Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice.

BACKGROUND: While tumour necrosis factor alpha (TNF-alpha) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. METHODS: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. RESULTS: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-alpha, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-alpha administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. CONCLUSIONS: Enhancement of the TNF-alpha/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Tooth development included in the multifocal jaw lesions of Langerhans cell histiocytosis.

Many cases demonstrating the oral manifestations of Langerhans cell histiocytosis (LCH) have been reported; however, tooth development in jaw lesions has rarely been mentioned. This paper reports the case of a 3-year-old Japanese girl with LCH suffering from multiple osteolytic lesions of the skull and jaw bones. She was referred to our paediatric clinic because of swelling of the mucogingival folds in the upper and lower primary molar regions. The patient responded well to steroid therapy and the osteolytic lesions resolved. There was an accompanying development of permanent tooth germs included in the lesions. Langerhans cell histiocytosis in children usually has a long-term clinical course and paediatric dentists should be involved with oral health care for affected patients.

Clinical observations of odontomas in Japanese children: 39 cases including one recurrent case.

Retrospective investigations of odontomas in Japanese children and one recurrent case were carried out. Thirty-nine cases of odontoma in 38 children were treated in the Paediatric Dentistry Clinic of Niigata University Dental Hospital between September 1979 and December 2002. The patients consisted of 23 males and 15 females and their ages ranged from 1 year 2 months to 14 years old. The chief complaints were delayed tooth eruption in 19 cases (five: primary teeth, 14: permanent teeth), retention of primary teeth in 11, incidentally found on the radiographic examination in eight cases, and swelling of the jaw in one case. Thirty-four cases (87%) were associated with tooth eruption disturbances. The most frequently affected region was the maxillary anterior region. Treatment consisted of surgical removal of odontomas in all cases, after which if the impacted teeth did not erupt, exposure of the crown and/or orthodontic traction was performed. Pathological diagnoses were compound odontoma in 30 cases, complex odontoma (n = 7), and compound and complex odontoma (n = 2). A retrospective study of the radiographs revealed the developing process of odontomas in four cases and odontoma disturbed tooth eruption since the early uncalcified developing stage. A recurrent case was a boy aged 6 years 5 months in whom the first surgical removal of odontoma was performed at the age of 1 year 8 months. Recurrence of an odontoma is very rare, but in very young children odontomas are in the early developing stages, containing uncalcified portions, so it is important to perform periodical observations until the succedaneous teeth erupt.

Identification of two new C4 alleles by DNA sequencing and evidence for a historical recombination of serologically defined C4A and C4B alleles.

Nucleotide polymorphisms of the C4 genes were investigated by direct sequencing of seven different homozygous typing cells from the 10IHW panels. Two novel sequences were identified within the C4d region of the C4 genes. Our sequencing analyses extend previous findings suggesting that a recombination hot spot is likely to have occurred between codon positions 1157 and 1186 within the C4d region. The classification of electrophoretically defined C4A and C4B alleles can be further subtyped by sequencing. Because the central major histocompatibility complex region that carries various copies of the C4 gene has been associated with a range of disorders; further analysis at the sequence level within the C4 locus may provide informative genetic markers for the investigation of disease-associated polymorphisms.

A promoter region of the midkine gene that is frequently expressed in human hepatocellular carcinoma can activate a suicide gene as effectively as the alpha-fetoprotein promoter.

We examined the expression of the midkine (MK) and alpha-fetoprotein (AFP) genes in 15 paired human specimens obtained from hepatocellular carcinoma (HCC) and the corresponding noncancerous regions of the same patients. A total of 14 HCC but none of the noncancerous specimens were positive for the MK mRNA. In contrast, three HCC specimens and one corresponding noncancerous sample out of the three AFP-positive HCC cases expressed the AFP gene. A 2.3-kb genomic fragment in the regulatory region of the MK gene could activate a fused reporter gene in both AFP-producing and -nonproducing HCC lines, and the MK fragment-mediated transcriptional activity was comparable to the AFP enhancer-linked AFP promoter in AFP-producing cell lines. The AFP-producing but not AFP-nonproducing HCC cell lines that were transfected with the MK promoter-linked herpes simplex virus-thymidine kinase (HSV-TK) gene became susceptible to a prodrug ganciclovir to a similar degree of the HCC transfected with the enhancer-linked AFP promoter-fused HSV-TK gene. These data suggest that the MK promoter can activate a therapeutic gene preferentially in HCC and is as useful as the AFP promoter in clinical settings.

