Structures of N-Glycans of Bothrops Venoms Revealed as Molecular Signatures that Contribute to Venom Phenotype in Viperid Snakes.

The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.

Evidence of facultative parthenogenesis in three Neotropical pitviper species of the Bothrops atrox group.

We examined four suspected cases of facultative parthenogenesis in three species of a neotropical lineage of pitvipers of the Bothrops atrox group. Reproduction without mating was observed in captive females of B. atrox, B. moojeni and B. leucurus housed alone for seven years (the two former species) and nine years (the latter one). In addition to the observation of captivity data, we investigated molecularly this phenomenon using heterologous microsatellites. DNA was extracted from the mothers' scales or liver, from embryo and newborn fragments, and yolked ova. Four of the microsatellites showed good amplification using Polymerase Chain Reaction and informative band segregation patterns among each mother and respective offspring. Captivity information, litter characteristics (comparison of the number of newborns, embryos and yolked ova) and molecular data altogether agreed with facultative parthenogenesis predictions in at least three out of the four mothers studied: B. atrox (ID#933) was heterozygous for three out of the four markers, and the sons S1 and S2 were homozygous; B. moojeni (BUT86) was heterozygous for two out of four markers, offspring S1, S3, E2, and E4, and O1 to O6 were homozygous; and B. leucurus (MJJS503) was heterozygous for three out of four markers, and son E1 and O1 were homozygous. B. moojeni (BUT44) was homozygous for all loci analyzed in the mother and offspring, which although not informative is also consistent with parthenogenesis. This study represents the first molecular confirmation of different pitviper species undergoing facultative parthenogenesis among Neotropical endemic snakes.

Eosinophilic Esophagitis: Latent Disease in Patients with Anaphylactic Reaction to Cow's Milk.

BACKGROUND: Food allergy and eosinophilic esophagitis are a substantial and evolving public health issue. Clinicians should know the relationship between these diseases and how one may predispose to the other. This can help minimize misdiagnosis. OBJECTIVE: The objective of this study was to assess esophageal eosinophilia and eosinophilic esophagitis frequency in patients with persistent cow's milk allergy and anaphylaxis manifestations. METHODS: Patients with persistent cow's milk allergy with anaphylaxis manifestations were enrolled from 2012 through 2016 at the São Paulo University Hospital, Brazil. All of them were submitted to endoscopy despite the presence or absence of gastrointestinal symptoms. Demographics data, atopic comorbidities, medication use, endoscopic findings, and esophageal eosinophilia frequency were evaluated. RESULTS: Eighty-nine patients were selected. The median age was 8 years. It was observed that 34 of 89 patients (38.2%; 95% confidence interval [CI]: 28.14%-49.16%) presented with esophageal eosinophilia. Five patients (7.1%) presented proton pump inhibitor-responsive esophageal eosinophilia, and 10 patients (14.2%) presented eosinophilic esophagitis. We found that 29.4% were asymptomatic patients, 23.5% had nonspecific symptoms, 23.5% had persistent typical symptoms, and 23.5% had intermittent typical symptoms. There was an association with inflammatory endoscopy findings in 21 patients (61.7%). CONCLUSIONS: This description demands scientific attention because it is the highest frequency of esophageal eosinophilia yet described in a group of patients with cow's milk allergy presenting with anaphylaxis. Eosinophilic esophagitis is a condition that can coexist "silently" with an IgE-mediated food allergy and is most often underestimated and underdiagnosed.

The ingestion of a large prey facilitating the capture of the predator: a study case in Micrurus corallinus (Serpentes Elapidae)

We report a case of predation of a large specimen of the Dipsas neuwiedi by a Micrurus corallinus, in the state of Santa Catarina, Brazil. The ingestion reported here is one of the greatest prey / predator ratios registered for genus Micrurus in nature. In this predation event, the ingestion of a large prey limited the M. corallinus locomotion, which did not offer resistance and was not able to move efficiently during the capture.

MAST CELLS DISTINGUISH EOSINOPHILIC ESOPHAGITIS IN PEDIATRIC PATIENTS.

BACKGROUND:: Mast cells exert a substantial role in gastrointestinal allergic diseases. Therefore, it is reasonable to presume that mast cell may aid diagnosis in eosinophilic gastroenteropathy. OBJECTIVE:: To evaluate whether mast cell count in the esophageal epithelium can discriminate eosinophilic esophagitis, proton-pump inhibitor (PPI)-responsive eosinophilic esophagitis and gastroesophageal reflux esophagitis. METHODS:: Retrospectively we reviewed the files of 53 consecutive patients (age: 7.8 years; range: 8-14 years) with definitive diagnose established during clinical follow up in a universitary outpatient clinic as follow: eosinophilic esophagitis (N=23), PPI-responsive eosinophilic esophagitis (N=15) and gastroesophageal reflux esophagitis (N=15). Eosinophil count in the esophageal epithelium in slides stained with H-E was reviewed and immunohistochemistry for mast cell tryptase was performed. RESULTS:: Count of eosinophils/high-power field (HPF) higher than 15 were found in 14 out of 15 reflux esophagitis patients. The mean count of eosinophils/HPF was similar in eosinophilic esophagitis patients and in those with PPI-responsive eosinophilic esophagitis (42 and 39 eosinophils/HPF, respectively, P=0.47). Values of mast cell tryptase (+) were higher in eosinophilic esophagitis [median: 25 mast cells/HPF; range (17-43) ] and in PPI-responsive eosinophilic esophagitis patients [25 (16-32) ], compared to reflux esophagitis [4 (2-14) ], P<0.001. There was no difference between the mean count of mast cells/HPF in the esophageal epithelium of eosinophilic esophagitis patients and PPI-responsive eosinophilic esophagitis patients, respectively, 26 and 24 mast cells/HPF, P=0.391. CONCLUSION:: Tryptase staining of mast cells differentiates eosinophilic esophagitis from reflux esophagitis.

Segmental muscle twitching behaviour in the flanks of lancehead vipers Bothrops spp in response to human approach

Bothrops species are known to have a wide range of defensive behaviours against potential threats. Herein we show a frequently observed, but to date undocumented, behaviour in male and female Bothrops spp of varying sizes in newly arrived specimens from the wild, short-or long-term captives as well as in captive born individuals. The behaviour consists of irregular, synchronous or asynchronous muscular twitches in segments of the body flanks in otherwise motionless snakes. We named this ‘Segmental Muscle Twitching Behaviour’ (SMTB). We observed fifteen Bothrops spp from six species groups for incidence of this behaviour and made videos of snakes during the ‘alertness’ and ‘threatening’ phases of response to human approach. We found the behaviour in only five species, these belong to the monophyletic Bothrops jararacussu and Bothrops atrox species groups, suggesting a single evolutionary origin of this behaviour. Macroscopically, SMTB varied in the number of segments involved and the twitches had uneven intensities. Although recurrent and often replicable in similar situations during human approach, the behaviour was not displayed consistently. In addition, SMTB can be interrupted, stay inactive for quite some time and then restart. Hypotheses to explain this behaviour are suggested

Venom profiling of the insular species Bothrops alcatraz: characterization of proteome, Glycoproteome, and N-Terminome using terminal amine isotopic labeling of substrates

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

Semen collection and evaluation in micrurus corallinus

The Painted Coral Snake Micrurus corallinus is one of the Brazilian species kept in captivity to obtain venom for antivenom production. Difficulties in establishing a sizeable breeding colony make it necessary to find alternatives that increase the reproductive efficiency of captive individuals. Here, we tested a semen collection protocol and characterize the seminal parameters of captive M. corallinus. We collected semen during the mating season of the species (spring-summer) and were successful at every first attempt. Spermatozoa of M. corallinus are elongated and filiform, and the midpiece is the longest part. Sperm motility and progressive motility reached values of 80% and 3.6%, respectively, during the reproductive period of this species. Our results will allow further studies to improve husbandry, reproductive rates, and conservation of captive M. corallinus.

