Treatment Invitro Research Articles

Interleukin-17 Receptor E and C-C Motif Chemokine Receptor 10 Identify Heterogeneous T Helper 17 Subsets in a Mouse Dry Eye Disease Model

Dry eye disease (DED) features the inflammatory response of the ocular surface. Pro-inflammatory T helper 17 (Th17) cells are important for the pathogenesis of DED. In the present study a mouse DED model was used to discover two Th17 subsets in draining lymph nodes and conjunctivae based on the expression of IL-17 receptor E (IL-17RE) and CCR10: IL-17RElowCCR10- Th17 and IL-17REhighCCR10+ Th17. IL-17REhighCCR10+ Th17 expressed more retinoic acid-related orphan receptor gamma t but fewer T-box-expressed-in-T-cells than IL-17RElowCCR10- Th17. In addition, the former expressed higher IL-17A, IL-21, and IL-22 but fewer IFN-γ than the latter. Further analysis showed that IL-17REhighCCR10+ Th17 did not express IFN-γ invivo, whereas IL-17RElowCCR10- Th17 contained IFN-γ-expressing Th17/Th1 cells. Moreover, IL-17REhighCCR10+ Th17 possessed more phosphorylated p38 mitogen-activated proteinkinase (MAPK) and Jnk than IL-17RElowCCR10- Th17, suggesting higher activation of MAPK signaling in IL-17REhighCCR10+ Th17. Invitro treatment with IL-17C effectively maintained IL-17A expression in Th17 cells through p38 MAPK rather than Jnk MAPK. Furthermore, the adoptive transfer of the two Th17subpopulations indicated their equivalent pathogenicity in DED. Interestingly, IL-17REhighCCR10+ Th17 cells were able to phenotypically polarize to IL-17RElowCCR10- Th17 cells invivo. In conclusion, the current study revealed novel Th17 subsets with differential phenotypes, functions, and signaling status in DED, thus deepening the understanding of Th17 pathogenicity, and exhibited Th17 heterogeneity in DED.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by invitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.

Citrus rootstock characterization against citrus canker and evaluation of antibiotics effect against Xanthomonas axonopodis pv. citri

Kamran khan, Sufian Ikram, Ammara Noreen, Muhammad Ashfaq, Muhammad Jafar Jaskani & Waqar Shafqat.2021.Citrus rootstock characterization against citrus canker and evaluation of antibiotics effect against Xanthomonas axonopodis pv. citri.Journal of Innovative Agriculture.volume 8, issue 2, Pages 1-8. Citrus canker (X. axonopodis pv. citri) widespread bacterial disease which limits citrus productivity and causes quality deterioration. Citrus canker is mostly a leaf-spotting and fruit rind-blemishing disease, but under highly favorable conditions, infections cause defoliation, shoot dieback, and fruit drop. Citrus rootstocks exert a high degree of influence to scions in fruit production and plant susceptibility to diseases including citrus canker. Keeping in view the importance of citrus rootstock present study was planned to evaluate the performance of citrus rootstocks against citrus canker and antibiotics response to control X. axonopodis pv. citri. Eleven exotic citrus rootstocks were selected to evaluate against canker which was inoculated by grafting. Disease index was observed after 9 months of inoculation. Five different commercially available antibiotics were selected for the Invitro treatment, applied at 300, 400 and 500ppm. Inhibition zone was observed at four time points (24, 48, 72 and 96 hours). Kirumakki showed maximum resistance followed by Rangpur Poona nucellar and X639 while Rough lemon was highly susceptible. In Invitro evaluation maximum inhibition zone was expressed by Kanamycin sulphate at 500 ppm followed by Streptomycin sulphate. Lincomycin showed minimum inhibition zone as compared with control (Distilled water, No inhibition). Biochemical analysis exhibited that the disease index (%) decrease with increase of total phenolics contents and total protein contents. Whereas disease index had increasing trend with TSS. This study concludes that according to rootstocks evaluation Kirumakki and Rangpur Poona nucellar showed resistant performance while, on other hand Kanamycin Sulphate having maximum inhabitation against canker.

