Epigenetic differences in the innate response after immune stimulation during zebrafish sex differentiation.

Infections are able to trigger epigenetic modifications; however, epigenetic-mediating infections in the immune system in fish is currently unavailable. Within this purpose, zebrafish were immune-stimulated with three lipopolysaccharides (LPS) during sex differentiation. Methylation patterns of three immune genes were studied by a candidate gene approach together with gene expression analysis, and in adulthood, sex ratios were determined. It was shown that the entrance of LPS was through the gills and accumulated in the pronephros. Significant hypomethylation levels of CASP9 and a significant CpG site for IL1ß after Pseudomonas aeruginosa LPS exposure were found. No methylation difference was observed for TNFα. Gene expression and correlation data differed among studied genes. Sex ratios showed a feminization in dose and LPS strain-dependent manner. Here, it is provided epigenetic regulatory mechanisms derived by innate response and the first evidence of possible epigenetic interactions between the immune and reproductive systems.

Orientational order induced by a polymer network in the isotropic phase of liquid crystal.

We studied the paranematic ordering induced by a polymer network in the isotropic phase of a liquid crystal (LC) that occurs in polymer-stabilized cells with bend configuration of the LC director (π cells) fabricated via photopolymerization of photoreactive monomer RM 82 added in small concentrations (3-5 wt %) to a nematic LC [4-cyano-4'-pentylbiphenyl (5CB)] when low voltage was applied across the cell. The polymer network formed in the nematic phase of the LC consists of fine fibrils that are aligned along the LC director and thus mirror the bend deformation of the LC at the time of polymerization. When heated to temperatures above the nematic-to-isotropic (N-I) phase transition such highly ordered polymer network anchors LC molecules providing ordering of the LC around the fibrils which results in unusually high optical retardation of the cell, R_{cell}. We present a theoretical model that relates R_{cell} to the degree of order of the fibrils, the anchoring energy of the LC molecules on the surface of the polymer fibrils, and the fibril radius r_{0}. Fitting of the experimental R_{cell}(T) curves with the developed model reveals correlation of r_{0} with the nematic correlation length ξ_{0} which characterizes penetration of the nematic order in the isotropic phase of the LC. Accepting ξ_{0} as a material constant of about 1 nm leads to a very small radius of the fibrils, r_{0}∼1nm, which is also supported by other reported experimental data. High optical retardation and fast electro-optical response of the cells at the temperatures deep into the isotropic phase point toward the enhancement of the polymer-induced paranematic order by a well-oriented layer of LC molecules that are absorbed on the surface of fibrils. Application of high voltage at the isotropic phase temperatures results in high variations of the optical retardation of the cells. Characteristic on and off response times were about 10-100 µs, independent of the cell gap. Combination of large voltage-driven changes of the optical retardation occurring in the low-viscosity isotropic state with switching times that are at least two orders of magnitude shorter than the typical relaxation times of the cells operating in the nematic phase make such polymer-stabilized π cells very promising for application in fast electro-optical switches and light modulators.

Immune genes, IL1ß and Casp9, show sexual dimorphic methylation patterns in zebrafish gonads.

There is crosstalk between the immune and reproductive systems in which sexual dimorphism is a common pattern in vertebrates. In recent years, epigenetics has emerged as a way to study the molecular mechanisms involved in gonadal development, those responsible for integrating environmental information that contribute to assigning a specific sexual phenotype (either an ovary or a testis). The knowledge of epigenetic mechanisms in certain molecular processes allows the development of epigenetic markers. In fish gonads, the existence of reproduction-immune system interactions is known, although the epigenetic mechanisms involved are far from clear. Here, we used the zebrafish (Danio rerio) as a model to study the DNA methylation patterns in gonads of two well-known innate immune genes: IL1ß and Casp9. DNA methylation levels were studied by a candidate gene approach at single nucleotide resolution and gene expression analyses were also carried out. Results showed that there was clear sexual dimorphism in the DNA methylation levels of the two immune genes studied, being significantly higher in the testes when compared to the ovaries. In summary, and although further research is needed, this paper presents sexual dimorphic methylation patterns of two immune-related genes, thus sex-biased differences in methylation profiles should considered when analyzing immune responses in fish. Data showed here can help to develop epimarkers with forthcoming applications in livestock and fish farming production, for example, in immune fish diseases or sexual control programs as epigenetic molecular tools to predict environmental pressure in the gonads.

Trees, fungi and bacteria: tripartite metatranscriptomics of a root microbiome responding to soil contamination.

