A Lutzomyia longipalpis Salivary Protein Induces Cross-Reactive Antibodies to Pemphigus Autoantigen Desmoglein 1.

Fogo selvagem (FS) is a blistering skin disease caused by pathogenic IgG4 autoantibodies to desmoglein 1 (DSG1). Preclinical FS and leishmaniasis are endemic to certain regions of Brazil and exhibit nonpathogenic anti-DSG1 antibodies. Recurring bites from Lutzomyia longipalpis, the sand fly vector of leishmaniasis, immunize individuals with L. longipalpis salivary antigens LJM17 and LJM11. We measured the antibody responses to LJM17, LJM11, and DSG1 in normal settlers and patients with FS from an endemic focus of FS and nonendemic control populations. We also immunized mice with these antigens and assessed the IgG response. Healthy individuals and patients with FS from endemic areas had significantly higher values of IgG4 anti-LJM17 antibodies than nonendemic controls (P < 0.001 for both). The levels of IgG anti-DSG1 and IgG4 anti-LJM17 and anti-LJM11 antibodies correlated positively in normal settlers and patients with FS. Mice immunized with recombinant LJM17 produced IgG1 antibodies (human IgG4 homolog) that strongly cross-reacted with recombinant DSG1; these IgG1 antibodies were inhibited by LJM17, LJM11, and DSG1 in a dose-dependent manner. However, they did not bind human or mouse epidermis by indirect immunofluorescence. Lastly, we identified short-sequence homologies of surface-exposed residues within the human DSG1 ectodomain and LJM17. Inoculation by LJM17 from L. longipalpis-elicited DSG1-cross-reactive IgG4 antibodies may lead to FS in genetically predisposed individuals.

Paratrygon aiereba irradiated anti-mucus serum reduce edematogenic activity induced in experimental model.

Accidents by freshwater stingrays are common in northern Brazil, there is no specific therapy for high morbidity and local tissue destruction. The irradiation of venoms and toxins by ionizing radiation has been used to produce appropriate immunogens for the production of antisera. We planned to study the efficacy of stinging mucus irradiation in the production of antisera, with serum neutralization assays of edematogenic activity and quantification of cytokines performed in animal models of immunization with native and irradiated mucus of Paratrygon aiereba, a large freshwater stingray. Antiserum potency and its cross-reactivity with mucus from other freshwater stingrays were detected by ELISA. Immunization models demonstrated the ability to stimulate a strong humoral response with elevated levels of serum IgG detectable by ELISA, and both native and irradiated mucus were immunogenic and capable of recognizing mucus proteins from other freshwater neotropical stingrays. Mucus P. aiereba causes cellular and humoral adaptive immune responses in cells of immunized mice producing antibodies and cytokines such as TNF-α, IL-6 and IL-17. Rabbit antisera immunized with mucus from P. aiereba irradiated at 2 kGy showed a significant reduction of mucus-induced edematogenic activity in mice. Our data suggest that the use of antisera against freshwater stingray mucus show the possibility of specific therapy for these accidents.

[Sensitivity and specificity of prick skin test with two concentrations of standardized extract of Culex quinquefasciatus in allergic children]. / Sensibilidad y especificidad de la prueba cutánea por punción con dos concentraciones del extracto estandarizado de Culex quinquefasciatus en niños alérgicos.

BACKGROUND: Diagnostic options for immune reactions to mosquito bites are limited. In Cuba, IgE-mediated reactions are frequently related to Culex quinquefasciatus bite. OBJECTIVE: To determine the sensitivity and specificity of skin prick test with two doses of standardized extract in nitrogen protein units (PNU of Culex quinquefasciatus (BIOCEN, Cuba). MATERIAL AND METHOD: An analytical study was conducted on 100 children between 2 and 15 years old. Fifty atopic patients with a history of allergy to mosquito bite and positive specific serum IgE Culex quinquefasciatus and fifty atopic patients without a history of allergy to mosquito bite and negative specific serum IgE to Culex quinquefasciatus. Skin prick tests (SPT) were performed by duplicates on the forearms of the patients. Investigated doses were 100 PNU/mL and 10 PNU/mL. RESULTS: SPT with the highest concentration obtained a mean wheal size of 22.09 mm2 and for lower doses of 8.09 mm2, a statistically significant difference (p=0.001, Student's t test). Positive skin test correlated in 100% of patients with the presence of specific IgE. Testing with both doses showed a 94% of specificity and 88% of sensitivity. CONCLUSION: The diagnostic accuracy of SPT using both doses of standardized extract was similar, which justifies its use for diagnosis of sensitization to Culex quinquefasciatus in patients with symptoms of allergy to mosquito bite.

