An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

TLR signaling adapter BCAP regulates inflammatory to reparatory macrophage transition by promoting histone lactylation.

Macrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a proinflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage-specific deletion of BCAP fail to recover from and succumb to dextran sulfate sodium-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP-deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3ß, which contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be a critical cell-intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.

TGF-ß Regulates Collagen Type I Expression in Myoblasts and Myotubes via Transient Ctgf and Fgf-2 Expression.

Transforming Growth Factor ß (TGF-ß) is involved in fibrosis as well as the regulation of muscle mass, and contributes to the progressive pathology of muscle wasting disorders. However, little is known regarding the time-dependent signalling of TGF-ß in myoblasts and myotubes, as well as how TGF-ß affects collagen type I expression and the phenotypes of these cells. Here, we assessed effects of TGF-ß on gene expression in C2C12 myoblasts and myotubes after 1, 3, 9, 24 and 48 h treatment. In myoblasts, various myogenic genes were repressed after 9, 24 and 48 h, while in myotubes only a reduction in Myh3 expression was observed. In both myoblasts and myotubes, TGF-ß acutely induced the expression of a subset of genes involved in fibrosis, such as Ctgf and Fgf-2, which was subsequently followed by increased expression of Col1a1. Knockdown of Ctgf and Fgf-2 resulted in a lower Col1a1 expression level. Furthermore, the effects of TGF-ß on myogenic and fibrotic gene expression were more pronounced than those of myostatin, and knockdown of TGF-ß type I receptor Tgfbr1, but not receptor Acvr1b, resulted in a reduction in Ctgf and Col1a1 expression. These results indicate that, during muscle regeneration, TGF-ß induces fibrosis via Tgfbr1 by stimulating the autocrine signalling of Ctgf and Fgf-2.

Transcriptional profiling identifies caspase-1 as a T cell-intrinsic regulator of Th17 differentiation.

Dendritic cells (DCs) are critical for the differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells remains elusive. Here, we used DCs stimulated with specific pathogens to prime CD4 T cells in vitro and found that these T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. More specifically, the transcriptome of in vitro C. rodentium-primed Th17 cells resembled that of Th17 cells primed following infection in vivo but was remarkably distinct from cytokine-polarized Th17 cells. We identified caspase-1 as a unique gene up-regulated only in pathogen-primed Th17 cells and discovered a critical role for T cell-intrinsic caspase-1, independent of inflammasome, in optimal priming of Th17 responses. T cells lacking caspase-1 failed to induce colitis or confer protection against C. rodentium infection due to suboptimal Th17 cell differentiation in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.

T cells instruct myeloid cells to produce inflammasome-independent IL-1ß and cause autoimmunity.

The cytokine interleukin (IL)-1ß is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1ß requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1ß production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1ß production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1ß synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1ß cleavage. The T cell-instructed IL-1ß resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR-Fas-caspase-8-dependent pathway provides a mechanistic explanation for IL-1ß production and its consequences in CD4+ T cell-driven autoimmune pathology.

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3-4 M KCl. However, specific activity values were two-fold higher in the presence of 3-4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

STING-associated vasculopathy develops independently of IRF3 in mice.

Patients with stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) develop systemic inflammation characterized by vasculopathy, interstitial lung disease, ulcerative skin lesions, and premature death. Autosomal dominant mutations in STING are thought to trigger activation of IRF3 and subsequent up-regulation of interferon (IFN)-stimulated genes (ISGs) in patients with SAVI. We generated heterozygous STING N153S knock-in mice as a model of SAVI. These mice spontaneously developed inflammation within the lung, hypercytokinemia, T cell cytopenia, skin ulcerations, and premature death. Cytometry by time-of-flight (CyTOF) analysis revealed that the STING N153S mutation caused myeloid cell expansion, T cell cytopenia, and dysregulation of immune cell signaling. Unexpectedly, we observed only mild up-regulation of ISGs in STING N153S fibroblasts and splenocytes and STING N154S SAVI patient fibroblasts. STING N153S mice lacking IRF3 also developed lung disease, myeloid cell expansion, and T cell cytopenia. Thus, the SAVI-associated STING N153S mutation triggers IRF3-independent immune cell dysregulation and lung disease in mice.

Mitotic Phosphorylation of TREX1 C Terminus Disrupts TREX1 Regulation of the Oligosaccharyltransferase Complex.

