Robert P. Marini

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Biology and diseases of the ferret.

Section I: Biology and Husbandry. Chapter 1 Taxonomy, History, and Use. (James G. Fox). Chapter 2 Anatomy of the Ferret. (Howard E. Evans and Nguyen Quoc An). Chapter 3 Neuroanatomy of the Ferret Brain. (I. Nigel C. Lawes and Paul L. R. Andrews). Chapter 4 Physiology of the Ferret. (Mark T. Whary and Paul L.R. Andrews). Chapter 5 Nutrition. (James G. Fox and Daniel E. McLain). Chapter 6 Housing and Management. (James G. Fox). Chapter 7 Normal Clinical and Biologic Parameters. (James G. Fox). Chapter 8 Growth, Reproduction, and Breeding. (James G. Fox and Judi A. Bell). Section II: Diseases and Clinical Applications. Chapter 9 Hematopoietic Diseases. (Susan E. Erdman, Xiantang Li, and James G. Fox). Chapter 10 Diseases of the Genitourinary System. (James G. Fox, Renee C. Pearson, and Jusi A. Bell). Chapter 11 Diseases of the Gastrointestinal System. (James G. Fox). Chapter 12 Diseases of the Endocrine System. (James G. Fox and Robert P. Marini). Chapter 13 Other Systemic Diseases. (James G. Fox). Chapter 14 Bacterial and Mycoplasmal Disease. (James G. Fox). Chapter 15 Viral Diseases. (James G. Fox, Renee C. Pearson, and John R. Gorham). Chapter 16 Parasitic Diseases. (James G. Fox). Chapter 17 Mycotic Diseases. (James G. Fox). Chapter 18 Neoplastic Diseases. (Xiantang Li and James G. Fox). Chapter 19 Anesthesia, Surgery, and Biomethodology. (Robert P. Marini and James G. Fox). Section III: Research and Applications. Chapter 20 Viral Disease Models. (Renne C. Pearson and John R. Gorham). Chapter 21 Use of the Ferret in Cardiovascular Research. (James P. Morgan and Kerry E. Travers). Chapter 22 use of the Ferret in Behavioral Research. (Michael J. Baum). Chapter 23 Use of the Ferret in Reproductive Neuroendocrinology. (Michael J. Baum). Chapter 24 use f the Ferret in Parasitologic Research. (Mark L. Eberhard). Index.

In Situ Cross-linkable Hyaluronan Hydrogels Containing Polymeric Nanoparticles for Preventing Postsurgical Adhesions

Objective:To develop a combined barrier method and drug delivery system (“hybrid system”) for preventing postoperative peritoneal adhesions, which could combine the biocompatibility and ease of application of in situ cross-linkable hydrogels with the controlled release features of polymeric nanoparticles. Methods:Poly(lactic-co-glycolic acid) nanoparticles were dispersed in aldehyde- and hydrazide-modified hyaluronic acids (HA), then combined via a double-barreled syringe. The material was subjected to mechanical testing and was assayed for in vitro cytotoxicity to murine mesothelial cells. Subsequently, it was tested for biocompatibility by intraperitoneal injection in mice. The hybrids effectiveness in preventing postsurgical adhesions was assessed using a rabbit sidewall defect-cecum abrasion model, where it was applied to both injured surfaces. Results:The in situ hybrid gel system formed a flexible and durable hydrogel in less than 10 seconds. It had low in vitro cytotoxicity. In the mouse, the cross-linked HA maintained the polymeric nanoparticles in the peritoneum for 1 week, which we had previously shown would have cleared in less than 2 days, and no animals developed adhesions. Notably, the hybrid gel, even in the absence of encapsulated drug, was highly effective in preventing peritoneal adhesions in the rabbit model employed. Animals treated with the hybrid (n = 8) had no adhesions in 62.5% of cases, and none had adhesions that could only be separated by sharp dissection. In contrast, only 4.2% of untreated animals (n = 24) had no adhesions, and 58.3% developed adhesions requiring sharp dissection. Conclusions:The hybrid cross-linked HA-nanoparticle system described here appears to be a biocompatible and highly effective adhesion barrier, which could also deliver antiadhesion drugs.

