Les mélanomes oculaires sont des tumeurs rares représentant environ 5% des mélanomes. Les mélanomes oculaires peuvent provenir de deux tissus : l’uvée (~500 cas/an en France) et la conjonctive (~30 cas/an). Les mélanomes uvéaux présentent un très faible taux de mutations somatiques. Ces tumeurs sont également porteuses d’altérations du nombre de copies caractéristiques (gains du 8q, 1q, 6p, pertes du 3, du 1p, du 6q ou du 8p). L’évolution du génome de ces tumeurs durant la progression métastatique est à ce jour mal décrit. Afin d’explorer l’évolution métastatique du mélanome uvéal, nous avons séquencé les exomes de 14 tumeurs primaires et 79 métastases provenant de 24 patients. Il existait une grande proximité génétique entre tumeurs primaires et métastases avec une médiane de 11,5 mutations dans les tumeurs primaires, et 14 dans les métastases. Bien que les mutations SF3B1 et EIF1AX soient des facteurs pronostiques majeurs dans les mélanomes uvéaux, leurs fréquences dans les métastases étaient similaires à celle observée dans les séries historiques de tumeurs primaires. Les métastases présentaient quelques altérations de nombre de copies supplémentaires par rapport aux tumeurs primaires correspondantes. Parmi les altérations du nombre de copies les plus souvent acquises lors du processus métastatique, les gains du 8q étaient présents dans 92% des métastases. Lors de ce travail, nous avons découvert un mélanome uvéal présentant un phénotype hypermuté CpG>TpG chez une patiente ayant présenté une réponse exceptionnelle à une immunothérapie anti-PD1. Ce phénotype hypermutateur a été expliqué par une mutation germinale délétère de MBD4 (Methyl-CpG Binding Domain 4) avec une inactivation bi-allélique dans la tumeur. Deux autres tumeurs hypermutées CpG>TpG porteuses d’une mutation de MBD4 germinale, un mélanome uvéal et un glioblastome, ont été identifiées dans les bases de données publiques. La biologie des mélanomes conjonctivaux et leurs profils génomiques sont mal connus. Nous avons séquencé les génomes de 6 tumeurs, puis procédé à un séquençage ciblé de 47 autres tumeurs. Nous avons montré que ces tumeurs présentent un profil hypermuté C>T induit par l’exposition aux ultra-violets. Ces tumeurs présentaient un profil de mutations proche des mélanomes cutanés avec une fréquence moindre de mutations BRAF (33%), et des mutations plus spécifiques des mélanomes muqueux telles que des mutations activatrices de KIT et SF3B1 dans les mélanomes conjonctivaux non exposés au soleil. Nous avons également identifié des mutations de CTNNB1 dans les tumeurs développées sur des nevi conjonctivaux. L’ensemble de ces travaux illustrent comment la description moléculaire des tumeurs rares permet d’envisager de nouvelles stratégies de médecine de précision. / Ocular melanomas are rare tumors representing about 5% of all melanomas. Ocular melanomas may arise from two tissues: the uvea (~ 500 cases / year in France) and the conjunctiva (~ 30 cases / year). Uveal melanomas have a very low rate of somatic mutations. These tumors also carry specific distinctive copy number alterations (gains of 8q, 1q, 6p, losses of 3, 1p, 6q or 8p). The evolution of the genome of these tumors during metastatic progression has been poorly described.To explore the metastatic evolution of uveal melanoma, we whole-exome sequenced 14 primary tumors and 79 metastases from 24 patients. Primary tumors and metastases presented close genetic profiles with a median of 11.5 mutations in primary tumors, and 14 in metastases. Although SF3B1 and EIF1AX mutations are major prognostic factors in uveal melanomas, their frequencies in metastases were similar to those observed in historical primary tumors. The metastases showed some additional copy number alterations compared to the corresponding primary tumors. Among the alterations acquired during the metastatic process, 8q gains were present in 92% of metastases.Thanks to this work, we found a uveal melanoma with a CpG> TpG hypermutated phenotype in a patient who had an exceptional response to anti-PD1 immunotherapy. This hypermutated phenotype was explained by a deleterious germline mutation of MBD4 (Methyl-CpG Binding Domain 4) with bi-allelic inactivation in the tumor. Two other hypermuted CpG> TpG tumors with germline MBD4 mutation, a uveal melanoma and a glioblastoma, were identified in public databases.The biology of conjunctival melanomas and their genomic profiles have been scarcely described. We sequenced the genomes of 6 tumors and then target-sequenced 47 other tumors. We showed that these tumors had a C> T hypermuted profile induced by ultraviolet exposure. These tumors presented a pattern of mutations close to cutaneous melanomas with a lower frequency of BRAF mutations (33%), and mutations that were more specific of mucosal melanomas such as activating mutations of KIT and SF3B1 in conjunctival melanomas not exposed to the sun. We also identified CTNNB1 mutations in tumors developed on conjunctival nevi.All of these works illustrate how the molecular description of rare tumors opens new avenues for precision medicine.

