Host Gasdermin D restrains systemic endotoxemia by capturing Proteobacteria in the colon of high-fat diet-feeding mice.

Gasdermin D (GSDMD) functions as a key pyroptotic executor through its secreted N-terminal domain (GSDMD-N). However, the functional relevance and mechanistic basis of the precise roles of host colonic GSDMD in high-fat diet (HFD)-induced gut dysbiosis and systemic endotoxemia remain elusive. In this study, we demonstrate that HFD feeding triggers GSDMD-N secretion of both T-lymphocytes and enterocytes in mouse colons. GSDMD deficiency aggravates HFD-induced systemic endotoxemia, gut barrier impairment, and colonic inflammation. More importantly, active GSDMD-N kills the Proteobacteria phylum via directly interacting with Cardiolipin. Mechanistically, we identify that the Glu236 (a known residue for GSDMD protein cleavage) is a bona fide important site for the bacterial recognition of GSDMD. Collectively, our findings explain the mechanism by which colonic GSDMD-N maintains low levels of HFD-induced metabolic endotoxemia. A GSDMD-N mimetic containing an exposed Glu236 site could be an attractive strategy for the treatment of HFD-induced metabolic endotoxemia.

Endocan, sepsis, pneumonia, and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) are major problems of public health in intensive care units (ICUs), occurring in 15% of critically ill patients. Among the factors explaining ARDS development, sepsis is known as a frequent cause. Sepsis, ARDS, and HAP increase morbidity, mortality, length of stay in the ICU, and the overall costs of healthcare. The major challenge remains to identify accurately among critically ill patients those at risk of poor outcomes who could benefit from novel therapies. Endocan is released by the pulmonary endothelium in response to local or systemic injury. It inhibits mainly leukocyte diapedesis rather than leukocyte rolling or adhesion to the endothelial cells both in vitro and in vivo. Endocan was evaluated in 25 clinical reports, including 2454 critically ill patients and 452 healthy controls. The diagnostic value of endocan for sepsis or sepsis severity was equal to procalcitonin but its prognostic value was better. A predictive value for postoperative pneumonia was evidenced in two studies, and a predictive value for ARDS in four studies from three independent centers. This review presents an overview of the structure, expression, and functions of endocan. We also hereby summarize the potential applications of endocan in the prediction and prognosis of ARDS and HAP, as well as in the prognosis of sepsis.

The clinical significance of CDX2 in leukemia: A new perspective for leukemia research.

CDX2 gene encodes a transcription factor involved in primary embryogenesis and hematopoietic development; however, the expression of CDX2 in adults is restricted to intestine and is not observed in blood tissues. The ectopic expression of CDX2 has been frequently observed in acute myeloid and lymphoid leukemia which in most cases is concomitant with poor prognosis. Induction of CDX2 in mice leads to hematologic complications, showing the leukemogenic origin of this gene. CDX2 plays significant role in the most critical pathways as the regulator of important transcription factors targeting cell proliferation, multi-drug resistance and survival. On the whole, the results indicate that CDX2 has the potential to be suggested as the diagnostic marker in hematologic malignancies. This review discusses the role of aberrant expression of CDX2 in the prognosis and the response to treatment in patients with different leukemia in clinical reports in the recent decades. The improvement in this regard could be of high importance in diagnosis and treatment methods.

Roles of PIP2 in the membrane binding of MIM I-BAR: insights from molecular dynamics simulations.

In order to probe the roles of PIP2 in the interactions between MIM I-BAR and model membranes, we performed a series of 10 µs-scale coarse-grained molecular dynamics simulations. Our results indicate that PIP2 plays predominant roles in the membrane binding of MIM I-BAR in a concentration-dependent manner and via electrostatic interactions. Besides, we find that the occurrence of the membrane curvature may induce the re-distribution of lipids in the membrane and result in the local enrichment of PIP2 at negatively curved membrane areas. Combining these roles of PIP2 in the membrane binding of MIM I-BAR helps explain how MIM I-BAR senses negative curvature and, thus, contributes to maintaining membrane protrusions.

Tetraspecific ligand for tumor-targeted delivery of nanomaterials.

