PBS Control Research Articles

Two Potential Syphilis Vaccine Candidates Inhibit Dissemination of Treponema pallidum.

Syphilis, caused by the spirochete Treponema pallidum subspecies pallidum, continues to be a major public health problem worldwide. Recent increases in the number of syphilis cases, in addition to the lack of an efficient vaccine against T. pallidum for humans, highlights an urgent need for the design and development of an efficacious syphilis vaccine. Here, we assess the vaccine potential of the adhesion protein Tp0136 and the outer membrane protein Tp0663. Rabbits were subcutaneously immunized with recombinant proteins Tp0136, Tp0663, or control PBS. Immunization with Tp0136 or Tp0663 generated a strong humoral immune response with high titers of IgG, as assessed by ELISA. Moreover, animals immunized with Tp0136 or Tp0663 exhibited attenuated lesion development, increased cellular infiltration at the lesion sites, and inhibition of treponemal dissemination to distant organs compared to the unimmunized animals. These findings indicate that Tp0136 and Tp0663 are promising syphilis vaccine candidates. Furthermore, these results provide novel and important information for not only understanding the pathogenic mechanisms of spirochetes, but also the development of spirochete-specific subunit vaccines.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1β, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Type II ice structuring protein (ISP II) gene and its potential role in low temperature tolerance in Manila clam, Ruditapes philippinarum

To elucidate the function of type II ice structuring protein (ISP II) in the cold tolerance and resistance of Ruditapes philippinarum, we investigated ISP II mRNA expression, the antifreeze effect of recombinant ISP II protein, and adaptation of R. philippinarum after acute and chronic cold stress. Bacterial freeze-thaw experiments showed that ISP II significantly increased the survival rate of Escherichia coli BL21 (DE3) under low temperature stress, suggesting that it had a potential antifreeze effect. Furthermore, rISP II protein injections significantly increased the survival rate of R. philippinarum compared with a PBS control group. In addition, succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), and glutathione peroxidase (GSH-Px) activities, Malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), and immune-related genes (Syk, SCD, and SOD) were measured in three populations of clams (northern, southern, and zebra). The results indicated that ISP II was likely to improve the cold resistance and performance of R. philippinarum under cold stress. These results not only provided molecular insights for improving the low-temperature resistance and immunity of shellfish but also provided useful information for improving the low temperature tolerance of R. philippinarum in aquaculture environments.

Abstract 12620: Myeloid Cell-Specific Arginase-1 is Essential for Cardiosphere-Derived Cell Extracellular Vesicle Mediated Angiogenesis Following Acute MI

Introduction: Following a myocardial infarction (MI), macrophages play a pivotal role in initiating and resolving inflammation. Extracellular vesicles (EVs) released by cardiosphere-derived cells (CDCs) have been shown to improve cardiac function in animal models of ischemia in part via effects on immunomodulation. We have recently shown that CDC-EVs polarize macrophages toward a unique pro-angiogenic phenotype, characterized by increased expression of Arginase-1 (Arg1) and enhanced release of pro-angiogenic factors. We hypothesized that CDC-EV dependent activation of Arg1 could underlie their therapeutic benefit in vivo . Methods: LysMCre mice were crossed with Arg1 fl/fl mice to generate a myeloid cell-specific Arg1 knockout model (Arg1-KO) according to institutional approved protocols. To assess the importance of myeloid-specific Arg1 in the therapeutic efficacy of CDC-EVs in vivo , Arg1-KO mice and their fl/fl littermates (wt) were subjected to permanent LAD ligation and randomized to receive 1) CDC-EVs or 2) vehicle control. Prior to euthanasia, mice were injected with BSL-1 Lectin to measure capillary density. Ejection fraction was measured by echocardiography at 5 days post-MI and tissue samples processed for western blot, PCR, flow cytometry, and histological analyses. Results: Arg1 protein expression was significantly repressed in splenic monocytes isolated from Arg1-KO mice compared with WT mice (fold change: 0.3 ± 0.1, p<0.05, t-test, n=3). CDC-EV therapy enhanced ejection fraction in both Arg1-KO mice and WT mice following MI (Arg1-KO: 49.0 ± 3.2% vs. 40.3 ± 1.9%, WT: 51.2 ± 2.9% vs. 39.0 ± 3.8%, p<0.05, t-test, n=6-7 mice/group). CDC-EV therapy significantly increased capillary density in WT mice compared with PBS control (3642 ± 632 vs. 2125 ± 290 capillaries/mm 2 , p<0.05, t-test, n=4-5), but was unable to increase capillary density in Arg1-KO mice above the level seen with PBS treatment (2979 ± 341 vs. 3033 ± 469 capillaries/mm 2 , ns, t-test, n=4-5). Conclusions: Myeloid-specific Arg1 expression is required for CDC-EV mediated angiogenesis but occurs independently of CDC-EV beneficial effects on LVEF following acute MI.

Abstract 12532: Extracellular Vesicles Released by Cardiosphere-Derived Cells Dampen the Pro-Inflammatory Monocyte Response Through Repression of C-C Chemokine Receptor Type 2 (CCR2)

Background: Following a myocardial infarction (MI), CCR2+ monocytes migrate toward the injury site along a CCL2 concentration gradient and play an integral role in removing debris and facilitating repair. Although required to orchestrate the pro-inflammatory response, prolonged activation of CCR2+ monocytes can lead to adverse outcomes. Extracellular vesicles (EVs) derived from cardiosphere-derived cells (CDCs) have been suggested to mediate cardioprotection in part via immunomodulation. We hypothesized that CDC-EVs influence CCR2+ monocyte recruitment and function. Methods: Blood samples were obtained from healthy human donors under approved institutional IRBs. Human monocytes were isolated from peripheral blood using density gradient centrifugation (Ficoll) followed by magnetic activated cell sorting (MACS). EVs were isolated from CDCs, normal human dermal fibroblasts (NHDFs, an inert population in models of ischemia), and CDCs transfected with a miR-146a antagomir. EVs were characterized according to established guidelines for size, morphology and protein expression. Monocytes were incubated with PBS, NHDF-EVs, CDC-EVs, or miR-146a deficient CDC-EVs and analyzed for CCR2 protein expression by flow cytometry and western blot and migration along a CCL2/MCP-1 gradient by in vitro chemotaxis assays. Results: CDC-EVs significantly reduced CD14 hi CD16 - monocyte surface expression of CCR2 by 50% as quantified by flow cytometry when compared with PBS and NHDF-EVs (p<0.05, one-way ANOVA, n=4). This appeared to be mediated by miR-146a as monocytes treated with CDC-EVs deficient in miR-146a had no significant difference in CCR2 levels compared with cells treated with PBS and NHDF-EV controls. Importantly, reduced levels of monocyte CCR2 expression following CDC-EV treatment led to a greater than 35% functional reduction in monocyte migration towards CCL2 compared to PBS control (42.8 ± 3.2% vs. 68.3 ± 4.9%, p<0.05, t-test, n=4). CCR2 expression remained suppressed for 8-12 hours and could be maintained with repeated doses. Conclusions: CDC-EVs modulate classical monocyte expression of CCR2 and migration towards CCL2/MCP-1 via miR-146a, elucidating a novel mechanism of CDC-EV immunomodulation.

Abstract 9769: Bicarbonate Treatment Before Reperfusion Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibition of Spleen-Mediated Inflammatory Response

Introduction: The spleen plays an important role in mediating myocardial ischemia/reperfusion injury (IRI). Sodium bicarbonate (NaHCO 3 ) administration is reported to inhibit spleen-mediated inflammatory responses via vagus nerve activation. Hypothesis: NaHCO 3 treatment attenuates myocardial IRI in a spleen dependent-manner. Methods: C57BL/6 mice underwent 40 minutes of LCA occlusion followed by 60 minutes of reperfusion. PBS (control), NaHCO 3 (1 mEq/ml), or 5.8% NaCl of equivalent osmolality were administered intravenously (IV) 10 minutes before reperfusion at a dose of 1 μl/g. Intraperitoneal (IP) NaHCO 3 was administered 10 minutes before reperfusion at low dose (0.25 mEq/ml, 20 μl/g), high dose (0.5 mEq/ml, 20 μl/g), or as a localized topical rinse of the spleen or liver (1 mEq/ml, 1μl/g). Infarct size (IF), denoted as percentage of risk region (RR, % of LV), was measured by TTC-Phthalo blue staining. Additional groups (n=4) without IRI received IV PBS, IV NaHCO 3 , or IP NaHCO 3 splenic rinse and underwent plasma and spleen harvest 10 minutes after treatment. Results: RR was comparable among all groups with IRI. Among IV-treated groups (Figure A), IF was similar with PBS and 5.8% NaCl (51±4% and 46±3%), while IV NaHCO 3 significantly reduced IF to 25±3% (p<0.05 vs. PBS & 5.8% NaCl). Among IP-treated groups (Figure B), low-dose NaCO3, high-dose NaHCO 3 , and topical NaHCO 3 spleen rinse equally attenuated IF compared to control (p<0.05), while topical NaHCO 3 liver rinse did not reduce IF (51±2%, p = NS vs. control). IV NaHCO 3 administration elevated base excess and HCO 3 - without changing pH, pO 2 or pCO 2 . IV and topical splenic NaHCO 3 elevated plasma GLP-1 and enhanced splenic monocyte M2 polarization. Conclusions: Both IV and IP NaHCO 3 treatment attenuated IF via inhibiting spleen-mediated inflammatory responses. Sodium bicarbonate administered prior to reperfusion may a potential therapeutic adjunct for ischemia/reperfusion injury in acute coronary syndrome.

