key: cord-0010430-go8cmgpo authors: Masihi, K.Noel; Kröger, Hans; Lange, Werner; Chedid, Louis title: Muramyl peptides confer hepatoprotection against murine viral hepatitis date: 2006-03-15 journal: Int J Immunopharmacol DOI: 10.1016/0192-0561(89)90109-4 sha: de41c31117f8648496781c8e271bbdef030282c5 doc_id: 10430 cord_uid: go8cmgpo The hepatoprotection induced by synthetic muramyl peptides was investigated using a model of lethal murine mouse hepatitis MHV-3 virus infection. MDP and a nonpyrogenic analog, Murametide, inhibited the steep elevation of serum transaminases induced by MHV-3 irrespective of whether the immunomodulators were administered before or after the infection. A significant proportion of MDP or Murametide-treated animals, in contrast to controls, survived the MHV-3 infection. The histopathological examination of the liver revealed marked necrosis of the hepatic parenchymal cells and infiltration of the inflammatory cells in controls but not in MDP-treated animals. due to hepatitis viruses and Mycobacterium tuberculosis are endemic in Southeast Asia and amongst refugees from that region. A study of immune responses to both infections in Indochinese refugees showed a significant association in the reactivity to purified tuberculin protein derivative (PPD) and the presence of hepatitis Be antigen (McGlynn, Lustbader & London, 1985) . Persons having a positive PPD skin test tended to be HBeAg negative suggesting that Mycobacterium tuberculosis infection may affect the outcome of viral hepatitis. Mouse hepatitis virus type 3 (MHV-3) belongs to the group of coronaviruses. Parenteral administration of MHV-3 to susceptible mice causes fatal hepatic necrosis culminating in death within a matter of few days. Hepatic necrosis liberates several enzymes that are usually present intracellularly within the liver into the blood circulation. The elevation of serum transaminases is an important biochemical manifestation of human and murine viral hepatitis and can be used diagnostically as a marker of liver damage. Measurements of serum alanine aminotransferase in Indochinese refugees showed normal transaminase levels in PPD-positive persons compared with PPD-negative individuals (McGlynn et al., 1985) . It would be of considerable interest if the protection against virus-mediated liver damage associated with tuberculosis infection could be duplicated by immunomodulators of mycobacterial origin. Mycobacteria contain on their cell walls, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), a small glycopeptide which represents the minimal structure essential for bacterial adjuvanticity. Synthetic MDP and its analogs are endowed with multifarious properties including the stimulation of nonspecific resistance against viral pathogens (Chedid, 1988) . Already in 1980 it was reported that MDP, in combination with trehalose dimycolate, could induce resistance against influenza virus infection (Masihi, Brehmer, Lange & Ribi, 1980 , 1983 . Several MDP analogs like 6-O-acyl, ubiquinone (Masihi, Brehmer, Azuma, Lange & Miiller, 1984a) , seryl and aminobutyryl (Masihi, Brehmer, Lange, Ribi & Schwartzman, 1984b ) conferred long-term resistance against aerogenic influenza virus in combination with trehalose dimycolate. Subsequently, MDP and analogs were shown to induce protection against various strains of influenza (Dietrich, Hochkeppel & Lukas, 1986) , herpes simplex virus (Dietrich et al., 1986; Koff, Showalter, Hampar & Fidler, 1985) , vaccinia virus (Ikeda, Negishi & Nishimura, 1985) , sendai virus (Yamamura, Ishihara, Hamada, Yamamoto & Azuma, 1986) and in combination with an interferon-inducer, against Semliki Forest virus (George, Jain, Gupta & Anand, 1986) . MDP can also protect rat hepatocytes against the in vitro toxic effects of acrolein, chloroform and carbon tetrachloride and decrease serum transaminases (Farghali, Machkovfi, Kameinikov~, Jank~ & Mgek, 1984) . In the present study, the effect of MDP and a nonpyrogenic analog, Murametide, on biochemical and other parameters was investigated using a model of lethal routine MHV-3 virus infection. K, NOEL MASIHI el al. Nuclei from liver cells were isolated using the technique previously described (Blobel & Potter, 1966) . The ADPR transferase activity was measured in the presence of DNAse (Kidwell & Burdette, 1974) . Five to six-week old NMRI mice were purchased from Zentralinstitut fur Versuchstiere, Hannover, F.R.G. For histological studies, mice were given saline or 1 mg of MDP 1 h, 24 h, and 48 h after the MHV-3 infection. Livers were removed on day 3 after the viral infection and fixed in 10% formalin. Histological sections were stained with hematoxylin and eosin. MDP and its analog, Murametide, were synthesized by P. Lefrancier, Institut Choay, Paris, France (Lefrancier, Derrien, Jamet, Choay, Lederer, Audibert, Parant, Parant & Chedid, 1982) . Desired amounts of muramyl peptides were dissolved in pyrogen-free physiological saline. All substances were administered by the intraperitoneal (i.p.) route. MHV-3 was passaged i.p. in young NMRI mice. Livers were removed 3 days after the infection and homogenized in 3 ml of medium/liver using a tissue grinder. Supernatant obtained after centrifugation was diluted and further passaged in mouse L-cells. Marked cytopathic effects could be observed in tissue cultures of L-cells using supernatant dilutions of 10 ~ to 10 3. Three-day old cultures infected with 1:100 dilution of the supernate were frozen and thawed three times. The supernate obtained after centrifugation was stored in liquid nitrogen. Various dilutions were injected i.p. into mice for the determination of lethal dose. Half a milliliter of 1:750 dilution injected i.p. consistently gave LD,o o in 6-week old NMRI mice and was used for all experiments. The GOT and GPT enzyme activities present in nonhemolytic sera collected at different intervals were determined using the standard method (Bergmeyer, 1974) . Reagents for the test were purchased from Boehringer Mannheim, F.R.G. The enzyme activity is presented in mU/ml. Thirty animals were administered a single dose of 300/~g of MDP by the i.p. route. The effect of MDP itself on liver enzymes was determined in a group of 10 pretreated mice. Twenty mice from the MDP-pretreated group and 20 normal mice injected with saline were infected i.p. with MHV-3 24 h after the MDP administration. Sera were collected everyday for four days. The results of enzyme activities are presented in Fig. 1 . Serum GOT and GPT activities were not induced after the administration of MDP alone. In contrast, the MHV-3 infection induced increased GOT and GPT activities on day 3 and the enzyme levels were elevated even further on day 4, a time period when many of the animals were dying. Pretreatment with MDP greatly reduced the rise in GOT and GPT levels observed after the MHV-3 infection. Sixteen mice were given 100/~g of MDP 1 h, 24 h, 48 h and 72 h after the MHV-3 infection. Another group of 16 mice was similarly treated with MDP but did not receive the viral infection. A third group of 16 mice received MHV-3 infection only. Sera were collected every day for 4 days after the infection. Results presented in Fig. 2 show that the multiple administration of MDP alone did not affect the liver transaminases. MDP given after the MHV-3 infection could inhibit the induction of serum GOT and GPT (Fig. 2) . The effect of MDP or its potent nonpyrogenic analog murametide was investigated at a higher dosage. Twenty-eight animals each were given 1 mg of MDP or 1 mg of Murametide 1 h, 24 h, and 48 h after the MHV-3 infection. Eight animals were each the virus controls (Fig. 3) . Nonpyrogenic Murametide was active and inhibited the GOT and the GPT induction (Fig. 3) . Neither MDP alone nor Murametide alone induced liver enzyme activities at this dosage. The activity of ADPR transferase in the nuclei of liver cells increases after infection with MHV-3 (Table 1 ). In contrast, treatment with 1 mg of MDP 1 h after infection resulted in a very small reduction of ADPR transferase activity. Additional treatments with MDP at 22 h and 42 h post-infection did not after the ADPR transferase activity at 24 h or 48 h. One milligram of MDP or Murametide administered three times after the MHV-3 infection conferred significant protection to respectively 42% and 40°7o of treated mice compared to only 7% survivors in the control group (