Avaliação da exposição a fungos e partículas em explorações avícolas e suinícolas: estudo

O projeto “Avaliação da Exposição a Fungos e Partículas em Explorações Avícolas e Suinícolas” contemplou um elevado número de colheitas ambientais e biológicas e respectivo processamento laboratorial, sendo apenas possível a sua concretização graças ao financiamento disponibilizado pela Autoridade para as Condições de Trabalho. Foi realizado um estudo transversal para avaliar a contaminação causada por fungos e partículas em 7 explorações avícolas e 7 explorações suinícolas. No que concerne à monitorização biológica, foram medidos os parâmetros espirométricos, utilizando o espirómetro MK8 Microlab, avaliada a existência de sintomas clínicos associados com a asma e outras doenças alérgicas, através de questionário adaptado European Community Respiratory Health Survey e, ainda, avaliada a sensibilização aos agentes fúngicos (IgE). Foram ainda adicionados dois objetivos ao estudo, designadamente: aferir a existência de três espécies/estirpes potencialmente patogénicas/toxinogénicas com recurso à biologia molecular e avaliar a exposição dos trabalhadores à micotoxina aflatoxina B1 por recurso a indicador biológico de exposição. Foram colhidas 27 amostras de ar de 25 litros nas explorações avícolas e 56 de 50 litros nas explorações suinícolas através do método de impacto. As colheitas de ar e a medição da concentração das partículas foram realizadas no interior e no exterior dos pavilhões, sendo este último considerado como local de referência. Simultaneamente, a temperatura e a humidade relativa também foram registadas. As colheitas das superfícies foram realizadas através da técnica de zaragatoa, tendo sido utilizado um quadrado de metal inoxidável de 10 cm de lado, de acordo com a International Standard ISO 18593 – 2004. As zaragatoas obtidas (20 das explorações avícolas e 48 das explorações suinícolas) foram inoculadas em malte de extract agar (2%) com cloranfenicol (0,05 g/L). Além das colheitas de ar e de superfícies, foram também obtidas colheitas da cama das explorações avícolas (7 novas e 14 usadas) e da cobertura do pavimento das explorações suinícolas (3 novas e 4 usadas) e embaladas em sacos esterilizados. Cada amostra foi diluída e inoculada em placas contendo malte extract agar. Todas as amostras foram incubadas a 27,5ºC durante 5 a 7 dias e obtidos resultados quantitativos (UFC/m3; UFC/m2; UFC/g) e qualitativos com a identificação das espécies fúngicas. Para a aplicação dos métodos de biologia molecular foram realizadas colheitas de ar de 300 litros utilizando o método de impinger com a velocidade de recolha de 300 L/min. A identificação molecular de três espécies potencialmente patogénicas e/ou toxinogénicas (Aspergillus flavus, Aspergillus fumigatus e Stachybotrys chartarum) foram obtidas por PCR em tempo real (PCR TR) utilizando o Rotor-Gene 6000 qPCR Detection System. As medições de partículas foram realizadas por recurso a equipamento de leitura direta (modelo Lighthouse, 2016 IAQ). Este recurso permitiu medir a concentração (mg/m3) de partículas em 5 dimensões distintas (PM 0.5; PM 1.0; PM 2.5; PM 5.0; PM10). Nas explorações avícolas, 28 espécies/géneros de fungos foram isolados no ar, tendo Aspergillus versicolor sido a espécie mais frequente (20.9%), seguida por Scopulariopsis brevicaulis (17.0%) e Penicillium sp. (14.1%). Entre o género Aspergillus, Aspergillus flavus apresentou o maior número de esporos (>2000 UFC/m3). Em relação às superfícies, A. versicolor foi detetada em maior número (>3 × 10−2 UFC/m2). Na cama nova, Penicillium foi o género mais frequente (59,9%), seguido por Alternaria (17,8%), Cladosporium (7,1%) e Aspergillus (5,7%). Na cama usada, Penicillium sp. foi o mais frequente (42,3%), seguido por Scopulariopsis sp. (38,3%), Trichosporon sp. (8,8%) e Aspergillus sp. (5,5%). Em relação à contaminação por partículas, as partículas com maior dimensão foram detectadas em maiores concentrações, designadamente as PM5.0 (partículas com a dimensão de 5.0 bm ou menos) e PM10 (partículas com a dimensão de 10 bm ou menos). Neste setting a prevalência da alteração ventilatória obstrutiva foi superior nos indivíduos com maior tempo de exposição (31,7%) independentemente de serem fumadores (17,1%) ou não fumadores (14,6%). Relativamente à avaliação do IgE específico, foi apenas realizado em trabalhadores das explorações avícolas (14 mulheres e 33 homens), não tendo sido encontrada associação positiva (p<0.05%) entre a contaminação fúngica e a sensibilização a antigénios fúngicos. No caso das explorações suinícolas, Aspergillus versicolor foi a espécie mais frequente (20,9%), seguida por Scopulariopsis brevicaulis (17,0%) e Penicillium sp. (14,1%). No género Aspergillus, A. versicolor apresentou o maior isolamento no ar (>2000 UFC/m3) e a maior prevalência (41,9%), seguida por A. flavus e A. fumigatus (8,1%). Em relação às superfícies analisadas, A. versicolor foi detetada em maior número (>3 ×10−2 UFC/m2). No caso da cobertura do pavimento das explorações suinícolas, o género Thicoderma foi o mais frequente na cobertura nova (28,0%) seguida por A. versicolor e Acremonium sp. (14,0%). O género Mucor foi o mais frequente na cobertura usada (25,1%), seguido por Trichoderma sp. (18,3%) e Acremonium sp. (11,2%). Relativamente às partículas, foram evidenciados também valores mais elevados na dimensão PM5 e, predominantes nas PM10. Neste contexto, apenas 4 participantes (22,2%) apresentaram uma alteração ventilatória obstrutiva. Destes, as obstruções mais graves encontraram-se nos que também apresentavam maior tempo de exposição. A prevalência de asma na amostra de trabalhadores em estudo, pertencentes aos 2 contextos em estudo, foi de 8,75%, tendo-se verificado também uma prevalência elevada de sintomatologia respiratória em profissionais não asmáticos. Em relação à utilização complementar dos métodos convencionais e moleculares, é recomendável que a avaliação da contaminação fúngica nestes settings, e, consequentemente, a exposição profissional a fungos, seja suportada pelas duas metodologias e, ainda, que ocorre exposição ocupacional à micotoxina aflatoxina B1 em ambos os contextos profissionais. Face aos resultados obtidos, é importante salientar que os settings alvo de estudo carecem de uma intervenção integrada em Saúde Ocupacional no âmbito da vigilância ambiental e da vigilância da saúde, com o objetivo de diminuir a exposição aos dois factores de risco estudados (fungos e partículas).

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG aginst T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytothropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

Quantitative Analysis of Immunoglobulin E Reactivity Profiles in Patients Allergic or Sensitized to Natural Rubber Latex (Hevea Brasiliensis)

Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy

Predicting positive food challenges in children sensitised to peanuts/tree nuts.

BACKGROUND: Children with atopic diseases in early life are frequently found with positive IgE tests to peanuts/tree nuts without a history of previous ingestion. We aimed to identify risk factors for reactions to nuts at first introduction. METHODS: A retrospective case-note and database analysis was performed. Recruitment criteria were: patients aged 3-16 yr who had a standardized food challenge to peanut and/or tree nuts due to sensitisation to the peanut/tree nut (positive spIgE or SPT) without previous consumption. A detailed assessment was performed of factors relating to food challenge outcome with univariate and multivariate logistic regression analysis. RESULTS: There were 98 food challenges (47 peanut, 51 tree nut) with 29 positive, 67 negative and 2 inconclusive outcomes. A positive maternal history of allergy and a specific IgE >5 kU/l were strongly associated with a significantly increased risk of a positive food challenge (OR 3.73; 95% CI 1.31-10.59; p = 0.013 and OR 3.35; 95% CI 1.23-9.11; p = 0.007, respectively). Adjusting for age, a three year-old with these criteria has a 67% probability of a positive challenge. There was no significant association between types of peanut/tree nut, other food allergies, atopic conditions or severity of previous food reactions and positive challenges. CONCLUSIONS: We have demonstrated an association between the presence of maternal atopic history and a specific IgE >5 kU/l, with a significant increase in the likelihood of a positive food challenge. Although requiring further prospective validation these easily identifiable components should be considered when deciding the need for a challenge.

