110: 259 -269, 2002). Heptanoate and C 5-ketone bodies derived from its partial oxidation in liver are precursors of anaplerotic propionyl-CoA in peripheral tissues. It was hypothesized that increasing anaplerosis in peripheral tissues would boost energy production. In the present study, we tested the potential of a triheptanoin emulsion as an intravenous nutrient. Normal rats were infused with triheptanoin intravenously or intraduodenally at up to 40% of caloric requirement. The blood concentration ratio (heptanoate/C 5-ketone bodies) was high with intravenous and low with intraduodenal triheptanoin infusion. During intravenous infusion of triheptanoin, lipolysis was stimulated but appeared compensated by fatty acid reesterification. During intraduodenal infusion of triheptanoin, lipolysis was not stimulated. Our data support the hypothesis that intravenous triheptanoin could be used to treat decompensated patients with long-chain fatty acid oxidation disorders.heptanoate; C 5-ketone bodies; fatty acid oxidation disorders; anaplerosis INHERITED FATTY ACID OXIDATION DISORDERS (FOD) include defects of the cell membrane carnitine transporter, the "carnitine cycle" (CPT I, translocase, CPT II), or the mitochondrial ␤-oxidation spiral. The most common disorders of ␤-oxidation affect very-long-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein, isolated long-chain hydroxyacylCoA dehydrogenase, and medium-and short-chain acyl-CoA dehydrogenase. Patients with long-chain FOD commonly present with recurrent hypoketotic hypoglycemia, hypertrophic or dilated cardiomyopathy, cardiac arrhythmias, rhabdomyolysis, muscle weakness, and hypotonia, (for a review, see Ref. 21). There is considerable phenotypic variation associated with nearly all of these disorders.The classical chronic treatment of long-chain FOD involves frequent feeding with a diet adjusted so as to lower long-chain fat intake from the usual 30 -35% of total kilocalories to ϳ20%, including essential fatty acids. The decrease in energy from long-chain fats is partly compensated by an increase in carbohydrates, often with cornstarch at bed time. In addition, even carbon medium-chain triglycerides (trioctanoin/tridecanoin) are added to the diet. This is because fatty acids with 8 -10 carbons enter the mitochondrion as carboxylates, which, after activation, require only those ␤-oxidative enzymes with medium-and short-chain length specificity. The dietary treatment with medium-chain triglycerides is obviously restricted to long-chain disorders and is contraindicated for medium-and short-chain deficiencies.A new strategy was recently conceived for the dietary treatment of long-chain FOD, i.e., providing about one-third of the calories as triheptanoin (22). The catabolism of heptanoate yields anaplerotic propionyl-CoA in addition to acetyl-CoA. It was hypothesized that part of the energy deficit in FOD patients results from a decrease in the concentration of citric acid cycle intermediates in muscle and heart cells. These intermediates carry the carb...

Complement per se has been shown to play an important role in demyelinating disease but controversy remains regarding the role of C3 in the development and progression of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. In this study we used C3 -/-mice to confirm previous findings that C3 is required for full development of EAE. Furthermore, C3 +/-mice (with serum C3 levels 50% that of wild type mice) developed EAE with a severity intermediate between wild type and C3 -/-mice. Importantly transfer of wild type encephalitogenic T cells to C3 -/-mice resulted in attenuated EAE. C3 -/-mice with EAE had fewer CD4 + and CD8 + T cells in the CNS and 50% fewer of these cells produced IFN-γ compared to wild type mice. When treated with anti-CD3 antibody, CD4 + T cell from wild type and C3 -/-mice had similar activation profiles as judged by IFN-γ production and CD25 and CD69 expression, indicating there is no gross or intrinsic defect in T cells from C3 -/-mice. T cells from primed C3 -/-mice proliferated comparably to that of control T cells on re-stimulation with MOG peptide. Our results confirm a requirement for C3 for maximal development of EAE and suggest that receptors for C3-derived activation fragments might be a viable therapeutic target for prevention and treatment demyelinating disease.

