Amir N. Hamir

Noninvasive Radiofrequency Field Destruction of Pancreatic Adenocarcinoma Xenografts Treated with Targeted Gold Nanoparticles

Purpose: Pancreatic carcinoma is one of the deadliest cancers with few effective treatments. Gold nanoparticles (AuNP) are potentially therapeutic because of the safety demonstrated thus far and their physiochemical characteristics. We used the astounding heating rates of AuNPs in nonionizing radiofrequency (RF) radiation to investigate human pancreatic xenograft destruction in a murine model. Experimental Design: Weekly, Panc-1 and Capan-1 human pancreatic carcinoma xenografts in immunocompromised mice were exposed to an RF field 36 hours after treatment (intraperitoneal) with cetuximab- or PAM4 antibody–conjugated AuNPs, respectively. Tumor sizes were measured weekly, whereas necrosis and cleaved caspase-3 were investigated with hematoxylin–eosin staining and immunofluorescence, respectively. In addition, AuNP internalization and cytotoxicity were investigated in vitro with confocal microscopy and flow cytometry, respectively. Results: Panc-1 cells demonstrated increased apoptosis with decreased viability after treatment with cetuximab-conjugated AuNPs and RF field exposure (P = 0.00005). Differences in xenograft volumes were observed within 2 weeks of initiating therapy. Cetuximab- and PAM4-conjugated AuNPs demonstrated RF field–induced destruction of Panc-1 and Capan-1 pancreatic carcinoma xenografts after 6 weeks of weekly treatment (P = 0.004 and P = 0.035, respectively). There was no evidence of injury to murine organs. Cleaved caspase-3 and necrosis were both increased in treated tumors. Conclusions: This study demonstrates a potentially novel cancer therapy by noninvasively inducing intracellular hyperthermia with targeted AuNPs in an RF field. While the therapy is dependent on the specificity of the targeting antibody, normal tissues were without toxicity despite systemic therapy and whole-body RF field exposure. Clin Cancer Res; 16(23); 5712–21. ©2010 AACR.

FIRST NORTH AMERICAN FIELD RELEASE OF A VACCINIA-RABIES GLYCOPROTEIN RECOMBINANT VIRUS

Following nearly 10 yr of extensive laboratory evaluation, a vaccinia-rabies glycoprotein (V-RG) vaccine was the first recombinant virus to undergo limited North American field release on 20 August 1990. The free-ranging raccoon population on Parramore Island (Virginia, USA) was exposed to a high density (10 baits/ha) of vaccine-laden baits distributed on a 300 ha vaccination area. An annual total of 887 raccoons were live-trapped for sedation, physical examination and blood collection for rabies antibody determination; there was no evidence of adverse effects or lesions due to the vaccine. Age and sex distributions, mean body weights, and live-capture histories of raccoons from the vaccination and non-baited control areas were compared. There were no statistically significant differences in survivorship between the baited and non-baited areas, nor between rabies antibody-positive and antibody-negative raccoons from the vaccination area. There was no trend in field mortality that suggested an association with either tetracycline or sulfadimethoxine, used as biomakers, or with vaccine contact determined by antibody status. No gross or histopathologic lesions due to the vaccine were demonstrated among a subsample of live-trapped raccoons collected for gross necropsy, biomarker analysis, histopathologic examination, and V-RG virus isolation attempts. Recovery of V-RG virus was limited to the tonsils of two biomarker-positive, clinically healthy raccoons collected from the vaccination area for postmortem examination on days 2 and 4 following bait distribution. These data reinforce the extensive body of safety data on the V-RG virus and extend it to include field evaluation where vaccine is offered free-choice in abundance, in baits designed to attract free-ranging raccoons, in a relatively simple ecosystem.

A High-Frequency Regulatory Polymorphism in the p53 Pathway Accelerates Tumor Development

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele.

PROPOSED FIELD EVALUATION OF A RABIES RECOMBINANT VACCINE FOR RACCOONS (PROCYON LOTOR): SITE SELECTION, TARGET SPECIES CHARACTERISTICS, AND PLACEBO BAITING TRIALS

Prior to a limited field application of an orally-administered vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine for wildlife, background data were obtained for the proposed site on Parramore Island, Virginia (USA). Mammalian target and nontarget species, potentially at risk for exposure to vaccine were inventoried. Placebo baiting trials with a fishmeal polymer bait resulted in high bait disturbance (88 to 100%), primarily by raccoons (Procyon lotor), with infrequent visitation and no evidence of bait consumption by deer (Odocoileus virginianus), small mammals or avian species. Definitive bait acceptance rates by raccoons (indicative of bait ingestion) were difficult to accurately determine based exclusively on premolar and vibrissae samples collected antemortem from live-trapped raccoons for tetracycline and rhodamine B biomarker analyses, respectively. Bait acceptance rate was more accurately determined during a pilot baiting trial conducted on North Island, South Carolina, when mandibles (postmortem samples) were examined for tetracycline incorporation. Parasitologic findings in raccoons on Parramore Island included Hepatozoan procyonis, Phagicola angrense and Physaloptera rata and a variety of incidental microscopic lesions, and provided baseline pathological data for comparison subsequent to V-RG vaccine application. A population density estimate of one raccoon/2.7 ha was calculated using mark–recapture data for comparison after vaccine deployment. Limited reproductive data, including estimates of pregnancy rates by palpation, the number of live kits/litter live-trapped with previously pregnant raccoons or observed in the dens of radio-collared raccoons, was gathered to assess the effect of proposed oral vaccination with V-RG vaccine. Home ranges were assessed by radio-telemetry of 15 raccoons; all radio-collared raccoons currently reside on Parramore Island. Longest straight line distance travelled by raccoons was <2 km, except when animals were translocated and were found to return to their original range.

