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Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension, and endocytosis. FRAP, 4-D imaging, and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.

The following document is based on the current knowledge of SARS-CoV-2 and the related disease (COVID-19). The Italian Institute of Health (ISS) is monitoring the virus spread throughout the country through daily reports sent by individual regions and regional reference laboratories. The diagnosis of infection is based on the use of standardized and validated molecular tests for the search for viral RNA in different sets of biological samples, from patients suspected of having contracted the infection. This document provides a specific guidance for the collection and storage of biological samples from deceased persons. It also provides recommendations on safety practices to be adopted during both collection and handling of specimens, and during autopsy procedures. With regard autopsy, it is recommended to follow the procedures for the execution of diagnostic findings in patients died with SARS-CoV-2 infection issued by ISS Working Group on Causes of Death from COVID-19, published on March 27, 2020.

Objective To report our clinical experience with bevacizumab in a cohort of Hereditary Hemorrhagic Telangiectasia (HHT) patients with severe hepatic involvement and/or refractory anemia. Methods Observational, ambispective study of the Institutional Registry of HHT at Hospital Italiano de Buenos Aires. Patients were treated with bevacizumab due to iron deficiency refractory anemia secondary to nasal/gastrointestinal bleeding and/or high output cardiac failure. We describe basal clinical data, bevacizumab schedules, efficacy outcomes and adverse events. Wilcoxon signed ranks test and longitudinal analysis were conducted. Results Twenty adult patients were included from July 2013 to June 2019. Clinical indications were: 13 for anemia, 4 for heart failure and 3 for both. In the anemia group, median pretreatment hemoglobin was 8.1 g/dl [IQR: 7.2–8.4] and median transfusion requirement was 4 units [2–6]. In heart failure group, pretreatment median cardiac index was 4.5 L/min/m2 [4.1–5.6] and cardiac output was 8.3 L/min [7.5–9.2]. Bevacizumab 5 mg/kg/dose every 2 weeks for 6 applications was scheduled. By the end of induction, median hemoglobin at 3 months was 10.9 g/dl [9.5–12.8] (p = 0.01) and median transfusion requirement 0 units [0–1] (p

Additional file 1.

An aryne insertion cascade reaction on oxindoles has been observed and constitutes a convenient "one pot" preparation of bioactive di- and triarylated oxindoles in good yields under mild conditions. A temperature controlled "reaction switch" enables ready access to dibenzo[b,e]azepin-6-one derivatives employing the same reaction regime. This tactic has been extended to a short synthesis of potent antiulcer agent darenzepine.

This article is based on analysis of data gathered from Social Science faculty in ten State Universities in Maharashtra that are conducting user education programs. 291 (82.97%) Social Science faculty responded to the questionnaire. Data analysis reveals that only 35.05% of the respondents attended user education programs. Most professors were 'Not aware' of the programs while more than half of 'Associate Professors' (54.29%) attended these programs. Majority (58.42%) of the Social Science faculty have 'not attended any program' - 'No time to attend' (43.20%), 'Lack of information' (36.80%) are the primary reasons for not attending these programmes. But majority of the faculty have indicated that these programs are useful to them (68.73%) and 97.50 percent of these respondents indicated that their 'accessing the Online Sources' will improve immensely.

