Masashi Kohanawa

Usefulness of 11C-Methionine for Differentiating Tumors from Granulomas in Experimental Rat Models: A Comparison with 18F-FDG and 18F-FLT

Many clinical PET studies have shown that increased 18F-FDG uptake is not specific to malignant tumors. 18F-FDG is also taken up in inflammatory lesions, particularly in granulomatous lesions such as sarcoidosis or active inflammatory processes after chemoradiotherapy, making it difficult to differentiate malignant tumors from benign lesions, and is the main source of false-positive 18F-FDG PET findings in oncology. These problems may be overcome by multitracer studies using 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) or l-11C-methionine. However, 18F-FLT or 11C-methionine uptake in granulomatous lesions remains unclarified. In this study, the potentials of 18F-FLT and 11C-methionine in differentiating malignant tumors from granulomas were compared with 18F-FDG using experimental rat models. Methods: Dual-tracer tissue distribution studies using 18F-FDG and 3H-FLT (groups I and III) or 18F-FDG and 14C-methionine (groups II and IV) were performed on rats bearing both granulomas (Mycobacterium bovis bacillus Calmette-Guérin [BCG]–induced) and hepatomas (KDH-8–induced) (groups I and II) or on rats bearing both turpentine oil–induced inflammation and hepatomas (groups III and IV). One hour after the injection of a mixture of 18F-FDG and 3H-FLT or of 18F-FDG and 14C-methionine, tissues were excised to determine the radioactivities of 18F-FDG, 3H-FLT, and 14C-methionine (differential uptake ratio). Results: Mature epithelioid cell granuloma formation and massive lymphocyte infiltration were observed in the granuloma tissue induced by BCG, histologically similar to sarcoidosis. The granulomas showed high 18F-FDG uptake comparable to that in the hepatomas (group I, 8.18 ± 2.40 vs. 9.13 ± 1.52, P = NS; group II, 8.43 ± 1.45 vs. 8.91 ± 2.32, P = NS). 14C-Methionine uptake in the granuloma was significantly lower than that in the hepatoma (1.31 ± 0.22 vs. 2.47 ± 0.60, P < 0.01), whereas 3H-FLT uptake in the granuloma was comparable to that in the hepatoma (1.98 ± 0.70 vs. 2.30 ± 0.67, P = NS). Mean uptake of 18F-FDG, 3H-FLT, and 14C-methionine was markedly lower in the turpentine oil–induced inflammation than in the tumor. Conclusion: 14C-Methionine uptake was significantly lower in the granuloma than in the tumor, whereas 18F-FDG and 3H-FLT were not able to differentiate granulomas from tumors. These results suggest that 14C-methionine has the potential to accurately differentiate malignant tumors from benign lesions, particularly granulomatous lesions, providing a biologic basis for clinical PET studies.

Endogenous Interleukin-6 Plays a Crucial Protective Role in Streptococcal Toxic Shock Syndrome via Suppression of Tumor Necrosis Factor Alpha Production

ABSTRACT During a Streptococcus pyogenes infection in interleukin-6 (IL-6)-deficient mice, there is elevation of serum tumor necrosis factor alpha (TNF-α) levels, muscular necrosis, and death compared with infection of C57BL/6 mice. Anti-TNF-α monoclonal antibody treatment decreased mortality and muscular necrosis in the infected IL-6-deficient mice. These results suggest that IL-6 plays a crucial protective role via suppression of TNF-α production in S. pyogenes infection.

A novel murine model for non-alcoholic steatohepatitis developed by combination of a high-fat diet and oxidized low-density lipoprotein

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome that is characterized by steatosis, inflammation, and fibrosis, and may progress to cirrhosis and carcinoma. To investigate its pathogenic processes, we established a novel murine model for NASH by combination of a high-fat diet (HFD) and oxidized low-density lipoprotein (oxLDL). Mice that received HFD for 23 weeks showed hepatic steatosis, slight fibrosis, and a high level of lipid peroxidation compared with a regular diet (RD)-fed mice. Hepatic injury and elevated tumor necrosis factor (TNF)-α mRNA expression were also detected in these mice. Moreover, oxLDL administration to HFD-fed mice during weeks 21–23 not only aggravated hepatic steatosis, fibrosis, and lipid metabolism, but also resulted in intense inflammation, including severe hepatic injury and inflammatory cell infiltration, which are the typical histological features of NASH. Inflammation was accompanied by increased gene expression of TNF-α and interleukin (IL)-6. Additionally, the livers of RD-fed animals treated with oxLDL during weeks 21–23 were characterized by foamy macrophages and inflammatory cell infiltration along with an elevated IL-6 mRNA level. These results suggest that an increased oxidative state, including HFD-induced intracellular lipid peroxidation and its extracellular source from oxLDL, is the actual trigger for hepatic inflammation in which liver injury is mediated by TNF-α and inflammatory cell accumulation is dependent on IL-6. HFD and oxLDL also induced insulin resistance in mice; additionally, oxLDL downregulated insulin secretion. In this model, CD36 overexpression was observed in the hepatocytes of HFD-fed mice and those treated with HFD and oxLDL, and in the hepatic macrophages of RD-fed mice immediately after oxLDL treatment. In vitro experiments indicated a rapid and transient elevation of CD36 on macrophage plasma membrane in response to oxLDL. Our findings demonstrate that CD36 expressed on hepatocytes and hepatic macrophages mediates the pathophysiology of NASH.

