Suppression of autoimmune retinal disease by lovastatin does not require Th2 cytokine induction

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In an acute mouse model of autoimmune retinal disease, we demonstrate that treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, lovastatin, suppresses clinical ocular pathology, retinal vascular leakage, and leukocytic infiltration into the retina. Efficacy was reversed by coadministration of mevalonolactone, the downstream product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not by squalene, which is distal to isoprenoid pyrophosphate metabolites within the cholesterol biosynthetic pathway. Lovastatin treatment (20 mg/kg/day i.p.) over 7 days, which resulted in plasma lovastatin hydroxyacid concentrations of 0.098 +/- 0.03 microM, did not induce splenocyte Th2 cytokine production but did cause a small reduction in Ag-induced T cell proliferation and a decrease in the production of IFN-gamma and IL-10. Thus, it is possible to dissociate the therapeutic effect of statins in experimental autoimmune uveitic mice from their activity on the Th1/Th2 balance. Statins inhibit isoprenoid pyrophosphate synthesis, precursors required for the prenylation and posttranslational activation of Rho GTPase, a key molecule in the endothelial ICAM-1-mediated pathway that facilitates lymphocyte migration. Consistent with inhibition of leukocyte infiltration in vivo, lovastatin treatment of retinal endothelial cell monolayers in vitro leads to inhibition of lymphocyte transmigration, which may, in part, account for drug efficacy. Unlike lovastatin, atorvastatin treatment showed little efficacy in retinal inflammatory disease despite showing significant clinical benefit in experimental autoimmune encephalomyelitis. These data highlight the potential differential activity of statins in different inflammatory conditions and their possible therapeutic use for the treatment of human posterior uveitis.     

P-Rex1 regulates neutrophil function

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.     

Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases

The p47 GTPases are essential for interferon-gamma-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-gamma-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-gamma-mediated T. gondii growth restriction in mouse astrocytes.     

The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh.

Potato mop-top virus (PMTV) was best detected in field soils by air-drying them for more than a week before remoistening and growing seedlings of Nicotiana tabacum or N. debneyi for a 6–10 week period. Infection of N. tabacum was assessed by inoculating sap from roots and shoots to Chenopodium amaranticolor. Similar inoculations from N. debneyi were far less convenient for detecting PMTV than recording leaf symptoms, but slightly more efficient. Air-dry soil retained PMTV infectivity for 9 months, when passed through a 50 μ sieve or when diluted with 10³ but not 10⁴ parts of steamed soil. Tobacco seedlings were not infected when their roots were steeped in PMTV-containing tobacco sap. Infective soils contained Spongospora subterranea, spore balls of which resisted air-drying for more than a year and passed a 50 μ sieve. Roots of susceptible seedlings were infected with PMTV when exposed to spore balls of S. subterranea taken from powdery scabs on PMTV-infected potato tubers, or to suspensions obtained by steeping, in nutrient solution, roots infected with virus-carrying cultures of S. subterranea. Plants in several families were hosts of S. subterranea, but probabilities of infection when exposed to spore balls differed greatly between families and only species of Solanaceae were good hosts. The ten species infected with PMTV when grown in infective soil or when exposed to spore balls of S. subterranea taken from PMTV-infected potato tubers are all members of this family. PMTV seems to be carried internally in S. subterranea spore balls and survived in them for at least a year. PMTV was transmitted by S. subterranea to Arran Pilot potato, causing yellow blotches in some leaves and spraing in many tubers. However, when newly infected with PMTV in the field, not all Arran Pilot tubers developed spraing. Also, although many spraing-affected or symptomless but PMTV-infected tubers carried PMTV-containing spore balls of S. subterranea, powdery scabs were rarely found near the centres of the rings of primary spraing. PMTV became established in virus-free soil when PMTV-infected tubers carrying S. subterranea were planted as ‘seed’ but not when virus-free tubers bearing powdery scabs were used. 5. subterranea seems the main, and possibly the only, vector of PMTV in the soils examined. S. subterranea did not transmit potato aucuba mosaic virus from potato to N. debneyi or Capsicum annuum.     

<Emphasis Type="Italic">Thyridariella</Emphasis>, a novel marine fungal genus from India: morphological characterization and phylogeny inferred from multigene DNA sequence analyses

A novel saprobic fungal genus, Thyridariella (Thyridariaceae), is herein described to include Thyridariella mangrovei, the type species and T. mahakoshae spp. nov. Both species were collected as saprobes on decaying wood of Avicennia marina, a common mangrove species found near Kaveri River Delta, Tamil Nadu, on the east coast of India. Thyridariella is diagnosed by having an exclusive combination of characters, such as ascomata with ostiolar necks thickened laterally, hyaline, and centrally constricted muriform ascospores with a single longitudinal septum in each segment and surrounded by a mucilaginous sheath. These characters demarcate these taxa from morphologically similar genera such as Halojulella and Julella. In addition, the new genus also differs from Parathyridaria and Thyridaria in having hyaline, muriform ascospores with distinct mucilaginous sheaths. The monophyly of Thyridariella is well supported in the phylogenetic analysis based on a concatenated dataset from two proteins and three nuclear gene regions. The phylogeny also depicts a sister group relationship of our new genus to Parathyridaria and Thyridaria and hence confirms its position within Thyridariaceae.     

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HIyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA