Transcripts Of Markers Research Articles

Characteristics of Infection Immunity Regulated by Toxoplasma gondii to Maintain Chronic Infection in the Brain.

To examine the immune environment of chronic Toxoplasma gondii infection in the brain, the characteristics of infection-immunity (premunition) in infection with T. gondii strain ME49 were investigated for 12 weeks postinfection (PI). The results showed that neuronal cell death, microglia infiltration and activation, inflammatory and anti-inflammatory cytokine expression, Stat1 phosphorylation, and microglia activation and inflammatory gene transcripts related to M1 polarization in the brain were increased during the acute infection (AI) stage (within 6 weeks PI), suggesting that innate and cellular inflammatory response activation and neurodegeneration contributed to excessive inflammatory responses. However, these immune responses decreased during the chronic infection (CI) stage (over 6 weeks PI) with reductions in phosphorylated STAT1 (pSTAT1) and eosinophilic neurons. Notably, increases were observed in transcripts of T-cell exhaustion markers (TIM3, LAG3, KLRG1, etc.), suppressor of cytokines signaling 1 protein (SOCS1), inhibitory checkpoint molecules (PD-1 and PD-L1), and Arg1 from the AI stage (3 weeks PI), implying active immune intervention under the immune environment of M1 polarization of microglia and increases in inflammatory cytokine levels. However, when BV-2 microglia were stimulated with T. gondii lysate antigens (strain RH or ME49) in vitro, nitrite production increased and urea production decreased. Furthermore, when BV-2 cells were infected by T. gondii tachyzoites (strain RH or ME49) in vitro, nitric oxide synthase and COX-2 levels decreased, whereas Arg1 levels significantly increased. Moreover, Arg1 expression was higher in ME49 infection than in RH infection, whereas nitrite production was lower in ME49 infection than in RH infection. Accordingly, these results strongly suggest that immune triggering of T. gondii antigens induces M1 polarization and activation of microglia as well as increase NO production, whereas T. gondii infection induces the inhibition of harmful inflammatory responses, even with M1 polarization and activation of microglia and Th1 inflammatory responses, suggesting a host–parasite relationship through immune regulation during CI. This is a characteristic of infection immunity in infection with T. gondii in the central nervous system, and SOCS1, a negative regulator of toxoplasmic encephalitis, may play a role in the increase in Arg1 levels to suppress NO production.

B Cell Activity Is Impaired in Human and Mouse Obesity and Is Responsive to an Essential Fatty Acid upon Murine Influenza Infection.

Obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. However, there is limited information about the influence of obesity on B cell function and underlying factors that modulate B cell responses. Therefore, we studied B cell cytokine secretion and/or Ab production across obesity models. In obese humans, B cell IL-6 secretion was lowered and IgM levels were elevated upon ex vivo anti-BCR/TLR9 stimulation. In murine obesity induced by a high fat diet, ex vivo IgM and IgG were elevated with unstimulated B cells. Furthermore, the high fat diet lowered bone marrow B cell frequency accompanied by diminished transcripts of early lymphoid commitment markers. Murine B cell responses were subsequently investigated upon influenza A/Puerto Rico/8/34 infection using a Western diet model in the absence or presence of docosahexaenoic acid (DHA). DHA, an essential fatty acid with immunomodulatory properties, was tested because its plasma levels are lowered in obesity. Relative to controls, mice consuming the Western diet had diminished Ab titers whereas the Western diet plus DHA improved titers. Mechanistically, DHA did not directly target B cells to elevate Ab levels. Instead, DHA increased the concentration of the downstream specialized proresolving lipid mediators (SPMs) 14-hydroxydocosahexaenoic acid, 17-hydroxydocosahexaenoic acid, and protectin DX. All three SPMs were found to be effective in elevating murine Ab levels upon influenza infection. Collectively, the results demonstrate that B cell responses are impaired across human and mouse obesity models and show that essential fatty acid status is a factor influencing humoral immunity, potentially through an SPM-mediated mechanism.

Signalling pathways associated with IL-6 production and epithelial-mesenchymal transition induction in prostate epithelial cells stimulated with Trichomonas vaginalis.

