Increased radiographic opacity in the region of the gallbladder can be identified in dogs with large amounts of gallbladder sediment, sludge balls, and gallbladder mucocele.

Increased soft-tissue opacity in the region of the canine gallbladder is incidentally detected on radiographs. We hypothesized that there is a difference in the detection of gallbladder sediment on radiographs depending on the amount or mobility of the sediment. In this retrospective and analytical study, we aimed to assess the ultrasonographic features of gallbladder sediment that were detected radiographically. We also aimed to assess the differences in the detection of increased opacity of the gallbladder between radiographic views. We included 223 dogs that underwent thoracic radiography, abdominal radiography, and gallbladder ultrasonography. Ultrasonographic images of the gallbladder were divided into five groups: group 1, gravity-dependent sediment occupying < 50% of the gallbladder; group 2, gravity-dependent sediment occupying ≥50%; group 3, sediment attached to the gallbladder wall; group 4, sludge ball; and group 5, gallbladder mucocele. Dogs showing increased opacity on subjective assessment of any radiographic view were recorded, and the sensitivity of radiographic views for detecting gallbladder sediment was analyzed. Of 168 dogs with gallbladder sediment, 37 had increased opacity on at least one radiographic projection. The frequency was compared as a percentage within each category, and Group 4 was the highest percentage with increased radiographic gallbladder opacity, followed by Groups 2 and 5. The sensitivity for detecting increased opacity was highest in the thoracic ventrodorsal view. Thus, in dogs with increased gallbladder opacity on radiographs, large amounts of gallbladder sediment, sludge balls, and gallbladder mucocele should be considered differential diagnoses. In addition, the thoracic ventrodorsal view is recommended to evaluate gallbladder opacity.

NAD+-boosting molecules suppress mast cell degranulation and anaphylactic responses in mice.

Nicotinamide adenine dinucleotide (NAD+) acts as a cofactor for multiple biological processes. While previous research has revealed that the NAD+ declines associated with aging contributes to an impairment of immune cells, its role in mast cell function, especially in response to an anaphylactic condition, has remained unexplored. We tested whether the restoration of cellular NAD+ concentration by the supplementation of NAD+ boosting molecules prevented mast cell degranulation and anaphylactic responses. Methods: Bone marrow derived mast cells (BMMCs) and human cord blood derived mast cells were treated with NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), and FcεRI downstream signaling was assessed. Animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) were used to investigate the effects of NAD+ precursors in the anaphylactic responses of mice. Results: Treatment of murine BMMCs and human cord blood derived mast cells with NAD+ precursors repressed intracellular signaling downstream of FcεRI, as well as the release of inflammatory cytokines and lipid mediators. The intraperitoneal administration of NMN or NR also markedly attenuated IgE-mediated anaphylactic responses in mouse models of PSA and PCA. These beneficial effects of NAD+ precursors, however, were attenuated in mast cell-specific Sirt6 knockout mice, indicating a Sirt6 dependency for their action. Conclusion: NAD+ precursors may serve as an effective therapeutic strategy that limits mast cell-mediated anaphylactic responses.

Sirtuin 6 is a negative regulator of FcεRI signaling and anaphylactic responses.