Subclinical vitamin A deficiency was assessed in 65 Hispanic children attending four migrant Head Start programs in Utah. Subjects aged 2 to 6 years (median 3 years 10 months) were examined for evidence of vitamin A deficiency by conjunctival impression cytology. Biochemical indices for serum vitamin A, retinol-binding protein, zinc, and iron were performed. Of eight children (12.5%) with subclinical vitamin A deficiency, one child had a marginal serum vitamin A of 11 μg/dl. Retinol-binding protein concentrations were significantly lower in two subjects with abnormal conjunctival impression cytology. Serum zinc, which when low can mimic signs of ocular vitamin A lesions, was normal for all 65 subjects. Fifteen children (23%) had iron-deficiency anemia. Logistic regression was the central method of analysis used in this study. The results of the statistical analyses indicated there was a correlation value (0.31) between abnormal conjunctival impression cytology and serum vitamin A, which supports the hypothesis that abnormal conjunctiva! impression cytology is concurrent with decreased serum vitamin A. Assessment of vitamin A status of Hispanic migrant children by impression cytology was effective in identifying children at risk for hypovitaminosis A. Beyond vitamin A's role in vision and maintenance of epithelium, it is also required for growth and hematopoiesis. The children of migrant workers may be suffering physiologically important consequences of vitamin A and iron deficiency that can be prevented by screening with biochemical and histological testing. Nutrition intervention for deficient children is warranted.

A leishmaniose visceral canina (LVC) é uma grave zoonose, causada pela Leishmania infantum (syn. chagasi). O ciclo deste parasito é heteroxeno e a transmissão acontece principalmente pela picada da fêmea do vetor, dípteros da espécie Lutzomyia longipapis. O cão doméstico é o principal alvo das campanhas de controle da doença por ser a principal fonte de infecção para o vetor no ambiente urbano. O Ministério da Saúde adota testes sorológicos para a detecção de animais positivos; no entanto a sensibilidade e especificidade desses testes são questionáveis. Além das falhas inerentes a qualquer teste diagnóstico, no caso da LVC existem alguns entraves, especialmente pela distribuição geográfica, comum a outras doenças causadas por tripanossomas, e pela similaridade genética com os outros parasitas da mesma família. Nessas áreas de sobreposição pode haver tanto reação cruzada quanto co-infecção, dificultando a interpretação dos testes. No presente estudo, foram coletadas amostras de suabe conjuntival e sangue de cães em inquérito soroepidemiológico realizado no município de Ilha Solteira - SP. A presença de Leishmania spp. e Leishmania infantum foram testadas por PCR convencional e PCR em tempo real com primers direcionados ao kDNA de Leishmania spp. A avaliação sorológica foi realizada através da RIFI, e a identificação e caracterização dos tripanossomatídeos foi realizada através da PCR com primers ITS1. A SC-qPCR foi o teste que detectou o maior número de animais. De 204 cães utilizados no estudo, 19,12% (30/204) foram positivos na SC-qPCR. Na SG-qPCR foram 12,74% (26/204) de animais positivos. O teste que