Corneodesmosin gene: no evidence for PSORS 1 gene in North-eastern Thai psoriasis patients.

Psoriasis vulgaris, a common inflammatory skin disorder, is known to be associated with the HLA-Cw*06 allele. It has been recently suggested by microsatellite mapping that a real susceptible gene for psoriasis resides in the approximately 100-kb genomic region telomeric of the HLA-C gene. In this respect, the corneodesmosin (CDSN) gene 160-kb telomeric of HLA-C is a strong candidate because of its location and its functional role in corneocyte cohesion and desquamation. In fact, a significant association between CDSN polymorphism and psoriasis was recently recognized in Caucasian populations. However, this association has not been replicated in other studies, being still controversial. In this study, we investigated the genetic polymorphism of the CDSN gene in 139 psoriasis patients and 144 healthy controls in the North-eastern Thai population. By direct sequencing technique, a total of 28 polymorphic sites were found, consisting of 26 single nucleotide polymorphisms (SNPs) and two indels (insertion/deletion). Among them, six SNPs have not been previously reported. Through this analysis, as many as 28 different SNP/indel haplotypes within the CDSN gene were identified. Seven SNPs and one indel, namely 9C, 614 A, 722T, 971T, 1215G, 1243C, 1331G and 1606AAG (deletion), revealed significant deviation in the allelic frequencies of the patients from those of the healthy controls. However, none of them are likely to be responsible for controlling the susceptibility of psoriasis, but these associations can be explained by a linkage disequilibrium to a real pathogenic allele of a nearby gene. Further, the large variations between the CDSN SNP/indel haplotypes and the psoriatic major histocompatibility complex (MHC) haplotypes also make it unlikely that CDSN is a major psoriasis-susceptible gene.

Localization of a non-melanoma skin cancer susceptibility region within the major histocompatibility complex by association analysis using microsatellite markers.

The major histocompatibility complex (MHC) is known to have a role in the development of non-melanoma skin cancer (NMSC), although the genes and mechanisms involved have yet to be determined. To identify the susceptibility locus for NMSC within the MHC, we used a collection of well-defined polymorphic microsatellite markers from the Human leucocyte antigen (HLA) region for an association analysis of 150 cases with NMSC and 200 healthy controls selected from the Busselton population in Western Australia. High-resolution mapping was undertaken using a total of 40 highly polymorphic markers located at regular intervals across the HLA region (3.6Mb). Polymerase chain reaction (PCR) analysis was initially performed on pooled DNA markers to detect those markers that showed different allele profiles. Statistically significant differences in allelic frequencies (differentiating alleles) were found between cases and controls at three polymorphic microsatellite loci within a 470-kb genomic susceptibility region ranging between 6 kb centromeric of the HLA-B gene and intron 5 of the DDR gene. Interestingly, this genome region corresponded completely with the psoriasis-susceptibility locus. The three differentiating alleles and another four markers outside the susceptibility region were then PCR tested by individual genotyping of cases and controls. The newly identified susceptibility locus for NMSC within the MHC was found to be significantly different between the cases and controls by comparisons of allele frequencies at the three differentiating loci estimated from DNA pools and then confirmed by individual genotyping. This is the first study using high density microsatellite markers to localize a NMSC susceptibility region within the human genome.

Corneodesmosin DNA polymorphisms in MHC haplotypes and Japanese patients with psoriasis.

In order to examine the relationship between corneodesmosin (CDSN) and psoriasis we have determined the presence of CDSN polymorphisms by DNA sequencing in (a) nine B-LCL cell lines of major histocompatibility complex ancestral haplotypes known to be associated with psoriasis vulgaris including 13.1AH, 46.1AH, 46.2 and 57.1AH, and in (b) a group of 267 unrelated individuals comprising Japanese psoriasis patients (n = 101) and Japanese subjects without the disease (n = 166). Three novel CDSN gene sequences were identified. In addition, we have classified the 18 alleles into seven main groups based on phylogeny of non-synonymous substitutions. However, we have found no statistically significant differences between the patients and the unaffected individuals in any of these groups. These findings indicate that CDSN is not a major psoriasis susceptibility gene.

Impacted supernumerary tooth developed under palatal polyp.

We present a rare case of an infant's palatal polyp associated with an impacted supernumerary tooth. We have previously reported three cases of palatal polyps in infants [1]. In one case, after surgical removal of the palatal polyp at the age of 1 year and 8 months, the lesion subsequently began swelling. A periapical radiograph at the age of 2 years and 9 months revealed a small calcified mass in the maxillary left incisor region. The swelling was kept under observation, the calcified mass developed gradually and was removed surgically at the age of 5 years and 5 months. The removed calcified mass was clinically diagnosed as an impacted supernumerary tooth and this was confirmed histologically. Histological findings did not indicate any relationship between the palatal polyp and the impacted supernumerary tooth.

Bilateral maxillary fused primary incisors accompanied by succedaneous supernumerary teeth: report of a case.

A rare case of fusion between maxillary primary central incisors and supplemental teeth occurring bilaterally, accompanied by succedaneous supernumerary teeth, is presented. The patient was an 8.5-year-old Japanese boy. Intraoral examination revealed fusion of left and right maxillary primary central incisors to supplemental teeth, which had labial and lingual grooves. The maxillary primary lateral incisors were present. Radiographs showed that the fused teeth had separate roots, pulp chambers and root canals. There were two impacted supernumerary teeth and eruption of the permanent maxillary central incisors was delayed. Treatment was performed and the fused primary teeth and the impacted supernumerary teeth were extracted. After 6 months observation, surgical exposure of the two crowns of the permanent maxillary central incisors was performed. The teeth began to erupt and have since been kept under observation.

Analgesic and toxic effects of neonicotinoid insecticides in mice.

Several nicotinic agonists with the 6-chloro-3-pyridinyl moiety are potent insecticides (e.g., the neonicotinoids imidacloprid and thiacloprid) while others are candidate nonopioid and nonantiinflammatory analgesics (i.e., epibatidine and several heterocyclic analogs). This study examines the hypothesis for the first time that the neonicotinoid insecticides and their imine metabolites and analogs display analgesic (antinociceptive) activity or adverse toxic effects associated with their action on binding to the alpha 4 beta 2 nicotinic acetylcholine receptor (AChR) subtype. Seven 6-chloro-3-pyridinyl compounds were studied, i.e., imidacloprid and thiacloprid, the corresponding imines and an olefin derivative, a nitromethylene analog, and (+/-)-epibatidine. Like (-)-nicotine and carbachol, they all act as full agonists in the (86)rubidium ion efflux experiment with intact mouse fibroblast M10 cells stably expressing the alpha 4 beta 2 nicotinic AChR. Their agonist action is correlated with binding affinity to the alpha 4 beta 2 receptor from M10 cells. Imidacloprid, thiacloprid, and their imine analogs are not antinociceptive agents in mice by abdominal constriction and hot plate analgesic tests. Their agonist actions at the alpha 4 beta 2 receptor correlate instead with their toxicity. Surprisingly, the nitromethylene analog, a weak agonist, is as potent as (-)-nicotine in inducing antinociception, and the effect persists longer than that caused by (-)-nicotine. However, mecamylamine (1 mg/kg) prevents antinociception induced by (-)-nicotine but not by the nitromethylene analog. Interestingly, this nitromethylene neonicotinoid insecticide gives 80-100% mortality within 15 min at 3 mg/kg with mecamylamine pretreatment at 2 mg/kg, doses at which each agent alone gives no lethality. Therefore, analgesic and toxic effects of the nitromethylene analog differ in their mechanism of action from (-)-nicotine and (+/-)-epibatidine.

Structure and diversity of insect nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is an agonist-regulated ion-channel complex responsible for rapid neurotransmission. The vertebrate nAChR, assembled from five homologous subunits, penetrates the synaptic membrane. Different subunit combinations lead to receptor subtypes with distinctive pharmacological profiles. In comparison with mammalian nAChRs, the insect receptor is poorly understood relative to functional architecture and diversity. Several genes for Drosophila, Locusta and Myzus encoding insect nAChR subunits have been identified, although the functional assembly and presence of different subtypes of these receptors are not defined. The insect nAChR is the primary target site for the neonicotinoid insecticides, thereby providing an incentive to explore its functional architecture with neonicotinoid radioligands, photoaffinity probes and affinity chromatography matrices. This review considers the current understanding of the structure and diversity of insect nAChRs based mainly on recent studies in molecular biology and protein biochemistry.