The role of the different lymphocyte populations in liver microenvironment of chronic hepatitis C (CHC) patients is still matter of debate. Since Th17 and Treg have opposite functions, their balance could affect disease progression. The aim was to explore liver microenvironment and its peripheral blood counterpart in adult CHC patients. CD4+ lymphocytes were predominant in the liver, with high Foxp3+ but low IL-17A+ frequency. IL-17A+ lymphocytes and IL-17A+/Foxp3+ ratio displayed association with advanced fibrosis (p = 0.0130; p = 0.0236, respectively), while Foxp3+ lymphocytes and IL-10 expression level inversely correlated with fibrosis severity (p = 0.0381, p = 0.0398, respectively). TGF-β/IL-6 ratio correlated with IL-17A+/Foxp3+ ratio (p = 0.0036, r = 0.5944) and with IL-17A+ lymphocytes (p = 0.0093; r = 0.5203). TNF-α and TGF-β were associated with hepatitis severity (p = 0.0409, p = 0.0321). Peripheral blood lymphocyte frequency was not associated with liver damage. There are functionally different immune cell populations actively involved in liver damage, but the liver cytokine milieu actually drives the pathogenesis. The intrahepatic Foxp3+ lymphocytes predominance beside the low IL-17A+ lymphocytes frequency, delineate a skewed IL-17A+/Foxp3+ balance towards Foxp3+ lymphocytes. However, the IL-17A+ lymphocytes association with advanced fibrosis denotes their role in the pathogenesis. Therefore, the interplay between Th17 and Treg conditions liver fibrogenesis. Fil: Rios, Daniela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Anatomía Patológica; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina Fil: Valva, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Anatomía Patológica; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina Fil: Casciato, Paola Cecilia. Hospital Italiano; Argentina Fil: Frias, Silvia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; Argentina Fil: Soledad Caldirola, María. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Área de Inmunología; Argentina Fil: Gaillard, María Isabel. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Área de Inmunología; Argentina Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Área de Inmunología; Argentina Fil: Bandi, Juan. Hospital Italiano; Argentina Fil: Galdame, Omar. Hospital Ramos Mejia; Argentina Fil: Ameigeiras, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; Argentina Fil: Krasniansky, Diana. Hospital General de Agudos “Carlos G. Durand”. Unidad de Hepatología; Argentina Fil: Brodersen, Carlos. Hospital General de Agudos “Carlos G. Durand”. Unidad de Hepatología; Argentina Fil: Mullen, Eduardo. Hospital Italiano; Argentina Fil: de Matteo, Elena Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Anatomía Patológica; Argentina Fil: Preciado, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Anatomía Patológica; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina

Background Around 47–74% of patients with hereditary hemorrhagic telangiectasia (HHT) have hepatic vascular malformations (HVMs); magnetic resonance images (MRI) of the central nervous system (CNS) might show in T1 sequences a hyper-intensity signal in different areas, mainly in the basal ganglia (BG) as consequence of manganese (Mn) deposits as observed in cirrhotic patients. These patients might suffer from different neuropsychiatric disorders (hepatic encephalopathy). In HHT patients, even in the presence of hepatic shunts, hepatocellular function is usually preserved. Additionally, Mn shares iron absorption mechanisms, transferrin and CNS transferrin receptors. In iron deficiency conditions, the Mn may harbor transferrin and access BG. The objectives were to describe frequency of BG Mn deposit-induced lesions (BGMnIL) in HHT patients, its relationship with iron deficiency anemia (IDA) and HVMs. Finally, explore the association between neuropsychological and motor consequences. We performed a cross-sectional study. We determined HHT patients with or without BG-MnIL by the MRI screening of the CNS. We included all patients with lesions and a random sample of those without lesions. All patients underwent standardized and validated neuropsychological assessment to evaluate BG actions. Results were analyzed with multiple logistic regression, adjusting for potential confounders. Results Among 307 participants from a cohort included in the Institutional HHT Registry, 179 patients had MRI performed and Curaçao Criteria ≥3. The prevalence of BG-MnIL was 34.6% (95%CI 27.69-42.09). While neuropsychological symptoms were present in all patients, BG-MnIL patients performed poorly in three of the neuropsychological tests (serial dotting, line tracing time, number connection test A). HVMs frequency in BG-MnIL was 95.1%, versus 71.4% in those without lesions (p

BACKGROUND: The incidence, prevalence, mortality and morbidity of ischemic heart disease are increasing all over the world. Early detection and prevention of ischemic heart disease is important. Hence, identification of biomarkers for cardiovascular disease and its risk factors is needed. AIMS AND OBJECTIVES: To evaluate the association of Gamaglutamyl transferase activity with lipid risk ratios in ischemic heart disease. MATERIALS & METHODS: This cross sectional comprises 50 healthy subjects and equal number of myocardial infarction cases. Serum gamaglutamyl transferase activity, lipid profile was estimated and lipid ratios were calculated. Data was statistically analyzed. RESULTS: Serum Gamaglutamyl transferase activity increased in myocardial infarction cases and is correlated with lipid ratios. CONCLUSION: Serum Gamaglutamyl Transferase activity may be used in clinical practice as an aid for prediction of ischemic heart disease.

