Variabilita' ed evoluzione dei prioni: Mutabilita' in vitro

I prioni, privi di acidi nucleici, esistono come ceppi e possono mutare, in particolare quando attraversano una barriera di specie. Numerosi studi convergono sulla conclusione che le caratteristiche ceppo-specifiche siano inscritte nella conformazione della PrPSc, con la variabilità di ceppo associata a varianti conformazionali della PrPSc. In questo studio ci siamo avvalsi del PMCA, tecnica che riproduce in vitro molti aspetti della biologia dei prioni, per mettere a punto condizioni sperimentali di replicazione che permettessero di osservare fenomeni di mutazione e selezione, onde investigare i meccanismi molecolari e di popolazione alla base della mutabilità dei prioni. In condizioni di replicazione eterologa, che mima la trasmissione tra diverse specie, è stato inizialmente possibile identificare un mutante difettivo della scrapie, caratterizzato da una diversa conformazione della PrPSc e capace di replicare in vitro ma non più in vivo. Le condizioni in cui tale mutante è emerso hanno permesso di sviluppare ulteriori ipotesi di lavoro, basate sul concetto della quasi-specie. Impartendo diversi regimi di replicazione e seguendo l’evoluzione di due ceppi, è stato possibile evidenziare fenomeni di mutazione anche in condizioni di replicazione omologa, in assenza di forti pressioni selettive. In entrambi i ceppi sono emerse varianti conformazionali di PrPSc durante passaggi replicativi ad ampia popolazione, mentre le popolazioni sottoposte a ripetuti colli di bottiglia hanno mostrato un rapido declino del tasso di replicazione. Sono stati infine investigati l’efficacia e il potenziale mutageno di molecole anti-prioniche, ottenendo importanti risultati preliminari sull’efficacia di molecole che legano la PrPC. Questi risultati evidenziano come la mutabilità sia una caratteristica intrinseca dei prioni e supportano l’idea che i prioni siano molto variabili, similmente alle quasi-specie virali, e perciò adattabili e proni a fenomeni di mutazione e selezione. Tali conclusioni hanno impatto su problematiche sanitarie quali lo studio del potenziale zoonotico e i fenomeni di farmaco-resistenza dei prioni.

A model of cerebral aspergillosis in non-immunosuppressed nursing rats

Central nervous system aspergillosis is an often fatal complication of invasive Aspergillus infection. Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop novel therapeutic approaches. This study presents a model of central nervous system aspergillosis that mimics important aspects of human disease. Eleven-day-old non-immunosuppressed male Wistar rats were infected by an intracisternal injection of 10 mul of a conidial suspension of Aspergillus fumigatus. An inoculum of 7.18 log(10) colony-forming units (CFU) consistently produced cerebral infection and resulted in death of all animals (n = 25) within 3-10 days. Median survival time was 3 days. Histomorphologically, all animals developed intracerebral abscesses (2-26 per brain) containing abundant fungal hyphae and neutrophils. Fungal culture of cortical homogenates yielded maximal growth on day 3 after infection (5.4 log(10) CFU/g, n = 15) that declined over time. Galactomannan concentrations in cortical homogenates, assessed as an index for hyphal burden, peaked on days 3-5. Fungal infection spread to peripheral organs in 83% of animals. Fungal burden in lung, liver, spleen and kidney was two orders of magnitude lower than in the brain. The successful establishment of a model of cerebral aspergillosis in a non-immunosuppressed host provides the opportunity to investigate mechanisms of disease and to develop novel treatment regimens for this commonly fatal infection.

Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Topical caspofungin for treatment of keratitis caused by Candida albicans in a rabbit model

Candida albicans is the most frequent cause of fungal keratitis in temperate regions. Caspofungin has potent activity against Candida spp. in a variety of clinical settings. Little is known, however, about its activity against fungal keratitis. We compared the efficacy of topical caspofungin with that of topical amphotericin B (AMB) in a rabbit model of experimental keratomycosis. Keratitis was induced with a standardized inoculum of Candida albicans (SC 5314) placed on the debrided cornea. Twenty-four hours after infection, animals were randomly assigned to treatment with 0.15% caspofungin, 0.5% caspofungin, 0.15% AMB, and a saline control (n = 12 rabbits in each group). For the first 12 h, treatment was repeated every 30 min and, after a 12-h pause, was resumed at hourly intervals for another 12 h. The animals were examined and killed 12 h after administration of the last dose. Treatment effects were evaluated by clinical assessment, fungal culture, and histopathology. Drug treatment significantly reduced corneal fungal recovery from 3.78 log10 CFU in saline-treated animals to 2.97, 1.76, and 1.18 log10 CFU in animals treated with 0.15% caspofungin, 0.5% caspofungin, and 0.15% AMB, respectively. By histopathology, the mean hyphal density was significantly lower in the corneas of treated animals than in those of the controls; there was no difference in hyphal densities between the different treatment groups. The depth of corneal invasion was not significantly reduced by the antifungal treatments. By clinical assessment, keratitis progressed in animals treated with saline, whereas disease progression was inhibited by all drug treatment regimens. In our rabbit model, 0.5% caspofungin was as effective as 0.15% AMB for the topical treatment of Candida keratitis. The potential clinical efficacy of caspofungin awaits further investigation.