Antecedentes: las opciones diagnósticas de las reacciones inmunológicas a la picadura del mosquito son limitadas. En Cuba, las reacciones mediadas por IgE más frecuentes son por picadura de Culex quinquefasciatus. Objetivo: determinar la sensibilidad y especificidad de la prueba cutánea por punción con dos dosis del extracto estandarizado en unidades de nitrógeno proteico (UNP) de Culex quinquefasciatus (BIOCEN, Cuba). Material y método: estudio analítico efectuado en 100 niños entre 2 y 15 años de edad: 50 pacientes atópicos con antecedentes de alergia a la picadura de mosquito e IgE sérica específica positiva a Culex quinquefasciatus y 50 pacientes atópicos sin antecedentes de alergia a la picadura de mosquito e IgE sérica específica negativa a Culex quinquefasciatus. La prueba cutánea por punción se realizó por duplicado en los antebrazos de los pacientes. Las dosis investigadas fueron 100 y 10 UNP/mL. Resultados: en la prueba cutánea por punción con el extracto de mayor concentración se obtuvo un tamaño del área del habón de 22.09 mm2 y con la menor concentración de 8.19 mm2; una diferencia estadísticamente significativa (p=0.001, prueba t de Student). La prueba cutánea positiva se correlacionó en el 100% de los pacientes con la existencia de IgE específica. La prueba con ambas dosis mostró 94% de especificidad y 88% de sensibilidad. Conclusión: la alta coincidencia en el resultado de la prueba cutánea nos muestra que puede sustituirse la concentración del extracto a 100 UNP/mL por la de menor concentración, sin perder confiabilidad en el diagnóstico de sensibilización al mosquito Culex quinquefasciatus, utilizando ese método in vivo.

Characterization of the early inflammatory infiltrate at the feeding site of infected sand flies in mice protected from vector-transmitted Leishmania major by exposure to uninfected bites.

BACKGROUND: Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN-γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+ T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. CONCLUSION/SIGNIFICANCE: The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection.

Characterization of guinea pig antibody responses to salivary proteins of Triatoma infestans for the development of a triatomine exposure marker.

BACKGROUND: Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure. METHODOLOGY AND PRINCIPAL FINDINGS: In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs. CONCLUSION: Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae).

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Specific pattern of flea antigen recognition by IgG subclass and IgE during the progression of papular urticaria caused by flea bite.

BACKGROUND: Papular urticaria caused by flea bite presents clinical symptoms of a hypersensitivity reaction accompanied by skin lesions. However, the pattern of recognition by different antibody isotypes during the progression of the disease is unknown. This study evaluated variations in immunoglobulin E and immunoglobulin G subclass antibody responses to flea antigens during the progression of papular urticaria caused by flea bite METHODS: Twenty-five patients clinically diagnosed with papular urticaria due to flea bite were included. Ten healthy children were included as controls. Recognition of antigens from complete flea body extract by patients and healthy controls was determined using immunoblot assays. RESULTS: The results revealed that patients with 2-5 years of papular urticaria evidenced more IgE bands than those with shorter or longer durations of symptoms. In contrast, healthy children showed a predominance of immunoglobulin G1 and immunoglobulin G3. The majority of the recognised antigens were low molecular weight proteins (<90 kDa). Proteins with molecular weights between 16-20, 21-25, and 31-35 kDa showed different patterns of recognition between patients and healthy children. CONCLUSION: The predominant specific antibody isotypes vary according to the time elapsed since the onset of symptoms in papular urticaria caused by flea bite.

In vivo protection against Tityus serrulatus scorpion venom by antibodies raised against a discontinuous synthetic epitope.

Scorpion stings cause human fatalities in numerous countries. Serotherapy is the only specific means to try to circumvent the noxious effects of venom toxins. TsNTxP is a natural anatoxin from the venom of the scorpion Tityus serrulatus that may be useful to raise therapeutic anti-venom sera. Linear epitopes recognized by anti-TsNTxP antibodies have previously been mapped. Here, we attempted to identify discontinuous epitopes in TsNTxP since neutralizing epitopes are often associated with such complex entities. One hundred and fifty-three octadecapeptides with the general formula (P1)-(Gly-Gly)-(P2) were synthesized by the Spot method on cellulose membranes. P1 and P2 were octapeptides from the TsNTxP N-terminal and C-terminal sections, respectively. Each sequence of eight amino acids was frameshifted in turn by three residues, in order to cover TsNTxP entire sequence. Binding of neutralizing anti-TsNTxP rabbit antibodies to spotted peptides revealed GREGYPADGGGLPDSVKI as the more reactive peptide sequence. This epitope was made from the first eight residues of the protein (GREGYPAD) and from residues 47 to 54 (GLPDSVKI) of the C-terminal part of TsNTxP. BALB/c mice were immunized with synthetic GREGYPADGGGLPDSVKI peptide conjugated to ovalbumin. One week after the last immunization, in vivo protection assays showed that immunized mice could resist a challenge by an amount of T.serrulatus whole venom equivalent to 1.75 LD(100), a dose that killed all control non-immune mice. Based on molecular models of TsNTxP and related Tityus toxins, we found that the above peptide matches with a discontinuous epitope, well exposed at the toxin molecular surface which contains residues known to be important for the bioactivity of toxins.

