O objetivo deste trabalho foi esclarecer qual das frações protéicas da soja é mais alergênica, avaliando-se sua imunogenicidade e alergenicidade por imunoensaios e teste de anafilaxia cutânea passiva. Além desse propósito, este estudo visou a produção de anticorpos monoclonais para utilizá-lo como ferramenta na padronização de um ensaio imunoenzimático com a finalidade de detectar proteínas da soja, em produtos comercializados. A purificação das frações 2S,7S e 11S da soja foi realizada com base em estudos prévios, padronizando-se a metodologia dentro das condições experimentais do laboratório. A constatação da pureza foi realizada por eletroforese em gel de poliacrilamida (gel de empilhamento a 5% e de separação em gradiente de 7 a 15%), onde se verificou as bandas protéicas características de cada fração. Evidenciou-se as subunidades α (63,17 KDa) α\' (58,06 KDa) e β (42,09 KDa), correspondentes à fração 7S da soja e as subunidades ácida (38,8 KDa) e básica (21,04 KDa), correspondentes à fração 11S da soja. A fração 2S da soja mostrou uma banda protéica de 20 KDa, nesse ensaio. Os resultados da cinética demonstraram que as frações 7S e 11S da soja são muito imunogênicas, visto que induziram uma grande produção de anticorpos das classes IgM e IgG, em camundongos BALB/c. Por outro lado, a fração 2S da soja induziu uma pequena produção de anticorpos IgG, principalmente no 30º dia após a imunização. No teste de anafilaxia cutânea passiva, quando se avaliou a capacidade das frações protéicas da soja de induzirem a produção de anticorpos IgE, em camundongos BALB/c, verificou-se que a fração 7S da soja é alergênica, nesse modelo experimental. Entretanto, as frações 2S e 11S não estimularam o sistema imunológico desses animais a produzir essa classe de anticorpo. Quando se investigou a presença de anticorpos IgE dirigidos contra as frações protéicas 2S, 7S e 11S da soja, no soro de pacientes alérgicos, obteve-se resultado negativo com todas as três proteínas estudadas. Entretanto, na análise da presença de IgG, tanto o soro dos pacientes alérgicos como o dos controles mostraram-se reativos às frações 2S e 7S da soja, sugerindo a presença do anticorpo IgG4 anafilático no soro teste. Os quatro anticorpos monoclonais reativos às frações 7S e 11S da soja, obtidos neste estudo, mostraram-se reativos até as diluições 1/8000 (2A8 e 1H4) e 1/10000 (1F9) para a fração 7S e 1/12000 (3F2) para a fração 11S da soja. A identificação dos determinantes antigênicos reconhecidos pelos anticorpos monoclonais foi realizado por Imunotransferência. Observou-se que os anticorpos monoclonais anti-fração protéica 7S da soja (1H4, 2A8 e 1F9) reconheceram as subunidades α, α e β (1F9), α\' e β (1H4) e β (2A8), além de outras proteínas. Por outro lado, o anticorpo monoclonal anti-fração protéica 11S da soja (3F2) reagiu apenas com a subunidade básica dessa proteína. A padronização do ensaio imunoenzimático com o objetivo de detectar proteínas de soja, em produtos comercializados, mostrou que os anticorpos monoclonais dirigidos contra as frações protéicas 7S e 11S reconhecem, apenas, as proteínas da soja. Em conclusão, esses resultados sugerem que as frações protéicas 7S e 11S da soja são imunogênicas em camundongos, enquanto que na espécie humana, essa resposta foi observada com as frações 2S e 7S da soja. No teste de anafilaxia cutânea passiva, a fração protéica 7S da soja mostrou a capacidade de induzir anticorpos da classe IgE em camundongos BALB/c, sugerindo que essa fração é alergênica nesse ensaio. Por outro lado, os anticorpos monoclonais reativos às frações 7S e 11S detectaram as proteínas da soja nos produtos comercializados testados. / The goal of this study was to elucidate which of the soy protein fractions is more allergenic by using enzyme immunoassays and passive cutaneous anaphylactic activity test. Furthermore, monoclonal antibodies were obtained in order to standardize immunoassays to detect soy protein fractions in commercial soy-derived products. Purification of 2S, 7S and 11S fractions from soy was based on previous studies which were adapted to our experimental conditions. Purity of the isolated fractions