Neutral endopeptidase depletion decreases colon cancer cell proliferation and TGF-ß1 synthesis in indirect co-cultures with normal colon fibroblasts.

PURPOSE: Although recent studies have suggested that neutral endopeptidase (NEP) is implicated in the regulation of colon cancer (CC) cell growth and metastasis, the influence of the tumor microenvironment on this role of NEP has not been investigated so far. Normal colon fibroblasts (NCFs) constitute a component of the stroma surrounding a tumor in an early stage of its development. NCFs can influence transformed cells via different paracrine factors, including TGF-ß1. This in vitro study was undertaken to evaluate the role of NEP in CC promotion in conditions of indirect co-culture of CC cells (LS180 and SW620) with NCFs (CCD-18Co) or their conditioned medium (CM-18Co). METHODS: We examined cell proliferation (with the BrdU assay) and invasiveness (using BME-coated inserts, 8 µm) of NEP-expressing, NEP-silenced (siRNA), and NEP-inhibited (with thiorphan, i.e. a NEP specific inhibitor) CC cells cultured alone or co-cultured with CCD-18Co or with their conditioned medium. The Western blot and ELISA methods were used to assess the level of TGF-ß1. RESULTS: The results showed that the co-culture of the NEP-depleted CC cells with NCFs or their conditioned medium resulted in a significant decrease in cell proliferation in comparison with the proliferative potential of NEP-silenced/inhibited CC cells cultivated alone. In contrast, the NEP depletion did not influence the invasiveness of CC cells in the co-cultures. The co-culture of CC cells with CCD-18Co or CM-C18Co resulted in increased synthesis of TGF-ß1, while the NEP downregulation decreased the synthesis of TGF-ß1 in CC cells and abolished the stimulatory effect of the co-cultures on TGF-ß1 production. CONCLUSIONS: The results suggest that the expression of NEP by colon cancer cells is essential for their proliferation and TGF-ß1 synthesis during paracrine interactions with NCFs.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects.

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect.

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.