Antibody response to third and fourth BNT162b2 mRNA booster vaccinations in healthcare workers in Tokyo, Japan.

BACKGROUND: Booster vaccinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being promoted worldwide to counter the coronavirus disease 2019 (COVID-19) pandemic. In this study, we analyzed the longitudinal effect of the third BNT162b2 mRNA vaccination on antibody responses in healthcare workers. Additionally, antibody responses induced by the fourth vaccination were analyzed. METHODS: The levels of anti-spike (S) IgG and neutralizing antibody against SARS-CoV-2 were measured at 7 months after the second vaccination (n = 1138), and at 4 (n = 701) and 7 (n = 417) months after the third vaccination using an iFlash 3000 chemiluminescence immunoassay analyzer. Among the 417 participants surveyed at 7 months after the third vaccination, 40 had received the fourth vaccination. A multiple linear regression analysis was performed to clarify which factors were associated with the anti-S IgG and neutralizing antibody. Variables assessed included sex, age, number of days after the second or third vaccination, diagnostic history of COVID-19, and anti-nucleocapsid (N) IgG level. RESULTS: At 7 months after the third vaccination, antibody responses were significantly higher than those at the same time after the second vaccination. Unlike the second vaccination, age had no effect on the antibody responses induced by the third vaccination. Furthermore, the fourth vaccination resulted in a further increase in antibody responses. The multiple linear regression analysis identified anti-N IgG level, presumably associated with infection, as a factor associated with antibody responses. CONCLUSIONS: Our findings showed that BNT162b2 booster vaccinations increased and sustained the antibody responses against SARS-CoV-2.

Serologic Survey of IgG Against SARS-CoV-2 Among Hospital Visitors Without a History of SARS-CoV-2 Infection in Tokyo, 2020-2021.

BACKGROUND: Tokyo, the capital of Japan, is a densely populated city of >13 million people, so the population is at high risk of epidemic severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. A serologic survey of anti-SARS-CoV-2 IgG would provide valuable data for assessing the city's SARS-CoV-2 infection status. Therefore, this cross-sectional study estimated the anti-SARS-CoV-2 IgG seroprevalence in Tokyo. METHODS: Leftover serum of 23,234 hospital visitors was tested for antibodies against SARS-CoV-2 using an iFlash 3000 chemiluminescence immunoassay analyzer (Shenzhen YHLO Biotech, Shenzhen, China) with an iFlash-SARS-CoV-2 IgG kit (YHLO) and iFlash-SARS-CoV-2 IgG-S1 kit (YHLO). Serum samples with a positive result (≥10 AU/mL) in either of these assays were considered seropositive for anti-SARS-CoV-2 IgG. Participants were randomly selected from patients visiting 14 Tokyo hospitals between September 1, 2020 and March 31, 2021. No participants were diagnosed with coronavirus disease 2019 (COVID-19), and none exhibited COVID-19-related symptoms at the time of blood collection. RESULTS: The overall anti-SARS-CoV-2 IgG seroprevalence among all participants was 1.83% (95% confidence interval [CI], 1.66-2.01%). The seroprevalence in March 2021, the most recent month of this study, was 2.70% (95% CI, 2.16-3.34%). After adjusting for population age, sex, and region, the estimated seroprevalence in Tokyo was 3.40%, indicating that 470,778 individuals had a history of SARS-CoV-2 infection. CONCLUSIONS: The estimated number of individuals in Tokyo with a history of SARS-CoV-2 infection was 3.9-fold higher than the number of confirmed cases. Our study enhances understanding of the SARS-CoV-2 epidemic in Tokyo.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 µM), DHA (100 µM), or siamycin I (2.5 µM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis.

Application of spatial transcriptomics analysis using the Visium system for the mouse nasal cavity after intranasal vaccination.