Background. Incidental hepatocellular carcinoma (iHCC) generates uncertainty over risk of recurrence after liver transplantation (LT). Aim. To compare recurrence between iHCC and confirmed HCC diagnosed prior to transplant based on imaging criteria (cHCC). Material and methods. Fifty-four HCC patients were analyzed from a series of 309 consecutive adult transplanted patients. We developed a recurrence predicting score (RPS) applying ORs based on pathologic risk variables. Results. Incidence of iHCC was 4.8% (n = 15) and overall recurrence 12.9% (cHCC 15.4% and iHCC 7%; P = 0.39). Variables included in the RPS were: microvascular invasion OR 17.8 (1.78-178.97; P = 0.014: 2 points), neural invasion OR 15.5 (1.13-212.17; P = 0.04: 1.5 points), nuclear grade > II OR 9.3 (1.17-74.84; P = 0.035: 1 point), and beyond Up-to 7 criteria OR 13.1 (1.66-103.67; P = 0.015: 1.5 points). Two risk groups were identified: low risk for recurrence (0-1 point) and intermediate-high risk groups (2-6 points). Low risk category remained an independent predictor of recurrence: OR 0.11 (0.01-0.67; P = 0.017); AUROC of 0.75 (0.54-0.96). A tendency towards more patients categorized as low risk group among iHCC patients was observed (69.2%; P = 0.13). Conclusions. In this series iHCC was not associated to lower risk of recurrence when compared to cHCC. We propose application of an RPS as a clinical tool for recurrence risk estimation.

Purpose To present a theoretical basis for noninvasively characterizing in vivo fluid-mechanical energy losses and to apply it in a pilot study of patients known to express abnormal aortic flow patterns. Methods Four-dimensional flow MRI was used to characterize laminar viscous energy losses in the aorta of normal controls (n = 12, age = 37 ± 10 yr), patients with aortic dilation (n = 16, age = 52 ± 8 yr), and patients with aortic valve stenosis matched for age and aortic size (n = 14, age = 46 ± 15 yr), using a relationship between the three-dimensional velocity field and viscous energy dissipation. Results Viscous energy loss was elevated significantly in the thoracic aorta in patients with dilated aorta (3.6 ± 1.3 mW, P = 0.024) and patients with aortic stenosis (14.3 ± 8.2 mW, P < 0.001) compared with healthy volunteers (2.3 ± 0.9 mW). The same pattern of significant differences was seen in the ascending aorta, where viscous energy losses in patients with dilated aortas (2.2 ± 1.1 mW, P = 0.021) and patients with aortic stenosis (10.9 ± 6.8 mW, P < 0.001) were elevated compared with healthy volunteers (1.2 ± 0.6 mW). Conclusion This technique provides a capability to quantify the contribution of abnormal laminar blood flow to increased ventricular afterload. In this pilot study, viscous energy loss in patient cohorts was significantly elevated and indicates that cardiac afterload is increased due to abnormal flow. Magn Reson Med 72:620–628, 2014. © 2013 Wiley Periodicals, Inc.