The polygenetic nature of most cancers emphasizes the necessity of cancer therapies that target multiple essential signaling pathways. However, there is a significant paucity of targeting ligands with multi-specificities for targeted delivery of biomaterials. To address this unmet need, we generated a tetraspecific targeting ligand that recognizes four different cancer biomarkers, including VEGFR2, αvß3 integrin, EGFR, and HER2 receptors, which have been implicated in numerous malignant tumors. The tetraspecific targeting ligand was constructed by sequentially connecting four targeting ligand subunits via flexible linkers, yielding a fusion protein that can be highly expressed in Escherichia coli and readily purified to near homogeneity. Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI) studies and extensive cellular binding analyses indicated that all the targeting ligand subunits in the tetraspecific fusion protein recognized their target receptors proximately to the corresponding monospecific ligands. The resulting tetraspecific targeting ligand was applied for the delivery of nanomaterials such as gold nanoparticles (AuNPs) for targeted hyperthermic killing of various cancer cell lines with biomarkers of interest expressed. We demonstrate that the tetraspecific ligand can be facilely introduced on the surface of AuNPs and efficient target-dependent killing of cancer cells can be achieved only when the AuNPs are conjugated with the tetraspecific ligand. Significantly, the tetraspecific ligand simultaneously interacts with more than one receptors, such as EGFR and HER2 receptors, when they are expressed on the surface of the same cell, as demonstrated by in vitro binding assays and cell binding analyses. Our results demonstrate that the tetraspecific ligand, through multivalency and synergistic binding, can be readily used to generate various 'smart' biomaterials with greatly broadened tumor targeting range for simultaneous targeting of multiple signaling pathways on many different cancer types.

MAGE-A3 with cell-penetrating domain as an efficient therapeutic cancer vaccine.

IMPORTANCE: In conjunction with chemotherapy, immunotherapy with dendritic cells (DCs) may eliminate minimal disease burden by generating cytotoxic T lymphocytes. Enhanced cytosolic bioavailability of tumor-specific antigens improves access to human leukocyte antigen (HLA) class I molecules for more efficient cytotoxic T lymphocyte generation. Various cell-penetrating domains (CPDs) are known to ferry covalently linked heterologous antigens to the intracellular compartment by traversing the plasma membrane. OBJECTIVE: To determine whether generating melanoma antigen family A, 3 (MAGE-A3), a tumor-specific cancer-testis antigen, as a fusion protein with CPD will enhance the cytosolic bioavailability of MAGE-A3. DESIGN: MAGE-A3 was amplified by polymerase chain reaction using complementary DNA from renal tissue and cloned in frame with a CPD (YARKARRQARR) at the amino-terminal end and hexahistidine at the carboxy-terminal end to generate CPD-MAGE-A3 in a pQE-70 expression vector. Cultures were grown in Escherichia coli BL21 Star (DE3-pLysS) cells followed by nickel-nitrilotriacetic acid affinity purification of recombinant proteins. MAIN OUTCOMES AND MEASURES: Measurement of DC membrane penetration of CPD-MAGE-A3 vs MAGE-A3 and determination of the effect of CPD-MAGE-A3 pulsing on DC phenotypic expression of cell-surface antigens. RESULTS: Media composition and isopropyl-d-thiogalactosidase induction were optimized to achieve high levels of protein expression followed by purification. Western blot analysis with MAGE-A3 antibodies recognized both MAGE-A3 and CPD-MAGE-A3 proteins, while CPD antibodies recognized only CPD-MAGE-A3. Purified CPD-MAGE-A3 exhibited more efficient DC membrane penetration than did MAGE-A3 alone as confirmed by immunofluorescence analysis. High-level expression of several unique DC markers (CD80, CD83, CD86, and HLA-DR) by flow cytometry was consistent with a mature DC phenotype, indicating that pulsing with CPD-MAGE-A3 did not alter specific cell-surface antigens required for T-cell activation. CONCLUSIONS AND RELEVANCE: We have demonstrated for the first time, to our knowledge, that cloning and purification of MAGE-A3 with CPD enhances its cytosolic bioavailability in DCs without altering cell-surface antigens, potentially making it a more potent therapeutic cancer vaccine compared with existing MAGE-A3 protein and peptide vaccines.

Antitumor activity and pharmacokinetic properties of ARS-interacting multi-functional protein 1 (AIMP1/p43).

Although AIMP1 was identified as a component of the macromolecular aminoacyl tRNA synthetase complex involved in the cellular translation process, it was also found to be secreted as a cytokine having complex physiological functions. Among these, AIMP1's angiostatic and immune stimulating activities suggest its potential use as a novel antitumor therapeutic protein. Here we evaluated its antitumor efficacy in a mouse xenograft model bearing human stomach cancer cells. Intravenous injection of recombinant AIMP1 for 6 days resulted in significant decreases in both tumor volume and weight. Tumor volume decreased 31.1% and 54.0% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Tumor weight decreased 29.1% and 52.2% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Proliferating cell nuclear antigen (PCNA) staining of tumor tissues from AIMP1-treated mice (at both 2mg/kg and 10mg/kg) showed a 53% reduction of cells exhibiting an active cell cycle progression. Blood levels of tumor-suppressing cytokines such as TNF-alpha and IL-1beta increased in AIMP1-treated mice, whereas IL-12p40 and IFN-gamma levels remained unaltered. Thus, this work suggests that AIMP1 may exert its antitumor activity by inducing tumor-suppressing cytokines. In a pharmacokinetic study in rats after a single intravenous injection, AMP1 exhibited a low clearance showing a one-compartmental disposition. However, due to a low volume of distribution, AIMP1 had a short half-life of 0.1h. In a serum stability test, AIMP1 showed a half life of >60 min in human serum, 52 min in dog serum and 32 min in rat serum.

Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells.

Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.

Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents.

PURPOSE: Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood-brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined. METHODS: To study substrate affinity of tested AEDs to BCRP, transport experiments were performed in epithelial BCRP-expressing MDCKII-BCRP and MDCKII-parent cell lines cultured on microporous membrane. For detection of inhibitory potency of AEDs to BCRP, accumulation assays were carried out in MEF3.8-BCRP cells with known BCRP substrates, BODIPY FL prazosin and mitoxantrone. RESULTS: No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. CONCLUSIONS: Based on our in vitro data we can conclude that resistance to treatment with the tested AEDs probably is not caused by the overexpression of BCRP in the BBB of epileptogenic brain tumors.

The transferrin homologue, melanotransferrin (p97), is rapidly catabolized by the liver of the rat and does not effectively donate iron to the brain.

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a membrane-bound transferrin (Tf) homologue that binds iron (Fe). This protein is also found as a soluble form in the plasma (sMTf) and was suggested to be an Alzheimer's disease marker. In addition, sMTf has been recently suggested to cross the blood-brain barrier (BBB) and accumulate in the brain of the mouse following intravenous infusion. Considering the importance of this observation to the physiology and pathophysiology of the BBB and the function of sMTf in vivo, we investigated the uptake and distribution of 59Fe-125I-sMTf and compared it to 59Fe-125I-Tf that were injected intravenously in rats. Studies were also performed to measure 59Fe and 125I-protein uptake by reticulocytes using these radiolabelled proteins. The results showed that sMTf was rapidly catabolized, mainly in the liver and to a lesser extent by the kidneys. The 59Fe was largely retained by these organs but the 125I was released into the plasma. Only a small amount of 125I-sMTf or its bound 59Fe was taken up by the brain, less than that from 59Fe-125I-Tf. There was much less 59Fe uptake by erythropoietic organs (spleen and femurs) from 59Fe-sMTf than from 59Fe-Tf, and no evidence of receptor-mediated uptake of sMTf was obtained using reticulocytes. It is concluded that compared to Tf, sMTf plays little or no role in Fe supply to the brain and erythropoietic tissue. However, a small amount of sMTf was taken up from the plasma by the brain and a far greater amount by the liver.

Synthesis of new pyrrolo[2, 3-b]pyridines as a potent inhibitor of tumour necrosis factor alpha.

The MAP kinase p38 plays a key role in the biosynthesis of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and 1L-1beta. Accordingly, new pyrrolo[2, 3-]pyridine derivatives 5a-d were prepared from 2-amino-3-cyanopyrroles 3a-d via the intermediate propenylaminopyrroles 4a-d. Then the compounds 5a-d were tested for their ability to inhibit the production of TNF-alpha in vivo in rats. The most potent compounds 5a and 5b possess enhanced ability to inhibit the production of TNF-alpha stimulated with bacterial lipopolysaccharide.

Effect of organic isothiocyanates on breast cancer resistance protein (ABCG2)-mediated transport.

PURPOSE: To investigate the effect of organic isothiocyanates (ITCs), dietary compounds with chemopreventive activity, on breast cancer resistance protein (BCRP)-mediated transport. METHODS: The effect of 12 ITCs on the cellular accumulation of mitoxantrone (MX) was measured in both BCRP-overexpressing and BCRP-negative human breast cancer (MCF-7) and large cell lung carcinoma (NCI-H460) cells by flow cytometric analysis. The ITCs showing activity in MX accumulation were examined for their effect on MX cytotoxicity, and the intracellular accumulation of radiolabeled phenethyl isothiocyanate (PEITC) was measured in both BCRP-overexpressing and BCRP-negative NCI-H460 cells. RESULTS: ITCs significantly increased MX accumulation and reversed its cytotoxicity in resistant cells, but had a small or no effect in sensitive cells. The effects of ITCs on MX accumulation and cytotoxicity were ITC-concentration dependent. Significant increases in MX accumulation were observed at ITC concentrations of 10 or 30 microM, and significant reversal of MX cytotoxicity was generally observed at ITC concentrations of 10 microM. Intracellular accumulation of radiolabeled PEITC in BCRP-overexpressing cells was significantly lower than that in BCRP-negative cells and was increased significantly by the BCRP inhibitor fumitremorgin C (FTC). CONCLUSIONS: Certain ITCs are BCRP inhibitors and PEITC and/or its cellular metabolite(s) may represent BCRP substrates, suggesting the potential for diet-drug interactions.

