Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND/OBJECTIVES: Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll-like receptor (TLR) 2- and 4-induced response in human periodontal ligament cells (hPDLCs). MATERIAL AND METHODS: Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 µg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 µg/mL); TLR4 agonist Escherichia coli LPS (0.1 µg/mL; 1 µg/mL); TLR2 agonist Pam3CSK4 (0.1 µg/mL; 1 µg/mL). The protein expression of interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 was analyzed with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction. RESULTS: Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL-6, IL-8 and monocyte chemotactic protein-1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non-pretreated cells. CONCLUSION: The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.

Silencing of essential genes by RNA interference in Haemonchus contortus.

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells.

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 µM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 µM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 µM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity.

Kinetics of blood flow during healing of excisional full-thickness skin wounds in pigs as monitored by laser speckle perfusion imaging.

BACKGROUND/PURPOSE: The laser speckle perfusion imaging (LSPI) system is a new, non-invasive technique for rapidly and reproducibly measuring tissue perfusion. The high resolution and frame rate of the LSPI overcome many of the limitations of traditional laser Doppler imaging techniques. Therefore, LSPI is a useful means for evaluating blood flow in a variety of situations. The present study investigates the ability of the LSPI system to detect temporal changes in blood flow during the healing of cutaneous wounds in a well-characterized animal model. METHODS: Full-thickness excisional skin wounds (2 x 2 cm) were created on the backs of juvenile female red Duroc pigs. Every week post-injury, the wounds were measured and photographed, and normalized blood flow values were determined using the LSPI system. RESULTS: Tissue perfusion values were available after complete re-epithelialization and removal of the eschar, at day 21. At this point, wound blood flow was significantly elevated as compared with the surrounding, uninvolved skin. Wound blood flow declined steadily during healing, and approached normal values by day 35 post-injury. CONCLUSION: The kinetics of blood flow during excisional wound healing in the red Duroc model are comparable with that previously observed in laser Doppler imaging of healing human skin wounds and hypertrophic scars. These results therefore confirm that the red Duroc is a good model of human wound healing, and further indicates that the LSPI is an excellent technique for evaluating angiogenesis and neovascularization during healing in this and other models.

Skin wound healing in the first generation (F1) offspring of Yorkshire and red Duroc pigs: evidence for genetic inheritance of wound phenotype.

Fibroproliferative scars in humans often demonstrate familial inheritance patterns, and genetics may contribute to healing and scarring. Genetic factors may also influence the scarring phenotype in a porcine model. Healing of full thickness excisional skin wounds in Yorkshire pigs closely resembles normal healing in humans, while identical wounds in red Duroc pigs form hypercontracted, hyperpigmented scars. The present study has evaluated the healing process in the first generation cross (F1) of red Duroc and Yorkshire pigs. Gross and histologic analysis revealed that the F1 animals exhibit an intermediate healing phenotype, with some features of each parent breed. F1 full thickness wounds were significantly hypercontracted and fibrotic, but apigmented. Analysis of mRNA expression patterns for a panel of relevant molecules (N=32) in the F1 animals revealed some similarities to each parent breed, as well as unique patterns for other molecules. Furthermore, a depth dependency to the healing response was observed at the gross, histologic, and molecular levels, with deep dermal wounds healing similar to Yorkshire wounds. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex. However, the results indicate that further understanding in this model may provide insights into risk factors for hypertrophic scarring in human burn patients.

Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes.

The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.

The flightless I protein and the gelsolin family in nuclear hormone receptor-mediated signalling.

The Drosophila melanogaster flightless I protein and its homologues in higher eukaryotes (FliI) are conserved members of the gelsolin family of actin-binding proteins. Members of the gelsolin family generally contain three or six copies of a 125-amino-acid residue gelsolin-related repeating unit, and may contain additional domains including the C-terminal villin-related 'headpiece' or N-terminal extensions such as the leucine-rich repeat of the FliI protein. Numerous studies including work done with mouse knockouts for gelsolin, villin and CapG support a role for the family in cytoskeletal actin dynamics. In both fruitfly and mouse, the FliI protein is also essential for early development. Recent studies indicate that supervillin, gelsolin and FliI are involved in intracellular signalling via nuclear hormone receptors including the androgen, oestrogen and thyroid hormone receptors. This unexpected role in signalling has opened a new area in research on the gelsolin family and is providing important new insights into the mechanisms of gene regulation via nuclear receptors.

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes.

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.

[Early results with a monorail-stent-balloon device for endovascular treatment of renal artery stenosis]. / Erste Erfahrungen mit einem Monorail-Stent-Ballon-System zur endovaskulären Behandlung von Nierenarterienstenosen.

