Xylella fastidiosa subsp. pauca and fastidiosa Colonize Arabidopsis Systemically and Induce Anthocyanin Accumulation in Infected Leaves.

The bacterium Xylella fastidiosa is a multihost pathogen that affects perennial crops such as grapevine, sweet orange, and olive tree worldwide. It is inherently difficult to study these pathosystems owing to the long-term growth habit of the host plant. Thus, the availability of model plants becomes essential to accelerate discoveries with economic impact. In this study, we uncovered evidence that the model plant Arabidopsis thaliana can be colonized by two different X. fastidiosa subspecies, pauca and fastidiosa. We observed that these bacteria are able to move away from the inoculation point as high bacterial populations were found in distant tissues. In addition, confocal laser scanning microscopy analysis of bacterial movement inside the petiole revealed the ability of the bacterium to move against the net xylem flow during the time course of colonization forming biofilm. These findings provide evidence for the capacity of X. fastidiosa to colonize Arabidopsis. Furthermore, leaves inoculated with X. fastidiosa showed a significant accumulation of anthocyanin. We propose that the X. fastidiosa subsp. pauca or fastidiosa colonization pattern and anthocyanin accumulation in the Arabidopsis ecotype Col-0 can be used as marker phenotypes to facilitate further studies aimed at improving genetic components involved in X. fastidiosa-host interaction.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

Expression of Xylella fastidiosa fimbrial and afimbrial proteins during biofilm formation.

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.

Use of an esophageal echo-Doppler device during liver transplantation: preliminary report.

Determination of cardiac output (CO) is crucial for perioperative monitoring of orthotopic liver transplant (OLT) recipients. A pulmonary artery catheter (PAC) has always been considered the "gold standard" of hemodynamic monitoring. The aim of this study was to evaluate the suitability of a transesophageal echo-Doppler device (ED) as a minimally invasive device to measure CO in OLT. ED was compared with the standard PAC technique taking into account the disease severity of OLT recipients as defined by the model for end-stage liver disease (MELD) score. We enrolled 42 cirrhotic patients scheduled for OLT 3 thermodilution CO measurements were taken by a PAC and the most recent ED measurement (CO(ED)) was also recorded. Paired measurements of CO were performed at standard times, unless there were additional clinical needs. Recipients were stratified into 3 groups according to MELD score: MELD score < or = 15 (14 patients); MELD score between 16 and 28 (17 patients); and MELD score > or = 29 (11 patients). We performed 495 paired measurements of CO. Mean bias was 0.34 +/- 0.9 L/min and limits of agreement were -1.46 and 2.14 L/min. In patients with MELD score <15, the bias was 0.12 +/- 0.55. The ED results were not interchangeable with PAC, because of the large limits of agreement. However, in cirrhotic patients with MELD scores <15, the precision of the new method was similar to that of PAC; therefore, in this subset of patients, it may represent a reliable alternative to PAC.

[Hypercoagulability during pregnancy: evidences for a thrombophilic state]. / Trombofilia in gravidanza: evidenze clinico-sperimentali di uno stato trombofilico.

AIM: The development of thrombotic disorders is a major threat for young women during pregnancy. It is one of the main causes of pregnancy-related disorders, which may also result in harm for the conceptus. Successful pregnancies require an even balance of coagulation and fibrinolysis, in order to secure stabilization of the basal plate as well as adequate placental perfusion. Broad spectrum assays which measure a range of thrombin/fibrin formation in serum have become an established means of identifying activation of blood coagulation and/or fibrinolysis. There is considerable interest in the application of these assays to the diagnosis of other hypercoagulable states, such as thrombophilia during pregnancy. We investigated coagulation/fibrinolysis parameters for significant differences between pregnant women during their gestation (first, second and third trimester) with or without pregnancy loss and healthy nonpregnant women. METHODS: Thirty-nine pregnant women, aged 24-39 years, were studied. They were subdivided according to pregnancy trimester: 15 patients in the first trimester; 13 in the second and 11 in the third. The selection of patients was carried out in cooperation with the Transfusion Center of the Second University of Naples in order to obtain a homogeneous sample group. The control group included 400 healthy patients. Biochemical and blood coagulation tests were performed for each patient and the results obtained were compared with the control group. RESULTS: A decrease in free protein S (PS) and fibrinolysis (t-PA/PAI-1) activities and an increase in Factor VII, Factor VIII, prothrombin fragment 1+2 (F1+2), D-dimer (D-dimer) were observed in pregnant women during the follow-up of gestation. However, there were statistical differences between the groups of women with one or more pregnancy loss where it was found the lowest values in t-PA and PAI and the highest values in FVII and F1+2. Among subjects with more than one abortion, coagulation/fibrinolysis derangements before the partum were more prominent. A significant association exists between consecutive recurrent abortions and pregnancy complications such as placental abruption, hypertensive disorders and CS. This association persists after controlling for variables considered to coexist with recurrent abortions. CONCLUSIONS: These findings suggest that an excessive hypercoagulable state is associated with the termination of pregnancy resulting into a moderate risk for thrombosis during the different trimesters of pregnancy. The follow-up of fibrinolytic markers could represent a useful diagnostic tool for termination of pregnancy.

