New promising Euphorbiaceae extracts with activity in human lymphocytes from primary cell cultures.

CONTEXT: Euphorbiaceae plants exhibit anti-inflammatory and immunomodulatory properties. METHODS: We evaluated the activity of 14 extracts from seven Euphorbiaceae plants on primary immune cell cultures from healthy individuals. Peripheral blood mononuclear cells (PBMC) were exposed to the extracts w/o phytohaemagglutinin A or cycloheximide as agents that induce proliferation or apoptosis in PBMC, respectively. RESULTS: We found that five up to 14 Euphorbiaceae's extracts had the ability to modulate at least one of the immune parameters evaluated in this study. However, only the latex extracts of Euphorbia cotinifolia and Euphorbia tirucalli strongly induced both proliferation and apoptosis in PBMC. These extracts were further subfractioned by silica gel column chromatography. Two subfractions with enhanced activity in comparison to the crude extracts were obtained. Although these subfractions induced proliferation on both CD3(+) and CD3(-) cells, the most prominent effects were observed in the former subpopulation. Interestingly, the subfraction from E. tirucalli induced lymphocyte proliferation without the need of accessory cells; this ability was not inhibited by the carbohydrates d-galactose and α-Methyl-D-Mannopyranoside. CONCLUSIONS: Altogether, these results reveal the presence of novel candidates within the Euphorbia plants to induce proliferation and apoptosis in human lymphocytes, mainly in CD3(+) T cells.

Adjuvant properties of AcF1, an immunostimulant fraction of Alchornea cordifolia extract.

As a result of strong experimental data supporting effectiveness and safety, herb-based immunomodulators are paving way as alternative sources of potent adjuvants for vaccines. In this study, the immunostimulatory and adjuvant properties of AcF1, a flavonoids-rich fraction of Alchornea cordifolia extract, was evaluated. In vitro, AcF1 was shown to activate total splenocytes, CD4+ T cells, and B cells, inducing remarkable increases in CD69 expression, profound proliferation, and increased IL-4 and IFN-gamma expression by the naïve splenic cells in a concentration-dependent manner. Lympho-activation and proliferation induced by AcF1 was partially inhibited by U0126, a selective mitogen activated protein kinase kinase (MKK) inhibitor. Additionally, AcF1 was shown to induce structural and functional maturation of bone marrow-derived dendritic cells (BM-DCs) and their specific-antigen presentation functions. Used as an adjuvant in a homologous prime-boost OVA immunisation in C57BL/6 mice, AcF1 significantly (P<0.05) increased the level of OVA-specific antibody titres in the sera of immunised mice, compared to the control group immunised with OVA alone. The results of this study show AcF1 as a potent immunostimulant and a potential adjuvant for further study in combination with other vaccine antigens.

Occupational rhinoconjunctivitis and bronchial asthma due to Acalypha wilkesiana allergy.

BACKGROUND: Acalypha wilkesiana, or copperleaf, is a plant of the Euphorbiaceae family. Although it is widely known as an outdoor ornamental plant, no cases of A. wilkesiana allergy have been reported to date. OBJECTIVE: To describe a patient with occupational respiratory allergy to A. wilkesiana. METHODS: Extracts from A. wilkesiana leaves and flowers were used for skin prick testing, specific conjunctival and bronchial challenge tests, and in vitro studies. These studies range from A. wilkesiana specific IgE determination to sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunodetection of A. wilkesiana protein bands in patient serum samples and immunoblot inhibition by preincubation with Salsola kali and Chenopodium album pollen extracts. RESULTS: Our patient had positive skin prick test reactions to A. wilkesiana leaf and flower extracts; negative reactions were found in a control group of 20 atopic patients. On immunodetection of A. wilkesiana extracts in patient serum samples, as many as 9 different IgE-binding proteins, with apparent molecular weights of 16 to 86 kDa, were revealed. Preincubation with S. kali and C. album pollen extracts completely inhibited IgE binding to the A. wilkesiana extract. Specific bronchial challenge resulted in a spirometric 30% decline in forced expiratory volume in 1 second with respect to baseline 1 minute after 1:100 (vol/vol) A. wilkesiana extract solution inhalation; 2 atopic controls had negative bronchial challenge test results. CONCLUSION: Acalypha wilkesiana is a new etiologic agent for IgE-mediated occupational respiratory allergy.