BACKGROUND: Binding IgE to a cognate allergen causes aggregation of Fcε receptor I (FcεRI) in mast cells, resulting in activation of receptor-associated Src family tyrosine kinases, including Lyn and Syk. Protein tyrosine phosphatase, receptor type C (PTPRC), also known as CD45, has emerged as a positive regulator of FcεRI signaling by dephosphorylation of the inhibitory tyrosine of Lyn. OBJECTIVE: Sirtuin 6 (Sirt6), a NAD+-dependent deacetylase, exhibits an anti-inflammatory property. It remains to be determined, however, whether Sirt6 attenuates mast cell-associated diseases, including anaphylaxis. METHODS: FcεRI signaling and mast cell degranulation were measured after IgE cross-linking in murine bone marrow-derived mast cells (BMMCs) and human cord blood-derived mast cells. To investigate the function of Sirt6 in mast cell activation in vivo, we used mast cell-dependent animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA). RESULTS: Sirt6-deficient BMMCs augmented IgE-FcεRI-mediated signaling and degranulation compared to wild-type BMMCs. Reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs received from Sirt6 knockout mice developed more severe PSA and PCA compared to mice engrafted with wild-type BMMCs. Similarly, genetic overexpression or pharmacologic activation of Sirt6 suppressed mast cell degranulation and blunted responses to PCA. Mechanistically, Sirt6 deficiency increased PTPRC transcription via acetylating histone H3, leading to enhanced aggregation of FcεRI in BMMCs. Finally, we recapitulated the Sirt6 regulation of PTPRC and FcεRI signaling in human mast cells. CONCLUSIONS: Sirt6 acts as a negative regulator of FcεRI signaling cascade in mast cells by suppressing PTPRC transcription. Activation of Sirt6 may therefore represent a promising and novel therapeutic strategy for anaphylaxis.

Inhibiting Protein Kinase Activity of Pyruvate Kinase M2 by SIRT2 Deacetylase Attenuates Psoriasis.

Signal transducer and activator of transcription 3 (STAT3) is crucial for the pathogenesis of psoriasis. Studies describe pleiotropic roles for a glycolytic enzyme pyruvate kinase M2 (PKM2) as a nuclear kinase of STAT3. However, little is known about the function of PKM2 in T helper type 17 cells in association with STAT3. In this study, we investigated whether and how SIRT2 deacetylase regulated the protein kinase function of PKM2 in T helper type 17 cell‒mediated inflammatory responses in psoriasis. Sirt2 knockout mice and wild-type littermates had psoriatic dermatitis induced by topical treatment of imiquimod or intradermal injection of recombinant IL-23. An initial downregulation of SIRT2 and an increase in PKM2 acetylation and STAT3 phosphorylation were observed in psoriasiform lesions of mice. SIRT2 directly interacted with and deacetylated PKM2 to suppress STAT3 phosphorylation. Consequently, psoriasiform skin inflammation was aggravated in Sirt2 knockout mice. Conversely, genetic re-expression of Sirt2 or pharmacological blockade of PKM2 decreased the disease severity. Flow cytometric analysis of skin tissues of Sirt2 knockout mice showed enhanced infiltration of T helper type 17 cells. Ex vivo experiments showed that SIRT2 deficiency accelerated T helper type 17 cell differentiation with the concomitant production of IL-17A and IL-22. The results suggest SIRT2-mediated PKM2 deacetylation as an effective option for psoriasis therapy.

Myeloid cell-specific sirtuin 6 deficiency delays wound healing in mice by modulating inflammation and macrophage phenotypes.

We recently reported that myeloid cell-expressed sirtuin 6 (Sirt6) plays a crucial role in M1 macrophage polarization and chemotaxis. Given the prominent role of macrophages during wound repair and macrophage heterogeneity, we hypothesized that a Sirt6 deficiency in myeloid cells would delay skin wound closure by affecting the phenotypes of macrophages in wounds. To address this question, a full-thickness excisional lesion was made in the dorsal skin of myeloid cell-specific Sirt6 knockout (KO) and wild-type mice. Wound closure was delayed in the KO mice, which exhibited less collagen deposition, suppressed angiogenesis, and reduced expression of wound healing-related genes compared to the wild-type mice. Using immunohistochemical, flow cytometric, and gene-expression analyses of macrophage subpopulations from wound tissue, we identified increased infiltration of M1 macrophages with a concomitant decrease in M2 macrophage numbers in the KO mice compared to the wild-type mice. Consistent with the in vivo wound closure defects observed in the KO mice, keratinocytes and fibroblasts treated with KO macrophage-derived conditioned medium migrated slower than those treated with wild-type macrophage-derived conditioned medium. An analysis of downstream signaling pathways indicated that impaired Akt signaling underlies the decreased M2 phenotypic switching in KO mice. These results suggest that a macrophage phenotypic switch induced by Sirt6 deficiency contributes to impaired wound healing in mice.

