William G. Henk

Regression of High-Grade Malignancy in Mice by Bleomycin and Interleukin-12 Electrochemogenetherapy

Purpose: Bleomycin electrochemotherapy has been successfully used in preclinical studies and clinical trials for treating squamous cell carcinoma (SCC) and adenocarcinoma; however, it is not effective for treating recurrent tumors or metastatic tumors, or for preventing tumor redevelopment. In this study, we explore the coadministration of bleomycin and interleukin-12 (IL-12) followed by electroporation for treating primary and metastatic tumors. Experimental Design: Bleomycin, IL-12 plasmid DNA, or a combination of both were injected into high-grade malignant mammary tumors and SCCVII followed by electroporation. The tumor growth, survival, metastasis in lungs, CTL activity, and vascular density were analyzed. The results were analyzed by the two-sided Students t test and Gehans Wilcoxon test. Results: Coadministration of bleomycin and IL-12 via electroporation eradicates preestablished 4T1 mammary tumors in up to 60% of mice, inhibits metastatic tumor development, and extends the long-term survival. Likewise, coadministration of bleomycin and IL-12 via electroporation eradicates squamous cell carcinoma (SCCVII) in 100% of mice and prevents tumor redevelopment in 80% of mice. Neither bleomycin nor IL-12 alone is able to achieve the same therapeutic potency. The primary role of bleomycin is to inhibit the tumor vessel development; the primary role of IL-12 is to increase the immune response that extends the survival of treated mice and inhibits the tumor redevelopment. Conclusions: This combination modality has great potential to be translated in a clinical setting for treating high-grade malignancies and for preventing tumor redevelopment.

Photobacterium damselae subsp. piscicida Is Capable of Replicating in Hybrid Striped Bass Macrophages

Abstract The purpose of this study was to use an in vitro assay to evaluate the ability of Photobacterium damselae subsp. piscicida to survive and replicate within macrophages obtained from hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). The results indicated that the number of bacteria recovered from macrophages after 3, 6, 12, and 18 h of incubation increased significantly from time 0 (105, 510, 1,091 and 1,385%, respectively). Replication of the intracellular bacteria was observed at a very low multiplicity of infection (1 bacterium to 100 macrophages). In contrast, the number of Escherichia coli recovered from macrophages declined 47, 80, 91, and 96%, respectively. Light and electron microscopy confirmed the internalization, uptake, and multiplication of bacteria within spacious, clear vacuoles in the macrophages. Using acid phosphatase as a lysosomal marker, we provide evidence that P. damselae subsp. piscicida inhibits phagolysosomal fusion. This study demonstrates that...

Observations on the Muscles of the Eye of the Bowhead Whale, Balaena mysticetus

The muscles of the eyelids and the extraocular muscles of mysticete whales are poorly described for a variety of reasons, including considerable difficulty in obtaining specimens. Our objective is to provide such a description for the bowhead whale, Balaena mysticetus. This study has examined the gross anatomy of the region in six specimens (five adults, one fetus) of the bowhead whale. Results show that the muscles associated with the eye are well developed and possess several distinctive features. For example, precise limits of each extraocular muscle are difficult to determine along their entire length because these muscles intermingle with one another near their insertion. Furthermore, some fibers from these muscles (except the retractor bulbi) also insert into the eyelids. Pulling on these muscles to simulate contraction results in movement of the eyelids and suggests a role for these muscles in palpebral retraction. Insertion of a large levator palpebrae superioris muscle into the upper eyelid further enhances opening of the palpebral fissure. Another unusual feature is the presence of tunnel‐like structures that redirect the dorsal and ventral oblique muscles. The dorsal oblique muscle is redirected caudally about 90 degrees, then directed medially by another 90 degrees. These directional changes are accomplished via a connective tissue tunnel derived in part from the fibrous connective tissue of the dorsal rectus and the levator palpebrae superioris muscles. In most terrestrial mammals, a similar change in direction is accomplished by a cartilaginous trochlea. The ventral oblique muscle originates via a slender tendon from the frontal bone and undergoes a similar radical change in direction. Its tendon of insertion undergoes about a 90‐degree change in direction that is accomplished through a tunnel‐like structure derived from fibrous connective tissue of the ventral rectus muscle. Based on the morphology of the musculature presented, it is likely that the eyeballs and eyelids of the bowhead whale are quite mobile and appear capable of complex movement. The possibility of retraction and protrusion of the eyeball is discussed. Anat Rec 259:189–204, 2000.