[The effects of phytoestrogen therapy on the endometrium in postmenopausal women]. / Effetti della terapia con fitoestrogeni sulla mucosa endometriale in postmenopausa.

AIM: The purpose of the present study was to carry out a comparative histological analysis of the endometrium in postmenopausal women who made use of phytoestrogens in order to assess the efficacy and possible side effects of this therapy. METHODS: This study was carried out by forming 2 groups in order to compare the results. One group was given a dietary supplement of phytoestrogens for 24 months, whereas the other was given a placebo for the same period of time. At the beginning of this study endometrial bioptical samples were taken from those patients who had been previously selected at our University Centre. This study was started only with those postmenopausal patients whose bioptical sample was histologically suitable, and it was neither hyperplastic, nor cancerous and nor secretive. During these 24 months there have been frequent contacts aimed at verifying the standard therapeutic behaviour, symptoms and appearance of side effects. At the end of the study new and final bioptical samples of endometrium were taken from both groups. RESULTS: One-hundred and forty-one patients completed the study. Five patients (3.4%) who were submitted to phytoestrogens therapy showed a weak proliferative endometrium bioptical sample. All the other biopsies at the beginning and at the end of the study showed an atrophic and inactive sample. Hot flushes, night sweats, vaginal dryness and dyspareunia improved at the end of the study for the group treated with phytoestrogens as compared to the one treated with a placebo. Although there have not been very significant differences ias to symptoms and side effects, it was noted that insomnia was the most common symptom in the group treated with non-hormonal therapy based on phytoestrogens. CONCLUSIONS: Phytoestrogens did not cause any sensitive and worrisome stimulation of the endometrial mucosa. Insomnia was more frequent in the group treated pharmacologically in the 24 months of the study, whereas hot flushes, night sweats, vaginal dryness and dyspareunia persisted or increased as compared to the beginning of the study in the group treated with a placebo, but this did not occur for the group treated with phytoestrogens.

Antenatal diagnosis and obstetric management of cystic hygroma occurring in twin pregnancies. A case report.

The lymphatic system has an early development in the embryo. Usually, its development is complete by the 40th postconceptional day. If this connection fails to develop, lymphatic stasis and dilatation of the lymphatic channels may occur, causing a number of pathologies such as: lymphangiomas, lymphangiectasis and cystic hygromas. Prenatal diagnosis can be made during the first trimester of pregnancy by ultrasonographic examination. A case of a twin pregnancy associated with cystic hygroma and bilateral hypoplasia of lower and upper limbs of both foetuses without chromosomal abnormalities is reported.

[Small ovarian cysts in postmenopause: assessment of their malignant potential with vaginal ultrasonography and tumor marker Ca125 titration]. / Piccole cisti ovariche in postmenopausa: valutazione del loro potenziale maligno mediante l'uso di ultrasonografia vaginale e titolazione del marcatore tumorale Ca125.