Synergism between CAR-T Cells and a Personalized Tumor Vaccine in Hematological Malignances

Synergism between CAR-T Cells and a Personalized Tumor Vaccine in Hematological Malignances

Single Agent CD117-Targeted Antibody Drug Conjugate in Combination with Lymphodepleting Antibodies Enables Allogenic Hematopoietic Stem Cell Transplantation in Mice without Chemotherapy or Radiation

Hematopoietic stem cell transplant (HSCT) is a highly effective and potentially curative treatment for malignant and non-malignant blood disorders. However, patient eligibility for this procedure can be limited due to the mortality and morbidity risks associated with current conditioning regimens, including organ toxicity, infertility, and secondary malignancies. We have developed a novel anti-CD117 antibody drug conjugate (ADC) that, in combination with lymphodepleting antibodies, can effectively condition mice to support a full allogeneic (allo) transplant. Specifically, we used a tool anti-mouse (anti-m) CD117-PBD ADC in combination with anti-mCD4 and CD8 depleting antibodies and assessed the ability of the combination to successfully condition in a murine model of allo-HSCT.Our tool ADC, anti-mCD117-PBD, was engineered for rapid clearance to enable a timely HSCT following conditioning. A single dose of 1 mg/kg robustly depleted long-term hematopoietic stem cells (LT-HSC) by 97% compared to PBS controls in C57BL/6 mice. We first evaluated the ability of single doses of 1 and 3 mg/kg anti-mCD117-PBD to condition for transplant in a congenic mouse model (C57BL/6 hosts [CD45.2+] with B6.SJL-Ptprca Pepcb/boyJ donors [CD45.1+]). We then evaluated conditioning with a single dose of 3 mg/kg anti-mCD117-PBD, in combination with 250 μg/mouse anti-mCD4 (GK1.5) and anti-mCD8 (YTS 169.4) antibody, in a fully mismatched allo transplant model (C57BL/6 hosts [CD45.2+] with CByJ.SJL(B6)-ptprca/J donors [CD45.1+]). In both studies, a dose matched non-targeted isotype-PBD (iso-PBD) was used as a negative control, while 9 Gy total body irradiation (TBI) was used as a fully myeloablative positive control. Anti-rat (anti-r) IgG isotype (LTF-2) was used as a negative lymphodepletion control antibody in the allo-HSCT study. Conditioned mice were transplanted with 2e7 whole BM cells. Lymphodepleting antibodies were dosed daily for three consecutive days before transplant. Peripheral blood chimerism was assessed over 16 weeks (congenic model) to 24 weeks (allo model), at which time donor HSC chimerism was evaluated in the terminal bone marrow.In the congenic HSCT model, conditioning recipient mice with a single dose of 3 mg/kg anti-mCD117-PBD enabled robust donor chimerism in the peripheral blood and bone marrow, as well as reconstitution of the T-, B- and myeloid cell compartments, that was comparable to the 9 Gy TBI positive control for myeloablative conditioning. Treatment with the iso-PBD control at 3 mg/kg was not effective at enabling HSC engraftment. In the fully mismatched allo-HSCT model, recipient mice conditioned with 3 mg/kg anti-mCD117-PBD in combination with 250 μg/mouse lymphodepleting anti-mCD4 + anti-mCD8 antibodies enabled full donor chimerism, achieving >90% engraftment by week 12 in the peripheral blood which was sustained through the end of the study at week 24 (Figure 1A). Multilineage reconstitution of immune cell subsets was also observed in this study, with >90% donor chimerism seen in the B cell and myeloid compartments and T cell reconstitution above 75% (Figure 1B-D). There was 99% donor HSC engraftment in the bone marrow at study termination (Figures 1E & F). These results were comparable to the chimerism observed in the 9 Gy TBI positive control mice. Groups conditioned with the non-targeting iso-PBD or anti-rIgG isotype antibody controls did not support donor engraftment in the model.In conclusion, conditioning with 3 mg/kg anti-mCD117-PBD, in combination with lymphodepleting antibodies anti-mCD4 and anti-mCD8, enables complete donor chimerism in a fully mismatched allo-HSCT murine model. This targeted conditioning approach could offer a more favorable risk-benefit profile over currently available conditioning regimens and could extend the curative potential of allo-HSCT to more patients with malignant and non-malignant diseases who otherwise would not be eligible for HSCT. [Display omitted] DisclosuresLanieri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lamothe: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

Capture the Tag: Mitigation of Biotin Interference in ELISA and Automated Immunoassays by Pre-Conjugating Biotinylated Antibodies to the Streptavidin Surface

Abstract Introduction Streptavidin to biotin binding is one of the strongest non-covalent interactions in nature, and is therefore, successfully incorporated into many immunoassays to facilitate antibody capture. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase the result depending on assay format. Currently, biotin-stripping methods capture free biotin in patient specimens by pre-incubation with streptavidin-coated microparticles. However, this approach increases turnaround time, test cost, and testing steps, which increases the chance of error. Our objective was to determine if pre-conjugating biotinylated antibodies to the assay’s streptavidin solid surface before adding patient specimen could mitigate biotin interference in ELISA and automated sandwich immunoassays. Methods We performed this study on 3 different ELISAs (CYFRA-21, NSE, S100B) and one automated assay (thyroglobulin); all are a sandwich immunoassay format. Serum pools were spiked with biotin (25 - 1000 µg/L) or PBS control. Manufacturer protocols were followed in both the ELISAs and automated assay to evaluate baseline concentration-dependent biotin interference. Mitigation of biotin interference by pre-incubation was then evaluated in the ELISAs by adding biotinylated antibody to the streptavidin-coated wells 0, 10, 15, or 60 min before adding biotin- or PBS-spiked serum specimens. For the automated assay, streptavidin-coated beads and biotinylated antibody were removed from the reagent cartridge, mixed, and incubated 4.5 hours. The mixture containing biotin-tagged antibody bound to streptavidin beads was added back to the cartridge and placed on the analyzer to evaluate spiked specimens. Lastly, we compared the pre-incubation method to a standard biotin-stripping protocol in the ELISAs to compare the effectiveness of mitigating biotin interference. Results We observed biotin interference across the three ELISAs, where 400 µg/L biotin spiked in serum pools reduced analyte detection to between 10 – 15% of the total activity using the standard assay format. Our time-course studies showed 84 – 95% recovery of the total activity when the biotinylated antibody was pre-incubated in the streptavidin-coated wells for 1 hour prior to addition of serum specimens, compared to 69 – 99% by a standard biotin stripping protocol. We extended this concept to the automated immunoassay where at 1000 µg/L biotin in the specimen, only 9 ±0.01% of the total analyte was measured by the conventional method. However, pre-mixing biotinylated antibody and streptavidin beads resulted in 97 ±0.01% of the total analyte recovery in the presence of 1000 µg/L biotin. Conclusion We have demonstrated that pre-conjugating the biotin antibody to streptavidin is as effective as biotin-stripping methods to avoid biotin interference in sandwich immunoassays that utilize the biotin-streptavidin system, with the additional benefit of optimizing turnaround times, cost, and labor. A simple change in manufacturer assay design could make immunoassays more robust against biotin interference in patient samples.

Combined therapy of platelet-rich plasma and basic fibroblast growth factor using gelatin-hydrogel sheet for rotator cuff healing in rat models

IntroductionExcellent outcomes of arthroscopic rotator cuff repair for small and medium tears have been recently reported. However, re-tears after surgery have been a common complication after surgical repair of large and massive rotator cuff tears and often occur in early postoperative phase. It was previously reported that basic fibroblast growth factor and platelet-rich plasma enhanced rotator cuff tear healing. We hypothesized that this combined therapy could enhance rotator cuff healing after rotator cuff repair in a rat model. This study aimed to evaluate the efficacy of combined therapy of platelet-rich plasma and basic fibroblast growth factor with gelatin-hydrogel sheet.MethodsTo create a rotator cuff defect, the infraspinatus tendon of Sprague Dawley rat was resected from the greater tuberosity. The infraspinatus tendons were repaired and covered with gelatin-hydrogel sheet impregnated with PBS (control group), basic fibroblast growth factor (bFGF group), platelet-rich plasma (PRP group), or both basic fibroblast growth factor and platelet-rich plasma (combined group). Histological examinations were conducted using hematoxylin and eosin, safranin O, and immunofluorescence staining, such as Isolectin B4, type II collagen at 2 weeks postoperatively. For mechanical analysis, ultimate failure load of the tendon-humeral head complex was evaluated at 6 weeks postoperatively.ResultsIn the hematoxylin and eosin staining, the tendon maturing score of the combined group was higher than that of the control group at postoperative 2 weeks. In the safranin O staining, stronger proteoglycan staining was observed in the combined group compared with the other groups at postoperative 2 weeks. Vascular staining with isolectin B4 in 3 treatment groups was significantly higher than that in the control group. Type II collagen expression in the combined group was significantly higher than those in the other groups. The ultimate failure load of the combined group was significantly higher than that of the control group.ConclusionCombined therapy of basic fibroblast growth factor and platelet-rich plasma promoted angiogenesis, tendon maturing and fibrocartilage regeneration at the enthesis, which could enhance the mechanical strength. It was suggested that combined basic fibroblast growth factor and platelet-rich plasma might enhance both tendon and bone–tendon junction healing, and basic fibroblast growth factor and platelet-rich plasma might be synergistic.