was determined by polyacrilamide gel electrophoresis (7-15%). The subunits α (63,17 KDa) α\' (58,06 KDa) and β (42,09 KDa), corresponding to 7S fraction as well as the acid (38,8 KDa) and basic (21,04 Kda) subunits of 11S fraction were visualized. The 2S fraction showed a 20 KDa band only. Data showed that the 7S and 11S soy fractions are immunogenic as they elicited IgM and IgG antibodies in BALB/c mice. In contrast, the 2S fraction was not immunogenic in this specie. The passive cutaneous anaphylactic activity test showed that 7S fraction is allergenic as it elicited IgE production in BALC mice. However, this test was negative for 2S and 7S fractions. No IgE reactive against the 2S, 7S e 11S was found in sera of patients (n=5) presenting previous food allergy symptoms. However, either these patients or controls (n=5) showed IgG reactive against 2S and 7S fractions in their sera. The four monoclonal antibodies obtained in this study were reactive up to 1/8000 (2A8 and 1H4) and 1/10000 (1F9) against 7S fraction, and up to 1/12000 (3F2) for 11S fraction. Immunoblot analysis showed that the monoclonal antibodies anti-7S fraction (1H4, 2A8 e 1F9) recognized the α α\' and β (1F9), α\' and β (1H4) and α (2A8) subunits. The monoclonal antibody anti-11S fraction (3F2) reacted with the basic subunit of this fraction only. By using ELISA and the monoclonal antibodies anti-7S and anti-11S it was possible to detect especifically these soy protein fractions in commercial soy-derived products. In conclusion, data suggest that 7S and 11S soy protein fractions are immunogenic in mice while 2S and 7S fraction are immunogenic in humans. The IgE response to 7S fraction in BALB/c mice showed by the passive cutaneous anaphylactic activity test indicates that this soy protein fraction is allergenic in this experimental model. Finally, the monoclonal antibodies anti-7S and anti-11S obtained in this study did not react with other vegetable or animal proteins being suitable to be used in ELISA for detection of these proteins in commercial soy-derived products.

Glyphosate (N-phosphonomethylglycine) is an organophosphate compound which was developed by the Monsanto Company in 1971 and is the active ingredient found in several herbicide formulations. The use of glyphosate-based herbicides in South Africa for the control of alien invasive plants and weeds is well established, extensive and currently unregulated, which vastly increases the likelihood of glyphosate contamination in environmental water systems. Although the use of glyphosate-based herbicides is required for economic enhancement in industries such as agriculture, the presence of this compound in natural water systems presents a potential risk to human health. Glyphosate and glyphosate formulations were previously considered safe, however their toxicity has become a major focal point of research over recent years. The lack of monitoring protocols for pesticides in South Africa is primarily due to limited financial capacity and the lack of analytical techniques.

Cyanobacteria are common inhabitants of freshwater lakes and reservoirs throughout the world. Under favourable conditions, certain cyanobacteria can dominate the phytoplankton within a waterbody and form nuisance blooms. Case reports and anecdotal references dating from 1949 describe a range of illnesses associated with recreational exposure to cyanobacteria: hay fever-like symptoms, pruritic skin rashes and gastro-intestinal symptoms (the latter probably related to ingestion of water) are most frequently reported. Some papers give convincing descriptions of allergic responses to cyanobacteria; others describe more serious acute illnesses, with symptoms such as severe headache, pneumonia, fever, myalgia, vertigo and blistering in the mouth. A U.S. coroner recently found that a teenage boy died as a result of accidentally ingesting a neurotoxic cyanotoxin from a golf course pond; this is the first recorded human fatality attributed to recreational exposure to cyanobacteria. One of the main public health concerns with exposure to freshwater cyanobacteria relates to the understanding that some blooms