Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence

Aggressive Multilocular Osteoblastoma in the Mandible: A Rare and Difficult Case to Diagnose

Osteoblastoma is a benign neoplasia and is uncommon in the jaws. In some cases, this lesion presents extremely aggressive local characteristics and is termed aggressive osteoblastoma. Because the clinical, radiographic and histopathologic characteristics are similar to those of a variety of benign and malignant tumors, it poses a diagnostic dilemma. This report presents a case of an aggressive osteoblastoma in the mandible and discusses the differential diagnosis of this lesion. A 13-year-old white male sought the Stomatology Clinic at the State University of Paraíba, Campina Grande, PB, Brazil, complaining of asymptomatic swelling on the left side of his face. Cone-beam computerized tomography showed a multilocular, hypodense bone lesion, located in the body of the left mandible and lower third of the ascending ramus. The initial diagnostic hypothesis was juvenile ossifying fibroma or osteosarcoma. After histopathologic examination, the final diagnosis was aggressive osteoblastoma. Surgical resection with a safety margin was performed. There was no evidence of recurrence after a follow-up period of 4 years.

O osteoblastoma é uma neoplasia benigna e incomum nos maxilares. Em alguns casos esta lesão apresenta características locais extremamente agressivas, sendo denominada osteoblastoma agressivo. Devido às características clínicas, radiográficas e histopatológicas serem similares a uma variedade de tumores benignos e malignos, o seu diagnóstico é um dilema. Este relato apresenta o caso de um osteoblastoma agressivo na mandíbula e discute o diagnóstico diferencial desta lesão. Paciente, branco, 13 anos de idade, foi atendido na Clínica de Estomatologia da Universidade Estadual da Paraíba, Campina Grande, PB, Brasil, queixando-se de aumento de volume assintomático do lado esquerdo de sua face. A tomografia computadorizada de feixe cônico revelou uma lesão óssea hipodensa multilocular, localizada no corpo do lado esquerdo da mandíbula e no terço inferior do ramo ascendente da mandíbula. A hipótese diagnóstica foi de fibroma ossificante juvenil e osteosarcoma. Após exame histopatológico, o diagnóstico final foi osteoblastoma agressivo. Foi realizada ressecção cirúrgica com margem de segurança. Não houve sinais de recorrência após 4 anos de acompanhamento.

Las trombopoyetinas en el tratamiento de la púrpura trombocitopénica inmunitaria y otras trombocitopenias / Thrombopoietines in the treatment of the thrombocytopenic purpura and other thrombocytopenics

La purpura trombocitopénica inmunitaria y las trombocitopenias secundarias representan condiciones patológicas graves cuyo tratamiento plantea diversos grados de dificultad. La aproximación terapéutica convencional ha sido la administración de esteroides, la esplenectomía y el uso de inmunoglobulina intravenosa u otros tipos de anticuerpos (e.g., anti-D). La mejor comprensión de la fisiología y fisiopatología de la trombopoyesis aunado a los avances en biología molecular ha permitido el desarrollo de una nueva aproximación terapéutica, la aplicación de las trombopoyetinas sintéticas o no inmunogénicas. Dentro de este grupo resaltan dos compuestos: el romiplostin (una proteína de fusión) y el eltrombopag (un compuesto sintético de bajo peso molecular). Ambas se encuentran disponibles comercialmente. Los estudios clínicos indican que estos medicamentos tienen un efecto satisfactorio en el tratamiento de las trombocitopenias, particularmente en los casos refractarios a los tratamientos convencionales.

Immune thrombocytopenic purpura and the secondary thrombocytopenias are conditions potentially severe with diverse degrees of treatment difficulties. Steroids administration, splenectomy and the use of intravenous immunoglobulin and other antibodies (e.g., anti-D) had been the conventional therapy. The better understanding of the thrombopoiesis physiology and physiopathology togetter with the biology advances have permitted the development of a new terapheutic approach: the use of synthetic or nonimmunogenic thrombopoietines. Among this group highlights composites: romiplostim (a fusion protein) and eltrombopag (a synthetic composite with low molecular wheigt). Both are already available and produce a satisfactory effect particularly in nonrespondent cases to the conventional treatment.

Effect of anti-KDR antibody on the proliferation of hemangioma vascular endothelial cells in vitro / 华中科技大学学报（医学）（英德文版）

The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells (HVECs) was investigated. HVECs from one case of hemangioma in proliferative phase were cultured. Both primary culture and sub-culture were conducted in M199 medium. The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody. Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference. The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody (50, 10 and 2 microg/mL) was 84%, 63% and 39% respectively at 9th day after interference, with the difference being significant. In the control group, the number of HVECs was increased significantly. In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR, indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.

Polyclonal antibody-mediated mitotic inhibition in Chinese hamster ovary cells.

The nucleus of a mammalian cell undergoes profound reorganization when the cell enters mitosis and a number of proteins involved at various levels of the cell cycle have been characterized. The presence of mitotic-specific proteins has been reported and their roles are important in understanding the mechanics of cell division. The ability of antibodies to recognize mitotic protein antigens and further inhibit mitosis is potentially valuable in their role as therapeutic and diagnostic agents in cancer therapy. In this study, we have aimed to analyze proteins isolated from mitotic cells of Chinese hamster ovary (CHO) cells and their significant role in inhibiting mitosis. The proteins extracted from mitotic cells were processed and antibodies produced. It was observed that the secondary response that yielded an antiserum of 1:8 titer was predominantly IgG. The antiserum was effective in inhibiting mitosis in CHO cells in culture in a dose-dependent manner. Although inhibition of mitosis was apparent by cell proliferation studies, there was no apparent effect of the antiserum on other cell morphology and culture characteristics. The unique molecular structure of the antibody by which it bivalently binds to a broad array of antigenic epitopes serves as the foundation of its utility. These antibodies, being polyclonal in nature, are targeted against a whole range of proteins; and their multiple epitopes involved in process of cell division might hence mediate recognition or inhibition of function of such proteins in a wholesome manner and thus accomplish inhibition of mitotic progression.

Studies on the effect of intratesticular administration of opioid peptides, naloxone or N-acetyl beta-endorphin antiserum on some testicular parameters in rats.

Opioid peptides have been localized in a variety of peripheral tissues like placenta, thyroid, pancreas, gastrointestinal tract, in the reproductive tract of male and female and in the testes of rats. Immunoassayable material was detected in extracts of gonads, reproductive tract and accessory reproductive organs. Studies with naloxone have suggested that beta-endorphin may have an important role in steroidogenesis and may have a role in regulating transport of luminal material. In our studies met-enkephalin, beta-endorphin, naloxone or N-acetyl beta-endorphin antiserum were microinjected intra testicularly once on alternate days for one week and autopsied 24 h after the last injection. Intratesticular administration of 25, 50 and 100 micrograms doses of naloxone induced significant decrease in in vitro secretion of testosterone per se, which was significantly greater with 50 micrograms dose than with those of the other two doses. A 25 micrograms dose had no effect on hyaluronidase or acid phosphatase activity while 50 micrograms dose significantly decreased the enzyme activity. One hundred micrograms dose also significantly decreased hyaluronidase activity. Intratesticular injection of 10 micrograms met-enkephalin or 1 microgram beta-endorphin significantly decreased hyaluronidase activity whereas 20 microliters N-acetyl beta-endorphin antiserum increased the specific activity of hyaluronidase. There was no change in the weight of the testes on treatment with the above agents.

Partial purification and characterization of a novel murine factor that augments the expression of class I MHC antigens on tumor cells

A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.

Expression of low density lipoprotein receptors in lymphoblasts induced by anti-CD3 antibody in patients with hypercholesterolemia

Familial hypercholesterolemia(FH) is a disease based on defects of low-density lipoprotein receptors(LDL-R). To interrupt and control the natural course of this disease, early identification of these patients is important. The routine lipid profile tests for hypercholesterolemia can not differentiate objectively FH from secondary hypercholesterolemia. The exact diagnosis of FH heterozygotes is especially essential because it is easier to develop premature coronary heart diseases compared with secondary hyper-cholesterolemia. A simplified rapid and precise method for the mass screening of FH patients and the differentiation between FH heterozygote and secondary hyperlipidemia was needed. For the test, lymphocytes were used as target cells in LDL-R assay. After a 5 day culture with anti-CD3 Ab as a mitogen, indirect immunofluorescence stain and flow cytometric analysis were applied. The results were as follows; 74 +/- 9% of the stimulated lymphoblasts from normal controls expressed LDL-R activity. Cultured, but unstimulated, lymphocytes of normal controls showed 27 +/- 8% positivity and total cultured lymphocytes showed positivity of 46 +/- 11% positivity. Lymphoblasts, unstimulated lymphocytes, and total cultured lymphocytes from hyper-cholesterolemia without FH showed 74 +/- 10%, 25 +/- 10% and 50 +/- 17%, respectively, which showed no significant differences from normal control groups. FH Heterozygotes showed LDL-R positivity, 21 +/- 11% in lymphoblasts, 11 +/- 6% in unstimulated lymphocytes and 18 +/- 7% in total cultured lymphocytes. These data imply that adequately stimulated lymphocytes might be used for detecting defects in LDL-R and used to differentiate FH from secondary hypercholesterolemia.