In Vitro Inhibitory Effect of Succinic Acid on T-Cell Acute Lymphoblastic Leukemia Cell Lines

Although several treatment regimens for T-cell acute lymphoblastic leukemia (T-ALL), trouble is still ongoing that relapse of disease after therapies in both pediatric and adult patients. Hence, the demand for new alternative therapeutics that are antiproliferative for cancer cells but do not harm healthy cells in treatments is increasing day by day. This study aimed to investigate whether succinic acid show anti-proliferative and apoptotic effect of on T-ALL cell lines. Time and dose-dependent effects of succinic acid on T-ALL cell lines were determined by using WST-1, caspase-3/ bicinchoninic acid (BCA), and Annexin V-Fluorescein isothiocyanate (FITC) assays. We included the MRC-5cell line in our study as a healthy control group. Based on our findings, 25 and 50mmol dosages of succinic acid has shown an apoptotic effect on T-ALL cell lines for 48h treatment. Also, it has shown that after 48h exposure of 25 and 50mmol dosages of succinic acid has no significant cytotoxic effect in healthy MRC-5cells. Apoptotic activity of succinic acid on CCRF-CEM cell line was caspase-3 dependent but not for MOLT-4. As a consequence, succinic acid was found to effect for T-ALL treatment invitro and might also enlighten new study fields for different cancer experiments.

CRIg+ Macrophages Prevent Gut Microbial DNA–Containing Extracellular Vesicle–Induced Tissue Inflammation and Insulin Resistance

Liver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown. We performed studies with CRIg-/-, C3-/-, cGAS-/-, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA-containing extracellular vesicles (mEVs) were adoptively transferred into CRIg-/-, C3-/-, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated. Gut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, invitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA-mediated inflammatory responses. Deficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

Restraint stress and elevation of corticotrophin-releasing hormone in female mice impair oocyte competence through activation of the tumour necrosis factor α (TNF-α) system.

Studies have observed that restraint stress (RS) and the associated elevation in corticotrophin-releasing hormone (CRH) impair oocyte competence by triggering apoptosis of ovarian cells but the underlying mechanisms are largely unclear. Although one study demonstrated that RS and CRH elevation triggered apoptosis in ovarian cells and oocytes via activating Fas/FasL signalling, other studies suggested that RS might damage cells by activating other pathways as well as Fas signalling. The objective of this study was to test whether RS and CRH elevation impairs oocytes by activating tumour necrosis factor α (TNF-α) signalling. Our invivo experiments showed that RS applied during oocyte prematuration significantly increased expression of TNF-α and its receptor (TNFR1) while inducing apoptosis in both oocytes and mural granulosa cells (MGCs). Invitro treatment of MGCs with CRH significantly increased their apoptotic percentages and levels of TNF-α and TNFR1 expression. Invitro knockdown by interfering RNA, invivo knockout of the TNF-α gene or injection of TNF-α antagonist etanercept significantly relieved the adverse effects of RS and CRH on apoptosis of MGCs and/or the developmental potential and apoptosis of oocytes. The results suggest that RS and CRH elevation in females impair oocyte competence through activating TNF-α signalling and that a TNF-α antagonist might be adopted to ameliorate the adverse effects of psychological stress on oocytes.

Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes

Keratitis-ichthyosis-deafness (KID) syndrome is a severe, untreatable condition characterized by ocular, auditory, and cutaneous abnormalities, with major complications of infection and skin cancer. Most cases of KID syndrome (86%) are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein Cx26, which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant allele-specific small interfering RNA could rescue the cellular phenotype in patient keratinocytes (KCs). A KID syndrome cell line (KID-KC) was established from primary patient KCs with a heterozygous p.D50N mutation. This cell line displayed impaired gap junction communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp, and neurobiotin uptake assays. A human-murine chimeric skin graft model constructed with KID-KCs mimicked patient skin invivo, further confirming the validity of these cells as a model. Invitro treatment with allele-specific small interfering RNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wild-type allele. This corrected both gap junction and hemichannel activity. Notably, allele-specific small interfering RNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of allele-specific small interfering RNA in treating KID syndrome and other dominant genetic conditions.