detectou o menor número de animais foi a SC-cPCR, com 10,78% (22/204). Enquanto na SG-cPCR obtivemos 13,23% (27/204) animais positivos. De 28 amostras selecionadas para o sequenciamento do gene ITS1, 19 (67,85%) foram 100 ou 99% similares à L. infantum, sugerindo que a maioria dos cães positivos para LVC estavam realmente infectados com esta espécie. Entretanto, 2 cães (7,14%), que tiveram suas amostras sequenciadas para tal gene, revelaram 99% de similaridade com Crithidia fasciculata. Dos testes avaliados, esses cães foram positivos apenas na SG-cPCR para Leishmania spp. Os resultados indicam que a SC-qPCR foi o teste mais eficaz em detectar amostras realmente positivas para L. infantum, e que se deve atentar ao fato de existirem outros tripanossomatídeos infectando os cães em área endêmica de LVC, podendo dificultar o diagnóstico adequado dos animais infectados por L. infantum. / Canine visceral leishmaniasis (CVL) is a serious zoonosis caused by Leishmania infantum (syn. chagasi). The life cycle of this parasite is heteroxenous and the transmission occurs through the bite of the female sandfly, diptera species Lutzomyia longipalpis. The domestic dog is the main focus disease control campaigns, since it is the most important source of infection for the vector in urban environment. For positive dogs detection the health ministry uses serological tests, however the sensitivity and specificity of these tests are questionable. In addition to flaws inherent in any diagnostic test, in case of CVL there are some obstacles, especially by geographic distribution, common to other diseases caused by trypanosomes, and also by genetic similarity with other parasites of the same family. In areas of disease overlap, cross-reaction or co-infection may occur, making it difficult to interpret the results. In this study, conjunctival swab samples and whole blood of dogs were collected in seroepidemiological survey conducted in Ilha Solteira - SP. The presence of Leishmania spp. and Leishmania infantum were tested by conventional PCR and real-time PCR with Leishmania spp. kDNA-targeted primers. The serological evaluation was carried out by RIFI, and the identification and characterization of trypanosomatids was performed by PCR with ITS1 primers. SC-qPCR was the test that detected the largest number of animals. Of 204 dogs used in this study, 19.12% (30/204) were positive in SC-qPCR. In SG-qPCR 12.74% (26/204) animals were positive. The test that detected the lowest number of animals was SC-cPCR, with 10.78% (22/204). While in the SG-cPCR we obtained 13.23% (27/204) positive animals. From 28 samples selected for ITS1 gene sequencing, 19 (67.85%) were 100 or 99% similar to L. infantum, suggesting that most CVL positive dogs were infected with this species. However, two dogs (7.14%), which had their samples sequenced for the same gene, showed 99% similarity with Crithidia fasciculata. From the evaluated tests, these dogs were only positive in SG-cPCR for Leishmania spp. The results indicate that SC-qPCR was the most effective test to detect L. infantum positive samples , and it should be noted that there are other trypanosomatids infecting dogs in an endemic CVL area, which can difficult to diagnose animals properly infected by L. infantum.