Trichomonas vaginalis (Tv) has been found in patient tissue of benign prostatic hyperplasia (BPH), and suggested to cause chronic prostatitis. IL-6 is known as one of the important factors of chronic inflammation in prostate cancer. Patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) had higher levels of IL-6 in seminal plasma. Furthermore, inflammatory conditions induced by pathogen infections have been shown to promote epithelial-mesenchymal transition (EMT). Here, we investigated the signals involved in IL-6 production by human prostate epithelial cells (PECs) stimulated with Tv and examined whether Tv induces EMT in PECs. We found that PECs stimulated with Tv increased the production of IL-6, as well as the expression of TLR2, TLR4, MAPKs (p38, JNK, ERK), NF-κB and JAK2/STAT3, and levels of ROS. Inhibition of TLR2 or TLR4 reduced IL-6 production as well as expression of these other factors, and agents inhibiting ROS, MAPKs, NF-κB and JAK reduced IL-6 production. However, when PECs were stimulated with Tv, transcripts of mesenchymal cell markers increased, and epithelial cell markers decreased. In addition, the induction of EMT was suppressed by inhibitors of JAK or NF-κB. These findings are the first evidence that Tv infection of prostate epithelial cells may induce EMT.

Abstract 3347: Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells

Abstract Background and Aim: Cancer of the cervix is the 2nd most common cancer in Indian women. Cell lines are essential in vitro models in cancer research. Till date, available cell lines in cervical cancer were highly passaged, developed from Caucasian, Black or Asian origin; hence, establishment of a low-passage cervical cancer derived cell line was the first aim of this study. Cancer stem cells (CSCs) are proposed to be responsible for metastasis and therapy resistance. Previously, we reported CD133 to be a putative marker for CSC, and they were further characterized. Materials and Methods: 25 women were recruited after informed consent and approval from the Institute Ethics committee. A small portion of the biopsy was collected in DMEM/F12 media and subjected to primary culture. Successful propagation was possible in 4 cases. Their morphology, epithelial nature and HPV status were evaluated. Variable Number Tandem Repeat (VNTR) assay, cell cycle and kinetic studies were performed. Tissue expression of CD133 and CD49f stem cell markers was evaluated by direct immunofluorescence on frozen tumor samples. Adherent cells were sorted into CD133+ vs CD133- tumor bulk from all 4 cell lines by FACS. These two populations were evaluated for differences in tumorosphere formation, chemosensitivity to cisplatinum, expression of stemness (SOX2, OCT4, NANOG) and EMT (SNAIL, SLUG, TWIST, VIMENTIN and E-CADHERIN) markers at the transcript level. Observations: Four cell lines designated RSBS-9, RSBS-14, RSBS-23 derived from non-keratinizing squamous cell carcinoma and RSBS-43 derived from adenocarcinoma cervix were established and passaged up to 50 times. The epithelial nature of cell lines was confirmed by cytokeratin and epithelial membrane antigen positivity. VNTR assay confirmed derivation of cell lines from respective parental tissue sample. All 4 cell lines were HPV-16positive. The cell doubling time was approximately 48 hours. Immunofluorescence on tissue samples revealed diffuse positivity for CD49f indicating that it may not represent a CSC marker as previously reported whereas CD133 staining showed scattered positive cells. No particular location of CD133+ CSC was observed. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorosphere formation; however, no significant difference in the chemosensitivity to cisplatinum was seen. Real time quantitation of transcripts of stemness and EMT markers revealed CD133+ cells showing upregulation of EMT markers VIMENTIN, SNAIL, SLUG in the RSBS-23 cell line and of VIMENTIN and TWIST in RSBS-9 cell line whereas there was no significant difference in the RSBS-14 and RSBS-43 cell lines for any of the markers studied. Conclusion: Low passage cell lines are useful model systems for understanding the biology of cervical cancer. CD133+ Cancer Stem cells exhibit some EMT markers in 2 of the 4 cell lines established. Citation Format: Shifa Javed, Bal Krishan Sharma, Sanjeev Kumar Sharma, Swati Sood, Rashmi Bagga, Shalmoli Bhattacharyya, Radhika Srinivasan. Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3347.

The Urokinase Receptor-Derived Peptide UPARANT Mitigates Angiogenesis in a Mouse Model of Laser-Induced Choroidal Neovascularization.

