Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6.

Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens. Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells. The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery. However, the signaling pathways involved are poorly understood. Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases. Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond. The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo.

Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

Induction of direct antimicrobial activity through mammalian toll-like receptors.

The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

MyD88 as a bottle neck in Toll/IL-1 signaling.

Myeloid differentiation factor 88 (MyD88) is an adaptor molecule composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-1R homology domain. Ligand binding to Toll-like receptor (TLR)/IL-1R family members results in the association of MyD88 to the cytoplasmic tail of receptors; this then initiates the signaling cascade that leads to the activation of nuclear factor-kappa B and mitogen-activated protein kinases. Analysis of MyD88-deficient mice revealed its essential role in TLR/IL-1R signaling as well as in both the innate and the adaptive immune response.

TLR6: A novel member of an expanding toll-like receptor family.

Drosophila Toll protein is shown to activate the innate immune system in adult and regulate the dorsoventral patterning in the developing embryo. Recently, five human homologs of Drosophila Toll, designated as Toll-like receptors (TLRs), have been identified and shown to play a role in the innate immune response. We report here the molecular cloning and characterization of a new member of Toll-like receptor family, Toll-like receptor 6 (TLR6). Human and murine TLR6 are type-I transmembrane receptors that contain both an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic Toll/IL-1 receptor (IL-1R)-like region. The amino acid sequence of human TLR6 (hTLR6) is most similar to that of hTLR1 with 69% identity. RT-PCR analysis revealed that murine TLR6 is expressed predominantly in spleen, thymus, ovary and lung. Like other TLR family members, constitutively active TLR6 activates both NF-kappaB and c-Jun N-terminal kinase (JNK). The TLR6 gene, as well as the TLR1 gene, mapped to the proximal region of murine chromosome 5 within 1.7cM of each other. These results suggest that TLR6 is a novel member of an expanding TLR family.

Effect of dopamine on pancreatic secretion in the dog.

1. Effects of L-dopa and dopamine on the secretion of pancreatic juice were investigated in preparations of the isolated blood-perfused canine pancreas.2. Dopamine (1-10 mug) given intra-arterially caused a profuse flow of juice.3. The secretory activity of dopamine (3 mug) was approximately equal to that of secretin (0.1 unit).4. L-Dopa (10-100 mug) given by a single intra-arterial injection was ineffective, but infusion at 100 mug/min for 10 min caused a marked increase of secretion after a delay of a few minutes.5. Intravenous administration of either L-dopa (3 mg/kg) or dopamine (10-100 mug/kg) elicited a marked increase of pancreatic secretion, but was definitely less effective than intra-arterial injection.6. Dopamine-induced secretion was not modified by atropine, phentolamine, propranolol, guanethidine or tetrodotoxin.7. It is concluded that dopamine acts directly on the exocrine cells in the pancreas.

Effects of enzyme inhibitors of catecholamine metabolism and of haloperidol on the pancreatic secretion induced by L-DOPA and by dopamine in dogs.

1. Effects of inhibitors of DOPA decarboxylase, dopamine beta-hydroxylase and monoamine oxidase, and haloperiodol on the secretion of pancreatic juice induced by L-DOPA and dopamine were studied in preparations of the isolated blood-perfused canine pancreas.2. The increased secretion induced by the infusion of L-DOPA (100 mug/min) was completely antagonized by Ro 4-4602 (300 mug), a DOPA decarboxylase inhibitor.3. The secretogogue effect of dopamine (1-10 mug) intra-arterially was not affected by Ro 4-4602, but was enhanced by the infusion of fusaric acid (100 mug/min), a dopamine beta-hydroxylase inhibitor.4. The increase in the secretion induced by dopamine (1-10 mug) was enhanced by treatment with nialamide (100 mg/kg), a monoamine oxidase inhibitor, given intravenously.5. Haloperidol (1 mg) intra-arterially attenuated the dopamine-induced pancreatic secretion.6. It is concluded that L-DOPA is converted to dopamine in the acinar cells which causes an increase in the secretion of pancreatic juice, thus the intracellular level of dopamine may be controlled by enzymatic equilibrium.

A case of renovascular hypertension due to bilateral renal artery microaneurysm who succeeded in baby delivery.

We report the case of a young pregnant woman with bilateral renovascular hypertension due to renal microaneurysms from an unknown cause, who had a successful delivery. Pregnancy did not affect the disease activity even in the postpartum period. Her blood pressure was maintained within the normal range by administration of labetalol. Although the angiographic appearance of the symmetrical aneurysms in both renal artery beds from the interlobular to arcuate artery levels suggested polyarteritis nodosa of multiple microaneurysms in the bilateral interlobular arteries, the clinical features suggested other causes of renovascular hypertension, such as fibromuscular dysplasia and/or congenital microaneurysms. We were thus unable to reach a definitive diagnosis.

Toll-like receptors; their physiological role and signal transduction system.