Vaccine strategies against schistosomiasis

In this review the authors analyze the effector and regulatory mechanisms in the immune response to schistosomiasis. To study these mechanisms two animal models were used, mouse and rat. The mouse totaly permissive host like human, show prominent-T cell control in the acquisition of resistance. But other mechanisms like antibody mediated cytotoxity (ADCC) involving eosinophils and IgG antibodies described in humans, are observed in rats. Also in this animal, it is observed specific IgE antibody high production and blood and tisssue eosinophilia. Using the rat model and schistosomula as target, some ADCC features have emerged: the cellular population involved are bone marrow derived inflammatory cell (mononuclear phagocytes, eosinophils and platelets), interacting with IgE through IgE Fc receptors. Immunization has been attempted using the recombinant protein Sm28/GST. Protection has been observed in rodents with significant decrease of parasite fecundity and egg viability affecting the number, size and volume of liver egg granulomas. The association of praziquantel and immunization with with Sm28/GST increases the resistance to infection and decreases egg viability. The authors suggest the possibility of the stablishment of a future vaccine against Schistosoma mansoni.

Interleukin-4 and interleukin-5 as targets for the inhibition of eosinophilic inflammation and allergic airways hyperreactivity

Clinical and experimental investigations suggest that allergen-specific CD4+ T-cells, IgE and the cytokines IL-4 and IL-5 play central roles in initiating and sustaining an asthmatic response by regulating the recruitment and/or activation of airways mast cells and eosinophils. IL-5 plays a unique role in eosinophil development and activation and has been strongly implicated in the aetiology of asthma. The present paper summarizes our recent investigations on the role of these cytokines using cytokine knockout mice and a mouse aeroallergen model. Investigations in IL-5-/- mice indicate that this cytokine is critical for regulating aeroallergen-induced eosinophilia, the onset of lung damage and airways hyperreactivity during allergic airways inflammation. While IL-4 and allergen-specific IgE play important roles in the regulation of allergic disease, recent investigations in IL4-/- mice suggest that allergic airways inflammation can occur via pathways which operate independently of these molecules. Activation of these IL-4 independent pathways are also intimately associated with CD4+ T-cells, IL-5 signal transduction and eosinophilic inflammation. Such IL-5 regulated pathways may also play a substantive role in the aetiology of asthma. Thus, evidence is now emerging that allergic airways disease is regulated by humoral and cell mediated processes. The central role of IL-5 in both components of allergic disease highlights the requirements for highly specific therapeutic agents which inhibit the production or action of this cytokine.

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemaglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

Clinical and immunological consequences of human T cell leukemia virus type-I and Schistosoma mansoni co-infection

Human T cell leukemia virus type-I (HTLV-I) infection is associated with spontaneous T cell activation and uncontrolled lymphocyte proliferation. An exacerbated type-1 immune response with production of pro-inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) is significantly higher in patients with myelopathy associated to HTLV-I than in HTLV-I asymptomatic carriers. In contrast with HTLV-I, a chronic Schistosoma mansoni infection is associated with a type-2 immune response with high levels of interleukin (IL-4, IL-5, and IL-10) and low levels of IFN-gamma. In this study, clinical and immunological consequences of the HTLV-I and S. mansoni infection were evaluated. The immune response in patients with schistosomiasis co-infected with HTLV-I showed low levels of IL-5 (p < 0.05) in peripheral blood mononuclear cells cultures stimulated with S. mansoni antigen (SWAP) and decreased SWAP-specific IgE levels when compared with patients with only schistosomiasis (p < 0.05). Liver fibrosis was mild in all HTLV-I co-infected patients. Immunological response was also compared in individuals who had only HTLV-I infection with those who were co-infected with HTLV-I and helminths (S. mansoni and Strongyloides stercoralis). In patients HTLV-I positive co-infected with helminths the IFN-gamma levels were lower than in individuals who had only HTLV-I. Moreover, there were fewer cells expressing IFN-gamma and more cells expressing IL-10 in individuals co-infected with HTLV-I and helminths. These dates indicate that HTLV-I infection decrease type 2-response and IgE synthesis and are inversely associated with the development of liver fibrosis. Moreover, helminths may protect HTLV-I infected patients to produce large quantities of pro-inflammatory cytokines such as IFN-gamma.

Allergologie: 3. Intérêts des allergènes recombinants dans la pratique de l'allergologie [Interests of recombinant allergens in the practice of allergy].