A metalloproteinase Tsdpy31 from Trichinella spiralis participates in larval molting and development

Trichinellosis is a serious food-borne zoonotic parasitic disease with global distribution, causing serious harm to public health and food safety. Molting is prerequisite for intestinal larval development in the life cycle of T. spiralis. Metalloproteinases play an important role in the molting process of T. spiralis intestinal infective larvae (IIL). In this study, the metalloproteinase Tsdpy31 was cloned, expressed and characterized. The results revealed that the Tsdpy31 was expressed at various T. spiralis stages and it was principally located in cuticle, hypodermis and embryos of the nematode. Recombinant Tsdpy31 (rTsdpy31) had the catalytic activity of natural metalloproteinase. Silencing of Tsdpy31 increased the permeability of larval new cuticle. When the mice were orally challenged with dsRNA treated- muscle larvae, the burden of intestinal adult and muscle larvae in Tsdpy31 dsRNA treatment group was significantly reduced, compared with the control green fluorescent protein (GFP) dsRNA and PBS groups (P < 0.05). Tsdpy31 may play a major role in the new cuticle synthesis and old cuticle shedding. Tsdpy31 also participates in T. spiralis embryonic development. We conclude that Tsdpy31 could be a candidate vaccine target molecule against intestinal T. spiralis ecdysis and development.

Jararhagin-C, a disintegrin-like protein, improves wound healing in mice through stimulation of M2-like macrophage, angiogenesis and collagen deposition

Jararhagin-C (Jar-C) is a disintegrin-like protein, isolated from the venom of B. jararaca, with affinity for α2β1 integrin and the ability to incite processes such as angiogenesis and collagen deposition in vivo. Thus, we raised the hypothesis that this protein could be used as a therapeutic strategy for stimulating the healing of excisional wounds in mice. Four wounds were made on the back of Swiss mice, treated with daily intradermal injections of PBS (control group) or Jar-C (200 ng). Ten animals from each experimental group were euthanized and the tissue from the wounds and skin around them were collected for further biochemical, histological and molecular analysis. Wounds treated with Jar-C showed a faster closure rate, accompanied by a reduction in neutrophil infiltrate (MPO), pro-inflammatory cytokine levels (TNF, CXCL1 and CCL2) and an accumulation of macrophages in the analyzed tissues. It was also observed a greater expression of genes associated with the phenotype of alternatively activated macrophages (M2). Concomitantly, the administration of Jar-C holds an angiogenic potential, increasing the density of blood vessels and the synthesis of pro-angiogenic cytokines (VEGF and FGF). We also observed an increase in collagen deposition, accompanied by higher levels of the pro-fibrogenic cytokine TGF-β1. Our data suggests Jar-C stimulates wound healing through stimulation of M2-like macrophage, angiogenesis and collagen deposition. Jar-C may be explored as a therapeutic strategy for wound healing, including the treatment of chronic wounds, where processes such as inflammation, angiogenesis and the deposition / remodeling of the matrix constituents are unregulated.

Effect of Monoclonal Antibody Blockade of Long Fragment Neurotensin on Weight Loss, Behavior, and Metabolic Traits After High-Fat Diet Induced Obesity.

