Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser + appendix.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

The innate immune system provides the first line of defense against infection. Pathogens are detected though a variety of Pattern Recognition Receptors (PRRs), which activate downstream signaling cascades. Effector molecules such as cytokines and chemokines are released upon activation and aid in cell recruitment, control of pathogen replication, and coordination of the adaptive immune response. Nucleic acids that are released into the cytosol during viral and bacterial infection are recognized through a special class of PRRs, coined cytosolic nucleic acid sensors. Upon recognition, these receptors induce the production of type I interferons and other cytokines to aid in pathogen clearance. Although many cytosolic nucleic acid sensors have been discovered, it is unclear how they work in concert to mediate these responses. The Interferon Gamma Inducible protein (IFI)16 and its proposed mouse orthologue IFI204 are cytosolic DNA sensors that have been linked to the detection of cytosolic DNA during infection with Herpes Simplex Virus (HSV-1). IFI16 binds dsDNA that has been released into the cytosol during viral infection and engages the adaptor molecule Stimulator of Interferon Genes (STING) leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I interferons and interferon stimulated genes. In addition to its role as a sensor, in chapter two of this thesis we describe a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in anti-viral immunity. In an effort to better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as other inducers of IFN such as cyclic-dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to cytosolic DNA ligands and DNA viruses. In contrast, expression of the NF-κB regulated cytokines such as IL-6 and IL-1β were unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of expression of IFN-α and IFN stimulated genes such as RIG-I in response to cyclic GMP-AMP (cGAMP), a second messenger produced in response to cGAS, as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in IFI16 knockdown cells suggesting that transcription of ISGs is dependent on IFI16. Since IFI16 knockdown compromised not only DNA virus driven pathways, we propose additional regulatory roles outside of DNA sensing. Collectively, these results indicate that IFI16 plays a role in the regulation of type I IFN gene transcription and production in response to both RNA and DNA viruses. The role of IFI16/IFI204 has been studied extensively in vitro, however the role of the receptors in vivo has yet to be determined. In chapter three of this thesis, we developed a mouse deficient in IFI204 to explore the role of IFI204 in in vivo immune responses to viruses. We investigated the ability of IFI204 deficient cells to induce type I interferons and other cytokines in response to a panel of DNA and RNA ligands in vitro. IFI204 deficient BMDMs displayed a partial defect in type I interferon induction in response to both DNA and RNA ligands and viruses as compared to WT mice. We also observed that this phenotype is time dependent, since there was no change in type I interferon induction after 12 hours post infection as compared to earlier time points. In contrast to these results, expression of the NF-κB regulated cytokines IL-6 and IL-1β were unaffected in IFI16 knockdown cells. These results suggest that IFI204 plays a partial role in the induction of type I interferons in response to both DNA and RNA ligands. Additionally, IFI204 may work in tandem with other receptors in a sequential manner to amplify the type I interferon response. We also studied the involvement of IFI204 in an in vivo model of HSV-1 infection. IFI204 knockout mice produce less brain and serum IFN-β, IL-6, and IL-1β 72 hours post intraperitoneal infection with HSV-1. Furthermore, IFI204 -/- mice are more susceptible to HSV-1 infection as compared to WT mice. These data indicate that IFI204 mediates the response to HSV-1 in vivo by inducing the production of cytokines that are necessary for the control of viral infection.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science (Medicine) in the branch of Anatomical Pathology Johannesburg, 2012 / Objective To determine the effectiveness of the cell block technique for immunocytochemical diagnosis by comparing cytomorphologic preservation and immunocytochemistry (ICC) stains in paired cell block and conventional fine needle aspiration (FNA) samples. Study Design This was a prospective study. Material for both conventional smears and cell blocks were collected simultaneously during fine needle aspiration of 50 lesions comprising lymph node, lung and liver masses. Grading of cellularity, morphological preservation, architectural preservation, immunocytochemical staining intensity and presence of background staining were compared on paired FNA smears and cell block samples derived from the same case. Each arm of the paired analysis was performed blindly without knowledge of the grading outcome of the other. The Kappa statistic (Κ) was used to measure inter-rater agreement. Results The fifty samples evaluated included FNAs from the lung, 24/50 (48%); liver, 23/50 (46%) and lymph node, 3/50 (6%). The immunocytochemistry stains consisted of 44/50 (88%) CK7, 44/50 (88%) CK20, 18/50 (36%) TTF1, 10/50 (20%) synaptophysin, 10/50 (20%) Hepar-1 and 7/50 (14%) AE1/3. There was no overall agreement in preservation of cytomorphological detail and ICC staining between the two methods. The Papanicolaou stained conventional FNA smears fared better then cell block for the vi evaluation of nuclear and cytomorphologic characteristics; cells in the cell block were poorly preserved in many cases. The ICC stains worked better on the cell block samples due to lack of background and aberrant staining. Conclusion Conventional FNA smears and cell blocks complement each other. Our results indicate that it would be optimal to use both modalities in the diagnostic work-up of mass lesions amenable to FNA diagnosis; the former to assess morphology, and the latter for optimal immunocytochemistry results. In resource constrained settings, the cost implications of performing both conventional and blocked smears on all FNA material warrants further evaluation.

