On the key role of secondary lymphoid organs in antiviral immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11(-)/-) mutant mice.

The role of the spleen and of other organized secondary lymphoid organs for the induction of protective antiviral immune responses was evaluated in orphan homeobox gene 11 knockout mice (Hox11(-/-)) lacking the spleen, and in homozygous alymphoplastic mutant mice (aly/aly) possessing a structurally altered spleen but lacking lymph nodes and Peyer's patches. Absence of the spleen had no major effects on the immune response, other than delaying the antibody response by 1-2 d. In aly/aly mice, the thymus-independent IgM response against vesicular stomatitis virus (VSV) was delayed and reduced, whereas the T-dependent switch to the protective IgG was absent. Therefore, aly/aly mice were highly susceptible to VSV infection. Since aly/aly spleen cells yielded neutralizing IgM and IgG after adoptive transfer into recipients with normally structured secondary lymphoid organs, these data suggest that the structural defect was mainly responsible for inefficient T-B cooperation. Although aly/aly mice generated detectable, but reduced, CTL responses after infection with vaccinia virus (VV) and lymphocytic choriomeningitis virus (LCMV), the elimination of these viruses was either delayed (VV) or virtually impossible (LCMV); irrespective of the dose or the route of infection, aly/aly mice developed life-long LCMV persistence. These results document the critical role of organized secondary lymphoid organs in the induction of naive T and B cells. These structures also provide the basis for cooperative interactions between antigen-presenting cells, T cells, and B cells, which are a prerequisite for recovery from primary virus infections via skin or via blood.

Sequential expression of macrophage anti-microbial/inflammatory and wound healing markers following innate, alternative and classical activation.

The present study examines the temporal dynamics of macrophage activation marker expression in response to variations in stimulation. We demonstrate that markers can be categorized as 'early' (expressed most abundantly at 6 h post-stimulation) or 'late' (expressed at 24 h post-stimulation). Thus nos2 and p40 (IL-12/IL-23) are early markers of innate and classical activation, while dectin-1 and mrc-1 are early markers and fizz1 (found in inflammatory zone-1) and ym1 are late markers of alternative activation. Furthermore, argI is a late marker of both innate and alternative activation. The ability of interferon (IFN)-gamma to alter these activation markers was studied at both the protein level and gene level. As reported previously, IFN-gamma was able to drive macrophages towards the classical phenotype by enhancing nos2 gene expression and enzyme activity and p40 (IL-12/IL-23) gene expression in lipopolysaccharide (LPS)-stimulated macrophages. IFN-gamma antagonized alternative macrophage activation, as evident by reduced expression of dectin-1, mrc-1, fizz1 and ym1 mRNA transcripts. In addition, IFN-gamma antagonized arginase activity irrespective of whether macrophages were activated innately or alternatively. Our data explain some apparent contradictions in the literature, demonstrate temporal plasticity in macrophage activation states and define for the first time 'early' and 'late' markers associated with anti-microbial/inflammatory and wound healing responses, respectively.

Correlation of changes in gravity, elevation, and strain in southern california.

Measurements made once or twice a year from 1977 through 1982 show large correlated changes in gravity, elevation, and strain in several southern California networks. Precise gravity surveys indicate changes of as much as 25 microgals between surveys 6 months apart. Repeated surveys show that annual elevation changes as large as 100 millimeters occur along baselines 40 to 100 kilometers long. Laser-ranging surveys reveal coherent changes in areal strain of 1 to 2 parts per million occurred over much of southern California during 1978 and 1979. Although the precision of these measuring systems has been questioned, the rather good agreement among them suggests that the observed changes reflect true crustal deformation.

Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia.

Molecular cloning of the t(10;14)(q24;q11) recurrent breakpoint of T cell acute lymphoblastic leukemia has demonstrated a transcript for the candidate gene TCL3. Characterization of this gene from chromosome segment 10q24 revealed it to be a new homeobox, HOX11. The HOX11 homeodomain is most similar to that of the murine gene Hlx and possesses a markedly glycine-rich variable region and an acidic carboxyl terminus. HOX11, while expressed in liver, was not detected in normal thymus or T cells. This lineage-restricted homeobox gene is deregulated upon translocation into the T cell receptor locus where it may act as an oncogene.