TREX1 mutations are associated with several autoimmune and inflammatory diseases. The N-terminal DNase domain of TREX1 is important for preventing self-DNA from activating the interferon response. The C terminus of TREX1 is required for ER localization and regulation of oligosacchariyltransferase (OST) activity. Here, we show that during mitosis TREX1 is predominately phosphorylated at the C-terminal Serine-261 by Cyclin B/CDK1. TREX1 is dephosphorylated quickly at mitotic exit, likely by PP1/PP2-type serine/threonine phosphatase. Mitotic phosphorylation does not affect TREX1 DNase activity. Phosphomimetic mutations of mitotic phosphorylation sites in TREX1 disrupted the interaction with the OST subunit RPN1. RNA-seq analysis of Trex1-/- mouse embryonic fibroblasts expressing TREX1 wild-type or phosphor-mutants revealed a glycol-gene signature that is elevated when TREX1 mitotic phosphorylation sites are disrupted. Thus, the cell-cycle-dependent post-translation modification of TREX1 regulates its interaction with OST, which may have important implications for immune disease associated with the DNase-independent function of TREX1.

Quality control validation for exogenous natural surfactant labeled with 99mTc.

OBJECTIVE: Exogenous natural surfactant (ENS) labeled with 99mTc shows an elevated lung specificity allowing the acquisition of high-quality images for ventilation scintigraphy. METHODS: The methods for 99mTc-ENS quality control (physical properties, pH determination, radiochemical studies, and biologic studies) were evaluated and validated. RESULTS: The physical properties of the nonradioactive precursor and of the radiopharmaceutical were analyzed as general descriptors of the product. The pH of the radiopharmaceutical was determined by using pH test papers, a method described and validated in the United States Pharmacopeia. Chromatographic studies performed using the acetone/Whatman-1 paper system were validated as a method to evaluate the radiochemical purity of the 99mTc-ENS. Biodistribution studies on rats after intratracheal administration were validated as a method to estimate the radiopharmaceutical biodistribution in humans. CONCLUSION: The proposed method for 99mTc-ENS quality control studies and stability studies was evaluated and validated following international standards.

Stabilized ferrous gluconate as iron source for food fortification: bioavailability and toxicity studies in rats.

The iron bioavailability and acute oral toxicity in rats of a ferrous gluconate compound stabilized with glycine (SFG), designed for food fortification, was studied in this work by means of the prophylactic method and the Wilcoxon method, respectively. For the former studies, SFG was homogeneously added to a basal diet of low iron content, reaching a final iron concentration of 20.1 +/- 2.4 mg Fe/kg diet. A reference standard diet using ferrous sulfate as an iron-fortifying source (19.0 +/- 2.1 mg Fe/kg diet) and a control diet without iron additions (9.3 +/- 1.4 mg Fe/kg diet) were prepared in the laboratory in a similar way. These diets were administered to three different groups of weaning rats during 23 d as the only type of solid nourishment. The iron bioavailability of SFG was calculated as the relationship between the mass of iron incorporated into hemoglobin during the treatment and the total iron intake per animal. This parameter resulted in 36.6 +/- 6.2% for SFG, whereas a value of 35.4 +/- 8.0% was obtained for ferrous sulfate. The acute toxicological studies were performed in two groups of 70 female and 70 male Sprague-Dawley rats that were administered increasing doses of iron from SFG. The LD50 values of 1775 and 1831 mg SFG/kg body wt were obtained for female and male rats, respectively, evidencing that SFG can be considered as a safe compound from a toxicological point of view.

Bioavailability of Petit-Suisse cheese as food vehicle for iron fortification estimated by the prophylactic method.

Microencapsulated ferrous sulfate (SFE-171) and ferric orthophosphate in Petit-Suisse cheese were examined for iron bioavailability by the prophylactic method. The iron sources were industrially added to different samples of Petit-Suisse cheese, which were mixed with other food components in our laboratory before use. A reference standard diet inclusive of nonmicroencapsulated ferrous sulfate and a control diet low in iron content were prepared in the laboratory. The final iron content in the fortified diets was approximately 15 mg Fe/kg diet. These diets were administered to weaning rats for 23 days. The iron bioavailability was evaluated as the ratio of iron incorporated into hemoglobin to oral iron intake, thereby being estimated as 62.6 +/- 8.8% for ferrous sulfate and 59.2 +/- 10.6% for SFE-171, which were significantly effective at p < 0.01 compared to 43.4 +/- 10.5% for ferric orthophosphate. It thus turned out that SFE-171 was stable through industrial processing with Petit-Suisse cheese as the food vehicle and served as an iron fortifier equal to ferrous sulfate in bioavailability.

Evidence of Helicobacter sp. in dental plaque of captive dolphins (Tursiops gephyreus).

