Selective release of miRNAs via extracellular vesicles is associated with house-dust mite allergen-induced airway inflammation.

BACKGROUND: MicroRNAs (miRNAs) may facilitate cell-to-cell communication via extracellular vesicles (EVs). The biological roles of miRNAs in EVs on allergic airway inflammation are unclear. METHODS: Airway-secreted EVs (AEVs) were isolated from bronchoalveolar lavage fluid (BALF) of control and house-dust mite (HDM) allergen-exposed HDM-sensitized mice. The expression of miRNAs in AEVs or miRNAs and mRNAs in lung tissue was analysed using miRNA microarray. RESULTS: The amount of AEV increased 8.9-fold in BALF from HDM-exposed mice compared with that from sham-control mice. HDM exposure resulted in significant changes in the expression of 139 miRNAs in EVs and 175 miRNAs in lung tissues, with 54 miRNAs being common in both samples. Expression changes of these 54 miRNAs between miRNAs in AEVs and lung tissues after HDM exposure were inversely correlated. Computational analysis revealed that 31 genes, including IL-13 and IL-5Ra, are putative targets of the miRNAs up-regulated in AEVs but down-regulated in lung tissues after HDM exposure. The amount of AEV in BALF after HDM exposure was diminished by treatment with the sphingomyelinase inhibitor GW4869. The treatment with GW4869 also decreased Th2 cytokines and eosinophil counts in BALFs and reduced eosinophil accumulation in airway walls and mucosa. CONCLUSION: These results indicate that selective sorting of miRNA including Th2 inhibitory miRNAs into AEVs and increase release to the airway after HDM exposure would be involved in the pathogenesis of allergic airway inflammation.

Expression of NF-kappaB in epidermis and the relationship between NF-kappaB activation and inhibition of keratinocyte growth.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-kappaB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation. OBJECTIVES: To evaluate the role of NF-kappaB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-kappaB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth. METHODS: The expression of subunits of the NF-kappaB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-kB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by 3H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-kappaB was assessed using sodium salicylate, which inhibits NF-kappaB activation. RESULTS: The NF-kappaB subunits, p50, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor alpha, and interferon gamma each activated NF-kappaB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-kappaB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca2+) and retinoic acid each failed to activate NF-kappaB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate. CONCLUSIONS: NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.

Epidermal Langerhans cell-targeted gene expression by a dectin-2 promoter.

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5'-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Molecular cloning of a dendritic cell-associated transmembrane protein, DC-HIL, that promotes RGD-dependent adhesion of endothelial cells through recognition of heparan sulfate proteoglycans.

We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells, but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore, DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial migration of DC.

Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms.

Using a subtractive cDNA cloning strategy, we isolated previously five novel genes that were expressed abundantly by the murine dendritic cell (DC) line XS52, but not by the J774 macrophage line. One of these genes encoded a unique, DC-associated C-type lectin, termed "dectin-1." Here we report the characterization of a second novel gene that was also expressed in a DC-specific manner. Clone 1B12 encoded a type II membrane-integrated polypeptide of 209 amino acids containing a single carbohydrate recognition domain motif in the COOH terminus. The expression pattern of this molecule, termed "dectin-2," was almost indistinguishable from that for dectin-1; that is, both were expressed abundantly at mRNA and protein levels by the XS52 DC line, but not by non-DC lines, and both were detected in spleen and thymus, as well as in skin resident DC (i.e. Langerhans cells). Interestingly, reverse transcriptase-polymerase chain reaction and immunoblotting revealed multiple bands of dectin-2 transcripts and proteins suggesting molecular heterogeneity. In fact, we isolated additional cDNA clones encoding two distinct, truncated dectin-2 isoforms. Genomic analyses indicated that a full-length dectin-2 (alpha isoform) is encoded by 6 exons, whereas truncated isoforms (beta and gamma) are produced by alternative splicing. We propose that dectin-2 and its isoforms, together with dectin-1, represent a unique subfamily of DC-associated C-type lectins.

Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

[Anatomical names of fossae and foveae in skeleton].

Latin anatomical names of Fossae and Foveae in the skeleton were analyzed and compared with Japanese anatomical names for better understanding of the structures of the human body and for possible revision in the future. The conclusions were as follows: 1. In general, round excavations were called Foveae (singular : Fovea), and nonround excavations were called Fossae (singular : Fossa). Some shallow excavations for articulation and some shallow excavations with the names which indicate their contents were called Foveae even though they were not round. 2. Each name of Fossae contained the word which indicates form, location or content of Fossa, the bone (or osseous structure) which articulates with Fossa, or the muscle which is attached to Fossa. 3. Each name of Foveae contained the word which indicates location, content or articulation of Fovea, the bone (or osseous structure) which articulates with Fovea, or the muscle (or muscular trochlea) which is attached to Fovea. 4. The Japanese name which corresponds to Fossa canina should be changed from Kenshi (canine tooth) = ka (fossa) to Kenshikin (canine muscle) = ka or Koukakukyokin (levator anguli oris muscle) = ka. 5. The Japanese name which corresponds to Fossa pterygopalatina should be changed from Yoku (wing) = kougai (palate) = ka (fossa) to Yokutotsu (pterygoid process) = kougaikotsu (palatine bone) = ka.