produce toxins that specifically affect the liver or the central nervous system. The route of exposure for these toxins is oral, from accidental or deliberate ingestion of recreational water, and possibly by inhalation. Cyanobacterial lipopolysaccharides (LPS) are also reported to be putative cutaneous, gastrointestinal, respiratory and pyrogenic toxins. The aims of this project were to enhance the understanding of public health issues relating to recreational exposure to cyanobacteria by conducting epidemiological and laboratory-based toxicology studies. A prospective cohort study of 1,331 recreational water users was conducted at various sites in southern Queensland, the Myall Lakes area of New South Wales, and central Florida. The study design sought to make improvements over previously published epidemiological studies, in that an unexposed group was recruited from cyanobacteria-free waters, cyanobacterial toxins were measured in site water samples, and respondents were asked to rate the severity of reported symptoms. This study has shown an increased likelihood of symptom reporting amongst bathers exposed to high cyanobacterial cell density (measured by total cell surface area) compared to those exposed to low cyanobacteria-affected waters. Mild respiratory symptoms appear to be the predominant symptom category. A clinical dermatology study to examine delayed-contact hypersensitivity reactions to cyanobacterial extracts was conducted. The study groups were 20 patients presenting for diagnostic skin patch testing at the Royal Brisbane Hospital's dermatology outpatient clinic; a convenience sample of 20 individuals was recruited from outside the hospital as a control group. One patient developed unequivocal reactions to several cyanobacteria extracts, with no dose-response pattern seen, indicating that the reactions were allergic in nature. A mouse model of delayed-contact hypersensitivity, the mouse ear swelling test, has demonstrated that the purified toxin cylindrospermopsin, a highly water-soluble compound, is capable of producing cutaneous injury. Encrusting lesions were seen on abdominal skin during the induction phase of these experiments. Delayed-contact hypersensitivity reactions were also demonstrated with this toxin. LPS from two non-axenic cyanobacterial samples - Cylindrospermopsis raciborskii and Microcystis aeruginosa - were extracted and purified. Thermoregulation studies were performed using a mouse model of rectal temperature measurement. Separate groups of mice were injected with these LPS extracts at two dose levels. Thermoregulation studies were also conducted with purified cyanobacterial toxins: microcystin-LR and cylindrospermopsin and anatoxin-a; cyanobacterial LPS samples purified by collaborators in Adelaide were also investigated for thermoregulation potential. These experiments have shown that the LPS extracts are weakly active when compared with responses to much lower doses of Escherichia coli LPS. Cylindrospermopsin also produces hypothermic responses in mice - comparable to pyrexia in larger mammals - although at a later stage than was seen with LPS extracts. Supplementing the experimental observations of the toxicology and immunotoxicology of cyanobacterial LPS are insights gained from beyond the cyanobacteria literature on the mechanisms of toxicity of different LPS structures. Cyanobacterial LPS was initially suggested to be toxic in the 1970s, at a time when lipid A, the active moiety of LPS, was thought to be identical across all Gram-negative bacteria. More recent work raises questions about the attribution of cutaneous and gastrointestinal illness to cyanobacterial LPS, with the understanding that some bacterial lipid A structures are LPS antagonists, LPS is not toxic by the oral route, and LPS is not reported as a toxin in the clinical dermatology literature. Gut-derived lipopolysaccharides, however, exert potent synergistic effects with a variety of xenobiotic hepatotoxins, and the well-known shock-like syndromes associated with severe cylindrospermopsin and microcystin poisoning deserve further scrutiny from the perspective of immunotoxicology.

