Anti-Human Tissue Factor Antibody Ameliorated Intestinal Ischemia Reperfusion-Induced Acute Lung Injury in Human Tissue Factor Knock-In Mice

BACKGROUND: Interaction between the coagulation and inflammation systems plays an important role in the development of acute respiratory distress syndrome (ARDS). Anti-coagulation is an attractive option for ARDS treatment, and this has promoted development of new antibodies. However, preclinical trials for these antibodies are often limited by the high cost and availability of non-human primates. In the present study, we developed a novel alternative method to test the role of a humanized anti-tissue factor mAb in acute lung injury with transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: Human tissue factor knock-in (hTF-KI) transgenic mice and a novel humanized anti-human tissue factor mAb (anti-hTF mAb, CNTO859) were developed. The hTF-KI mice showed a normal and functional expression of hTF. The anti-hTF mAb specifically blocked the pro-coagulation activity of brain extracts from the hTF-KI mice and human, but not from wild type mice. An extrapulmonary ARDS model was used by intestinal ischemia-reperfusion. Significant lung tissue damage in hTF-KI mice was observed after 2 h reperfusion. Administration of CNTO859 (5 mg/kg, i.v.) attenuated the severity of lung tissue injury, decreased the total cell counts and protein concentration in bronchoalveolar lavage fluid, and reduced Evans blue leakage. In addition, the treatment significantly reduced alveolar fibrin deposition, and decreased tissue factor and plasminogen activator inhibitor-1 activity in the serum. This treatment also down-regulated cytokine expression and reduced cell death in the lung. CONCLUSIONS: This novel anti-hTF antibody showed beneficial effects on intestinal ischemia-reperfusion induced acute lung injury, which merits further investigation for clinical usage. In addition, the use of knock-in transgenic mice to test the efficacy of antibodies against human-specific proteins is a novel strategy for preclinical studies

A ring-opening mechanism for DNA binding in the central channel of the T7 helicase–primase protein

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A′ helicase–primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein–DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding.

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases

Anti-hTF mAb treatment reduced inflammatory response.

<p>Inflammatory cytokines were measured in the lung tissues and BAL fluid with a cytometric bead array. The expression levels of IL-6 (A), TNFα (B), and MCP-1 (C) in the lung tissues were significantly reduced by CNTO859 in comparison to saline group. In the BAL fluid CNTO859 also reduced the IL-6 levels (D), but the IL-10 level remained unchanged between the two groups (E). Un-paired t-test was used, *: <i>p</i><0.05, n = 4 animals/group.</p

Anti-hTF mAb treatment reduced cell death.

<p>IIR-induced cell death in the lungs was determined by TUNEL staining. CNTO859 effect on the cell death was shown in representative slides (400x) (A, B), and quantified by counting the TUNEL positive cells from 10 randomly chosen fields (C). Caspase 3 activity in the lung tissue was also reduced by the anti-hTF antibody treatment (D). Un-paired t-test was used, 4 animals per group, *: <i>p</i><0.05.</p

Administration of anti-hTF mAb ameliorated intestinal ischemia-reperfusion (IIR)-induced acute lung injury in hTF-KI mice.

<p>IIR challenged hTF-KI mice were treated with CNTO859 (5 mg/kg, i.v.) or saline. The representative histology (H&E, x400) from the intestine (A, B) and lung (C, D) was shown. The lung injury was scored by a pathologist in a blind fashion (E) (▪ inflammatory cells infiltration; ▪ alveolar wall edema; ▪ hemorrhage; atelectasis). □ Lung injury scores of 4 categories were analyzed with Kruskall-Wallis test, n = 4 animals/group, *: <i>p</i><0.05. The pulmonary permeability was determined by Evans Blue dye assay (F, G, H). Administration of CNTO859 also reduced the wet/dry lung weight ratio (I), albumin concentration (J), and total cell counts (K) in the BAL fluid. Panels I-K: n = 4 animals/group,*: <i>p</i><0.05, un-paired <i>t</i>-test.</p