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Cross-reactivity between Cupressus arizonica and Cupressus sempervirens pollen extracts.

BACKGROUND: Cupressus arizonica and C. sempervirens, two species belonging to the Cupressaceae family, are recognized as an important cause of respiratory allergies in countries with a Mediterranean climate. OBJECTIVE: The relationship between pollen extracts from these two species was studied by evaluating the reactivity with polyclonal rabbit antisera and human IgE. METHODS: The two extracts were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Cross-reactivity was evaluated by ELISA and immunoblotting inhibition experiments. RESULTS: The electrophoretic patterns of the two extracts are quite different, although some components display identical molecular weights. The immunoblotting developed with human IgE from subjects allergic to members of the Cupressaceae family indicated that two major IgE-reactive components, displaying molecular weights of about 43,000 and 36,000 d, were similarly detected in both extracts. Inhibition experiments showed a high degree of crossreactivity between the two extracts when tested with rabbit polyclonal antibodies against C. arizonica and C. sempervirens. When tested with human IgE inhibition methods, both extracts were able to reciprocally inhibit all of the IgE-reactive bands, although C. arizonica extract was always a better inhibitor. CONCLUSIONS: C. arizonica and C. sempervirens extracts are highly cross-reactive at the IgE level and share a number of common epitopes also identified by polyclonal rabbit antisera.

Allergens↗

Sensitivity to Cupressus: allergenic significance in Córdoba (Spain).

The detection of high levels of Cupressaceae pollen concentration in the air from January to April for several years in our area prompted analysis of the incidence and allergenic significance of sensitivity to this pollen. Furthermore, this is the highest winter-blooming taxa in the city of Córdoba. Skin prick tests were carried out over a one-year period on 1532 patients suffering from respiratory disorders (asthma and/or rhinoconjunctivitis). A total of 42 variables were studied in Cupressus-positive and Cupressus-negative patients; the data obtained were analyzed using a statistical software package. Sensitivity to Cupressus was found in 13% of all outpatients attending the unit, 18% of patients with respiratory disorders and 35% of patients with pollinosis. No significant differences were found between Cupressus-positive (C+) and Cupressus-negative (C-) patients with regard to mean age, sex, patient environment (i.e., rural, semi-rural, urban), personal or family history of atopy, clinical symptoms or evolution after immunotherapy (which did not include this antigen). More C+ patients were found in the higher age brackets (over 25 years old; p < 0.05); C+ patients showed greater duration (p < 0.05) and slower development (p < 0.05) of symptoms, and were also found to be more sensitive to other pollens (p < 0.001). All the Cupressus-sensitive patients also reacted positively to Olea and Fraxinus, compared to 77% and 51% in the two Cupressus-negative groups.

Adolescent↗

Efficacy of sublingual specific immunotherapy in Cupressaceae allergy using an extract of Cupressus arizonica. A double blind study.

BACKGROUND: non-injective routes of immunotherapy in respiratory allergy have being proposed as an alternative to conventional immunotherapy. We carried out a study to evaluate the clinical efficacy and effects sublingual immunotherapy (SLIT) in patient with Cupressaceae pollen respiratory allergy. METHODS: twenty patients with Cupressaceae pollen (C. sempervirens) rhinoconjunctivitis, mild asthma or both were randomly chosen for sublingual immunotherapy (10 patients) or placebo treatment (10 patients) using a double blind placebo controlled technique. We have used an extract of Cupressus arizonica because of its better stability in solution and its crossreactivity with Cupressus sempervirens. The patients underwent treatment for 12 months (from april 1999 to april 2000). Symptoms and drug scores as well as nasal provocation tests were recorded. The pollen counts were carried out, during all the same period. RESULTS: we found significantly lower symptom scores (p < 0.05) and drug consumption scores (p < 0.05) in the immunotherapy group than in the placebo group, during the pollen season, after the first year of therapy. Besides threshold of allergen reactivity in nasal provocation tests was increased in the active group (p < 0.01). No untoward reactions have been observed. CONCLUSIONS: SLIT with Cupressus arizonica (pollen extracts) produced, after one year treatment, a significant improvement of allergic symptoms and a decrease of drug consumption scores and allergen-specific nasal reactivity. SLIT with Cupressus arizonica appeared to be effective and safe in the treatment of Cupressaceae pollen respiratory allergy.