BackgroundObesity is a major public health problem of our time as a risk factor for cardiometabolic disease and the available pharmacological tools needed to tackle the obesity pandemic are insufficient. Neurotensin (NTS) is a 13 amino acid peptide, which is derived from a larger precursor hormone called proneurotensin or Long Form NTS (LF NTS). NTS modulates neuro-transmitter release in the central system nervous, and facilitates intestinal fat absorption in the gastrointestinal tract. Mice lacking LF NTS are protected from high fat diet (HFD) induced obesity, hepatic steatosis and glucose intolerance. In humans, increased levels of LF NTS strongly and independently predict the development of obesity, diabetes mellitus, cardiovascular disease and mortality. With the perspective to develop therapeutic tools to neutralize LF NTS, we developed a monoclonal antibody, specifically inhibiting the function of the LF NTS (LF NTS mAb). This antibody was tested for the effects on body weight, metabolic parameters and behavior in mice made obese by high-fat diet.MethodsC57bl/6j mice were subjected to high-fat diet (HFD) until they reached an obesity state, then food was switched to chow. Mice were treated with either PBS (control therapy) or LF NTS mAb at the dose of 5 mg/kg once a week (i.v.). Mice weight, plasma biochemical analysis, fat and muscle size and distribution and behavioral tests were performed during the losing weight period and the stabilization period.ResultsObese mice treated with the LF NTS mAb lost weight significantly faster than the control treated group. LF NTS mAb treatment also resulted in smaller fat depots, increased fecal cholesterol excretion, reduced liver fat and larger muscle fiber size. Moreover, mice on active therapy were also less stressed, more curious and more active, providing a possible explanation to their weight loss.ConclusionOur results demonstrate that in mice subjected to HFD-induced obesity, a blockade of LF NTS with a monoclonal antibody results in reduced body weight, adipocyte volume and increased muscle fiber size, possibly explained by beneficial effects on behavior. The underlying mechanisms as well as any future role of LF NTS mAb as an anti-obesity agent warrants further studies.

516 Late-Breaking: Iturin a Protects the Intestinal Mucosal Integrity Against E. coli Containing the Gene Coding for Heat-stable Enterotoxin B

Abstract Enterotoxigenic E. coli causes severe infectious diarrhea with high morbidity and mortality in weanling pigs mainly through the production of heat-stable enterotoxins. Iturin A, originally isolated from the insect Hyalophora cecropia, exerts strong antibiotic activity against Gram-positive and Gram-negative bacteria. Thus, our study investigated whether iturin A could protect the intestinal epithelium from the insults of E. coli Rosetta containing the gene coding for heat-stable enterotoxin b (STb-Rosetta). Twenty-eight piglets were divided into four groups and were orally administered PBS (control), 0.16 g/kg BW iturin A, 2 × 109 CFU STb-Rosetta, and 0.16 g/kg BW iturin A + 2 × 109 CFU STb-Rosetta respectively for 8 days. The intestinal epithelial morphology and barrier function were assessed by H&amp;E staining and Ussing chamber, and the growth advantage in enteroids derived from jejunal crypts of piglets in each group was used to evaluate the intestinal stem cell (ISC) activity. The results showed that iturin A increased the average daily gain (ADG) and average daily feed intake (ADFI) of piglets exposed to STb-Rosetta (P &amp;lt; 0.05). Meanwhile, STb-Rosetta-induced decrease in jejunum weight and damage to jejunal morphology and barrier function were significantly improved after iturin A supplementation (P &amp;lt; 0.05). Furthermore, the jejunal crypt cells from piglets in the iturin A + STb-Rosetta group had greater growth advantages of ISCs compared with the STb-Rosetta group, including enteroid forming efficiency, surface area, budding efficiency, and branching coefficient (P &amp;lt; 0.05). Our results indicate that iturin A protects against STb-Rosetta-induced intestinal mucosal injury, which provides an intervention strategy that regulates the function of ISCs in the high turnover-rate crypt-villous axis under ETEC infection. This study was supported by the National Natural Science Foundation of China (31872389, 32072777) and Basic and Applied Basic Research Foundation of Guangdong Province (2019B1515210021).

Cholecystokinin Receptor Antagonist Improves Efficacy of Chemotherapy in Murine Models of Pancreatic Cancer by Altering the Tumor Microenvironment.

