Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

A cost-effective colourimetric assay for quantifying hydrogen peroxide in honey.

Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (H2O2) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of H2O2 in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that H2O2 production by these honeys varies greatly, with some samples producing negligible levels of H2O2. We assessed the effect of colour on the assay by measuring the recovery of spiked H2O2 from light and dark honey and from serially diluted dark corn syrup, and found the amount of H2O2 that could be detected was lower in dark corn syrup and darker honey samples.

Regulatory Architecture of the Neuronal Cacng2/Tarpγ2 Gene Promoter: Multiple Repressive Domains, a Polymorphic Regulatory Short Tandem Repeat, and Bidirectional Organization with Co-regulated lncRNAs.

CACNG2 (TARPγ2, Stargazin) is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts (Genetic Absence Epilepsy Rats, Strasbourg [GAERS] and non-epileptic control [NEC] rats). These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3' UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further (bidirectional) complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs.

Viscerosensory input drives angiotensin II type 1A receptor-expressing neurons in the solitary tract nucleus.

Homeostatic regulation of visceral organ function requires integrated processing of neural and neurohormonal sensory signals. The nucleus of the solitary tract (NTS) is the primary sensory nucleus for cranial visceral sensory afferents. Angiotensin II (ANG II) is known to modulate peripheral visceral reflexes, in part, by activating ANG II type 1A receptors (AT1AR) in the NTS. AT1AR-expressing NTS neurons occur throughout the NTS with a defined subnuclear distribution, and most of these neurons are depolarized by ANG II. In this study we determined whether AT1AR-expressing NTS neurons receive direct visceral sensory input, and whether this input is modulated by ANG II. Using AT1AR-GFP mice to make targeted whole cell recordings from AT1AR-expressing NTS neurons, we demonstrate that two-thirds (37 of 56) of AT1AR-expressing neurons receive direct excitatory, visceral sensory input. In half of the neurons tested (4 of 8) the excitatory visceral sensory input was significantly reduced by application of the transient receptor potential vallinoid type 1 receptor agonist, capsaicin, indicating AT1AR-expressing neurons can receive either C- or A-fiber-mediated input. Application of ANG II to a subset of second-order AT1AR-expressing neurons did not affect spontaneous, evoked, or asynchronous glutamate release from visceral sensory afferents. Thus it is unlikely that AT1AR-expressing viscerosensory neurons terminate on AT1AR-expressing NTS neurons. Our data suggest that ANG II is likely to modulate multiple visceral sensory modalities by altering the excitability of second-order AT1AR-expressing NTS neurons.

Novel Rbfox2 isoforms associated with alternative exon usage in rat cortex and suprachiasmatic nucleus.

Transcriptome diversity in adult neurons is partly mediated by RNA binding proteins (RBPs), including the RBFOX factors. RBFOX3/NeuN, a neuronal maturity marker, is strangely depleted in suprachiasmatic nucleus (SCN) neurons, and may be compensated by a change in Rbfox2 expression. In this study, we found no superficial changes in Rbfox2 expression in the SCN, but mRNA population analysis revealed a distinct SCN transcript profile that includes multiple novel Rbfox2 isoforms. Of eleven isoforms in SCN and cerebral cortex that exhibit exon variation across two protein domains, we found a 3-fold higher abundance of a novel ('-12-40') C-terminal domain (CTD)-variant in the SCN. This isoform embraces an alternative reading frame that imparts a 50% change in CTD protein sequence, and functional impairment of exon 7 exclusion activity in a RBFOX2-target, the L-type calcium channel gene, Cacna1c. We have also demonstrated functional correlates in SCN gene transcripts; inclusion of Cacna1c exon 7, and also exclusion of both NMDA receptor gene Grin1 exon 4, and Enah exon 12, all consistent with a change in SCN RBFOX activity. The demonstrated regional diversity of Rbfox2 in adult brain highlights the functional adaptability of this RBP, enabling neuronal specialization, and potentially responding to disease-related neuronal dysfunction.

Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT1AR) most widely expressed in adult neurons. Activation of the AT1R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT1R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT1AR-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT1AR promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT1AR-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT1AR-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT1AR-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT1AR-GFP neurons and TH-GFP neurons showed similar AT1AR-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT1AR-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing.

