Enteric Neuropathy Can Be Induced by High Fat Diet In Vivo and Palmitic Acid Exposure In Vitro

Objective

Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model.

Design

Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 – 10⁻³ M) and / or oleic acid (OA) (0 – 10⁻³ M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry.

Results

HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival.

Conclusions

HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.     

Parallel Changes in H2O2 and Catalase during Thermotolerance Induced by Salicylic Acid or Heat Acclimation in Mustard Seedlings

Spraying mustard (Sinapis alba L.) seedlings with salicylic acid (SA) solutions between 10 and 500 μm significantly improved their tolerance to a subsequent heat shock at 55°C for 1.5 h. The effects of SA were concentration dependent, with higher concentrations failing to induce thermotolerance. The time course of thermotolerance induced by 100 μm SA was similar to that obtained with seedlings acclimated at 45°C for 1 h. We examined the hypothesis that induced thermotolerance involved H₂O₂. Heat shock at 55°C caused a significant increase in endogenous H₂O₂ and reduced catalase activity. A peak in H₂O₂ content was observed within 5 min of either SA treatment or transfer to the 45°C acclimation temperature. Between 2 and 3 h after SA treatment or heat acclimation, both H₂O₂ and catalase activity significantly decreased below control levels. The lowered H₂O₂ content and catalase activity occurred in the period of maximum thermoprotection. It is suggested that thermoprotection obtained either by spraying SA or by heat acclimation may be achieved by a common signal transduction pathway involving an early increase in H₂O₂.     

In Vitro,In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC₁₀₀: 50; IC₅₀: 8.84 µM) and female adult worms (LC₁₀₀: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter''s ‘Metadrug’ tool screening predictions.     

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.     

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.     

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during Brucella abortus infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.     

In Vitro Matured Oocytes Are More Susceptible than In Vivo Matured Oocytes to Mock ICSI Induced Functional and Genetic Changes

Background

Concerns regarding the safety of ICSI have been intensified recently due to increased risk of birth defects in ICSI born children. Although fertilization rate is significantly higher in ICSI cycles, studies have failed to demonstrate the benefits of ICSI in improving the pregnancy rate. Poor technical skill, and suboptimal in vitro conditions may account for the ICSI results however, there is no report on the effects of oocyte manipulations on the ICSI outcome.

Objective

The present study elucidates the influence of mock ICSI on the functional and genetic integrity of the mouse oocytes.

Methods

Reactive Oxygen Species (ROS) level, mitochondrial status, and phosphorylation of H2AX were assessed in the in vivo matured and IVM oocytes subjected to mock ICSI.

Results

A significant increase in ROS level was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P<0.05-0.001) whereas unique mitochondrial distribution pattern was found only in IVM oocytes (P<0.01-0.001). Importantly, differential H2AX phosphorylation was observed in both in vivo matured and IVM oocytes subjected to mock ICSI (P <0.001).

Conclusion

The data from this study suggests that mock ICSI can alter genetic and functional integrity in oocytes and IVM oocytes are more vulnerable to mock ICSI induced changes.     

Lactobacilli Inactivate Chlamydia trachomatis through Lactic Acid but Not H2O2

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H₂O₂, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H₂O₂ concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H₂O₂ production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H₂O₂, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection.     

In Vitro and In Vivo Expression of Opioid and σ Receptors in Rat C6 Glioma and Mouse N18TG2 Neuroblastoma Cells

Abstract: Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for δ opioid binding using [³H][ d -Ala², d -Leu⁵]enkephalin and for σ₁ and σ₂ binding with 1,3-[³H]di- o -tolylguanidine in the presence and absence of 1 µ M pentazocine. Receptor density ( B _max) and affinity ( K _D) were estimated by homologous competition binding assays. Opioid and σ B _max values in the solid tumors were significantly lower than their original levels in vitro. K _D values for opioid/σ ligands were similar in vitro and in vivo. With successive passages in the murine host, δ opioid and σ₁ binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, σ₂ receptor B _max values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of δ opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for σ binding was more complex, with the σ₂ response in late passages of the glioma being reminiscent of the formerly observed increase in number of σ sites in transformed human meninges, kidney, and colon tissue.     