Irradiation with ultrasound of low output intensity increased chemosensitivity of subcutaneous solid tumors to an anti-cancer agent.

Ultrasound is a possible mechanical method to deliver small molecules into target cells. In order to evaluate the therapeutic potentials provided by ultrasound irradiation, we compared anti-tumor effects of electroporation- and ultrasound-mediated chemotherapy and efficacy of gene transfer by the two methods. Electric pulses (5 Hz, 100 V/cm, 8 square-wave/100 microsec) or ultrasound (1 MHz, 2 W/cm(2), 5 min) was delivered to subcutaneous solid tumors of murine lymphoma after the tumor-bearing mice received an intraperitoneal injection of bleomycin (BLM) (2.5 mg) or intratumoral injection of plasmid DNA containing the luciferase reporter gene. Administration of BLM alone did not affect the subsequent tumor growth but additive treatment with ultrasound irradiation suppressed the growth to the same level as electroporation. The luciferase activity of the DNA-injected tumors showed that ultrasound irradiation achieved better transfection efficiency than plasmid DNA injection alone but the efficacy was not as great as that by electroporation. The low energy level of ultrasound that is currently used for a diagnostic purpose and physical therapy in clinical fields can thereby increase the in vivo chemosensitivity of treated tumors but further modifications are necessary to achieve better efficacy of the ultrasound-mediated gene transfer.

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.

Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.

New polymorphic microsatellite markers in the human MHC class III region.

The human major histocompatibility complex (MHC) class III region spanning approximately 760 kb is characterized by a remarkably high gene density with 59 expressed genes (one gene every 12.9 kb). Recently, susceptibility loci to numerous diseases, such as Graves disease, Crohn disease, and SLE have been suggested to be localized to this region, as assessed by associations mainly with genetic polymorphisms of TNF and TNF-linked microsatellite loci. However, it has been difficult to precisely localize these susceptibility loci to a single gene due to a paucity to date of polymorphic markers in the HLA class III region. To facilitate disease mapping within this region, we have analyzed 2 approximately 5 bases short tandem repeats (microsatellites) in this region. A total of 297 microsatellites were identified from the genomic sequence, consisting of 69 di-, 62 tri-, 107 tetra-, and 59 penta-nucleotide repeats. It was noted that among them as many as 17 microsatellites were located within the coding sequence of expressed genes (NOTCH4, PBX2, RAGE, G16, LPAAT, PPT2, TNXB, P450-CYP21B, G9a, HSP70-2, HSP70-1, HSP-hom, MuTSH5 and BAT2). Eight microsatellite repeats were collected as polymorphic markers due to their high number of alleles (11.9 on average) as well as their high polymorphic content value (PIC) (0.63). By combining the 38 and the 22 polymorphic microsatellites we have previously collected in the HLA class I and class II regions, respectively, we have now established a total of 68 novel genetic markers which are uniformly interspersed with a high density of one every 63.3 kb throughout the HLA region. This collection of polymorphic microsatellites will enable us to search for the location of any disease susceptible loci within the HLA region by association analysis.

[Antimicrobial susceptibility of Pseudomonas aeruginosa isolated in Fukushima Prefecture].

