Interleukin 33 is induced by tumor necrosis factor alpha and interferon gamma in keratinocytes and contributes to allergic contact dermatitis.

BACKGROUND: Interleukin (IL) 33, a novel member of the IL-1 family, is produced mainly by epithelial cells and endothelial cells in response to various types of stress, including necrosis. The effects of IL-33 on the immune cells involved in allergic contact dermatitis have recently been revealed in vitro. However, in vivo, the induction mechanism and function of IL-33 are not fully understood. OBJECTIVES: Our objectives were to investigate induction of IL-33 in keratinocytes and to evaluate the functions of IL-33 and its inducers in a murine model of allergic contact dermatitis. MATERIAL AND METHODS: KERTr cells, a human keratinocyte cell line, were cultured with various cytokines, including tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. IL-33 expression was detected using quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry, and Western blotting. The functions of IL-33, TNF-a, and IFN-y in allergic contact dermatitis were evaluated using a murine model. RESULTS: TNF-alpha and IFN-gamma induced expression of IL-33 mRNA and protein in KERTr cells. Blockade of IL-33 attenuated swelling in the ears of the experimental mice. Similar effects were noted for blockade of TNF-alpha and IFN-gamma in these mice. CONCLUSIONS: TNF-alpha and IFN-gamma induce expression of IL-33, and IL-33 produced by keratinocytes contributes to allergic contact dermatitis. Blockade of IL-33, TNF-alpha, and IFN-gamma could represent novel and potent strategies to treat allergic contact dermatitis.

Efficacy and safety of multitarget therapy with mizoribine and tacrolimus for systemic lupus erythematosus with or without active nephritis.

The prognosis of lupus nephritis (LN) has improved since the introduction of immunosuppressant therapies, but the safety and effectiveness of treatments can also be improved. We retrospectively assessed the treatment courses of 12 patients with systemic lupus erythematosus who were treated with glucocorticoid, mizoribine (MZR) and tacrolimus. This regimen was used as initial therapy for active LN in six patients (mean glucocorticoid dose, 66.6 mg); four of these six patients also received pulse methylprednisolone therapy. The starting doses of MZR and tacrolimus were 150 and 3 mg, respectively, and they were titrated as required. Five of six patients achieved complete remission and one achieved partial remission at 6 months. Five patients who completed 12-month analysis achieved complete remission. Another six patients were given the combination regimen for treating minor flares or for steroid sparing. The mean prednisolone doses were reduced from 11.0 mg at baseline to 6.6 mg at 12 months. Six patients experienced minor adverse events, including three minor infections. One patient stopped tacrolimus because of suspected toxicity. All 12 patients were successfully treated, and none experienced severe adverse events. Multitarget therapy combining glucocorticoid, MZR and tacrolimus may have the potential to become a treatment option which is effective and safe.

Lifestyle, weight perception and change in body mass index of Japanese workers: MY Health Up Study.

OBJECTIVES: To investigate the effect of weight perception and lifestyle on body mass index (BMI) over a 2-year period. STUDY DESIGN: Longitudinal study to compare the change in BMI (kg/m(2)) according to weight perception and lifestyle at baseline. METHODS: Study subjects were 6029 men and 18,567 women aged 20-69 years who worked at a large financial firm in Japan. Subjects' weight and height were measured in 2004 and 2006. The data in 2004 were used as baseline data. Weight perception and lifestyle factors, including eating, physical exercise, hours of sleep, smoking and alcohol consumption, were determined by a self-administered questionnaire in 2004. RESULTS: The age-adjusted mean change in BMI over the 2-year period was -0.0593 among men and 0.0890 among women. In men, subjects who perceived themselves to be overweight had a reduced BMI 2 years later compared with subjects who perceived themselves to be 'just right' or underweight. Multiple regression analysis of lifestyle factors, adjusted for age and BMI at baseline, indicated that less time spent commuting, not having a hobby, not having a fixed lunch time, consumption of sweets, smoking and colleagues' smoking were associated with increased BMI among men. Fewer hours of sleep, no fixed lunch time and frequent soft drink consumption were associated with increased BMI among women. CONCLUSIONS: A perception of being overweight was associated with a decrease in BMI for Japanese male workers. Positive lifestyle factors associated with a decrease in BMI in both men and women include having a fixed lunch time and being older. These factors should therefore be highlighted in future health promotion activities in workplaces.

Expression of keratinocyte growth factor and its receptor in odontogenic keratocysts.

BACKGROUND: The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. METHODS: The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. RESULTS: KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1alpha increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. CONCLUSION: KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1alpha.

Development of immune and microbial environments is independently regulated in the mammary gland.

Breastfeeding is important for mammals, providing immunological and microbiological advantages to neonates, together with the nutritional supply from the mother. However, the mechanisms of this functional diversity in the mammary gland remain poorly characterized. Here, we show that, similar to the gastrointestinal tract, the mammary gland develops immune and microbial environments consisting of immunoglobulin A (IgA) and the microflora, respectively, both of which are important for protecting neonates and the mother from infectious diseases. The IgA production and microflora development are coordinated in the gastrointestinal tract but seem to be independently regulated in the mammary gland. In particular, the chemokine (C-C motif) ligand 28 and poly-Ig receptor, crucial molecules for the IgA production in milk, were expressed normally in germ-free lactating mice but were almost undetectable in postweaning mothers, regardless of the microflora presence. Our findings offer insights into potentially improving the quality of breastfeeding, using both immunological and microbiological approaches.

Linear mitochondrial genomes: 30 years down the line.

At variance with the earlier belief that mitochondrial genomes are represented by circular DNA molecules, a large number of organisms have been found to carry linear mitochondrial DNA. Studies of linear mitochondrial genomes might provide a novel view on the evolutionary history of organelle genomes and contribute to delineating mechanisms of maintenance and functioning of telomeres. Because linear mitochondrial DNA is present in a number of human pathogens, its replication mechanisms might become a target for drugs that would not interfere with replication of human circular mitochondrial DNA.

Signaling pathways regulating IL-1alpha-induced COX-2 expression.

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.

Surgical results of combined pars plana vitrectomy, phacoemulsification, and intraocular lens implantation.

PURPOSE: To evaluate the results and complications of combined pars plana vitrectomy (PPV), phacoemulsification and aspiration (PEA), and intraocular lens (IOL) implantation. METHODS: A total of 117 eyes from 114 patients who had undergone PPV combined with PEA and IOL implantation were retrospectively analyzed. Combined surgery was performed for a wide variety of vitreoretinal diseases. Intraoperative and postoperative complications were also reviewed. RESULTS: The postoperative BCVA improved by 2 lines or more in 85 eyes (72.6%). Intraoperative complications consisted of retinal tears in 14 eyes (12.0%) and posterior capsular rupture in 2 eyes (1.7%). Iatrogenic retinal tears occurred more frequently in eyes with a macular hole than in eyes with any other disease (p=0.005, chi-square test). Postoperative complications consisted of posterior capsule opacification (PCO) (21 eyes), transient IOP elevation (29 eyes), vitreous hemorrhage (6 eyes), anterior chamber fibrin exudation (11 eyes), posterior iris synechia (8 eyes), neovascular glaucoma (1 eye), and recurrent retinal detachment (RD) (2 eyes). Fibrin exudation occurred more frequently in eyes with proliferative diabetic retinopathy (PDR) and RD than in eyes with any other disease (p=0.03, chi-square test). PCO occurred more frequently in eyes with PDR than in eyes with any other disease (p=0.03, chi-square test). CONCLUSIONS: The present study suggests that a high success rate can be achieved when recently improved PPV techniques are combined wi th PEA and IOL implantation. The complications that were observed following this combined treatment varied with respect to the vitreoretinal disease present prior to surgery.

Regulated tRNA import in Leishmania mitochondria.

The genes for three new tRNA and a 5S RNA were identified from a genomic DNA clone of 917 nucleotide pairs from the protozoon Leishmania tarentolae. They were encoded in the following order. The transcriptional directions and anticodons are in parentheses: tRNA(Val) (CAC-->)-5SRNA (-->)-tRNA(His) (<--GUG)-tRNA(Phe) (GAA-->). The tRNA(His) and tRNA(Phe) sequences have not been reported previously in trypanosomatid organisms. By northern analysis, tRNA(Val) and tRNA(Phe) were equally distributed between the cytosol and mitochondria, while tRNA(His) was less abundant in mitochondria than in the cytosol. Accordingly, the latter tRNA is classified as Import restricted (Impr). As shown before, 5S RNA was not imported. Recently, Mahapatra and Adhya [S. Mahapatra, T. Ghosh, S. Adhya, Nucl. Acids Res. 22 (1994) 3381-3386; S. Mahapatra, S. Adhya, J. Biol. Chem. 271 (1996) 20432-20437] have developed an in vitro import system in Leishmania and suggested that the D-loop sequence could serve as the import determinant. We examined all available tRNA gene sequences in trypanosomatids but found no apparent consensus within the D-loop that might account for tRNA-import regulation.

