Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model.

BACKGROUND AND PURPOSE: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. EXPERIMENTAL APPROACH: Binding assay with [(3)H]-TAK-242 and nuclear factor-kappaB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. KEY RESULTS: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. CONCLUSIONS AND IMPLICATIONS: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis.

An anti-c-Fms antibody inhibits orthodontic tooth movement.

Orthodontic force induces osteoclastogenesis in vivo. It has recently been reported that administration of an antibody against the macrophage-colony-stimulating factor (M-CSF) receptor c-Fms blocks osteoclastogenesis and bone erosion induced by tumor necrosis factor-alpha (TNF-alpha) administration. This study aimed to examine the effect of an anti-c-Fms antibody on mechanical loading-induced osteoclastogenesis and osteolysis in an orthodontic tooth movement model in mice. Using TNF receptor 1- and 2-deficient mice, we showed that orthodontic tooth movement was mediated by TNF-alpha. We injected anti-c-Fms antibody daily into a local site, for 12 days, during mechanical loading. The anti-c-Fms antibody significantly inhibited orthodontic tooth movement, markedly reduced the number of osteoclasts in vivo, and inhibited TNF-alpha-induced osteoclastogenesis in vitro. These findings suggest that M-CSF plays an important role in mechanical loading-induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-alpha.

Familial amyloidotic polyneuropathy (ATTR Val30Met) with widespread cerebral amyloid angiopathy and lethal cerebral hemorrhage.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) with cerebral hemorrhage. A 38-year-old woman with a typical FAP pedigree started developing severe diarrhea and sensori-motor polyneuropathy at the age of 28 years; autonomic nervous system, heart and renal dysfunction manifested themselves in the following years. Genetic analysis revealed a single amino acid substitution at codon 30 of transthyretin (ATTR Val30Met). Ten years after her initial symptoms, the patient died of a sudden convulsive attack and respiratory failure. Autopsy revealed lethal cerebral hemorrhages and uremic lungs. Histochemical and immunohistochemical analyses revealed TTR-derived amyloid protein in every tissue examined, particularly in glomeruli and peripheral vessels. Severe meningo-cerebrovascular amyloidosis was also detected. Because uremia causes oxidative damage to the vascular system and amyloid formation is closely associated with oxidative stress, it is possible that uremic endothelial damage facilitated an unusual cerebral amyloid deposition. In typical FAP (ATTR Val30Met), cerebral amyloid angiopathy does not usually have clinical manifestations. However, cerebral amyloid angiopathy should be considered to explain FAP symptoms when some risk factors such as uremic vascular damage are accompanying features.

First synthesis of a alpha-trifluoromethyl allenol ether via the Julia-Lythgoe process.

Alpha-trifluoromethyl allenol ethers 9a-e were prepared in moderate to good yields by the Julia-Lythgoe process using beta-ethoxy-beta-trifluoromethyl vinylic sulfone 3. Several reactions of 9c were examined to give alpha,beta-unsaturated trifluoromethyl ketone derivatives 11 and 12.

Direct lithiation and alkylation of trifluoromethyl enol ethers having a beta-sulfur substituent.

The direct lithiation and alkylation of beta-sulfur-alpha-trifluoromethyl substituted enol ethers 1-3 proceeded to give the alkylated products 4a-f, 5, 6a-c in moderate to high yields.

Pathogenic role of glutamate in hyperthermia-induced seizures.

Hyperthermia induces seizures in both humans and rodents, but the underlying mechanism remains unknown. The present study showed that hyperthermia, causing rapid increase in body temperature, increases the concentration of glutamate (Glu) released into a cortical perfusate before onset of seizures in rats and that this increase in Glu concentration correlated with a decrease in seizure threshold temperature. These results indicate that increased cortical extracellular Glu induced by hyperthermia contributes to onset of seizures. The same mechanism may be involved in clinical seizures induced by fever in patients with febrile convulsions or epilepsy.

Three types of hyperthermic seizures in rats.

Three types of rat hyperthermic seizures were observed. The first comprised generalized clonic convulsions preceded by intermittent myoclonus. Ictal EEG showed diffuse intermittent spikes and sequential rapid spike-wave bursts (type 1 seizures). In the other types of seizures, the paroxysmal discharges originated in the occipital region without (type 2a seizures) or with (type 2b seizures) secondary generalization. Type 2a seizures involved no convulsive movement whereas type 2b seizures involved clonic convulsions. The threshold temperatures for type 1, 2a and 2b seizures were 44.1 +/- 0.60, 40.9 +/- 1.47 and 42.1 +/- 0.75 degrees C, respectively. Of these seizures, the type 2 seizures (2a and 2b) did not severely affect the general condition of rats and thus may be an appropriate model for the investigation of febrile seizures.

[Magnetic resonance imaging of skeletal muscle in patients with Duchenne muscular dystrophy--serial axial and sagittal section studies].

Magnetic resonance imaging of skeletal muscles in thirteen patients with Duchenne muscular dystrophy was performed to estimate pathological changes. Serial axial and sagittal sections of the right lower extremity were recorded. In the early stage, the T1 values of gastrocnemius and soleus muscles were slightly lower than the control values, and in the late stage, the values were much lower in all muscles examined. In sagittal sections, the gastrocnemius muscles in the early stage showed a high density area at the distal region adjacent to soleus muscle, and the soleus muscle showed a high density area adjacent to the gestrocnemius muscle. In serial axial sections, high density areas of the anterior and posterior tibialis muscles appeared first at their proximal and peripheral regions. It was concluded that the sequence of appearance of pathological changes was different not only among individual muscles but also among various regions of each muscle; the high density changes appeared first at myotendon junctions.

Wheat germ agglutinin binding sites in the organ of Corti as revealed by lectin-gold labeling.

The distribution of wheat germ agglutinin(WGA)-binding sites in the organ of Corti of the guinea pig and mongolian gerbil was studied. WGA was conjugated with gold particles and applied on thin sections of the cochlea embedded in Spurr's resin and in Lowicryl K4M. WGA-binding sites were found on the plasma membrane, lysosomes and cytoskeletons of hair and supporting cells as well as on the tectorial and basilar membranes. No distinct difference was discovered between hair cells and supporting cells in terms of WGA-binding activities.

UFT for head and neck cancers: its tissue concentrations and effects on lymphocyte subpopulations.

UFT, a combination of the masked compound of 5-fluorouracil (FT-207) and uracil, was given to head and neck cancer patients for 1 week preoperatively and for 8 weeks postoperatively. Drug concentrations were examined in the surgically removed tissues. The concentrations of FT-207, 5-fluorouracil, and uracil were higher in tumor tissues than in normal tissues. The lymphocyte subpopulations were assessed by cytofluorometry with monoclonal antibodies. There was no evidence that adjuvant chemotherapy with UFT specifically suppresses immunocompetent cells. We therefore conclude that further clinical evaluation of adjuvant chemotherapy with UFT would be worthwhile.