Murray Potter

Chronic Gastrointestinal Inflammation Induces Anxiety-Like Behavior and Alters Central Nervous System Biochemistry in Mice

BACKGROUND & AIMS Clinical and preclinical studies have associated gastrointestinal inflammation and infection with altered behavior. We investigated whether chronic gut inflammation alters behavior and brain biochemistry and examined underlying mechanisms. METHODS AKR mice were infected with the noninvasive parasite Trichuris muris and given etanercept, budesonide, or specific probiotics. Subdiaphragmatic vagotomy was performed in a subgroup of mice before infection. Gastrointestinal inflammation was assessed by histology and quantification of myeloperoxidase activity. Serum proteins were measured by proteomic analysis, circulating cytokines were measured by fluorescence activated cell sorting array, and serum tryptophan and kynurenine were measured by liquid chromatography. Behavior was assessed using light/dark preference and step-down tests. In situ hybridization was used to assess brain-derived neurotrophic factor (BDNF) expression in the brain. RESULTS T muris caused mild to moderate colonic inflammation and anxiety-like behavior that was associated with decreased hippocampal BDNF messenger RNA (mRNA). Circulating tumor necrosis factor-α and interferon-γ, as well as the kynurenine and kynurenine/tryptophan ratio, were increased. Proteomic analysis showed altered levels of several proteins related to inflammation and neural function. Administration of etanercept, and to a lesser degree of budesonide, normalized behavior, reduced cytokine and kynurenine levels, but did not influence BDNF expression. The probiotic Bifidobacterium longum normalized behavior and BDNF mRNA but did not affect cytokine or kynurenine levels. Anxiety-like behavior was present in infected mice after vagotomy. CONCLUSIONS Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry, which can be normalized by inflammation-dependent and -independent mechanisms, neither of which requires the integrity of the vagus nerve.

Core-Cross-Linked Alginate Microcapsules for Cell Encapsulation

Self-cross-linkable polyelectrolyte pairs comprised of poly(methacrylic acid, sodium salt-co-2-[methacryloyloxy]ethyl acetoacetate) (70:30 mol ratio, A70) and poly-L-lysine are incorporated into CaAlg beads to form either a covalently cross-linked shell or a core-cross-linked bead. In both cases the reactive polyanion is added to a solution of sodium alginate that may contain live cells and dropped into a calcium chloride gelling bath. Subsequent exposure to poly-L-lysine (15-30 kDa) leads to formation of a cross-linked shell, while exposure to lower molecular weight poly-L-lysine (4-15 kDa) leads to formation of an interpenetrating matrix of covalently cross-linked synthetic polymer within the CaAlg template. The resulting spherical composites are resistant to chemical and mechanical stress yet remain cyto-compatible. This approach to cell-encapsulation may be useful for cell immuno-isolation in therapeutic cell transplants.

The Relationship Between Trimethylamine-N-Oxide and Prevalent Cardiovascular Disease in a Multiethnic Population Living in Canada

BACKGROUND Microflora-dependent trimethylamine-N-oxide (TMAO) formation, which results from intake of choline and L-carnitine-rich food, shows promise as a predictor of cardiovascular disease (CVD) risk, but these associations have not been examined in ethnically diverse populations. In a multiethnic population-based study of adults in Canada, we assessed the stability of TMAO and L-carnitine in stored serum samples and their association with intimal medial thickness, prevalent risk factors, and clinical events. METHODS In a randomly sampled cross-sectional study of 1286 Canadians, fasting serum samples were collected and stored. In 292 consecutive individuals (99 CVD cases and 193 unmatched control subjects), L-carnitine and TMAO concentrations were assessed using validated analytical approaches. RESULTS The mean (± SD) TMAO level was 1.998 ± 3.13 μM and L-carnitine was 42.29 ± 11.35 μM. The relative levels of the samples did not appreciably change after 3 freeze-thaw cycles (coefficient of variation, 5.6% and 4.7%, respectively). No significant association between L-carnitine levels and prevalent CVD was found, with adjustment for covariates (odds ratio, 1.57; 95% confidence interval, 0.58-4.26; P trend = 0.65), for highest vs lowest quintile group. TMAO levels showed a significant, graded association with prevalent CVD (odds ratio, 3.17; 95% confidence interval, 1.05-9.51; P trend = 0.02). After further adjustment for diabetes status, meat, fish, and cholesterol intake, the association remained significant. No significant association between carotid intimal medial thickness and L-carnitine (P = 0.64) or TMAO (P = 0.18) was found. CONCLUSIONS Serum TMAO and L-carnitine analysis on stored samples is reliable. Our findings support an association between TMAO with prevalent CVD in a multiethnic population. This finding requires replication in larger studies in which dietary intake and stored serum samples exist.

Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation.

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.

Review--the use of immunosuppressive agents to prevent neutralizing antibodies against a transgene product.

ABSTRACT: A potential obstacle to successful gene therapy for some patients is the in vivo production of neutralizing antibodies against the recombinant therapeutic product delivered. This is a problem inherent to all gene therapy methods, regardless of the vector used to deliver the protein. This clinical situation can be mimicked in animal models by delivering a foreign protein (i.e., a human protein) to the animal to provoke anti‐human protein antibody production. The efficacy of different immunosuppressive treatments to inhibit the development of neutralizing antibodies can then be investigated. The immunosuppressive agents examined here include drugs (e.g., cyclophosphamide, FK506), cytokines (e.g., interferon‐γ, interleukin‐12), and monoclonal antibodies (e.g., anti‐CD4, anti‐gp39, CTLA4‐Ig). It has been found that a high level of antibody suppression is necessary to allow prolonged delivery of a foreign protein. Immunosuppressive agents capable of this high level of suppression will be important adjuncts to prevent treatment failures in situations where patients are at risk of developing neutralizing antibodies.

Poly(methyl vinyl ether-alt-maleic acid) polymers for cell encapsulation.

Polyanions based on poly(methyl vinyl ether-alt-maleic acid) were investigated as materials for cell encapsulation. These water-soluble polyanions having molecular masses ranging from 20 to 1980 kDa were prepared by functionalization of poly(methyl vinyl ether-alt-maleic anhydride) with 5-aminofluorescein and/or α-methoxy-ω-amino-poly(ethylene glycol), followed by base hydrolysis of the residual anhydride groups to form the corresponding poly(methyl vinyl ether-alt-sodium maleate). Their potential to replace alginate both in the core and, in particular, the outer shell of calcium alginate-poly(L-lysine)-alginate (APA) capsules was determined using confocal fluorescence microscopy, osmotic pressure tests, permeability studies, protein binding and cell viability assays. These polymers were shown to be able to replace the outer layer of alginate, forming more resilient capsule shells. The resulting capsules showed similar permeability and resistance to bovine serum albumin binding, as well as superior viability for encapsulated cells, when compared to standard APA capsules. In addition, these polymers showed promise for use as functional additives to the capsule cores.

Pregnancy in a healthy woman with untreated citrullinemia

We report the clinical and biochemical data on a second successful pregnancy in a woman with citrullinemia due to argininosuccinate synthetase deficiency (CTLN1). Despite very elevated plasma and urine citrulline and little or no measurable argininosuccinate synthetase enzyme activity on cultured skin fibroblasts, this 29‐year‐old woman, who was identified through newborn screening, has remained asymptomatic throughout her life. Mutation analysis has recently revealed that she is a compound heterozygote for a known and a novel mutation (IVS15‐1G > C and K310Q, respectively). Many newborn screening programs have recently been expanded to include citrullinemia and numerous asymptomatic hypercitrullinemic infants and children have been identified. It is now important to define prognostic indicators that will help with treatment decisions and genetic counseling for these patients. This patient, as the only citrullinemic adult who has been followed prospectively, contributes important information in this regard. In addition, her child was unaffected by the high citrulline levels demonstrated in amniotic fluid and breast milk suggesting that citrulline is not teratogenic. Although pregnancy is an important risk factor for women with CTLN1, it appears that females with citrullinemia can have normal pregnancy outcomes, as long as metabolic crisis is avoided.