No abstract

We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal β-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the 13 Clabelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio < 1.0) or in both mitochondria and peroxisomes (ratio > 1.0). The goals of the present study were to test, in rat livers perfused with [1-13 C]octanoate or [3-13 C]octanoate, (i) whether peroxisomal β-oxidation contributes acetyl groups for malonylCoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, β-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal β-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1 + 2 of β-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle.

The fourth member of the beta(2)-integrin family of adhesion molecules, CD11d (alpha(D)beta(2)), is expressed on a wide variety of immune cells, however its function in autoimmune diseases, including EAE remains unknown. We induced EAE in wild-type and CD11d(-/-) C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide. The clinical course and histopathology of EAE were identical in both groups of mice throughout the disease course. There were no significant differences in the infiltration of leukocyte subsets into the central nervous system or in the production of cytokines from T cells isolated from the spleen or spinal cord from both groups of mice. Our data demonstrate that CD11d is not required for the development of EAE and, to date, is the only beta(2)-integrin molecule whose deletion does not result in attenuated disease.

Aflatoxin B 1 (AFB 1 ) is a risk factor for hepatocellular carcinoma in humans. Infant, but not adult, mice are sensitive to AFB 1 -induced liver carcinogenesis; a single dose during the neonatal period leads to hepatocellular carcinoma in adulthood. Earlier work defined the mutational spectrum in the gpt gene of gpt delta B6C3F1 mice 3 weeks after exposure to aflatoxin. In the present study, we examined the gpt spectrum 10 weeks postdosing and expanded the study to examine, at 3 and 10 weeks, the spectrum at a second locus, the red/gam genes of the mouse λEG10 transgene. Whereas the gpt locus is typically used to define local base changes, the red/gam genes, via the Spi -assay, often are used to detect more global mutations such as large deletions and rearrangements. Three weeks after dosing with AFB 1 , there was a 10-fold increase over the control in the Spi -mutant fraction (MF) in liver DNA; after 10 weeks, a further increase was observed. The MF in the gpt gene was also increased at 10 weeks compared with the MF at 3 weeks. No gender-specific differences were found in the Spi -or gpt MFs. Whereas Spi -mutations often signal large genetic changes, they did not in this specific case. The Spi -spectrum was dominated by GC to TA transversions, with one exceptionally strong hotspot at position 314. Using two genetic loci, the data show a strong preference for the induction of GC to TA mutations in mice, which is the dominant mutation seen in people exposed to aflatoxin.

Aflatoxin B1 (AFB1) is a potent mutagen and an important risk factor for hepatocellular carcinoma (HCC) in humans. Transgenic mouse strains and cells in culture have been used to detect different types of mutations caused by AFB1 and investigate the molecular determinants of their location and frequency. The AFB1 mutational spectrum in the gpt gene was markedly different in AS52 cells compared to the liver in gpt delta B6C3F1 transgenic mice. The results demonstrate the importance of metabolism, chromosomal location, transcription and selection conditions on mutational spectra.

It is well established that CD11a (a.k.a. LFA-1) functions in T cell activation by providing a co-stimulatory signal and by participating in the formation of the immunological synapse. The role of other β2-integrins in T cell activation remains unknown. Recent EAE studies have suggested a role for CD11b and CD11c in T cell biology. These findings prompted us to simultaneously stimulate naïve T cells in vitro, using anti-CD3ε and anti-CD11c antibodies. We found that treatment of T cells with anti-CD3ε and anti-CD11c (clones N418 or HL3) antibodies substantially suppressed IFN-γ production at 72 hours. T cells treated with anti-CD3ε and anti-CD11c antibodies do not produce detectable amounts of IL-4 and IL-10 at 12, 24, and 48 hours. In these same cultures, IFN-γ production was only detected at 12h post-stimulation, but at a concentration 3 logs lower than that observed with anti-CD3ε treatment alone. Interestingly, TGF-β1 production was increased 3 fold on treatment with anti-CD11c and anti-CD3ε compared to anti-CD3ε treatment alone. The suppression of IL-4, IL-10, and IFN-γ production could not be rescued by co-stimulation with anti-CD28 antibodies. Our data suggest that CD11c may be a novel negative regulator in T cell activation. Supported by NH46032 from the NIH.