The Cryptocephalus marginellus (Coleoptera: Chrysomelidae) complex is composed by six species that are supposed to have originated by events of allo- or parapatric speciation. In the present study we investigated the alternative hypotheses that the bacterial communities associated with six populations of this species complex are shaped by environmental factors, or reflect the proposed pattern of speciation. The microbiota associated with the six populations, from five species of the complex, have been characterized through 16S rRNA pyrotag sequencing. Based on a 97% sequence similarity threshold, data were clustered into 381 OTUs, which were analyzed using a variety of diversity indices. The microbiota of C. acquitanus and C. marginellus (Calanques) were the most diverse (over 100 OTUs), while that from C. zoiai yielded less bacterial diversity (45 OTUs). Taxonomic assignment revealed Proteobacteria, Tenericutes and Firmicutes as the dominant components of these beetles’ microbiota. The most abundant genera were Ralstonia, Sphingomonas, Rickettsia, and Pseudomonas. Different strains of Rickettsia were detected in C. eridani and C. renatae. The analysis of β-diversity revealed high OTU turnover among the populations of C. marginellus complex, with only few shared species. Hierarchical clustering taking into account relative abundances of OTUs does not match the phylogeny of the beetles, therefore we hypothesize that factors other than phylogenetic constraints play a role in shaping the insects’ microbiota. Environmental factors that could potentially affect the composition of bacterial communities were tested by fitting them on the results of a multi-dimensional scaling analysis. No significant correlations were observed towards the geographic distances or the host plants, while the composition of the microbiota appeared associated with altitude. The metabolic profiles of the microbiotas associated with each population were inferred from bacterial taxonomy, and interestingly, the obtained clustering pattern was consistent with the host phylogeny., This work was partially supported by “Accordo di Programma, affermazione in Edolo del Centro di Eccellenza Università della Montagna” MIUR-Università degli Studi di Milano, prot. n. 1293–05/08/2011. JGZ has received support from the Spanish Ministry of Economy and Competitivity (project no. CGL2011–23820/BOS, with contribution from FEDER Funds).

This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology(WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification,diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.

A recent survey on the microflora associated with the leafhopper Scaphoideus titanus Ball (Hemiptera Cicadellidae), the vector of “flavescence dorée” (FD), showed the presence of bacteria belonging to the genus Asaia. These bacteria are symbionts of insects of the genus Anopheles, malarial mosquito vectors. We focused on the association between Asaia sp. and S. titanus with the aim to evaluate a possible role of this microorganism as a symbiotic control agent. After development of artificial feeding systems for S. titanus, quantitative PCR on insects or feeds DNA was performed with specific primers. Results underlined that Asaia sp. was dominant in the insect microflora. A strain of Asaia expressing a green fluorescent protein was used to perform recolonization of the body of S. titanus, showing the colonization by the bacterium of salivary glands, guts, female and male reproductive organs of the insect. In situ hybridization with specific probes was performed on insect dissections, confirming the presence of the bacterium in spermatic bundles and in Malpighian tubules. Easy cultivability, dominance within the bacterial population in the insect body, cryogenic preservability and easiness for genetic manipulation demonstrated by the overall data make Asaia sp. an optimal candidate for carrying factors for FD control.

Several wine production areas in Europe are afflicted by “flavescence dorée” (FD), a grape yellows caused by ‘Candidatus Phytoplasma vitis’, transmitted by the phloem-feeder leafhopper Scaphoideus titanus Ball (Hemiptera Cicadellidae). Symbiotic control of disease transmission, exploiting natural symbionts of the vector, could represent an innovative strategy against FD diffusion. A study was conducted to characterize the microflora associated to S. titanus specimens, collected from FD-contaminated vineyards in the Italian region of Piedmont. By length heterogeneity PCR we identified a major peak associated with almost all the individuals examined. Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band corresponding to sex-ratio-distorting endosymbionts of the ‘Candidatus Cardinium hertigii’ group; electron microscopy of tissues of S. titanus showed bacterial cells with the typical morphology of ‘Ca. Cardinium hertigii’ together with cell morphologies resembling those of ‘Ca. Phytoplasma vitis’. Our findings suggest that this endosymbiont, named ST1-C, described for the first time in Cicadellidae, is transovarially transmitted and could have a complex life cycle in the insect body; in addition it colocalizes with ‘Ca. Phytoplasma vitis’ several host tissues. These results make ST1-C an interesting candidate for the symbiotic control of FD diffusion.