Synthesis, further biological evaluation and pharmacodynamics of newly discovered inhibitors of TNF-alpha production.

(1S,2R)-2-Acylamino-1-methyl-2-phenylethyl phosphate derivatives 2a, 2b, 3a, and 5a, which are conformationally restricted and metabolically stable analogues of (2R)-2-acylamino-2-phenylethyl phosphate derivatives 1a and 1b, are a new class of inhibitors of TNF-alpha production. More efficient alternative synthesis of a key intermediate, (1R,2S)-1-amino-1-(3-methoxyphenyl)propan-2-ol hydrochloride (9), was achieved using one-step, three-component coupling of 3-methoxyphenyl boronic acid (13), (5S)-2,2,5-trimethyl-1,3-dioxolan-4-ol (14), and amino diphenyl methane (15), [as reported in J. Am. Chem. Soc. 1998, 120, 11798]. Evaluation of the hypotensive activity of these compounds was done to assess one of their side effects. Among the compounds tested, the above-mentioned four compounds (2a, 2b, 3a, and 5a) were identified as inhibitors with both sufficient potency and an acceptable safety margin regarding their hypotensive activity. The pharmacodynamics of these compounds were also investigated. Single-dose pharmacokinetic data for compounds 2a, 2b, 3a, and 5a are displayed. These compounds were estimated to be mainly metabolized by the liver in the species tested based on their in vitro stability in tissue homogenates and plasma. A representative compound, 2a, showed good linearity of its plasma concentration after intravenous injection.

Regulation of plasminogen activation: a role for melanotransferrin (p97) in cell migration.

We recently reported that human recombinant melanotransferrin (p97) presents a high transport rate across the blood-brain barrier that might involve the low-density lipoprotein receptor-related protein (LRP). We now report new interactions between p97 and another LRP ligand, the urokinase plasminogen activator (uPA) complex. By using biospecific interaction analysis, both pro-uPA and plasminogen are shown to interact with immobilized p97. Moreover, the activation of plasminogen by pro-uPA is increased by soluble p97. Because the uPA system plays a crucial role in cell migration, both in cancer and in angiogenesis, we also measured the impact of both endogenous membrane-bound and exogenous p97 on cell migration. The monoclonal antibody L235 (which recognizes a conformational epitope on p97) inhibited the migration of human microvascular endothelial cells (HMECs-1) and of human melanoma SK-MEL-28 cells, indicating that endogenous membrane-bound p97 could be associated with this process. In addition, low concentrations of exogenous p97 (10 and 100 nM) inhibited HMEC-1 and SK-MEL28 cell migration by more than 50%. These results indicate that membrane-bound and soluble p97 affect the migration capacity of endothelial and melanoma cells and suggest that p97 could be involved in the regulation of plasminogen activation by interacting with pro-uPA and plasminogen.

Identification of a novel route of iron transcytosis across the mammalian blood-brain barrier.

OBJECTIVE: This study was undertaken to assess the role of p97 (also known as melanotransferrin) in the transfer of iron into the brain, because the passage of most large molecules is limited by the presence of the blood-brain barrier, including that of the serum iron transporter transferrin. METHODS: To study the function of the soluble form of p97, we followed the uptake of radioiodinated and 55Fe loaded p97 and transferrin by the brain during a 24-hour period. RESULTS: We show that the soluble form of p97 has the ability to transcytose across the murine blood-brain barrier, and its transcytosis can be inhibited in a specific manner. We also provide evidence that p97 transports iron into the brain more efficiently than transferrin. CONCLUSIONS: These data support the idea that p97 is an important iron transporter across the blood-brain barrier in normal physiology and possibly in neurodegenerative diseases, such as Alzheimer disease, in which iron homeostasis in the brain becomes disrupted.

High transcytosis of melanotransferrin (P97) across the blood-brain barrier.

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.

Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endothelial cells.

Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% (P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II (P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls (P < 0.003). Tumors from human breast carcinoma-derived MDA-MB 468 cells were suppressed by >80% in EMAP II-treated animals (P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, approximately 80% were inhibited with maximum diameter <2 mm (P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature.