Lymphaticovenular anastomosis to prevent cellulitis associated with lymphoedema.

BACKGROUND: One of the complications of lymphoedema is recurrent cellulitis. The aim was to determine whether lymphaticovenous anastomosis (LVA) was effective at reducing cellulitis in patients with lymphoedema. METHODS: This was a retrospective review of patients with arm/leg lymphoedema who underwent LVA. The frequency of cellulitis was compared before and after surgery. The diagnostic criteria for cellulitis were a fever of 38·5°C or higher, and warmth/redness in the affected limb(s). RESULTS: A total of 95 patients were included. The mean number of episodes of cellulitis in the year preceding surgery was 1·46, compared with 0·18 in the year after surgery (P < 0·001). CONCLUSION: LVA reduced the rate of cellulitis in these patients with lymphoedema.

Close relationship between T helper (Th)17 and Th2 response in murine allergic contact dermatitis.

BACKGROUND: Repeated exposure to allergens induces chronic allergic lesions in the skin and a shift in the cutaneous cytokine milieu to T helper (Th)2. AIM: To assess the relationships between Th17 and Th2 response during allergic contact dermatitis (ACD) in mice. METHODS: ACD was induced in C57BL/6 mice by single or repeated epicutaneous challenge of 2,4,6-trinitro-1-chlorobenzene. Relationships between Th17 and Th2 response were analyzed by immunohistochemical observations and activity of cytokines on days 8 (first challenge), 18 (11th challenge), 28 (21st challenge) and 38 (31st challenge). RESULTS: On day 8, tissue levels of interleukin (IL)-17 and IL-22 were high, whereas tissue levels of IL-4 and serum IgE concentration were low. Following acute contact dermatitis, mice developed chronic eczematous lesions on day 18, and gradually improved on days 28 and 38. Tissue IL-4 and serum IgE levels corresponded to the development and improvement of chronic eczematous lesions. Numbers of Th17 cells and tissue levels of IL-17 and IL-22 rapidly decreased as IL-4 and IgE levels increased on day 18. As levels of IL-4 and IgE decreased, the number of Th17 cells and tissue levels of IL-17 and IL-22 increased again on days 28 and 38. On day 18, tissue levels of Th17 response-inducing cytokines (IL-6, IL-23 and transforming growth factor-ß) were high, and IL-23-expressing cells appeared in abundance, when Th2 response was extremely high. IL-17 injection decreased tissue IL-4 and serum IgE levels. CONCLUSIONS: Th17 correlates closely with Th2 in murine chronic ACD induced by repeated epicutaneous challenge.

In vitro screening of lactobacilli isolated from chicken excreta to control Salmonella Enteritidis and Typhimurium.

1. The aim of this work was to select lactic acid bacteria (LAB) strains from chicks and hens of egg-laying strains for potential use to control Salmonellae. 2. Nineteen LAB strains obtained from culture collections, and 24 strains isolated from excreta of laying hens and chicks, were evaluated for inhibitory capacities against two Salmonella serotypes using a "Spot-the-lawn" technique and other in vitro properties that could be predictive of antimicrobial activity. 3. The size of the inhibition zone differed slightly between Salmonella serotypes, however, the mean size of the Salmonella inhibition zone differed greatly among the LAB strains. Lactobacillus salivarius, L. plantarum, L. rhamnosus and L. reuteri exhibited powerful inhibitory effects to each Salmonella strain. 4. The result of the acid tolerance test showed that all L. salivarius, L. kitasatonis strains and each of L. ingluviei cannot survive in a low pH environment. In the bile acid tolerance assay, growth was inhibited in all strains, except L. kitasatonis HE4, and a large inhibition was observed in most of the L. salivarius and L. crispatus strains. 5. The results demonstrate that some LAB of poultry origin were able to inhibit the growth of Salmonella and survive simulated passage through the gastrointestinal tract. The selected LAB could act in the lower gastrointestinal tract to prevent salmonellosis in poultry.

Histamine H(4) receptor antagonist ameliorates chronic allergic contact dermatitis induced by repeated challenge.

