Morphology, ultrastructure, and molecular phylogeny of the ciliate Sonderia vorax with insights into the systematics of order Plagiopylida

BACKGROUND: Ciliates of the family Sonderiidae are common members of the eukaryotic communities in various anoxic environments. They host both ecto- and endosymbiotic prokaryotes (the latter associated with hydrogenosomes) and possess peculiar morpho-ultrastructural features, whose functions and homologies are not known. Their phylogenetic relationships with other ciliates are not completely resolved and the available literature, especially concerning electron microscopy and molecular studies, is quite scarce. RESULTS: Sonderia vorax Kahl, 1928 is redescribed from an oxygen-deficient, brackish-water pond along the Ligurian Sea coastlines of Italy. Data on morphology, morphometry, and ultrastructure are reported. S. vorax is ovoid-ellipsoid in shape, dorsoventrally flattened, 130 x 69 μm (mean in vivo); it shows an almost spherical macronucleus, and one relatively large micronucleus. The ventral kinetom has a “secant system” including fronto-ventral and fronto-lateral kineties. A distinctive layer of bacteria laying between kineties covers the ciliate surface. Two types of extrusomes and hydrogenosomes-endosymbiotic bacteria assemblages are present in the cytoplasm. The phylogeny based on 18S rRNA gene sequences places S. vorax among Plagiopylida; Sonderiidae clusters with Plagiopylidae, although lower-level relationships remain uncertain. The studied population is fixed as neotype and the ciliate is established as type species of the genus, currently lacking. CONCLUSIONS: This is the first description of a representative of Sonderiidae performed with both morphological and molecular data. To sum up, many previous hypotheses on this interesting, poorly known taxon are confirmed but confusion and contradictory data are as well highlighted

Candidatus Megaira polyxenophila' gen. nov., sp. nov.: Considerations on Evolutionary History, Host Range and Shift of Early Divergent Rickettsiae

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria

MHV-68 producing mIFN␣1 is severely attenuated in vivo and effectively protects mice against challenge with wt MHV-68

Corrigendum Corrigendum to "MHV-68 producing mIFN␣1 is severely attenuated in vivo and effectively protects mice against challenge with wt MHV-68" [Vaccine 29 (2011) In this study, we focused on the effects of interferon-␣ (IFN-␣) on both the lytic and latent phase of MHV-68 infection, as exerted by the constitutive release of IFN-␣1 by a clone of MHV-68 genetically modified to produce this cytokine (MHV-68mIFN␣1). Although the MHV-68mIFN␣1 recombinant virus exhibited in vitro replication features indistinguishable from those of the wild type MHV-68, its pathological properties were severely attenuated in vivo in immunocompetent mice and not in mice rendered genetically unresponsive to type I IFN, suggesting that a stronger immune response was primed in the presence of the cytokine. Notably, MHV-68mIFN␣1 attenuation did not result in a reduced level of longterm spleen latency establishment. These results prompted us to evaluate the efficacy of MHV-68mIFN␣1 in a prophylactic vaccination regimen aimed at inhibiting the symptoms of acute virus infection and the establishment of long-term latency after MHV-68 challenge. Our results show that mice vaccinated with MHV-68mIFN␣1, administered as a live-attenuated or partially inactivated (by Psoralen and UV treatment) vaccine, were protected against the challenge with wt MHV-68 from all phases of infection. The ability of MHV-68mIFN␣1 to produce IFN-␣ at the site of the infection, thus efficiently stimulating the immune system in case of virus reactivation from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the previously reported latency-deficient clones

Interferon-α-Conditioned Human Monocytes Combine a Th1-Orienting Attitude with the Induction of Autologous Th17 Responses: Role of IL-23 and IL-12

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α

"Studio preliminare delle associazioni simbiotiche fra protisti ciliati e procarioti in un ambiente salmastro costiero: caratterizzazione morfologica e molecolare"