Simple SummaryOur research team has identified the cholecystokinin-B receptor (or CCK-BR) as a novel target for treatment of pancreatic cancer. CCK-BRs are over-expressed in pancreatic cancer and activation stimulates growth in cell culture and in animal models. CCK-BRs are also expressed on pancreatic stellate cells—or the fibroblasts that are responsible for the dense fibrosis in the tumor microenvironment that impedes the penetration of chemotherapy. We show strong evidence that treatment with a CCK receptor antagonist, proglumide, decreases fibrosis in the pancreatic tumor microenvironment and increases the influx of T-cells rendering chemotherapy-resistant cancer, sensitive to gemcitabine. Mice treated with the combination therapy had fewer metastases and greater survival. Tumors from these mice had higher gemcitabine levels and novel differentially expressed genes by RNA-Seq. Proglumide is an older drug that was developed many years ago for peptic ulcer disease but is no longer in use. Repurposing this older drug may improve survival from this recalcitrant malignancy.Pancreatic cancer is resistant to chemotherapy in part due to the dense desmoplastic fibrosis surrounding the tumor, the immunosuppressive cells in the tumor microenvironment (TME), and the early rate of metastases. In this study, we examined the effects of a CCK receptor antagonist, proglumide, alone and in combination with gemcitabine in murine models of pancreatic cancer. Tumor growth rate, metastases, and survival were assessed in mice bearing syngeneic murine or human pancreatic tumors treated with PBS (control), gemcitabine, proglumide, or the combination of gemcitabine and proglumide. Excised tumors were evaluated histologically for fibrosis, immune cells, molecular markers, and uptake of chemotherapy by mass spectroscopy. Peripheral blood was analyzed with a microRNAs biomarker panel associated with fibrosis and oncogenesis. Differentially expressed genes between tumors of mice treated with gemcitabine monotherapy and combination therapy were compared by RNAseq. When given in combination the two compounds exhibited inhibitory effects by decreasing tumor growth rate by 70%, metastases, and prolonging survival. Proglumide monotherapy altered the TME by decreasing fibrosis, increasing intratumoral CD8+ T-cells, and decreasing arginase-positive cells, thus rendering the tumor sensitive to chemotherapy. Proglumide altered the expression of genes involved in fibrosis, epithelial–mesenchymal transition, and invasion. CCK-receptor antagonism with proglumide renders pancreatic cancer susceptible to chemotherapy.

Chitosan nanoparticle formulation attenuates poly (I:C) induced innate immune responses against inactivated virus vaccine in Atlantic salmon (Salmo salar)

Many vaccine formulations, in particular vaccines based on inactivated virus, needs adjuvants to boost immunogenicity. In aquaculture, mineral and plant oil are used as adjuvant in commercial vaccines, and the advent of oil-adjuvanted vaccines was crucial to aquaculture development. Nevertheless, some of these approved vaccines display suboptimal performance in the field compared to experimental conditions. Therefore, there is a need to improve adjuvants and delivery methods for fish vaccines against viruses. We used RNA sequencing of Atlantic salmon head kidney to analyse the difference in gene expression 24 h after injection of different experimental vaccine formulations. We compared five different formulations in addition to a PBS control: inactivated virus alone (group V), soluble poly (I:C) (group P), nanoparticles containing poly (I:C) (group N), soluble poly (I:C) + inactivated virus (group PV) and finally nanoparticles containing poly (I:C) + inactivated virus (group NV). Our results showed poly (I:C)’s ability as adjuvant and its capacity influence innate immune genes expression in Atlantic salmon. Soluble poly (I:C) upregulated multiple immune related genes and was more effective compared to poly (I:C) formulated into chitosan nanoparticles (more than 10 fold increase in differentially expressed genes, DEGs). However, inclusion of inactivated ISA virus in the nanoparticle vaccine, increased the number of DEGs fivefold suggesting a synergistic effect of adjuvant and antigen. Our results indicate that the way poly (I:C) is formulated and the presence of antigen is important for the magnitude of the innate immune response in Atlantic salmon.

Effects of four different adjuvants separately combined with Aeromonas veronii inactivated vaccine on haematoimmunological state, enzymatic activity, inflammatory response and disease resistance in crucian carp

The purpose of the current study was to explore the immunomodulatory effects of different adjuvants combined with inactivated vaccines under Aeromonas veronii TH0426 infection in crucian carp. This study explored the best conditions for A. veronii as an inactivated vaccine, and included an animal safety test. Furthermore, we expressed the flagellin FlaA of the A. veronii TH0426 strain for use as an adjuvant supplemented in the diet. Crucian carp were fed 12 different experimental diets for 35 days, including the administration of 10 different adjuvants and inactivated vaccine combinations (50% aluminum hydroxide gel and inactivated vaccine combination, and inactivated vaccine with 20%, 30%, or 50% glucan, astragalus polysaccharide or flagellin), inactivated vaccine alone, and PBS control without adjuvant and inactivated vaccine. After the 42 day feeding trials, the fish were challenged with A. veronii TH0426, and the survival rate over 14 days was recorded. In addition, flagellin FlaA can be expressed normally in large amounts. All experimental groups produced higher levels of IgM serum titres than the control group in the different feeding cycles. Moreover, the activity of serum ACP, AKP, SOD, and LZM, and the expression of inflammatory factors were significantly increased in the experimental groups compared with the control group. The results of qRT-PCR analysis showed that the transcription levels of the IL-10, IL-1β, IFN-γ and TNF-α genes in heart, liver, spleen and kidney tissues were significantly enhanced by adjuvant treatment, indicating that the addition of adjuvants can significantly promote the body's inflammatory response. In addition, the phagocytic activity of leukocytes in each adjuvant treated group was significantly enhanced compared to that in the groups without adjuvant. After the A. veronii challenge, the survival rate of all adjuvant-treated groups was significantly higher than that of the control group, and the 50% flagellin adjuvant group had the highest rate of 78.37%. Overall, our findings strongly indicate that adjuvants not only significantly improve the body's immunity, but also exhibit a strong anti-infection ability. Importantly, this work provides a new perspective for the prevention and control of aquaculture diseases.