Cyanobacteria are common inhabitants of freshwater lakes and reservoirs throughout the world. Under favourable conditions, certain cyanobacteria can dominate the phytoplankton within a waterbody and form nuisance blooms. Case reports and anecdotal references dating from 1949 describe a range of illnesses associated with recreational exposure to cyanobacteria: hay fever-like symptoms, pruritic skin rashes and gastro-intestinal symptoms (the latter probably related to ingestion of water) are most frequently reported. Some papers give convincing descriptions of allergic responses to cyanobacteria; others describe more serious acute illnesses, with symptoms such as severe headache, pneumonia, fever, myalgia, vertigo and blistering in the mouth. A U.S. coroner recently found that a teenage boy died as a result of accidentally ingesting a neurotoxic cyanotoxin from a golf course pond; this is the first recorded human fatality attributed to recreational exposure to cyanobacteria. One of the main public health concerns with exposure to freshwater cyanobacteria relates to the understanding that some blooms produce toxins that specifically affect the liver or the central nervous system. The route of exposure for these toxins is oral, from accidental or deliberate ingestion of recreational water, and possibly by inhalation. Cyanobacterial lipopolysaccharides (LPS) are also reported to be putative cutaneous, gastrointestinal, respiratory and pyrogenic toxins. The aims of this project were to enhance the understanding of public health issues relating to recreational exposure to cyanobacteria by conducting epidemiological and laboratory-based toxicology studies. A prospective cohort study of 1,331 recreational water users was conducted at various sites in southern Queensland, the Myall Lakes area of New South Wales, and central Florida. The study design sought to make improvements over previously published epidemiological studies, in that an unexposed group was recruited from cyanobacteria-free waters, cyanobacterial toxins were measured in site water samples, and respondents were asked to rate the severity of reported symptoms. This study has shown an increased likelihood of symptom reporting amongst bathers exposed to high cyanobacterial cell density (measured by total cell surface area) compared to those exposed to low cyanobacteria-affected waters. Mild respiratory symptoms appear to be the predominant symptom category. A clinical dermatology study to examine delayed-contact hypersensitivity reactions to cyanobacterial extracts was conducted. The study groups were 20 patients presenting for diagnostic skin patch testing at the Royal Brisbane Hospital's dermatology outpatient clinic; a convenience sample of 20 individuals was recruited from outside the hospital as a control group. One patient developed unequivocal reactions to several cyanobacteria extracts, with no dose-response pattern seen, indicating that the reactions were allergic in nature. A mouse model of delayed-contact hypersensitivity, the mouse ear swelling test, has demonstrated that the purified toxin cylindrospermopsin, a highly water-soluble compound, is capable of producing cutaneous injury. Encrusting lesions were seen on abdominal skin during the induction phase of these experiments. Delayed-contact hypersensitivity reactions were also demonstrated with this toxin. LPS from two non-axenic cyanobacterial samples - Cylindrospermopsis raciborskii and Microcystis aeruginosa - were extracted and purified. Thermoregulation studies were performed using a mouse model of rectal temperature measurement. Separate groups of mice were injected with these LPS extracts at two dose levels. Thermoregulation studies were also conducted with purified cyanobacterial toxins: microcystin-LR and cylindrospermopsin and anatoxin-a; cyanobacterial LPS samples purified by collaborators in Adelaide were also investigated for thermoregulation potential. These experiments have shown that the LPS extracts are weakly active when compared with responses to much lower doses of Escherichia coli LPS. Cylindrospermopsin also produces hypothermic responses in mice - comparable to pyrexia in larger mammals - although at a later stage than was seen with LPS extracts. Supplementing the experimental observations of the toxicology and immunotoxicology of cyanobacterial LPS are insights gained from beyond the cyanobacteria literature on the mechanisms of toxicity of different LPS structures. Cyanobacterial LPS was initially suggested to be toxic in the 1970s, at a time when lipid A, the active moiety of LPS, was thought to be identical across all Gram-negative bacteria. More recent work raises questions about the attribution of cutaneous and gastrointestinal illness to cyanobacterial LPS, with the understanding that some bacterial lipid A structures are LPS antagonists, LPS is not toxic by the oral route, and LPS is not reported as a toxin in the clinical dermatology literature. Gut-derived lipopolysaccharides, however, exert potent synergistic effects with a variety of xenobiotic hepatotoxins, and the well-known shock-like syndromes associated with severe cylindrospermopsin and microcystin poisoning deserve further scrutiny from the perspective of immunotoxicology.