O objetivo do presente estudo foi detectar Leishmania spp. pela PCR em tempo real (qPCR) em amostras de DNA extraído de sangue e suabe conjuntival de cães, gatos e equinos. E também, verificar a positividade dessas amostras pela PCR convencional (cPCR), utilizando oligonucleotídeos específicos para L. infantum (inf.cPCR). Para isso, amostras de sangue e suabe conjuntival de 204 cães, 108 gatos e 54 equinos saudáveis foram testadas pela qPCR para Leishmania spp. e os resultados comparados pelo índice kappa a resultados de cPCR para Leishmania spp. (ssp.cPCR) previamente obtidos. A qPCR de sangue não detectou nenhum animal positivo. Já os resultados da qPCR de suabe conjuntival (qPCR-SC), revelaram 0,98% (2/204) de cães positivos. Ao comparar os resultados obtidos pela qPCR-SC com os resultados obtidos pela cPCR de suabe conjuntival (ssp.cPCR-SC), observou-se uma concordância baixa entre os métodos, k=0,32. Em relação aos gatos, 1,85% (2/108) desses animais foram detectados positivos para Leishmania spp. pela qPCR-SC, esse resultado corroborou com o resultado obtidos pela ssp.cPCR-SC o que resultou em uma excelente concordância entre os métodos comparados, k=1. Em relação aos equinos, 12,96% (7/54) dos animais foram detectados positivos para o parasito pela qPCR-SC. Esses resultados não possuem concordância com os resultados obtidos pela ssp.cPCR-SC, uma vez que por esse método, 66,66% (36/54) foram detectados infectados pela Leishmania spp. Ao realizar a inf.cPCR, 11,11% (6/54) dos equinos foram positivos. Submetidas ao sequenciamento, dois fragmentos apresentaram 99% de similaridade com sequencias de L. infantum disponíveis no GenBank. Enquanto a qPCR-SG não foi capaz de detectar Leishmania spp. em cães, gatos e equinos, a qPCR-SC foi capaz de detectar o parasito nas três espécies avaliadas. A associação entre a qPCR e o uso de um método prático, fácil e não invasivo, como o suabe conjuntival, representa um grande avanço no diagnóstico da doença em cães, gatos e equinos uma vez que, a qPCR-SC, foi capaz de detectar um maior número de animais infectados pela Leishmania spp. em relação a qPCR-SG. Além disso, a detecção de Leishmania spp. nas diferentes espécies avaliadas reforça a possível atuação desses animais como reservatórios de agentes da leishmaniose cutânea e a confirmação de L. infantum em equinos, associado à inexistência de sinais clínicos nestes animais revela a possibilidade de equinos serem reservatórios desse parasito. / The aim of this study was to detect Leishmania spp. by real-time PCR (qPCR) on DNA extracted from blood samples and conjunctival swab dogs, cats and equines. Also, check the positivity of these samples by conventional PCR (cPCR) using specific oligonucleotídeos for L. infantum (inf.cPCR). For this, blood samples and conjunctival swab of 204 dogs, 108 cats and 54 horses healthy were tested by qPCR for Leishmania spp. and the results compared by kappa index to cPCR results for Leishmania spp (ssp.cPCR). previously obtained. The blood qPCR detected no positive animal. Already the qPCR results of conjunctival swab (CS-qPCR), revealed 0.98% (2/204) of positive dogs. Comparing the results obtained by CS-qPCR with the results obtained by conjunctival swab cPCR (ssp.CS-cPCR), there was a low correlation between the methods, k = 0.32. In relation to cats, 1.85% (2/108) of these animals were detected positive for Leishmania spp. by CS-qPCR, this results corroborated with the results obtained by ssp.CS-cPCR which resulted in excellent agreement between the methods compared, k = 1. Already in horses, 12.96% (7/54) of the animals were found positive for parasites by CS-qPCR. These results not have agreement with the results obtained by ssp.CS-cPCR, since by this method, 66.66% (36/54) were found to be infected by Leishmania spp. When performing inf.cPCR, 11.11% (6/54) of the horses were positive. Submitted to sequencing, two fragments showed 99% similarity to L. infantum sequences available in the GenBank. While blood qPCR was not able to detect Leishmania spp. in dogs, cats and horses, CS-qPCR was able to detect the parasite in the three species evaluated. The association between the use of qPCR and a practical, easy and non-invasive method, such as conjunctival swab, is a great advance in the diagnosis of disease in dogs, cats and horses since CS-qPCR was able to detect a greater number of animals infected with Leishmania spp. in respect of blood qPCR. Furthermore, the detection of Leishmania spp. the different species evaluated reinforces the possible role of these animals as reservoirs of cutaneous Leishmaniasis agents and confirmation of L. infantum in equines associated with the absence of clinical signs in these animals reveals the possibility of equines being reservoirs of this parasite.

