Jun-ichi Yamamura

THERAPEUTIC BASIS OF GLYCYRRHIZIN ON CHRONIC HEPATITIS B

Glycyrrhizin, a major component of a herb (licorice), has been intravenously used for the treatment of chronic hepatitis B in Japan and improves liver function with occasional complete recovery from hepatitis. This substance modifies the intracellular transport and suppresses sialylation of hepatitis B virus (HBV) surface antigen (HBsAg) in vitro. This study was designed to clarify the pharmacological basis for its effectiveness. The structure-bioactivity relationship of glycyrrhizin, glycyrrhetic acid 3-O-monoglucuronide and glycyrrhetic acid was determined, and glycyrrhetic acid was found to be the most active of them. The amounts of three substances bound to the liver were evaluated in guinea pigs after intravenous administration of glycyrrhizin. Glycyrrhizin and glycyrrhetic acid 3-O-monoglucuronide were detected at concentrations of 31.8-1.3 micrograms/g of liver, but glycyrrhetic acid was not detected. When glycyrrhizin attained these concentrations in the cellular fraction of the PLC/PRF/5 cell culture, it suppressed the secretion of HBsAg as reported previously. These results indicated that glycyrrhizin administered intravenously might bind to hepatocytes at the concentration at which glycyrrhizin could modify the expression of HBV-related antigens on the hepatocytes and suppress sialylation of HBsAg.

Antipyretic activity of cinnamyl derivatives and related compounds in influenza virus-infected mice

Kakkon-to is composed of seven medicinal herbs and exhibited novel antipyretic activity by suppressing interleukin-1alpha production responsive to interferon in a murine intranasal influenza virus infection model. Using this model, antipyretic compounds with such novel biological activities were characterized from the herbs. The organic solvent-extractable fractions of Cinnamomum cassia among the herbs showed antipyretic activity. We selected six antipyretic compounds from 48 cinnamyl derivatives and related compounds that may be mainly involved in the fractions. Their antipyretic activity was significantly correlated with interleukin-1alpha regulatory activity. Four of them suppressed interleukin-1alpha production to a basal level and showed different mode of antipyretic action from that of aspirin in interleukin-1alpha-injected mice. Structure-bioactivity relationship of the four suggested that an ester bond played an important role for both antipyretic and interleukin-1alpha regulatory activities. These compounds may be useful in analyzing interleukin-1alpha-producing cells in fever production and the mechanism of defervescence by suppressing interferon-induced interleukin-1alpha production.

Emergence of resistance to acyclovir and penciclovir in varicella-zoster virus and genetic analysis of acyclovir-resistant variants.

We have characterized the differential actions of acyclovir and penciclovir against varicella-zoster virus (VZV) in cell culture by comparing the frequency of appearance of resistant viruses followed by their characterization. Cells were infected with cell-free virus and the cultures were successively treated with increasing concentrations of acyclovir or penciclovir. Drug-resistant viruses were selected in the presence of 6 microg/ml of acyclovir or penciclovir. The emergence frequency of resistant viruses was significantly higher following acyclovir exposure than following penciclovir exposure (Fishers exact test, P<0.0001), possibly reflecting virus growth differences under these experimental conditions. Based on antiviral drug susceptibility and thymidine kinase (TK) activity assays, 11 acyclovir-resistant variants from seven experiments using three virus strains (Kawaguchi strain, Oka varicella vaccine strain and a clinical isolate from a zoster patient) were found to be TK-deficient. Sequence analysis of TK-deficient variants of the Kawaguchi strain revealed deletions that caused frameshifts, resulting in premature termination in the TK gene.

Long-term gene expression in the anterior horn motor neurons after intramuscular inoculation of a live herpes simplex virus vector

To clarify the feasibility of the herpes simplex virus (HSV) vector in expressing the foreign gene in the motor neuron, we inoculated a live attenuated HSV expressing β-galactosidase (β-gal) activity under a latency-associated transcript promoter in the right gastrocnemius muscle of rats. Expression of β-gal activity was observed 5 days after inoculation in the bilateral anterior horn cells of the spinal cord that innervates the inoculation muscle. However, the spread of β-gal activity was not observed in the inoculation muscle. Without significant pathological changes, the spread of β-gal-expressing neurons was observed in the lumbosacral spinal cord until 14 days after inoculation with staining concentrated in the anterior horn cells. Ninety percent of the anterior horn motor neurons expressed β-gal activity with expression continuing to at least 182 days after inoculation. Thus β-gal activity was expressed in the bilateral anterior horn cells at the lumbosacral spinal cord that innervates the inoculated muscle for a long time, possibly a life-long period. This indicates that this recombinant HSV vector system to motor neurons may further improve the understanding and treatment of neurological diseases in motor neurons of the spinal cord.

Vasculitis induced by immunization with Bacillus Calmette‐Guérin followed by atypical mycobacterium antigen: a new mouse model for Kawasaki disease

Abstract Kawasaki disease causes systemic vasculitis. The development of skin lesions at the vaccination site with Bacillus Calmette‐Guérin (BCG) is an important diagnostic symptom. We hypothesized that infection with ubiquitous microorganisms immunogenically related to BCG might induce an immunopathologic reaction leading to the development of Kawasaki disease. Mice were first inoculated with BCG, and then secondarily inoculated 4 weeks later with crude extract from Mycobacterium intracellulare (cMI), an abundant atypical mycobacterium. Animals inoculated with BCG followed by cMI developed coronary arteritis with infiltration of inflammatory cells, whereas control animals inoculated with only cMI or BCG did not, suggesting that the immune response to the mycobacteria induced autoimmunity to the vascular wall. Intravenous injection with antibodies to peroxiredoxin II, a modulator of vascular remodeling and a suggested target for autoimmune vasculitis, also resulted in coronary arteritis, but only after prior inoculation with BCG. Tumor necrosis factor‐α, MCP1 and interferon‐γ production were significantly higher in the animals inoculated with BCG than in the control groups (P<0.05). BCG immunization was required for the development of coronary arteritis, suggesting that these cytokines might play important roles. The results indicate that BCG induces primary autoimmunity and stimulates cytokine induction, and that atypical mycobacterial infection boosts the autoimmunity resulting in coronary arteritis.

Immune response to varicella-zoster virus glycoproteins in guinea pigs infected with Oka varicella vaccine.

A varicella skin test antigen has been developed based on the induction of delayed-type hypersensitivity to varicella-zoster virus (VZV), and has been used to evaluate the immune status to VZV. The authors have purified gB, gE:gI and gH:gL and examined their cutaneous reactivity in guinea pigs infected with Oka varicella vaccine. The cutaneous reaction to each glycoprotein was observed and the maturation process of cutaneous reaction was examined in infected guinea pigs. Cutaneous reaction to gH:gL, a major target of virus-neutralizing antibody, appeared first on day 3 among three glycoprotein complexes and the reaction to gE:gI, the most abundant glycoprotein, became strongest three weeks after infection. The earliest recognition of gH:gL may contribute to minimizing the spread of viral infection. Thus, the skin test may be a suitable marker to assess the cell-mediated immunity in varicella, including vaccine recipients and zoster, in relation to the immune status of glycoproteins.

Functions of purified gB, gE:gI, and gH:gL, and their sialyl residues in varicella-zoster virus infection

SummeryVaricella-zoster virus glycoproteins were purified by using monoclonal antibodies and analyzed for their effects on cell-free virus infection. Preinfection treatment of cells with gH:gL reduced the infection efficiency and increased the number of unadsorbed virus. Postinfection treatment of cells with gB increased the infection efficiency, but that with gE:gI reduced it. Treatment of gE:gI and gH:gL with neuraminidase (NA) abolished their inhibitory activity and the plaque formation was enhanced by NA treatment of glycoproteins and cells. Glycoproteins exhibited their diverse activities despite their common role in viral penetration, and sialyl residues were responsible for their function in cell-free virus infection.

Superiority of varicella skin test antigen over purified varicella-zoster virus glycoproteins in monitoring booster response to Oka varicella vaccine

Varicella skin test antigen has been developed based on the induction of delayed-type hypersensitivity (DTH) to varicella-zoster virus (VZV). The booster immune response to Oka varicella vaccine was assessed by cutaneous reactivity to purified VZV glycoprotein complexes, gB, gE:gI, gH:gL, and varicella skin test antigen. Skin tests with these antigens significantly augmented antibody production to glycoproteins and VZV antigen resulting in no further augmentation by the subsequent vaccination. All glycoprotein complexes induced the cutaneous reaction similarly to varicella skin test antigen. Cutaneous reaction to glycoproteins and varicella skin test antigen was boosted after vaccination. However, the magnitude of cutaneous reaction to each glycoprotein complex before and after vaccination was rich in variety. These results indicated that skin test with varicella skin test antigen is a more suitable indicator in monitoring cell-mediated immunity to VZV than that using purified glycoproteins.

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real‐time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real‐time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR‐negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real‐time PCR is suitable for rapid diagnosis as a first screening test to determine first‐line antibacterial agents to be used against these infectious diseases.

A live non-neurovirulent herpes simplex virus vector expresses β-galactosidase in the nervous system of the Wistar and Sprague-Dawley strain rat for a prolonged period

We have constructed a live non-neurovirulent herpes simplex virus vector expressing beta-galactosidase under the control of the latency associated transcript promoter without inducing inflammation. Pathogenicity of the recombinant virus (betaH1) was not observed in the cutaneous, intravenous and intracerebral infection in mice. When betaH1 was inoculated at the caudate putamen of rats, beta-galactosidase activity was observed in neurons at the inoculation site and its projecting frontal cortex. Expression of beta-galactosidase was observed in the neurons of the innervating dorsal root ganglia 45 days after inoculation of betaH1 into the hind paws of the rats. Neither inflammation nor tissue destruction was observed in both neural tissues in this study. Thus this non-neurovirulent recombinant virus is a suitable vector for expressing the foreign genes in the nervous system for the prolonged period.