Neuropsychiatric disease and Toxoplasma gondii infection.

Toxoplasma gondii infects approximately 30% of the world's population, but causes overt clinical symptoms in only a small proportion of people. In recent years, the ability of the parasite to manipulate the behaviour of infected mice and rats and alter personality attributes of humans has been reported. Furthermore, a number of studies have now suggested T. gondii infection as a risk factor for the development of schizophrenia and depression in humans. As T. gondii forms cysts that are located in various anatomical sites including the brain during a chronic infection, it is well placed anatomically to mediate these effects directly. The T. gondii genome is known to contain 2 aromatic amino acid hydroxylases that potentially could directly affect dopamine and/or serotonin biosynthesis. However, stimulation of the immune response has also recently been associated with mood and behavioural alterations in humans, and compounds designed to alter mood, such as fluoxetine, have been demonstrated to alter aspects of immune function. Herein, the evidence for T.-gondii-induced behavioural changes relevant to schizophrenia and depression is reviewed. Potential mechanisms responsible for these changes in behaviour including the role of tryptophan metabolism and the hypothalamic-pituitary-adrenal axis are discussed.

The Toxoplasma gondii plastid replication and repair enzyme complex, PREX.

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii.

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.

Toward effective school-based substance abuse prevention "breaking the cycle" programme in Antigua and Barbuda.

The "Breaking the Cycle" programme, based on the Project Charlie programme, was developed for Antigua and Barbuda third grade students and was implemented in 2001. Aspects of the programme are compared with aspects recently proven effective in randomized studies in developed countries. The "Breaking the Cycle" programme includes life-skills training, teaches decision making skills, includes peer resistance training, uses trained teachers, interactive teaching methods, effective content and delivery, targets students prior to onset of drug use, teaches drug harm, teaches community values and is culturally sensitive, all aspects of successful programmes overseas. The cost of about $7 US per student would suggests cost-benefit effectiveness compared with overseas programmes. The "Breaking the Cycle" school-based drug and alcohol use prevention programme includes most aspects of evidence-based successful programmes overseas, appears cost effective and could serve as a model for programmes in the Caribbean region.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

A brief consideration of the SOS inducing signal.

SOS functions are induced in E. coli by treatments that damage cellular DNA or interrupt its synthesis. The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor. We discuss the development of the inducing signal in the cell.

The effect of ionizing radiation on intraocular lenses.

BACKGROUND: The native crystalline lens is the principal shield against ultraviolet radiation (UV), damage to the human retina. Every year in the United States, more than one million patients undergo removal of the natural lens in the course of cataract surgery (phakectomy), at which time an intraocular lens (IOL) is placed in the lens capsule. The IOL thenceforth serves as the principal barrier to ultraviolet radiation over the life of the implant, potentially for decades. The synthetic organic molecules of which IOLs are composed offer little UV protection unless ultraviolet-absorbing chromophores are incorporated into the lens material during manufacture. However, chromophores are alkenes potentially subject to radiolytic degradation. It is unknown whether ionizing radiation at clinical doses (e.g., to the brain or in the head-and-neck region) affects the UV-absorbing capacity of chromophore-bearing IOLs and consequently exposes the retina to potentially chronic UV damage. In addition, the polymers of which IOLs are composed are themselves subject to radiation damage, which theoretically might result in optical distortion in the visible light range. OBJECTIVE: To determine whether megavoltage photon ionizing radiation alters the absorption spectra of ultraviolet-shielding polymethylmethacrylate (PMMA) and organopolysiloxane (silicone) intraocular lenses (IOLs) in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), and low-end near-infrared (700 nm < lambda < or = 830 nm) ranges. DESIGN: Prospective, nonrandomized trial of dose-paired IOL cohorts. METHODS: Fourteen IOLs, seven of PMMA (Chiron 6842B) and seven of silicone (IOLAB L141U), were paired and examined for absorption spectra in 1-nm intervals over the range lambda = 280-830 nm on a Cary 400 deuterium and quartz halogen source-lamp UV/visible spectrophotometer before and after undergoing megavoltage ionizing irradiation to doses of 2, 5, 10, 20, 40, 60, and 100 Gray, respectively. Because of artifactual aberrations inherent in analyzing convex lenses on a conventional flat-plate spectrophotometer, post-irradiation absorption spectra were subsequently reanalyzed on a Cary 300 spectrophotometer outfitted with a Labsphere Diffused Reflectance Accessory (DRA-CA-30-I) incorporating a Spectralon-coated integrating sphere. MAIN OUTCOME MEASURES: Primary: Changes in UV absorbance after irradiation. Secondary: Changes in visible and low-end near-infrared absorbance after irradiation. RESULTS: Photon ionizing radiation in the 2-Gy to 100-Gy range produced no detectable alterations in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), or low-end near-infrared (700 nm < lambda < or = 830 nm) absorption spectra of any of the lenses irradiated. However, silicone IOLs as a group revealed peak post-irradiation UV absorption at a shorter wavelength than did PMMA IOLs, with marginally greater UV transmission at the uppermost extreme of the UV spectrum (lambda = 384.5-400 nm). CONCLUSIONS: At clinically relevant doses used in radiation therapy, megavoltage photon ionizing radiation produces no significant alterations in the absorption spectra of PMMA and silicone IOLs over the range lambda = 280- 830 nm. These findings indicate that, even at supraclinical doses, the UV-absorbing capacity of chromophore-bearing PMMA and silicone IOLs remains unimpaired. It is not clear whether the lower UV peak of silicone lenses represents a radiation effect or a peculiarity of the chromophore used in the lenses tested.

Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa.

Current treatments for diseases caused by apicomplexan and trypanosomatid parasites are inadequate due to toxicity, the development of drug resistance and an inability to eliminate all life cycle stages of these parasites from the host. New therapeutics agents are urgently required. It has recently been demonstrated that type II fatty acid biosynthesis occurs in the plastid of Plasmodium falciparum and Toxoplasma gondii and inhibitors of this pathway such as triclosan and thiolactomycin restrict their growth. Furthermore, Trypanosoma brucei has recently been demonstrated to use type II fatty acid biosynthesis for myristate synthesis and to be susceptible to thiolactomycin. As this pathway is absent from mammals, it may provide an excellent target for novel antimicrobial agents to combat these diverse parasites. Leishmania and Trypanosoma parasites produce ergosterol-related sterols by a biosynthetic pathway similar to that operating in pathogenic fungi and their growth is susceptible to sterol biosynthesis inhibitors. Thus, inhibition of squalene 2,3-epoxidase by terbinafine, 14alpha-methylsterol 14-demethylase by azole and triazole compounds and delta(24)-sterol methyl transferase by azasterols all cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytoxic consequences. However, Leishmania parasites can survive with greatly altered sterol profiles induced by continuous treatment with low concentrations of some inhibitors and they also have some ability to utilise and metabolise host sterol. These properties may permit the parasites to evade treatment with sterol biosynthesis inhibitors in some clinical situations and need to be taken into account in the design of future drugs.

Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii.

Oocysts of Cryptosporidium parvum were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) similar to those previously described for Eimeria tenella and Toxoplasma gondii. PPi-PFK of C. parvum displayed simple hyperbolic kinetics with respect to its substrate fructose 6-phosphate and was not affected by fructose 2,6-bisphosphate, the major allosteric activator of most ATP-PFKs. Inorganic pyrophosphatase was not detectable in any of the three parasites. T. gondii tachyzoites and C. parvum cysts both contained a pyruvate kinase (PK) specific for ADP rather than PPi/AMP. The PK of T. gondii was similar to that of E. tenella in that it displayed strong positive cooperativity with respect to its substrate phosphoenolpyruvate and was heterotropically activated by glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. PK of C. parvum showed no evidence of allosteric properties. The results suggest that the three coccidia are similar in depending heavily on anaerobic energy production via glycolysis but that the mechanisms for regulating glycolysis are not common to all species.

Optical zone diameters for photorefractive corneal surgery.