O objetivo do presente estudo foi detectar Leishmania spp. pela PCR em tempo real (qPCR) em amostras de DNA extraído de sangue e suabe conjuntival de cães, gatos e equinos. E também, verificar a positividade dessas amostras pela PCR convencional (cPCR), utilizando oligonucleotídeos específicos para L. infantum (inf.cPCR). Para isso, amostras de sangue e suabe conjuntival de 204 cães, 108 gatos e 54 equinos saudáveis foram testadas pela qPCR para Leishmania spp. e os resultados comparados pelo índice kappa a resultados de cPCR para Leishmania spp. (ssp.cPCR) previamente obtidos. A qPCR de sangue não detectou nenhum animal positivo. Já os resultados da qPCR de suabe conjuntival (qPCR-SC), revelaram 0,98% (2/204) de cães positivos. Ao comparar os resultados obtidos pela qPCR-SC com os resultados obtidos pela cPCR de suabe conjuntival (ssp.cPCR-SC), observou-se uma concordância baixa entre os métodos, k=0,32. Em relação aos gatos, 1,85% (2/108) desses animais foram detectados positivos para Leishmania spp. pela qPCR-SC, esse resultado corroborou com o resultado obtidos pela ssp.cPCR-SC o que resultou em uma excelente concordância entre os métodos comparados, k=1. Em relação aos equinos, 12,96% (7/54) dos animais foram detectados positivos para o parasito pela qPCR-SC. Esses resultados não possuem concordância com os resultados obtidos pela ssp.cPCR-SC, uma vez que por esse método, 66,66% (36/54) foram detectados infectados pela Leishmania spp. Ao realizar a inf.cPCR, 11,11% (6/54) dos equinos foram positivos. Submetidas ao sequenciamento, dois fragmentos apresentaram 99% de similaridade com sequencias de L. infantum disponíveis no GenBank. Enquanto a qPCR-SG não foi capaz de detectar Leishmania spp. em cães, gatos e equinos, a qPCR-SC foi capaz de detectar o parasito nas três espécies avaliadas. A associação entre a qPCR e o uso de um método prático, fácil e não invasivo, como o suabe conjuntival, representa um grande avanço no diagnóstico da doença em cães, gatos e equinos uma vez que, a qPCR-SC, foi capaz de detectar um maior número de animais infectados pela Leishmania spp. em relação a qPCR-SG. Além disso, a detecção de Leishmania spp. nas diferentes espécies avaliadas reforça a possível atuação desses animais como reservatórios de agentes da leishmaniose cutânea e a confirmação de L. infantum em equinos, associado à inexistência de sinais clínicos nestes animais revela a possibilidade de equinos serem reservatórios desse parasito. / The aim of this study was to detect Leishmania spp. by real-time PCR (qPCR) on DNA extracted from blood samples and conjunctival swab dogs, cats and equines. Also, check the positivity of these samples by conventional PCR (cPCR) using specific oligonucleotídeos for L. infantum (inf.cPCR). For this, blood samples and conjunctival swab of 204 dogs, 108 cats and 54 horses healthy were tested by qPCR for Leishmania spp. and the results compared by kappa index to cPCR results for Leishmania spp (ssp.cPCR). previously obtained. The blood qPCR detected no positive animal. Already the qPCR results of conjunctival swab (CS-qPCR), revealed 0.98% (2/204) of positive dogs. Comparing the results obtained by CS-qPCR with the results obtained by conjunctival swab cPCR (ssp.CS-cPCR), there was a low correlation between the methods, k = 0.32. In relation to cats, 1.85% (2/108) of these animals were detected positive for Leishmania spp. by CS-qPCR, this results corroborated with the results obtained by ssp.CS-cPCR which resulted in excellent agreement between the methods compared, k = 1. Already in horses, 12.96% (7/54) of the animals were found positive for parasites by CS-qPCR. These results not have agreement with the results obtained by ssp.CS-cPCR, since by this method, 66.66% (36/54) were found to be infected by Leishmania spp. When performing inf.cPCR, 11.11% (6/54) of the horses were positive. Submitted to sequencing, two fragments showed 99% similarity to L. infantum sequences available in the GenBank. While blood qPCR was not able to detect Leishmania spp. in dogs, cats and horses, CS-qPCR was able to detect the parasite in the three species evaluated. The association between the use of qPCR and a practical, easy and non-invasive method, such as conjunctival swab, is a great advance in the diagnosis of disease in dogs, cats and horses since CS-qPCR was able to detect a greater number of animals infected with Leishmania spp. in respect of blood qPCR. Furthermore, the detection of Leishmania spp. the different species evaluated reinforces the possible role of these animals as reservoirs of cutaneous Leishmaniasis agents and confirmation of L. infantum in equines associated with the absence of clinical signs in these animals reveals the possibility of equines being reservoirs of this parasite.