A mouse model of age-related macular degeneration (AMD) was used to investigate the anti-angiogenic and anti-inflammatory role of UPARANT in laser-induced choroidal neovascularization (CNV). Choroidal neovascularization was induced by laser photocoagulation, and UPARANT was intravitreally injected. Some experiments were also performed after either intravitreal injection of anti-VEGF drugs or systemic administration of UPARANT. Immunohistochemistry using CD31 antibodies was used to evaluate the area of CNV. Evans blue dye extravasation was quantitatively assessed. Transcripts of markers of outer blood retinal barrier were measured by quantitative RT-PCR, also used to evaluate angiogenesis and inflammation markers. Western blot was used to determine levels of transcription factors encoding genes involved in angiogenesis and inflammation. Levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and formyl peptide receptors (FPRs) were determined at the transcript and the protein level. Intravitreal UPARANT reduced the CNV area and the leakage from the choroid. The uPA/uPAR/FPR system was upregulated in CNV, but was not influenced by UPARANT. UPARANT recovered laser-induced upregulation of transcription factors encoding angiogenic and inflammatory markers. Accordingly, angiogenic and inflammatory factors were also reduced. UPARANT as compared to anti-VEGF drugs displayed similar effects on CNV area. UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming.

Abstract 11465: Ultrafast Cooling With Total Liquid Ventilation Attenuates Multi-organ Failure and Sepsis-like Response After Experimental Shock Induced by Aortic Cross Clamping

Introduction: Total liquid ventilation (TLV) provides ultrafast cooling and potent neuro- and cardioprotection after experimental cardiac arrest. Hypothesis: We determined whether it could also mitigate multi-organ failure and sepsis-like response after abdominal ischemia/reperfusion and subsequent shock. Methods: Anesthetized rabbits were submitted to 30 min of supracoeliac aortic cross-clamping and 300 min of reperfusion. They underwent a normothermic procedure (Control group) or rapid cooling toward 32-33°C with TLV started either before, during or after aortic clamping (PRE, PER and POST groups, respectively). TLV was maintained during 75 min after which rabbits were rapidly rewarmed and switched to conventional mechanical ventilation. Results: Control animals elicited a dramatic shock state with a low cardiac output, high demand in norepinephrine, liver failure and acute kidney injury. Cardiac output was gradually improved in POST, PER and PRE groups as compared to Control (53±3, 64±5, 90±10 and 36±8 ml/min/kg after 300 min, respectively; all p<0.05). A significant protection was also observed in the TLV groups regarding liver enzymes (ASAT, ALAT, L-FABP), acute kidney injury markers (nNAG, KIM-1, NGAL, β2-microglobulin) and renal function parameters (creatinine clearance and fractional Na+ excretion). The protection was maximal in the PRE group and achieved a lower extent in PER and POST groups when compared to Control. As example, urinary clearance of creatinine achieved 4.8±1.2, 1.3±0.9, 0.9±1.4 in PRE, PER and POST groups vs 0.5±0.4 ml/kg/min in Control (all p<0.05), respectively. Importantly, multi-organ failure was associated with elevated blood transcripts of inflammatory markers (e.g., IL-1β, IL-8, IL-10). This was attenuated in all groups submitted to TLV with no difference between PRE, PER and POST groups. As example, changes in IL-1β achieved 2.7±0.4, 3.8±0.4 and 3.2±1.1 in PRE, PER and POST groups vs 5.6±0.7 in Control (fold-change from baseline; p<0.05), respectively. Conclusions: Ultra-fast cooling with TLV attenuates shock and multi-organ failure in a model of abdominal ischemia/reperfusion. The cardiac, liver and renal protections but not anti-inflammatory effects depend upon the window of application.

High dietary cholesterol and ovariectomy in rats repress gene expression of key markers of VLDL and bile acid metabolism in liver.