BACKGROUND: One method for rejuvenating land polluted with anthropogenic contaminants is through phytoremediation, the reclamation of land through the cultivation of specific crops. The capacity for phytoremediation crops, such as Salix spp., to tolerate and even flourish in contaminated soils relies on a highly complex and predominantly cryptic interacting community of microbial life. METHODS: Here, Illumina HiSeq 2500 sequencing and de novo transcriptome assembly were used to observe gene expression in washed Salix purpurea cv. 'Fish Creek' roots from trees pot grown in petroleum hydrocarbon-contaminated or non-contaminated soil. All 189,849 assembled contigs were annotated without a priori assumption as to sequence origin and differential expression was assessed. RESULTS: The 839 contigs differentially expressed (DE) and annotated from S. purpurea revealed substantial increases in transcripts encoding abiotic stress response equipment, such as glutathione S-transferases, in roots of contaminated trees as well as the hallmarks of fungal interaction, such as SWEET2 (Sugars Will Eventually Be Exported Transporter). A total of 8252 DE transcripts were fungal in origin, with contamination conditions resulting in a community shift from Ascomycota to Basidiomycota genera. In response to contamination, 1745 Basidiomycota transcripts increased in abundance (the majority uniquely expressed in contaminated soil) including major monosaccharide transporter MST1, primary cell wall and lamella CAZy enzymes, and an ectomycorrhiza-upregulated exo-ß-1,3-glucanase (GH5). Additionally, 639 DE polycistronic transcripts from an uncharacterised Enterobacteriaceae species were uniformly in higher abundance in contamination conditions and comprised a wide spectrum of genes cryptic under laboratory conditions but considered putatively involved in eukaryotic interaction, biofilm formation and dioxygenase hydrocarbon degradation. CONCLUSIONS: Fungal gene expression, representing the majority of contigs assembled, suggests out-competition of white rot Ascomycota genera (dominated by Pyronema), a sometimes ectomycorrhizal (ECM) Ascomycota (Tuber) and ECM Basidiomycota (Hebeloma) by a poorly characterised putative ECM Basidiomycota due to contamination. Root and fungal expression involved transcripts encoding carbohydrate/amino acid (C/N) dialogue whereas bacterial gene expression included the apparatus necessary for biofilm interaction and direct reduction of contamination stress, a potential bacterial currency for a role in tripartite mutualism. Unmistakable within the metatranscriptome is the degree to which the landscape of rhizospheric biology, particularly the important but predominantly uncharacterised fungal genetics, is yet to be discovered.

Ultra-light extensometer for the assessment of the mechanical properties of the human skin in vivo.

BACKGROUND/PURPOSE: This paper aims to present an ultra-light extensometer device dedicated to the mechanical characterization of the human skin in vivo. METHODS: The device developed was conceived to be non-invasive, to work without any stand and to perform various uniaxial tensile tests with either effort or displacement control. We also use specific guarding tabs to make in vivo extension tests analogous to traction tests. RESULTS: Force-displacement curves are derived from the data provided by the device's sensors. The latter are converted into stress-strain curves thanks to complementary measurements of the skin thickness. We present typical experimental data and results that demonstrate the device ability to built stress-strain curves characteristic of the human skin behavior. An additional imaging unit records a sequence of images of the solicited skin area for further calculations of the displacement fields by digital image correlation. CONCLUSION: The analysis of the displacement and deformation fields validates the guarding tab efficiency and the capacity of the device to characterize the mechanical behavior of the human skin in vivo.

[Hypercalcemia revealing sarcoidosis in a child]. / Hypercalcémie révélant une sarcoïdose chez un enfant.

Sarcoidosis is a systemic granulomatosis disease with a classic triad of presentation: typical clinical and radiological signs, presence of tuberculoid granuloma without caseum in histopathology, and exclusion of other causes of granulomatosis, especially tuberculosis. Sarcoidosis is rare in the general population, and even more so in children. In the literature, few cases of sarcoidosis associated with hypercalcemia have been reported in children. We report here the case of a 14-year-old boy with bone marrow and lymph node sarcoidosis suspected, based on poor general condition with hypercalcemia. The patient was treated with hydration, diuretics, and bisphosphonates with good results. We also performed a literature review of published cases of hypercalcemia since 1990 with a diagnosis of sarcoidosis in children, comparing 23 cases (including ours) on clinical and epidemiological, biological, imaging, and histopathological diagnosis. When hypercalcemia is present in the initial clinical presentation, the diagnosis of sarcoidosis is usually made in younger children. Classical locations of the lesions, including lung, skin, and lymph nodes, were highly suggestive of sarcoidosis. Corticosteroids are commonly used to treat sarcoidosis lesions including hypercalcemia. In conclusion, sarcoidosis in children remains difficult to diagnose because the disease is rare and it is common to have nonspecific symptoms in the clinical picture (with diagnosis delayed between 3 months and several years). The classic triad is not always present. Sarcoidosis should be systematically considered and investigated in case of hypercalcemia of unknown cause in children.

Pattern of Dermatophytes in Patients Attend in Mymensingh Medical College Hospital.