Este trabalho consistiu numa avaliação, sob um ponto de vista físico, da aplicabilidade da sílica mesoporosa ordenada tipo SBA-15 como adjuvante imunológico. Inicialmente foi estudado o método de preparação e reprodutibilidade das propriedades do material, condição necessária para a síntese de grandes quantidades (N 100g). Mostrou-se que a calcinação em vácuo, comparada com o processo em N2 e ar, resulta em material com estrutura mesoporosa mais bem ordenada. Para aplicações biológicas foi analisado o potencial de encapsulação de antígerios no material, através de estudos de incorporação de Albumina Bovina (BSA) e vacina contra Hepatite A. Foi observada uma incorporação bem-sucedida de BSA na sílica, com essa proteína alojando-se dentro da estrutura de poros. Resultado semelhante foi observado para a vacina contra hepatite A. O processo mais eficiente de incorporação foi determinado para uma mistura em repouso e seca através de evaporação. A aplicabilidade da sílica como adjuvante para uso animal foi avaliada através de análises, pelo método PIXE, da acumulação do material no organismo de camundongos. A sílica foi administrada a camundongos Swiss por via oral e intra-muscular, e o teor de silício em diferentes órgáos foi comparado aos teores em um grupo controle. Foi detectada a presença de sílica em determinados órgãos dos camundongos, cuja eliminação total se processa num período de 70 dias. Além disso, em testes toxicológicos realizados no Instituto Butantan, a sílica mostrou-se eficaz na indução de resposta humoral e atóxica. / This work consisted of an evaluation, from a physical standpoint, of the applicability of SB.4-15 type ordered rnesoporous silica as an inmunological adjuvant. The method of preparation and reproducibility of the material properties were initially studied. Those conditions are necessary to synthesize large quantities of the material (N 100g). Vacuum calcination, whem compared to the process executed in A5 and air, results in a better ordered mesoporous structure. For biological applications, the potential to encapsulate antigens in the material was analyzed through studies of incorporation of Bovine Serum Albumin (BSA) and vaccine for Hepatitis A. A successful incorporation of BSA in the silica was observed, with that protein being lodged inside the porous structure. A similar result was obtained for the vaccine for Hepatitis A. The most efficient incorporation process was determined by keeping the solution at rest and drying it through evaporation. The applicability of silica as an immunological adjuvant for animal use was evaluated through PIXE analyses of the silicon accumulation in mice\'s organs. Silica was administrated to Swiss mice through oral and intramuscular ways and the silicon content of different organs was compared to the figures of the control group. The silica´s presence was detected on certain organs, and it was completely eliminated after 70 days. Besides that, toxicological studies accomplished at the Butantan Institute showed that the silica is efficient for inductíon of humoral response and it is non-toxic.