Optimized conditions for the supplementation of human-induced pluripotent stem cell cultures with a GSK-3 inhibitor during embryoid body formation with the aim of inducing differentiation into mesodermal and cardiac lineage

We optimized the conditions for the differentiation of human induced pluripotent stem cells (hiPSCs) into mesoderm lineage-committed cells by supplementing the cultures with CHIR, a selective GSK-3 inhibitor, during embryoid body (EB) formation. Invitro treatment with 4μM CHIR during the late 2 days of a 4-day suspension culture period was most effective at promoting mesodermal differentiation. The resulting EBs showed a significant increase in the expression levels of mesoderm-associated genes (WNT3A, T, DKK1, GATA4, FOXC1, and MESP1) and a maintenance of OCT3/4 and NANOG expressions. Upon subsequent differentiation into a cardiac cell lineage, these EBs were shown to generate contractile cardiomyocytes. When shortening the CHIR treatment period to 1 day, the resulting EBs showed reduced expression of mesoderm-associated genes in comparison to the 2-day CHIR treatment. In particular, the expression level of FOXC1 in the 1-day CHIR-treated EBs was much lower than that of the 2-day CHIR-treated EBs. When the treatment period with CHIR was extended to 4 days, the resulting EBs presented significantly reduced expression of WNT3A, OCT3/4, and NANOG upon CHIR concentrations above 4μM. Similarly, when CHIR treatment was conducted after the formation of EBs, the effectiveness of the GSK-3 inhibitor was reduced compared to a treatment performed during EB formation. Our results indicate that spatiotemporal constraints associated with EB formation, i.e., three-dimensional structuration and cell development in EBs, should be taken into account when designing EB formation-based differentiation protocol involving CHIR treatment.

Age-induced oxidative stress impairs adipogenesis and thermogenesis in brown fat.

It is well-established that the mass and function of human brown adipose tissue (BAT) declines with age. A key factor involved in age-related impairment of BAT is oxidative stress; however, there is a paucity of studies to date that have explored this relationship. Here, we characterized the age-related molecular and functional alterations in BAT invivo in mice of different ages, and treated human brown adipocytes with H2 O2 to dissect the direct effect of oxidative stress invitro. We further explored the structural and functional changes in BAT in an oxidative stress-induced mouse model of aging. We found that the progressive deterioration of BAT was linked to oxidative stress, and observed that the adipogenesis and thermogenic program were significantly impaired upon H2 O2 treatment invitro. Moreover, antioxidant supplementation (e.g., vitamin E) attenuated oxidative stress and rescued BAT activity decline, suggesting that age-related injury in BAT function can be partly alleviated by antioxidant treatment. Finally, we found that oxidative stress-induced BAT dysfunction is linked to the enhancement of autophagy. These results point to oxidative stress as being an important factor in age-dependent functional impairment of BAT.

Invitro Treatment of Blue Ticks Using a Phytosubstance

Ticks are some of the most devastating ectoparasites of livestock causing several tick borne diseases. Management of ticks is increasing getting difficulty using synthetic acaricides due their rapid tolerance to the synthetic acaricides. Botanical acaricides are now used as an alternative management strategy to curb such problems. The blue tick, Rhiphicephalus (Boophilus) decolaratus, is a one-host tick that parasitizes cattle and vectors a debilitating protozoan parasite that causes babesiosis. Acaricidal properties of thyme were investigated on the cattle blue tick, R.B. decolaratus using various concentrations to establish the lethal dose concentration. The acaridal activity of thyme on ticks was monitored and recorded at two-hour intervals for 48 hours. Tick mortality was observed at all concentrations except the control. The lowest mortality rate was observed for 10μl/cm3 while 40μl/cm3 resulted in total tick mortality. The LC50 was established as 1,9μl/cm3. Significant differences (p<0.01) were observed between concentrations, however the mortality was directly proportional to thyme concentration. The thyme oil dehydrated the ticks leading to death within 48 hours. The study thus concludes that thyme oil can be used as a complimentary or alternative acaricide in an integrated livestock program or where organic means are enforced or to as a way of minimizing tolerance of ticks to conventional acaricides.

PML/RARA inhibits expression of HSP90 and its target AKT.

Essential for cell survival, the 90kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that invitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.

Targeting the Epichaperome As an Effective Precision Medicine Approach in a Novel PML-SYK Fusion Acute Myeloid Leukemia

The epichaperome is a new cancer target defined, in part, by changes in the interaction strength between chaperone and co-chaperone proteins to form stable hyperconnected networks that support oncoprotein stability and are vital for tumor survival (Nature 2016, Nature Rev Cancer 2018 and Nature Med 2018). Cancers with this altered chaperone configuration may become susceptible to drugs that target the epichaperome, such as the inhibitor PU-H71. We have developed a novel flow cytometry-based test, the PU-FITC binding assay, to evaluate epichaperome levels at the single cell level and identify patients who are most likely to respond to PU-H71 treatment.A 61-year-old woman was diagnosed with an accelerated phase myeloproliferative neoplasm in 2013 after years of leukocytosis, arthritis, urticaria, and vasculitis. Cytogenetic analysis revealed t(9;15) involving the PML gene. Molecular studies were negative for BCR-ABL, PDGFRA/B and FGFR1 rearrangements as well as JAK2 mutation. Mutations in ETV6 and multiple ASXL1 subclones were present. She was treated with hydroxyurea and in Aug 2013 underwent matched unrelated donor allogeneic stem cell transplantation conditioned with Fludarabine/Melphalan, followed by 3 cycles of vidaza in 2014 for early recurrence. In Jan 2016, she relapsed and was treated with hydroxyurea. She also had painful ulcerations of toes, thought to be an atypical presentation of graft versus host disease complicated by her underlying Raynaud's disease and treated successfully with ruxolitinib. During 2017, she had bone marrow (BM) evidence of recurrent disease, skin lesions consistent with recurrent disease, and significant splenomegaly, but since neutrophils and platelets were preserved, she did not require transfusions and she had no bleeding or infections, she was maintained on hydrea, prednisone and ruxolitinib. In 2017, BM biopsy showed progression to AML with fibrosis. She developed progressive splenomegaly and weight loss and was treated with 2 cycles of decitabine without response. The patient was sent for precision medicine evaluation. Whole-exome sequencing and RNA-seq was performed with a control buccal swab. No somatic alterations in clinically relevant genes were found. However, a novel fusion protein, PML-SYK, was detected and validated by FISH and PCR. We found constitutive Syk, Stat5, Erk and ribosomal S6 kinase phosphorylation. Elevated epichaperome levels were found in the cell populations bearing the translocation, suggesting sensitivity to PU-H71. Also, invitro treatment of patient's cells with PU-H71 resulted in cell death and decreased colony formation. These findings confirm preclinical data in AML in which a relationship between a hyperactive signalosome and epichaperome expression was observed (Cell Reports 2015). Based on the poor prognosis, lack of effective therapies, and laboratory data suggesting sensitivity to PU-H71, the patient was granted compassionate access to this medication by the FDA. After 16 doses of PU-H71 at 300 mg/m2 over 3 months, the patient has attained complete remission, with normalization of peripheral blood counts and <5% marrow blasts. Splenomegaly and all constitutional symptoms have completely resolved. Therapy is ongoing. Phosphorylation of SYK in blast as stem cell populations decreased after treatment.In summary, a novel flow cytometry-based PU-FITC binding assay to evaluate epichaperome levels at the single cell level successfully identified a patient predicted to respond to treatment with PU-H71. This poor prognosis AML patient who had relapsed after allogeneic stem cell transplantation is presently in remission and the assay is being used to screen other potential patients. [Display omitted] DisclosuresRoboz:Sandoz: Consultancy; Janssen Pharmaceuticals: Consultancy; AbbVie: Consultancy; Eisai: Consultancy; Celgene Corporation: Consultancy; Sandoz: Consultancy; Celgene Corporation: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Orsenix: Consultancy; Aphivena Therapeutics: Consultancy; Novartis: Consultancy; Orsenix: Consultancy; Roche/Genentech: Consultancy; AbbVie: Consultancy; Argenx: Consultancy; Bayer: Consultancy; Roche/Genentech: Consultancy; Otsuka: Consultancy; Astex Pharmaceuticals: Consultancy; Otsuka: Consultancy; Argenx: Consultancy; Cellectis: Research Funding; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Cellectis: Research Funding; Astex Pharmaceuticals: Consultancy; Celltrion: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Daiichi Sankyo: Consultancy; Bayer: Consultancy; Eisai: Consultancy; Daiichi Sankyo: Consultancy. Morgan:Samus Therapeutics, Inc: Employment, Equity Ownership. Chiosis:Samus Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guzman:Cellectis: Research Funding.

