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.

Heterogeneity in Dental Tissue-Derived MSCs Revealed by Single-Cell RNA-seq.

Mesenchymal stromal cells (MSCs) are multipotent, progenitor cells that reside in tissues across the human body, including the periodontal ligament (PDL) and gingiva. They are a promising therapeutic tool for various degenerative and inflammatory diseases. However, different heterogeneity levels caused by tissue-to-tissue and donor-to-donor variability, and even intercellular differences within a given MSCs population, restrict their therapeutic potential. There are considerable efforts to decipher these heterogeneity levels using different "omics" approaches, including single-cell transcriptomics. Previous studies applied this approach to compare MSCs isolated from various tissues of different individuals, but distinguishing between donor-to-donor and tissue-to-tissue variability is still challenging. In this study, MSCs were isolated from the PDL and gingiva of 5 periodontally healthy individuals and cultured in vitro. A total of 3,844 transcriptomes were generated using single-cell mRNA sequencing. Clustering across the 2 different tissues per donor identified PDL- and gingiva-specific and tissue-spanning MSCs subpopulations with unique upregulated gene sets. Gene/pathway enrichment and protein-protein interaction (PPI) network analysis revealed differences restricted to several cellular processes between tissue-specific subpopulations, indicating a limited tissue-of-origin variability in MSCs. Gene expression, pathway enrichment, and PPI network analysis across all donors' PDL- or gingiva-specific subpopulations showed significant but limited donor-to-donor differences. In conclusion, this study demonstrates tissue- and donor-specific variabilities in the transcriptome level of PDL- and gingiva-derived MSCs, which seem restricted to specific cellular processes. Identifying tissue-specific and tissue-spanning subpopulations highlights the intercellular differences in dental tissue-derived MSCs. It could be reasonable to control MSCs at a single-cell level to ensure their properties before transplantation.

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.

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.

Characterization of the oligomycin-sensitivity properties of the F1F0-ATPase in mitochondria from rats infected with the liver fluke Fasciola hepatica.

The F1F0-ATPase activity of liver mitochondria isolated from rats infected with Fasciola hepatica at 3 and 4 weeks post-infection showed a marked loss of sensitivity to oligomycin and to N,N'-dicyclohexylcarbodiimide. A loss of sensitivity to diethylstilbestrol was also demonstrated at 4 weeks post-infection. Recovery was apparent in most cases by 6 weeks post-infection. No significant difference in latent ATPase activity was observed between mitochondria from control and infected livers at any stage of the infection. The mitochondria from infected livers were therefore considered to have a full complement of the F1 moiety of the F1F0-ATPase complex. Purification of the mitochondrial ATPase from 4-week infected livers resulted in a very low yield of an oligomycin-insensitive complex. This was due to a failure to enrich specific activity during purification. The evidence presented indicates that infection with Fasciola hepatica gives rise to alterations in the function of the host liver mitochondrial ATPase, namely loss of inhibitor sensitivity and apparent structural alterations of the ATPase complex.

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.

Restoration of mitochondrial energy-linked reactions following dexamethasone treatment of rats infected with the liver fluke Fasciola hepatica.

Mitochondria isolated from male Wistar rats experimentally infected with the common liver fluke, Fasciola hepatica, exhibit loss of respiratory control from 2 weeks post-infection (Rule, et al. (1989) Biochem. J. 260, 517-523). We now report that subcutaneous injections of the anti-inflammatory drug, dexamethasone, during the final week of infection prevented the mitochondrial uncoupling and restored respiratory control almost to the levels of uninfected controls. Further investigations have shown that mitochondria from infected rat livers are unable to synthesize ATP and that abnormal respiration is also evident in hepatocytes isolated from infected rats. These abnormalities were absent when infected rats were treated with dexamethasone. In addition, liver mitochondrial function in infected, congenitally athymic, nude rats (CBH/R nu/nu) was not significantly different from that in uninfected nude or Wistar controls. These results provide evidence that the mitochondrial dysfunction in fascioliasis is host-mediated and that T lymphocytes in particular may be involved.

Beneficial effect of dexamethasone on attenuated hormone-induced uptake of calcium and glycogenolysis by perfused liver of rats infected with Fasciola hepatica.

Infection of rats with the liver fluke, Fasciola hepatica, impaired the responses of the perfused liver to calcium uptake and glucose release induced by the synergistic action of glucagon and vasopressin. Treatment of infected rats with dexamethasone prevented the impairment of each of these two responses.