Il lavoro ha come obiettivo l’identificazione e la caratterizzazione morfologica e molecolare di protozoi ciliati mesopsammici di uno specifico ambiente salmastro confinato e dei loro eventuali batteri simbionti. L’attenzione è stata focalizzata sui ciliati quantitativamente dominanti nell’ambiente e che, ad uno screening preliminare, risultavano associati a batteri ecto- od endosimbionti. Tali caratterizzazioni rappresentano un punto di partenza indispensabile per possibili studi futuri di tipo ecologico volti a determinare la variabilità spaziale e temporale delle associazioni simbiotiche all’interno della comunità in esame. Sono stati effettuati tentativi di coltivabilità su ciascuna specie di ciliati presa in esame, ed ove possibile sono state allestite colture monoclonali. La caratterizzazione morfologica è stata effettuata mediante osservazioni in vivo (binoculare, D.I.C.), colorazioni cellulari (Feulgen, impregnazione argentica). osservazione al S.E.M. La microscopia a trasmissione (T.E.M.) ha consentito una descrizione ultrastrutturale dei batteri simbionti. Come marcatori molecolari sono stati impiegati il 18S rDNA per i ciliati ospiti ed il 16S rDNA per i batteri simbionti, caratterizzati tramite PCR e sequenziamento diretto (ove possibile) o PCR, clonaggio e sequenziamento dei cloni rappresentativi. Sulla base delle sequenze del 16S rRNA desunte, sono state fatte sintetizzare specifiche sonde oligonucleotidiche marcate con fluorocromi al fine di poter associare con certezza, tramite ibridazione in situ, una sequenza di 16S rRNA ad uno specifico batterio simbionte. Sei tipi di batteri simbionti sono stati ritrovati in quattro diversi ciliati: Euplotes parawoodruffi (Spirotrichea, Hypotrichia), che ne contiene due, Frontonia salmastra (Oligohymenophorea, Peniculia), anch’esso contenente due simbionti, Sonderia cfr. vorax (Plagiopylea, Plagiopylida), Pseudomicrothorax dubius (Nassophorea, Microthoracida). Sono state messe a punto specifiche tecniche per il mantenimento di colture monoclonali di F. salmastra e di popolazioni di Sonderia cfr. vorax e P. dubius. Gli organismi sono stati caratterizzati morfologicamente e molecolarmente. La scarsità di letteratura e l’ambiguità di interpretazione di alcune caratteristiche morfologiche non ha consentito di poter giungere in tutti i casi alla determinazione della specie. Sono state ottenute le sequenze del 16S rDNA del simbionte principale di E. parawoodruffi, un β-proteobatterio vicino al gen. Polynucleobacter, del simbionte di F. salmastra., un β-proteobatterio vicino al gen. Holospora, e del simbionte di P. dubius., un β-proteobatterio vicino al gen. Rickettsia; tali risultati sono stati confermati da esperimenti di ibridazione in situ utilizzando sonde genere-specifiche. La caratterizzazione dei simbionti di Sonderia sp. e del simbionte secondario di E. parawoodruffi, appartenenti alla cl. Gammaproteobacteria, è tuttora in corso

Facultative Associations between Protists and Rickettsial Symbionts: Morphological and Molecular Characterization Functional Implications, and Insights on Emerging Intracellular Parasites

