Indospicine cytotoxicity and transport in human cell lines.

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera. Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat.

MHY218-induced apoptotic cell death is enhanced by the inhibition of autophagy in AGS human gastric cancer cells.

We previously reported the anticancer effects of MHY218, which is a hydroxamic acid derivative, in HCT116 human colon cancer cells. In the present study, the involvement of autophagy in the MHY218-induced apoptotic cell death of AGS human gastric cancer cells was investigated. MHY218 treatment induced growth inhibition and apoptotic cell death in a concentration- and time-dependent manner. The induction of apoptosis was confirmed by observations of decreased viability, DNA fragmentation, and an increase in late apoptosis and sub-G1 DNA, which were detected with a flow cytometric analysis. Western blot analyses showed that MHY218 treatment resulted in decreased protein levels of procaspase-8, -9, and -3; cleavage of poly(ADP-ribose) polymerase (PARP); and alterations in the ratio of Bax/Bcl-2 protein expression. Apoptosis induced by MHY218 was involved in the activation of caspase-8, -9, and -3, and it was blocked by the addition of Z-VAD­FMK, a pan-caspase inhibitor. In addition, autophagy-inducing effects of MHY218 were indicated by cytoplasmic vacuolation, the accumulation of acidic vesicular organelles, the appearance of green fluorescent protein-light-chain 3 (LC3) punctate dots, and increased levels of Beclin-1 and LC3-II protein expression. Pretreatment with the autophagy inhibitors LY294002, 3-methyladenine, chloroquine, and bafilomycin A1 enhanced the induction of apoptosis by MHY218, and this was accompanied by an increase in PARP cleavage. Taken together, these results provide new insights into the role of MHY218 as a potential antitumor agent. The combination of MHY218 with an autophagy inhibitor might be a useful candidate for the chemoprevention and/or treatment of gastric cancer.

Oral administration of the pimelic diphenylamide HDAC inhibitor HDACi 4b is unsuitable for chronic inhibition of HDAC activity in the CNS in vivo.

Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich's ataxia and Huntington's disease, based on efficacy in cell and mouse models. These studies' authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi 4b, previously described to show efficacy in vivo in the R6/2 mouse model of Huntington's disease. Based on our data reported here, we conclude that while the in vitro selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b render this compound suboptimal to investigate central Class I HDAC inhibition in vivo in mouse per oral administration. A drug administration regimen using HDACi 4b dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington's disease. We highlight physicochemical stability and metabolic issues with 4b that are likely intrinsic liabilities of the benzamide chemotype in general.

A new synthetic HDAC inhibitor, MHY218, induces apoptosis or autophagy-related cell death in tamoxifen-resistant MCF-7 breast cancer cells.

Acquired resistance to tamoxifen (Tam) is a critical problem in breast cancer therapy. Therefore, new potential strategies for Tam-resistant breast cancer are needed recently. In this study, we synthesized a novel histone deacetylase (HDAC) inhibitor, MHY218, for the development of potent inhibitors of HDAC and evaluated its biological activities by monitoring the anticancer effects in Tam-resistant MCF-7 (TAMR/MCF-7) cells via in vitro and in vivo studies. MHY218 significantly inhibited the proliferation of TAMR/MCF-7 cells in a dose-dependent manner. The total HDAC enzyme activity was significantly inhibited, corresponding with inhibition of acetylated H3 and H4 expression in TAMR/MCF-7 cells. HDAC1, 4, and 6 expression levels were decreased in response to MHY218 treatment. Cell cycle analysis indicated that MHY218 induced G2/M phase cell cycle arrest. As expected, apoptotic cell death was observed in response to MHY218 treatment. Interestingly, levels of beclin-1 and LC3-II, the markers of autophagy, were increased in TAMR/MCF-7 cells treated with MHY218. The efficacy of MHY218 was also compared with that of SAHA in vivo in a xenograft model of nude mice bearing a TAMR/MCF-7 cells. MHY218 (10 mg/kg, twice a week for 21 days) completely inhibited tumor growth and MHY218 markedly inhibited the expression of proliferative cell nuclear antigen (PCNA) in tumor tissue. These results indicate that MHY218 can induce caspase-independent autophagic cell death rather than apoptotic cell death. The MHY218-induced autophagic cell death could be a new strategy in the treatment of Tam-resistant human breast cancer.

Anticancer effects of the MHY218 novel hydroxamic acid-derived histone deacetylase inhibitor in human ovarian cancer cells.