Characterization and application of chemical-resistant polyurethane-based enzyme and whole cell compartments.

This study presents the preparation and physical-chemical characterization of chemical resistant polyurethane-based compartments for biocatalytic application. The artificial compartments were prepared from an emulsion of polymer precursor and an aqueous phase that includes a biocatalytic reaction system. After curing, highly dispersed aqueous domains were obtained, which still contain the entire biocatalytic reaction system and remain fixed in the solid polymer preparation. The tensile and compression behavior of the prepared polymeric material is not significantly affected by the incorporation and facilitates excellent stability against various organic solvents and acid solutions. Thereby, the compartments can be used not only for enantioselective alcohol-dehydrogenase catalyzed reduction but also for a whole cell catalyzed hydrolysis of esters. Moreover, the compartmented whole-cell system was considerably stable to allow multiple reuses without a noticeable loss of catalytic activity of the incorporated whole cell catalytic reaction system.

Atopic dermatitis-like skin lesions are suppressed in fat-1 transgenic mice through the inhibition of inflammasomes.

Previous clinical trials have addressed the beneficial effects of fish oil supplementation on atopic dermatitis. Recently, we reported that fat-1 mice, which can convert n-6 to n-3 polyunsaturated fatty acids (PUFAs), are protected against allergic airway inflammation because their Th2 immune responses are suppressed. Here, we examined the effects of endogenously synthesized n-3 PUFAs on atopic dermatitis, a representative Th2-dominant allergic inflammatory disease. Mouse models of atopic dermatitis-like skin lesions were prepared by epicutaneous application of 2,4-dinitrochlorobenzene (DNCB) or house dust mite (HDM) extract to the ears. DNCB-treated fat-1 mice exhibited markedly reduced epidermal thickening, lower mast cell infiltration, and lower serum IgE and histamine compared with wild-type mice. The draining lymph nodes of fat-1 mice were substantially smaller and contained significantly smaller proportions of activated CD4+ T cells and IL-4-producing Th2 cells than those of wild-type mice. Consistent with these findings, the mRNA levels of Th2 cytokines were significantly decreased in DNCB-sensitized skin lesions of fat-1 mice. Lastly, inflammasome activation, IL-1ß production, and pyroptotic cell injury were suppressed in fat-1 mice. Similar results were observed in HDM-challenged fat-1 mice. This study confirms the results of previous clinical studies and suggests fish oil supplementation as a therapeutic strategy for atopic dermatitis-like skin lesions.

Polyphenols isolated from Broussonetia kazinoki prevent cytokine-induced ß-cell damage and the development of type 1 diabetes.

The axis of nuclear factor κB (NF-κB)-inducible NO synthase (iNOS)-nitric oxide plays a key role in cytokine- and streptozotocin-mediated pancreatic ß-cell damage. In this study, we investigated the effects of kazinol C and isokazinol D isolated from Broussonetia kazinoki on the ß-cell viability and function. RINm5F cells and primary islets were used for in vitro and ex vivo cytokine toxicity experiments, respectively. For type 1 diabetes induction, mice were injected with multiple low-dose streptozotocin (MLDS). Cytokine-induced toxicity was completely abolished in both RINm5F cells and islets that were pretreated with either kazinol C or isokazinol D. Both kazinols inhibited the NF-κB signaling pathway, thereby inhibiting cytokine-mediated iNOS induction, nitric oxide production, apoptotic cell death and defects in insulin secretion. Moreover, the occurrence of diabetes in MLDS-treated mice was efficiently attenuated in kazinol-pretreated mice. Immunohistochemical analysis revealed that the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cells and nuclear p65-positive cells were significantly decreased in kazinol-pretreated mice. Our results suggest that kazinol C and isokazinol D block the NF-κB pathway, thus reducing the extent of ß-cell damage. Therefore, kazinol C and isokazinol D may have therapeutic value in delaying pancreatic ß-cell damage in type 1 diabetes.

SPA0355 suppresses T-cell responses and reduces airway inflammation in mice.