Gastrointestinal lesions have been extensively reported in wild and captive marine mammals. However, their etiology remains unclear. In humans and other animals, chronic gastritis and peptic ulcers have been associated with Helicobacter sp. Therefore, the aim of our study was to investigate the presence of Helicobacter sp. in the gastric juice, dental plaque, and saliva of marine mammals living in a controlled environment. Five dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), three sea lions (Otaria flavescens), two elephant seals (Mirounga leonina), and two fur seals (Arctocephalus australis) were studied. Saliva, dental plaque, and gastric juice samples were examined for Helicobacter sp. using polymerase chain reaction. None of the gastric juice or saliva samples were positive for Helicobacter sp. However, Helicobacter sp. DNA was detected in dental plaque from two dolphins, suggesting the oral cavity might be a reservoir of this bacterium.

The role of zinc in the growth and development of children.

This review concerns the importance of zinc in growth, development, and cognitive function in children and the deleterious consequences of its deficiency on children's health. Possible strategies to overcome zinc deficiency and the results of some supplementation trials are discussed.

Bioavailability of microencapsulated ferrous sulfate in powdered milk produced from fortified fluid milk: a prophylactic study in rats.

OBJECTIVE: We investigated the iron bioavailability of microencapsulated ferrous sulfate (SFE-171) in a diet based on powdered milk by using the prophylactic method in rats. METHODS: The SFE-171 was added into fluid milk and industrially processed into powdered milk, which was then mixed in our laboratory with a normalized diet (17.2 +/- 2.1 mg Fe/kg). A reference standard diet using ferrous sulfate as iron-fortifying source (19.8 p+/- 2.9 mg Fe/kg) and a control diet without added iron (4.6 +/- 0.8 mg Fe/kg) were prepared in the laboratory in a similar way. These diets were administered to different groups of weaning rats for 28 d as the only solid nourishment. The iron bioavailability of the different sources was calculated as the relation between the mass of iron incorporated into hemoglobin during the treatment and the total iron intake per animal. RESULTS: The iron bioavailability values of SFE-171 and ferrous sulfate in the fortified diets were 41.6 +/- 6.6% and 42.6 +/- 4.2%, respectively; these results were significantly higher (P < 0.01) than the iron bioavailability of the control diet (28.8 +/- 8.1%). CONCLUSION: These results showed that iron-fortified powdered milk can be produced from fluid milk fortified with SFE-171. The bioavailability of SFE-171 in this rat model was not altered by the manufacturing process.

Bioavailability of microencapsulated ferrous sulfate in fluid milk studies in human beings

Iron deficiency is one of the most important nutritional problems in the world. The best method to overcome this problem is the fortification of foods with highly bioavailable iron. Fluid milk is a massive consumption food with an easy access and which is generally the only food intake during the first months of life. Therefore the fortification of fluid milk with highly biovalable iron and no detectable alterations of its sensorial characteristics was studied in the present work. This procedure was made possible using a new type of ferrous sulfate, stabilized and microencapsulated with soy lecithin (SFE-171). The iron concentration of the fortified milk is 12 mg per liter. In order study the iron absorption from milk fortifield with this product, SFE-171 was labeled with59 Fe and given to 29 volunteers with a normal iron status, each of which received an iron quantily of 3 mg in 250 ml of fluid milk. The average iron absorption was (10.2+4.7) per cent. This result shows that the iron given in this physiocochemical form has the advantage of a high biovailability and it is possible that this product will be the first attempt for an adequate solution of iron deficiency.

Bioavailability of microencapsulated ferrous sulfate in fluid milk studies in human beings

Iron deficiency is one of the most important nutritional problems in the world. The best method to overcome this problem is the fortification of foods with highly bioavailable iron. Fluid milk is a massive consumption food with an easy access and which is generally the only food intake during the first months of life. Therefore the fortification of fluid milk with highly biovalable iron and no detectable alterations of its sensorial characteristics was studied in the present work. This procedure was made possible using a new type of ferrous sulfate, stabilized and microencapsulated with soy lecithin (SFE-171). The iron concentration of the fortified milk is 12 mg per liter. In order study the iron absorption from milk fortifield with this product, SFE-171 was labeled with59 Fe and given to 29 volunteers with a normal iron status, each of which received an iron quantily of 3 mg in 250 ml of fluid milk. The average iron absorption was (10.2+4.7) per cent. This result shows that the iron given in this physiocochemical form has the advantage of a high biovailability and it is possible that this product will be the first attempt for an adequate solution of iron deficiency. (AU)