Gastric Helicobacter species as a cause of feline gastric lymphoma: A viable hypothesis

Gastric Helicobacter spp. are associated with chronic inflammation and neoplastic transformation in humans as well as domestic and laboratory species. The present study examined the association of Helicobacter heilmannii (Hhe) infection in pet cats with feline gastric mucosa associated lymphoid tissue (MALT) lymphoma. Tissues were collected via gastric biopsy or at necropsy from 47 pet cats with clinical signs of gastrointestinal disease, including vomiting and inappetance, and classified as gastritis (14/47), lymphoma (31/37), or normal (2/47). Tissues positive for argyrophilic organisms with Warthin-Starry stain (29/47) were assessed by fluorescent in situ hybridization (FISH) for the presence of Hhe strains 1-4 as well as with a fifth probe that detected Helicobacter salomonis, Helicobacter bizzozeronii, or Helicobacter felis. A significant association of positive Warthin-Starry status with Hhe infection was found in cases of sick cats (22/29; p<0.05 by Chi-square; chi(2)=7.034). Interestingly, a significant association between Hhe status and a diagnosis of lymphoblastic or lymphocytic lymphoma was observed as well in a subset of 24 Warthin-Starry positive lymphoma cases: of lymphoblastic lymphoma cases, 13/17 were positive for Hhe (p<0.05; chi(2)=4.854). Hhe strains 2 and 4 were most commonly found (18/29 and 17/29, respectively) among sick cats, although a higher than expected number of cats was also positive for Hhe1, which initial reports have described as rare in cats and common in humans. The association found between a positive Hhe status with the presence of feline gastric lymphoma, especially lymphoblastic lymphoma, argues for the need to conduct prospective studies to better identify the frequency and strain distribution of Hhe infection in both healthy and clinically ill cats, particularly those cats with gastric lymphoma.

A Naturally Occurring Rabbit Model of Enterohemorrhagic Escherichia coli–Induced Disease

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans. The exact mechanism by which EHEC induces disease remains unclear because of the lack of a natural animal model for the disease. An outbreak of bloody diarrhea and sudden death was investigated in a group of Dutch belted rabbits. Two of these rabbits harbored enteropathogenic E. coli O145:H(-), and 1 rabbit was coinfected with EHEC O153:H(-). A partial Shiga toxin 1 gene (stx1) fragment from E. coli O153:H(-) was confirmed by Southern blot and sequence analysis. Toxin production was demonstrated by a HeLa cell cytotoxicity assay. Histopathologic findings in all affected rabbits included erosive and necrotizing enterocolitis with adherent bacterial rods, proliferative glomerulonephritis, tubular necrosis, and fibrin thrombi within small vessels and capillaries. Our findings provide evidence for a naturally occurring animal model of EHEC-induced systemic disease that closely resembles human HUS.

Helicobacter pylori gastritis in cats with long-term natural infection as a model of human disease.

A natural infection with Helicobacter pylori (H. pylori) in domestic cats (Felis cattus) less than 2 years of age has been well described in a closed colony of animals. Six cats from this colony that were serially evaluated by culture, polymerase chain reaction, and light and electron microscopy for a period of 3 years demonstrated persistent gastric colonization with a single cag(-) vac(+) strain of H. pylori. In these cats, as well as five other 5- to 6-year-old cats that were examined, a long-term infection resulted in chronic diffuse lymphofollicular atrophic gastritis with areas of mucosal dysplasia in the antrum and predominantly midsuperficial gastritis in the body and cardia. Topographically, the distribution of lesions was similar in both young and older cats and closely resembled that found in humans, with the most severe changes occurring in the gastric antrum. Few granulocytes and no significant elevation in mast cells were seen in older H. pylori-infected cats compared with uninfected controls; however, marked increases in interepithelial globule leukocytes and numerous active mucosal lymphoid follicles were present in infected animals. Indices of gastritis were significantly greater in older infected cats when compared with uninfected controls and younger cats (P < 0.05). The antral cell proliferation index of infected older cats was significantly (P = 0.021) greater than that of uninfected controls. Apoptotic indices of the gastric antrum and body of infected cats were significantly (P = 0.01) increased versus controls. Chronic infection with H. pylori in cats shares many features of long-term H. pylori infection in humans, including the development of preneoplastic processes. This similarity provides useful, comparative insights into host-pathogen interactions.