Adult Hepatocytes Are Hedgehog-Responsive Cells in the Setting of Liver Injury: Evidence for Smoothened-Mediated Activation of NF-κB/Epidermal Growth Factor Receptor/Akt in Hepatocytes that Counteract Fas-Induced Apoptosis

Although hedgehog (Hh) signaling pathway is inactive in adult healthy liver, it becomes activated during acute and chronic liver injury and, thus, modulates the reparative process and disease progression. We developed a novel mouse model with liver-specific knockout of Smoothened (Smo LKO), and animals were subjected to Fas-induced liver injury invivo. Results showed that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Hh signaling in hepatocytes in the setting of Fas-induced injury was indicated by the fact that Jo2 treatment enhanced hepatic expression of Ptch1, Smo, and its downstream target Gli1 in control but not Smo LKO mice. Primary hepatocytes from control mice showed increased Hh signaling activation in response to Jo2 treatment invitro. On the other hand, the Smo KO hepatocytes were devoid of Hh activation and were more susceptible to Jo2-induced apoptosis. The levels of NF-κB and related signaling molecules, including epidermal growth factor receptor and Akt, were lower in Smo KO livers/hepatocytes than in control livers/hepatocytes. Accordingly, hydrodynamic gene delivery of active NK-κB prevented Jo2-induced liver injury in the Smo LKO mice. Our findings provide important evidence that adult hepatocytes become responsive to Hh signaling through up-regulation of Smo in the setting of Fas-induced liver injury and that such alteration leads to activation of NF-κB/epidermal growth factor receptor/Akt, which counteracts Fas-induced hepatocyte apoptosis.

Resveratrol ameliorates depressive disorder through the NETRIN1-mediated extracellularsignal-regulated kinase/cAMP signal transduction pathway.

Depressive disorder is a mental health disorder caused by the dysfunction of nerve regeneration, neuroendocrine and neurobiochemistry, which frequently results in cognitive impairments and disorder. Evidence has shown that resveratrol offers benefits for the treatment of depressive disorder. In the present study, the therapeutic effects of resveratrol were investigated and the potential mechanisms mediated by resveratrol were analyzed in hippocampal neuron cells. The anti‑oxidative stress and anti‑inflammatory properties of resveratrol were also examined invitro and invivo. The results revealed that resveratrol administration inhibited the inflammation in hippocampal neuron cells induced by ouabain. Oxidative stress in the hippocampal neuron cells was ameliorated by resveratrol treatment invitro and invivo. In addition, the apoptosis of hippocampal neuron cells was inhibited by the upregulation of anti‑apoptotic genes, including P53, B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2‑associated death promoter, and the downregulation of the cleaved caspase‑3 and caspase‑9. The analysis of the mechanism revealed that that resveratrol treatment suppressed the apoptosis of hippocampal neuron cells through the NETRIN1‑mediated extracellularsignal‑regulated kinase/cAMP signal transduction pathway. The results of the invivo assay showed that resveratrol treatment led to improvements in cognitive competence, learning memory ability and anxiety in a mouse model of depressive disorder induced by ouabain. In conclusion, these results indicated that resveratrol treatment had protective effects against oxidative stress and neuroinflammatory pathogenesis through the NETRIN1‑mediated extracellularsignal‑regulated kinase/cAMP signal transduction pathway, suggesting that resveratrol treatment may be a potential antidepressant agent for the treatment of depressive disorder.

Effects of Cynanchum wilfordii on osteoporosis with inhibition of bone resorption and induction of bone formation.