Contribution of toll-like receptor 2 to the innate response against Staphylococcus aureus infection in mice.

Staphylococcus aureus is a common pathogen that causes a wide range of infectious diseases. The function of TLRs, specifically TLR2, during S. aureus infection is still debated. In this study, we investigated the extent to which TLR2 contributes to the host innate response against the bacterial infection using TLR2-deficient mice. Intravenous inoculation with S. aureus resulted in all TLR2-deficient mice dying within 4 d, along with a high bacterial burden in the livers. However, histological examination showed the same degree of macrophage and neutrophil accumulation in the livers of infected TLR2-deficient mice as that in infected wild-type (WT) mice. TLR2-deficient mouse macrophages also showed normal phagocytic activity, although they failed to express CD36 that appeared on the surface of WT mouse cells upon challenge with heat-killed S. aureus. These data indicate that TLR2, as well as CD36, does not directly affect S. aureus clearance and that CD36 expression on macrophages depends on the presence of TLR2. In vivo infection with S. aureus caused significantly elevated production of TNF-α and IL-6 in the livers and blood of TLR2-deficient mice compared with those in WT mice, while the hepatic and serum levels of IL-10 decreased in these mice. In contrast, lower expression of IL-6 and IL-10, but not of TNF-α, at both the gene and protein levels was found in TLR2-deficient mouse macrophages compared to that in WT mouse cells, in response to challenge with heat-killed S. aureus. These findings suggest that the S. aureus-induced pro-inflammatory cytokine response is not dependent on macrophages and that TLR2 deficiency results in decreased IL-10 release by macrophages, which contributes to dysregulated cytokine balance, impaired bacterial clearance, and mouse death. Therefore, TLR2 possesses a protective function during S. aureus infection by regulating pro- and anti-inflammatory cytokine responses.

Endogenous gamma interferon produced in central nervous system by systemic infection infection with Theiler's virus in mice

Abstract Theilers virus GD VII strain causes acute encephalomyelitis by intracerebral inoculation. We established acute encephalomyelitis in mice by the intravenous (i.v.) inoculation of Theilers virus GD VII strain. Replication of Theilers virus injected i.v. could be observed in both the brain and spinal cord of mice, and interferon (IFN)-γ could be detected in the extracts of brain and spinal cord in parallel with viral replication. Furthermore, by the injection of anti-IFN-γ monoclonal antibody (mAb) on Day 1 post-infection (p.i.), mortality and virus titres in the spinal cord increased compared with the control mice treated with normal rat globulin. The histological exacerbation of inflammation was observed in spinal cord of anti-IFN-γ mAb-treated mice. These results indicate that endogenous IFN-γ, produced locally in the brain and spinal cord of mice through both antiviral action and anti-inflammatory action of IFN-γ in central nervous system, plays an important role in Theilers virus infection.

Detection of high levels of immunoreactive human beta-1 interferon in sera from HIV-infected patients

We have examined human interferon beta-1 (HuIFN-beta 1) levels in the sera from patients with various diseases by using a newly developed highly sensitive enzyme-linked immunosorbent assay system (ELISA). High levels of HuIFN-beta 1 were detected in the sera from Human Immunodeficiency Virus-I (HIV-I) infected patients, especially of asymptomatic carriers. The serum level of HuIFN-beta 1 may be a good indicator of HIV-I infection and of other immunological disorders.

A Regulatory Effect of the Balance between TNF-α and IL-6 in the Granulomatous and Inflammatory Response to Rhodococcus aurantiacus Infection in Mice