OBJECTIVE: To evaluate the technical feasibility of a new monorail-stent-balloon device for treatment of renal artery stenosis (RAS). PATIENTS AND METHODS: During a study period of 18 months, 38 patients with proven RAS in 41 cases (hypertension n = 36, renal insufficiency n = 13) and indication for stenting (calicified ostial lesions n = 35, insufficient PTA n = 4, dissection n = 2) were enrolled into this prospective evaluation. Pre-mounted stents (Rx-Herculink(TM) 5 mm = 13, 6 mm = 34, 7 mm = 1) were implanted a transfemoral (n = 35) or transbrachial approach (n = 6). Mean grade and lengths of stenosis measured were 88 % plus minus 10 and 9 mm plus minus 5. RESULTS: Renal stent implantation was technically successful in all cases (100 %). In 7 cases a second stent had to be implanted to cover the entire lesion. The transstenotic pressure drop decreased from 88 mmHg plus minus 10 before to 1 mmHg plus minus 1.8 after the procedure. Remaining stenosis measured 0.7 % plus minus 4.2. Serum creatine levels decreased from 1.9 mm/dl to 1.5 mg/dl (n. s.), blood pressure decreased from 178/94 mmHg to 148/79 mmHg (p < 0.0001) after the intervention. Primary and secondary patency rates at 6 months were 72 % (Standard Error 9.8 %) and 77 (% (Standard Error 9.2 %), respectively. CONCLUSION: With the used monorail-stend-balloon device a technically easy, secure and exact renal stent placement is guaranteed, patency rates are similar to those described in the current literature.

The role of eosinophils in parasitic helminth infections: insights from genetically modified mice.

Eosinophilia - an increase in the number of eosinophils in the blood or tissues - has historically been recognized as a distinctive feature of helminth infections in mammals. Yet the precise functions of these cells are still poorly understood. Many scientists consider that their primary function is protection against parasites, although there is little unequivocal in vivo evidence to prove this. Eosinophils are also responsible for considerable pathology in mammals because they are inevitably present in large numbers in inflammatory lesions associated with helminth infections or allergic conditions. In this review, Carolyn Behm and Karen Ovington outline some of the cellular and biological properties of eosinophils and evaluate the evidence for their role(s) in parasitic infections.

Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils.

The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change.

Regulation of primary Strongyloides ratti infections in mice: a role for interleukin-5.

C57BL/6 mice genetically deficient in interleukin-5 (IL-5-/-) and normal C57BL/6 (IL-5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL-5-/- mice released two to four times more eggs and larvae than IL-5+/+ mice. IL-5-/- mice harboured about 60% more intestinal worms, which were more fecund, than IL-5+/+ mice. The duration of the infection was similar in normal and IL-5-deficient mice. Both IL-5-/- and IL-5+/+ mice resisted a secondary infection. IL5-/- mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL-5+/+ mice, but not IL-5-/- mice, following infection. No significant differences between infected IL-5+/+ and IL-5-/- mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL-5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL-5-/- mice.

Factors influencing the development and carbohydrate metabolism of Echinococcus granulosus in dogs.

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities, worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development.

Eosinophilia, parasite burden and lung damage in Toxocara canis infection in C57Bl/6 mice genetically deficient in IL-5.

C57Bl/6 mice genetically deficient in interleukin (IL)-5 (IL-5-/-) and mice with the normal IL-5 gene (IL-5+/+) were infected with embryonated eggs of Toxocara canis. IL-5+/+ mice developed a marked eosinophilia in their peripheral bloods and bone marrows after infection. In contrast, the number of eosinophils at these sites actually decreased during the acute phase of infection in IL-5-/- mice. A smaller number of eosinophils infiltrated the lung, liver, heart and skeletal muscle of infected IL-5-/- mice than those of infected IL-5+/+ mice. Eosinophils were not produced in cultures of bone marrow cells from either IL-5+/+ or IL-5-/- mice which were stimulated with excretory secretory antigen of T. canis larvae. The capacity of cells from the bone marrow to differentiate into eosinophils when stimulated in vitro with recombinant murine IL-5 was the same whether the cells were from IL-5+/+ or IL-5-/- mice. Taken together, these results show that an IL-5-like molecule is not produced by the T. canis larvae and that IL-5 produced by host cells is solely responsible for the eosinophilia in mice infected with this nematode. The number and location of T. canis larvae were not altered in the absence of IL-5. In contrast, lung damage in infected IL-5-/- mice was less extensive than that in infected IL-5+/+ mice, although structures resembling Charcot-Leyden crystals were seen in the lungs of both IL-5+/+ and IL-5-/- mice. These results suggest that eosinophils play a role in the pathology in mice infected with T. canis.

The role of trehalose in the physiology of nematodes.