A leishmaniose visceral canina (LVC) é uma grave zoonose, causada pela Leishmania infantum (syn. chagasi). O ciclo deste parasito é heteroxeno e a transmissão acontece principalmente pela picada da fêmea do vetor, dípteros da espécie Lutzomyia longipapis. O cão doméstico é o principal alvo das campanhas de controle da doença por ser a principal fonte de infecção para o vetor no ambiente urbano. O Ministério da Saúde adota testes sorológicos para a detecção de animais positivos; no entanto a sensibilidade e especificidade desses testes são questionáveis. Além das falhas inerentes a qualquer teste diagnóstico, no caso da LVC existem alguns entraves, especialmente pela distribuição geográfica, comum a outras doenças causadas por tripanossomas, e pela similaridade genética com os outros parasitas da mesma família. Nessas áreas de sobreposição pode haver tanto reação cruzada quanto co-infecção, dificultando a interpretação dos testes. No presente estudo, foram coletadas amostras de suabe conjuntival e sangue de cães em inquérito soroepidemiológico realizado no município de Ilha Solteira - SP. A presença de Leishmania spp. e Leishmania infantum foram testadas por PCR convencional e PCR em tempo real com primers direcionados ao kDNA de Leishmania spp. A avaliação sorológica foi realizada através da RIFI, e a identificação e caracterização dos tripanossomatídeos foi realizada através da PCR com primers ITS1. A SC-qPCR foi o teste que detectou o maior número de animais. De 204 cães utilizados no estudo, 19,12% (30/204) foram positivos na SC-qPCR. Na SG-qPCR foram 12,74% (26/204) de animais positivos. O teste que detectou o menor número de animais foi a SC-cPCR, com 10,78% (22/204). Enquanto na SG-cPCR obtivemos 13,23% (27/204) animais positivos. De 28 amostras selecionadas para o sequenciamento do gene ITS1, 19 (67,85%) foram 100 ou 99% similares à L. infantum, sugerindo que a maioria dos cães positivos para LVC estavam realmente infectados com esta espécie. Entretanto, 2 cães (7,14%), que tiveram suas amostras sequenciadas para tal gene, revelaram 99% de similaridade com Crithidia fasciculata. Dos testes avaliados, esses cães foram positivos apenas na SG-cPCR para Leishmania spp. Os resultados indicam que a SC-qPCR foi o teste mais eficaz em detectar amostras realmente positivas para L. infantum, e que se deve atentar ao fato de existirem outros tripanossomatídeos infectando os cães em área endêmica de LVC, podendo dificultar o diagnóstico adequado dos animais infectados por L. infantum. / Canine visceral leishmaniasis (CVL) is a serious zoonosis caused by Leishmania infantum (syn. chagasi). The life cycle of this parasite is heteroxenous and the transmission occurs through the bite of the female sandfly, diptera species Lutzomyia longipalpis. The domestic dog is the main focus disease control campaigns, since it is the most important source of infection for the vector in urban environment. For positive dogs detection the health ministry uses serological tests, however the sensitivity and specificity of these tests are questionable. In addition to flaws inherent in any diagnostic test, in case of CVL there are some obstacles, especially by geographic distribution, common to other diseases caused by trypanosomes, and also by genetic similarity with other parasites of the same family. In areas of disease overlap, cross-reaction or co-infection may occur, making it difficult to interpret the results. In this study, conjunctival swab samples and whole blood of dogs were collected in seroepidemiological survey conducted in Ilha Solteira - SP. The presence of Leishmania spp. and Leishmania infantum were tested by conventional PCR and real-time PCR with Leishmania spp. kDNA-targeted primers. The serological evaluation was carried out by RIFI, and the identification and characterization of trypanosomatids was performed by PCR with ITS1 primers. SC-qPCR was the test that detected the largest number of animals. Of 204 dogs used in this study, 19.12% (30/204) were