Tutorial: Triheptanoin and Nutrition Management for Treatment of Long-Chain Fatty Acid Oxidation Disorders.

BACKGROUND: Patients with severe long-chain fatty acid oxidation disorders (LC-FAODs) experience serious morbidity and mortality despite traditional dietary management including medium-chain triglyceride (MCT)-supplemented, low-fat diets. Triheptanoin is a triglyceride oil that is broken down to acetyl-coenzyme A (CoA) and propionyl-CoA, which replenishes deficient tricarboxylic acid cycle intermediates. We report the complex medical and nutrition management of triheptanoin therapy initiated emergently for 3 patients with LC-FAOD. METHODS: Triheptanoin (Ultragenyx Pharmaceutical, Inc, Novato, CA, USA) was administered to 3 patients with LC-FAOD on a compassionate-use basis. Triheptanoin was mixed with non-MCT-containing low-fat formula. Patients were closely followed with regular cardiac and laboratory monitoring. RESULTS: Cardiac ejection fraction normalized after triheptanoin initiation. Patients experienced fewer hospitalizations related to metabolic crises while on triheptanoin. Patient 1 has tolerated oral administration without difficulty since birth. Patients 2 and 3 experienced increased diarrhea. Recurrent breakdown of the silicone gastrostomy tube occurred in patient 3, whereas the polyurethane nasogastric tube for patient 2 remained intact. Patient 3 experiences recurrent episodes of elevated creatine kinase levels and muscle weakness associated with illness. Patient 3 had chronically elevated C10-acylcarnitines while on MCT supplementation, which normalized after initiation of triheptanoin and discontinuation of MCT oil. CONCLUSIONS: Triheptanoin can ameliorate acute cardiomyopathy and increase survival in patients with severe LC-FAOD. Substituting triheptanoin for traditional MCT-based treatment improves clinical outcomes. MCT oil might be less effective in carnitine-acylcarnitine translocase deficiency patients compared with other FAODs and needs further investigation.

Magnesium-Dependent Promotion of H2O2 Production Increases Ecological Competitiveness of Oral Commensal Streptococci.

The pyruvate oxidase (SpxB)-dependent production of H2O2 is widely distributed among oral commensal streptococci. Several studies confirmed the ability of H2O2 to antagonize susceptible oral bacterial species, including caries-associated Streptococcus mutans as well as several periodontal pathobionts. Here we report a potential mechanism to bolster oral commensal streptococcal H2O2 production by magnesium (Mg2+) supplementation. Magnesium is a cofactor for SpxB catalytic activity, and supplementation increases the production of H2O2 in vitro. We demonstrate that Mg2+ affects spxB transcription and SpxB abundance in Streptococcus sanguinis and Streptococcus gordonii. The competitiveness of low-passage commensal streptococcal clinical isolates is positively influenced in antagonism assays against S. mutans. In growth conditions normally selective for S. mutans, Mg2+ supplementation is able to increase the abundance of S. sanguinis in dual-species biofilms. Using an in vivo biophotonic imaging platform, we further demonstrate that dietary Mg2+ supplementation significantly improves S. gordonii oral colonization in mice. In summary, our results support a role for Mg2+ supplementation as a potential prebiotic to promote establishment of oral health-associated commensal streptococci.

Fatty acid oxidation disorders.

Fatty acid oxidation disorders (FAODs) are inborn errors of metabolism due to disruption of either mitochondrial ß-oxidation or the fatty acid transport using the carnitine transport pathway. The presentation of a FAOD will depend upon the specific disorder, but common elements may be seen, and ultimately require a similar treatment. Initial presentations of the FAODs in the neonatal period with severe symptoms include cardiomyopathy, while during infancy and childhood liver dysfunction and hypoketotic hypoglycemia are common. Episodic rhabdomyolysis is frequently the initial presentation during or after adolescence; although, these symptoms may develop at any age for most of the FAODs The treatment of all FAOD's include avoidance of fasting, aggressive treatment during illness, and supplementation of carnitine, if necessary. The long-chain FAODs differ by requiring a fat-restricted diet and supplementation of medium chain triglyceride oil and often docosahexaenoic acid (DHA)-an essential fatty acid, crucial for brain, visual, and immune functions and prevention of fat soluble vitamin deficiencies. The FAOD are a group of autosomal recessive disorders associated with significant morbidity and mortality, but early diagnosis on newborn screening (NBS) and early initiation of treatment are improving outcomes. There is a need for clinical studies including randomized, controlled, therapeutic trials to continue to evaluate current understanding and to implement future therapies.

A cognitively normal PDH-deficient 18-year-old man carrying the R263G mutation in the PDHA1 gene.