BACKGROUND: The present study observed effects of the histamine H(4) receptor on chronic allergic contact dermatitis induced by repeated challenge in mice. METHODS: Acute contact dermatitis was induced by single epicutaneous challenge of 2,4,6-trinitro-1-chlorobenzene (TNCB) to the ear. Chronic allergic contact dermatitis was developed by repeated epicutaneous challenge using TNCB on the dorsal back skin. H(4) receptor antagonist JNJ7777120 was administered to wild-type mice, while H(4) receptor agonist 4-methylhistamine was administered to histidine decarboxylase (HDC) (-/-) mice that synthesized no histamine. RESULTS: HDC (-/-) mice did not differ phenotypically from HDC (+/+) mice, and H(4) receptor antagonist/agonist did not have clinical effects in terms of acute contact dermatitis reactions. H(4) receptor antagonist ameliorated skin eczematous lesions induced by repeated TNCB challenge in HDC (+/+) mice. On the contrary, H(4) receptor agonist exacerbated skin lesions exclusively in HDC (-/-) mice. Application of H(4) receptor agonist induced migration of mast cells and eosinophils in skin lesions, and H(4) receptor antagonist suppressed these changes. H(4) receptor was immunohistochemically detected on mast cells in eczematous lesions. Levels of interleukin (IL)-4, -5, and -6 in lesions were decreased, whereas levels of interferon-gamma and IL-12 were increased by H(4) receptor antagonistic activity. Serum Immunoglobulin E levels rapidly increased with repeated challenge, but decreased with H(4) receptor antagonist. CONCLUSION: Because chronic allergic contact dermatitis is developed by H(4) receptor stimulation, H(4) receptor antagonists might represent new candidate drugs for treating chronic allergic contact dermatitis.

Histamine plays an essential regulatory role in lung inflammation and protective immunity in the acute phase of Mycobacterium tuberculosis infection.

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Recent data suggest a regulatory role of histamine not only in the innate but also in the adaptive immune response. We used a model of pulmonary Mycobacterium tuberculosis infection in histamine-deficient mice lacking histidine decarboxylase (HDC(-/-)), the histamine-synthesizing enzyme. To confirm that mycobacterial infection induced histamine production, we exposed mice to M. tuberculosis and compared responses in C57BL/6 (wild-type) and HDC(-/-) mice. Histamine levels increased around fivefold above baseline in infected C57BL/6 mice at day 28 of infection, whereas only small amounts were detected in the lungs of infected HDC(-/-) mice. Blocking histamine production decreased both neutrophil influx into lung tissue and the release of proinflammatory mediators, such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), in the acute phase of infection. However, the accumulation and activation of CD4(+) T cells were augmented in the lungs of infected HDC(-/-) mice and correlated with a distinct granuloma formation that contained abundant lymphocytic infiltration and reduced numbers of mycobacteria 28 days after infection. Furthermore, the production of IL-12, gamma interferon, and nitric oxide, as well as CD11c(+) cell influx into the lungs of infected HDC(-/-) mice, was increased. These findings indicate that histamine produced after M. tuberculosis infection may play a regulatory role not only by enhancing the pulmonary neutrophilia and production of IL-6 and TNF-alpha but also by impairing the protective Th1 response, which ultimately restricts mycobacterial growth.

Pravastatin attenuates allergic airway inflammation by suppressing antigen sensitisation, interleukin 17 production and antigen presentation in the lung.

BACKGROUND: Statins are widely used to treat hyperlipidaemia. Their immunosuppressive effect has recently been confirmed in various immune mediated disease models. However, relatively few studies have been conducted on allergic inflammation, so the precise mechanisms of their actions against allergies have not been fully clarified. On the other hand, the role of interleukin (IL)17 in immune responses has been recently highlighted, but whether statins affect IL17 production has not been well studied. The effect of pravastatin on allergic airway inflammation in a mouse model was examined to elucidate the mechanism of action, focusing on its effect on IL17 production. METHODS: BALB/c mice were immunised with ovalbumin (OVA) and then challenged with OVA aerosol. Pravastatin was delivered by intraperitoneal injection during either sensitisation or the challenge. RESULTS: When delivered during systemic sensitisation, pravastatin suppressed OVA induced proliferation and production of Th2 type cytokines such as IL5 in spleen cells ex vivo and in vitro. IL17 production was also suppressed. Furthermore, pavastatin delivered during the inhalation of OVA attenuated eosinophilic airway inflammation, OVA specific IgE production in serum and OVA induced IL17 production in the thoracic lymph node. We also found that pravastatin attenuated the antigen presenting capacity of CD11c(+) cells obtained from the OVA challenged lung. CONCLUSION: Pravastatin suppresses the systemic sensitisation to allergen with downregulation of IL17 production. It also suppresses an ongoing immune response in the airway partly by suppressing antigen presentation in the lung. Therefore, statins could be a novel therapeutic option for treatment of asthma.