Accumulation of nuclear-encoded tRNA(Thr) (AGU) in mitochondria of the liverwort Marchantia polymorpha.

The mitochondrial genome of the liverwort Marchantia polymorpha does not encode the full complement of tRNAs for the threonine and isoleucine codon boxes. To find the missing tRNA genes specifically for tRNA(Thr) in mitochondria, we have searched the genomic library and identified two clones (pTT1 and pTT2), encoding the identical tRNA(Thr) (AGU) gene copy with different 5'- and 3'-flanking sequences. By northern analysis, we demonstrate considerable accumulation of the nuclear encoded tRNA(Thr) and moderate expression of native tRNA(Thr) (GGU) in mitochondria. Nonetheless, the imported and native tRNA(Thr) species together are not sufficient to translate all four threonine codons used in liverwort mitochondria, implicating mitochondrial import of at least one additional threonine isoacceptor tRNA.

Scanning electron microscopy of the epidermal lamina densa in normal human skin.

The lamina densa of normal human epidermis was exposed by treatment with 1 M sodium chloride and was examined by high-power scanning electron microscopy before and after trypsinization. Localization of type IV collagen in the lamina densa was also studied by transmission and scanning immunoelectron microscopy. Before trypsinization, the surface of the lamina densa consisted of microridges and microvalleys. The microridges varied in height and were connected with each other. They were arranged in a concentric fashion around the tips of the dermal microprojections. At a higher magnification, the surface of the lamina densa was composed of densely packed cobblestone-like structures approximately 7-15 nm in size, between which were interspaces 4-11 nm wide. These structures expressed type IV collagen. After trypsinization, the lamina densa was found to be composed of microfilaments approximately 10 nm thick showing beaded appearances. These microfilaments exhibited the same cobblestone-like structures as the lamina densa surface. Observation of the torn lamina densa demonstrated anchoring fibrils and oxytalan fibers that were attached to the lamina densa itself. Another kind of filament about 7 nm thick linked the anchoring fibrils and the oxytalan fibers. Beneath the lamina densa was a network of fibers about 40-50 nm thick, which was composed of collagen fibers and possibly also elaunin fibers. In conclusion, this study revealed the detailed surface ultrastructure of the epidermal lamina densa and its underlying filamentous elements.

Sodium excretion in relation to calcium and hydroxyproline excretion in a healthy Japanese population.

To evaluate whether habitual excess sodium intake is a significant risk factor for calcium loss, we studied the relation between calcium excretion and sodium excretion in 410 male and 476 female Japanese aged 20-79 y. They were apparently healthy, free-living, and consuming diets of their own choosing. We divided the subjects into two groups: 20-49 y olds and 50-79 y olds. In each group, we observed significant positive correlation between daily calcium excretion and daily sodium excretion in both sexes. Multivariate analyses revealed that in each age group the relation was still significant after sex, age, body weight, and protein, calcium, and phosphorus intakes were adjusted for. The increases in urinary calcium excretion were estimated to be approximately 0.6 and 1.0 mmol for a 100-mmol increment in urinary sodium excretion for the 20-49 y olds and 50-79-y olds, respectively. We also observed significant positive correlations between daily hydroxyproline excretion and daily sodium excretion in both sexes for both age groups. The relation was still significant after sex, age, body weight, and protein intake from meat and fish were adjusted for. The results suggest that individuals with high sodium intake may lose more calcium in their urine than those with low sodium intake.

Dietary protein intake and urinary excretion of calcium: a cross-sectional study in a healthy Japanese population.

To evaluate whether habitual excess protein intake is a significant risk factor for calcium loss, we studied the relation between urinary excretion of calcium and protein intakes, in 349 male and 406 female Japanese aged 20-79 y. The subjects were apparently healthy, free-living, and consuming diets of their own choosing. We divided the subjects into two groups: those aged 20-49 y and those aged 50-79 y. In each group, we observed a significant positive correlation between daily urinary excretion of calcium and protein intake. Calcium excretion also correlated positively with daily urinary excretion of urea. Multivariate analyses revealed that in each age group the relation between calcium excretion and urea excretion remained significant even after sex, age, body weight, urinary sodium excretion, and calcium intake were adjusted for. The correlation of calcium excretion with animal protein intake was significantly positive in both sexes and in each age group whereas that with plant protein was not. We observed a significant positive correlation between daily calcium excretion and daily urinary excretion of sulfate. The correlation in 50-79-y old subjects remained significant even after sex, age, body weight, sodium excretion, and calcium intake were adjusted for. Our findings suggest that excess protein, especially that rich in sulfur-containing amino acids, in habitual diets may augment calcium excretion in the urine, at least in the elderly.