Cynanchum wilfordii Hemsley has been used for the treatment of musculoskeletal diseases in traditional Republic of Korean medicine. The present study investigated the effects of C. wilfordii water extract (CW) on postmenopausal osteoporosis. Female mice were used and randomly assigned into a normal group and three ovariectomized (OVX) groups: OVX with vehicle (OVX + vehicle); OVX with 17β‑estradiol (E2; 10µg/kg/day); and OVX with CW (1mg/kg/day). Oral administration of CW or E2 intraperitoneal injection began 9weeks after OVX and continued for 3weeks. Following sacrifice, bone histology, bone mineral density (BMD) and bone mineral content (BMC) of the femur were observed. Serum osteocalcin concentration was analyzed. In addition, the expression levels of osteoprotegerin (OPG) and osterix were evaluated in human osteoblast‑like Saos‑2 cells. In the lateral and medial epicondyles of the CW‑administrated group, dense and well‑formed bone marrow cells with reduced bone marrow pores were observed. CW decreased the number of tartrate resistant acid phosphatase‑positive multinucleated osteoclasts. BMD and BMC were increased following increased serum osteocalcin levels by CW treatment. The expression levels of OPG and osterix were upregulated by CW treatment invitro. The results suggested that C. wilfordii has an advantageous effect on osteoporosis and possesses the potential to be used in osteoporosis treatment.

Lipopolysaccharide inhibits hepatic gluconeogenesis in rats: The role of immune cells.

Aims/IntroductionBacterial septicemia has diverse clinical symptoms including severe hypoglycemia. However, sepsis‐induced hypoglycemia has not yet been examined in detail. The aim of the present study was to investigate the mechanisms underlying hypoglycemia in sepsis.Materials and MethodsWe induced endotoxin shock in rats using lipopolysaccharide (LPS). After an intraperitoneal injection of LPS, we measured gluconeogenesis using the pyruvate tolerance test. The effects of LPS on glucose metabolism were investigated in perfused livers and isolated hepatocytes. Furthermore, its effects on the production of inflammatory cytokines were examined in isolated splenocytes. The interaction between splenocytes and hepatocytes in response to LPS was investigated in vitro using a co‐culture of splenocytes and hepatocytes.ResultsIn the pyruvate tolerance test, the pretreatment with LPS decreased gluconeogenesis. The in vivo pretreatment of rats with LPS did not inhibit glucose production in perfused livers. The in vitro treatment of isolated hepatocytes with LPS did not decrease hepatic gluconeogenesis. Although LPS increased the production of inflammatory cytokines (tumor necrosis factor‐α, interferon‐γ, interleukin‐1β, interleukin‐6 and interleukin‐10) and nitric oxide in isolated splenocytes, only nitric oxide significantly inhibited gluconeogenesis in isolated hepatocytes. When splenocytes and hepatocytes were co‐cultured in medium containing LPS, the messenger ribonucleic acid expression of glucose‐6‐phosphatase in hepatocytes was suppressed.ConclusionsLPS reduced hepatic gluconeogenesis, at least in part, by stimulating the production of nitric oxide in splenocytes. This effect could contribute to the mechanisms responsible for septicemia‐induced hypoglycemia.

NF-κB signalling is involved in immune-modulation, but not basal functioning, of the mouse suprachiasmatic circadian clock.

Circadian rhythms are recurring near-24 hour patterns driven by an endogenous circadian timekeeping system. The master pacemaker in this system is the hypothalamic suprachiasmatic nucleus (SCN). Recently interest has been drawn to how the SCN clock responds to immune system stimulation. A major signalling component in the immune system is nuclear factor (NF)-κB. In the present study we examined the role of NF-κB in SCN function. Whilst serum shocked fibroblasts showed rhythmic nuclear localisation of p65 and p65-dependent transcription, there were no circadian changes in the SCN in expression of the NF-κB components p65, c-Rel, p-IκB or p-IKK. Chronic treatment with the NF-κB inhibitor PDTC did not impact on circadian or diurnal rhythms. Phase-shifting light pulses did not impact on SCN expression of p65, and PDTC treatment did not attenuate the behavioural or molecular response to light pulses. Peripheral treatment with lipopolysaccharide resulted in increased NF-κB component expression in the SCN. In vitro experiments with SCN slice cultures showed that treatment with NF-κB inhibitors did not markedly alter rhythmic changes in PER2::LUC expression. Further, SCN slices from nf-κb::luc mice did not show any evidence for circadian rhythms in NF-κB-mediated transcription. Experiments utilising older mice (~16months old) showed that SCN treatment invitro with PDTC resulted in increased amplitude of rhythmic PER2::LUC expression, and LPS treatment resulted in altered PER2::LUC rhythm acrophase. Overall, we interpret our results as providing evidence for the involvement of NF-κB in the suprachiasmatic circadian clock following immune stimulation, but not under basal conditions.