To investigate the anticancer effects of the novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor MHY218, its efficacy was compared to that of suberoylanilide hydroxamic acid (SAHA) in human ovarian cancer cells. The anticancer effects of MHY218 on cell viability, cell cycle regulation and apoptosis were investigated. In addition, MHY218 or SAHA was administered for 28 days in a tumor carcinomatosis model with SKOV-3 cells. MHY218 significantly reduced the expression of HDAC4 and HDAC7 in SKOV-3 cells. Similarly, MHY218 also inhibited total HDAC, HDAC1, HDAC4 and HDAC7 enzyme activity in a concentration-dependent manner. The anticancer effect of MHY218 (IC50, 3.2 microM) was more potent than SAHA (IC50, 3.9 microM) in suppressing the SKOV-3 cell viability. Moreover, MHY218 markedly increased expression of p21WAF1/CIP1, which acts as a cell cycle inhibitor. Cell cycle analysis showed that the high dose (5 microM) of MHY218 significantly increased the proportion of cells in the G2/M phase. In particular, MHY218 and SAHA significantly increased the sub-G1 population and the number of TUNEL-positive apoptotic cells compared with those in the untreated control. These results were confirmed by analysis of poly-ADP ribose polymerase (PARP), where MHY218 and SAHA increased the level of an 85-kDa fragment resulting from PARP cleavage as well as caspase-3 activity. Likewise, MHY218-induced apoptosis through caspase-3 activation was confirmed by the increase in the release of cytochrome c and Bax/Bcl-2 ratio. In an in vivo tumor carcinomatosis model, the growth of transplanted SKOV-3 cells was inhibited by 71% after treatment with MHY218 (10 mg/kg), whereas SAHA (25 mg/kg) suppressed growth by 48%. These results indicate that MHY218 is a potent HDAC inhibitor that targets regulating multiple aspects of cancer cell death and might have preclinical value in ovarian cancer chemotherapy, warranting further investigation.

Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich's ataxia patients and in a mouse model.

BACKGROUND: Friedreich's ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems. CONCLUSIONS/SIGNIFICANCE: Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.

Chemical probes identify a role for histone deacetylase 3 in Friedreich's ataxia gene silencing.

We recently identified a class of pimelic diphenylamide histone deacetylase (HDAC) inhibitors that show promise as therapeutics in the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease. Here, we describe chemical approaches to identify the HDAC enzyme target of these inhibitors. Incubation of a trifunctional activity-based probe with a panel of class I and class II recombinant HDAC enzymes, followed by click chemistry addition of a fluorescent dye and gel electrophoresis, identifies HDAC3 as a unique high-affinity target of the probe. Photoaffinity labeling in a nuclear extract prepared from human lymphoblasts with the trifunctional probe, followed by biotin addition through click chemistry, streptavidin enrichment, and Western blotting also identifies HDAC3 as the preferred cellular target of the inhibitor. Additional inhibitors with different HDAC specificity profiles were synthesized, and results from transcription experiments in FRDA cells point to a unique role for HDAC3 in gene silencing in Friedreich's ataxia.

Pimelic diphenylamide 106 is a slow, tight-binding inhibitor of class I histone deacetylases.

Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC(50) values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K(i) of approximately 14 nm, 15 times lower than the K(i) for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.

Conformationally constrained diketopimelic acid analogues as inhibitors of dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and a potential antibiotic target. The enzyme catalyses the condensation of (S)-aspartate semi-aldehyde (ASA) and pyruvate to form dihydrodipicolinate. Constrained diketopimelic acid derivatives have been designed as mimics of the acyclic enzyme-bound condensation product of ASA and pyruvate. Several of the compounds are shown to be active, slow-binding inhibitors with improved inhibition of DHDPS.

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells.

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Inhibition of uptake and release of a novel mGluR agonist (L-F2CCG-I) by anion transport blockers in the rat spinal cord.