BackgroundThe purpose of the study was to evaluate the effects of high dietary cholesterol in ovariectomized (Ovx) rats on several key markers of hepatic cholesterol and bile acid metabolism.MethodOvx and sham operated (Sham) rats were given either a standard diet (SD), a SD diet supplemented with 0.25 % cholesterol (SD + Chol), or a high fat diet supplemented with 0.25 % cholesterol (HF + Chol) for 5 weeks.ResultsOvx was associated with higher (P < 0.05) liver total cholesterol (TC) under the SD and the SD + Chol diet, while liver triglyceride (TG) content was higher in Ovx than in Sham rats in all 3 diet conditions. Surprisingly, the SD + Chol diet was associated with lower (P < 0.001) plasma TC and TG levels in Ovx than in Sham rats, suggesting a decrease in VLDL secretion. Accordingly, several transcripts of key markers of VLDL synthesis including microsomal TG transfer protein (Mttp) and Apob-100 were decreased (P < 0.05) in Ovx compared to Sham rats under the three dietary conditions and even more so for Mttp and Apob-100 when rats were fed the SD + Chol diet. Transcripts of bile acid transporters including bile salt export pump (Bsep) and Na + −taurocholate cotransporting polypeptide (Ntcp) were decreased by the addition of cholesterol to the SD diet in both Ovx and Sham rats.ConclusionThese results indicate that a high cholesterol feeding and ovariectomy combine to reduce the gene expression of key markers of VLDL synthesis suggesting a reduction in excretion of cholesterol from the liver.

Transcriptional profiling of dividing tumor cells detects intratumor heterogeneity linked to cell proliferation in a brain tumor model

Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2′-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate ∼2–6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and ∼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design.

Effects of moderate intensity static magnetic fields on human bone marrow-derived mesenchymal stem cells.

This study aimed to explore effects of static magnetic fields (SMFs) of moderate intensity (3-50 mT) as biophysical stimulators of proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). MSCs were exposed to SMFs of three intensities: 3, 15, and 50 mT. Proliferation was assessed by cell counting and bromodeoxyuridine incorporation, and differentiation by measuring alkaline phosphatase (ALP) activity, calcium content, mineralized nodule formation, and transcripts of osteogenic markers. Moderate intensity SMFs increased cell proliferation, ALP activity, calcium release, and mineralized nodule formation in a dose- and time-dependent manner, which peaked at 15 mT. In the same manner, they upregulated expression of osteogenic marker genes such as ALP, bone sialoprotein 2 (BSP2), collagen1a1 (COL1a1), osteocalcin (OCN), osteonectin (ON), osteopontin (OPN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) with peak at 15 mT after 14 or 21 days of exposure. Results demonstrate that moderate intensity SMFs promote proliferation and osteoblastic differentiation of MSCs. This effect could help to improve MSC responses during osseointegration between a dental implant and surrounding bone.

CGRP receptor blockade by MK-8825 alleviates allodynia in infraorbital nerve-ligated rats.

Previous data showed that, in rats, anti-migraine drugs (triptans, olcegepant) significantly reduced mechanical allodynia induced by infraorbital nerve (ION) ligation but not that evoked by sciatic nerve (SN) ligation. Whether this also occurs with MK-8825, a novel anti-migraine drug also acting through CGRP receptor blockade (but chemically unrelated to olcegepant) was tested in the present study, which also investigated possible anti-neuroinflammatory effects of this drug. Adult male Sprague-Dawley rats underwent unilateral chronic constriction injury (CCI) to either the ION or the SN, and mechanical allodynia was assessed 2 weeks later within the ipsilateral vibrissae territory or hindpaw, respectively. Transcripts of neuroinflammatory markers were quantified by real-time quantitative RT-PCR in ipsilateral trigeminal ganglion and spinal trigeminal nucleus in CCI-ION rats. Acute as well as repeated (for 4 days) administration of MK-8825 (30-100 mg/kg, i.p.) significantly reduced CCI-ION-induced mechanical allodynia but was ineffective in CCI-SN rats. CCI-ION was associated with marked up-regulation of neuronal and glial inflammatory markers (ATF3, IL6, iNOS, COX2) in ipsilateral trigeminal ganglion but not spinal trigeminal nucleus. MK-8825-induced inhibition of iNOS mRNA up-regulation probably underlay its anti-allodynic effect because pharmacological blockade of iNOS by AMT (6 mg/kg, s.c.) mimicked this effect. These data further support the idea that CGRP receptor blockade might be a valuable approach to alleviate trigeminal, but not spinal, neuropathic pain through, at least partly, an inhibitory effect on neuropathic pain-associated increase in NO production in trigeminal ganglion.

Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes

Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5(mu/mu)) and dysbindin (Bloc1s8(sdy/sdy)). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5(mu/mu) and Bloc1s8(sdy/sdy) mice. Unlike Bloc1s8(sdy/sdy), elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders.

Induction of heme oxygenase-1 with hemin reduces obesity-induced adipose tissue inflammation via adipose macrophage phenotype switching.

Adipose macrophages with the anti-inflammatory M2 phenotype protect against obesity-induced inflammation and insulin resistance. Heme oxygenase-1 (HO-1), which elicits antioxidant and anti-inflammatory activity, modulates macrophage phenotypes and thus is implicated in various inflammatory diseases. Here, we demonstrate that the HO-1 inducer, hemin, protects against obesity-induced adipose inflammation by inducing macrophages to switch to the M2 phenotype. HO-1 induction by hemin reduced the production of proinflammatory cytokines (TNF-α and IL-6) from cocultured adipocytes and macrophages by inhibiting the activation of inflammatory signaling molecules (JNK and NF-κB) in both cell types. Hemin enhanced transcript levels of M2 macrophage marker genes (IL-4, Mrc1, and Clec10a) in the cocultures, while reducing transcripts of M1 macrophage markers (CD274 and TNF-α). The protective effects of hemin on adipose inflammation and macrophage phenotype switching were confirmed in mice fed a high-fat diet, and these were associated with PPARγ upregulation and STAT6 activation. These findings suggest that induction of HO-1 with hemin protects against obesity-induced adipose inflammation through M2 macrophage phenotype switching, which is induced by the PPARγ and STAT6 pathway. HO-1 inducers such as hemin may be useful for preventing obesity-induced adipose inflammation.

Differential pharmacological alleviation of oxaliplatin-induced hyperalgesia/allodynia at cephalic versus extra-cephalic level in rodents

Previous data showed that neuropathic pain induced by mechanical lesion of peripheral nerves responds differently to alleviating drugs at cephalic versus extracephalic level. Because neuropathic pain evoked by anti-cancer drugs differs from that triggered by mechanical nerve lesion, we investigated whether differences between cephalic and extracephalic levels could also be characterized in rodents rendered neuropathic by treatment with the anti-cancer platinum derivative oxaliplatin.C57BL/6J mice received two injections and Sprague-Dawley rats three injections of oxaliplatin (10 mg/kg, i.p.) or its vehicle, with three days intervals. Supersensitivity to mechanical (von Frey filaments), cold (acetone drop) and chemical/inflammatory (formalin) stimulations was assessed in vibrissae and hindpaw territories. Transcripts of neuroinflammatory markers were quantified by real-time RT-qPCR in rat ganglia and central tissues.Oxaliplatin induced mechanical allodynia, cold hyperalgesia and chemical/inflammatory supersensitivity at both hindpaw and vibrissal levels in mice and rats. Acute treatment with gabapentin (30 mg/kg i.p.), morphine (3 mg/kg s.c.) or the 5-HT1A receptor agonist 8-OH-DPAT (0.16 mg/kg s.c.) significantly reduced oxaliplatin-induced supersensitivity in hindpaw but not vibrissal territory. In contrast, the antimigraine drugs naratriptan (0.1 mg/kg s.c.) and olcegepant (0.6 mg/kg i.v.) decreased oxaliplatin-induced supersensitivity in vibrissal territory only. Among the various markers investigated, only TRPA1 transcript was upregulated in ganglia of oxaliplatin-treated rats.These data showed that oxaliplatin induced supersensitivity to various stimuli in both cephalic and extra-cephalic territories in rodents. Regional differences in the efficacy of drugs to alleviate oxaliplatin-induced allodynia/hyperalgesia further support the idea that mechanisms underlying neuropathic pain have peculiarities at cephalic versus extra-cephalic level.