Immuno-isolation in cancer gene therapy.

The implantation of genetically-modified non-autologous cells in immuno-protected microcapsules is an alternative to ex vivo gene therapy. Such cells delivering a recombinant therapeutic product are isolated from the hosts immune system by being encapsulated within permselective microcapsules. This approach has been successful in pre-clinical animal studies involving delivery of hormone or enzymes to treat dwarfism, lysosomal storage disease, or hemophilia B. Recently, this platform technology has shown promise in the treatment for more complex diseases such as cancer. One of the earliest strategy was to augment the chemotherapeutic effect of a prodrug by implanting encapsulated cells that can metabolise prodrugs into cytotoxic products in close proximity to the cancer cells. More recent approaches include enhancing tumor cell death through immunotherapy, or suppressing tumor cell proliferation through anti-angiogenesis. These can be achieved by delivering single molecules of cytokines or angiostatin, respectively, by implanting microencapsulated cells engineered to secrete these recombinant products. Recent refinements of these approaches include genetic fusion of cytokines or angiostatin to additional functional groups with tumor targeting or tumor cell killing properties, thus enhancing the potency of the recombinant products. Furthermore, a COMBO strategy of implanting microencapsulated cells to deliver multiple products targeted to diverse pathways in tumor suppression also showed much promise. This review will summarise the application of microencapsulation of genetically-modified cells to cancer treatment in animal models, the efficacy of such approaches, and how these studies have led to better understanding of the biology of cancer treatment. The flexibility of this modular system involving molecular engineering, cellular genetic modification, and polymer chemistry provides potentially a huge range of application modalities, and a tremendous multi-disciplinary challenge for the future.

β-Alanine Supplementation Does Not Augment the Skeletal Muscle Adaptive Response to 6 Weeks of Sprint Interval Training

Sprint interval training (SIT), repeated bouts of high-intensity exercise, improves skeletal muscle oxidative capacity and exercise performance. β-alanine (β-ALA) supplementation has been shown to enhance exercise performance, which led us to hypothesize that chronic β-ALA supplementation would augment work capacity during SIT and augment training-induced adaptations in skeletal muscle and performance. Twenty-four active but untrained men (23 ± 2 yr; VO2peak = 50 ± 6 mL · kg(-1) · min(-1)) ingested 3.2 g/day of β-ALA or a placebo (PLA) for a total of 10 weeks (n = 12 per group). Following 4 weeks of baseline supplementation, participants completed a 6-week SIT intervention. Each of 3 weekly sessions consisted of 4-6 Wingate tests, i.e., 30-s bouts of maximal cycling, interspersed with 4 min of recovery. Before and after the 6-week SIT program, participants completed a 250-kJ time trial and a repeated sprint test. Biopsies (v. lateralis) revealed that skeletal muscle carnosine content increased by 33% and 52%, respectively, after 4 and 10 weeks of β-ALA supplementation, but was unchanged in PLA. Total work performed during each training session was similar across treatments. SIT increased markers of mitochondrial content, including cytochome c oxidase (40%) and β-hydroxyacyl-CoA dehydrogenase maximal activities (19%), as well as VO2peak (9%), repeated-sprint capacity (5%), and 250-kJ time trial performance (13%), but there were no differences between treatments for any measure (p < .01, main effects for time; p > .05, interaction effects). The training stimulus may have overwhelmed any potential influence of β-ALA, or the supplementation protocol was insufficient to alter the variables to a detectable extent.

Isolation of human foetal myoblasts and its application for microencapsulation

Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non‐collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi‐nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18–24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell‐based therapy in human clinical applications with microencapsulation technology.