Administration, Sublingual↗

Vasculitis during immunotherapy treatment in a patient with allergy to Cupressus arizonica.

UNLABELLED: Allergen immunotherapy dates back to 1911 and has been used successfully to treat large numbers of patients throughout the last century. CASE REPORT: a 66-year-old woman presented with symptoms of allergic rhinitis and asthma due to sensitization to Cupressus arizonica. Specific immunotherapy was prescribed as a continuous 2-year treatment with a depot preparation of standarized and characterized allergen extracts of Cupressus arizonica pollen. Forty-eight hours after one maintenance dose of 0.8 cc, the patient presented palpable violaceous purpuric lesions and pruritus on both legs. We performed skin prick and intradermal tests with Cupressus arizonica. Twenty-four hours later, the 1/1 dilution intradermal skin test was positive. Biopsy showed leukocytoclastic vasculitis. CONCLUSIONS: A middle-aged woman experienced cutaneous non-necrotizing vasculitis after 2 years of maintenance immunotherapy. The interval between injections and the first appearance of cutaneous lesions suggests a type III hypersensitivity immune reaction. Skin biopsy of the positive intradermal test also supports this hypothesis.

Aged↗

Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1.

The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies. Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients. To obtain a full-length complementary DNA for Cup a 1, the major allergen of Cupressus arizonica pollen. It was cloned and sequenced and the recombinant protein was expressed. Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3'-RACE system and primers based on the N-terminal amino acid sequence. Recombinant Cup a 1 was cloned in pBluescript and expressed in a glycosylated form in rabbit reticulocytes. The cDNA was subcloned in pGEX-5X-1 and expressed in Escherichia coli as a fusion protein with GST. Recombinant Cup a 1 is highly homologous with the major allergens of mountain cedar (Jun a 1), Japanese cypress (Cha o 1) and Japanese cedar (Cry j 1). Cup a 1 contains three potential N-glycosylation sites that are different from those found in Jun a 1 and Cry j 1. The cloned protein contains a pectate lyase active site identical to those of Cry j 1 and Jun a 1. The IgE from patients' sera recognizes recombinant Cup a 1, and this reactivity is higher with the glycosylated protein. Cup a 1 has been cloned and sequenced. As expected, the high degree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross-reactivity of conifer pollens. Different IgE reactivity with the glycosylated and non-glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.

Allergens↗

How to reduce air pollution by Cupressus pollen?

Cypress trees are among the most famous trees of the Mediterranean region. The origin of Cupressus sempervirens is apparently in the East Mediterranean. A nice description of the site of origin of Cupressus was provided by the Roman philosopher Plinius: "cypress trees sprout spontaneously on Mt. Ida on the island of Crete, whenever the soil there is disturbed". The aesthetic shape of the trees, the quality of their timber and their adaptation to harsh environmental conditions has attracted people. Certain varieties of the tree (e.g., var. Pyramidalis) were spread rapidly all over the Mediterranean and since the 16th century were introduced into the Western Hemisphere and other parts of the world. During historical times, Cupressus sempervirens was so widely used in the East Mediterranean countries that it reached the brink of extinction. Nowadays it is planted on a large scale as an ornamental plant as a windbreak and as a forest tree. Such plantations have various impacts on their environment, many positive ones, some negative ones, and some disastrous ones. This last aspect is where cypresses come in.

Air Pollution↗

Cross-reactivity between the pollens of Cupressus sempervirens (common cypress) and of Cryptomeria japonica (Japanese cedar).