The brain H3-receptor as a novel therapeutic target for vigilance and sleep-wake disorders.

Brain histaminergic neurons play a prominent role in arousal and maintenance of wakefulness (W). H(3)-receptors control the activity of histaminergic neurons through presynaptic autoinhibition. The role of H(3)-receptor antagonists/inverse agonists (H(3)R-antagonists) in the potential therapy of vigilance deficiency and sleep-wake disorders were studied by assessing their effects on the mouse cortical EEG and sleep-wake cycle in comparison to modafinil and classical psychostimulants. The H(3)R-antagonists, thioperamide and ciproxifan increased W and cortical EEG fast rhythms and, like modafinil, but unlike amphetamine and caffeine, their waking effects were not accompanied by sleep rebound. Conversely, imetit (H(3)R-agonist) enhanced slow wave sleep and dose-dependently attenuated ciproxifan-induced W, indicating that the effects of both ligands involve H(3)-receptor mechanisms. Additional studies using knockout (KO) mice confirmed the essential role of H(3)-receptors and histamine-mediated transmission in the wake properties of H(3)R-antagonists. Thus ciproxifan produced no increase in W in either histidine-decarboxylase (HDC, histamine-synthesizing enzyme) or H(1)- or H(3)-receptor KO-mice whereas its waking effects persisted in H(2)-receptor KO-mice. These data validate the hypothesis that H(3)R-antagonists, through disinhibition of H(3)-autoreceptors, enhancing synaptic histamine that in turn activates postsynaptic H(1)-receptors promoting W. Interestingly amphetamine and modafinil, despite their potent arousal effects, appear unlikely to depend on histaminergic mechanism as their effects still occurred in HDC KO-mice. The present study thus distinguishes two classes of wake-improving agents: the first acting through non-histaminergic mechanisms and the second acting via histamine and supports brain H(3)-receptors as potentially novel therapeutic targets for vigilance and sleep-wake disorders.

Histamine H3R receptor activation in the dorsal striatum triggers stereotypies in a mouse model of tic disorders.

Tic disorders affect ~5% of the population and are frequently comorbid with obsessive-compulsive disorder, autism, and attention deficit disorder. Histamine dysregulation has been identified as a rare genetic cause of tic disorders; mice with a knockout of the histidine decarboxylase (Hdc) gene represent a promising pathophysiologically grounded model. How alterations in the histamine system lead to tics and other neuropsychiatric pathology, however, remains unclear. We found elevated expression of the histamine H3 receptor in the striatum of Hdc knockout mice. The H3 receptor has significant basal activity even in the absence of ligand and thus may modulate striatal function in this knockout model. We probed H3R function using specific agonists. The H3 agonists R-aminomethylhistamine (RAMH) and immepip produced behavioral stereotypies in KO mice, but not in controls. H3 agonist treatment elevated intra-striatal dopamine in KO mice, but not in controls. This was associated with elevations in phosphorylation of rpS6, a sensitive marker of neural activity, in the dorsal striatum. We used a novel chemogenetic strategy to demonstrate that this dorsal striatal activity is necessary and sufficient for the development of stereotypy: when RAMH-activated cells in the dorsal striatum were chemogenetically activated (in the absence of RAMH), stereotypy was recapitulated in KO animals, and when they were silenced the ability of RAMH to produce stereotypy was blocked. These results identify the H3 receptor in the dorsal striatum as a contributor to repetitive behavioral pathology.

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region.

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.

Induction of L-histidine decarboxylase in a human mast cell line, HMC-1.

Histamine is an important mediator in allergic reactions, gastric acid secretions, and neurotransmission in the central nervous system. Basophils and mast cells are the main sources of histamine, which is formed from L-histidine by histidine decarboxylase (HDC). However, the regulatory mechanism of HDC in these cells remains unclear. We examined the regulation of HDC activity and gene expression using a unique human mast cell line, HMC-1, after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin. HDC activity was increased from 52.1+/-0.4 (mean+/-standard deviation) to 154+/-6.9, or 105.6+/-6.2 pmol/min/mg protein (n = 3), 4 hours after stimulation with PMA (10 ng/mL) or ionomycin (10[-6] M). Although actinomycin D had no effect on this increase, cycloheximide completely inhibited the increase caused by these stimuli. The population of HMC-1 cells containing HDC protein was increased after stimulation with either PMA or ionomycin as evaluated by immunocytochemical analysis with anti-HDC antibody as a marker. HMC-1 constitutively expressed HDC mRNA, and its level was not increased with these stimuli. These results suggest that the increase of HDC activity in HMC-1 induced by PMA or ionomycin is regulated at the translational level.