Metabolic studies on the new fasciolicidal drug, closantel.

Studies of the metabolic disturbances caused in Fasciola hepatica by closantel have been carried out in vitro and in fluke recovered from treated sheep. Fluke exposed to the anthelmintic under both conditions exhibit increased carbohydrate mobilisation, increased end-product formation, especially of succinate, diminished ATP synthesis, increased oxaloacetate/malate ratios, and increased internal concentrations of pyruvate. No specific enzyme inhibition was detected. These results are consistent with the view that closantel acts as an uncoupler of oxidative phosphorylation by increasing mitochondrial permeability.

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 role of the host in the regulation of end-product formation in two strains of the rat tapeworm, Hymenolepis diminuta.

Individual worms from rats infected with different strains of Hymenolepis diminuta were incubated in vitro and the products lactate, succinate, acetate and ammonia assayed. Variability in excretion was not confined to differences between strains. Two metabolic types were identified. Where succinate was above 20 mumol g-1 h-1, lactate excretion was low. Where succinate was not detected, lactate excretion was high. Acetate excretion was variable. Lactate and ammonia excretion were positively correlated. All worms from one rat were of the same type but could be of either type from different rats. The host strain had no effect. A relationship was shown between lactate excretion and the number of worms from a standard inoculum present at 21 days of infection. The incidence of high lactate excretion was increased in worms from secondary infections. Components of the host immune response may thus exert effects on the metabolism of H. diminuta, manifest as shifts in emphasis on cytosolic and mitochondrial metabolism.

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.

A study of hepatic mitochondrial respiration and microsomal cytochrome P450 content in mice infected with the liver fluke Fasciola hepatica.

Previous studies of the effects of infection of Wistar rats with the common liver fluke, Fasciola hepatica, on liver bioenergetic and drug metabolism have demonstrated a loss of respiratory control in isolated mitochondria and reduced microsomal cytochrome P450 content, respectively, from 2 weeks post-infection throughout the acute phase of the infection. In the present study male Balb/c mice infected with F. hepatica showed a loss of respiratory control in isolated liver mitochondria only at 4 weeks post-infection. A similar time course was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. Preparations from infected CBA mice showed similar changes to Balb/c mice but mitochondrial respiration in preparations from infected Swiss outbred mice was normal. A host difference between strains of mice and between mice and rats is therefore evident in the timing and extent of liver mitochondrial dysfunction and in the timing of the decrease in the cytochrome P450 content of hepatic microsomes. This difference between hosts may be related to the reported differences in cellular inflammatory responses to the migrating juvenile flukes in the livers of rats and mice.

Aberrant energy-linked reactions in mitochondria isolated from the livers of sheep infected with the liver fluke Fasciola hepatica.

Respiration by mitochondria isolated from the livers of sheep following infection up to 15 weeks with F. hepatica was measured with the respiratory substrates pyruvate (plus malate) and succinate in the absence and presence of ADP; the rates were compared with those obtained by mitochondria isolated from livers of uninfected sheep. It was found that respiration supported by both substrates in mitochondria isolated from the left lobe but not the middle lobe of 4-week infected sheep exhibited abnormalities such that the acceptor control ratios were only marginally above one. Some, but not total, recovery was seen in the later stages of infection. The aberrant respiratory behaviour is similar to that observed with infected rats.

Genetic characterization of three strains of Hymenolepis diminuta at 39 enzyme loci.

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.

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.

Evaluation of the SOS chromotest.

In the present investigation, the SOS chromotest with E. coli PQ37 was evaluated. The potential to identify different kinds of bacterial mutagens was examined. 124 chemicals of different chemical classes were tested. Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test.

[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.

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.

Mebendazole concentrations in sheep plasma.

Formulated mebendazole was administered to sheep by intraruminal injection at dose rates of 12.5, 25, 50 or 100 mg/kg bodyweight. The concentrations of mebendazole and two major metabolites were measured by high-performance liquid chromatography in plasma taken at intervals up to 48 hours after treatment. At 12.5 mg/kg the peak plasma concentration was 0.22 +/- 0.03 microM mebendazole, rising to 0.76 +/- 0.04 microM at 100 mg/kg. Peak plasma concentrations occurred between nine and 24 hours for all dose rates and declined rapidly. Two major metabolites were detected; their concentrations exceeded that of mebendazole at all dose rates.