Introdução: A síndrome de Wiskott-Aldrich (SWA) é uma imunodeficiência primária rara ligada ao X caracterizada pela presença de eczema, trombocitopenia com ou sem microplaquetas e infecções recorrentes. Mais de 300 tipos de mutações associadas com o gene WAS foram descritas, incluindo oito hotspots. A diversidade destas mutações pode levar ao aparecimento de grande variabilidade nas apresentações clínicas, o que dificulta a previsão da evolução da doença baseada apenas nas manifestações iniciais. Além disso, há escassez de informações sobre a população brasileira. Objetivos: Descrever as características clínicas e genéticas de pacientes brasileiros com diagnóstico clínico de SWA. Resultados: Dezoito pacientes foram avaliados. Dezessete pacientes apresentaram os primeiros sintomas no primeiro ano de idade. Idade média para o início dos sintomas foi de 4,5 meses e média de tempo para o diagnóstico foi de 31,2 meses. Três pacientes (16,3%) foram diagnosticados após 10 anos de início dos sintomas. Dezessete pacientes (94,5%) apresentaram eczema. Os níveis plaquetários variaram entre 1.000 e 65.000/mm3 e nove pacientes (50%) apresentaram microtrombocitopenia. Dois pacientes (11,1%) apresentaram macroplaquetas. Dezesseis pacientes (88,9%) tiveram eventos hemorrágicos ao longo de suas vidas, especialmente sangramentos intestinais, urinários e petéquias. Em relação às manifestações infecciosas, otite média aguda foi a infecção mais frequente, relatada por 13 pacientes (72,2%), seguido por infecções cutâneas (66,7%). Três pacientes(16,6%) apresentaram manifestações autoimunes, incluindo nefropatia por IgA, trombose isquêmica e vasculite. A maioria dos pacientes (55,5%) não apresentou alterações nos níveis IgG. Níveis elevados de IgA só foram observados em 4 pacientes (23,5%). Redução dos níveis IgM foi observada em 7 pacientes (38,9%). Os pacientes foram classificados de acordo com um escore clínico previamente descrito. A maioria apresentou pontuações de 3 (33,3%) e 4 (27,8%). Quatro pacientes (22,2%) foram classificados com pontuação 5 devido às manifestações autoimunes ou neoplasias. Em relação à análise genética, foram encontradas mutações em 10 pacientes (55,5%). Apenas três das mutações encontradas neste estudo foram descritas previamente. Conclusão: As características clínicas dos pacientes brasileiros foram semelhantes às características observadas em outras populações, porém a análise genética revelou mutações ainda não descritas. / Introduction: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by eczema, thrombocytopenia with or no small sized platelets and recurrent infections. More than 300 types of mutations associated with the WAS gene have been described, including eight hotspots. The diversity of these mutations can lead to the appearance of great variation in the clinical presentations, which makes it difficult to predict the evolution of the disease based only on the initial manifestations. Furthermore, there is paucity of information on WAS from the Brazilian population. Objectives: To describe the clinical and genetic characteristics of Brazilian patients with clinical diagnose of WAS. Results: Eighteen patients were evaluated. Seventeen patients presented first symptoms within first year of age. Mean age for initiating symptoms was 4,5 months and mean time for diagnosis was 31,2 months. Three patients (16.6%) were diagnosed after 10 years of initiating symptoms. Seventeen patients (94.5%) had eczema. The platelet levels ranged from 1,000 to 65,000/mm3 and nine patients (50%) presented microthrombocytopenia. Two patients (11.1%) had macroplatelets. Sixteen patients (88.9%) had hemorrhagic events throughout their lives, especially intestinal, urinary and petechial bleedings. In relation to infectious manifestations, acute media otitis was the most frequent infection, reported by 13 patients (72.2%), followed by skin infections (66.7%). Three patients (16.6%) had autoimmune manifestations including IgA nephropathy,ischemic stroke and vasculitis. Most patients (55.5%) did not present alterations in IgG levels. Twelve patients (70.6%) did not present alterations in IgA levels and elevated levels of IgA were only observed in 4 patients (23.5%). Reduction of IgM levels was observed in 7 patients (38.9%). Patients were classified according to a previously described clinical score. Most patients presented scores of 3 (33.3%) and 4 (27.8%). Four patients (22.2%) were classified with score 5 due to autoimmune or neoplastic manifestations. Regarding genetic analysis, mutations were found in 10 patients (55.5%). Only three of mutations found in this study were previously described. Conclusion: Clinical characteristics of Brazilian patients were similar to medical features observed in other populations, however genetic analysis showed undescribed mutations yet