Rickettsiae and Rickettsia-like organisms (RLOs) are Gram-negative prokaryotes known as obligate intracellular parasites of a variety of eukaryotic hosts, including humans. Recent studies reported the presence of these organisms also in protists, although no specific researches were accomplished. This work was aimed to investigate the diffusion of RLOs among protists, and to get insights about their host range and host shift capabilities. The first goal was achieved by systematical screenings of natural populations of ciliated protists from different habitats trough the “full-cycle rRNA approach” (individuation of symbionts through FISH, 16S rDNA characterization and use of specifically designed oligonucleotide probes for in situ detection). Descriptions of symbionts were refined through electron microscopy techniques and phylogenetic analysis. Six novel species of RLOs were identified in five different ciliates, namely Pseudomicrothorax dubius (Nassophorea), Spirostomum minus (Heterotrichea), Euplotes octocarinatus (Spirotrichea), Paramecium multimicronucleatum (Oligohymenophorea) and Diophrys oligothrix (Spirotrichea), which harbours two different symbionts. Phylogenetic inferences supported their belonging to the family Rickettsiaceae as candidate novel genera, except for symbionts of S. minus and P. multimicronucleatum that branches within the genus Rickettsia. These latter also share motile flagella, which were never reported among Rickettsiaceae. Analysis revealed the belonging of symbionts of E. octocarinatus and of one of that of D. oligothrix to the same novel species, which was further retrieved in three unrelated ciliate hosts. The finding accounted for possible host shifts through horizontal transfer. Both inter- and intraspecific transmissibility were tested using the other symbiont of D. oligotrix as infector and uninfected conspecific and allospecific (E. harpa) strains as hosts. Experiments evidenced its capability to undergo horizontal host shifts at both levels. Preliminary results indicate that some RLOs (symbionts of P. multimicronucleatum and one of that of D. oligothrix) can survive in association with lab-cultured metazoan cell lines. The present work contributed to reveal an unexpected, intriguing phylogenetic and morphological diversity among ciliate-borne Rickettsiaceae. The frequency of occurrence of symbioses between these latter and protists, together with the retrieval of the same RLO species from different hosts and the capability of horizontal shift (in case of symbionts of D. oligothrix) account for their polyxenic and likely opportunistic nature. Given the parasitic/pathogenic nature of the majority of known Rickettsiaceae, ciliated protists could effectively play the role of both natural reservoir and vectors for potentially hazardous rickettsial pathogens

Biotherapy of Cancer: Break the Barriers to Foster Translation oF knowledge

Biotherapy of cancer holds great promise for its potential to lead to the identification of novel, selective, and effective treatments against cancer. However, the clinical development of biopharmaceuticals and biotherapy products is hampered by several and diverse barriers. Herein, we will address some of the critical issues identified both at the national and European level as the major obstacles for the translation of knowledge into clinical applications in the field of biotherapy and immunotherapy of cancer. We will also illustrate specific initiatives undertaken both in Europe and in Italy in order to support the translational and clinical research and that are expected to have a favorable impact on the process of clinical development of novel and more effective therapeutic interventions against cancer. The contents of this article are directly referred to the event “International Clinical Trials’ Day on Biotherapy of Cancer” organized in the context of the OECI Genoa 2008, with the sponsorship of Alliance Against Cancer (ACC) and the Istituto Superiore di Sanità (ISS, the Italian National Institute of Health), and under the auspices of the European Clinical Research Infrastructures Network (ECRIN). This event sees the active participation of representatives of the ISS and of the Italian Network for Tumor Biotherapy, both involved in a project recently funded by ACC and aimed at the promotion of clinical research in the field of cancer biotherapy and immunotherapy, through the creation of a national network of clinical cancer research centers and GMP facilities dedicated to the production of biological drugs and advanced medicinal products. </jats:p

“Candidatus Hafkinia simulans” gen. nov., sp. nov., a Novel Holospora-Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host

We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, BCandidatus Hafkinia simulans^, was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus ofHolospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra’s phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature

“Candidatus Anadelfobacter veles” and “Candidatus Cyrtobacter comes,” Two New Rickettsiales Species Hosted by the Protist Ciliate Euplotes harpa (Ciliophora, Spirotrichea)▿

The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “Candidatus Midichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “Candidatus Midichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the “Candidatus Midichloria” clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “Candidatus Midichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “Candidatus Midichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “Candidatus Anadelfobacter veles” and “Candidatus Cyrtobacter comes.