Evidence of facultative parthenogenesis in three Neotropical pitviper species of the Bothrops atrox group

We examined four suspected cases of facultative parthenogenesis in three species of a neotropical lineage of pitvipers of the Bothrops atrox group. Reproduction without mating was observed in captive females of B. atrox, B. moojeni and B. leucurus housed alone for seven years (the two former species) and nine years (the latter one). In addition to the observation of captivity data, we investigated molecularly this phenomenon using heterologous microsatellites. DNA was extracted from the mothers’ scales or liver, from embryo and newborn fragments, and yolked ova. Four of the microsatellites showed good amplification using Polymerase Chain Reaction and informative band segregation patterns among each mother and respective offspring. Captivity information, litter characteristics (comparison of the number of newborns, embryos and yolked ova) and molecular data altogether agreed with facultative parthenogenesis predictions in at least three out of the four mothers studied: B. atrox (ID#933) was heterozygous for three out of the four markers, and the sons S1 and S2 were homozygous; B. moojeni (BUT86) was heterozygous for two out of four markers, offspring S1, S3, E2, and E4, and O1 to O6 were homozygous; and B. leucurus (MJJS503) was heterozygous for three out of four markers, and son E1 and O1 were homozygous. B. moojeni (BUT44) was homozygous for all loci analyzed in the mother and offspring, which although not informative is also consistent with parthenogenesis. This study represents the first molecular confirmation of different pitviper species undergoing facultative parthenogenesis among Neotropical endemic snakes.

Functional variability of Bothrops atrox venoms from three distinct areas across the Brazilian Amazon and consequences for human envenomings

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca.

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.

Growth assessment in children with extra-hepatic portal vein obstruction and portal hypertension.

BACKGROUND: Several studies carried out to examine the growth of children with extra-hepatic portal vein obstruction and portal hypertension have reported a variety of findings. AIM: To assess anthropometric indices in children with portal hypertension due to extra-hepatic portal vein obstruction and who were treated by endoscopic variceal sclerotherapy. METHODS: Anthropometric data were obtained retrospectively from the medical records of 24 patients who had been followed for 3.8 +/- 2.5 years at the Pediatric Gastroenterology Outpatient Clinic of the University Hospital, Campinas, SP, Brazil. The mean Z scores of weight for age, height for age and body mass index at diagnosis and at the last consultation were compared to reference data of the National Center for Health Statistics. The two recordings were compared to assess the long-term effect of the condition. RESULTS: The mean Z scores at diagnosis and at the last follow-up visit were all within normal ranges when compared to the reference population, with the following respective values: weight for age, 0.042 +/- 1.09 and 0.132 +/- 1.29; height for age, 0.200 +/- 1.04 and 0.466 +/- 1.24, and body mass index, -0.223 +/- 0.98 and -0.198 +/- 0.98. In addition, there were no significant differences between the Z score values obtained in the two recordings. CONCLUSION: Portal hypertension due to extra-hepatic portal vein obstruction was not associated with growth impairment in the group of children studied.

Structures of N-glycans of Bothrops venoms revealed as molecular signatures that contribute to venom phenotype in viperid snakes

The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.

Functional variability of Bothrops atrox venoms from three distinct areas across the Brazilian Amazon and consequences for human envenomings

Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.

Coagulotoxicity of Bothrops (Lancehead Pit-Vipers) venoms from Brazil: differential biochemistry and antivenom efficacy resulting from prey-driven venom variation

Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter– and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.

Simophis rhinostoma (Squamata: Colubridae): feeding habits and defensive behavior

The biology of Simophis rhinostoma (Schlegel, 1837) is poorly known.There are no enough information about its diet, just about feeding on anurans and, in captivity, there are records of lizards ingestion. We present the first record of fish ingestion by S. rhinostoma, a colubrid inhabiting the Atlantic Forest and Cerrado areas, also some behavior aspects during feeding.

Structures of N-glycans of Bothrops venoms revealed as molecular signatures that contribute to venom phenotype in viperid snakes

The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.

Coagulotoxicity of Bothrops (Lancehead Pit-Vipers) venoms from Brazil: differential biochemistry and antivenom efficacy resulting from prey-driven venom variation

Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter– and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.