Histochemistry and ultrastructure of the heart in experimental cobalt cardiomyopathy in the dog

Abstract Experimental cobalt cardiomyopathy was studied in dogs given cobalt twice weekly through a chronic indwelling iv catheter. The dogs became dyspneic and lethargic and had exercise intolerance. Cardiac failure developed in some animals. The hearts in these dogs were pale and flabby with dilatation of the atria. All had scattered myocardial degeneration characterized by diffuse cytoplasmic vacuolation, loss of cross striations, and interstitial edema. No inflammation was seen. Histochemical reactions for succinic dehydrogenase and reduced diphosphopyridine nucleotide (DPN) diaphorase were done on frozen heart muscle. There was no differences in activity of reduced DPN diaphorase when muscle from control dogs was compared to muscle from treated dogs. The activity of succinic dehydrogenase was decreased in cardiac muscle taken from dogs in the two cobalt-treated groups. Ultrastructurally, the mitochondria were extremely swollen with loss of cristae. Electron-dense particles were seen in some damaged mitochondria. The sarcoplasmic reticulum and the T system contained numerous dilated vesicles in cells with mitochondrial and myofibrillar damage. Loss of cross striations and Z-band thickening were present in degenerating myofibers. Lipid droplets of varying sizes, some containing dense osmiophilic particles at the periphery, were observed in the myocytes.

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin.

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed.

Observations on the thermoreversible gelation of two‐directional arborols in water–methanol mixtures

Reversible gels of two-directional cascade polymers with hydrophilic groups covalently attached by an hydrophobic center chain were studied by light and small- angle X-ray scattering, differential scanning calorimetry, and freeze-fracture transmis- sion electron microscopy. The long, self-assembled fibers interact side-by-side over ex- tended regions to form bundles. A given fiber may participate in several bundles, thus forming a three-dimensional gel network. q 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 2787-2793, 1997

Observations on the external morphology and vasculature of a fetal heart of the bowhead whale, Balaena mysticetus

Specialized demands within the aquatic environment for over some 60 million years have shaped unique morphological expressions in the whales, dolphins, and porpoises (Cetacea). Detailed consideration of these features, particularly in the great whales, has often been constrained by difficulties in securing adequate specimens for study. We had the opportunity to examine external heart morphology in a rarely obtained and prepared specimen from the bowhead whale, Balaena mysticetus.

The anatomy of the larynx of the bowhead whale, Balaena mysticetus, and its sound-producing functions.

This study describes the morphology of the laryngeal apparatus in bowhead whales (Balaena mysticetus) with respect to respiration, deglutition, and vocalization. We also examined the intrinsic cricoarytenothyroid muscle (Musculus (M.) diverticuli laryngei) which forms the laryngeal diverticulum, to ascertain its interactions with the laryngeal cartilages during respiration and sound production. Five fetal larynges and four from adult whales were studied using noninvasive imaging, as well as macroscopic and microscopic techniques. The larynx extends from the skull base into the thoracic inlet. The dorsally curved laryngeal stalk, supported by epiglottis and the corniculate processes of arytenoid cartilages, is situated within the nasopharynx. The epiglottic cartilage exhibits a prominent medial ridge. The arytenoid cartilages are rod‐shaped, and extend through the laryngeal cavity. The thyroid cartilage possesses a prominent caudal horn with a fibrous articulation to the ventrally incomplete cricoid cartilage. The M. thyroepiglotticus forms the connection between epiglottic and thyroid cartilages. The M. cricothyroideus lateralis connects the caudal horn of the thyroid cartilage with the cricoid cartilage and the M. cricothyroideus medialis connects the cricoid and thyroid cartilage. An extensive laryngeal diverticulum (Diverticulum laryngis), formed by the laryngeal mucosa and M. diverticuli laryngei, is positioned caudo‐ventral to the laryngeal vestibule. The mucosa thickens into a fold medial to the vocal processes of the arytenoid cartilages. Experiments with airflow combined with histological and anatomical evidence strongly suggest a sound producing function for these (vocal) folds. This analysis provides the first account of sound producing structures and function in bowhead whales. Anat Rec, 297:1316–1330, 2014.

Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting

Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2–5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host’s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production.