A clinical and immunologic study was performed comparing a group of French patients allergic to the pollens of cypress (Cupressus sempervirens, which belongs to the Cupressaceae family) and a group of Japanese patients allergic to the pollens of Sugi (Cryptomeria japonica, which belongs to the Taxodiaceae family). By skin testing, RAST, and RAST inhibition, clear cross-reactivity was detected between the two pollens. No cross-reactivity was detected between the pollens of Cupressus sempervirens and Sugi and the Pinaceae family. In addition, one can speculate that an antigen in Cupressus sempervirens is cross-reactive with SBP, the major allergen of Sugi, suggesting that there is a closer relationship between the Taxodiaceae family and the Cupressaceae family than between these two families and the other families of the gymnosperms. This finding throws new light on the taxonomy of the gymnosperms.

Cross Reactions↗

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid.

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.

Allergens↗

Effects of Cupressus sempervirens cone extract on lipid parameters in Wistar rats.

BACKGROUND: Lipid-lowering drugs have been developed to treat hyperlipidemia, but they are expensive, while serious side-effects may occasionally occur. In Albania, a hydroalcoholic extract of the cones of Cupressus sempervirens is used to treat a variety of disorders, including hyperlipidemia. The purpose of our study was to assess the effects of Cupressus sempervirens cone extract (CSE) on the lipid profile of Wistar rats. MATERIALS AND METHODS: The animals were divided into two groups. Group I (30 animals) served as a control group. Serum lipid parameters, muscle and liver enzymes, red and white blood count, platelets, and serum concentrations of uric acid and creatinine were determined at baseline and at weeks 6, 12, 18 and 24 of the study. RESULTS: The oral administration of the extract resulted in a substantial decrease of serum total cholesterol, which was significant even after 6 weeks of treatment. Moreover, these animals exhibited lower total cholesterol levels compared to the controls after the initiation of treatment (p < 0.001) during the study period. The administration of the extract led to a substantial reduction in serum triglycerides (p < 0.05) in Group I, comparing 0 week to 6-24 weeks. However no significant differences in triglyceride levels were observed between CSE animals (Group I) and controls (Group II) during the entire study period. No significant changes in HDL-cholesterol in the other parameters occurred in either group. CONCLUSION: The administration of CSE has an important lipid-lowering effect in Wistar rats.

Animals↗

Allergens of Arizona cypress (Cupressus arizonica) pollen: characterization of the pollen extract and identification of the allergenic components.

Species of the Cupressaceae family are an important cause of respiratory allergies in countries with a Mediterranean climate. An allergenic extract from Cupressus arizonica pollen was prepared with two extraction steps followed by ammonium sulfate precipitation, giving a protein yield of about 3%. Cupressus arizonica pollen extract was also characterized by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by IgE and IgG immunoblotting and lectin blotting. IgE reactivity was restricted to six components, whereas IgG binding showed a more complex pattern. A 43 kd component, predominant both in its intensity and frequency of recognition by human IgE antibodies, was identified as the major allergen of C. arizonica. Four of the six IgE-binding components, including the major allergen, seem to be glycoproteins, as confirmed by the lectin blotting analysis. The extract produced inhouse was used to set up an immunoenzymatic test to evaluate the specific IgE binding in a panel of sera from 33 immunotherapy-free subjects who were monosensitized to cypress pollen. The percent of positivity obtained was much higher than that reported in the literature for commercial immunoassays.

Adult↗

Effect of the plant Cupressus macro-carpa (Cupressacea) on some haematolo-gical and biochemical parameters of Biomphalaria alexandrina snails.

The dry powder of the plant aereal part; Cupressus macro-carpa (Cupressacea) was tested against Biomphalaria alexandrina. LC50 & LC90 values were 59.5 & 98.8 ppm, respec-tively. Exposure of B. alexandrina to sublethal concentrations (LC0, LC10 & LC25) of C. macrocarpa for three weeks signi-ficantly decreased the number of circulating hemocytes. The magnitude of reduction was increased with increasing of the tested concentration. The main type of cell in the hemolymph of B. alexandrina was the granulocyte (71.8%), followed by large round cells or hyalinocytes (19.0%) and small round cells or undifferentiate cells (9.2%). The percentage of different hemocyte categories was changed in treated snails. In snails maintained at LC25, showed significantly higher percentages of small round cells than controls, 56.2% & 9.2% respectively. Maintainence of B. alexandrina in sublethal concentrations for three weeks significantly reduced protein & hemoglobin content in the hemolymph. Reduction in enzyme activities occurred in the hemolymph and tissues of treated snails. The enzymes were pyruvate kinase (PK), lactat dehydrogenase (LDH), hexokinase (HK) and phosphoenol pyruvate carboxy kinase (PEPCK) which are very important in metabolism of the protein and carbohydrate. The infectivity of Schistosoma mansoni miracidia was greatly reduced by exposure to the sublethal concentrations (LC0, LC10 & LC25) of Cupressus. Infection rate of B. alexandrina reached to 54.5%, 37.5% & 16.7%, respectively compared to control (81.25%). Duration of cercarial shedding and the total periodic cercarial production/snail showed significant reduction while the parasite incubation period was significantly longer (p<0.05).