Tocilizumab improves the proatherothrombotic profile of rheumatoid arthritis patients modulating endothelial dysfunction, NETosis, and inflammation

Tocilizumab (TCZ) is an effective treatment for rheumatoid arthritis (RA). However, the changes that occurred after TCZ therapy on endothelial dysfunction, monocyte activity, NETosis, and oxidative stress, the principal effectors of atherosclerosis and cardiovascular disease, have not been analyzed yet. A total of 20 RA patients received 162mg per week subcutaneous TCZ for 6months. Endothelial function was measured through postocclusive hyperemia using Laser Doppler. Oxidative stress markers in monocytes and neutrophils were analyzed by flow cytometry. NETosis was measured through SYTOX staining of DNA fibers and the expression of myeloperoxidase and neutrophil elastase. Percentage of low-density granulocytes was analyzed through flow cytometry. Gene expression and phosphorylation of intracellular pathways was analyzed in monocytes. TCZ improved endothelial function and decreased oxidative stress in RA leukocytes. Percentage of low-density granulocytes and NETosis generation were reduced. The proinflammatory and prothrombotic status of RA monocytes was also reversed through a modulation of specific intracellular pathways. All these results were recapitulated after invitro treatment with TCZ of monocytes and neutrophils purified from RA patients and cocultured with endothelial cells. TCZ might reduce the proatherothrombotic profile in RA patients through the restoration of the endothelial function, oxidative stress reduction, inhibition of monocytes' prothrombotic and inflammatory profile, and abridged NETosis generation.

Hematopoietic-Derived Galectin-3 Causes Cellular and Systemic Insulin Resistance

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. Invitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.

Cardiotrophin-1 decreases intestinal sugar uptake in mice and in Caco-2 cells.

Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines with a key role in glucose and lipid metabolism. In the current investigation, we examined the invivo and invitro effects of CT-1 treatment on intestinal sugar absorption in different experimental models. rCT-1 effects on α-Methyl-D-glucoside uptake were assessed in everted intestinal rings from wild-type and CT-1(-/-) mice and in Caco-2 cells. rCT-1 actions on SGLT-1 expression in brush border membrane vesicles and the identification of the potential signalling pathways involved were determined by Western blot. In vivo administration (0.2mgkg(-1) ) of rCT-1 caused a significant decrease on α-Methyl-D-glucoside uptake in everted intestinal rings from wild-type and CT-1(-/-) mice after short-term and long-term treatments. Similarly, invitro treatment (1-50ngmL(-1) ) with rCT-1 reduced α-Methyl-D-glucoside uptake in everted intestinal rings. In Caco-2 cells, rCT-1 treatment (20ngmL(-1) , 1 and 24h) lowered apical uptake of α-Methyl-D-glucoside in parallel with a decrease on SGLT-1 protein expression. rCT-1 promoted the phosphorylation of STAT-3 after 5 and 15min treatment, but inhibited the activation by phosphorylation of AMPK after 30 and 60min. Interestingly, pre-treatment with the JAK/STAT inhibitor (AG490) and with the AMPK activator (AICAR) reversed the inhibitory effects of rCT-1 on α-Methyl-D-glucoside uptake. AICAR also prevented the inhibition of SGLT-1 observed in rCT-1-treated cells. CT-1 inhibits intestinal sugar absorption by the reduction of SGLT-1 levels through the AMPK pathway, which could also contribute to explain the hypoglycaemic and anti-obesity properties of CT-1.