Introdução: Diabetes tipo 1A(DM1A) é uma doença causada pela destruição autoimune das células beta. Em adição aos linfócitos T helper 1(Th1) e Th2, um subtipo específico de células T helper recentemente descrito, Th17, caracterizado pela produção da interleucina 17(IL-17A), IL-17F e IL-22, está também envolvido na imunidade adaptativa e autoimunidade, incluindo DM1A. A IL-23 tem função fundamental na expansão e sobrevivência das células Th17. A mesma é composta por 2 subunidades: a p19-específica (IL-23A) e a p40. Variantes dos genes IL-23A e de seu receptor (IL-23R) ou o aumento das concentrações séricas da IL-23 estão associados a várias doenças autoimunes, mas seus efeitos no DM1A não estão definidos. Com o intuito de avaliar a importância da IL-23 na patogênese do DM1A, as variantes dos genes IL23A e IL23R foram analisadas. Metodologia: A região codificadora e os regiões intrônicas proximais do gene IL23A, incluindo a região 5 proximal foram sequenciadas. Duas variantes do gene IL23A (rs2066808 e rs11171806) e duas do gene IL-23R (rs11209026 e rs10889677) foram também genotipadas. A amostra contou com 370 pacientes com DM1A e 314 indivíduos controles saudáveis. As medidas das concentrações séricas da IL-23 e os autoanticorpos pancreáticos e extra-pancreáticos foram determinados. Resultados: Nós observamos somente uma das seis variantes da IL-23 descritas nos bancos de dados (rs11171806 G>A localizada no exon 3) e descrevemos uma nova variante no gene IL-23A, que consistiu na substituição da citosina por timina na posição c.-403 (C>T) na região 5 proximal deste gene (encontrada em heterozigose em apenas uma paciente com DM1A, do sexo feminino, com 28 anos ao diagnóstico).Os alelos G dessas duas variantes estiveram em forte desequilíbrio de ligação (D\' = -0,825 para controles, p<2,0X10-6 e D\' = -0,902, p<2,0X10-17 para pacientes). Em consequência, a análise dos haplótipos destas variantes foi realizada. O haplótipo GG foi mais frequente nos controles (16.7%) do que nos pacientes com DM1A (9.5%), conferindo proteção à doença (pc = 0,0009, OR = 0,53) . A presença do haplótipo GG diferiu de acordo com a etnia no conjunto de pacientes e controles, sendo menor naqueles de etnia caucasóide (18%) em relação aos outros grupos (39%); p<0.0001. Entretanto, o efeito protetor da haplótipo GG foi independente da etnia. As duas variantes do gene IL23R (rs10889677 e rs11209026) tinham frequência alélica e genotípica semelhante entre pacientes com DM1A e controles. Não foi observada diferença significante nas concentrações da IL-23 entre 135 pacientes com DM1A (5,65 ± 14,0 pg/mL) e 112 indivíduos controles (9,06 ± 23,7pg/mL) (p =0,18). , mesmo quando analisamos apenas o pacientes com duração do diabetes inferior a dois anos, nos quais a resposta imune contra as células beta ainda está presente, (4.65 ± 6.94 pg/mL e 9.07 ± 23.62 pg/mL, p = 0.076). Não foi encontrada associação entre as variantes do gene IL23A com a idade diagnóstica, presença do peptídeo C residual e auto-anticorpo anti-descarboxilase do ácido glutâmico em pacientes com diagnóstico recente de DM1A. Estas variantes também não influenciaram na freqüência dos auto anticorpos extrapancreáticos: anti-tireoglobulina, anti-peroxidase, anti-21 hidroxilase, fator anti-núcleo, fator reumatóide e anti-citoplasma de neutrófilos. Conclusões: O haplótipo GG das variantes do gene lL23A (rs11171806 e rs2066808) foi associado a proteção ao DM1A. As variantes do gene IL23R (rs11209026 e rs10889677) não foram associadas ao DM1A. As concentrações séricas da IL-23 foram semelhantes entre os grupos. / Introduction: Type 1 diabetes mellitus (T1D) is a disorder caused by the immune-mediated destruction of insulin-secreting pancreatic beta cells. In addition to T helper 1 (Th1) and Th2 cells, a recently discovered subset of T helper cells, Th17, characterized by the production of interleukin 17 A (IL-17A), IL-17F, and IL-22 is also involved in adaptive immunity and autoimmunity, including T1D. The Interleukin IL-23 has a central role in the expansion and survival of Th 17 cells. It is composed of