In vivo expression and mitochondrial import of normal and mutated tRNA(thr) in Leishmania.

Evidence suggests that mitochondria of protozoans and plants contain nuclear-encoded tRNAs. In trypanosomatids, the entire set of tRNAs in the mitochondria are presumably imported from the nucleus, but the mechanism of tRNA import is not presently understood. In this study, we have employed a plasmid-encoded nuclear tRNA gene as a means of investigating tRNA expression and mitochondrial import in vivo in Leishmania tarentolae. Using a Leishmania plasmid, we cloned a 1-kb or 250-bp restriction fragment carrying the nuclear tRNA(thr) gene and three in vitro mutagenized derivatives: Tac6 (an insertion of 6 nucleotides at the anticodon loop), Td4 (a 4-nt insert at the D-loop) and Tv4 (a 4-nt insert at the variable arm). Leishmania cells stably transfected with these plasmids were then examined for tRNA expression and import by Northern analysis. The results show that the plasmid-encoded wild type tRNA(thr) gene produced a significantly elevated level of expression in the cytosol. Similarly, the Tac6-transfected cells exhibited a large abundance of the mutant RNA relative to the normal tRNA (chromosome-encoded gene transcripts) in the cytosol. Furthermore, the mutant Tac6 RNA was found imported into mitochondria, although the proportion of the mutant vs. normal tRNA in mitochondria was greatly reduced as compared to that in the cytosol. We suggest that the mitochondrial import machinery is capable of discriminating against the mutant RNA in favor of the normal tRNA for import. In another example, we found that the Tv4 gene showed expression, albeit somewhat reduced, but its import into mitochondria was completely blocked. Unexpectedly, the 4-base addition mutation (Td4) at the D-loop showed neither expression nor import. While these results clearly signify the importance of various segments within the tRNA gene for in vivo expression, our data underscore the significance of the variable loop for mitochondrial import. It is our belief that this plasmid-encoded tRNA gene expression system in Leishmania may be useful in gaining further insights on tRNA import.

A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria.

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA(leu)(CAG) and tRNA2(arg)(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA(ile)(TAT) and tRNA1(lys)(TTT) are abundant in mitochondria, but relatively scarce in the cytosol. tRNA(val)(TAC) and tRNA2(thr)(TGT) are barely detectable in either cellular compartment, while tRNA(gln)(TTG), tRNA1(arg)(ACG), tRNA(gly)(TCC), and tRNA(trp)(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA(trp)(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.

Genomic organisation of nuclear tRNAGly and tRNALeu genes in Trypanosoma brucei.

We have isolated a 0.3-kb HaeIII restriction fragment from Trypanosoma brucei which contains two tRNA genes. Secondary structure models predict that the two genes identified encode tRNA molecules which specify glycine (anticodon UCC) and leucine (anticodon CAG). The two genes are separated by 86 nucleotides, transcribed in the same direction and contain features of conventional RNA polymerase III transcription units. Southern blot analysis indicates the presence of multicopy tRNa gene families in T. brucei.

Selective import of nuclear-encoded tRNAs into mitochondria of the protozoan Leishmania tarentolae.

The trypanosomatid mitochondrial genome does not encode tRNA genes at all and experimental evidence obtained with Leishmania tarentolae shows that tRNAs in mitochondria represent a selected set of imported nuclear-encoded tRNAs. In this paper we present the data showing that tRNAs derived from the clustered genomic tRNA genes are invariably imported into mitochondria, while tRNA from the solitary gene is not. By sequencing a cosmid DNA clone of L. tarentolae genomic DNA, we have identified a 1.5-kb subclone encoding a duplicate set of the closely linked tRNA(Tyr) (GTA) and tRNA(Thr) (AGT) genes. Northern analysis shows that these tRNAs are imported into mitochondria. In contrast, when the tRNA gene [tRNA(Gln) (CUG)] located alone in a 40-kb DNA fragment was examined, the corresponding tRNA was not detected in the mitochondrion. This "loner" tRNA gene is highly unusual since the 3'-flanking putative RNA polymerase III transcription termination signal sequence is characterized by a long string of 8 Ts followed by an A and a stretch of 7 Cs, while all other trypanosomatid tRNA genes whose tRNA transcripts are imported are terminated by a possible transcription termination signal of only 4-6 Ts. Whether the correlation found between the gene organization and tRNA-import characteristics is of general significance needs to be investigated further. A simple computer analysis presented in this paper rules out the possibility that tRNAs found in the trypanosomatid mitochondrion are the products of the U-addition type 'RNA editing' of maxicircle DNA.

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi.

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.