CXCL10 Controls Inflammatory Pain via Opioid Peptide-Containing Macrophages in Electroacupuncture

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund''s adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3⁺ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3⁺ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.     

Modelling the effect of water activity and temperature on growth rate and aflatoxin production by two isolates of Aspergillus flavus on paddy

Aims: This study was conducted to characterize the growth of and aflatoxin production by Aspergillus flavus on paddy and to develop kinetic models describing the growth rate as a function of water activity (a_w) and temperature. Methods and Results: The growth of A. flavus on paddy and aflatoxin production were studied following a full factorial design with seven a_w levels within the range of 0·82–0·99 and seven temperatures between 10 and 43°C. The growth of the fungi, expressed as colony diameter (mm), was measured daily, and the aflatoxins were analysed using HPLC with a fluorescence detector. The maximum colony growth rates of both isolates were estimated by fitting the primary model of Baranyi to growth data. Three potentially suitable secondary models, Rosso, polynomial and Davey, were assessed for their ability to describe the radial growth rate as a function of temperature and a_w. Both strains failed to grow at the marginal temperatures (10 and 43°C), regardless of the a_w studied, and at the a_w level of 0·82, regardless of temperature. Despite that the predictions of all studied models showed good agreement with the observed growth rates, Davey model proved to be the best predictor of the experimental data. The cardinal parameters as estimated by Rosso model were comparable to those reported in previous studies. Toxins were detected in the range of 0·86–0·99 a_w with optimal a_w of 0·98 and optimal temperature in the range of 25–30°C. Conclusions: The influences of a_w and temperature on the growth of A. flavus and aflatoxin production were successfully characterized, and the models developed were found to be capable of providing good, related estimates of the growth rates. Significance and Impact of the Study: The results of this study could be effectively implemented in minimizing the risk of aflatoxin contamination of the paddy at postharvest.     

Pharmacodynamic modeling of anti-cancer activity of tetraiodothyroacetic acid in a perfused cell culture system

Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.     

Cloning and expression of a new bacteriophage (SHPh) DNA ligase isolated from sewage

During the last 50 years, major advances in molecular biology and biotechnology have been attributed to the discovery of enzymes that allow molecular cloning of important genes. One of these enzymes that has been widely acknowledged for its role in the development of biotechnology is the T4 DNA ligase. This enzyme joins the break in the DNA backbone structure by creating a phosphodiester bond between 5′ PO₄ and 3′ OH ends, in an ATP dependent multi-step reaction, thus allowing the ligation of related and foreign DNA sequences. Due to its role in modern DNA recombinant technology, there is a high demand on DNA ligase to allow the ligation of target DNA inserts into a chosen vector as part of DNA cloning technology. To closely look at ligase sequence diversity, a bacteriophage that infects DH5α (commercial lab strain of Escherichia coli) was isolated from sewage system in Hebron, Palestine. The DNA ligase gene of this phage was cloned and its sequence was compared to the NCBI database. The new bacteriophage ligase, named (South Hebron Phage, SHPh) DNA ligase, shows homology to T even bacteriophage DNA ligases posted in the NCBI database with 35 nucleotide differences, an indication of existed diversity among T even DNA ligation enzymes that can be used as markers in phage classification.     

The influence of low power microwave on the properties of DPPC vesicles

The effect of microwave exposure on liposome at non-thermal level are studied. Dipalmitoyl phosphatidylcholine (DPPC) liposomes were exposed to 950 MHz at power densities of 2.5 mW/cm², which is equivalent to specific absorption rate (SAR) of 0.238 W/K. The interaction of microwave with liposomes was investigated by membrane solubilization measurements using a non-ionic detergent, octylglucoside (OG), as well as Fourier transform infrared (FTIR) spectroscopy and flow activation energy measurements. The amount of detergent needed to completely solubilize the liposomal membrane was increased after exposure of liposomes to microwave irradiation, indicating an increased membrane resistance to the detergent and hence a change in the natural membrane permeation properties. In the analysis of FTIR spectra the symmetric and antisymmetric CH₂ (at 2070 cm^?1) band and the CO (at 1640 cm^?1) stretching bands were investigated after liposomal exposure to microwave irradiation. It is clearly shown from the flow activation energy measurements, that low-power microwave induce changes in the liposomes deformability (decreases the liposome fluidity and increases the liposome rigidity). Finally it could be concluded that low-power microwave of 950 MHz induced structural and functional changes in liposomes as a membrane model system.     

VI. Measurement of proenkephalin A-derived peptides in biological tissues by high pressure liquid chromatography coupled with amperometric electrochemical detection

We describe an analytic method for the separation and quantitation of a number of proenkephalin A-derived peptides using high pressure liquid chromatography coupled with amperometric electrochemical detection (HPLC-AECD). Initially, we coupled our HPLC separation system with AECD in series with a UV detector for additional confirmation of peak specificity. AECD provided a 10⁶ - fold increase in sensitivity over UV detection for these peptides. In addition to Met-enkephalin (ME), ME-Arg, ME-Arg-Phe, ME-Arg-Gly-Leu, Leu-enkephalin (LE) and LE-Arg (Dyn 1–6), we separated and detected the sulfoxides of ME and its extended peptides. Subsequently, we used minor modifications of the isocratic mobile phase to separate and detect enkephalin-related peptides with greater sensitivity and shorter chromatographic run times; each of these mobile phases was used to separate and detect two to three peptides. We have applied this HPLC-AECD methodology to quantitate ME, ME-Arg-Phe, ME-Arg-Gly-Leu and LE in pheochromocytoma tumors.     

Galectin-3 is a negative regulator of lipopolysaccharide-mediated inflammation

Galectin-3 is a beta-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, beta-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.     

Synthesis and biological evaluation of nonpeptide integrin antagonists containing spirocyclic scaffolds

Analogues of isoxazoline alpha(v)beta(3) antagonist 1 designed to further restrict the four carbon alkyl tether were prepared by incorporating two spirocyclic scaffolds, 1-oxa-2-azaspiro[4,5]dec-2-ene and 1-oxa-2,7-diazaspiro[4,4]non-2-ene. Additional optimization provided potent antagonists of both alpha(v)beta(3) and alpha(5)beta(1) which are selective over GPIIb/IIIa.