two subunits: p19-specific (IL-23A) and p40. Single nucleotide polymorphisms (SNPs) of IL-23A and IL-23 receptor (IL-23R) genes or increased IL-23 serum concentrations were associated with several autoimmune diseases, but their role in T1D has not been defined. We therefore searched for variants of IL-23A and IL-23R genes that could predispose to T1D. Methods:The coding regions and boundary intron sequences of IL-23A gene, including the 5 proximal region were sequenced. Two variants (rs2066808 and rs 11171806) of IL-23A and two of IL-23R (rs11209026 and rs10889677) genes were also genotyped. IL-23 serum levels and pancreatic and extra-pancreatic auto-antibodies were also determined. The cohort involved 370 patients with T1D and 314 healthy control subjects.Results: We observed only 1 out of 6 IL-23A coding variants (rs11171806 G>A localized in exon 3) described in a database repository . A new allelic variant of the IL-23A gene, consisting of the substitution of a cytosine by a thymine at position c.-403 (C>T) in the 5 proximal region of the IL-23A gene (found in heterozygosis in only 1 female patient with T1D) was described. The G alleles of rs11171806 and rs2066808 variants of IL-23A gene were in strong linkage disequilibrium (D\' = -0,825 for controls, p<2,0X10-6 and D\' = -0,902, p<2,0X10-17 for patients). So, further analyses were performed with the haplotypes instead of separated SNPs. The GG haplotype was more frequent in controls (16,7%) than in T1D patients (9,5%), conferring a protection to the disease (pc= 0,0009, OR = 0.53). The presence of haplotype GG was also different according to the ethnic group in the overall sample (patients+controls), when we pooled the Caucasians (18%) against the other groups (39%); p<0.0001. However, the lower susceptibility to T1D conferred by GG haplotype was independent of the ethnic group. Two IL-23R gene variants (rs10889677 and rs11209026) were also analyzed. The allelic and genotypic frequency of the variants did not differ between patients with T1D and control subjects. No significant differences were observed between the plasma IL-23 concentrations of 135 T1D patients (5.65 ± 14.0) and 112 control subjects (9.06 ± 23.7) (p = 0.18), even when we only the patients with less than 2 years disease duration (n = 43), when the immune attack to beta cells is still present, were included (4.65 ± 6.94 pg/mL and 9.07 ± 23.62 pg/mL, p = 0.076). No association was found between IL-23A variants with age at diagnosis of diabetes, presence of residual C-peptide levels or frequency of glutamic acid anti-decarboxilase antibody in patients with recent-onset T1D. Furthermore, these variants were not related to the presence of the extrapancreatic autoantibodies such as thyroid peroxidase (TPO) Ab, thyroglobulin (TG) Ab, 21-Hydroxilase (21OH) Ab, Anti nuclear factor (ANA) Ab, rheumatoid factor (FR) Ab and Neutrophil cytoplasmic (ANCA) Ab. Conclusions : The GG haplotype of lL23A gene variants( rs11171806 and rs2066808) was protective against T1D. The IL23R variants (rs11209026 and rs10889677) were not associated with susceptibility toT1D . IL-23 serum concentrations did not differ between T1D patients and controls.

A hipersensibilidade a veneno de Hymenoptera representa importante problema do ponto de vista de saúde da população, uma vez que pacientes alérgicos aos componentes do veneno podem desenvolver reações graves, às vezes fatais. A única profilaxia efetiva em pacientes sensibilizados é a imunoterapia veneno específica. Objetivos: avaliar as evidências científicas a respeito dos efeitos da imunoterapia específica utilizada na profilaxia secundária das reações graves em pacientes sensibilizados a veneno de Hymenoptera, por meio da realização de uma revisão sistemática. Métodos: a estratégia de busca seguiu as recomendações do Grupo de Pele da Colaboração Cochrane. A pesquisa foi realizada nas seguintes bases de dados: MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), EMBASE, LILACS, SciSEARCH e nas referências de artigos mais relevantes. Todos os ensaios clínicos controlados e randomizados envolvendo imunoterapia com veneno de Hymenoptera versus imunoterapia com placebo ou apenas seguimento dos pacientes foram avaliados. Dois revisores de forma independente (ASW e LAMF) avaliaram a elegibilidade e a qualidade metodológica de cada ensaio clínico e extraíram os dados. O risco de reações sistêmicas, conseqüentes a ferroada acidental ou a teste de provocação com o inseto responsável, após a imunoterapia específica, foi avaliado por meio do cálculo do odds ratio e do respectivo intervalo de confiança de 95%. Resultados: 2267 resumos foram identificados. A maioria dos artigos foi excluída pelas seguintes razões: os estudos não eram controlados e randomizados ou não satisfaziam alguns critérios de inclusão. Apenas quatro estudos foram analisados. A idade dos pacientes variou entre dois e 65 anos. Apenas um estudo comparou imunoterapia com veneno de formiga contra placebo (Brown et al.) e três estudos (Hunt et al., Schubert et al. e Valentine et al.) comparam imunoterapia com venenos de abelha e vespa contra placebo ou seguimento de pacientes. Em cada estudo, o odds ratio para reações sistêmicas foi: Hunt et al.- Grupo I (veneno) x III (placebo): 0,10 (0,01 <OR < 0,68) Schubert et al.: 0,35 (0,05<OR<2,56); Valentine et al.: 0,16 (0,02 < OR < 1,21) e Brown et al.: 0,04 (0,01<OR<0,28). Após o teste de heterogeneidade, apenas dois estudos (Schuberth 1983 e Valentine 1990) se mostraram homogêneos o suficiente e assim puderam ser incluídos na meta-análise (p = 0,623). Ao combinar os dois estudos, o odds ratio passou a ser significativo: 0.29 (0.10 a 0.87). Entretanto, ao analisar a gravidade das reações ocorridas após a imunoterapia, observou-se que os benefícios podem não ser tão relevantes, pois as reações foram, na grande maioria, ou mais leves ou semelhantes à reação original. Conclusões: A imunoterapia específica deve ser recomendada para adultos que apresentaram reações sistêmicas e para crianças com reações moderadas a graves, porém não é necessária em crianças que apresentaram apenas reações cutâneas após ferroada de abelha ou vespa, principalmente se a exposição for esporádica. / Background: Hymenoptera venom hypersensitivity is a significant public health problem. For patients who are allergic to components of the venom, reactions can be severe and sometimes fatal. The only effective treatment in the management of those patients is the specific venom immunotherapy. Objective: to assess the effects of the specific venom immunotherapy for Hymenoptera venom hypersensitivity through a systematic review. Methods: the standard search strategy of the Cochrane Skin Group was used for searches of electronic and other databases. These included MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), EMBASE, LILACS, SciSEARCH and the references of relevant articles. All randomized controlled trials involving Hymenoptera venom immunotherapy versus immunotherapy with placebo or only follow-up of the patients were included. Two independent reviewers (ASW and LAMF) assessed the eligibility, and the methodological quality of each trial, and extracted the data. Post immunotherapy risk of systemic reactions after either challenge or accidental stings was calculated through the odds ratio and respective 95 percent confidence interval. Main results: 2,267 abstracts, identified through electronic sources, were assessed. Most articles were excluded for the following reasons: the studies did not satisfy all the inclusion criteria or they were not randomized controlled trials. Four studies were included on this review. The age of the participants varied between two and 65 years. Only one study compared ant venom immunotherapy with placebo (Brown et al.) and three studies (Hunt et al., Schubert et al. and Valentine et al.) compared bees and wasps immunotherapy with placebo or simply patient follow-up. In each study, the odds ratio to a systemic reaction was: Hunt et al.- Group I (venom) x III(placebo): 0,10 (0,01 < OR < 0,68) Schubert et al.: 0,35 (0,05 < OR < 2,56); Valentine et al.: 0,16 (0,02 < OR < 1,21) and Brown et al.: 0,04 (0,01 < OR < 0,28). After the heterogeneity test (p = 0,623), only two studies (Schuberth 1983 and Valentine 1990) were homogeneous enough as to be included in a meta-analysis. The summary odds ratio was 0.29 (IC 95%: 0.10 - 0.87). However, when the severity of the reaction occurring after the sting challenge or accidental sting was taken into account, the benefits were not so relevant because the reactions were, for the most, milder than the original one. Conclusions: The specific-venom immunotherapy must be recommended for adults with systemic reactions and for children with moderate to severe reactions, but there is no need to prescribe it for children who present only skin reactions after insects sting, particularly if the exposure is sporadic.