Identification of novel mast cell genes by serial analysis of gene expression in cord blood-derived mast cells.

The gene expression profile of human cord blood-derived mast cells (MCs) was investigated using serial analysis of gene expression (SAGE). A total of 22914 tags, representing 9181 unique transcripts, were sequenced. By selecting tags that were detected more frequently in MCs than in other tissues, genes characteristic of MCs were enriched. Reverse transcription-PCR and the high-density oligonucleotide array hybridization confirmed the validity of our SAGE result. About 70% of the selected genes were previously uncharacterized. Northern blot analysis showed the MC-specific expression of selected genes. This inventory will be useful to identify novel genes with important functions in MCs.

Mice lacking histidine decarboxylase exhibit abnormal mast cells.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Synaptotagmin I expression in mast cells of normal human tissues, systemic mast cell disease, and a human mast cell leukemia cell line.

Synaptotagmin I (STG I) is a Ca(2+) sensor and one of the synaptic vesicle proteins that mediate exocytosis. To determine the mechanism of release of large granules from mast cells, we studied by immunohistochemistry the presence of STG I in mast cells in normal human tissues simultaneously with the mast cell markers mast cell tryptase (tryptase) and c-kit. The tumor cells of systemic mast cell disease (SMCD) and a human mast cell leukemia cell line (HMC-1) were also examined. Human mast cells in normal tissues and the tumor cells of SMCD expressed STG I as well as mast cell tryptase (tryptase) and c-kit. STG I mRNA and its products in HMC-1 were examined by RT-PCR analysis and immunocytochemistry, respectively. STG I expression in HMC-1 cells was compared with that in cells stimulated and non-stimulated by phorbol 12-myristate 13-acetate and also with that in NB-1 and PC12 cells, known to express STG I. STG I mRNA was detected in both non-stimulated and stimulated HMC-1 cells and in NB-1 and PC12 cells. STG I immunoreactivity was weaker than NB-1 or PC12 immunoreactivity. However, it increased in the stimulated HMC-1 cells. Mast cells expressed STG I in various states. STG I may mediate exocytosis of large granules in mast cells.

Cancer chemopreventive agents, labdane diterpenoids from the stem bark of Thuja standishii (Gord.) Carr.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

Histidine decarboxylase deficiency in gene knockout mice elevates male sex steroid production.

Histamine is synthesized in cells by histidine decarboxylase (HDC). HDC-deficient knockout (KO) mice lack functional HDC and histamine in the tissues. In the present study we used this in vivo model for studying the role of HDC deficiency in the regulation of male steroid hormone metabolism. In agreement with earlier studies showing the lack of effects of central histamine on the basal secretion of gonadotrope hormones, we found no difference with in situ hybridization in the expression of GnRH in the hypothalamus of wild type and KO mice. The tissue concentrations of testosterone and several androgenic steroids were significantly elevated in the testes but not in the adrenal glands of HDC-KO mice. In contrast, serum estradiol levels failed to show a significant difference between the two groups. The weight of the testes was significantly smaller in both 7-day-old and adult KO mice. The ultrastructure of the adult testis indicated elevated steroid synthesis with more tightly coiled membranous whorls in Leydig cells. The present results suggest that changes in reproductive functions and sex steroid secretion in male HDC-KO mice are not due to altered hypothalamic GnRH expression but are probably related to definite modifications during fetal development of KO mice reinforced later by the lack of the effect of peripheral histamine. This may provide in vivo evidence that peripheral histamine is an important regulatory factor of male gonadal development during embryogenesis and of sex steroid metabolism later in adulthood.

Identification of multiple regulatory elements of human L-histidine decarboxylase gene.

Previously, we reported the structure of human L-histidine decarboxylase gene. To identify the regions that regulate the tissue-specific expression of HDC, we constructed a fusion DNA with the 5'-flanking region from -1003 to +99 of the HDC gene and chloramphenicol acetyltransferase (CAT) gene, which was then transfected into human basophilic leukemia KU-812-F cells or human epithelial carcinoma HeLa cells. The 1102 bp DNA fragment stimulated the CAT activity in KU-812-F cells, but not in HeLa cells. CAT analysis with a series of 5'-deletion constructs of the HDC-CAT gene revealed the existence of two positive and one negative regulatory elements at -855 to -841 and -532 to -497 and -829 to -821, respectively. Sequence analysis showed a nuclear factor c-Myb binding motif, TAACTG, at position -520. Gel mobility shift analysis showed that the nuclear extract of KU-812-F cells, but not that of HeLa cells, contains a factor which can bind to this motif. These results suggest that the 5'-flanking region of the HDC gene contains multiple regulatory elements for HDC gene expression and that at least one element, including a c-Myb binding motif, is responsible for the tissue-specific expression of HDC.