Animals↗

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P(3) phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P(3) occur simultaneously and that the Ins(1,4,5)P(3) level may be regulated by both PtdIns(4,5)P(2)-PLC and Ins(1,4,5)P(3) phosphatases. Studies on the properties of PLC and Ins(1,4,5)P(3) phosphatases indicate that PLC activity toward PtdIns(4,5)P(2) was optimal at a lower Ca(2+) concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P(3) phosphatase activity is inhibited by high Ca(2+) concentration. This suggests that Ins(1,4,5)P(3) biosynthesis and degradation may be regulated by free cytosolic Ca(2+). In addition, a relationship between Ins(1,4,5)P(3) signaling and accumulation of a phytoalexin (beta-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P(3) accumulation by neomycin or LiCl affects elicitor-induced production of beta-thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of beta-thujaplicin while thapsigargin alone induces beta-thujaplicin accumulation. These results suggest that Ca(2+) released from intracellular calcium stores may mediate elicitor-induced accumulation of beta-thujaplicin via an Ins(1,4,5)P(3) signaling pathway, since it is widely accepted that Ins(1,4,5)P(3) can mobilize Ca(2+) from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P(3) turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P(3) in elicitor-induced phytoalexin accumulation via a Ca(2+) signaling pathway.

Calcium↗

Microsporogenesis in the endangered species Cupressus dupreziana A. Camus: evidence for meiotic defects yielding unreduced and abortive pollen.

To understand the reproductive biology of Cupressus dupreziana A. Camus (Cupressaceae), a highly endangered Mediterranean conifer, the processes of microsporogenesis and pollen differentiation were investigated cytologically. Pre-meiotic development proved to be similar to the coniferous pattern: the microsporangia differentiated sporogenous tissue in which microsporocytes separated and underwent meiosis. As the meiotic steps proceeded, unexpected irregularities were observed concerning chromosomal and nuclear behaviour. This mainly included: abnormal chromosome segregation and cytokinesis, and nuclear fusion of the meiotic products. The result was the formation, in the same microsporangium, of heterogeneous microspore populations arranged in monads, dyads, triads, tetrads, and polyads, and cytoplasts giving rise to pollen grains of different sizes. This indicates that in C. dupreziana both abortive and unreduced pollen grains are generated. The significance of the finding is discussed in relation to reproductive biology and vulnerability to extinction.

Cell Differentiation↗

A study of conservation genetics in Cupressus chengiana, an endangered endemic of China, using ISSR markers.

ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (He) was 0.3120, effective number of alleles (Ae) was 1.5236, and Shannon's information index (I) was 0.4740. At the population level, PPB = 48%, Ae = 1.2774, He = 0.1631, and I = 0.2452. Genetic differentiation (Gst) detected by Nei's genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (Nm) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r = 0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity.

China↗

Chemical composition and antidermatophytic properties of volatile fractions of hexanic extract from leaves of Cupressus lusitanica Mill. from Cameroon.