Cyanobacteria are common inhabitants of freshwater lakes and reservoirs throughout the world. Under favourable conditions, certain cyanobacteria can dominate the phytoplankton within a waterbody and form nuisance blooms. Case reports and anecdotal references dating from 1949 describe a range of illnesses associated with recreational exposure to cyanobacteria: hay fever-like symptoms, pruritic skin rashes and gastro-intestinal symptoms (the latter probably related to ingestion of water) are most frequently reported. Some papers give convincing descriptions of allergic responses to cyanobacteria; others describe more serious acute illnesses, with symptoms such as severe headache, pneumonia, fever, myalgia, vertigo and blistering in the mouth. A U.S. coroner recently found that a teenage boy died as a result of accidentally ingesting a neurotoxic cyanotoxin from a golf course pond; this is the first recorded human fatality attributed to recreational exposure to cyanobacteria. One of the main public health concerns with exposure to freshwater cyanobacteria relates to the understanding that some blooms produce toxins that specifically affect the liver or the central nervous system. The route of exposure for these toxins is oral, from accidental or deliberate ingestion of recreational water, and possibly by inhalation. Cyanobacterial lipopolysaccharides (LPS) are also reported to be putative cutaneous, gastrointestinal, respiratory and pyrogenic toxins. The aims of this project were to enhance the understanding of public health issues relating to recreational exposure to cyanobacteria by conducting epidemiological and laboratory-based toxicology studies. A prospective cohort study of 1,331 recreational water users was conducted at various sites in southern Queensland, the Myall Lakes area of New South Wales, and central Florida. The study design sought to make improvements over previously published epidemiological studies, in that an unexposed group was recruited from cyanobacteria-free waters, cyanobacterial toxins were measured in site water samples, and respondents were asked to rate the severity of reported symptoms. This study has shown an increased likelihood of symptom reporting amongst bathers exposed to high cyanobacterial cell density (measured by total cell surface area) compared to those exposed to low cyanobacteria-affected waters. Mild respiratory symptoms appear to be the predominant symptom category. A clinical dermatology study to examine delayed-contact hypersensitivity reactions to cyanobacterial extracts was conducted. The study groups were 20 patients presenting for diagnostic skin patch testing at the Royal Brisbane Hospital's dermatology outpatient clinic; a convenience sample of 20 individuals was recruited from outside the hospital as a control group. One patient developed unequivocal reactions to several cyanobacteria extracts, with no dose-response pattern seen, indicating that the reactions were allergic in nature. A mouse model of delayed-contact hypersensitivity, the mouse ear swelling test, has demonstrated that the purified toxin cylindrospermopsin, a highly water-soluble compound, is capable of producing cutaneous injury. Encrusting lesions were seen on abdominal skin during the induction phase of these experiments. Delayed-contact hypersensitivity reactions were also demonstrated with this toxin. LPS from two non-axenic cyanobacterial samples - Cylindrospermopsis raciborskii and Microcystis aeruginosa - were extracted and purified. Thermoregulation studies were performed using a mouse model of rectal temperature measurement. Separate groups of mice were injected with these LPS extracts at two dose levels. Thermoregulation studies were also conducted with purified cyanobacterial toxins: microcystin-LR and cylindrospermopsin and anatoxin-a; cyanobacterial LPS samples purified by collaborators in Adelaide were also investigated for thermoregulation potential. These experiments have shown that the LPS extracts are weakly active when compared with responses to much lower doses of Escherichia coli LPS. Cylindrospermopsin also produces hypothermic responses in mice - comparable to pyrexia in larger mammals - although at a later stage than was seen with LPS extracts. Supplementing the experimental observations of the toxicology and immunotoxicology of cyanobacterial LPS are insights gained from beyond the cyanobacteria literature on the mechanisms of toxicity of different LPS structures. Cyanobacterial LPS was initially suggested to be toxic in the 1970s, at a time when lipid A, the active moiety of LPS, was thought to be identical across all Gram-negative bacteria. More recent work raises questions about the attribution of cutaneous and gastrointestinal illness to cyanobacterial LPS, with the understanding that some bacterial lipid A structures are LPS antagonists, LPS is not toxic by the oral route, and LPS is not reported as a toxin in the clinical dermatology literature. Gut-derived lipopolysaccharides, however, exert potent synergistic effects with a variety of xenobiotic hepatotoxins, and the well-known shock-like syndromes associated with severe cylindrospermopsin and microcystin poisoning deserve further scrutiny from the perspective of immunotoxicology.