PURPOSE: To examine the physiological optics of photorefractive corneal surgery and to study the effect on glare production of the optical zone diameter. METHODS: An optical analysis computer program was used to generate rays that define the edge of the optical zone for any given pupil size and glare-free field. RESULTS: The optical zone diameter must be based on the postoperative corneal curvature because the determines magnification of the pupil. The minimal optical zone diameter of uniform optical power was determined both for myopic and hyperopic surgery and for two values of anterior chamber depth. CONCLUSIONS: Optical zone diameters must be at least as large as the entrance pupil diameter to preclude glare at the fovea, and larger than the entrance pupil to preclude parafoveal glare.

The role of IL-4 in adult acquired and congenital toxoplasmosis.

The course of Toxoplasma gondii infection was studied in IL-4-deficient mice from three genetic backgrounds and their wild-type counterparts following peroral inoculation of tissue cysts. Survival rates were significantly reduced in disease-susceptible C57 BL/6 mice and F1 (C57BL/6 x 129Sv) mice deficient in IL-4 compared with wild-type controls. In contrast, this difference was not observed in T. gondii-resistant BALB/c mice. However, brain tissue cyst burdens in IL-4-deficient mice were either equivalent to (C57BL/6 and BALB/c mice) or significantly less (B6/129 mice) than similarly infected wild-type mice. Thus strain-specific differences in the course of T. gondii were demonstrated in the absence of IL-4. The course of T. gondii infection was also compared between B6/129 IL-4-deficient mice and their wild-type counterparts following peroral challenge with 20 tissue cysts on day 12 of pregnancy. Age-matched non-pregnant IL-4-/- and IL-4+/+ mice were also infected to assess the role of IL-4 on T. gondii infection during pregnancy. Disease phenotypes, as measured by mortality, were reversed if infections were initiated during pregnancy compared with non-pregnant infection. Thus significant mortality occurred immediately post partum in IL-4+/+ mothers, while all IL-4-/- mothers survived. Cyst burdens 28 days p.i. were significantly lower in IL-4-/- mothers than IL-4+/+ mothers and both IL-4-/- and IL-4+/+ non-pregnant mice. Congenital disease transmission as measured by foetal death or vertical disease transmission was independent of the presence or absence of IL-4. These studies demonstrate a role for IL-4 in pregnancy-induced immunosuppression and the associated increased susceptibility to T. gondii infection.

HLA-class II genes modify outcome of Toxoplasma gondii infection.

Associations between Human Leukocyte Antigen (HLA) (i.e. human major histocompatibility complex [MHC]) genes and susceptibility to infections and inflammatory processes have been described, but causal relationships have not been proven. We characterized effects of HLA-DQ alleles on outcome of congenital toxoplasma infection and found that among Caucasians, the DQ3 gene frequency was significantly higher in infected infants with hydrocephalus (0.783) than infected infants without hydrocephalus (0.444) or published normal controls (0.487). We then developed a novel animal model to definitively determine the effect of these HLA DQ molecules on the severity of toxoplasmosis. Human MHC-Class II transgenes reduced parasite burden and necrosis in brains of mice infected with Toxoplasma gondii. Consistent with the observed association between DQ3 and hydrocephalus in human infants, in the murine model the DQ3(DQ8; DQB1*0302) gene protected less than DQ1 (DQ6; DQB1*0601). Our findings definitively prove a cause and effect relationship between human MHC genes and resistance to infection, provide novel means to characterise human immune responses that are protective or pathogenic in infections, and are important for vaccine development.

A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation.

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

A comparison of topical diclofenac with prednisolone for postcataract inflammation.

OBJECTIVE: To compare diclofenac sodium with prednisolone acetate for the control of postoperative inflammation after cataract surgery. DESIGN: Fifty-two patients undergoing phacoemulsification with posterior chamber intraocular lens implantation were randomly assigned to receive either 0.1% diclofenac eye drops or 1% prednisolone eye drops as their postoperative anti-inflammatory medication. The patients were examined 1 day (baseline), 1 week, and 1 month after surgery. Postoperative inflammation was evaluated subjectively by slit-lamp assessment of cell and flare and objectively by measurement of cell and flare with a laser cell and flare meter. RESULTS: At each visit, there was no statistically significant difference in postoperative inflammation either by slit-lamp assessment or with the laser cell and flare meter for the two treatment groups. CONCLUSION: In the dosage used, diclofenac was as effective an anti-inflammatory agent for uncomplicated post-cataract inflammation as prednisolone.