Dermatophytosis is a common fungal disease which involves the keratinized tissue. This is an attempt to observe the spectrum of dermatophytes among the clinically suspected cases of dermatophytosis attending the outpatient department of Dermatology and Venereology, Mymensingh Medical College Hospital, Mymensingh during the period of July 2013 to December 2014. Two hundred thirty (230) clinically suspected cases of dermatophytosis were subjected in this study. Sixty three cases (27.39%) were positive for fungus in direct microscopy while 53(23.04%) were culture positive. Among the clinical types tinea unguium was the most common followed by tinea corporis. Dermatophytosis was more common in the age group of 21-30 years. The male to female ratio was 1.53:1. Trichophyton rubrum 44(83.04%) was found common etiological dermatophyte species followed by Trichophyton mentagrophytes 5(9.43%) and Epidermophyton floccosum 4(7.55%).

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve.

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.

Sex differences in the GSK3ß-mediated survival of adherent leukemic progenitors.

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3ß (GSK3ß) by serine 9-dephosphorylation. Strikingly, GSK3ß-mediated survival was restricted to leukemic progenitors from female patients. GSK3ß inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)ß(1)-integrin engagement, was specifically upregulated and controlled GSK3ß activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3ß-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3ß as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina.

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.

Retinal degeneration modulates intracellular localization of CDC42 in photoreceptors.

PURPOSE: Rho GTPases such as RAS-related C3 botulinum substrate 1 (RAC1) and cell division cycle 42 homolog (S. cerevisiae; CDC42) have been linked to cellular processes including movement, development, and apoptosis. Recently, RAC1 has been shown to be a pro-apoptotic factor in the retina during light-induced photoreceptor degeneration. Here, we analyzed the role of CDC42 in the degenerating retina. METHODS: Photoreceptor degeneration was studied in a mouse model for autosomal dominant retinitis pigmentosa (VPP) with or without a rod-specific knockdown of Cdc42, as well as in wild-type and Cdc42 knockdown mice after light exposure. Gene and protein expression were analyzed by real-time PCR, western blotting, and immunofluorescence. Retinal morphology and function were assessed by light microscopy and electroretinography, respectively. RESULTS: CDC42 accumulated in the perinuclear region of terminal deoxynucleotidyl transferase dUTP nick end labeling-negative photoreceptors during retinal degeneration induced by excessive light exposure and in the rd1, rd10, and VPP mouse models of retinitis pigmentosa. The knockdown of Cdc42 did not affect retinal morphology or function in the adult mice and did not influence photoreceptor apoptosis or molecular signaling during induced and inherited retinal degeneration. CONCLUSIONS: Retinal degeneration induces the accumulation of CDC42 in the perinuclear region of photoreceptors. In contrast to RAC1, however, lack of CDC42 does not affect the progression of degeneration. CDC42 is also dispensable for normal morphology and function of adult rod photoreceptor cells. RECEIVED: May 25, 2011 ACCEPTED: November 10, 2011.

Interleukin-1 and interleukin-8 in nicotine- and lipopolysaccharide-exposed gingival keratinocyte cultures.

BACKGROUND AND OBJECTIVE: Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin-1 alpha (IL-1 alpha) and interleukin-8 (IL-8). MATERIAL AND METHODS: Gingival keratinocyte cultures were established from 10 healthy, non-tobacco-using subjects. The cells were stimulated for 24 h with 1 mum or 1 mm nicotine and/or 10 microg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin-1 alpha and IL-8 proteins were quantified using ELISAs. RESULTS: Compared with untreated cultures, 1 mm nicotine stimulated production of IL-1 alpha (p < 0.001); E. coli and P. gingivalis LPS increased IL-8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL-1 alpha and IL-8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin-8 was also responsive to 0.1 mum nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. CONCLUSION: These results demonstrate that nicotine and LPS differentially regulate IL-1 and IL-8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy.

BACKGROUND AND OBJECTIVE: Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. MATERIAL AND METHODS: Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. RESULTS: Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. CONCLUSION: This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

Elastic limit and strain hardening of thin wires in torsion.

A theory for the size effect in the strength of wires under torsion is reported and compared with data from thin copper wires. Critical thickness theory is solved rigorously and used to validate a useful approximation which is combined with slip-distance theory modified for a finite structure size. Experimental data with high accuracy around and above the elastic limit show excellent agreement with the theory. The results strongly imply that the physical principle is the constraint that size, whether grain size or structure size, puts on allowed dislocation curvature.

Loss of human beta-defensin 1, 2, and 3 expression in oral squamous cell carcinoma.