Brain injury in experimental neonatal meningitis due to group B streptococci

We have characterized the pattern of brain injury in a rat model of meningitis caused by group B streptococci (GBS). Infant rats (12-14 days old; n = 69) were infected intracisternally with 10 microliters of GBS (log10(2.3) to 4.5 colony-forming units). Twenty hours later, illness was assessed clinically and cerebrospinal fluid was cultured. Animals were either immediately euthanized for brain histopathology or treated with antibiotics and examined later. Early GBS meningitis was characterized clinically by severe obtundation and seizures, and histopathologically by acute inflammation in the subarachnoid space and ventricles, a vasculopathy characterized by vascular engorgement, and neuronal injury that was most prominent in the cortex and often followed a vascular pattern. Incidence of seizures, vasculopathy and neuronal injury correlated with the inoculum size (p < 0.01). Early injury was almost completely prevented by treatment with dexamethasone. Within days after meningitis, injured areas became well demarcated and showed new cellular infiltrates. Thirty days post-infection, brain weights of infected animals treated with antibiotics were decreased compared to uninfected controls (1.39 +/- 0.18 vs 1.64 +/- 0.1 g; p < 0.05). Thus, GBS meningitis in this model caused extensive cortical neuronal injury resembling severe neonatal meningitis in humans.

Experimental pneumococcal meningitis causes central nervous system pathology without inducing the 72-kd heat shock protein

We examined whether experimental pneumococcal meningitis induced the 72-kd heat shock protein (HSP72), a sensitive marker of neuronal stress in other models of central nervous system (CNS) injury. Brain injury was characterized by vasculitis, cerebritis, and abscess formation in the cortex of infected animals. The extent of these changes correlated with the size of the inoculum (P less than 0.003) and with pathophysiologic parameters of disease severity, i.e., cerebrospinal fluid (CSF) lactate (r = 0.61, P less than 0.0001) and CSF glucose concentrations (r = -0.55, P less than 0.0001). Despite the presence of numerous cortical regions having morphologic evidence of injury, HSP72 was not detected in most animals. When present, only rare neurons were HSP72 positive. Western blot analysis of brain samples confirmed the paucity of HSP72 induction. The lack of neuronal HSP72 expression in this model suggests that at least some of the events leading to neuronal injury in meningitis are unique, when compared with CNS diseases associated with HSP72 induction.

IL-21 restricts virus-driven Treg cell expansion in chronic LCMV infection

Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune homeostasis and tolerance. During viral infections, Treg cells can limit the immunopathology resulting from excessive inflammation, yet potentially inhibit effective antiviral T cell responses and promote virus persistence. We report here that the fast-replicating LCMV strain Docile triggers a massive expansion of the Treg population that directly correlates with the size of the virus inoculum and its tendency to establish a chronic, persistent infection. This Treg cell proliferation was greatly enhanced in IL-21R-/- mice and depletion of Treg cells partially rescued defective CD8+ T cell cytokine responses and improved viral clearance in some but not all organs. Notably, IL-21 inhibited Treg cell expansion in a cell intrinsic manner. Moreover, experimental augmentation of Treg cells driven by injection of IL-2/anti-IL-2 immune complexes drastically impaired the functionality of the antiviral T cell response and impeded virus clearance. As a consequence, mice became highly susceptible to chronic infection following exposure to low virus doses. These findings reveal virus-driven Treg cell proliferation as potential evasion strategy that facilitates T cell exhaustion and virus persistence. Furthermore, they suggest that besides its primary function as a direct survival signal for antiviral CD8+ T cells during chronic infections, IL-21 may also indirectly promote CD8+ T cell poly-functionality by restricting the suppressive activity of infection-induced Treg cells.

Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates.