Allergen cross-reactivity between proteins of the latex from Hevea brasiliensis, seeds and pollen of Ricinus communis, and pollen of Mercurialis annua, members of the Euphorbiaceae family.

Allergen cross-reactions among three strongly sensitizing Euphorbiaceae species, i.e., the rubber tree (Hevea brasiliensis), castor bean (Ricinus communis), and the Mediterranean weed Mercurialis annua were studied in Finnish patients (n = 25) allergic to natural rubber latex (NRL), but with no known exposure to castor bean or M. annua, and French patients allergic to castor bean (n = 26) or to M. annua (n = 9), but not to NRL. In immunoglobulin E (IgE)-immunoblotting, 28% of NRL-allergic patient sera recognized castor bean seed and 48% reacted to castor bean pollen proteins. Likewise, 35% of the NRL-allergic patient sera bound to M. annua pollen allergens. Nineteen percent of castor bean-allergic patients showed IgE to NRL and 8% to M. annua proteins. Sera from patients allergic to M. annua reacted in 44% to NRL, in 56% to castor bean seed, and in 78% to castor bean pollen proteins. In immunoblotting, castor bean seed extract inhibited the binding of NRL-reactive IgE to 20 kDa, 30 kDa of NRL, and 55 kDa of proteins; NRL extract, in turn, inhibited the binding of castor bean-reactive IgE to 14, 21-22, 29, and 32-34 kDa of castor bean proteins. In ELISA inhibition, NRL extract inhibited 33% of the binding of M. annua--reactive IgE of pooled sera to M. annua pollen. In conclusion, allergen cross-reactivity in vitro was observed among three botanically related Euphorbiaceae members, H. brasiliensis, R. communis, and M. annua, but the molecular specificity of the observed cross-reactions as well as their clinical significance remains to be elucidated. Allergen cross-reactivity should be taken into account in diagnostic work.

[Latex allergy. Diagnosis and therapeutic aspects]. / Alergia al látex. Diagnóstico y aspectos terapéuticos.

In the last two decades of the 20th century, latex allergy has reached epidemic proportions. Epidemiological studies demonstrate that 3-25 % of health personnel is allergic to latex. The main risk groups are health workers, machine operators in latex factories, and children with spina bifida and urogenital anomalies. From the allergenic point of view, latex contains 240 peptides but approximately 50 are able to react to IgE. Latex elongation factor Hevdl is the relevant allergen in patients with spina bifida. Prohevein (hev B6) behaves as a major allergen, since it reacts to IgE in most of the sera of patients with latex allergy. The nature of latex is complex; it is an allergenic mixture that depends on chemical, immunological and epidemiological variables. Latex proteins show strong cross reactivity with several proteins from fruit and vegetable grains such as avocado, potato, banana, tomato, chestnut, and kiwi. In vivo studies have shown that class I chitinase from avocado and chestnut behave as major allergens in allergic patients with latex-fruit syndrome. The clinical manifestations related to the use of latex products depend on the type of exposure, the amount of the allergen, and individual variability. The most useful diagnostic method is the skin prick test. Several perioperative guidelines are recommended in patients sensitized to latex as well as various alternatives to rubber gloves. An increasing number of studies describe the efficacy of etiological treatment (immunotherapy), using different guidelines and routes of administration. These preliminary data encourage the hope that in the near future immunomodulatory therapy will be available to mitigate against the latex allergy epidemic.

[Latex: a complex allergy]. / Le latex: une allergie complexe.

The authors review several of the most important aspects of latex allergy, an increasing problem in Public Health, which should be understood by all health professionals. After briefly presenting the history of the origin latex, from Hevea brasiliensis the authors describe the antigens of latex: Hev b1 to Hev b8, major allergens. They also note the crossed reactivity not only with foods, exotic fruits, but also with pneumoallergens and in particular the pollens. The groups at risk are essentially workers in the latex industry, health professionals and finally infants with spina bifida or other severe urological anomaly. The clinical signs are reactions of type 1 hypersensitivity, to urticaria and/or angio-oedema and anaphylactic shock. Diagnosis is based on a search for specific serum IgE, skin tests and provocation tests. Prophylaxis depends on removal of all substances that are based on latex, especially replacement of gloves with vinyl, but also on a food diet that excludes all foods that have a cross-reactivity with latex.

