphangiogenesis.

Theranostic agents are critical for improving the diagnosis and treatment of non-Hodgkin Lymphoma (NHL). The peptidomimetic LLP2A is a novel peptide receptor radiotherapy candidate for treating NHL that expresses the activated α4β1 integrin. Tumor-bearing dogs are an excellent model of human NHL with similar clinical characteristics, behavior, and compressed clinical course. Canine in vivo imaging studies will provide valuable biodistribution and affinity information that reflects a diverse clinical population of lymphoma. This may also help to determine potential dose-limiting radiotoxicity to organs in human clinical trials. To validate this construct in a naturally occurring model of NHL, we performed in-vivo molecular targeted imaging and biodistribution in 3 normal dogs and 5 NHL bearing dogs. (99m)Tc-LLP2A-HYNIC-PEG and (99m)Tc-LLP2A-HYNIC were successfully synthesized and had very good labeling efficiency and radiochemical purity. (99m)Tc-LLP2A-HYNIC and (99m)Tc-LLP2A-HYNIC-PEG had biodistribution in keeping with their molecular size, with (99m)Tc-LLP2A-HYNIC-PEG remaining longer in the circulation, having higher tissue uptake, and having more activity in the liver compared to (99m)Tc-LLP2A-HYNIC. (99m)Tc-LLP2A-HYNIC was mainly eliminated through the kidneys with some residual activity. Radioactivity was reduced to near-background levels at 6 hours after injection. In NHL dogs, tumor showed moderately increased activity over background, with tumor activity in B-cell lymphoma dogs decreasing after chemotherapy. This compound is promising in the development of targeted drug-delivery radiopharmaceuticals and may contribute to translational work in people affected by non-Hodgkin lymphoma.

The neuropeptide oxytocin is part of a neuroendocrine system that has physiological effects ranging from ensuring uterine myometrial contractions at parturition and post-partum mammary gland milk ejection to the modulation of neural control of social relationships. This initial study was performed to investigate the potential use of positron emission tomography (PET) for localizing oxytocin receptors in two New World primates. Three biomarkers for PET (1-3) that are known to have high affinity and selectivity for the human oxytocin receptor were investigated in the common marmoset (Callithrix jacchus) via PET imaging. Brain penetration, and uptake in the salivary gland area were both observed with biomarkers 2 and 3. No brain penetration was observed with 1, but uptake was observed more specifically in several peripheral endocrine glands compared to 2 or 3. Biomarker 2, which displayed the best brain penetration of the three biomarkers in the marmoset, was then investigated in the monogamous coppery titi monkey (Callicebus cupreus) in a brain scan and a limited full body scan. No significant brain penetration of 2 was observed in the titi monkey, but significant uptake was observed in various locations throughout the periphery. Metabolism of 2 was suspected to have been significant based upon HPLC analysis of blood draws, but parent compound was still present near the end of the scan. Follow-up investigations will focus on next generation biomarkers bearing improved binding characteristics and brain penetrability as well as investigating tissue in regions where biomarker uptake was observed.

Women exposed to a variety of viral and bacterial infections during pregnancy have an increased risk of giving birth to a child with autism, schizophrenia or other neurodevelopmental disorders. Preclinical maternal immune activation (MIA) models are powerful translational tools to investigate mechanisms underlying epidemiological links between infection during pregnancy and offspring neurodevelopmental disorders. Our previous studies documenting the emergence of aberrant behavior in rhesus monkey offspring born to MIA-treated dams extends the rodent MIA model into a species more closely related to humans. Here we present novel neuroimaging data from these animals to further explore the translational potential of the nonhuman primate MIA model. Nine male MIA-treated offspring and 4 controls from our original cohort underwent in vivo positron emission tomography (PET) scanning at approximately 3.5-years of age using [¹⁸F] fluoro-l-m-tyrosine (FMT) to measure presynaptic dopamine levels in the striatum, which are consistently elevated in individuals with schizophrenia. Analysis of [¹⁸F]FMT signal in the striatum of these nonhuman primates showed that MIA animals had significantly higher [¹⁸F]FMT index of influx compared to control animals. In spite of the modest sample size, this group difference reflects a large effect size (Cohen's d = 0.998). Nonhuman primates born to MIA-treated dams exhibited increased striatal dopamine in late adolescence-a hallmark molecular biomarker of schizophrenia. These results validate the MIA model in a species more closely related to humans and open up new avenues for understanding the neurodevelopmental biology of schizophrenia and other neurodevelopmental disorders associated with prenatal immune challenge.

Background

Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can trigger convulsions that progress to life-threatening status epilepticus. Survivors face long-term morbidity including mild-to-severe decline in memory. It is posited that neuroinflammation plays a key role in the pathogenesis of OP-induced neuropsychiatric deficits. Rigorous testing of this hypothesis requires preclinical models that recapitulate relevant phenotypic outcomes. Here, we describe a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) that exhibits persistent neuroinflammation and cognitive impairment.

Methods

Neuroinflammation, neurodegeneration, and cognitive function were compared in adult male Sprague Dawley rats injected with an acutely toxic dose of DFP vs. vehicle controls at multiple time points up to 36 days post-exposure. Neuroinflammation was quantified using immunohistochemical biomarkers of microglia (ionized calcium-binding adapter molecule 1, IBA1) and activated astrocytes (glial fibrillary acidic protein, GFAP) and positron emission tomography (PET) imaging of [¹¹C]-(R)-PK11195, a ligand for the 18-kDa mitochondrial membrane translocator protein (TSPO). FluoroJade-B staining was used to assess neurodegeneration; Pavlovian conditioning, to assess cognitive function.

Results

Animals exhibited moderate-to-severe seizures within minutes of DFP injection that continued for up to 6 h post-injection. As indicated by IBA1 and GFAP immunoreactivity and by PET imaging of TSPO, acute DFP intoxication triggered neuroinflammation in the hippocampus and cortex during the first 3 days that peaked at 7 days and persisted to 21 days post-exposure in most animals. Neurodegeneration was detected in multiple brain regions from 1 to 14 days post-exposure. All DFP-intoxicated animals exhibited significant deficits in contextual fear conditioning at 9 and 20 days post-exposure compared to vehicle controls. Whole-brain TSPO labeling positively correlated with seizure severity score, but did not correlate with performance in the contextual fear-conditioning task.

Conclusions

We describe a preclinical model in which acute DFP intoxication causes seizures, persistent neuroinflammation, neurodegeneration, and memory impairment. The extent of the neuroinflammatory response is influenced by seizure severity. However, the observation that a subset of animals with moderate seizures and minimal TSPO labeling exhibited cognitive deficits comparable to those of animals with severe seizures and significant TSPO labeling suggests that DFP may impair learning and memory circuitry via mechanisms independent of seizures or neuroinflammation.