We investigated the susceptibility of Pseudomonas aeruginosa (isolated from the sputum of patients with respiratory infection in 4 medical institutions in Fukushima Prefecture) to 8 beta-lactam antibiotics including three carbapenems and relationships among MICs of antibiotics tested. The MIC90 values for a total of 216 strains were 6.25 micrograms/ml for meropenem, 12.5 micrograms/ml for imipenem and ceftazidime, 25 micrograms/ml for panipenem and cefsulodin, 50 micrograms/ml for cefpirome and over than 200 micrograms/ml for cefoperazone and piperacillin. The frequency of resistance of these strains to each antibiotic was as follows: The resistant strains were 19 (8.8%) for meropenem, 34 (15.7%) for imipenem and ceftazidime, 50 (23.1%) for cefsulodin, 72 (33.3%) for panipenem, 76 (35.2%) for piperacillin and 90 (41.7%) for cefpirome. Eighteen strains (18.3%) of 19 meropenem resitant straisn were resistant to imipenem and panipenem, but 16 strains of the 34 imipenem-resistant strains and 54 strains of the 72 panipenem-resistant strains were susceptible to meropenem. In investigation of isolation of multi-resistant Pseudomonas aeruginosa, the susceptibility of strains tested to 7 antibiotics except cefoperazone was as follows: The strains susceptible to all the 7 antibiotics were 92 strains (42.6%), and 33 strains (15.2%) were resistant to 2 antibiotics, 31 strains (14.4%) were resistant to 1 antibiotic, 21 strains (9.7%) were resistant to 3 antibiotics, 13 strains (6.0%) were resistant to 5 antibiotics, 9 (4.2%) were resistant to 4 and 7 antibiotics, and 8 strains (3.7%) were reistant to 6 antibiotics. Since the emergence of these multi-resistant strains is closely related to frequent use of antibiotics for nosocomial infections, special attention should be paid to the antimicrobial susceptibility of Pseudomonas aeruginosa and the situation of antibiotic resistant strains.

A case of inability to belch.

A 22-year-old man was unable to belch. He could sense intraesophageal gas, but had no chest pain. An upper gastrointestinal X-ray series and endoscopic examination showed no abnormalities. Esophageal manometry showed normal relaxation of both the upper and lower esophageal sphincters with primary peristalsis during deglutition. However, bolus injection of air into the middle esophagus failed to initiate the belch reflex.

Quantitative and qualitative abnormalities in HIV-1-specific T cells.

OBJECTIVE: To assess the characteristics of CD4 and CD8 T cells specific for HIV-1 and cytomegalovirus (CMV) antigens in untreated and treated HIV-1-infected patients. METHODS: Antigen-specific T cell frequencies were determined by flow cytometric detection of antigen-induced intracellular cytokines. RESULTS: In untreated patients, HIV-1-specific CD4 T cell counts in peripheral blood were less than one tenth of CMV-specific CD4 T cell counts, while the number of specific CD8 T cells was approximately the same for both HIV-1 and CMV. In patients treated with highly active antiretroviral therapy (HAART) for less than 1.5 years, HIV-1-specific CD4 and CD8T cell counts were significantly lower than those in untreated patients. Perforin expression in HIV-1-specific CD8 T cells was significantly lower than that in CMV-specific CD8 T cells. CONCLUSION: These data indicate that HIV-1-specific T cells in HIV-1-infected patients have quantitative and qualitative abnormalities.

A review of clinical features in 13 cases of impacted primary teeth.

AIM: The aim of this study was to review clinical findings in 14 impacted primary teeth in 13 cases treated at a Paediatric Dental Clinic over a period of 18 years. METHOD: The retrospective study used clinical records, radiographs and oral photographs. Data included age, gender, presenting complaints, location, radiographic findings, aetiological factors, treatment and prognosis of impacted primary teeth and their permanent successors. RESULTS: The patients included five males and eight females aged from one year two months to seven years five months. One case had impacted bilateral, mandibular primary central incisors and the remaining 12 cases each had one impacted tooth. The maxillary second primary molar was the tooth most frequently involved. Permanent successor tooth germs were identified in 12 teeth but not in two. Five cases were impacted because of odontomas, in the case with bilaterally affected mandibular primary central incisors these were malpositioned and were erupting ectopically. In seven cases, aetiology was unknown. Four impacted primary teeth were extracted because eruption was unlikely. In four cases, odontomas were surgically removed and the teeth kept under observation. The remaining six were surgically exposed. Traction was applied in two of the six. Eight of the teeth erupted. In two teeth in which traction was used, one was subsequently extracted, and one erupted. In the cases of seven, permanent successors erupted. These were hypoplastic teeth and were delayed in development and eruption. CONCLUSIONS: Impacted primary teeth may be associated with defects in development and eruption of their permanent successors, long-term observation is therefore necessary until the permanent successors erupt.