Cyanobacteria are common inhabitants of freshwater lakes and reservoirs throughout the world. Under favourable conditions, certain cyanobacteria can dominate the phytoplankton within a waterbody and form nuisance blooms. Case reports and anecdotal references dating from 1949 describe a range of illnesses associated with recreational exposure to cyanobacteria: hay fever-like symptoms, pruritic skin rashes and gastro-intestinal symptoms (the latter probably related to ingestion of water) are most frequently reported. Some papers give convincing descriptions of allergic responses to cyanobacteria; others describe more serious acute illnesses, with symptoms such as severe headache, pneumonia, fever, myalgia, vertigo and blistering in the mouth. A U.S. coroner recently found that a teenage boy died as a result of accidentally ingesting a neurotoxic cyanotoxin from a golf course pond; this is the first recorded human fatality attributed to recreational exposure to cyanobacteria. One of the main public health concerns with exposure to freshwater cyanobacteria relates to the understanding that some blooms produce toxins that specifically affect the liver or the central nervous system. The route of exposure for these toxins is oral, from accidental or deliberate ingestion of recreational water, and possibly by inhalation. Cyanobacterial lipopolysaccharides (LPS) are also reported to be putative cutaneous, gastrointestinal, respiratory and pyrogenic toxins. The aims of this project were to enhance the understanding of public health issues relating to recreational exposure to cyanobacteria by conducting epidemiological and laboratory-based toxicology studies. A prospective cohort study of 1,331 recreational water users was conducted at various sites in southern Queensland, the Myall Lakes area of New South Wales, and central Florida. The study design sought to make improvements over previously published epidemiological studies, in that an unexposed group was recruited from cyanobacteria-free waters, cyanobacterial toxins were measured in site water samples, and respondents were asked to rate the severity of reported symptoms. This study has shown an increased likelihood of symptom reporting amongst bathers exposed to high cyanobacterial cell density (measured by total cell surface area) compared to those exposed to low cyanobacteria-affected waters. Mild respiratory symptoms appear to be the predominant symptom category. A clinical dermatology study to examine delayed-contact hypersensitivity reactions to cyanobacterial extracts was conducted. The study groups were 20 patients presenting for diagnostic skin patch testing at the Royal Brisbane Hospital's dermatology outpatient clinic; a convenience sample of 20 individuals was recruited from outside the hospital as a control group. One patient developed unequivocal reactions to several cyanobacteria extracts, with no dose-response pattern seen, indicating that the reactions were allergic in nature. A mouse model of delayed-contact hypersensitivity, the mouse ear swelling test, has demonstrated that the purified toxin cylindrospermopsin, a highly water-soluble compound, is capable of producing cutaneous injury. Encrusting lesions were seen on abdominal skin during the induction phase of these experiments. Delayed-contact hypersensitivity reactions were also demonstrated with this toxin. LPS from two non-axenic cyanobacterial samples - Cylindrospermopsis raciborskii and Microcystis aeruginosa - were extracted and purified. Thermoregulation studies were performed using a mouse model of rectal temperature measurement. Separate groups of mice were injected with these LPS extracts at two dose levels. Thermoregulation studies were also conducted with purified cyanobacterial toxins: microcystin-LR and cylindrospermopsin and anatoxin-a; cyanobacterial LPS samples purified by collaborators in Adelaide were also investigated for thermoregulation potential. These experiments have shown that the LPS extracts are weakly active when compared with responses to much lower doses of Escherichia coli LPS. Cylindrospermopsin also produces hypothermic responses in mice - comparable to pyrexia in larger mammals - although at a later stage than was seen with LPS extracts. Supplementing the experimental observations of the toxicology and immunotoxicology of cyanobacterial LPS are insights gained from beyond the cyanobacteria literature on the mechanisms of toxicity of different LPS structures. Cyanobacterial LPS was initially suggested to be toxic in the 1970s, at a time when lipid A, the active moiety of LPS, was thought to be identical across all Gram-negative bacteria. More recent work raises questions about the attribution of cutaneous and gastrointestinal illness to cyanobacterial LPS, with the understanding that some bacterial lipid A structures are LPS antagonists, LPS is not toxic by the oral route, and LPS is not reported as a toxin in the clinical dermatology literature. Gut-derived lipopolysaccharides, however, exert potent synergistic effects with a variety of xenobiotic hepatotoxins, and the well-known shock-like syndromes associated with severe cylindrospermopsin and microcystin poisoning deserve further scrutiny from the perspective of immunotoxicology.