Mastócitos discriminam esofagite eosinofílica em pacientes pediátricos / Mast cells distinguish eosinophilic esophagitis in pediatric patients

ABSTRACT BACKGROUND: Mast cells exert a substantial role in gastrointestinal allergic diseases. Therefore, it is reasonable to presume that mast cell may aid diagnosis in eosinophilic gastroenteropathy. OBJECTIVE: To evaluate whether mast cell count in the esophageal epithelium can discriminate eosinophilic esophagitis, proton-pump inhibitor (PPI)-responsive eosinophilic esophagitis and gastroesophageal reflux esophagitis. METHODS: Retrospectively we reviewed the files of 53 consecutive patients (age: 7.8 years; range: 8-14 years) with definitive diagnose established during clinical follow up in a universitary outpatient clinic as follow: eosinophilic esophagitis (N=23), PPI-responsive eosinophilic esophagitis (N=15) and gastroesophageal reflux esophagitis (N=15). Eosinophil count in the esophageal epithelium in slides stained with H-E was reviewed and immunohistochemistry for mast cell tryptase was performed. RESULTS: Count of eosinophils/high-power field (HPF) higher than 15 were found in 14 out of 15 reflux esophagitis patients. The mean count of eosinophils/HPF was similar in eosinophilic esophagitis patients and in those with PPI-responsive eosinophilic esophagitis (42 and 39 eosinophils/HPF, respectively, P=0.47). Values of mast cell tryptase (+) were higher in eosinophilic esophagitis [median: 25 mast cells/HPF; range (17-43) ] and in PPI-responsive eosinophilic esophagitis patients [25 (16-32) ], compared to reflux esophagitis [4 (2-14) ], P<0.001. There was no difference between the mean count of mast cells/HPF in the esophageal epithelium of eosinophilic esophagitis patients and PPI-responsive eosinophilic esophagitis patients, respectively, 26 and 24 mast cells/HPF, P=0.391. CONCLUSION: Tryptase staining of mast cells differentiates eosinophilic esophagitis from reflux esophagitis.

RESUMO CONTEXTO: Os mastócitos detêm papel fundamental na resposta imuno-alérgica gastrintestinal. Assim, é razoável admitir que essas células sejam úteis no diagnóstico diferencial das gastroenteropatias eosinofílicas. OBJETIVO: Determinar se a análise quantitativa de mastócitos na mucosa esofágica permite discernir esofagite eosinofílica, esofagite eosinofílica responsiva ao inibidor de bomba de prótons e esofagite péptica por doença de refluxo gastroesofágico. MÉTODOS: Revisamos retrospectivamente os prontuários 53 crianças (idade: 7,8 anos; variação: 8-14 anos), atendidas consecutivamente, num serviço terciário e cujos diagnósticos definitivos estabelecidos após seguimento clínico foram esofagite eosinofílica (N=23), esofagite eosinofílica responsiva ao inibidor de bomba de prótons (N=15) e esofagite péptica por doença de refluxo gastroesofágico (N=15). As amostras histológicas foram revisadas quanto à contagem de eosinófilos na coloração de H-E e processadas para imunoistoquímica da triptase de mastócitos. RESULTADOS: Valores de eosinófilos/campo de maior aumento (CMA; 400X) >15 foram encontrados em 14 dos 15 pacientes com refluxo gastroesofágico. A média de eosinófilos/CMA foi similar nos pacientes com esofagite eosinofílica e com esofagite eosinofílica responsiva ao inibidor de bomba de prótons, respectivamente, 42 e 39 eosinófilos/CMA, P=0,47). Os valores de mastócitos triptase (+) foram superiores no epitélio esofágico dos pacientes com esofagite eosinofílica [mediana: 25 mastócitos/CMA; variação (17- 43) ] e na esofagite eosinofílica responsiva ao inibidor de bomba de prótons [25 (16-32) ], comparados aos pacientes com refluxo gastroesofágico [4(2-14) ], P<0,001. Não houve diferença entre a média de mastócitos/CMA nos pacientes com esofagite eosinofílica comparados aos com esofagite eosinofílica responsiva ao inibidor de bomba de prótons, respectivamente, 26 e 24 mastócitos/CMA, P=0,391. CONCLUSÃO: A coloração para mastócitos pela imunoistoquímica da triptase diferencia as esofagites eosinofílicas da esofagite péptica.