After i.v. inoculation with Rhodococcus aurantiacus, wild-type (WT) mice develop nonnecrotic, epithelioid granulomas. Because a high level of TNF-α is observed during the initial phase postinfection, we examined the extent to which TNF-α contributes to granulomatous inflammation using TNF-α gene-deficient (TNF-α−/−) mice. Despite a lack of R. aurantiacus proliferation, TNF-α−/− mice displayed high mortality rates within 5 days postinfection, as well as a high level of IL-6 in their spleens. Histological examination showed an absence of granuloma formation in TNF-α−/− mice. Pretreatment of TNF-α−/− mice with rTNF-α failed to restore this granuloma formation but accelerated bacterial removal and cellular recruitment. This rTNF-α administration also attenuated IL-6 production, resulting in increased survival rates of TNF-α−/− mice. Heat-killed R. aurantiacus induced in vitro enhanced mRNA expression and production of IL-6 in macrophages and DCs from TNF-α−/− mice when compared with WT controls, and treatment of TNF-α−/− mouse cells with rTNF-α decreased the IL-6 secretion. Moreover, anti-TNF-α or anti-IL-6 treatment increased IL-6 or TNF-α production by WT mouse cells, respectively. These data suggest that the production of TNF-α and IL-6 can be negatively regulated by each other. Administration of rIFN-γ to TNF-α−/− mice caused immature granulomas in livers, and treatment with both rTNF-α and rIFN-γ led to the formation of mature granulomas. Overall, TNF-α appears crucial for bacterial clearance, cellular recruitment, and granuloma formation. The balance between TNF-α and IL-6 during the early phase of infection controls the development of the inflammatory response to R. aurantiacus infection.

Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus

After intravenous injection of Rhodococcus aurantiacus normal mice develop non‐necrotic granulomas, the formation of which is dependent on endogenous interferon‐γ (IFN‐γ). In the early phase of R. aurantiacus infection a high level of endogenous interleukin‐6 (IL‐6) is detected in the spleen extracts, though its importance is unknown. Using IL‐6 knockout (IL‐6−/−) mice, we studied the role of IL‐6 in granulomatous inflammation induced by R. aurantiacus. The size of granulomas generated in IL‐6−/− mice was significantly larger than that of wild‐type (IL‐6+/+) mice at 2 weeks postinjection (p.i). Moreover, central necrosis of the granuloma was observed in IL‐6−/− mice but not in IL‐6+/+ controls. Titres of endogenous IFN‐γ and tumour necrosis factor‐α (TNF‐α) were markedly increased in the spleens and livers of IL‐6−/− mice in comparison with IL‐6+/+ mice at days 1 through 3 p.i. In vivo administration of either an anti‐IFN‐γ monoclonal antibody (mAb) or anti‐TNF‐α mAb to IL‐6−/− mice reduced the number and size of granulomas, and prevented formation of necrotic granulomas. In addition, the production of endogenous IFN‐γ and TNF‐α in the early phase of R. aurantiacus infection by IL‐6−/− mice was suppressed by treatment with recombinant IL‐6 (rIL‐6). This suppression of IFN‐γ and TNF‐α production was followed by a reduction in the number and size of central necrotic granulomas at 2 weeks p.i. These findings suggest that overproduction of IFN‐γ and TNF‐α induces central necrotic granuloma formation in IL‐6−/− mice, and that IL‐6 down‐regulates granulomatous inflammation reaction in response to R. aurantiacus infection by modulating production of IFN‐γ and TNF‐α.

Sequential involvement of NK cells and CD8+ T cells in granuloma formation of Rhodococcus aurantiacus-infected mice

We investigated the effect of in vivo administration of antibodies against T‐cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN‐γ) production and granuloma formation in Rhodococcus aurantiacus‐infected mice. High titers of endogenous IFN‐γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN‐γ production was reduced by in vivo administration of anti‐NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1+ cells. Endogenous IFN‐γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN‐γ at 1 and 3 weeks was reduced by in vivo administration of anti‐CD8 MAb, but not by anti‐CD4 MAb or anti‐NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti‐IFN‐γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8+ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN‐γ is attributable to NK cells in the early phase of the infection and CD8+ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8+ T cells through the secretion of endogenous IFN‐γ.

A possible role for NKT-like cells in patients with chronic hepatitis B during telbivudine treatment.

Natural killer T-like (NKT-like) cells are a source of different pro-inflammatory cytokines and therefore may be involved in inflammatory processes. However, little is known about NKT-like cells during antiviral therapy. In this study, we observed significantly higher numbers of CD3(+)CD56(+) cells in patients with chronic hepatitis B (CHB) than healthy controls. Importantly, CD3(+)CD56(+) NKT-like cells markedly decreased during telbivudine treatment in patients with CHB, and a positive correlation between NKT-like cell frequency and the serum HBV DNA level was observed during early antiviral therapy. Interestingly, NKT-like cell frequency significantly reduced in well-responders at week 12 of telbivudine therapy compared to baseline, but did not significantly change in non-responders after treatment. Previous studies have shown that interleukin (IL)-17 plays a role in the pathogenesis of CHB. Serum IL-17 levels reduced significantly during early antiviral therapy, however, interferon (IFN)-γ, IL-6 and tumor necrosis factor (TNF)-α levels did not change significantly. A positive correlation was observed between the NKT-like cell frequency and serum IL-17 level in CHB patients, and NKT-like cells isolated from patients with CHB secreted substantial amounts of IL-17 in vitro. These results suggest that the NKT-like cell frequency may be one of useful immunologic marker for evaluating the efficacy of anti-HBV therapy, and that NKT-like cells are also an important source of IL-17 (in addition to conventional T cells) in patients with CHB.