Mitochondrial function in Babesia microti and Babesia rodhaini.

The role of mitochondria in the energy metabolism of Babesia microti and Babesia rodhaini was investigated. A variety of mitochondrial inhibitors showed greater sensitivity to B. microti than to B. rodhaini. Additionally, alpha-glycerophosphate- and succinate-cytochrome c reductase activities in the crude mitochondrial fraction from B. microti were substantially higher than those from B. rodhaini. Our results suggest that the mitochondria of these parasites possess a series of "classical" apparati for energy production and their relative functional role may be quantitatively greater in B. microti when compared with B. rodhaini.

[Anatomical names of foramina and canales in skeleton].

Latin anatomical names of Foramina and Canales in skeleton were analyzed and compared with Japanese anatomical names for better understanding of the structures of the human body and for possible revision in the future. The conclusions were as follows: 1. In general, short tunnels were called Foramina (singular: Foramen), and long tunnels Canales (singular: Canalis). 2. One end of Canalis was sometimes called Foramen. In this case, Canalis and Foramen were usually modified by the same words. 3. Each name of Foramina contained the word which means form, state, absolute size, region of existence, one of the contents or function of Foramina. 4. Each name of Canales contained the word which means region of existence, one of the contents or function of Canales. 5. Some names of Foramina and Canales that were supposed to mean the region of existence meant one of the contents of the structures. 6. As for Latin anatomical names, the relation between words were relatively clear by the proper use of noun, adjective, nominative, and genitive. 7. Since different Chinese characters were sometimes pronounced similarly in Japanese anatomical names, different structures might be confused. 8. It seemed that some Japanese anatomical names needed partial correction.

Adjectives that have different meanings in various Latin anatomical names, and expression of these adjectives in Japanese anatomical names.

In order to understand the role of Latin adjectives, international anatomical names containing the same adjectives were chosen from among the names of structures in the head and neck: these names were assorted into groups according to the actual meanings of the adjectives. When the Latin adjectives indicated the name of the structure, they signified: belonging to the structure, entering into the formation of the structure, articulating with the structure, transmitting the structure, giving attachment to the structure, or some other relationship to the structure. And, furthermore, the structures that were indicated by the same Latin adjectives might be different. In the Japanese language, those adjectives that have different meanings in other internationally accepted anatomical names were sometimes translated into different words. Because of this, some Japanese anatomical names were more concrete than the corresponding Latin anatomical names. It seemed that compound words and abbreviations, which can be formed easily in the Japanese language, made such expression possible.

Advances in soil microbial ecology and the biodiversity.

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection (EC). Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections. A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.

The official nomenclature of the articular facets for the rib on the thoracic vertebra: a case for revision.

In Nomina Anatomica 6th ed., the articular facets for the rib on the thoracic vertebra are called Fovea costalis superior, Fovea costalis inferior, and Fovea costalis processus transversi respectively. But, there is a little problem about the names of Fovea costalis superior and Fovea costalis inferior on the body of vertebra, because usually only one facet exists on each side of the body of 10th to 12th (or 9th to 12th) thoracic vertebra. This single facet on the body of vertebra should be called just Fovea costalis.

[Histological observations on structure of bundle bone in area of mylohyoid line].

The purpose of this study was to investigate the structure and the function of the part of the mandible where mylohyoid muscle originates. The bundle bone in the area of mylohyoid line and the tendon fibers on the surface of the bundle bone were observed by light microscopy and scanning and transmission electron microscopy. The conclusions were as follows: 1. It seemed that remodeling occurs frequently in bundle bone in the area of the mylohyoid line. 2. Sharpey's fibers in bundle bone in the area of the mylohyoid line seemed to be flexible. This suggested that Sharpey's fibers can be adapted to the tension from directions that are different to a certain degree. 3. Both directions of Sharpey's fibers in bundle bone in the area of the mylohyoid line and the tendon fibers on the surface of bundle bone were fundamentally the same as that of the muscle fibers of the mylohyoid muscle. But a few existing fibers crossed these fibers. Such Sharpey's fibers and tendon fibers seemed to prevent the exfoliation of the periosteum, and seemed to support main tendon fibers of the mylohyoid muscle when it is contracting.