Mortality and antibody responses of mice to three successive episodes of experimental scorpion (Centruroides limpidus limpidus) envenomation and immunological rescue.

Mortality rates of mice and their levels of anti-venom and anti-F(ab')2 antibodies were assessed after three episodes of subcutaneous envenomations with or without treatment with horse F(ab')2. Soluble venom from the Mexican scorpion Centruroides limpidus limpidus was used for these experiments. Repetition of episodes did not induce different mortality rates in untreated mice. F(ab')2 rescued about 85% of the mice in the first two episodes and 66% in the third, without distinction of gender or ostensible side-effects: a suggestion of selection of the most resistant mice. Surviving mice produced in vitro neutralizing antibodies to the scorpion venom and also antibodies to F(ab')2, when injected alone but more so if combined: a possible immunological adjuvant or alarm effect of the venom or of the cascading physiopathology of envenomation. In the few surviving mice, both anti-venom and anti-F(ab')2 antibodies increased significantly after the first envenomation but not thereafter, showing no correlation with mortality rates: a suggestion of their clinical irrelevance, the few hard-to kill mice appeared to resist envenomation by mechanisms other than antibody response. Injection of F(ab')2 alone induced production of detectable anti-venom antibodies in a few mice and injection of venom alone induced that of anti-F(ab')2 antibodies, perhaps due to trace amounts of venom in the high affinity fraction of F(ab')2 and to anti-idiotypic antibodies or polyclonal activity in the envenomation episode, respectively.

Cytokine and nitric oxide production following severe envenomation.

Venom is a complex mixture of many substances such as toxins, enzymes, growth factor activators, and inhibitors are particularly responsible for the deleterious effects of cells. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Envenomation by bees, scorpions, snakes, spiders and wasps involves the activation of the inflammatory response with the release and activation of pro-inflammatory cytokines and other mediators, such as nitric oxide. Recently, a battery of cytokines produced by activated T cells or macrophages have been added to in envenomations. Cytokines are important for the interactions between cells in the immune and inflammatory responses. Although the pathophysiology of envenomation is not fully understood, venom and immune responses are known to trigger the release of cytokines and nitric oxide. The cytokines initiate a cascade of events that lead to illness behaviors such as fever, anorexia, and, as well as a host of physiologic events such as activation of vasodilation, hypotension and increased nitric oxide production. Accumulating evidence indicates that these cytokines play important roles in mediating cell recruitment and activation necessary for inflammation and the repair of tissue damage. A better understanding of the involvement of the inflammatory system in different envenoming syndromes may have future therapeutic benefits.

Advances in equine immunology: Havemeyer workshop reports from Santa Fe, New Mexico, and Hortobagy, Hungary.

The horse has been human kind's most important partner throughout history. Similarly, in the field of immunology, many critical scientific advances have depended on the horse. Equine immunology today is an active and important field of study, with a focus on control of many common infectious diseases and immunopathologic conditions of broad comparative interest. In 2001 two major equine immunology workshops were held, in Santa Fe, USA, and in Hortobagy, Hungary, with major sponsorship from the Havemeyer Foundation. This report summarizes the scientific themes and foci of those meetings.

Standartization of an enzyme linked immunonosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus

Neste trabalho foram determinadas a sensibilidade e a especificidade da tecnica imunoenzimatica (ELISA) desenvolvida por CHAVEZ-OLORTEGUI et al. para detectar antigenos circulantes de veneno em pacientes picados por Tityus serrulatus. A media mais dois desvios padrao da observancia do soro de 100 pacientes controles foi utilizada como limite entre teste positivo e teste negativo ("cutoff"). A especificidade do ELISA foi igual a 97,0 por cento . A sensibilidade do metodo, quando incluidos pacientes classificados como casos leves, moderados e graves de escorpionismo, foi de 39,3 por cento e aumentou para 94,7 por cento quando considerados apenas os casos moderados e graves. Estes resultados mostram que o ELISA pode ser utilizado para deteccao de antigenos toxicos circulantes em pacientes com manifestacoes sistemicas de envenenamento escorpionico mas nao deve ser empregado no estudo de pacientes que apresentam apenas dor no local da picada (casos leves). O tempo necessario para a realizacao do ELISA e superior a 1 hora. Portanto, o teste tem sua utilizacao limitada para o diagnostico de envenenamento, mas pode construir um instrumento util para o estudo da cinetica de neutralizacao do veneno pelo antiveneno especifico.

Clinical perspectives on seabather's eruption, also known as 'sea lice'.

Seabather's eruption is usually a benign clinical syndrome that resolves spontaneously, although severe symptoms and long-term sequelae have been identified. Recent research has implicated the larvae of a jellyfish, Linuche unguiculata, as the cause of this syndrome; confirmation by serological and experimental studies is pending. Clinical signs and symptoms are consistent with this etiology. Outbreaks occur when jellyfish larvae are transported to shore by ocean currents. Treatment is symptomatic and involves use of antihistamines and steroids.