BACKGROUND: The aim of this study was to determine the risk of malignancy in cystic ovarian tumors < 10 cm in diameter in asymptomatic postmenopausal women. METHODS: All cystic ovarian tumors, detected by abdominal and transvaginal sonography screening, in asymptomatic postmenopausal women were evaluated with respect to size and morphology. Follow-up data were available both on patients undergoing surgery and on those who elected to be followed without operative intervention. Titration of the tumoral marker Ca125 was carried out, too. RESULTS: Unilocular cystic tumors were detected in 32 of 352 postmenopausal patients (9%), of 45-65 years of age arrived at the "Centre for diagnosis and therapy of menopausal diseases" of the III Divisione di Ginecologia e Ostetricia della Seconda Università degli Studi di Napoli from the 1st January to the 31st December 1999. All tumors were < 10 cm in diameter and 98% were < 5 cm in diameter; just one tumor was hardly > 5 cm in diameter (5.8), 14 of these cystic ovarian tumors (49%) resolved spontaneously within 60 days while 18 (51%) persisted. Seven patients with persistent cystic ovarian tumors underwent operative tumor removal. Five of these patients had serous cystadenoma and 2 other women had cystoadenofibroma. Not even one case of ovarian carcinoma was found in this group. The remaining 11 patients with unilocular cystic ovarian tumors underwent sonography control every 3 months for one year and no one of these patients developed ovarian carcinoma. In all these patients the dosage of the tumoral marker Ca125 remained under the suspicious threshold of malignant ovaric tumor (Ca125 = 35 U/ml). CONCLUSIONS: Unilocular cystic ovarian tumors < 5 cm in diameter in asymptomatic postmenopausal women were associated with minimal risk for ovarian cancer. In contrast, complex ovarian cysts wall abnormalities or solid areas are associated with a significant risk for malignancy. These date are important in determining therapeutic optimal strategies in these patients.

[Thrombophilic syndrome associated to phenotypic resistance to activated protein C in postmenopausal women]. / Sindrome trombofilica associata a resistenza fenotipica alla proteina C attivata in donne in postmenopausa.

AIM: Hormone replacement therapy (HRT) may reduce the risk of cardiovascular events in healthy postmenopausal women. However recent studies suggest a 2-4 fold increased risk of idiopathic venous thromboembolism (VTE) among users of HRT. Our aim was to evaluate the overall effect of HRT on hemostatic variables probably related to increased VTE risk reported in epidemiological studies. METHODS: Therefore, 100 healthy postmenopausal women aged 45-60 years divided into 50 HRT non-users and 50 HRT users were examined. The authors assayed on the automated coagulometer ACL7000 (Instrumentation Laboratory, Milan) the procoagulant proteins: factor VIII (VIII:C) and factor VII (VII:C); the natural anticoagulant proteins: antithrombin (ATIII), protein C (PC), protein S (PS) and the resistance to anticoagulant action of activated protein C (APC-Resistance). The free tissue factor pathway inhibitor (TFPI) was measured with an ELISA method (Diagnostica Stagò; France, Roche). The in vivo coagulation and fibrinolysis activation was evaluated by the assays of prothrombin fragment 1+2 (F1+2) and plasmin- antiplasmin complexes (PAP) using ELISA techniques. RESULTS: Increased levels of FVIII:C and FVII:C were observed in HRT users and HRT non-users women compared to controls (FVIII:C= 126+/-58%, 120+/-59% vs 85+/-15% p=0.0001; FVII: C 113+/-23%, 103+/-19% vs 90+/-16% p=0.0001). The activation peptides were significantly different compared to those found in control subjects; higher values were observed in HRT users compared to HRT non-users (F1+2=1.11+/-0.44 nM, 077+/-0.31 nM vs 0.45+/-0.35 p=0.00001; P-AP= 606+/-406 ng/ml, 514+/-205 ng/ml vs 235+/-59 p=0.0001). The ATIII and the PC were similar among the 3 different groups of subjects, but reduced levels of PS were observed in HRT users (PS 93+/-23%, 105+/-22% vs 109+/-12 p=0.0001). The mean normalized APC sensitivity ratio (APC-SR) was lower in the two populations of women as compared with that of controls (nAPC-SR 1.02+/-0.7, 1.02+/-0.8 vs 1.1+/-25 p=0.02). The values of free TFPI were reduced in HRT users compared to HRT non-users (9.1+/-1.9 ng/ml, 10.1+/-2.3 ng/ml vs 4.6+/-1.5 ng/ml p<0.0001). CONCLUSION: HRT appears to be associated to a shift in the procoagulant-anticoagulant balance towards a procoagulant state. The changes in hemostatic system could explain the increased risk of VTE in healthy postmenopausal women during HRT, nevertheless this risk could be higher in women known to have a congenital or acquired thrombophilic state.