Vitamin E enhances the lymphatic transport of β-carotene and its conversion to vitamin A in the ferret

BACKGROUND/AIMS beta-Carotene and alpha-tocopherol may have either antagonistic or synergistic effects on each others absorption and metabolism. The effects of both physiological and pharmacological concentrations of alpha-tocopherol on the absorption and metabolism of beta-carotene in ferret intestine were determined. METHODS A high concentration of beta-carotene was perfused through the upper portion of the small intestine of ferrets in vivo with varying levels of alpha-tocopherol. The effluent of a mesenteric lymph duct cannulation, the intestinal mucosal scraping, and portal vein blood were sampled and analyzed by high-performance liquid chromatography. RESULTS The lymphatic transport of beta-carotene was enhanced 4-fold by alpha-tocopherol at a physiological dose and 12-21-fold at a pharmacological dose. The lymphatic transport of alpha-tocopherol was linearly (r = 0.8; P < 0.05) related to the luminal alpha-tocopherol concentration even in the presence of a high concentration of beta-carotene. Furthermore, alpha-tocopherol increased the conversion of beta-carotene into retinol in the intestine in a dose-dependent manner. CONCLUSIONS alpha-Tocopherol has a positive effect on the intestinal absorption of intact beta-carotene and may modulate the metabolic conversion of beta-carotene into retinoids.

Three-dimensional conductive constructs for nerve regeneration.

The unique electrochemical properties of conductive polymers can be utilized to form stand-alone polymeric tubes and arrays of tubes that are suitable for guides to promote peripheral nerve regeneration. Noncomposite, polypyrrole (PPy) tubes ranging in inner diameter from 25 microm to 1.6 mm as well as multichannel tubes were fabricated by electrodeposition. While oxidation of the pyrrole monomer causes growth of the film, brief subsequent reduction allowed mechanical dissociation from the electrode mold, creating a stand-alone, conductive PPy tube. Conductive polymer nerve guides made in this manner were placed in transected rat sciatic nerves and shown to support nerve regeneration over an 8-week time period.

Isolation and characterization of a novel helicobacter species, "Helicobacter macacae," from rhesus monkeys with and without chronic idiopathic colitis.

ABSTRACT Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, “Helicobacter macacae” (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

A technique for catheterization of the urinary bladder in the ferret

The technique of catheterization of the urinary bladder, an important clinical skill for the diagnosis of urinary tract disorders, has not been described for the ferret. The bladder was catheterized in 23 ferrets (10 intact females; 11 spayed females; and 2 intact males) using a 3½ French, red rubber urethral catheter fitted with a steel wire stylet. Ferrets were anaesthetized with isoflurane or ketamine (30mg/kg IM) and xylazine (3mg/kg IM). Females were positioned in ventral recumbency with the rear quarters elevated by a rolled surgical towel. The urethra was catheterized by direct visualization of the external urethral orifice using a vaginal speculum. The orifice was approximately 1 cm cranial to the clitoral fossa on the ventral floor of the vestibule. Blind passage was used in several spayed females. In males, the distal end of the penis was exteriorized from the prepuce and the external urethral orifice cannulated without stylet. No difficulty was encountered in advancing the catheter past the os penis. This catheterization technique allows urinary tract access for urine collection, pneumocystography, contrast cystography, double contrast cystography, and urine output determination in pharmacologic studies or in critical care of debilitated animals.