Identification of hevein (Hev b 6.02) in Hevea latex as a major cross-reacting allergen with avocado fruit in patients with latex allergy.

BACKGROUND: Recent studies demonstrated that allergy to natural rubber latex is frequently associated with hypersensitivity to avocado fruit. The responsible cross-sensitizing allergen has not been identified. OBJECTIVE: The purpose of this study was to investigate the cross-reactivity of a latex major allergen, hevein, with avocado proteins. METHODS: Serum samples from 118 health care workers (HCWs) allergic to latex (HCW group) and 78 patients with spina bifida (SB) allergic to latex (SB group) were included in this study. Anti-hevein and anti-avocado IgE antibodies were measured by enzyme-linked allergosorbent assay. Cross-reactivity of hevein to avocado proteins was assessed by inhibition of the IgE binding in individual patients' sera containing IgE antibodies to both hevein and avocado. RESULTS: The prevalence of seropositive IgE antibodies to avocado was found to be strongly associated with the presence of hevein-specific IgE antibodies in subjects of both groups (P < .001). Sixty-seven of 91 (73%) subjects from the HCW group and all 19 subjects in the SB group with positive IgE antibodies to hevein also had elevated IgE values to avocado. Competitive RAST inhibition with 42 sera showed that IgE binding to avocado could be completely inhibited in 27 (64%) sera by preincubation with hevein. By contrast, the degrees of inhibition of IgE to hevein by avocado extract ranged from 0% to 36% (n = 16). These results indicate that sensitization to avocado in most patients allergic to latex is caused exclusively by IgE-binding epitopes present in hevein. Results of immunoblots and immunoblot inhibition with 11 serum samples confirmed that a 30-kd protein in avocado was the major IgE-binding component; the IgE-binding reactivity to this protein could be inhibited by hevein in all sera tested. CONCLUSION: Hevein is the major cross-reacting allergen with avocado in subjects with latex allergy.

Diagnosis of natural rubber latex allergy: multicenter latex skin testing efficacy study. Multicenter Latex Skin Testing Study Task Force.

BACKGROUND: No characterized diagnostic natural rubber latex skin testing material is licensed for use in the United States. OBJECTIVE: We have conducted a multicenter clinical skin testing study to document the safety and diagnostic sensitivity and specificity of a candidate Hevea brasiliensis nonammoniated latex (NAL) extract. These data are intended to support the licensing of this reagent for the diagnosis of latex allergy in high-risk populations. METHODS: Three hundred twenty-four subjects (304 adults and 20 children) were classified by their clinical history as having latex allergy (LA group, 124 adults and 10 children) or having no latex allergy (NLA group, 180 adults and 10 children). All subjects provided blood samples and then received sequential puncture skin tests (PSTs) at 1, 100, or 1000 microg/mL protein with a bifurcated needle and NAL (Greer Laboratories) from Malaysian Hevea brasiliensis (clone 600) sap. A 2-stage glove provocation test was used to clarify latex allergy status of individuals with positive history/negative PST result and negative history/positive PST result mismatches. RESULTS: Twenty-four subjects (15%) originally designated as having LA on the basis of their initial clinical history were reclassified to the NLA group on the basis of a negative glove provocation test result. Of the 134 subjects with LA, 54 (40%) were highly sensitive to latex, with a positive PST result at 1 microg/mL NAL. The Greer NAL reagent produced a positive PST rate (sensitivity) of 95% and 99% in subjects with LA at 100 microg/mL and 1 mg/mL, respectively. The negative PST rate (specificity) in 190 subjects with a negative history with the NAL extract at 100 microg/mL and 1 mg/mL, was 100% and 96%, respectively. Immediately after the PST, mild systemic reactions (mainly pruritus) were recorded in 16.1 % of the adults in the LA group and 4.4% of the adults in the NLA group. No reactions required treatment with epinephrine. Only mild delayed reactions were observed in 9.6% (LA group) and 2.8% (NLA group) of subjects 24 to 48 hours after PST. Mean wheal and erythema diameters measured in the 10 children in the LA group with spina bifida at 100 microg/mL and 1 mg/mL were similar to those observed in the adults in the LA group, suggesting that children are not at increased risk for systemic reactions compared with adults. CONCLUSIONS: A suggestive clinical history is necessary but not sufficient for a definitive diagnosis of IgE-dependent latex allergy. These data support the safety and diagnostic efficacy of the Greer NAL, skin test reagent at 100 micro/mL and 1 mg/mL for confirmatory PSTs.