A new metabotropic glutamate receptor (mGluR) agonist, (2S,1'S,2'S)-2-(2-carboxy-3,3-difluorocyclopropyl)glycine (L-F2CCG-I), induces a priming effect on (RS)-alpha-aminopimelate in the isolated spinal cord of newborn rats. Similar to (RS)-alpha-aminopimelate, L-glutamate (30-100 microM) neither affected spinal reflexes nor the resting membrane potentials of motoneurones, but preferentially potentiated the depression of monosynaptic excitation caused by L-F2CCG-I (0.4 microM). Following L-F2CCG-I treatment (1-2 microM), L-glutamate decreased the monosynaptic spinal reflexes in a concentration dependent manner, indicating a priming' effect of L-F2CCG-I. Thus L-glutamate is completely compatible with (RS)-alpha-aminopimelate in revealing the priming effect. An anion transport blocker, 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) (100 microM), markedly inhibited both the response to (RS)-alpha-aminopimelate and the induction of the L-F2CCG-I priming effect. The data suggest that L-F2CCG-I is Cl- -dependently incorporated into certain stores, and that (RS)-alpha-aminopimelate or L-glutamate must stimulate the release of L-F2CCG-I from the storage site. There were pharmacological similarities between the quisqualate and L-F2CCG-I priming effect. The physiological significance of the quisqualate or L-F2CCG-I priming is not yet established. L-F2CCG-I would be expected to be a useful pharmacological probe for elucidating the mechanism of the priming.

Potentiation by DL-alpha-aminopimelate of the inhibitory action of a novel mGluR agonist (L-F2CCG-I) on monosynaptic excitation in the rat spinal cord.

1. Neuropharmacological actions of all the possible stereoisomers of 3',3'-difluoro-2-(carboxycyclopropyl)glycine (3',3'-difluoro-CCG) were compared with those of the corresponding 2-(carboxycyclopropyl)glycine (CCG) isomers in the isolated spinal cord of newborn rats. (2S,1'S,2'S)- and (2S,1'R,2'S)-2-(2-carboxy-3,3-difluorocyclopropyl)glycine (L-F2CCG-I and L-F2CCG-IV) were the most potent in causing depolarization, their threshold concentrations being approximately 1 microM. 2. The depolarization evoked by L-F2CCG-I (30 microM) was depressed by (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM (n=4)) to 17+/-3% of the control: this depolarizing action was not decreased by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM), and only slightly decreased by high concentrations of D-2-amino-5-phosphonopentanoic acid (D-AP5, 100 microM), suggesting that L-F2CCG-I activates mainly metabotropic glutamate receptors. 3. L-F2CCG-I preferentially depressed the monosynaptic component of the spinal reflex approximately 3 times more effectively than (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). The depressant action of L-F2CCG-I (0.2 microM-0.7 microM) on monosynaptic excitation was antagonized by (2S,1'S,2'S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG, 0.3 mM-1 mM) and (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4, 0.3 mM). 4. DL-alpha-aminopimelate (10 and 100 microM) selectively potentiated the depression of monosynaptic excitation caused by L-CCG-I (0.2 microM) and L-F2CCG-I (0.1 microM). The actions of (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) (50 nM-0.2 microM), L-2-amino-4-phosphonobutanoic acid (L-AP4) (0.3-1 microM), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) (1-7 microM) and baclofen (0.1-0.7 microM) were unaffected by DL-alpha-aminopimelate. The threshold concentration for the potentiating actions of DL-alpha-aminopimelate was 3 microM. 5. The depolarization induced by quisqualate (3 microM, 10 s application) was increased to 115+/-2% and 137+/-5% of the control values during combined application of quisqualate with either 30 microM or 100 microM DL-alpha-aminopimelate, respectively. 6. Following the application and subsequent washout of L-F2CCG-I, DL-alpha-aminopimelate (3-100 microM) decreased the amplitude of the monosynaptic component of spinal reflexes in a concentration-dependent manner, indicating a 'priming' effect of L-F2CCG-I.

Regulation of glutamate in cultures of human monocytic THP-1 and astrocytoma U-373 MG cells.

Glutamate, an excitatory neurotransmitter, is neurotoxic at high concentrations. Neuroglial cells, including astrocytes and microglia, play an important role in regulating its extracellular levels. Cultured human monocytic THP-1 cells increased their glutamate secretion following 18 and 68 h exposure to the inflammatory mediators zymosan, phorbol myristate acetate (PMA), lipopolysaccharide, interferon-gamma, tumor-necrosis factor-alpha and interleukin-1beta. Cultured astrocytoma U-373 MG cells increased their glutamate secretion following similar exposure to zymosan and PMA. DL-Alpha-aminopimelic acid, an inhibitor of the glutamate secretion system, reduced extracellular glutamate in both cell culture systems, while the high-affinity glutamate uptake inhibitors D-Aspartic acid, DL-threo-beta-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular glutamate in U-373 MG, but not THP-1 cell cultures. In co-cultures of THP-1 and U-373 MG cells, extracellular glutamate levels were increased significantly by the Alzheimer beta-amyloid peptide (1-40) and were decreased significantly by the anti-inflammatory drug dexamethasone. These data indicate that inflammatory stimuli may increase extracellular glutamate while antiinflammatory drugs decrease it.

Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors.

The classical view of fast chemical synaptic transmission is that released neurotransmitter acts locally on postsynaptic receptors and is cleared from the synaptic cleft within a few milliseconds by diffusion and by specific reuptake mechanisms. This rapid clearance restricts the spread of neurotransmitter and, combined with the low affinities of many ionotropic receptors, ensures that synaptic transmission occurs in a point-to-point fashion. We now show, however, that when transmitter release is enhanced at hippocampal mossy fibre synapses, the concentration of glutamate increases and its clearance is delayed; this allows it to spread away from the synapse and to activate presynaptic inhibitory metabotropic glutamate receptors (mGluRs). At normal levels of glutamate release during low-frequency activity, these presynaptic receptors are not activated. When glutamate concentration is increased by higher-frequency activity or by blocking glutamate uptake, however, these receptors become activated, leading to a rapid inhibition of transmitter release. This effect may be related to the long-term depression of mossy fibre synaptic responses that has recently been shown after prolonged activation of presynaptic mGluRs (refs 2, 3). The use-dependent activation of presynaptic mGluRs that we describe here thus represents a negative feedback mechanism for controlling the strength of synaptic transmission.

Immunostimulating lipopeptide, LtriP (RP 56142): comparison of the effect on hepatic cytochrome P 450 modulation and radioprotection in male and female of three mouse strains.

The sex-dependent effect of lauroyl-L-Ala-D-gamma-Glu-L,L-A2pmNH2 (LtriP, RP 56142) on hepatic microsomal cytochromes P 450 (cyt P 450) was studied in three mouse strains NMRI, C3H/OuJ and C3H/HeJ. In NMRI and C3H/OuJ, strains which are responsive to bacterial lipopolysaccharides (LPS-responsive), regardless of the sex of the mouse, significant decrease in the amount of cyt P 450 was observed after LtriP treatment, with a concomitant reduction in ethoxyresorufin-O-deethylase (cyt P 450 1A-dependent) and 7-ethoxycoumarin-O-deethylase activities. This was not seen in C3H/HeJ (LPS-hyporesponsive) mice. These effects may be related to LtriP-dependent cytokine induction, since neither LtriP nor LPS stimulated interleukin-1 (IL-1) secretion by C3H/HeJ macrophages. 11- and 12-hydroxylations (11- and 12-OH) of lauric acid were compared in C3H/OuJ and C3H/HeJ mice. LtriP depressed the total enzymatic conversion of lauric acid in the two strains without modification of the 11/12-OH ratio for C3H/OuJ or male C3H/HeJ mice. However, in females C3H/HeJ mice this decrease was particularly significant and concerned especially the 12-OH activity (a marker of cyt P450 4A family). Although males of the three strains were more sensitive to irradiation than females, LtriP exerted a sex-independent radioprotection on NMRI and C3H/OuJ mice. Its radioprotective effect was illustrated by the preservation of all the enzymatic activities studied in treated NMRI mice, contrary to irradiated control animals. In contrast, for the C3H/HeJ strain, males were not protected by LtriP treatment and, furthermore, females showed a marked sensitization to irradiation. The effects in CH3/HeJ strain implicate LtriP in the control of cyt P 450 induction and of sensitivity to irradiation independently of IL-1 induction.

Radioprotective effects of the immunostimulating lauroylpeptide LtriP (RP 56142).

The lipopeptide lauroyl-L-Ala-gamma-D-Glu-L,L-A2pm (LtriP) increased the resistance of mice to the lethal effect of gamma-ray irradiation. The radioprotective effect was dependent on the doses of LtriP and of radiation. Maximum survival was observed when the lipopeptide was injected on two successive days before irradiation. This activity seems to be related to immunostimulating functions, since the non-immunostimulating analog lauroyl-L-Ala-gamma-D-Glu-D,D-A2pm-Gly, containing D,D-diaminopimelic acid, was not radioprotective. The protective activity might result from an induction of cytokines, such as IL-1, TNF and M-CSF, since LtriP induced the mRNA expression and the secretion of these immunomodulators.

A comparative study on the activation of J-774 macrophage-like cells by gamma-interferon, 1,25-dihydroxyvitamin D3 and lipopeptide RP-56142: ability to kill intracellularly multiplying Mycobacterium tuberculosis and Mycobacterium avium.