In Vitro and In Vivo Models of Cerebral Ischemia Show Discrepancy in Therapeutic Effects of M2 Macrophages

The inflammatory response following ischemic stroke is dominated by innate immune cells: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.     

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background

Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).

Methods

The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.

Results

Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.

Conclusions

BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.     

In Vitro and In Vivo Enhancement of Adipogenesis by Italian Ryegrass (Lolium multiflorum) in 3T3-L1 Cells and Mice

Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as α-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-γ2, CCAAT/enhancer binding protein-α, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPARγ-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals.     

Bortezomib Resistance Can Be Reversed by Induced Expression of Plasma Cell Maturation Markers in a Mouse In Vitro Model of Multiple Myeloma

Multiple myeloma (MM), the second most common hematopoietic malignancy, remains an incurable plasma cell (PC) neoplasm. While the proteasome inhibitor, bortezomib (Bz) has increased patient survival, resistance represents a major treatment obstacle as most patients ultimately relapse becoming refractory to additional Bz therapy. Current tests fail to detect emerging resistance; by the time patients acquire resistance, their prognosis is often poor. To establish immunophenotypic signatures that predict Bz sensitivity, we utilized Bz-sensitive and -resistant cell lines derived from tumors of the Bcl-X_L/Myc mouse model of PC malignancy. We identified significantly reduced expression of two markers (CD93, CD69) in “acquired” (Bz-selected) resistant cells. Using this phenotypic signature, we isolated a subpopulation of cells from a drug-naïve, Bz-sensitive culture that displayed “innate” resistance to Bz. Although these genes were identified as biomarkers, they may indicate a mechanism for Bz-resistance through the loss of PC maturation which may be induced and/or selected by Bz. Significantly, induction of PC maturation in both “acquired” and “innate” resistant cells restored Bz sensitivity suggesting a novel therapeutic approach for reversing Bz resistance in refractory MM.     

In Vivo T Cell Activation Induces the Formation of CD209+ PDL-2+ Dendritic Cells

Two critical functions of dendritic cells (DC) are to activate and functionally polarize T cells. Activated T cells can, in turn, influence DC maturation, although their effect on de novo DC development is poorly understood. Here we report that activation of T cells in mice, with either an anti-CD3 antibody or super antigen, drives the rapid formation of CD209⁺CD11b⁺CD11c⁺ MHC II⁺ DC from monocytic precursors (Mo-DC). GM-CSF is produced by T cells following activation, but surprisingly, it is not required for the formation of CD209⁺ Mo-DC. CD40L, however, is critical for the full induction of Mo-DC following T cell activation. T cell induced CD209⁺ Mo-DC are comparable to conventional CD209^- DC in their ability to stimulate T cell proliferation. However, in contrast to conventional CD209^- DC, CD209⁺ Mo-DC fail to effectively polarize T cells, as indicated by a paucity of T cell cytokine production. The inability of CD209⁺ Mo-DC to polarize T cells is partly explained by increased expression of PDL-2, since blockade of this molecule restores some polarizing capacity to the Mo-DC. These findings expand the range of signals capable of driving Mo-DC differentiation in vivo beyond exogenous microbial factors to include endogenous factors produced following T cell activation.     

RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis In Vitro and In Vivo

This study aimed to investigate the anti-tumor activity of RY10-4, a small molecular that was designed and synthesized based on the structure of protoapigenone. A previous screening study showed that RY10-4 possessed anti-proliferative effects against HepG2 human hepatocellular carcinoma cells. However, the full range of RY10-4 anti-cancer effects on liver tumors and the underlying mechanisms have not been identified. Herein, employing flow cytometry, and Western blot analysis, we demonstrate that RY10-4 can induce cell cycle arrest, intracellular reactive oxygen species (ROS) production and apoptosis in HepG2 cells. In HepG2 cell xenograft tumor model, RY10-4 significantly inhibited the growth of tumors and induced apoptosis in tumor cells, with little side effects. Moreover, RY10-4 caused the suppression of STAT3 activation, which may be involved the apoptosis induction. In addition, RY10-4 inhibited the proliferation of Hep3B and HuH-7 human hepatocellular carcinoma cells in a concentration-dependent manner. Taken together, our results suggest that RY10-4 has a great potential to develop as chemotherapeutic agent for liver cancer.     

Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.