Cloning and characterization of a latex allergen (Hev b 7): homology to patatin, a plant PLA2.

We previously identified a 46-kD protein allergen in latex as having amino acid sequence homology to the patatin gene family. The objective of this study was to characterize this protein by molecular techniques. RNA was isolated from the latex or leaf material from Hevea brasiliensis and from potato tubers. Specific polymerase chain reaction (PCR) primers were designed from the amino acid sequence and reverse transcriptase (RT)-PCR amplified a specific product from latex RNA that was subsequently cloned and sequenced. This product was 1493 bp in length with an 1167 bp open reading frame. The deduced amino acid sequence encodes for a 389 aa protein, pI 4.82 with 43% homology to tobacco patatin. Northern analysis of potato, Hevea leaf, and latex RNA demonstrated the message to be most abundant in latex, weakly present in Hevea leaf, but no hybridization occurred with potato RNA. Patatin has lipid acyl-transferase and PLA2-like activity, suggesting it plays a role as a defence-related protein. Other defence-related proteins in latex such as hevein, glucanase, and hevamine are also allergens. Increased production of defence-related proteins as a result of increased tapping of the rubber trees to meet the demand for latex may explain the increased allergenicity of latex.

A lectin from the bark of the rubber tree (Hevea brasiliensis).

A protein isolated and purified from extracts of bark strips from mature rubber (Hevea brasilinsis) agglutinated erythrocytes from rabbits and all human blood types except AB. No agglutination was detected with erythrocytes from sheep, rates or mice. Proteolytic treatment of Hevea bark lectin (HBL) abolished haemagglutinin activity. The M(r) determined by SDS-PAGE was 40 k and that estimated from gel filtration was 140 k. Fetuin, asialofetuin, bovine submaxillary mucin and asialosubmaxillary mucin inhibited HBL-induced agglutination of rabbit erythrocytes. The HBL showed maximum haemagglutination activity over the pH range 4.5-9.5 and heat stability up to 60 degrees.

Immunobiochemical characterization of Putranjiva roxburghii pollen extract and cross-reactivity with Ricinus communis.

Putranjiva roxburghii (PR) pollen has been found to be an important aeroallergen for type I hypersensitivity. In the present study, the IgE binding proteins of PR pollen have been characterized and compared with pollen allergens of Ricinus communis (RC) belonging to family Euphorbiaceae. On isoelectric focusing, PR pollen extract resolved into 35 bands (pI 3-9), whereas SDS-PAGE separated it into 18 protein components (MW 14-100 kD). Pooled patient's sera (ID +ve to PR) recognized 12 allergenic proteins in Putranjiva and five of them (MWs 92, 80, 55, 43 and 30 kD) showed immunologic reactivity to most of the sera samples tested individually by immunoblot. A number of shared allergenic proteins (MWs 92, 80, 66, 50, 43 and 14 kD) were observed between PR and RC pollen extracts on immunoblot using Putranjiva allergic serum pool. Inhibition in the binding for most of PR pollen allergenic proteins was obtained with higher concentration of RC extract than PR itself, depicting the presence of cross-reacting allergens in both. Putranjiva pollen extract was fractionated by a combination of DEAE Sephadex-A 50 and Sephadex-G 200 column chromatography. Periodate deglycosylation of western blotted PR extract and Put I fraction indicated the involvement of carbohydrate moieties in the allergenic activity. Of the two fractions from Put I (Ia and Ib), Put Ib was found to be the most allergenic protein by ELISA inhibition. Dot blot analysis with individual patients sera identified it as a major allergen of PR.

Latex allergen database.

Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.