INTRODUCTION: Human beta-defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of beta-defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of beta-defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD-1 (also published as DEFB1), HBD-2 (DEFB4) and HBD-3 (DEFB103A) (http://www.genenames.org/index.html) and HBD-1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes. METHODS: beta-defensin expression was quantitatively assessed using real-time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD-1 region spanning the 5' untranslated region to the first intron was sequenced and analysed for SNP identification and distribution. RESULTS: HBD-1 and HBD-2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three beta-defensins. Four HBD-1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD-1 locus also suggested loss of heterozygosity in OSCC. CONCLUSIONS: The genetic variation observed in OSCC compared with that in control cell lines may account for differences in beta-defensin expression. These results suggest a putative role for beta-defensins in carcinogenesis and indicate that beta-defensins may be useful markers of OSCC.

The effect of oxygen and light on the structure and function of the neonatal rat retina.

The neonatal rat is born with its eyes closed and an immature visual system, that some say is equivalent to that of a human fetus at 26 weeks of gestation. From birth, the visual system of the newborn rat will gradually mature, the first manifestation of that being the opening of the eye which usually take place at postnatal day 14. Complete maturation of the retina and visual pathways is normally reached at the end of the first month of life. The neonatal rat model thus represents a unique paradigm to study the normal and abnormal maturation of the primary visual pathways that normally occurs in utero in human subjects. Our laboratory has, over the past decade, developed two animal models of postnatally induced retinopathy, namely the Oxygen-Induced Retinopathy (OIR) that share several common features with the human Retinopathy of Prematurity (ROP) and the Light-Induced Retinopathy that is viewed by some as a valid model of some forms of Retinitis Pigmentosa (RP). The following pages review what is known of the pathophysiological processes taking place and suggest possible therapeutic avenues that could be explored in order to halt the degenerative process.

SMAP29 congeners demonstrate activity against oral bacteria and reduced toxicity against oral keratinocytes.

INTRODUCTION: Cathelicidins are antimicrobial peptides found in epithelial and mucosal tissues as well as the secondary granules of neutrophils. SMAP29, a sheep cathelicidin, has differential antimicrobial properties against various pathogens, including periodontal organisms. The purpose of this study was to evaluate the antimicrobial properties and cytotoxicity of SMAP29, SMAP28, and three congeners (SMAP18A, SMAP18D, and SMAP14A). METHODS: The peptides at concentrations ranging from 0.25 to 250 microg/ml were tested for their activity against multiple strains of Streptococcus mutans, Streptococcus sanguis, Actinomyces israelii, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Peptostreptococcus micros, and Porphyromonas gingivalis using a radial diffusion assay. Cytotoxicity of keratinocytes was evaluated by measuring lactate dehydrogenase release after incubation with the individual peptides. RESULTS: SMAP28, thought to be the biologically active peptide, was the most potent antimicrobial (range of minimum inhibitory concentrations 0.06-7.03 microg/ml, P < 0.05); however, the activity of SMAP28 and SMAP29 was strongly associated (r = 0.933). The congeners also demonstrated antimicrobial activity against the bacteria tested (range of minimum inhibitory concnetrations 0.21-79 microg/ml). Overall, F. nucleatum was the most susceptible organism, while P. gingivalis was the least susceptible. Keratinocyte cytotoxicity was dependent on peptide length and dose. SMAP28 was the most cytotoxic, while SMAP14A was the least cytotoxic. CONCLUSION: The antimicrobial activities against oral microorganisms and the minimal toxicity seen in this study suggest that the congeners of SMAP29 may serve as an alternative to traditional antibiotics in the prevention and treatment of periodontal and other oral diseases.

Variations in extracellular levels of dopamine, noradrenaline, glutamate, and aspartate across the sleep--wake cycle in the medial prefrontal cortex and nucleus accumbens of freely moving rats.

We used intracerebral microdialysis coupled with electrophysiologic recordings to determine relative changes in the concentrations of several neurotransmitters in the medial prefrontal cortex and nucleus accumbens of freely moving rats during waking, slow-wave sleep, and rapid eye movement (REM) sleep. The concentrations of noradrenaline, dopamine, glutamate, and aspartate in 2-min dialysate samples were analyzed by capillary electrophoresis combined with laser-induced fluorescence detection. Changes in glutamate and aspartate concentrations were found only in the nucleus accumbens, in which a decrease was obtained during both slow-wave sleep and REM sleep compared to waking. A progressive reduction in the release of noradrenaline was observed from waking to REM sleep in both structures. In contrast, dopamine concentrations were higher during waking and REM sleep compared to that during slow-wave sleep. The latter results demonstrate that contrary to the findings of earlier electrophysiologic studies carried out on ventral tegmental area dopaminergic neurons, changes in the release of dopamine in projection areas occur across the sleep-wake cycle. The elevated levels of dopamine during waking and REM sleep in the medial prefrontal cortex and the nucleus accumbens could result from changes during these two states in afferent modulation at the level of cell bodies or at the level of dopaminergic terminals.