Pyruvate dehydrogenase (PDH) is a crucial multienzyme system linking glycolysis to the tricarboxylic acid cycle by catalysing the decarboxylation of pyruvate to acetyl-CoA. Deficiency in pyruvate dehydrogenase is most commonly secondary to mutations in the X-linked PDHA1 gene encoding the E1 alpha subunit. There is a wide range of clinical presentations from severe neonatal lactic acidosis to chronic encephalopathy (Leigh syndrome). In recent years, a small subset of patients was recognized with less severe involvement, presenting initially only with intermittent symptoms, mainly of ataxia. Most of these patients remain stable for a number of years before developing progressive neurological deterioration around puberty at the latest. There does not appear to be a reliable correlation between genotype, phenotype, or enzyme activity. This makes counselling in a clinical setting challenging. We report a case with a previously known common mutation in PDHA1 (R263G) with an excellent outcome at 18 years of age. Previous patients with this mutation have presented with mental retardation and/or Leigh syndrome, while our patient's clinical outcome is exceptional. He is cognitively normal and has normal brain MRI. His management includes a stringent carbohydrate-free diet, as well as supplementation with thiamine, carnitine and vitamin E. This case further broadens the clinical spectrum, including now an example of a cognitively normal adult with PDH deficiency.

Effects of high peak power microwaves on the retina of the rhesus monkey.

We studied the retinal effects of 1.25 GHz high peak power microwaves in Rhesus monkeys. Preexposure fundus photographs, retinal angiograms, and electroretinograms (ERG) were obtained to screen for normal ocular structure and function and, after exposure, as endpoints of the study. Histopathology of the retina was an additional endpoint. Seventeen monkeys were randomly assigned to receive sham exposure or pulsed microwave exposures. Microwaves were delivered anteriorly to the face at 0, 4.3, 8.4, or 20.2 W/kg spatially and temporally averaged retinal specific absorption rates (R-SAR). The pulse characteristics were 1.04 MW ( approximately 1.30 MW/kg temporal peak R-SAR), 5.59 micros pulse length at 0, 0.59, 1. 18, and 2.79 Hz pulse repetition rates. Exposure was 4 h per day and 3 days per week for 3 weeks, for a total of nine exposures. The preexposure and postexposure fundus pictures and angiograms were all within normal limits. The response of cone photoreceptors to light flash was enhanced in monkeys exposed at 8.4 or 20.2 W/kg R-SAR, but not in monkeys exposed at 4.3 W/kg R-SAR. Scotopic (rod) response, maximum (combined cone and rod) response, and Naka-Rushton R(max) and log K of scotopic b-waves were all within normal range. Retinal histopathology revealed the presence of enhanced glycogen storage in photoreceptors among sham (2/5), 8.4 W/kg (3/3), and 20.2 W/kg (2/5) exposed monkeys, while enhanced glycogen storage was not observed in the 4.3 W/kg (0/4) exposed group. Supranormal cone photoreceptor b-wave was R-SAR dependent and may be an early indicator of mild injury. However no evidence of degenerative changes and ERG depression was seen. We concluded that retinal injury is very unlikely at 4 W/kg. Functional changes that occur at higher R-SAR are probably reversible since we saw no evidence of histopathologic correlation with ERG changes. Bioelectromagnetics 21:439-454, 2000. Published 2000 Wiley-Liss, Inc.

Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

Chronic, low-level (1.0 W/kg) exposure of mice prone to mammary cancer to 2450 MHz microwaves.

In a previous study (Frei et al., Bioelectromagnetics 19, 20-31, 1998), we showed that low-level (0.3 W/kg), long-term exposure of mice prone to mammary tumors to 2450 MHz radiofrequency (RF) radiation did not affect the incidence of mammary tumors, latency to tumor onset, tumor growth rate or animal survival when compared to sham-irradiated animals. In the current study, the specific absorption rate (SAR) was increased from 0.3 W/kg to 1.0 W/kg. The same biological end points were used. One hundred C3H/HeJ mice were exposed in circularly polarized waveguides for 78 weeks (20 h/day, 7 days/week) to continuous-wave, 2450 MHz RF radiation; 100 mice were sham-exposed. There was no significant difference between exposed and sham-exposed groups with respect to the incidence of palpated mammary tumors (sham-exposed = 30%; irradiated = 38%), latency to tumor onset (sham-exposed = 62.0 +/- 2.3 weeks; irradiated = 62.5 +/- 2.2 weeks) and rate of tumor growth. Histopathological evaluations revealed no significant difference in numbers of malignant, metastatic or benign neoplasms between the two groups. Thus long-term exposures of mice prone to mammary tumors to 2450 MHz RF radiation at SARs of 0.3 and 1.0 W/kg had no significant effects when compared to sham-irradiated animals.

Establishing orally self-administered cocaine as a reinforcer in rats using home-cage pre-exposure.

1. Rats were force-exposed to a cocaine + saccharin solution in their home cage water bottles for five days. They were then given 5 h home-cage access to both cocaine and cocaine-free solutions for 40 days. 2. The subjects consumed large doses of the cocaine solution despite the ad libitum availability of water. 3. The animals were then trained on a task consisting of operant bar pressing rewarded on an intermittent schedule with a liquid cocaine reinforcer. 4. All subjects performed the operant task and consumed doses of cocaine solution which are preferred over water in other paradigms. 5. Levels of responding were significantly reduced in three of four subjects when vehicle was substituted for liquid cocaine as the reward. 6. This demonstrates that orally self-administered cocaine can be used as a reinforcer in rats.