Effects of depletion of T cell subpopulations on the course of infection and anti-parasite delayed type hypersensitivity response in mice infected with Babesia microti and Babesia rodhaini.

To elucidate the role of T cell subpopulations in the protective cell-mediated immune response at the initial phase of infection with Babesia microti (BM) and B. rodhaini (BR), the changes in the course of infection and anti-parasite delayed type hypersensitivity (DTH) response after BM or BR inoculation were investigated in Lyt-2+ T cell or L3T4+ T cell-depleted mice. Depletion of Lyt-2+ T cells strongly enhanced the resistance to BM infection, whereas it increased the susceptibility to BR infection. In contrast, depletion of L3T4+ T cells increased susceptibility to BM infection, while it enhanced resistance to BR infection. The anti-parasite DTH response in BM-infected mice was significantly enhanced by depletion of Lyt-2+ T cells, while significantly reduced by depletion of L3T4+ T cells. No effects of depletion of either Lyt-2+ or L3T4+ cells on DTH response was observed in BR-infected mice. From these results, it was suggested that the roles of Lyt-2+ and L3T4+ T cells in the protective cell-mediated immune response at the initial phase of infection were different between BM- and BR-infected mice, resulting in the difference in their course of infection.

A short term in vitro cultivation of Babesia rodhaini and Babesia microti.

In vitro cultivation of Babesia rodhaini (BR) and Babesia microti (BM) was attempted. When RPMI1640 was supplemented with 30 or 40% of non-treated fetal bovine serum (FBS), the gas mixture of 3% CO2-8% O2 best supported the growth of both parasites. Under this optimized condition, the percent parasitized erythrocytes peaked to approximately 4- and 2-times initial values for Br and BM, respectively. The cultivated parasites retained the infectivity to the host mice. BM showed the characteristic feature of division during cultivation. However, the lots of FBS will have to be taken into consideration, since the FBS lots were shown to give large varieties to the results. Selection of the appropriate FBS lot may yield the better growth of these protozoa.

Isoenzyme patterns of glucose-6-phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase in Babesia rodhaini and Babesia microti.

Glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) activities and their isoenzyme patterns in B. rodhaini (BR) and B. microti (BM), the two major causative species of murine babesiosis, were examined. G6PD and LDH activities were higher in BR than those in BM, whereas MDH activity was lower in BR than that in BM. No differences were observed between BR and BM in the mobility of isoenzyme bands of G6PD and MDH. On LDH isoenzyme pattern, at least 5 bands were detected in BR, while only one band in BM. Since each subunit of LDH is known to be coded by different gene, these results suggests that BR and BM are able to be differentiated genetically.

Enzyme activities related to glucose metabolism in Babesia microti and Babesia rodhaini.

A comparative study was carried out on the glucose metabolism in Babesia microti (BM) and Babesia rodhaini (BR) by analyzing the enzyme activities. The lactate dehydrogenase (LDH) activity in BM showed significantly lower values than that in BR, whereas citrate synthase (CS) and malate dehydrogenase (MDH) activities were remarkably higher in BM. In addition, pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), alpha-ketoglutarate dehydrogenase (KGDH), and succinate dehydrogenase (SDH) activities also tended to be higher in BM. Then, the change of enzyme activities related to the proliferation of parasites was examined. In BM infected mice, the parasitemia increased from day 15 to day 19 after inoculation (a.i.). While BM showed decrease of G6PD and LDH activities at day 19 a.i., it showed remarkably increased activities in CS and MDH (368 and 8,842 nmol/min.mg protein, respectively). In addition, PDH, ICDH, KGDH, and SDH activities also tended to increase from day 15 to 19 a.i. In BR infected mice, parasitemia increased from day 9 to day 12 a.i. LDH activity showed a considerable increase at day 12 a.i. (12,920 IU/mg.protein). Although CS and MDH activities also showed a slight increase at day 12 a.i., the activities of PDH, ICDH, KGDH and SDH didn't change from day 9 to 12 a.i. Since these changes observed in the enzyme activities of BM and BR seemed to be correlated with their proliferation, it was suggested that BM and BR depended on aerobic and anaerobic pathways, respectively, for their glucose metabolism.

Tissue residues of sulfadimethoxine following dietary administration to broiler-chickens.

Broilers were divided into four groups and the first group served as the control. The second, third and fourth groups were given feed containing 25, 50 and 100 mg/kg of sulfadimethoxine (SDM), respectively, for 21 days, and thereafter each group received the SDM free feed. On certain days during the experiment period, three broilers in each group were sacrificed and tissues, including blood, heart, liver, spleen, gizzard, thigh muscle, breast muscle and fat, were collected and residual SDM were determined by HPLC. Two days after withdrawal, SDM in each tissue had decreased to below the detection limit of 0.01 microgram/g.