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of

Does proximity to conspecific adults influence the establishment of ectomycorrhizal trees in rain forest?

Three ectomycorrhizal legume trees, Microberlinia bisulcata, Tetraberlinia bifoliolata and T. moreliana, form discrete groves in the southern part of Korup National Park, in southwest Cameroon and contribute c. 45–70% of stand basal area locally in a matrix of otherwise species-rich arbuscular mycorrhizal forest. A transplant experiment was performed to assess the importance of ectomycorrhizal infection associated with proximity to parents in seedling establishment of the grove-forming species. Nonectomycorrhizal seedlings of the three species were transplanted into plots of two forest types, one of high (HEM, within-grove) and one of very low (LEM, outside the grove) abundance of all three species as adult trees. For two species (T. moreliana and M. bisulcata) there was no difference in survival over 16 months, but for the third (T. bifoliolata) survival was best in HEM forest, and correlated with the basal area of adult trees of ectomycorrhizal species. Only one species (T. moreliana) increased in biomass over the experimental period; the others declined. There was no effect of forest type on overall growth of any species, but the survivors of two (T. moreliana and M. bisulcata) had heavier stems in the HEM forest. Differences in survival and growth of transplants between the three species were in accord with the ecology of the species as inferred from the frequency distributions of adult tree size in the forest. Seedlings became infected with ectomycorrhizas in both forest types; where there was a difference in extent of infection (T. moreliana) this was not related to survival or growth; and where there was a difference in survival (T. bifoliolata) this was not related to extent of infection. These results confirm that mycorrhizal inoculum associated with conspecific adults is neither a prerequisite nor a guarantee of seedling establishment, but indicates that in some circumstances there might be benefits of being close to parents. Further research is required to unravel the complexities of ectomycorrhizal community structure in this spatially and temporally heterogeneous forest, and to clarify the extent to which the various hosts share ectomycorrhizal partners.

Enteroaggregative Escherichia coli as a cause of acute diarrhea

Enteroaggregative Escherichia coli (EAEC) are considered an important emerging enteric and food-borne pathogen. The groups importantly affected by EAEC include international travelers, children in the developing world, and patients with HIV infection. EAEC does not commonly cause diarrheal illness in all hosts. ^ The reasons for the observed clinical variation in EAEC infection are multifactorial and are dependant on the pathogen, the inoculum ingested and the host susceptibility. A major obstacle in identifying the mechanism of pathogenesis for EAEC is the heterogeneity in virulence of strains. No EAEC virulence gene is consistently present in all diarrheagenic strains. However, a recent report suggests that a package of plasmid borne and chromosomal virulence factors are under the control of the described transcriptional activator aggR. Although the exact inoculum required for EAEC diarrheal illness is not known, a volunteer study has shown that oral ingestion of 10 10 cfu of virulent EAEC elicited diarrhea. Ongoing studies are being conducted to better define the exact infectious dose. There are also host factors associated with increased susceptibility of persons to diarrheal illness with EAEC. ^ The following three manuscripts: (1) review EAEC as an emerging enteric pathogen; (2) identify EAEC as a cause of acute diarrhea among different subpopulations worldwide; (3) identify virulence characteristics and the molecular epidemiology of EAEC isolates among travelers with diarrheal illness and describe the pathogenesis of EAEC infection. ^

Oidiopsis taurica : nuevo patógeno de pimiento (Capsicum annum L.) y ají (Capsicum frutescens L.) en Mendoza (Argentina)

En febrero de 1998 se observó en cultivos de pimiento y ají de Mendoza (Argentina) muerte anticipada de plantas con severos daños en la producción. La magnitud del problema motivó una prospección en los principales oasis de la provincia, a fin de describir la sintomatología, conocer su dispersión, estimar los daños causados y recolectar material para realizar estudios en laboratorio tendientes a confirmar su etiología. Se constató que la afección se encontraba distribuida en todos los oasis del centro y norte. La sintomatología en hojas se caracterizó por la presencia en la cara adaxial de manchas amarillentas, de forma circular de 15 a 20 mm de diámetro, con puntuaciones necróticas, con tendencia a presentarse en anillos concéntricos. En la cara abaxial, generalmente las lesiones eran acompañadas de un micelio blanco tenue. Posteriormente siguió una severa defoliación. La producción disminuyó notablemente y además en pimiento los frutos fueron afectados por escaldaduras de sol y perdieron su valor comercial. A partir de aislamientos de pimiento y ají, mantenidos en plantas de brincos (Impatiens balsamina L.), se inocularon plantas sanas y se logró reproducir la enfermedad en condiciones de invernáculo.

Physalis viscosa : un nuevo hospedero de Leveillula taurica en Argentina

Leveillula taurica (Lév.) Salm. es un hongo patógeno que se encuentra actualmente en expansión en nuestro país y en particular en la provincia de Mendoza. Physalis viscosa (camambú) es una especie muy difundida en Argentina. La misma es principalmente una maleza que crece espontáneamente en numerosos cultivos de interés económico y en sus alrededores. El hallazgo de L. taurica sobre una especie tan difundida fue el motivo de su estudio. Los síntomas característicos observados fueron parches cuneiformes amarillos en la cara adaxial de las hojas y en correspondencia con los mismos, en la abaxial se presentó un afelpado blanquecino formado por las fructificaciones asexuales del patógeno. El estudio etiológico realizado mediante la histopatología de los órganos afectados y la morfometría del patógeno confirmó la presencia del hongo parásito obligado Leveillula taurica (Lév.) Salm., en su forma anamórfica Oidiopsis taurica Sal. [= Oidiopsis sicula (Scal)]. La enfermedad en el camambú se ha difundido en forma importante en la provincia de Mendoza desde su primer hallazgo en el departamento de Tupungato en 1996. Se encuentra presente desde diciembre hasta mayo. Este prolongado período puede representar una importante fuente de inóculo para cultivos susceptibles al patógeno. La presente constituye la primera cita, no sólo a nivel nacional sino también mundial, de L. taurica sobre Physalis viscosa.

Estudio de una modificación del medio de Fowell para la esporulación de levaduras

Se describe la influencia del agregado de agua de levaduras autorizadas al medio de Fowell, sobre la esporulación de levaduras del género Saccharomyces. Los resultados indican, en general, un aumento en el número de ascos en casi todos los casos. No obstante, la respuesta de las distintas cepas a esta modificación del Fowell es distinta, lo cual indica que esas cepas tienen requerimientos diferentes para la producción de esporas. Algunas cepas han sido ensayadas mediante siembras leves y densas en el medio de Fowell, y en líneas generales puede decirse que un aumento de la densidad de células en ese medio da resultados parecidos al agregado de agua de levaduras, probablemente por que los productos excretados por las células que se lisan alcanzan una concentración que favorece la esporulación del resto de las células. Los resultados permiten aconsejar el uso de inóculos densos sobre el medio de Fowell para obtener los mejores resultados en la esporulación.

Diversity and identification of heterotrophic bacteria from Antarctic Rocks of the McMurdo Dry Valleys (Ross Desert)

Diversity of endolithic Dry Valley rock microorganisms was studied by evaluating the presence of morphotypes in enrichments. Storage of rock samples for 16 h over dry ice affected the diversity of endolithic organisms, especially that of algae and fungi. Diversity in various samples depended on rock location and exposure, on the rock type, and to some extent on the pH of the pulverized rock samples. In most cases sandstone contained more morphotypes than dolerite or granite. Presence of many different phototrophs resulted in greater diversity of the heterotrophs in the enrichments. Samples from Linnaeus Terrace and Battleship Promontory had higher morphotype (MT) numbers than those from more exposed sites such as New Mountain, University Valley, Dais, or Mt. Fleming. Beacon sandstone (13 samples) from Linnaeus Terrace varied greatly with respect to MT numbers, although the pH values ranged only from 4.2-5.3. The highest MT number of 24 per sample was obtained from the upper surface of a flat boulder tilted to the North. Only two MT's were found in a hard sandstone sample from the wind-exposed and more shaded east side of the Terrace. 15 sandstone samples from Battleship Promontory contained more diverse populations: there occurred a total of 131 different MT's in these samples as compared to only 68 in Linnaeus Terrace samples. Cysts of colorless flagellates were found in some Battleship Promontory samples; rnost samples were populated with a wealth of different cyanobacteria. Studies on the distribution of actinomycete morphotypes in Linnaeus Terrace sandstone revealed great differences between individual boulders. Identification tests and lipid analyses made with representative strains of the isolated 1500 pure cultures led to genus names such as Caulobacter, Blastobacter, Hyphomicrobium, Micrococcus, Arthrobacter, Brevibacterium, Corynebacterium, Bifidobacterium, Mycobacterium, Nocardia (Amycolata), Micromonospora, Streptomyces, Blastococcus, and Deinococcus. Our data demonstrate the great diversity of Antarctic endolithic microbial populations.