Purpose : Aortic valve disease (AVD) in the form of stenosis, insufficiency, or congenital defect will disrupt normal function beyond the valve itself. This includes an increase in cardiac afterload and a drastic alteration in post-valvular 3D blood flow patterns 1, 2. The current AHA/ACC standard-of-care guidelines, however, assess disease severity based on simplified measurements local to the valve, such as: peak velocity, effective orifice area, regurgitation, aortic diameter and transvalvular pressure gradient 3. Paradoxically, it is known that similarly classified AVD patients under these guideline metrics can exhibit radically divergent outcomes — implying an incomplete characterization of the disease 4. For this reason, functional assessment and risk-stratification may benefit from a robust methodology capable of quantifying the energetic load placed on the left ventricle (LV) due to the presence of AVD. The measurement of viscous energy loss, a parameter which is directly responsible for increased cardiac afterload and is independent of pressure recovery effects, is a promising candidate to quantify LV loading. With this in mind, the 4D flow technique (time-resolved 3D phase-contrast MRI with all principal velocity directions encoded) provides the necessary information to calculate this parameter. Therefore, we present a theoretical basis for the use of 4D flow MRI to characterize in-vivo energy loss and apply the technique in a pilot study of patients with aortic valve stenosis (n = 13) or aortic dilation (n = 17) as compared to normal controls (n = 12).

Liver transplantation success is limited by the availability of donors. To overcome this limitation, anti-core-positive donors are increasingly being accepted, but underutilization of this resource still occurs. We performed the current study to determine the prevalence of anti-core-positive donors in our region and to describe the management of these donors and their recipients. Between January 2005 and July 2011, the national transplant database included 2,262 registered liver donors among whom 106 (4.7%) were anti-core-positive including 59 (56%) discarded and 47 (44%) implanted organs. A median of 14.5 offers (range 4-60) were rejected before harvesting and implanting the accepted grafts. The only difference between the implanted and the discarded grafts was found for the alanine aminotransferase level, which was higher among the discarded ones (50 ± 59 UI/L vs 25 ± 16, P < .05). Among 40 recipients included in the study, 5 (12.5%) did not receive any prophylaxis; 18 (45%) a nucleos(t)ide analog 11 (25.5%), heptitis B immunoglobulin and nucleos(t)ide analogs and 6 (15%) pretransplant hepatitis B vaccination. Over a mean follow-up of 871 ± 585 days, 4 de novo hepatitis B cases were identified at 545, 720, 748, and 1,080 days posttransplantation. None of these patients had received any prophylaxis. In all cases entecavir successfully controlled viral replication. We believe that better utilization of these donors and careful management of their recipients represent safe strategies to expand the liver donor pool in Argentina.

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.

Wolbachia pipientis is an intracellular bacterial endosymbiont of arthropods and filarial nematodes. Six main supergroups of W. pipientis have been described: supergroups A, B, E, and F encompass arthropod wolbachiae; supergroups C and D encompass nematode wolbachiae. The description of these six supergroups has been based on the analysis of only two genes (ftsZ and 16S rDNA) and before decisions are taken on the taxonomic status of the six supergroups, analysis of further genes is required. In addition, the branching order of the six supergroups is still unresolved. Sequence information from other genes is also needed to allow phylogenesis to be addressed through the analysis of a higher number of characters. Here we report sequences from a portion of the gene coding for the DNAA protein of W pipientis, generated from the endosymbionts of 22 host species. Phylogenies based on dnaA gene sequences are congruent with the existence of at least six supergroups of W pipientis. In addition, subtrees generated for nematode wolbachiae in supergroups C and D were compared to the trees based on the already available gene sequences (ftsZ, 16S rDNA and wsp). The congruence observed among the trees based on the different genes agrees with the hypothesis that recombination does not occur in nematode wolbachiae.

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

Filarial nematodes harbour intracellular symbiotic bacteria belonging to the genus Wolbachia. Wolbachia is thought to play an important role in the biology of the nematode. Moreover, Wolbachia appears to be involved in the immunopathogenesis of filariasis and in the onset of the side-effects of antifilarial therapy. Investigations in these research areas require reliable methods to quantify Wolbachia both in nematodes and in vertebrate tissues. To this purpose, we designed a quantitative real-time PCR targeted on the ftsZ gene of the Wolbachia of Brugia pahangi, a model filarial species maintained in gerbils. The method was applied to quantify Wolbachia in Brugia pahangi, from animals with or without tetracycline treatment. Our results show that tetracycline treatment leads to dramatic reduction or clearance of Wolbachia from the nematode. Results obtained from different replicates were reproducible and the method appeared very sensitive compared to other PCR protocols for Wolbachia detection. Real-time PCR is thus an appropriate method for investigations on the biological role of Wolbachia and on the implication of these bacteria in the pathogenesis of filariasis. With slight modifications of the primers and probe, the protocol we have developed could be applied in studies of the human pathogen Brugia malayi and on the model filarial species Litomosoides sigmodontis.