The Nogo-B-PirB Axis Controls Macrophage-Mediated Vascular Remodeling

ObjectiveNogo-B mediates vascular protection and facilitates monocyte- and macrophage-dependent vascular remodeling. PirB is an alternate receptor for Nogo-B, but a role for the Nogo-PirB axis within the vascular system has not been previously reported. We examined whether Nogo-B or PirB play a role in regulating macrophage-mediated vascular remodeling and hypothesized that endothelial Nogo-B regulates vein graft macrophage infiltration via its alternate receptor PirB.MethodsVein grafts were performed using Nogo and PirB wild type and knockout mice. Human vein grafts were similarly analyzed. The hindlimb ischemia model was performed in PirB wild type and knockout mice. Accompanying in vitro work included isolation of macrophages from PirB wild type and knockout mice.ResultsIncreased Nogo-B and PirB mRNA transcripts and protein expression were observed within mouse and human vein grafts. Both Nogo knockout and PirB knockout vein grafts showed increased wall thickness and increased numbers of F4/80-positive macrophages. Macrophages derived from PirB knockout mice had increased adhesion to fibronectin, increased EC-specific binding, and increased numbers of mRNA transcripts of M2 markers as well as MMP3 and MMP9. PirB knockout vein grafts had increased active MMP9 compared to wild type vein grafts. PirB knockout mice had increased recovery from hindlimb ischemia and increased macrophage infiltration compared to wild type mice.ConclusionsVein graft adaptation shows increased expression of both Nogo-B and PirB. Loss of PirB, or its endothelial ligand Nogo-B, results in increased inflammatory cell infiltration and vein graft wall thickening. These findings suggest that PirB regulates macrophage activity in vein grafts and that Nogo-B in the vein graft limits macrophage infiltration and vein graft thickening. PirB may play a more general role in regulating macrophage responses to vascular injury. Macrophage inhibition via Nogo-PirB interactions may be an important mechanism regulating vein graft adaptation to the arterial circulation.

Identification of CD147 (basigin) as a mediator of trophoblast functions

Does CD147 regulate trophoblast functions in vitro? CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast. CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear. A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts. CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs. CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA. Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast. This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation. This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

Nuclear transcription factor κ B activation and protein turnover adaptations in skeletal muscle of patients with progressive stages of lung cancer cachexia

Experimental models of cancer cachexia have indicated that systemic inflammation induces muscle-protein breakdown and wasting via muscular nuclear transcription factor κB (NF-κB) activation. This process may limit the efficacy of nutritional intervention. We assessed muscle NF-κB activity and protein turnover signaling in progressive stages of clinical lung cancer cachexia and assessed whether circulating factors can induce muscular NF-κB activity. Patients with lung cancer precachexia (n = 10) and cachexia (n = 16) were cross-sectionally compared with 22 healthy control subjects. mRNA transcripts of muscle proteolytic (ubiquitin proteasome system and autophagy lysosomal pathway) and myogenic markers and protein expression of PI3K/Akt, myostatin, and autophagy signaling were measured. A multiplex analysis showed the systemic inflammatory status, whereas plasma exposure to stable NF-κB-luciferase-reporter muscle cells revealed NF-κB inducibility. Compared with healthy control subjects, cachectic patients had reduced (appendicular) muscle mass (-10%), muscle fiber atrophy (-27%), and decreased quadriceps strength (-31%). Subtle alterations in the muscle morphology were also detectable in precachectic patients, without changes in body composition. Despite increased Akt phosphorylation, downstream phosphosubstrates glycogen synthase kinase 3β, mammalian target of rapamycin, and Forkhead box protein were unaltered. The expression of autophagy effectors B cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3 and microtubule-associated proteins 1A/1B light chain 3B gradually increased from precachectic to cachectic patients, without differences in E3 ubiquitin ligases. Systemic and local inflammation was evident in cachexia and intermediate in precachexia, but the plasma of both patients groups caused ex vivo muscle NF-κB activation. In lung cancer, muscular NF-κB activity is induced by factors contained within the circulation. Autophagy may contribute to increased muscle proteolysis in lung cancer cachexia, whereas the absence of downstream changes in phosphosubstrates despite increased Akt phosphorylation suggests impaired anabolic signaling that may require targeted nutritional intervention.

Adverse effect of demineralized bone powder on osteogenesis of human mesenchymal stem cells

Demineralized bone powder (DBP) has been used by clinicians for years to treat bone defects. Although DBP treatment often leads to successful bone healing, a number of studies using DBP have demonstrated poor bone formation. It is known that soluble factors released from DBP modulate bone formation. We hypothesized that DBP releases or interacts with soluble factors that modulate osteogenesis of mesenchymal stem cells (MSCs). Our in vitro study demonstrated that the expression of mRNA transcripts of bone-related markers decreased in osteogenic culture of human MSCs (hMSCs) with DBP compared to that without DBP. Using a high-throughput protein array, we identified insulin-like growth factor binding protein-1, thrombospondin, and angiostatin that were found abundant in the medium cultured with DBP. Separately, we detected a significant reduction of soluble calcium and phosphate in the DBP-present medium compared to that in the DBP-absent medium, and showed that hMSC osteogenesis was regulated by the amounts of soluble calcium and phosphate in the medium. Moreover, DBP was shown to sequester soluble calcium and phosphate in the medium, thereby depleting them from interacting with hMSCs during osteogenesis. This study provides a possible explanation to an important question associated with the use of DBP in clinical treatments.

Stem-bark of Terminalia arjuna attenuates human monocytic (THP-1) and aortic endothelial cell activation

Ethnopharmacological relevanceTerminalia arjuna – stem bark extract is traditionally used as cardiotonic in Ayurvedic medicine. Aim of the studyThe present study was aimed to evaluate the molecular basis for cardioprotective potential of Terminalia arjuna (TA) stem bark, using cell cultures of human monocytic (THP-1) and human aortic endothelial cells (HAECs). Materials and methodsInhibitory effect of alcoholic (TAAE) and aqueous (TAWE) extracts of TA-stem bark was assessed on human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LpL) and lipid peroxidation in rat (wistar) liver and heart homogenates. The patterns of H2O2 induced reactive oxygen species (ROS) generation were observed by confocal microscopy. The activities of antioxidant enzymes and reducing power of the cells were measured in a microplate reader. Gene transcripts of proinflammatory markers in THP-1 and HAECs were assayed by real time PCR and levels of inflammatory protein markers by ELISA or flow cytometry. Phytochemical analyses of TAAE and TAWE were done using liquid chromatography, coupled to mass spectrometry (LC-MS). ResultsTAAE and TAWE inhibited the lipid peroxidation and HMG-CoA reductase but had no effect on LpL. Both the extracts attenuated H2O2 mediated ROS generation in THP-1 cells by promoting catalase (CAT), glutathione peroxidase (GPx) activities, and by sustaining cellular reducing power. TAAE was highly effective in attenuating proinflammatory gene transcripts in THP-1 cells and HAECs, whereas the response to TAWE depended on the type of transcript and cell type. Both extracts decreased the levels of typical inflammatory marker proteins, viz. LPS induced tumor necrosis factor (TNF)-α secreted by THP-1 cells and TNF-α induced cell surface adhesion molecules on HAECs, namely vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Phytochemical analyses indicated the richness in phenolic compounds and terpenes of TAAE and TAWE, while revealing variability in their metabolite profile. ConclusionOur study scientifically validates the antioxidative and antiinflammatory properties of Terminalia arjuna stem bark. The marked effects on cultured human monocytic and aortic endothelial cells (HAEC) provide the biochemical and molecular basis for therapeutic potential of TA-stem bark against cardiovascular diseases (CVD).

Ovariectomy Stimulates Hepatic Fat and Cholesterol Accumulation in High-fat Diet-fed Rats

This study was designed to determine how estrogens withdrawal during a high-fat (HF) diet regimen affects liver triacylglycerol (TAG) and cholesterol accumulation. Female Sprague-Dawley rats were submitted to a HF (42% energy as fat) or a standard (SD) diet for 6 weeks before being either ovariectomized (Ovx) or sham operated (Sham). Thereafter, Ovx and Sham rats were kept on the same diet for another 6 weeks leading to euthanasia. Liver TAG content was increased (p<0.01) in Ovx rats but not by the HF diet alone. However, the combination of HF diet and Ovx resulted in a greater liver TAG accumulation (p<0.06) than that observed in Ovx-SD/SD. Measurement of molecular markers of liver lipid metabolism revealed an increase in transcripts of markers of lipid oxidation (CPT-1 and PGC1; p<0.05) in rats fed the HF diet. This increase was, however, substantially less if HF fed rats were Ovx. Liver total cholesterol levels were increased (p<0.01) only in the Ovx-HF/HF rats while plasma cholesterol levels were increased in Ovx-SD/SD and in SHAM-HF/HF and Ovx-HF/HF rats. Transcripts of molecular markers of cholesterol metabolism suggest that biliary acids synthesis (CYP7a-1) was reduced in Ovx-SD/SD and Sham-HF/HF rats and even more so in Ovx-HF/HF rats. It is concluded that the effects of a HF diet on liver TAG accumulation are especially observed in Ovx rats possibly through a reduction in hepatic lipid oxidation. The combination of Ovx and HF diet also acts synergistically to favor liver cholesterol accumulation.