In recent studies, SPA0355, a thiourea analog, has been demonstrated to possess strong anti-inflammatory activity. However, the mechanisms underlying the effects of SPA0355 on immune-mediated diseases have not been fully defined. The present study was designed to investigate the immunological and molecular mechanisms by which SPA0355 modulates cluster of differentiation of (CD4)(+) T-cell-mediated immune responses in allergic airway inflammation. In vitro studies have shown that SPA0355 suppresses CD4(+) T-cell activation, proliferation, and differentiation via modulation of T-cell receptor (TCR) signal transduction and cytokine-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Next, we investigated the efficacy of SPA0355 in ovalbumin (OVA)-induced allergic airway inflammation. Intraperitoneal administration of SPA0355 inhibited inflammatory cell recruitment into the airways as well as the production of Th2 cytokines in bronchoalveolar fluid and suppressed OVA-induced IgE production in serum. Additionally, SPA0355 suppressed mucin production and smooth muscle hypertrophy and prevented the development of airway hyperresponsiveness. Given that allergic airway inflammation is mainly driven by Th2 cell responses, it is highly possible that the defects in CD4(+) T-cell activation and Th2 cell differentiation in the draining lymph nodes and suppressed NF-κB activation in the lungs of SPA0355-treated mice illustrate an immunological mechanism of the preventive effect of SPA0355 on the aforementioned asthmatic characteristics. Collectively, our results suggest that SPA0355 directly modulates Th1 and Th2 responses through the suppression of multiple signaling pathways triggered by TCR or cytokine receptor stimulation, and that SPA0355 has protective effects in a murine model of allergic airway inflammation.

Effects of n-3 PUFA on the CD4⁺ type 2 helper T-cell-mediated immune responses in Fat-1 mice.

SCOPE: It has been suggested that n-3 PUFA can be used as a preventive or therapeutic strategy to control allergic asthma. But little is known about the exact mechanisms by which n-3 PUFA modulates it. Here, the effects of elevated n-3 PUFA on ovalbumin (OVA) induced airway inflammation were investigated using Fat-1 transgenic mice that can convert n-6 PUFA to n-3 PUFA endogenously. METHODS AND RESULTS: First, we tested whether Fat-1 expression modulates CD4⁺ T-cell activation, proliferation, and differentiation in vitro and found that the Fat-1 expression attenuated all of these CD4⁺ T-cell responses by suppression of T-cell receptor mediated signaling and cytokine-mediated phosphorylation of STATs. When the Fat-1 mice were sensitized and challenged with the OVA, they showed a significant decrease in the recruitment of inflammatory cells into airway, the production of Th2 cytokines, eotaxin, and mucin in the lung, and the concentration of OVA-specific IgE in the serum. Furthermore, the differentiation of CD4⁺ T cells into Th2 was also decreased in the spleen of Fat-1 mice. CONCLUSION: Our results showed that an elevated level of n-3 PUFA was effective in preventing allergic airway inflammation by modulating the activation and differentiation of CD4⁺ T cells in Fat-1 mice.

The efficacy of SPA0355 in protecting ß cells in isolated pancreatic islets and in a murine experimental model of type 1 diabetes.

Cytokines activate several inflammatory signals that mediate ß-cell destruction. We recently determined that SPA0355 is a strong anti-inflammatory compound, thus reporting its efficacy in protecting ß cells from various insults. The effects of SPA0355 on ß-cell survival were studied in RINm5F cells and primary islets. The protective effects of this compound on the development of type 1 diabetes were evaluated in non-obese diabetic (NOD) mice. SPA0355 completely prevented cytokine-induced nitric oxide synthase (iNOS) expression and cytotoxicity in RINm5F cells and isolated islets. The molecular mechanism of SPA0355 inhibition of iNOS expression involves the inhibition of nuclear factor κB and Janus kinase signal transducer and activator of transcription pathways. The protective effects of SPA0355 against cytokine toxicity were further demonstrated by normal insulin secretion and absence of apoptosis of cytokine-treated islets. In experiments with NOD mice, the occurrence of diabetes was efficiently reduced when the mice were treated with SPA0355. Therefore, SPA0355 might be a valuable treatment option that delays the destruction of pancreatic ß cells in type 1 diabetes.

Multistep enzymatic synthesis of long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils.

A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid.

Guggulsterone inhibits melanogenesis in B16 murine melanoma cells by downregulating tyrosinase expression.

In the present study, we investigated the effect of guggulsterone on melanogenesis in B16 melanoma cells and elucidated its possible mechanism of action. The effects of guggulsterone on melanogenesis were determined by assaying melanin synthesis and cellular tyrosinase activity in B16/F10 mouse melanoma cells. Guggulsterone dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis and cellular tyrosinase activity with no cytotoxicity. Decreased melanin biosynthesis was accompanied by the reduced expression of melanogenesis-related genes, such as tyrosinase, microphthalmia-associated transcription factor, tyrosinase-related protein (TRP)-1 and TRP-2. Guggulsterone also inhibited α-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. Co-incubation with chenodeoxycholic acid, a well-known farnesoid-X receptor agonist, did not affect IBMX-induced melanogenesis. These results suggest that guggulsterone exerts a melanogenic inhibitory effect through the downregulation of tyrosinase expression.

Sulfuretin protects against cytokine-induced beta-cell damage and prevents streptozotocin-induced diabetes.

NF-kappaB activation has been implicated as a key signaling mechanism for pancreatic beta-cell damage. Sulfuretin is one of the main flavonoids produced by Rhus verniciflua, which is reported to inhibit the inflammatory response by suppressing the NF-kappaB pathway. Therefore, we isolated sulfuretin from Rhus verniciflua and evaluated if sulfuretin could inhibit cytokine- or streptozotocin-induced beta-cell damage. Rat insulinoma RINm5F cells and isolated rat islets were treated with IL-1 beta and IFN-gamma to induce cytotoxicity. Incubation of cells and islets with sulfuretin resulted in a significant reduction of cytokine-induced NF-gamma B activation and its downstream events, iNOS expression, and nitric oxide production. The cytotoxic effects of cytokines were completely abolished when cells or islets were pretreated with sulfuretin. The protective effect of sulfuretin was further demonstrated by normal insulin secretion of cytokine-treated islets in response to glucose. Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining of islets. However, the diabetogenic effects of streptozotocin were completely prevented when mice were pretreated with sulfuretin. The anti-diabetogenic effects of sulfuretin were also mediated by suppression of NF-kappaB activation. Collectively, these results indicate that sulfuretin may have therapeutic value in preventing beta-cell damage.

Biosorption of nanoparticles to heterotrophic wastewater biomass.

Sorption to activated sludge is a major removal mechanism for pollutants, including manufactured nanoparticles (NPs), in conventional activated sludge wastewater treatment plants. The objectives of this work were to (1) image sorption of fluorescent NPs to wastewater biomass; (2) quantify and compare biosorption of different types of NPs exposed to wastewater biomass; (3) quantify the effects of natural organic matter (NOM), extracellular polymeric substances (EPS), surfactants, and salt on NP biosorption; and (4) explore how different surface functionalities for fullerenes affect biosorption. Batch sorption isotherm experiments were conducted with activated sludge as sorbent and a total of eight types of NPs as sorbates. Epifluorescence images clearly show the biosorption of fluorescent silica NPs; the greater the concentration of NPs exposed to biomass, the greater the quantity of NPs that biosorb. Furthermore, biosorption removes different types of NPs from water to different extents. Upon exposure to 400 mg/L total suspended solids (TSS) of wastewater biomass, 97% of silver nanoparticles were removed, probably in part by aggregation and sedimentation, whereas biosorption was predominantly responsible for the removal of 88% of aqueous fullerenes, 39% of functionalized silver NPs, 23% of nanoscale titanium dioxide, and 13% of fullerol NPs. Of the NP types investigated, only aq-nC(60) showed a change in the degree of removal when the NP suspension was equilibrated with NOM or when EPS was extracted from the biomass. Further study of carbonaceous NPs showed that different surface functionalities affect biosorption. Thus, the production and transformations in NP surface properties will be key factors in determining their fate in the environment.