Introdução: A síndrome de Wiskott-Aldrich (SWA) é uma imunodeficiência primária rara ligada ao X caracterizada pela presença de eczema, trombocitopenia com ou sem microplaquetas e infecções recorrentes. Mais de 300 tipos de mutações associadas com o gene WAS foram descritas, incluindo oito hotspots. A diversidade destas mutações pode levar ao aparecimento de grande variabilidade nas apresentações clínicas, o que dificulta a previsão da evolução da doença baseada apenas nas manifestações iniciais. Além disso, há escassez de informações sobre a população brasileira. Objetivos: Descrever as características clínicas e genéticas de pacientes brasileiros com diagnóstico clínico de SWA. Resultados: Dezoito pacientes foram avaliados. Dezessete pacientes apresentaram os primeiros sintomas no primeiro ano de idade. Idade média para o início dos sintomas foi de 4,5 meses e média de tempo para o diagnóstico foi de 31,2 meses. Três pacientes (16,3%) foram diagnosticados após 10 anos de início dos sintomas. Dezessete pacientes (94,5%) apresentaram eczema. Os níveis plaquetários variaram entre 1.000 e 65.000/mm3 e nove pacientes (50%) apresentaram microtrombocitopenia. Dois pacientes (11,1%) apresentaram macroplaquetas. Dezesseis pacientes (88,9%) tiveram eventos hemorrágicos ao longo de suas vidas, especialmente sangramentos intestinais, urinários e petéquias. Em relação às manifestações infecciosas, otite média aguda foi a infecção mais frequente, relatada por 13 pacientes (72,2%), seguido por infecções cutâneas (66,7%). Três pacientes(16,6%) apresentaram manifestações autoimunes, incluindo nefropatia por IgA, trombose isquêmica e vasculite. A maioria dos pacientes (55,5%) não apresentou alterações nos níveis IgG. Níveis elevados de IgA só foram observados em 4 pacientes (23,5%). Redução dos níveis IgM foi observada em 7 pacientes (38,9%). Os pacientes foram classificados de acordo com um escore clínico previamente descrito. A maioria apresentou pontuações de 3 (33,3%) e 4 (27,8%). Quatro pacientes (22,2%) foram classificados com pontuação 5 devido às manifestações autoimunes ou neoplasias. Em relação à análise genética, foram encontradas mutações em 10 pacientes (55,5%). Apenas três das mutações encontradas neste estudo foram descritas previamente. Conclusão: As características clínicas dos pacientes brasileiros foram semelhantes às características observadas em outras populações, porém a análise genética revelou mutações ainda não descritas. / Introduction: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by eczema, thrombocytopenia with or no small sized platelets and recurrent infections. More than 300 types of mutations associated with the WAS gene have been described, including eight hotspots. The diversity of these mutations can lead to the appearance of great variation in the clinical presentations, which makes it difficult to predict the evolution of the disease based only on the initial manifestations. Furthermore, there is paucity of information on WAS from the Brazilian population. Objectives: To describe the clinical and genetic characteristics of Brazilian patients with clinical diagnose of WAS. Results: Eighteen patients were evaluated. Seventeen patients presented first symptoms within first year of age. Mean age for initiating symptoms was 4,5 months and mean time for diagnosis was 31,2 months. Three patients (16.6%) were diagnosed after 10 years of initiating symptoms. Seventeen patients (94.5%) had eczema. The platelet levels ranged from 1,000 to 65,000/mm3 and nine patients (50%) presented microthrombocytopenia. Two patients (11.1%) had macroplatelets. Sixteen patients (88.9%) had hemorrhagic events throughout their lives, especially intestinal, urinary and petechial bleedings. In relation to infectious manifestations, acute media otitis was the most frequent infection, reported by 13 patients (72.2%), followed by skin infections (66.7%). Three patients (16.6%) had autoimmune manifestations including IgA nephropathy,ischemic stroke and vasculitis. Most patients (55.5%) did not present alterations in IgG levels. Twelve patients (70.6%) did not present alterations in IgA levels and elevated levels of IgA were only observed in 4 patients (23.5%). Reduction of IgM levels was observed in 7 patients (38.9%). Patients were classified according to a previously described clinical score. Most patients presented scores of 3 (33.3%) and 4 (27.8%). Four patients (22.2%) were classified with score 5 due to autoimmune or neoplastic manifestations. Regarding genetic analysis, mutations were found in 10 patients (55.5%). Only three of mutations found in this study were previously described. Conclusion: Clinical characteristics of Brazilian patients were similar to medical features observed in other populations, however genetic analysis showed undescribed mutations yet