Active specific immunotherapy of metastatic melanoma with an antiidiotype vaccine: a phase I/II trial of I-Mel-2 plus SAF-m.

PURPOSE: To determine the toxicity and immunologic activity of an antiidiotype melanoma vaccine that consists of monoclonal antibody I-Mel-2 (MELIMMUNE-2, IDEC Pharmaceuticals, La Jolla, CA) and an immunologic adjuvant SAF-m. PATIENTS AND METHODS: Twenty-six patients with metastatic melanoma, 17 of whom had previously received chemotherapy, were given 2 mg of I-Mel-2 and either 100 micrograms (n = 6) or 250 micrograms (n = 20) of SAF-m. Antiidiotype vaccine was given intramuscularly (IM) biweekly for 4 weeks, and then bimonthly until disease progression. Human antimurine antibodies (HAMA), anti-I-Mel-2 antibodies, and specific antibody (Ab)3 against the melanoma epitope mimicked by the vaccine were titrated before treatment, biweekly from weeks 4 to 12, and every 4 to 8 weeks thereafter. Computed tomographic (CT) scans of the chest, abdomen, and pelvis and magnetic resonance imaging (MRI) of the brain were obtained before and bimonthly during treatment to evaluate responses. RESULTS: Elevated titers of human antimouse antibodies and anti-I-Mel-2 antibodies were associated with clinical antitumor effect (P = .02 and P = .05, respectively). Ab3 was absent in most patients, but was found in the best clinical responder. Fever, myalgias/arthralgias, fatigue, nausea, and headaches were the most common toxicities. Grade III myalgias/arthralgias and headaches required dose reduction of SAF-m in eight patients at the 250-microgram dose. No treatment-related death occurred. Six patients had an antitumor effect: one complete response in liver and lung, two minor responses, and three stable disease. The patient with a complete response has survived nearly 5 years. CONCLUSION: I-Mel-2 antiidiotype vaccine was safe, tolerated best at the 100-microgram dose of SAF-m, and had immunologic and clinical activity.

Expression of cholera toxin B subunit oligomers in transgenic potato plants.

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

Intake, digestion and gastrointestinal transit time in resting donkeys and ponies and exercised donkeys given ad libitum hay and straw diets.

Four donkeys and four ponies maintained in climate rooms, were provided with meadow hay or barley straw supplemented with minerals and vitamins ad libitum. Both diets were given to all animals for periods of 21 days. Measurements were made during the last seven days following single doses of two non-absorbable markers (Cr-fibre and Co-EDTA). After six weeks the donkeys repeated the trial, walking 14 km and ascending 260 m five days per week. At rest ponies ate more than donkeys, however the donkeys showed a higher digestibility of dry matter (DM), organic matter (OM) and acid detergent fibre (ADF) than the ponies. Both species lost weight, ate less and had a lower digestibility of DM, OM and energy on the straw than on the hay ration. Fibre digestibility (neutral detergent fibre [NDF] and ADF) was higher on the straw than the hay diet. Gastrointestinal transit time was slower in the donkeys than in the ponies on both diets. Both species showed a higher mean retention time of digesta on straw than on hay. Exercise had no significant effect on food intake and liveweight changes in the donkeys. Although digestibility coefficients were higher in exercised animals, the effect was not significant. We conclude that, although it is generally considered that donkeys can thrive better on poor feeds than ponies, when the animals are allowed ad libitum access to hay and straw better intakes by ponies may compensate for any differences in digestibility seen in donkeys. Intake of barley straw by both species was not sufficient to meet maintenance requirements.

Relative afferent pupillary defects in primary open-angle glaucoma--five years' experience.

Afferent pupillary defects may accompany asymmetric primary open-angle glaucoma, though the exact incidence has not been reported. Charts were reviewed on 89 patients attending the Glaucoma/Uveitis Clinic at the North Carolina Memorial Hospital in Chapel Hill, North Carolina over a five-year period. All patients had primary open-angle glaucoma diagnosed by: (1) increased ocular tensions (22 mmHg) in the presence of open-anterior-chamber angles and (2) optic-nerve cupping and atrophy compatible with (3) pressure-dependent, visual-field loss. No subjects with secondary glaucomas, primary-angle-closure glaucoma, or ocular hypertension are included.The presence of the relative afferent pupillary defect was noted in 21 of 89 patients (23 percent). Sixteen of 70 black patients had relative afferent pupillary defect in the more severely affected eye, while five of 19 white patients demonstrated afferent pupils. Other demographic characteristics of this population are described. Two typical primary-open-angle glaucoma patients are discussed to demonstrate comparable changes within the optic nerves and Goldmann visual fields. The presence of the relative afferent pupillary defect best correlates with asymmetric, visual-field loss in the more severely affected eye.

Orientation effects on microwave-induced hyperthermia and neurochemical correlates.

This paper describes the effect of field orientation on the hyperthermia produced by microwave irradiation. Rats exposed in the E-orientation (long-axis of animal parallel to E-field) to 1600 tmhz radiation become significantly hotter than those exposed in the H-field at the same power density. Norepinephrine and dopamine, but not serotonin content of the basal hypothalamus is reduced as a concomitant of microwave-induced brain hyperthermia.