Abietane diterpenoids from the cones of larix kaempferi

Three new abietane-type diterpenes, 7alpha,15-dihydroxyabieta-8,11, 13-trien-18-al (1); 15,18-dihydroxyabieta-8,11,13-trien-7-one (2); and 18-nor-4,15-dihydroxyabieta-8,11,13-trien-7-one (3), were isolated from the cones of Larix kaempferi, together with three known diterpenes, abieta-8,11,13-trien-18-yl succinate, 16-nor-15-oxoabieta-8,11,13-trien-18-oic acid, and 7beta-hydroxyabieta-8,11,13-trien-18-oic acid. The structures of 1-3 were determined on the basis of chemical and spectral evidence.

18-nor-Abietatrienes from the Cones of Larix kaempferi

Two novel norditerpenediols, 18-nor-abieta-8,11,13-triene-4,15-diol (1) and 18-nor-abieta-8,11,13-triene-4,7alpha-diol (2), were isolated from the cones of Larix kaempferi, together with two known diterpenes, abieta-8,11,13-triene-15,18-diol and abieta-8,11,13-triene-7alpha,18-diol. The structures of 1 and 2 were determined on the basis of chemical and spectral evidence.

Immediate-early, transient induction of the interleukin-1 beta gene in mouse spleen macrophages by ionizing radiation.

In murine spleen cells, x ray irradiation induces the expression of the IL-1 beta gene at multiple phases of the peak time. We analyzed the immediate-early phase of IL-1 beta mRNA accumulation. To determine the lineage of cells that showed the immediate response to irradiation, normal spleen cells were analyzed by Northern blotting and in situ hybridization after separation by magnetic antibodies against specific cell-surface antigens. Although most of the spleen macrophages continuously expressed a low level of IL-1 beta mRNA, a portion of the macrophage population transiently accumulated large amounts of IL-1 beta message immediately after irradiation. A macrophage-like leukemia cell line that resembles these inducible macrophages was identified. A similar immediate-early and transient increase in the IL-1 beta mRNA level occurred when cultured spleen cells were irradiated with a low dose (3 Gy) of x rays. In contrast, the x ray-inducible expression of the IL-1 beta gene was immediate and continuous, not transient, in spleen cells from whole-body irradiated mice. Results of the run-on transcription assay and the determination of the decrease in the message using cultured spleen and macrophage-like leukemia cells indicated that x ray irradiation appears to activate the transcription of the IL-1 beta gene and partially stabilize the message. The results show that the x ray-induced immediate-early accumulation of IL-1 beta mRNA is regulated at both the transcriptional and post-transcriptional levels in an as yet unidentified population of spleen macrophages.

Expression of IL-1 beta mRNA in mice after whole body X-irradiation.

IL-1 beta is a stimulator of hematopoietic and inflammatory systems, and also acts as a radioprotector. After whole-body exposure to sublethal doses of ionizing radiation, the IL-1 beta mRNA level in spleen cells increases for a short time prior to regeneration of the spleen. We analyzed spleen cells of C3H/He mice after whole-body irradiation with 3 Gy x-rays to determine the cause of this short-term increase in the transcription level. An increase in the level of the message in spleen cells, found by Northern blot hybridization, reached its peak 5 to 7 days after irradiation. There was a low correlation between the curves of the mRNA level and the ratio of monocyte/macrophage lineage cells; a typical source of the message. Spleen macrophages that produce a large amount of the message were found 7 days after irradiation in an in situ hybridization experiment in which heterogeneous spleen cell populations were used. In contrast, spleen cells had no detectable levels of macrophages rich in IL-1 beta mRNA before and 17 days after irradiation. Additionally, the population of message-rich cells was 9.4% of the total number of monocytes/macrophages in the spleen. These results suggest that the short-term increase in IL-1 beta mRNA is a result of the heterogeneous differentiation of a subpopulation of spleen macrophages before regeneration of the spleen.