The small-subunit (SSU) rDNA of the Neospora sp. NC-PV1 strain isolated in Italy from cattle has been sequenced and compared to the other five N. caninum strains SSU rDNA sequences deposited in the data bases. The NC-PV1 strain sequence is identical to three published sequences. Minor differences, respectively four nucleotide bases and one nucleotide base, have been found when comparing the NC-PV1 sequence with two other available sequences of N. caninum. According to these results, the Neospora sp. NC-PV1 strain is assigned to the species N. caninum.

Increased nitric oxide formation has been shown to be involved in the hyperdynamic circulation of portal hypertension. It has been proposed that it could be related to stimulation of the inducible nitric oxide synthase by endotoxin. Therefore, the aim of the present study was to evaluate whether dexamethasone treatment, an inhibitor of the expression of the inducible enzyme, ameliorates the hyperdynamic circulation observed in cirrhotic rats due to chronic bile duct ligation. Systemic and splanchnic hemodynamic parameters were measured after administration of dexamethasone (3 mg/kg/day during 3 days, i.p.) or its vehicle. In cirrhotic rats dexamethasone treatment caused a mild but not significantly higher mean arterial pressure in comparison with vehicle while similar values of cardiac output, peripheral vascular resistance, portal blood flow and portal pressure were observed in both group of animals. A significant body weight loss over the three days of treatment was observed in rats receiving dexamethasone. In sham-operated rats, dexamethasone administration caused similar changes as observed in cirrhotic animals. Endotoxemia was observed in five of six cirrhotic rats while it was not detected in the control group. Our results show that dexamethasone administration does not modify systemic and splanchnic hemodynamic parameters in endotoxemic cirrhotic rats. This finding suggests that stimulation of the inducible nitric oxide synthase may not play a role in the increased nitric oxide production in portal hypertension.

The aim of this study was to investigate the role of portal hypertension determining the severity of bleeding in portal hypertensive rats. The effects of section of branches of the ileocolic vein were studied in sham-operated (SO), partial portal vein-ligated (PPVL), and common bile duct-ligated (CBDL) rats. The ensuing hemorrhage was compared with that caused by section of femoral vein, where the portal hypertensive factor is excluded. In PPVL rats, section of branches of increasing size (divided into fourth, third, second, and first order) resulted in increasingly severe bleeding (arterial pressure: / +/- 4%, / 6 +/- 12%, / /15 +/- 8%, and / 28 +/- 13%; P.005; hematocrit / 4 +/- 2%, / 6 +/- 1%, / 7 +/- 2%, and / 10 +/- 4%; P.005). Bleeding from first-order branches was mild in SO, moderate in PPVL, and severe in CBDL rats, as shown by increasing changes in arterial pressure (/ 3 +/- 3%, / 12 +/- 16% and, / 43 +/- 23%; P.01), hematocrit (/ 4 +/- 1%, / 12 +/- 2%, and / 32 +/- 19%; P.01), and mortality (0%, 0%, and 56%; P.001). Greater blood loss in CBDL rats was associated with higher portal pressure (16.6 +/- 2.7 vs. 13. 1 +/- 1.1 mm Hg in PPVL; P.01) and more prolonged bleeding time (70 +/- 4 vs. 35 +/- 3 seconds in PPVL; P.001). Vessels were similarly dilated in CBDL and PPVL (0.7 +/- 0.2 and 0.7 +/- 0.1 vs. 0.4 +/- 0.1 mm in SO; P.05). Section of femoral vein caused equal blood loss in SO, PPVL, and CBDL rats, assessed by falls in hematocrit (/ 8 +/- 2%, / 7 +/- 1%, / 8 +/- 1%, respectively; NS) and by the blood loss (3.6 +/- 0.7, 3.5 +/- 0.9, and 3.8 +/- 0.7 g; NS). The study shows that the degree of portal pressure elevation is a major determinant of the severity of portal hypertension-related bleeding in PPVL and CBDL rats.