The sugar trehalose, an alpha-1-linked non-reducing disaccharide of glucose, is important in the physiology of many micro-organisms as well as in some groups of metazoan organisms, including insects and nematodes. Trehalose is a stress protectant in biological systems as it interacts with and directly protects lipid membranes and proteins from the damage caused by environmental stresses such as desiccation and freezing. Trehalose is present in many nematode species where its concentration often exceeds that of glucose but is usually lower than that of glycogen. In Ascaris suum it is found in all tissues, with highest concentrations in muscle, haemolymph and the female and male reproductive organs. Trehalose acts as an energy reserve in some nematodes and their eggs, and may be important in uptake of glucose; it appears to function as the major circulating blood sugar. Trehalose accumulates in nematodes that can withstand dehydration and may be important in supercooling of nematodes or eggs that can withstand freezing. In many nematodes trehalose is also important in the process of egg hatching. The combined action of 2 enzymes, trehalose 6-phosphate (T6P) synthase and T6P phosphatase, catalyses the synthesis of trehalose in most organisms. Hydrolysis of trehalose glucose is catalysed by trehalase. These enzymes to have been detected in nematodes but the processes regulating their activity are unknown. Trehalose metabolism may provide new molecular targets for attack in nematodes parasitic in mammals.

The enigmatic eosinophil: investigation of the biological role of eosinophils in parasitic helminth infection.

In many helminth infected hosts the number of eosinophils increases dramatically, often without any concurrent increases in the number of other leukocytes, so that eosinophils become the dominant cell type. Many experimental investigations have shown that the eosinophilia is induced by interleukin-5 (IL-5) but its functional significance remains unclear. Mice genetically deficient in IL-5 (IL-5-/-) have been used to evaluate the functional consequences of the IL-5 dependent eosinophilia in helminth infected hosts. Host pathology and level of infection were determined in IL-5-/- and wild type mice infected with a range of species representative of each major group of helminths. The effects of IL-5 deficiency were very heterogeneous. Of the six species of helminth examined, IL-5 dependent immune responses had no detectable effect in infections with three species, namely the cestodes Mesocestoides corti and Hymenolepis diminuta and the trematode Fasciola hepatica. In contrast, IL-5 dependent immune responses were functionally important in mice infected with three species, notably all nematodes. Damage to the lungs caused by migrating larvae of Toxocara canis was reduced in IL-5-/- mice. Infections of the intestine by adult stages of either Strongyloides ratti or Heligmosomoides polygyrus were more severe in IL-5-/- mice. Adult intestinal nematodes were clearly deleteriously affected by IL- 5 dependent processes since in its presence there were fewer worms which had reduced fecundity and longevity. The implications of these results for the viability of using inhibitors of IL-5 as a therapy for asthma are considered.

[Behm's T-piece for reducing anesthesia gas pollution at the anesthesia work site]. / Behm'sches T-Stück zur Verminderung der Narkosegasbelastung am Anästhesiearbeitsplatz.

The safety standards demanded for performing anaesthesia and the increasing burden of work to be handled by OP personnel entail continuous supervision and elimination of hazards and leaks. One example illustrates this necessity: Anaesthetic agents in the circular system of the induction are pollute the atmosphere after disconnection of one patient and transport into the OP suite and are a potential risk to personnel and next patient. C. Behm developed a T-piece which connects the Y-piece with the scavenging system leading to elimination of residual anaesthetics from the circular system. A useful byproduct is the longer life of the galvanic O2 sensor.

Fasciola hepatica infection in sheep: changes in liver metabolism.

Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previously reported respiratory abnormalities in mitochondria isolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in nonesterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in common with the rat, a substantial loss of cytochrome P450 was recorded in microsomes prepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in glucose 6-phosphatase activity. All these changes were localised effects, confined to areas of fluke infiltration.

IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses.

Mice deficient in interleukin-5 (IL-5-/- mice) were generated by gene targeting in embryonal stem cells. Contrary to previous studies, no obligatory role for IL-5 was demonstrated in the regulation of conventional B (B-2) cells, in normal T cell-dependent antibody responses or in cytotoxic T cell development. However, CD5+ B cells (B-1 cells) in the peritoneal cavity were reduced by 50%-80% in 2-week-old IL-5-/- mice, returning to normal by 6-8 weeks of age. The IL-5-/- mice did not develop blood and tissue eosinophilia when infected with the helminth Mesocestoides corti, but basal levels of eosinophils with normal morphology were produced in the absence of IL-5. IL-5 deficiency did not affect the worm burden of infected mice, indicating that increased eosinophils do not play a significant role in the host defence in this parasite model.

Importance of T-cell-dependent inflammatory reactions in the decline of microsomal cytochrome P450 concentration in the livers of rats infected with Fasciola hepatica.

The concentration of cytochrome P450, measured spectrophotometrically in microsomal preparations from the livers of rats infected with 30 metacercariae of Fasciola hepatica, declined by approximately 50% at 3 weeks post-infection. Treatment of infected rats with the anti-inflammatory agent dexamethasone (2 mg/kg at 48 h intervals for 8 days prior to assay) abolished the decline in P450 content. Assay of P450 in infected congenitally athymic (nude) rats showed normal levels. These results demonstrate that the T-cell-dependent inflammatory response in the liver of the host is a necessary factor in the development of the decline in hepatic P450, which is known to compromise the metabolism of certain drugs in infected hosts.