positive in SC-qPCR. In SG-qPCR 12.74% (26/204) animals were positive. The test that detected the lowest number of animals was SC-cPCR, with 10.78% (22/204). While in the SG-cPCR we obtained 13.23% (27/204) positive animals. From 28 samples selected for ITS1 gene sequencing, 19 (67.85%) were 100 or 99% similar to L. infantum, suggesting that most CVL positive dogs were infected with this species. However, two dogs (7.14%), which had their samples sequenced for the same gene, showed 99% similarity with Crithidia fasciculata. From the evaluated tests, these dogs were only positive in SG-cPCR for Leishmania spp. The results indicate that SC-qPCR was the most effective test to detect L. infantum positive samples , and it should be noted that there are other trypanosomatids infecting dogs in an endemic CVL area, which can difficult to diagnose animals properly infected by L. infantum.

Hund, bakterieller/ zytologischer Konjunktivalstatus, Konjunktivitis, Klinikaufenthalt

In this investigation of immunotherapy (IT) children 6-16 years old with rhinoconjunctivitis due to birch polli­nosis were included. I. Methodological studies. To monitor IT a reliable provocation test is desirable. The conjunctival provocation test (CPT) was evaluated in 20 children with four repeated challenges. The test was found to have a good preci­sion, it was simple and appeared to be clinically safe. After repeated tests the levels of IgE antibodies against birch increased considerably in three children, indicating an immunological response. A pollen peak affects the symptoms of an atopic individual for several days. Thus pollen counts for previous days must be taken into account when relating symptom scores with the counts. A dynamic time series model was therefore developed by which groups of atopic patients could be compared when exposed to different amounts of pollens. II: Cross-reactivity between deciduous trees during IT. Immunotherapy with pollen allergen preparations made from either birch (B) or a mixture of birch, alder and hazel (M) were compared. As measured with symptom scores the children in the M group improved at least as much as those in the B group. In the B group but not in the M group the improvement correlated with immunochemical findings before IT or early during the treatment, probably an unsignificant finding. Otherwise there was little difference between the two groups. Analysis of sera with crossed radioimmunoelectrophoresis in 20 children revealed that 60% of the children below 13 years had de­veloped IgE antibodies during IT against allergens against which they had not been allergic before IT. This had no appearent clinical implications. III: Oral immunotherapy (OIT). A pilot study of 18 children treated with high doses of a birch pollen allergen preparation in enteric coated capsules and 8 untreated controls indicated that OIT was effective as shown by lower symptom scores, less conjuctival sensitivity and increased levels of IgE antibodies against birch. However, the gastrointestinal side-effects were pronounced. Therefore a second double-blind study, in 30 children, was performed reducing the side-effects through a different dose schedule. Compared with the placebo group, the ac­tively treated children had lower symptom scores (p = 0.04), reduced skin sensitivity (p = 0.01), increasing levels of IgE (p = 0.001) and IgG (p = 0.007) antibodies against birch before the birch pollen season and a suppression of the seasonal increase in levels of IgE antibodies against birch (p <0.001). After three months of OIT but not after ten months they also had a lower sensitivity in CPT than the controls (p = 0.01). The intestinal permeability as assessed by the urinary recovery of differently-sized polyethyleneglycols was studied in 24 of the children during IT. No changes were seen in the group of actively treated children. In two ad­ditional children openly treated with OIT small bowel biopsies were taken with normal morphological findings. Thus OIT did not result in a generalized inflammation of the small bowel. / digitalisering@umu