The distribution and phylogeny of Wolbachia in filarial species suggests that these endosymbiotic bacteria may be important in the biology of their filarial hosts. An experiment to falsify this hypothesis would be to treat filarial worms with antibiotics which are active against intracellular bacteria. Indeed, it has already been shown that tetracycline treatment inhibits development in a model filarial species (Brugia pahangi) at different stages of the life cycle, in both mosquito and mammalian hosts. Here we discuss these previous data and present new results on the effect of tetracycline on the embryogenesis of the canine filaria Dirofilaria immitis.

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.

From small subunit ribosomal RNA gene sequences, a pair of PCR primers were designed to amplify a portion of this gene from five species of microsporidia. The amplified fragments encompass polymorphic restriction sites for the Hphl enzyme, resulting in different restriction patterns in the different species. We tested this identification method both on cultured microsporidia and on clinical samples. On cultured microsporidia the expected amplification bands were obtained even when DNA preparations from only ten spores were analysed. On clinical samples, identification of microsporidia was obtained from crude DNA preparations. This method allows for a rapid and easy diagnosis of human microsporidioses.

Physical exercise increases portal pressure (hepatic venous pressure gradient [HVPG]) in patients with cirrhosis. It is unknown if this deleterious effect is associated with changes in gastroesophageal collateral blood flow and if these can be prevented by propranolol administration. The aim of this study was to characterize the effects of propranolol on the splanchnic hemodynamic response to exercise in patients with cirrhosis. Twenty-three patients with cirrhosis and portal hypertension had hemodynamic measurements in baseline conditions, and during moderate cycling exercise (40 W) under double-blind propranolol or placebo administration. In patients receiving placebo, HVPG significantly increased during exercise (from 16.7 +/- 0.9 to 19.0 +/- 1.0 mm Hg; P.01), hepatic blood flow (HBF) decreased (-18% +/- 4%; P.01), while azygos blood flow (AzBF) was unchanged (4% +/- 12%; ns). In patients receiving propranolol, portal pressure did not increase during exercise, but decreased from 16.3 +/- 1.0 to 12.9 +/- 1.1 mm Hg (P.01). The lack of increase in HVPG in response to exercise in patients receiving propranolol may be related to a more pronounced decrease in HBF, as compared with patients receiving placebo, and to a blunted increase in cardiac output (CO). Moderate physical exercise adversely influences the hepatic hemodynamics in patients with cirrhosis, causing a significant increase in portal pressure. This is effectively prevented by propranolol pretreatment.

Little information exists on the effects of transjugular intrahepatic portosystemic shunts (TIPS) in the management of cirrhotic patients with hepatorenal syndrome (HRS). The current study was aimed to prospectively evaluate the effects of TIPS on renal function and vasoactive systems in patients with type I HRS. Glomerular filtration rate (GFR) (inulin clearance), renal plasma flow (RPF) (para-aminohippurate clearance), plasma renin activity (PRA), aldosterone (ALDO), norepinephrine (NE), and endothelin (ET) were determined in baseline conditions and at different time intervals after TIPS in 7 patients with type I HRS. TIPS induced a marked reduction of portal pressure gradient (PPG) (20 +/- 1 to 10 +/- 1 mm Hg; P.05). Renal function improved in 6 of the 7 patients. Serum creatinine and blood urea nitrogen (BUN) decreased from 5 +/- 0.8 and 109 +/- 7 to 1.8 +/- 0.4 mg/dL and 56 +/- 11 mg/dL, respectively (P.05 for both), and GFR and RPF increased from 9 +/- 4 and 103 +/- 33 to 27 +/- 7 mL/min and 233 +/- 40 mL/min, respectively (P.05 for both), 30 days after TIPS. These beneficial effects on renal function were associated with a significant (P.05) reduction of PRA (18 +/- 5 to 3 +/- 1 ng/mL x h), ALDO (279 +/- 58 to 99 +/- 56 ng/dL), and NE (1,257 +/- 187 to 612 +/- 197 pg/mL). ET did not change significantly (28 +/- 8 to 27 +/- 11 pg/mL). Mean survival was 4.7 +/- 2 months (0.3-17 months). Three patients remained alive more than 3 months after TIPS insertion. In conclusion, TIPS improves renal function and reduces the activity of the renin-angiotensin and sympathetic nervous systems in cirrhotic patients with type I HRS. Nevertheless, the efficacy of TIPS in the management of these patients should be confirmed in controlled investigations.

Cockroaches are known to harbour intracellular bacteria in specialised cells (mycetocytes, or bacteriocytes) of the fat body. In termites, mycetocyte bacteria have been observed only in Mastotermes darwiniensis. These symbionts are thought to have originated from a bacterium that infected an ancestor common to cockroaches and termites. Thus, loss of the infection should have occurred during evolution in all termite lineages, with the exception of that leading to M. darwiniensis. One might suspect that traces of the ancient infection may be present in some termites, in the form of non-mycetocyte intracellular bacteria (e.g. a small number of bacteria within normal cells). Indeed, circumstantial evidence for the presence of intracellular bacteria in two termite species has been reported. However, no data are available on the actual distribution of these bacteria in termites, or on their relationships with the mycetocyte bacteria of cockroaches and M. darwiniensis. In this paper we report results indicating that non-mycetocyte intracellular bacteria are widespread in termites. These results were obtained by electron microscopy on representatives of nine termite species. In addition, sequence analysis of the 16S rRNA genes indicated that the non-mycetocyte bacteria of termites belong to the wolbachia group of the alpha-2 subclass of the proteobacteria. These latter bacteria are not related to the mycetocyte bacteria of cockroaches and M. darwiniensis, which belong to the blattabacterium group of the flavobacteria-bacteroides. PCR analyses with primers specific for wolbachia or blattabacterium provided further support for the identification of the observed non-mycetocyte bacteria as members of the wolbachia group.

OBJECTIVE To evaluate the influences of blood viscosity changes, mediated by a haemorheological agent, on splanchnic and systemic haemodynamic parameters in rats with cirrhosis due to chronic bile duct ligation. METHODS Blood viscosity was measured using a cone-plate viscometer and whole blood filterability was determined by a filtration method. Cardiac index and portal venous inflow were measured using radioactive microspheres. Measurements were performed 30 min after double-blind administration of placebo or pentoxifylline (25 mg/kg, intravenously). RESULTS As compared with placebo, pentoxifylline-treated cirrhotic rats had a lower portal pressure (13.8 +/- 1.4 vs. 12.1 +/- 1.6 mmHg, P < 0.05) and blood viscosity at shear rates of 115/s (6.6 +/- 0.8 vs. 5.8 +/- 0.3 mPas, P < 0.05), associated with an improvement of whole blood filterability (45.0 +/- 12.9 vs. 20.0 = 3.9 s/ml, P < 0.01). Similar values of mean arterial pressure, cardiac index and portal venous inflow were observed in both groups. A significant correlation was found between portal pressure and blood viscosity at a shear rate of 115/s (r = 0.71, P < 0.01). CONCLUSION These results suggest that portal pressure can be modified by pentoxifylline in an experimental model of cirrhosis. These haemodynamic changes are associated with a lower blood viscosity and whole blood filterability. Pentoxifylline may be a new approach in the treatment of portal hypertension.

Colonic inflammation was produced in rats by chemotactic peptides acting on polymorphonuclear leucocytes. Instillation during one hour of formylated tripeptide: formylmethionyl-leucyl-phenylalanine (FMLP) and a tetrapeptide: alanine-glycine-sefine-glutamine (AGSG) into rat colon caused erosions and exulcerations. Neutrophils increased secondary to instillation, predominantly with FMLP, and mucus depletion was marked in the cecum. Chloride ion secretion and mucosal permeability were significatively greater in the colonic lumen with the chemotactic peptides. Histamine and serotonin concentration were greater in the colonic fluid in animals treated with the peptides. These observations could suggest that the presence of chemotactic peptides at the colonic lumen produce changes at the mucosal wall, that would participate in generation and perpetuation of the colonic inflammation.