Point mutation of tyrosine 759 of the IL-6 family cytokine receptor, gp130, augments collagen-induced arthritis in DBA/1J mice

<p>Abstract</p> <p>Background</p> <p>Knock-in mice (gp130F759) with a Y759F point mutation in gp130, a signal transducing receptor subunit shared by members of the IL-6 cytokine family, show sustained activation of STAT3, enhanced acute-phase or immune responses, and autoimmune arthritis. We conducted a detailed analysis of collagen-induced arthritis (CIA) in gp130F759 with a DBA/1J background (D/J.gp130F759).</p> <p>Methods</p> <p>We backcrossed gp130F759 to C57BL/6 and DBA/1J, and compared the pathologic changes, including occurrence of arthritis, in the two distinct genetic backgrounds. We analyzed CIA in D/J.gp130F759 and investigated the effects of methotrexate (MTX) on CIA.</p> <p>Results</p> <p>C57BL/6 background gp130F759 mice, but not D/J.gp130F759, spontaneously developed polyarthritis and glomerulonephritis. On the other hand, keratitis of the eyes only developed in D/J.gp130F759, indicating the influence of genetic background on disease development in gp130F759 mice. Resistance of the DBA/1J background against spontaneous arthritis urged us to examine CIA in D/J.gp130F759. CIA in D/J.gp130F759 was more severe, with greater bone destruction, than the control mice. After collagen immunization, splenomegaly and serum levels of rheumatoid factor and anti-DNA antibody were augmented in D/J.gp130F759. Bio-Plex analysis of serum cytokines revealed increased IL-12p40 and PDGF-BB before immunization, and increased levels of IFN-γ, IL-17, TNF-α, IL-9, and MIP-1β 8 days after the booster dose. IL-6 and PDGF-BB in D/J.gp130F759 showed distinct kinetics from the other cytokines; higher levels were observed after arthritis development. MTX partially attenuated the development of arthritis and inhibited bone destruction in D/J.gp130F759, with reduction of anti-type II collagen antibody levels, suggesting that MTX mainly affects antigen-specific immune responses in CIA.</p> <p>Conclusion</p> <p>The Tyr-759 point mutation of the IL-6 family cytokine receptor subunit, gp130, caused autoimmune disease, and this was also influenced by the genetic background. CIA in D/J.gp130F759 is useful for evaluating drugs in a relatively short period because sustained activation of STAT3 may enhance the disease symptoms.</p

Unc5B Interacts with FLRT3 and Rnd1 to Modulate Cell Adhesion in Xenopus Embryos

The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development

Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

BACKGROUND: Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias) – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. METHODS: Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX) for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. RESULTS: We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc). In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II) and Npr3 (natriuretic peptide decoy receptor) genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor), as well as the Npr2 gene (encoding the CNP receptor). CONCLUSION: Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine factors

Normative references of heart rate variability and salivary alpha-amylase in a healthy young male population

<p>Abstract</p> <p>Background</p> <p>This study aimed to present normative reference values of heart rate variability and salivary alpha-amylase in a healthy young male population with a particular focus on their distribution and reproducibility.</p> <p>Methods</p> <p>The short-term heart rate variability of 417 young healthy Japanese men was studied. Furthermore, salivary alpha-amylase was measured in 430 men. The average age of the subjects were 21.9 years with standard deviation of 1.6 years. Interindividual variations in heart rate variability indices and salivary alpha-amylase levels were plotted as histograms. Data are presented as the mean, median, standard deviation, coefficient of variation, skewness, kurtosis, and fifth and 95th percentiles of each physiological index.</p> <p>Results</p> <p>Mean recorded values were heart period 945.85 ms, log-transformed high frequency component 9.84 ln-ms², log-transformed low frequency component 10.42 ln-ms², log-transformed low frequency to high frequency ratio 0.58 ln-ratio, standard deviation of beat-to-beat interval 27.17 ms and root mean square of successive difference 37.49 ms. The mean value of raw salivary alpha-amylase was 17.48 U/mL, square root salivary alpha-amylase 3.96 sqrt[U/mL] and log-transformed salivary alpha-amylase 2.65 ln[U/mL]. Log-transformed heart rate variability indices exhibited almost symmetrical distributions; however, time-domain indices of heart rate variability (standard deviation of beat-to-beat interval and root mean square of successive difference) exhibited right-skewed (positive skewness) distributions. A considerable right-skewed distribution was observed for raw salivary alpha-amylase. Logarithmic transformation improved the distribution of salivary alpha-amylase, although square root transformation was insufficient. The day-to-day reproducibility of these indices was assessed using intraclass correlation coefficients. Intraclass correlation coefficients of most heart rate variability and salivary indices were approximately 0.5 to 0.6. Intraclass correlation coefficients of raw salivary markers were approximately 0.6, which was similar to those of heart rate variability; however, log transformation of the salivary markers did not considerably improve their reproducibility. Correlations between sympathetic indicators of heart rate variability and salivary alpha-amylase were not observed.</p> <p>Conclusion</p> <p>Because the sample population examined in this study involved limited age and gender variations, the present results were independent of these factors and were indicative of pure interindividual variation.</p

Creatine Protects against Excitoxicity in an In Vitro Model of Neurodegeneration

Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H2O2. In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H2O2-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity

Effect of thrombin peptide 508 (TP508) on bone healing during distraction osteogenesis in rabbit tibia

