We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature-sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and ...encodes a protein with an estimated molecular mass of 64,460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP-dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.
Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are ...defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) α/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-α–deficient (Retnla−/−) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-α. After challenge with Schistosoma mansoni (Sm) eggs, Retnla−/− mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg–challenged Retnla−/− mice exhibited elevated expression of pathogen-specific CD4+ T cell–derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM-α could bind to macrophages and effector CD4+ Th2 cells and inhibited Th2 cytokine production in a Bruton's tyrosine kinase–dependent manner. Additionally, Retnla−/− AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM-α in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4+ T cell responses.
Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a ...potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to βKlotho with high affinity and specifically activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on βKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.
The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified ...(SIRT1–SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2–3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.
BackgroundInterleukin-2 (IL-2) is a driver of T and NK cell proliferation and activation, and has produced remarkable clinical efficacy in a few cancer patients. However, its clinical use is limited ...by its narrow therapeutic index, and potentially by its preferential stimulation of immune suppressing Treg cells. TNRX-257, a novel multi-specific LAG3 antagonist with unique LAG3-conditional partial agonism of the IL2Rγ/β receptors, was developed using Tentarix’s propriety Tentacles™ platform, which is based on fully human, stabilized antibody VH domains. LAG3 expression is restricted to antigen-experienced and tumor-reactive immune cells with little expression on peripheral PBMC or immune cells in normal tissues. Thus, TNRX-257 is anticipated to drive the proliferation of this tumor-antigen positive pool of cells, while minimizing the dose limiting toxicity seen in the pre-clinical models and human clinical trials, as well as the bias towards Treg proliferation seen with WT IL-2.MethodsTNRX-257 was assessed for the affinity for human and cynomolgus LAG3, IL2Rγ and IL2Rβ affinities by Bio-Layer Interferometry, and by cytometry for binding and pSTAT5 induction on CD3/CD28 activated human and cynomolgus monkey PBMCs. To determine the pharmacokinetics and pharmacodynamics of TNRX-257, the molecule was dosed intravenously at 2 dose levels, 2.5mg/kg or 9mg/kg, to 2 cynomolgus monkeys each, and blood was harvested at multiple timepoints over 14 days for analysis. Pharmacokinetics was determined by ELISA, cytokine release by MSD assays, and Ki67 positivity of NK and T cells by cytometry.ResultsTNRX-257 was shown to have equivalent affinities for human and cynomolgus LAG3, IL2Rγ, IL2Rβ. The bioactivity of TNRX-257 on cynomolgus monkey PBMCs is highly similar to its human responses, retaining its unique IL2Rγ/β partial agonism and full antagonist activity for LAG3. At both dose levels, TNRX-257 was well tolerated and blood levels remained above the minimal concentration required for IL2Rγ/β agonism throughout the study period. TNRX-257 bioactivity was seen at both day 5 and day 7 post-dosing, as increases in the percentage of Ki67 positive NK cells, CD4 and CD8 T cells over the pre-dose level with the higher dose.ConclusionsThese data show that TNRX-257 has pharmacokinetics, bioactivity and a unique safety profile that support its clinical development for treating multiple cancer indications.Ethics ApprovalThis study was approved by Labcorp institution’s IACUC Board; study number 8486120
Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout ...mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.
The secreted goblet cell-derived protein resistin-like molecule beta (RELMbeta) has been implicated in divergent functions, including a direct effector function against parasitic helminths and a ...pathogenic function in promoting inflammation in models of colitis and ileitis. However, whether RELMbeta influences CD4(+) T cell responses in the intestine is unknown. Using a natural model of intestinal inflammation induced by chronic infection with gastrointestinal helminth Trichuris muris, we identify dual functions for RELMbeta in augmenting CD4(+) Th1 cell responses and promoting infection-induced intestinal inflammation. Following exposure to low-dose Trichuris, wild-type C57BL/6 mice exhibit persistent infection associated with robust IFN-gamma production and intestinal inflammation. In contrast, infected RELMbeta(-/-) mice exhibited a significantly reduced expression of parasite-specific CD4(+) T cell-derived IFN-gamma and TNF-alpha and failed to develop Trichuris-induced intestinal inflammation. In in vitro T cell differentiation assays, recombinant RELMbeta activated macrophages to express MHC class II and secrete IL-12/23p40 and enhanced their ability to mediate Ag-specific IFN-gamma expression in CD4(+) T cells. Taken together, these data suggest that goblet cell-macrophage cross-talk, mediated in part by RELMbeta, can promote adaptive CD4(+) T cell responses and chronic inflammation following intestinal helminth infection.
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to ...chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide–antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide–antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
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