Antonello Bufalari

Transplantation of Pancreatic Islets Contained in Minimal Volume Microcapsules in Diabetic High Mammalians

ABSTRACT: To minimize technical problems relating to excessive size (600–800μ in diameter) of standard alginate microcapsules (CSM) for pancreatic islet graft immunoisolation, we have developed two novel minimal volume, chemically identical, capsule prototypes (MVC): 1) coherent microcapsules (CM), and 2) medium‐size microcapsules (300–400μ, MSM). CM, which envelop each individual islet within a thin alginate hydrogel cast, are prepared by emulsification, whereas MSM are made by atomizing the islet‐alginate suspension through a special microdroplet generator. Upon graft into diabetic rodents, CM have shown to immunoprotect both allo‐ and xenogeneic nondiscordant islets, and restored normoglycemia. In higher mammals, at sub‐therapeutic doses, CM fully immunoprotected islet allografts (pig→pig), but only temporarily xenografts (dog→pig). We then used MSM to immunoisolate canine islet allografts in the peritoneal cavity of dogs with spontaneous insulin‐dependent diabetes. Of three grafted dogs, two showed full remission of hyperglycemia with insulin withdrawal. MSM could represent an intermediate solution between CSM and CM for peritoneal immunoisolated islet transplants.

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy

Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre‐clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti‐inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre‐transplant storage stability of specific pathogen‐free pSCs (SPF‐pSCs) and evaluated the in vivo long‐term viability and safety of grafts.

Transplantation of allogeneic/xenogeneic pancreatic islets containing coherent microcapsules in adult pigs.

WE HAVE PREVIOUSLY described a method for fabrication of alginate/polyaminoacidic CM. Thereafter, we completed assessment of either morphological properties of structural as well as ultrastructural level, or in vitro immunoselectivity, or in vivo post-transplant (Tx) functional performance in diabetic rodents of these new biomembranes for islet Tx immunoprotection in full absence of the recipient’s pharmacological immunosuppression. Because CM, unlike conventional-size microcapsules (CSM), measuring an average 600 to 800 mm in equatorial diameter, hold the unique advantage of occupying a volume which is almost coincident with that of naked islets, these new microimmunobarriers could address a number of unsolved problems with regard to encapsulated islet cell Tx in diabetic large mammalians. One of the most formidable hurdles to this approach for the therapy of insulin-dependent diabetes mellitus has consisted of inadequacy of implant sites for encapsulated islets. Because of the final excessive CSM’s Tx size, these capsules had been only implanted in the peritoneal cavity. Unfortunately, even if highly biocompatible, intraperitoneally grafted, islet-containing microcapsules had often provoked severe, possibly mass-related (60 to 80 mL/diabetic dog) inflammatory cell reaction. Dense connective tissue infiltration invariably resulted in impairment of biochemical exchange, ultimately leading to Tx failure. After testing the new CM in either in vitro functional and immunological or in vivo rodent diabetes-correction studies, we had embarked on assessment of CM biocompatibility, after multiple-site transplantation into adult pigs. Preliminarily, we have shown that empty CM, per se, indeed were biocompatible in this large-size mammal animal model, with a very low degree of sensitization being induced by repeated CM-Tx booster. In an attempt to determine whether CM, because of their minimal size, would permit access to Tx sites so far interdicted to CSM, including parenchymatous organs, with full retention of the encapsulated allogeneic/xenogeneic islet cell viability, we have transplanted porcine or canine islet containing CM into multiple Tx sites, and examined them histologically, at 30 days post-Tx, in normal, adult pigs. MATERIALS AND METHODS Animals

The Use of Alpha-2 Agonists in the Equine Practice: Comparison between Three Molecules

Nannarone, S., Gialletti, R., Veschini, I., Bufalari, A. and Moriconi, F., 2007. The use of alpha-2 agonists in the equine practice: comparison between three molecules. Veterinary Research Communications, 31(Suppl. 1), 309–312

Intraperitoneal injection of microencapsulated Sertoli cells restores muscle morphology and performance in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration leading to impaired locomotion, respiratory failure and premature death. In DMD patients, inflammatory events secondary to dystrophin mutation play a major role in the progression of the pathology. Sertoli cells (SeC) have been largely used to protect xenogeneic engraftments or induce trophic effects thanks to their ability to secrete trophic, antiinflammatory, and immunomodulatory factors. Here we have purified SeC from specific pathogen-free (SPF)-certified neonatal pigs, and embedded them into clinical grade alginate microcapsules. We show that a single intraperitoneal injection of microencapsulated SPF SeC (SeC-MC) in an experimental model of DMD can rescue muscle morphology and performance in the absence of pharmacologic immunosuppressive treatments. Once i.p. injected, SeC-MC act as a drug delivery system that modulates the inflammatory response in muscle tissue, and upregulates the expression of the dystrophin paralogue, utrophin in muscles through systemic release of heregulin-β1, thus promoting sarcolemma stability. Analyses performed five months after single injection show high biocompatibility and long-term efficacy of SeC-MC. Our results might open new avenues for the treatment of patients with DMD and related diseases.

Fentanyl or sufentanil continuous infusion during isoflurane anaesthesia in dogs: clinical experiences.

Bufalari, A., Di Meo, A., Nannarone, S., Padua, S. and Adami, C., 2007. Fentanyl or sufetanil continuous infusion during isoflurane anaesthesia in dogs: Clinical experiences. Veterinary Research Communications,31(Suppl. 1), 277–280

Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice.

We report data about the effects of intraperitoneal (i.p.) injection of specific pathogen-free (SPF) porcine Sertoli cells (SeC) encapsulated into clinical grade alginate-based microcapsules (SeC-MC) on muscles of chronic and presymptomatic dystrophic, mdx mice. Mdx mouse is the best characterized animal model of Duchenne muscular dystrophy (DMD), an X-linked lethal myopathy due to mutation in the gene of dystrophin, which is crucial for myofiber integrity during muscle contraction. Our data show that three weeks after i.p. injection of SeC-MC significantly reduced adipose and fibrous tissue deposition, reduced macrophage infiltrate, and reduced numbers of damaged myofibers are found in muscles of 12-month-old mdx mice, which reproduce chronic DMD conditions. Compared with muscles of mock-treated mdx mice muscles of SeC-MC-treated mice show upregulation of the dystrophin paralogue, utrophin which is localized to the periphery of myofibers. Moreover, our data show that i.p. injection of SeC-MC into presymptomatic, 2-week-old mdx mice, although not fully preventing myofiber degeneration, results in protection against myofiber necrosis and muscle inflammation. Extensive discussion of these data can be found in Ref. [1].

IMAGING DIAGNOSIS—TRANSESOPHAGEAL ULTRASOUND‐GUIDED REMOVAL OF A MIGRATING GRASS AWN FOREIGN BODY IN A DOG

A 3-year-old English Setter dog was presented for an acute onset of coughing. Tracheobronchoscopic examination allowed localization and removal of one grass awn foreign body. A second migrated grass awn was suspected to be present in the left caudal lung lobe. Transesophageal ultrasound revealed an area of pulmonary consolidation in the dorsomedial portion of left caudal lobe and a linear hyperechoic structure consistent with a grass awn foreign body within the area of consolidation. Transesophageal ultrasonography was also used to provide anatomical landmarks that facilitated successful thoracoscopic removal of the foreign body.

Pancreatic regeneration after subtotal distal resection in rats. Effects of bombesin and octreotide by the in vivo bromodeoxyuridine uptake technique.

Forty male Wistar rats were randomly allocated to four treatment groups after 90% distal pancreatectomy: group A (control) received saline (0.5 ml subcutaneously); group B received bombesin (BBS; 10 μg/kg intraperitoneally); group C received octreotide (2.5 μg/kg subcutaneously), and group D received BBS and octreotide. All substances were injected three times a day until sacrifice after 28 days. BBS increased pancreas weight (p = 0.003) and DNA synthesis (p < 0.001), as measured by a bromodeoxyuridine nuclear-labeling index (BrdU LI). The simultaneous administration of octreotide significantly decreases the remnant pancreas weight (p = 0.016) as compared to group B rats; however the BrdU LI is not significantly reduced in group D as compared to group B. BBS administration promotes regeneration of the remnant pancreas in terms of hypertrophy and hyperplasia. Although octreotide appears to significantly reduce the pancreatic weight increase induced by BBS, it does not reduce DNA synthesis and cell proliferation.

Intra-articular administration of lidocaine plus adrenaline in dogs: Pharmacokinetic profile and evaluation of toxicity in vivo and in vitro

The aim of this study was to evaluate the safety of intra-articular (IA) lidocaine plus adrenaline for improving peri-operative analgesia in anaesthetized dogs undergoing arthroscopy of the elbow. A solution of lidocaine (L) 1.98% plus adrenaline 1:100.000 was administered via the IA route and its safety evaluated in terms of cardio-, neuro-, and chondro-toxicity. No bradycardia or hypotension was recorded from induction to the last observational time point. Signs of toxicity of the nervous system could have been masked by the general anaesthesia but lidocaine concentrations detected in the blood were lower than those thought to be capable of producing toxicity. The assessment of in vitro chondrotoxicity showed a dose- and time-dependent effect of lidocaine on the viability of articular cells. Adrenaline appeared to reduce the chondrotoxicity of 1% lidocaine, following an exposure of up to 30 min.