The unusual antibacterial activity of medical-grade Leptospermum honey: antibacterial spectrum, resistance and transcriptome analysis.

There is an urgent need for new, effective agents in topical wound care, and selected honeys show potential in this regard. Using a medical-grade honey, eight species of problematic wound pathogens, including those with high levels of innate or acquired antibiotic resistance, were killed by 4.0-14.8% honey, which is a concentration that can be maintained in the wound environment. Resistance to honey could not be induced under conditions that rapidly induced resistance to antibiotics. Escherichia coli macroarrays were used to determine the response of bacterial cells to a sub-lethal dose of honey. The pattern of gene expression differed to that reported for other antimicrobial agents, indicating that honey acts in a unique and multifactorial way; 78 (2%) genes were upregulated and 46 (1%) genes were downregulated more than two-fold upon exposure to the medical-grade honey. Most of the upregulated genes clustered into distinct functional regulatory groups, with many involved in stress responses, and the majority of downregulated genes encoded for products involved in protein synthesis. Taken together, these data indicate that honey is an effective topical antimicrobial agent that could help reduce some of the current pressures that are promoting antibiotic resistance.

Egr1 involvement in evening gene regulation by melatonin.

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE's in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE's contribute to evening gene expression profiles in this pituitary melatonin target tissue.

Compliance in the neck structures of the guinea pig spermatozoon, as indicated by rapid freezing and electron microscopy.

Electron microscopy has been used to investigate whether the transversely striated columns of the connecting piece in the neck region of guinea pig spermatozoa, undergo lengthening and shortening as a result of the forces generated during motility. Motile spermatozoa were subjected to near-instantaneous rapid freezing, followed by freeze-substitution fixation and epoxy embedment. Thin sections passing longitudinally through the striated columns revealed that the periodicity was indeed variable. The repeat period, taken to have an unstressed width of 60 nm, could be found extended to 75 nm in some specimens, and reduced to 54 nm in others. The estimates of the coefficients of variation were 6.6% for the width of the 'dense' band and 33.5% for the 'pale' band. The 'pale' band in the extended state showed longitudinal striae. Such variations in length, which - it is suggested - are physiological, and passively induced, would have functional implications for the flagellum - for both bend initiation and bend growth. Also, hypothetically, any mechanism that could increase the degree of compliance in these columns, such as perhaps phosphorylation of the constituent proteins, could permit the flagellum to develop the exaggerated bend angles and asymmetries of the 'hyperactivated' state.

The pregnancy-induced increase in baseline circulating growth hormone in rats is not induced by ghrelin.

The elevation in baseline circulating growth hormone (GH) that occurs in pregnant rats is thought to arise from increased pituitary GH secretion, but the underlying mechanism remains unclear. Distribution, Fourier and algorithmic analyses confirmed that the pregnancy-induced increase in circulating GH in 3-week pregnant rats was due to a 13-fold increase in baseline circulating GH (P < 0.01), without any significant alteration in the parameters of episodic secretion. Electron microscopy revealed that pregnancy resulted in a reduction in the proportion of mammosomatotrophs (P < 0.01) and an increase in type II lactotrophs (P < 0.05), without any significant change in the somatotroph population. However, the density of the secretory granules in somatotrophs from 3-week pregnant rats was reduced (P < 0.05), and their distribution markedly polarised; the granules being grouped nearest the vasculature. Pituitary GH content was not increased, but steady-state GH mRNA levels declined progressively during pregnancy (P < 0.05). In situ hybridisation revealed that pregnancy was accompanied by a suppression of GH-releasing hormone mRNA expression in the arcuate nuclei (P < 0.05) and enhanced somatostatin mRNA expression in the periventricular nuclei (P < 0.05), an expression pattern normally associated with increased GH feedback. Although gastric ghrelin mRNA expression was elevated by 50% in 3-week pregnant rats (P < 0.01), circulating ghrelin, GH-secretagogue receptor mRNA expression and the GH response to a bolus i.v. injection of exogenous ghrelin were all largely unaffected during pregnancy. Although trace amounts of 'pituitary' GH could be detected in the placenta with radioimmunoassay, significant GH-immunoreactivity could not be observed by immunohistochemistry, indicating that rat placenta itself does not produce 'pituitary' GH. Although not excluding the possibility that the pregnancy-associated elevation in baseline circulating GH could arise from alternative extra-pituitary sources (e.g. the ovary), our data indicate that this phenomenon is most likely to result from a direct alteration of somatotroph function.

Anterior thalamic lesions produce chronic and profuse transcriptional de-regulation in retrosplenial cortex: A model of retrosplenial hypoactivity and covert pathology.

Anterior thalamic lesions are thought to produce 'covert pathology' in retrosplenial cortex, but the causes are unknown. Microarray analyses tested the hypothesis that thalamic damage causes a chronic, hypo-function of metabolic and plasticity-related pathways (Experiment 1). Rats with unilateral, anterior thalamic lesions were exposed to a novel environment for 20 minutes, and granular retrosplenial tissue sampled from both hemispheres 30 minutes, 2h, or 8h later. Complementary statistical approaches (analyses of variance, predictive patterning and gene set enrichment analysis) revealed pervasive gene expression differences between retrosplenial cortex ipsilateral to the thalamic lesion and contralateral to the lesion. Selected gene differences were validated by QPCR, immunohistochemistry (Experiment 1), and in situ hybridisation (Experiment 2). Following thalamic lesions, the retrosplenial cortex undergoes profuse cellular transcriptome changes including lower relative levels of specific mRNAs involved in energy metabolism and neuronal plasticity. These changes in functional gene expression may be largely driven by decreases in the expression of multiple transcription factors, including brd8, c-fos, fra-2, klf5, nfix, nr4a1, smad3, smarcc2, and zfp9, with a much smaller number (nfat5, neuroD1, RXRγ) showing increases. These findings have implications for conditions such as diencephalic amnesia and Alzheimer's disease, where both anterior thalamic pathology and retrosplenial cortex hypometabolism are prominent.

Egr-1-d2EGFP transgenic rats identify transient populations of neurons and glial cells during postnatal brain development.

The inducible transcription factor Egr-1 has been extensively studied in the adult brain but potential roles during development are largely unexplored. Here we describe the analysis of a new transgenic rat model (egr-1 promoter driving a destabilized GFP molecule) that has provided novel information about the postnatal roles of Egr-1. We show that Egr-1 is more widely expressed in the neonatal brain than was previously appreciated, and is not restricted to neurons; it is expressed in glial cells in the postnatal neocortex and hippocampus. This pattern of expression has been revealed due to cellular filling by GFP, permitting co-localization with glial markers. The transgene/Egr-1 is also expressed in a novel population of cells associated with Cajal-Retzius-like neurons within the marginal zone of the postnatal neocortex. Both of these cellular populations are transient, being limited to the neonatal period, before Egr-1 expression becomes established in an adult-like pattern within neocortical neurons, CA1 hippocampus, and striatum. Another transient population of transgene/Egr-1 cells in the bed nucleus of the stria terminalis is maintained until pre-adolescence. The transient phenotype of these cells involves a low relative expression of the neuronal marker NeuN, perhaps indicating a failure to achieve full neuronal differentiation. Egr-1 is therefore present in a diverse range of cell-types during postnatal development. Transgenic expression of a destabilized fluorescent marker has permitted identification of these novel cell populations and will facilitate further analysis of the transcriptional mechanisms that underlie the specific functions and fate of these cells during postnatal brain development.

Protein/DNA interaction profiling reveals novel regulators of the pineal transcriptome.

The rat pineal gland transcriptome exhibits dynamic daily variation that reflects nocturnally restricted hormone production. Here we have used a protein/DNA interaction array to screen for day-night changes in DNA binding activity that are associated with transcriptional rhythms. Overall, 47 of 54 potential consensus binding sequence activities were detected, and of these, 29 (62%) were found to exhibit day:night differences in level. In addition to known, rhythmic pineal DNA binding activities (CRE and AP-1), multiple novel activities were observed including nocturnally elevated AP-2 consensus sequence binding activity. This array result was validated using conventional DNA binding assays, and we have also demonstrated AP-2beta and AP-2gamma proteins in the pineal gland, in addition to a nocturnally elevated AP-2alpha isoform. Our results have confirmed the presence of a complex assembly of transcriptional rhythms in the rat pineal gland and have provided details of more factors that contribute to this aspect of circadian neuroendocrine function.

Neural progenitor cells from postmortem adult human retina.

BACKGROUND: Given the presence of neural progenitor cells (NPC) in the retina of other species capable of differentiating into multiple neural components, the authors report the presence of NPC in the adult human retina. A resident population of NPC suggests that the retina may constitutively replace neurons, photoreceptors, and glia. METHODS: Adult human postmortem retinal explants and cell suspensions were used to generate cells in tissue culture that display the features of NPC. The phenotype of cells and differentiation into neurons was determined by immunocytochemistry. Dividing cells were labelled with 5-bromo-2-deoxyuridine (BrdU) and neurospheres were generated and passaged. RESULTS: Cells labelled with nestin, neurofilament M (NFM), rhodopsin, or glial fibrillary acidic protein (GFAP) grew out from explant cultures. BrdU labelling of these cells occurred only with basic fibroblast growth factor (FGF-2). Dissociated retina and pars plana generated primary neurospheres. From primary neurospheres, NPC were passaged to generate secondary neurospheres, neurons, photoreceptors, and glia. BrdU labelling identified dividing cells from neurospheres that differentiated to express NFM and rhodopsin. CONCLUSION: The adult human retina contains NPC and may have the potential to replace neurons and photoreceptors. This has implications for the pathogenesis and treatment of retinal disorders and degenerations, including glaucoma, and those disorders associated with retinal scarring.

Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct.

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

A comparison of the nature and abundance of microsatellites in 14 fungal genomes.

An overview of the character of microsatellites in 14 fungal genomes was obtained by analyzing databases containing complete or nearly complete genome sequences. Low GC content, rather than genome size, was the best predictor of high microsatellite density, although very long iterations of tandem repeats were less common in small genomes. Motif type correlated with %GC in that low-GC genomes were more likely to be dominated by A/T-rich motifs, and vice versa, although some exceptions were noted. The experimentally useful dinucleotide and trinucleotide arrays were analyzed in greater detail. Although these varied in sequence and length among fungal species, some that are likely to be universally useful were identified. This information will be useful for researchers wanting to identify the most useful microsatellites to analyze for the fungi included in this survey and provides a platform for choosing microsatellites to target in fungi that are not yet sequenced.

Nestin positive cells in adult human retina and in epiretinal membranes.

BACKGROUND/AIM: Nestin is an intermediate filament marker for neural progenitor cells. The authors aimed to identify nestin positive cells in adult human retina and within surgically removed epiretinal membranes. METHODS: Adult human retina and epiretinal membranes were studied. Tissue was fixed and processed for semithin sections or whole mount preparations for immunohistochemical detection of nestin and glial fibrillary acidic protein (GFAP) expression. RESULTS: Nestin positive cells are most prominent at the ora serrata, possess fibrillary processes, small amounts of perinuclear cytoplasm, and are arranged radially within or superficially on the retina. In the posterior retina, speckled cytoplasmic nestin staining is seen around the nuclei of neurons. In the peripapillary retina most of the cells in the retinal ganglion cell layer are nestin positive. These cells appear to represent nestin positive neurons. Speckled cells are also seen in the myelinated portion of the optic nerve. In epiretinal membranes patches of elongated nestin positive cells were found. These cells were also positive for GFAP. CONCLUSIONS: Some neurons and glia in the adult human retina are nestin positive. Their pattern in anterior retina suggests an analogy with the ciliary marginal zone found in many other species. The role of these cells in pathological responses to retinal disease is suggested by the presence of large numbers of ectopic nestin positive cells in epiretinal membranes. The authors hypothesise that nestin positive cells represent a population of progenitor cells from normal adult human retina that differentiate to make up retinal scar tissue.

Oestrogenic regulation of an egr-1 transgene in rat anterior pituitary.

The C(2)H(2) zinc-finger transcription factor Egr-1 has previously been shown to play an essential role within the endocrine system as a molecular determinant of LH beta-subunit synthesis in anterior pituitary gonadotrophs. The extent to which Egr-1 may be a dynamic mediator of changes in gonadotroph function during the oestrous cycle is currently unclear. We have recently produced a novel rat transgenic model of egr-1 gene function in which proximal regions of the rat egr-1 gene drive expression of a reporter gene. In the present study, we have investigated the expression and physiological regulation of our egr-1/d4 enhanced green fluorescent protein (EGFP) transgene in the female rat pituitary gland. In situ hybridization analysis has revealed anterior pituitary-specific expression that is limited to a sub-population of cells that includes immunohistochemically defined gonadotrophs. Expression of the transgene is up-regulated 5-fold following ovariectomy. The transgene also exhibits regulated expression during the oestrous cycle, mRNA levels being significantly raised on pro-oestrus. Using an explant culture system, we have also demonstrated a direct stimulatory effect of 17beta-oestradiol on anterior pituitary transgene and egr-1 expression. The acute response of egr-1 to an oestrogenic stimulus is attenuated by the MEK (MAPK kinase) inhibitor U0126, and is accompanied by increased levels of phospho-p44/42 MAPK protein, suggesting regulation of egr-1 through a MAPK-linked pathway in the pituitary. These findings provide further evidence of cyclical endocrine regulation of egr-1 in the rat, demonstrate that proximal sequences of the egr-1 gene mediate endocrine-regulated expression, and indicate a novel pathway through which pituitary transcriptional responses to oestrogen may be mediated.

Lipopolysaccharide/interferon-gamma and not transforming growth factor beta inhibits retinal microglial migration from retinal explant.

BACKGROUND: /aims: The retina possesses a rich network of CD45(+) positive myeloid derived cells that both surround inner retinal vessels and lie within the retina (microglia). Microglia migrate and accumulate in response to neurodegeneration and inflammation. Although microglia express MHC class II, their role remains undefined. The aims of this study are to investigate changes in human microglia phenotype, migration, and activation status in response to pro-inflammatory and anti-inflammatory stimulation. METHODS: Donor eyes were obtained from the Bristol Eye Bank with consent and whole retina was removed. 5 mm retinal trephines were cultured in glucose enhanced RPMI on cell culture insert membranes for up to 72 hours. The effects of lipopolysaccharide/interferon-gamma (LPS/IFNgamma) and transforming growth factor beta inhibits (TGFbeta) stimulation, alone or in combination, on migration, phenotype, and activation status (iNOS expression) of microglia were studied using immunofluorescence and cytokine analysis by ELISA. RESULTS: CD45(+) MHC class II(+) retinal microglia were observed within retinal explants, and in culture microglia readily migrated, adhered to culture membrane, downregulated MHC class II expression, and produced interleukin 12 (IL-12) and tumour necrosis factor alpha (TNFalpha). Following LPS/IFNgamma stimulation microglia remained MHC class II(-) iNOS(-), and secreted IL-10. Migration was suppressed and this could be reversed by neutralising IL-10 activity. TGFbeta did not affect ability of microglia to migrate and was unable to reverse LPS/IFNgamma induced suppression. CONCLUSIONS: Microglia readily migrate from retinal explants and are subsequently MHC class II(-), iNOS(-), and generate IL-12. In response to LPS/IFNgamma microglia produce IL-10, which inhibits both their migration and activation. TGFbeta was unable to counter LPS/IFNgamma effects. The data infer that microglia respond coordinately, dependent upon initial cytokine stimulation, but paradoxically respond to classic myeloid activation signals.

Clonal reproduction and limited dispersal in an environmental population of Cryptococcus neoformans var gattii isolates from Australia.

Cryptococcus neoformans var. gattii is a causative agent of cryptococcosis and is thought to have a specific ecological association with a number of Eucalyptus species in Australia. However, the role that the tree plays in the life cycle of the fungus and the nature of the infectious propagule are not well understood. This study set out to examine whether sexual recombination is occurring in a natural population of C. neoformans var. gattii and whether the fungus disseminates between colonized trees. Thirty C. neoformans var. gattii isolates, consisting of both the alpha and a mating types, were collected from 13 Eucalyptus camaldulensis trees growing along a riverbank in Renmark, South Australia. The genetic diversity within the population was studied by using amplified fragment length polymorphism fingerprinting, and each isolate was assigned a unique multilocus genotype. Population genetic analyses of the multilocus data found no evidence of genetic exchange between members of the population, indicating a clonal population structure. Canonical variate analysis was then used to study the relationship between isolates from different colonized trees. Isolates from individual trees were strongly correlated, and it appeared that dispersal between trees was not occurring to any appreciable extent. These results suggest that the eucalypt may not be the primary niche for C. neoformans var. gattii but that the decaying wood present in hollows on these trees may provide a favorable substrate for extensive clonal propagation of the yeast cells.