Intranasal vaccines that elicit mucosal immunity are deemed effective against respiratory tract infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but their ability to induce humoral immunity characterized by immunoglobulin A (IgA) and IgG production is low. It has been reported that vaccination with a mixture of a viscous base carboxyvinyl polymer (CVP) and viral antigens induced robust systemic and mucosal immune responses. In this study, we analyzed the behavior of immunocompetent cells in the nasal cavity over time by spatial transcriptome profiling induced immediately after antigen vaccination using CVP. We established a method for performing spatial transcriptomics using the Visium system in the mouse nasal cavity and analyzed gene expression profiles within the nasal cavity after intranasal vaccination. Glycoprotein 2 (Gp2)-, SRY-box transcription factor 8 (Sox8)-, or Spi-B transcription factor (Spib)-expressing cells were increased in the nasal passage (NP) region at 3-6 hr after SARS-CoV-2 spike protein and CVP (S-CVP) vaccination. The results suggested that microfold (M) cells are activated within a short period of time (3-6 hr). Subsequent cluster analysis of cells in the nasal cavity showed an increase in Cluster 9 at 3-6 hr after intranasal vaccination with the S-CVP. We found that Il6 in Cluster 9 had the highest log2 fold values within the NP at 3-6 hr. A search for gene expression patterns similar to that of Il6 revealed that the log2 fold values of Edn2, Ccl20, and Hk2 also increased in the nasal cavity after 3-6 hr. The results showed that the early response of immune cells occurred immediately after intranasal vaccination. In this study, we identified changes in gene expression that contribute to the activation of M cells and immunocompetent cells after intranasal vaccination of mice with antigen-CVP using a time-series analysis of spatial transcriptomics data. The results facilitated the identification of the cell types that are activated during the initial induction of nasal mucosal immunity.

Molecular insights of a CBP/ß-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

B cell receptor repertoire analysis from autopsy samples of COVID-19 patients.

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.

Inhibition of CBP/ß-catenin signaling ameliorated fibrosis in cholestatic liver disease.

Chronic cholestatic liver diseases are characterized by injury of the bile ducts and hepatocytes caused by accumulated bile acids (BAs) and inflammation. Wnt/ß-catenin signaling is implicated in organ fibrosis; however, its role in cholestatic liver fibrosis remains unclear. Therefore, we explored the effect of a selective cAMP response element-binding protein-binding protein (CBP)/ß-catenin inhibitor, PRI-724, on murine cholestatic liver fibrosis. PRI-724 suppressed liver fibrosis induced by multidrug resistance protein 2 knockout (KO), bile duct ligation, or a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet; it also suppressed BA synthesis and macrophage infiltration. The expression of early growth response-1 (Egr-1), which plays a key role in BA synthesis, was increased in the hepatocytes of patients with cholestatic liver disease. PRI-724 inhibited Egr-1 expression induced by cholestasis, and adenoviral shEgr-1-mediated Egr-1 knockdown suppressed BA synthesis and fibrosis in DDC diet-fed mice, suggesting that PRI-724 exerts its effects, at least in part, by suppressing Egr-1 expression in hepatocytes. Hepatocyte-specific CBP KO in mice suppressed BA synthesis, liver injury, and fibrosis, whereas hepatocyte-specific KO of P300, a CBP homolog, exacerbated DDC-induced fibrosis. Intrahepatic Egr-1 expression was also decreased in hepatocyte-specific CBP-KO mice and increased in P300-KO mice, indicating that Egr-1 is located downstream of CBP/ß-catenin signaling. Conclusion: PRI-724 inhibits cholestatic liver injury and fibrosis by inhibiting BA synthesis in hepatocytes. These results highlight the therapeutic effect of CBP/ß-catenin inhibition in cholestatic liver diseases.

An attenuated vaccinia vaccine encoding the severe acute respiratory syndrome coronavirus-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human angiotensin-converting enzyme 2 transgenic mice from severe acute respiratory syndrome coronavirus-2 and its variants.

As long as the coronavirus disease-2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently required. We have developed a vaccine consisting of the attenuated vaccinia virus Dairen-I (DIs) strain platform carrying the SARS-CoV-2 S gene (rDIs-S). rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin-converting enzyme 2 (hACE2) transgenic mice, and the mouse model showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA.1 variant (TY38-873). Using a tandem mass tag (TMT)-based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that vaccination with rDIs-S maintains S protein-specific antibody titers for at least 6 months after a first vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current variants such as Omicron BA.1 and possibly future variants.

Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.

Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models.IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.

Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models.

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

Up-regulation of hepatic heme oxygenase-1 expression by locally induced interleukin-6 in rats administered carbon tetrachloride intraperitoneally.

We previously reported that interleukin-6 (IL-6) was locally produced in the early period after intraperitoneal (i.p.) or subcutaneous carbon tetrachloride (CCl4) administration, but not after oral (p.o.) administration. In the present study, we focused on the up-regulation of stress-inducible proteins induced by IL-6 after i.p. CCl4 administration. The expression of heme oxygenase-1 (HO-1) (EC 1.14.99.3) mRNA and protein were induced more in rats administered CCl4 via the i.p. route, compared with the p.o. route; however, expression of heat shock protein (HSP) 72 and HSP90 mRNA were increased to similar extents in both experimental groups. The induction of HO-1 mRNA and protein after i.p. CCl4 administration were significantly reduced after pretreatment with anti-rat IL-6 antibody. Activation of the signal transducer and activator of transcription factor 3 (STAT3), which promotes HO-1 expression, peaked together with plasma levels of IL-6 after i.p. CCl4 administration, suggesting that hepatic HO-1 expression was increased by IL-6 via the Janus kinase/STAT3 pathway. The present data indicate that hepatic HO-1 is up-regulated by endogenously produced IL-6, in addition to its up-regulation by heme derived from cytochrome P450 which has already been reported in rats administered i.p. CCl4. The up-regulation of hepatic HO-1 expression may reduce the tissue injury to livers caused by CCl4.

Early induced, high-level interleukin-6 expression in the rat peritoneal cavity into which a hepatotoxicant carbon tetrachloride was administered.

IL-6 induction depending on the mode of carbon tetrachloride (CCl4) administration was investigated in rats. After the intraperitoneal (i.p.) administration of CCl4 in 50% corn oil at 1.0 ml/kg body weight, IL-6 level markedly increased in plasma and peaked at 4h. TNF-alpha and IL-1beta levels gradually increased, reaching the maximum at 24h. IL-10 level transiently peaked at 4h and then decreased, but later further increased, reaching the second peak at 24h. Plasma alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities peaked at 24h. As the vehicle-to-CCl4 ratio increased, the level of IL-6 decreased and the activities of ALT and SDH increased. After oral CCl4 administration, IL-6 was not significantly detected. IL-6 level in peritoneal exudate fluid (PEF) increased simultaneously with plasma IL-6 level after i.p. CCl4 administration, but the total amount of PEF IL-6 was 37-fold as much as that of plasma IL-6, in contrast to the result that the total amount of plasma IL-6 was 19-fold as much as that of PEF IL-6 after i.p. lipopolysaccharide administration. These results suggest that i.p. administration of CCl4 dissolved in a small amount of vehicle selectively induces a high production of IL-6 in the peritoneal cavity early after the administration. Since IL-6 is a protective cytokine against hepatotoxicity, its induction should be taken into consideration during analysis of data obtained using the CCl4-induced liver injury model.

Selective inhibitor of Wnt/ß-catenin/CBP signaling ameliorates hepatitis C virus-induced liver fibrosis in mouse model.

Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/ß-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a ß-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of ß-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.

Trehangelins A, B and C, novel photo-oxidative hemolysis inhibitors produced by an endophytic actinomycete, Polymorphospora rubra K07-0510.

Three new natural products, designated trehangelins A, B and C, were isolated by solvent extraction, silica gel and octadecylsilyl silica gel column chromatographies and subsequent preparative HPLC from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The trehangelins consisted of a trehalose moiety and two angelic acid moieties. Trehangelins A (IC50 value, 0.1 mg ml(-1)) and C (IC50 value, 0.4 mg ml(-1)), with symmetric structures, showed potent inhibitory activity against hemolysis of red blood cells induced by light-activated pheophorbide a. However, trehangelin B, with an asymmetric structure, displayed only a slight inhibition (IC50 value, 1.0 mg ml(-1)).

Interleukin-6 production by peritoneal mesothelial cells and its regulation by inflammatory factors in rats administered carbon tetrachloride intraperitoneally.

We previously reported that a high level of interleukin-6 (IL-6), which is protective against CCl(4)-induced hepatotoxicity, is produced in the peritoneal cavity in the early period after ip carbon tetrachloride (CCl(4)) administration. The objective of this study was to identify the tissues and cells involved in IL-6 production and clarify the mechanisms underlying its regulation. IL-6 mRNA levels increased significantly in the serous membranes of the mesentery and peritoneum, but not in the parenchymal organs including liver, kidney and spleen, 3 h after ip CCl(4) administration. Peritoneal mesothelial cells (PMCs), a major cell population in serous membranes, were isolated from rat peritoneal walls by trypsin digestion and cultured with peritoneal exudate fluid (PEF) from CCl(4)-administered rats. PMCs produced a high level of IL-6 in the presence of PEF recovered 0.5 h after ip CCl(4) administration. Analyses of PEF revealed that the levels of prostaglandin E(2) (PGE(2)), histamine, IL-1alpha, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased immediately after ip CCl(4) administration. These inflammatory factors, except for histamine, stimulated IL-6 production to varying degrees, in the following order: IL-1alpha>IL-1beta>TNF-alpha>>PGE(2). In summary, the present study indicates that the high level of IL-6 observed in the rat peritoneal cavity after ip CCl(4) administration is at least partially produced by PMCs stimulated cooperatively with IL-1alpha, IL-1beta, TNF-alpha and PGE(2). These inflammatory factors may be released from tissues or cells either stimulated or injured directly by CCl(4).