Orientador: Cláudia Valéria Seullner Brandão / Resumo: O estudo objetivou descrever valores de referência para importantes testes diagnósticos oftalmológicos, como sensibilidade corneal, produção lacrimal, pressão intraocular, espessura corneal e microbiota conjuntival em saguis de tufo preto (Callithrix penicillata) saudáveis e sem alterações oculares. Foram utilizados 13 saguis de tufo preto, totalizando 26 olhos, provenientes do CEMPAS da Faculdade de Medicina Veterinária e Zootecnia - UNESP, Botucatu, São Paulo, Brasil. Os saguis foram submetidos a contenção química com anestesia dissociativa à base de cetamina e midazolam, para realização dos exames. Os resultados obtidos para sensibilidade corneal com estesiômetro Cochet-Bonnet foi de 5,8 ± 0,6cm; produção lacrimal com ponta de papel absorvente endodôntica estéril de 13 ± 3mm/min; pressão intraocular com tonômetro de rebote Tonovet Plus Icare® de 22,2 ± 2,9mmHg; espessura corneal 340,7 ± 25,5µm; e predomínio de bactérias Gram-positivas nas amostras de microbiota conjuntival. Não houve diferença significativa considerando lateralidade e sexo dos animais (P>0,05). Os resultados obtidos nos testes diagnósticos oftalmológicos poderão auxiliar médicos veterinários oftalmologistas a diagnosticar e tratar doenças oculares em saguis de tufo preto, além de contribuir para pesquisas na área de visão. / Abstract: The study aimed to describe reference values for important ophthalmic diagnostic tests, such as corneal sensitivity, tear production, intraocular pressure, corneal thickness and conjunctival microbiota in black tufted marmosets (Callithrix penicillata). Thirteen black tuft marmosets were used, totaling 26 eyes, followed by CEMPAS by the Faculty of Veterinary Medicine and Zootechnics - UNESP, Botucatu, São Paulo, Brazil. The procedures were used for chemical containment with dissociative anesthesia based on ketamine and midazolam, to perform the exams. The results obtained for corneal sensitivity with a Cochet-Bonnet esthesiometer were 5.8 ± 0.6 cm; tear production with sterile endodontic absorbent paper tip of 13 ± 3mm / min; intraocular pressure with Tonovet Plus Icare® rebound tonometer of 22.2 ± 2.9mmHg; corneal thickness 340.7 ± 25.5 µm; and predominance of Gram-positive bacteria in conjunctival microbiota. There was no significant difference considering the laterality and sex of the animals (P> 0.05). The results obtained in ophthalmological diagnostic tests can help veterinary ophthalmologists to diagnose and treat eye diseases in the black tufted marmosets, in addition to contributing to research in the field of vision. / Mestre

In this study, we aimed to determine the levels of cytokines IL-1β, IL-4, IL-10, IFN-γ, MIF and VEGF in tears derived from healthy subjects. We tested cytokines as potential markers of inflammation for their potential use in clinical practice. Having reliable method for measuring cytokine levels in tears would enable an early diagnosis of eye diseases. In two phases, cytokines in tears of healthy individuals were analyzed using Bio-Plex Cytokine Assay (Bio-Rad). We assessed the suitability of methods for diagnostic purposes as well as the suitability of our selected cytokines. Statistically significant positive correlations of cytokines were confirmed: IL-10 with IFN-γ (r = 0,81), MIF with VEGF (r = 0,42 / r = 0,49), IL-1β with IL-10 (r = 0,52), IL-1β with IFN-γ (r = 0,55), IL-1β with VEGF (r = 0,38), IFN-γ with VEGF (r = 0,45) and IL-4 with VEGF (r = 0,48) in healthy subjects in tears. IL-4 (r = -0,37) and IFN-γ (r = -0,42) correlate negatively with age. In healthy individuals, there seem to be no differences with regard to gender, BMI, body fat, time of meal consumption prior to tear collection, eye strain when using a computer, dry eyes. Thus, studied cytokines are suitable for diagnostic purposes. Significant differences in concentrations of four (IL-1β, IL-10, IFN-γ a VEGF) of the five...