Thrombin-related peptide 508 (TP508) accelerates bone regeneration during distraction osteogenesis (DO). We have examined the effect of TP508 on bone regeneration during DO by immunolocalization of Runx2 protein, a marker of osteoblast differentiation, and of osteopontin (OPN) and bone sialoprotein (BSP), two late markers of the osteoblast lineage. Distraction was performed in tibiae of rabbits over a period of 6 days. TP508 (30 or 300 μg) or vehicle was injected into the distraction gap at the beginning and end of the distraction period. Two weeks after active distraction, tissue samples were harvested and processed for immunohistochemical analysis. We also tested the in vitro effect of TP508 on Runx2 mRNA expression in osteoblast-like (MC3T3-E1) cells by polymerase chain reaction analysis. Runx2 and OPN protein were observed in preosteoblasts, osteoblasts, osteocytes of newly formed bone, blood vessel cells and many fibroblast-like cells of the soft connective tissue. Immunostaining for BSP was more restricted to osteoblasts and osteocytes. Significantly more Runx2- and OPN-expressing cells were seen in the group treated with 300 μg TP508 than in the control group injected with saline or with 30 μg TP508. However, TP508 failed to increase Runx2 mRNA levels significantly in MC3T3-E1 cells after 2–3 days of exposure. Our data suggest that TP508 enhances bone regeneration during DO by increasing the proportion of cells of the osteoblastic lineage. Clinically, TP508 may shorten the healing time during DO; this might be of benefit when bone regeneration is slow

The pharmacokinetics of the interstitial space in humans

BACKGROUND: The pharmacokinetics of extracellular solutes is determined by the blood-tissue exchange kinetics and the volume of distribution in the interstitial space in the different organs. This information can be used to develop a general physiologically based pharmacokinetic (PBPK) model applicable to most extracellular solutes. METHODS: The human pharmacokinetic literature was surveyed to tabulate the steady state and equilibrium volume of distribution of the solutes mannitol, EDTA, morphine-6-glucuronide, morphine-3-glucuronide, inulin and β-lactam antibiotics with a range of protein binding (amoxicillin, piperacillin, cefatrizine, ceforanide, flucloxacillin, dicloxacillin). A PBPK data set was developed for extracellular solutes based on the literature for interstitial organ volumes. The program PKQuest was used to generate the PBPK model predictions. The pharmacokinetics of the protein (albumin) bound β-lactam antibiotics were characterized by two parameters: 1) the free fraction of the solute in plasma; 2) the interstitial albumin concentration. A new approach to estimating the capillary permeability is described, based on the pharmacokinetics of the highly protein bound antibiotics. RESULTS: About 42% of the total body water is extracellular. There is a large variation in the organ distribution of this water – varying from about 13% of total tissue water for skeletal muscle, up to 70% for skin and connective tissue. The weakly bound antibiotics have flow limited capillary-tissue exchange kinetics. The highly protein bound antibiotics have a significant capillary permeability limitation. The experimental pharmacokinetics of the 11 solutes is well described using the new PBPK data set and PKQuest. CONCLUSIONS: Only one adjustable parameter (systemic clearance) is required to completely characterize the PBPK for these extracellular solutes. Knowledge of just this systemic clearance allows one to predict the complete time course of the absolute drug concentrations in the major organs. PKQuest is freely available

Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

<p>Abstract</p> <p>Background</p> <p>Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated.</p> <p>Methods</p> <p>MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy.</p> <p>Results</p> <p>MSI was found in 43 (13.8%) tumors. Absence of repair protein expression was assessed in 52 (17.0%) tumors, which had primarily lost hMLH1 in 39 (12.7%), hMSH2 in 5 (1.6%), and hMSH6 in 8 (2.6%) tumors. In multivariate analysis MSI (instable) compared to MSS (stable) tumors were significantly associated with lower risk of recurrence (hazard ratio (HR) = 0.3; 95% CI: 0.2–0.7; P = 0.0007) and death (HR = 0.4; 95% CI: 0.2–0.9; P = 0.02) independently of the TS and DPD expressions. A direct relationship between MSI and TS intensity (P = 0.001) was found, while there was no significant association with DPD intensity (P = 0.1).</p> <p>Conclusion</p> <p>The favourable outcome of MSI colorectal carcinomas is ascribed mainly to the tumor biology and to a lesser extent to antitumor response to 5-fluorouracil therapy. There is no evidence that differential TS or DPD expression may account for these outcome characteristics.</p

The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice.

BACKGROUND: In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses. METHODOLOGY/ PRINCIPAL FINDINGS: Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen-specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2. CONCLUSIONS/SIGNIFICANCE: The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans

Plant ecology meets animal cognition: impacts of animal memory on seed dispersal

We propose that an understanding of animal learning and memory is critical to predicting the impacts of animals on plant populations through processes such as seed dispersal, pollination and herbivory. Focussing on endozoochory, we review the evidence that animal memory plays a role in seed dispersal, and present a model which allows us to explore the fundamental consequences of memory for this process. We demonstrate that decision-making by animals based on their previous experiences has the potential to determine which plants are visited, which fruits are selected to be eaten from the plant and where seeds are subsequently deposited, as well as being an important determinant of animal survival. Collectively, these results suggest that the impact of animal learning and memory on seed dispersal is likely to be extremely important, although to date our understanding of these processes suffers from a conspicuous lack of empirical support. This is partly because of the difficulty of conducting appropriate experiments but is also the result of limited interaction between plant ecologists and those who work on animal cognition