Chlorophyll catabolism and gene expression in the peel of ripening banana fruits

The biochemical and molecular basis of chlorophyll (Chl) catabolism in bananas was investigated during ripening at 20°C and at an elevated temperature (35°C) where degreening is inhibited. Biochemical analysis showed that Chl breakdown products could be isolated from fruit ripened at both temperatures. The coloured breakdown products, chlorophyllide and pheophorbide, were not detected at any stage of ripening in the two treatments; however, a non-fluorescent Chl catabolite accumulated to a higher concentration at 20 than at 35°C. To investigate the ripening-related gene expression associated with these changes, a cDNA library was generated from the peel of fruit ripened at 20°C. Differential screening of this library produced 20 non-redundant families of clones including those encoding enzymes involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation and other metabolic events. The expression of these genes was followed by northern analysis in fruit ripened at 20 and 35°C.

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.

Penetration of ASM 981 in canine skin: a comparative study

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin. To make penetration of ASM 981 visible in dog skin, tritium labelled ASM 981 was applied to a living dog and to defrosted skin of the same dog. Using qualitative autoradiography the radioactive molecules were detected in the lumen of the hair follicles until the infundibulum, around the superficial parts of the hair follicles and into a depth of the dermis of 200 to 500 microm. Activity could not be found in deeper parts of the hair follicles, the dermis or in the sebaceous glands. Penetration of ASM 981 is low in canine skin and is only equally spread in the upper third of the dermis 24 hours after application. Penetration in frozen skin takes even longer than in living canine skin but shows the same distribution.

Intradermal injection of heat-killed Mycobacterium vaccae in dogs with atopic dermatitis: a multicentre pilot study

Canine atopic dermatitis (cAD) is a common disease with a multifactorial aetiology associated with impaired immunoregulation. The immunopathogenesis has similarities to that of human atopic dermatitis. Clinical signs of allergic disease in humans and mice are reduced by administration of saprophytic mycobacteria that amplify regulatory cytokines and hence the effect of Mycobacterium vaccae on the clinical severity of cAD was investigated. Sixty-two dogs with cAD, selected according to strict criteria, were treated with a single intradermal injection and evaluated monthly for 3 months in a placebo-controlled double-blind clinical trial. Clinical severity was quantified using standardized scores and by owner assessment of pruritus. A single injection of a heat-killed suspension of M. vaccae was found to be well tolerated and effective in treating mild to moderate cases of cAD demonstrable for 3 months, but was insignificant in more severely affected dogs.

Lymphocyte blastogenic response to ovalbumin in a model for canine allergy

Lymphocyte stimulation tests (LST) were performed in five dogs sensitised with ovalbumin (OVA) and seven healthy dogs. In addition, all five OVA-sensitised and two control dogs were tested after two in vivo provocations with OVA-containing eye drops. The isolated cells were suspended in culture media containing OVA and were cultured for up to 12 days. Proliferation was measured as reduction in 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) intensity by flow cytometry on days 0, 3, 6, 9 and 12. A cell proliferation index (CPI) for each day and the area under the curve (AUC) of the CPI was calculated for each dog. All OVA-sensitised dogs demonstrated increased erythema after conjunctival OVA application. The presence of OVA-specific lymphocytes was demonstrated in 2/5 OVA-sensitised dogs before and 4/5 after in vivo provocation. Using the AUC, the difference between OVA-sensitised and control dogs was significant in all three LST before in vivo provocation (P<0.05) and borderline significant (P=0.053) in 2/3 LST after provocation. The most significant difference in CPI was observed after 9 days of culture (P=0.001). This pilot study indicates that the LST allows detection of rare antigen specific memory T-cells in dogs previously sensitised to, but not concurrently undergoing challenge by a specific antigen.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

OBJECTIVE To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Effect of carprofen, etodolac, meloxicam, or butorphanol in dogs with induced acute synovitis

OBJECTIVE: To compare the analgesic and anti-inflammatory effect of single doses of carprofen, etodolac, meloxicam, and butorphanol in dogs with induced acute synovitis (acute pain model) via kinetic gait analysis and orthopedic evaluation and examine measurement of serum C-reactive protein (CRP) concentration as an indicator of treatment efficacy. ANIMALS: 12 Beagles and 6 additional Beagles that were used only in serum CRP analyses. PROCEDURE: Acute synovitis was induced in right stifle joints of dogs via intra-articular injection of monosodium urate solution. Treatments included butorphanol (0.2 mg/kg, i.v.), carprofen (4 mg/kg, PO), etodolac (17 mg/kg, PO), or meloxicam (0.2 mg/kg, PO); control dogs received no treatment. The procedure was repeated (3-week intervals) until all dogs received all treatments including control treatment. Lameness was assessed on a biomechanical force platform and via orthopedic evaluations of the stifle joints; blood was collected to monitor serum CRP concentration. RESULTS: Compared with control dogs, treated dogs had significantly different vertical ground reaction forces and weight-bearing scores. Greatest improvement in lameness was observed in carprofen-treated dogs. Etodolac had the fastest onset of action. Compared with butorphanol treatment, only carprofen and etodolac were associated with significantly lower pain scores. An increase in serum CRP concentration was detected after intra-articular injection in all dogs; this change was similar among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Carprofen, etodolac, and meloxicam had greater efficacy than butorphanol in relief of acute pain. Carprofen was most effective overall. In this acute pain model, serum CRP analysis was not useful to assess drug efficacy.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Pulmonary lymphangioleiomyomatosis: analysis of disease manifestation by region-based quantification of lung parenchyma

PURPOSE Lymphangioleiomyomatosis (LAM) is characterized by proliferation of smooth muscle tissue that causes bronchial obstruction and secondary cystic destruction of lung parenchyma. The aim of this study was to evaluate the typical distribution of cystic defects in LAM with quantitative volumetric chest computed tomography (CT). MATERIALS AND METHODS CT examinations of 20 patients with confirmed LAM were evaluated with region-based quantification of lung parenchyma. Additionally, 10 consecutive patients were identified who had recently undergone CT imaging of the lung at our institution, in which no pathologies of the lung were found, to serve as a control group. Each lung was divided into three regions (upper, middle and lower thirds) with identical number of slices. In addition, we defined a "peel" and "core" of the lung comprising the 2 cm subpleural space and the remaining inner lung area. Computerized detection of lung volume and relative emphysema was performed with the PULMO 3D software (v3.42, Fraunhofer MEVIS, Bremen, Germany). This software package enables the quantification of emphysematous lung parenchyma by calculating the pixel index, which is defined as the ratio of lung voxels with a density <-950HU to the total number of voxels in the lung. RESULTS Cystic changes accounted for 0.1-39.1% of the total lung volume in patients with LAM. Disease manifestation in the central lung was significantly higher than in peripheral areas (peel median: 15.1%, core median: 20.5%; p=0.001). Lower thirds of lung parenchyma showed significantly less cystic changes than upper and middle lung areas combined (lower third: median 13.4, upper and middle thirds: median 19.0, p=0.001). CONCLUSION The distribution of cystic lesions in LAM is significantly more pronounced in the central lung compared to peripheral areas. There is a significant predominance of cystic changes in apical and intermediate lung zones compared to the lung bases.