The chemical composition of five column fractions of hexanic leaf extract of Cupressus lusitanica were analysed by gas chromatography and gas chromatography-mass spectrometry and then tested for their antidermatophytic activities using the agar dilution method. The first fraction (F(1)) has only hydrocabon monoterpenes with alpha-pinene (80.0%) as major component. The main constituents of the second fraction (F(2)) were epi-bicyclosesquiphellandrene (35.3%), epi-zonarene (10.3%), 1S, cis-calamenene (13.1%) and beta-himachalene (10.4%). The third fraction (F(3)) was rich in hydrocarbon sesquiterpenes (45.4%) and a relatively high amount of diterpenes (29.8%) with epi-bicyclosesquiphellandrene (14.3%), pimaric acid (7.5%), kaurenoic acid (6.9%) and 8-beta-hydroxysandaracopimarane (3.5%) as main components. The last two fractions contain high molecular weight aliphatic hydrocarbons, their main constituents been eicosane (41.1%) and tricosane (37.3%) and heptacosane (22.1%). The agar dilution method was used to evaluate the antifungal properties of the crude extract and its fractions. These fractions showed several degrees of antidermatophytic activities against Microsporum audouinii, Microsporum Langeronii, Microsporum canis, Trichophyton rubrum and Trichophyton tonsurans. Fractions F(1) and F(3) exhibited the highest antidermatophytic activities with repective MICs of 250 and 125 mug/ml while the fractions F(4) and F(5) did not prevent the growth of the tested fungi up to dose 2,500 mug/ml.

Antifungal Agents↗

Sesquiterpenes from Cupressus macrocarpa foliage.

Ten sesquiterpenes, many with unusual carbon skeletons, were identified in foliage of Cupressus macrocarpa. These are (-)-10-epi-beta-acoradiene; ent-widdra-2,4(14)-diene; (E)-iso-gamma-bisabolene, i.e., (4E)-4-(1,5-dimethylhex-5-enylidene)-1-methylcyclohexene; (-)-cumacrene, i.e., (4S)-4-[(1R,2S)-2-isopropenyl-1-methylcyclobutyl]-1-methylcyclohexene; (-)-alpha-chamipinene, i.e., (1S,6S,7S)-2,2,6,8-tetramethyltricyclo[5.3.1.01,6]undec-8-ene; and five sesquiterpenes with a 3,3,4'-trimethyl-1,1'-bi(cyclohexyl) skeleton for which the trivial name macrocarpane is proposed. The possible single-enzyme biogenesis of these sesquiterpenes is discussed.

Cupressus↗

Feedback regulation of beta-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica.

Suspension cell cultures of Cupressus lusitanica produce beta-thujaplicin, a tropolone found mostly in Cupressaceae heartwood. The factors controlling beta-thujaplicin accumulation in this cell culture system were investigated. Initial cell density of the cultures did not affect beta-thujaplicin levels, though initial addition of beta-thujaplicin suppressed its de novo production. When beta-thujaplicin accumulation reached a certain level (ca. 40 mg/l) in the medium, the cultures seemed to cease beta-thujaplicin production. However, beta-thujaplicin productivity was restored when the beta-thujaplicin-containing medium was exchanged for fresh medium; the formation of 2-methoxy-6-(methylethyl)cyclohepta-2,4,6-trien-1-one, an isomer of methylated beta-thujaplicin, in medium was also observed. These results suggest that beta-thujaplicin synthesis was regulated by product feedback mechanism in this cell line, and that excess accumulation of beta-thujaplicin is relieved by conversion of beta-thujaplicin to its methyl ether.

Cell Count↗

Cypress tree (Cupressus semervirens L.) bark as an indicator for heavy metal pollution in the atmosphere of Amman City, Jordan.

Bio-monitoring of air quality in Amman City was investigated by analyzing 36 cypress tree (Cupressus semervirens L.) bark samples from three sites of different anthropogenic activities at the end of summer season 2001. Cypress barks were found to be a good bio-indicator for air pollution in arid regions. Variation in Pb, Zn, Mn, Cr, Ni, Cd, and Cu contents between sites was observed due to different types of activities. Traffic emissions were found to be the main source of heavy metal pollution in the atmosphere of Amman. Lead content was found to be the highest in highly traffic density areas. The industrial part of the city was characterized by high Zn, Mn, Cr, Ni, and Co contents. No significance variations were found in pH values of the bark between the sites. This was attributed to buffering effect of carbonate in the atmosphere originated from soil of the area.

Cupressus↗