Drosophila Toll protein is a transmembrane receptor whose function is to recognize the invasion of microorganisms as well as to establish dorso-ventral polarity. Recently, mammalian homologues of Toll, designated as Toll-like receptors (TLRs) have been discovered. So far, six members (TLR1-6) have been reported and two of these, TLR2 and TLR4, have been shown to be essential for the recognition of distinct bacterial cell wall components. TLR2 discriminates peptidoglycan (PGN), lipoprotein, lipoarabinomannan (LAM) and zymosan, whereas TLR4 recognizes lipopolysaccharide (LPS), lipoteichoic acid (LTA) and Taxol. Bacterial components elicit the activation of an intracellular signaling cascade via TLR in a similar way to that occurs upon ligand binding to IL-1 receptor (IL-1R). This signaling pathway leads to the activation of a transcription factor NF-kappaB and c-Jun N-terminal kinase (JNK), which initiate the transcription of proinflammatory cytokine genes. Particularly, analysis of knockout mice revealed a pivotal role for MyD88 in the signaling of the TLR/IL-1R family. Taken together, TLRs and the downstream signaling pathway play a key role in innate immune recognition and in subsequent activation of adaptive immunity.

Involvement of MyD88 in host defense and the down-regulation of anti-heat shock protein 70 autoantibody formation by MyD88 in Toxoplasma gondii-infected mice.

This study investigated the influence of TLR (toll-like receptor)4, TLR2, and MyD88 in Toxoplasma gondii-infected wild-type (WT) mice and TLR4-, TLR2-, and MyD88-deficient mice. Ninety-five percent of MyD88-deficient mice died 10-16 days after intraperitoneal infection with 100 cysts of T. gondii Fukaya strain, whereas 95-100% of TLR4- and TLR2-deficient mice and WT C57BL/6 (B6) mice survived for more than 7 wk after T. gondii infection. The distribution of T. gondii in various organs of TLR4-, TLR2-, and MyD88-deficient mice and WT B6 mice was assessed 2 wk after T. gondii intraperitoneal infection using quantitative competitive polymerase chain reaction. In MyD88-deficient mice, high levels of T. gondii load were observed in the brain, tongue, heart, lungs, spleen, liver, mesenteric lymph node, and kidneys after infection. The T. gondii load was significantly increased in the lungs in both TLR4- and TLR2-deficient mice compared with WT B6 mice. High levels of anti-mouse heat shock protein (mHSP)70 autoantibody and anti-T. gondii HSP70 antibody production were detected in the sera from MyD88-deficient mice.

A case of classical carcinoid tumor of the gallbladder: review of the Japanese published works.

A 58 year-old man was admitted to Kimitsu Chuo Hospital complaining of epigastralgia. Abdominal ultrasound and computed tomography revealed a polypoid lesion at the neck of the gallbladder. Given the pre-operative diagnosis of gallbladder carcinoma, we resected the gallbladder along with the extrahepatic bile duct. There was a papillary tumor (25 x 16 mm) at the neck of the gallbladder. Histopathological examinations showed a subserosal nodular proliferation of uniform small tumor cells. Grimelius staining was slightly positive and Fontana-Masson staining was negative. Most of the tumor cells stained positively for chromogranin A and neuron-specific enolase (NSE), and some of the tumor cells were positive for pancreatic polypeptide. The presence of neurosecretory intracytoplasmic granules was proven ultrastructurally. It was diagnosed as a classical carcinoid tumor of the gallbladder. We reviewed the Japanese reported cases and discussed the difference in clinicopathological findings between classical and atypical carcinoid tumors of the gallbladder. Classical carcinoids of the gallbladder have neither a metastatic nor invasive character, and an extremely favorable prognosis compared with atypical carcinoids. The difference in character between classical and atypical carcinoids of the gallbladder is thought to be derived from their histogenetic origin.

[Efficacy of immunized milk for preventing viral infection].

This paper investigated the efficacy of passive protection provided by milk (immunized milk) against enterovirus infection in mice experimentally infected with enterovirus. Milk with a high antibody titer against six enterovirus serotypes was prepared from hyperimmunized goat. In vivo and in vitro experiments were performed and the results showed that immunized milk has an antiviral activity against enterovirus infection. Further observation was performed using Coxsackie B 3 virus (CVB 3). When immunized milk was orally applied to mice prior to oral inoculation with CVB 3, preventive effects against viral infection such as reduction of histopathological changes in the heart and reduced detection of the virus genome in the organs were seen. The antiviral effect was also indicated by the increase of CD4+T cells proportion in the i-IEL. The proportion of virus specific CD4+T cells was increased in mice treated with immunized milk, whereas no such increase was detected in control mice. These results suggest that oral application of immunized milk is not only capable of preventing viral infection but also induces specific immunological responses. These phenomena may play an important role in host defense mechanisms.

[Comparative studies and application of a simple method of extraction of RNA from hepatitis C virus].

Conventional RNA extraction methods, such as SDS/phenol and AGPC methods involve time consuming and complicated manipulation procedures, which could be a cause of contamination. In this study, we compared the conventional methods with a method using a newly developed reagent, "SepaGene-RV" to extract RNA. The results obtained by the new method were the same as those obtained by the conventional methods in the comparative experiments with the sera of patients infected with HCV and detection limit tests using recombinant HCV genome. The sensitivity to detect HCV-RNA with SepaGene-RV was equivalent to that of conventionally used methods to extract RNA, and the manipulation procedure was simpler and it was less time consuming. In addition, we could detect plus-stranded and minus-stranded HCV-RNA by polymerase chain reaction assay (PCR) from liver tissue obtained from liver biopsy and peripheral blood mononuclear cells (PBMC). We confirmed that HCV infects not only liver tissue but also PBMC where replication occurs.