Characterization of a novel cysteine protease in Trichinella spiralis and its role in larval intrusion, development and fecundity

The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.

Mechanism of high expression of high mobility group protein 1 in a rat model of knee osteoarthritis

To investigate the molecular mechanism triggering pyroptosis of synovial fibroblast-like synoviocytes(FLSs)and the release of high mobility group protein 1(HMGB1)in a rat model of knee osteoarthritis(KOA). Twelve SD rats were randomized equally into blank control group without any treatment and KOA group with anterior cruciate ligament amputation (ACLT) to induce KOA.HE staining and Mankin score were used to evaluate the damage of knee cartilage.Western blotting was used to detect the expression of pyroptosis-related proteins and HMGB1 in the synovial tissue.In the cell experiment, rat FLSs were treated with PBS (control group), LPS+ATP (to induce cell pyroptosis), or LPS+ATP+siRNAs (to inhibit pyroptosis of the FLSs), and the cellular expressions of apoptosis-related proteins and HMGB1 were detected using Western blotting; the level of HMGB1 in the culture supernatant was detected with ELISA. In the rat models of KOA, the expressions of pyroptosis-related proteins and HMGB1 in the synovial tissue and Mankin score were significantly increased as compared with those in the control group(P < 0.05).In cultured rat FLSs, the expressions of apoptosis related proteins and HMGB1 were significantly higher in the pyroptosis group than in the control group and in cells transfected with the siRNAs targeting NLRP1, NLRP3, ASC and caspase-1(P < 0.05).The protein level of HMGB1 in the culture supernatant was significantly higher in pyroptosis group than in the control and siRNA groups (P < 0.05). In the pathological process of KOA, NLRPs inflammasome-mediated FLS pyroptosis causes massive release of HMGB1, which is associated with the activation of the downstream molecule caspase-1.

Blocking Jak/STAT signalling using tofacitinib inhibits angiogenesis in experimental arthritis

ObjectiveDuring rheumatoid arthritis (RA), the angiogenic processes, occurring with pannus-formation, may be a therapeutic target. JAK/STAT-pathway may play a role and the aim of this work was to investigate the inhibiting role of a JAK-inhibitor, tofacitinib, on the angiogenic mechanisms occurring during RA.MethodsAfter ethical approval, JAK-1, JAK-3, STAT-1, STAT-3 and VEGF expression was evaluated on RA-synovial-tissues. In vitro, endothelial cells (ECs), stimulated with 20 ng/ml of VEGF and/or 1 μM of tofacitinib, were assessed for tube formation, migration and proliferation, by Matrigel, Boyden chamber assay and ki67 gene-expression. In vivo, 32 mice received collagen (collagen-induced arthritis (CIA)) and 32 mice PBS (control). At day 19, CIA and controls mice were divided: 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day 35, the arthritis score, the thickness of paw joints and the serum levels of VEGF and Ang-2 were evaluated.ResultsThe expression of JAK-1, JAK-3, STAT-1, STAT-3 and VEGF in synovial tissue of RA-patients were significantly higher than healthy controls. In vitro, tofacitinib inhibited the ECs ability to form vessels, to proliferate and to migrate. In vivo, administration of tofacitinib prevented the increase of the arthritis score, the paw thickness, the synovial vessels and VEGF and Ang-2 serum-accumulation, when compared to CIA without tofacitinib.ConclusionsWe explored the anti-angiogenic role of tofacitinib, reporting its ability to inhibit in vitro the angiogenic mechanisms of ECs and in vivo the formation of new synovial vessels, occurring in CIA model. These findings suggest that the therapeutic effect of tofacitinib during RA may be also related to its anti-angiogenic activity.