Compared to total allergenic extracts, recombinant allergens available for specific IgE measurement represent an important advance in the diagnosis and treatment of IgE-mediated allergies. Recombinant allergens lead to define the sensitization profile of allergic patients, to identify markers of sensitization and to understand better polysensitivities related to cross-reactions and markers of severity of allergic reactions. They also contribute to the decision to establish tolerance induction (allergen specific immunotherapy) and the optimal selection of the allergenic composition of the vaccine.

Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon).

BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. CONCLUSIONS Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Predicting positive food challenges at the introduction of nuts in sensitised children

Rationale: Children with atopic diseases in early life are frequently found with positive IgE tests to nuts, without a history of previous ingestion. We aimed to identify risk factors for reactions to nuts at their first introduction. Methods: A detailed retrospective case note and database analysis was performed. Inclusion criteria were: patients aged 3 to 16 years who had had a standardized food challenge to peanut and/or tree nuts due to primary sensitisation to the nut (positive specific IgE or SPT). A detailed assessment was performed of factors relating to food challenge outcome with univariate and multivariate logistic regression analysis. Results: There were 98 food challenges (48% peanut, 52% tree nut) with 29 positive, 67 negative and 2 inconclusive challenges. A positive maternal history and a specific IgE > 2 kU/l were strongly associated with a significantly increased risk of a positive food challenge (OR 3.54; 95% CI 1.28 to 9.81; and OR 4.82; 95% CI 1.57 to 14.86; respectively). There was no significant association between the type of nut, age, presence of other food allergies, paternal or sibling atopic history, other atopic conditions or severity of previous reaction to other foods. Conclusions: We have demonstrated an association between the presence of a maternal atopic history and a specific IgE > 2 kU/l, and a significant increase in the likelihood of a positive food challenge in children with primary sensitisation to nuts. Although requiring further prospective validation we suggest these easily identifiable components should be considered when deciding the need for a nut challenge.

Allergie aux venins d'hyménopteres: nouveautés diagnostiques et prise en charge [Hymenoptera venom allergy: new diagnostic tools and management].

Anaphylactic reactions to hymenoptera venoms are common and, in our latitudes, mainly concern wasps and bees. Recently, molecular biology techniques have contributed to identifying and to sequencing the major allergens of insect venoms and led to the production of recombinant allergens. Assays for specific IgE directed against these recombinant allergens have recently been made available in clinical practice. They provide considerable assistance in identifying the insect responsible for an anaphylactic reaction, in particular when standard tests are positive for both wasp and bee. This article focuses on these new laboratory tests and also reviews the management of patients experiencing an anaphylactic reaction after hymenoptera sting.

Allergens Induce the Release of Lactoferrin by Neutrophils from Asthmatic Patients.

BACKGROUND Despite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question. METHODS Neutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed. RESULTS Lf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients.

Splenic hypereosinophilia in anaphylaxis-related death: different assessments depending on different types of allergens?

The aim of this study was to evaluate splenic eosinophil and mast cell accumulation using pagoda red stain in a series of anaphylaxis-related deaths that underwent medico-legal investigations. Our goal was to assess whether fatal reactions to insect stings, intramuscularly administered antibiotics and intravenously injected contrast media are responsible for specific patterns of eosinophil and mast cell accumulation. Two study groups were prospectively formed, an anaphylaxis-related death group and a control group. Autopsy, histology (haematoxylin-eosin stain, pagoda red stain and immunohistochemistry using anti-tryptase antibodies), toxicology and postmortem biochemistry (beta-tryptase, total IgE and specific IgE) were performed in all cases. All tested parameters (spleen weight, beta-tryptase and total IgE levels as well as eosinophil, mast cell and degranulated mast cell numbers in the spleen) were significantly higher in the anaphylaxis-related death group. No statistically significant differences were observed among the various groups (intramuscular antibiotic injection, intravenous contrast medium administration and stinging insects) in any combination, suggesting that mast cell and eosinophil accumulation in the spleen during anaphylaxis does not have any specific pattern related to the triggering allergen. Despite a lower sensitivity than immunohistochemical staining in discriminating eosinophil and mast cells, pagoda red stain allowed these cells to be identified and could therefore be proposed as a low-cost, first-line diagnostic procedure in those situations where immunohistochemistry is not systematically performed or cannot be carried out.