PREREQUISITES FOR ORAL IMMUNIZATION OF FREE-RANGING RACCOONS (PROCYON LOTOR) WITH A RECOMBINANT RABIES VIRUS VACCINE: STUDY SITE ECOLOGY AND BAIT SYSTEM DEVELOPMENT

A model baiting system suitable for the delivery of an oral rabies vaccine to free-ranging raccoons (Procyon lotor) was developed and tested on barrier islands in South Carolina (USA). Features of barrier island physiography and ecology were studied relative to selective bait deployment and site biosecurity. Capture-mark-recapture data were obtained from 228 raccoons. Raccoon density estimates, using a modified census assessment technique, were one raccoon per 1.8 to 2.7 ha. Mean (±SE) and range home area estimates of radio-collared raccoons were 84 (±15.6) ha (27 to 176 ha) by a minimum convex polygon method and 138 (±22.8) ha (43 to 241 ha), by a harmonic mean transformation method. Habitat utilization determinations of radio-collared raccoons were conducted to identify study areas to potentially maximize selectivity of bait towards raccoons and to reduce the absolute number of baits deployed. Island raccoons showed a habitat preference for maritime forest, maritime shrub and marsh areas. Additionally, there was no evidence of inter-island or mainland exchange of ear-tagged or radio-collared raccoons. A disease and mortality survey was conducted to identify baseline pathology and incidental lesions in the target raccoon population, prior to actual vaccination initiation. Thirty-eight percent of 30 clinically suspect raccoons sampled had intracytoplasmic eosinophilic inclusions diagnostic of canine distemper; no other lesions suggestive of viral etiologies were found. Serological surveys for raccoon poxvirus and rabies virus antibodies were negative. Antibody titers to canine adenovirus 1 and 2 indicated a moderate level of exposure (approximately 10 to 16%) in the raccoon population. Overall, 93 to 100% of placebo baits were consistently disturbed by 7 days post-bait deployment, and bait acceptance rates by raccoons ranged from 49 to 85%, by using a modular systems approach to select the optimum combination of bait attractant, biomarker, matrix, density, and distribution. These results suggest that a large proportion (up to 85%) of a free-ranging island raccoon population can be selectively and safely targeted, marked and monitored utilizing a proposed oral bait delivery system for recombinant or other rabies vaccines.

Biodistribution and acute toxicity of naked gold nanoparticles in a rabbit hepatic tumor model

Abstract There is a paucity of data regarding the safety of administering solid gold nanoparticles (AuNPs) in large animal tumor models. We assessed the acute toxicity and biodistribution of 5 nm and 25 nm solid AuNPs in New Zealand White rabbits (n = 6 in each) with implanted liver Vx2 tumors 24 h after intravenous injection. Gold concentration was determined by inductively coupled plasma atomic emission spectrometry (ICP) and imaged with transmission electron microscopy (TEM). There was no clinico-pathologic evidence of renal, hepatic, pulmonary, or other organ dysfunction. After 25 nm AuNP administration, the concentration of white blood cells increased after treatment (p = 0.001). Most other blood studies were unchanged. AuNPs were distributed to the spleen, liver, and Vx2 tumors, but not to other tissues. The urinary excretion of AuNPs was bimodal as measured by ICP. 25 nm AuNPs were more evenly distributed throughout tissues and may be better tools for medical therapy.

A Sarcocystis neurona-like organism associated with encephalitis in a striped skunk (Mephitis mephitis)

A Sarcocystis neurona-like organism was associated with granulomatous encephalitis in an ataxic male juvenile striped skunk (Mephitis mephitis) from Cape Cod, Massachusetts. Various stages of schizonts and merozoites of S. neurona were seen within some of the granulomata.

First Report of a Demodex sp. in Raccoons (Procyon lotor)

Demodex spp. mites were seen in skin sections of 5 of 53 raccoons (Procyon lotor) necropsied on Parramore Island, Virginia (USA). In all infections, mites were present in the skin over the lower legs; in one raccoon they also were located in a follicle of a vibrissa. None of the raccoons had Demodex-related gross lesions. Histopathologically, minimal lesions were seen in the affected follicles. This is the first documentation of Demodex spp. in raccoons.

Dirofilaria immitis in a river otter (Lutra canadensis) from Louisiana.

A single adult male river otter (Lutra canadensis) from Louisiana was found naturally infected with Dirofilaria immitis. One adult male nematode was found in the heart; lesions attributable to the presence of the parasite were not found. This is the first report of D. immitis in the river otter in North America.

Lesions associated with pulmonary parasites in bobcats (Felis rufus) from Arkansas.

Two of five bobcats (Felis rufus) from southwestern Arkansas had natural pulmonary infections of Paragonimus kellicotti and Filaroides rostratus. Pairs of P. kellicotti were found in spherical cyst-like structures approximately 1 cm in diameter. Filaroides rostratus were seen as serpentine pale white areas on the pleural surface and also firmly embedded in fibrous capsules in the pulmonary parenchyma. Histologic lesions associated with the presence of these parasites consisted primarily of a verminous bronchitis. Clinical signs or compromised pulmonary function were not associated with these infections.