The J-774 macrophage-like cell line has been established as a model for intracellular multiplication of pathogenic mycobacteria, permitting assessment of the intracellular bactericidal action of the macrophages after addition of both the drugs and immunomodulators. In this study, the action of immunomodulators was investigated. Significant morphological changes were demonstrated under the optical and scanning electron microscope (SEM), and the degree of macrophage activation was also measured by acid phosphatase (AcPase) cytochemistry, release of free oxygen radicals and by their ability to hinder the intracellular multiplication of virulent strains of Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av). For This purpose, the macrophages were left to multiply during 3 days in the presence of 50 U/ml of recombinant murine gamma-interferon (INF), 4 micrograms/ml of 1,25 dihydroxyvitamin D3 (D3) and 50 micrograms/ml of lipopeptide RP-56142 (RP) added separately or in various possible combinations, and these "activated" cells were then challenged with viable bacteria. Parallel controls included bacterial multiplication in nonactivated macrophages and also extracellularly but under the same experimental conditions as in the macrophage experiments. Transmission electron microscopy (TEM) using the AcPase marker to localize phagosome-lysosome fusion (PLF) in infected cells was also performed. Although all the immunomodulators used significantly changed the morphology of treated cells and increased the % of AcPase-positive cells, none had any effect on the release of oxygen radicals. On the other hand, guinea-pig alveolar macrophages which served as a parallel positive control, were activated by INF and D3 (but not RP) to release superoxide anions. Our data suggest that differential killing mechanisms for intracellular M.tb and M.av may exist. The results obtained also showed that established mycobactericidal mechanisms of the host could not solely account for the antimycobacterial effects observed. Consequently, mechanisms not yet revealed may account for some of the antimycobacterial effects observed.

The "survival hormones": azelaic and pimelic acids, suppress the stress elicited by isolation conditions on the steroids and phospholipids of adult worker honeybees.

The kinetics of abdomen, haemolymph and thoracic-muscle steroid and phospholipid concentrations have been determined in adult worker-bees kept for 0 to 12 hrs starving in darkness, either grouped by 8 (controls) or strictly isolated, or isolated in presence of a piece of cotton impregnated with 1 microgram azelaic acid and 1 microgram pimelic acid, the so-called "survivones" which restore the lifespan of isolated bees. The dynamics of both steroids and phospholipids strongly deviates in isolated bees relatively to controls. The introduction of survivones completely restored the variations of haemolymph steroids of haemolymph and thorax phospholipids of isolated bees to exactly similar features as in controls. The action of the lipoic hormones "survivones" thus involves the participation of lipid metabolism.

In vivo effects of immunostimulating lipopeptides on mouse liver microsomal cytochromes P-450 and on paracetamol-induced toxicity.

Immunomodulating lipopeptides lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2-Gly (RP 44.102) and lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2 (RP 56.142) were found to protect mice against the hepatotoxicity of paracetamol, which is due to cytochrome P-450 dependent formation of toxic metabolites and radicals. In fact they decreased the amount of hepatic microsomal cytochrome P-450, and the level of CCl4-induced lipid peroxidation. In contrast lauroyl-L-Ala-gamma-D-Glu-DD-A2pmNH2 (RP 53.204), which only differs by the configuration of the two chiral carbons of A2pm (diaminopimelic acid) and is not an immunomodulating agent, failed to protect against poisoning by paracetamol and had no effect on the level of hepatic cytochrome P-450 or the microsomal CCl4-induced lipid peroxidation. This provides a clear connection between the immunostimulating properties of a compound and its effects on xenobiotic biotransformations.

Immunomodulating and antitumor activities of a synthetic lauroyltripeptide (RP 56 142).

Lauroyltripeptide (RP 56 142) (N2-[N-(N-lauroyl-L-alanyl)-gamma-D-glutamyl]-L,L-2,6-diaminopimelami c acid) was shown in murine models to activate several immune mechanisms involved in host defense against tumors. RP 56 142 induced macrophage activation and enhanced cytotoxicity of natural killer cells in spleen, blood, and liver. These activities correlated with prophylactic and therapeutic effects of RP 56 142 on artificial liver metastases of M5076 histiocytosarcoma. RP 56 142 alone did not inhibit spontaneous liver metastases of M5076 sarcoma; however, in combination with surgery or suboptimal doses of cisplatin, the compound exerted synergistic antimetastatic effects in the same model. These findings suggest that RP 56 142 could be used in cancer patients as an immunotherapeutic agent in combination with surgery, radiotherapy and/or conventional chemotherapy.