Targeting the Transforming Growth Factor-β pathway inhibits human basal-like breast cancer metastasis

<p>Abstract</p> <p>Background</p> <p>Transforming Growth Factor β (TGF-β) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-β antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-β pathway antagonists (1D11, a mouse monoclonal pan-TGF-β neutralizing antibody and LY2109761, a chemical inhibitor of TGF-β type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR).</p> <p>Results</p> <p>Both 1D11 and LY2109761 effectively blocked TGF-β-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones <it>in vitro</it>. Moreover, both antagonists inhibited TGF-β stimulated <it>in vitro </it>migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-β. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones <it>in vivo</it>, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-β antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.</p> <p>Conclusions</p> <p>In aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.</p

Perioperative Applications of Therapeutic Plasma Exchange in Cardiac Surgery: A Narrative Review

Therapeutic plasma exchange (TPE) has a number of applications in cardiac surgical patients and has been used increasingly in high-risk heart and lung transplant patients. In this narrative review, the authors describe TPE principles, complications, and specific indications for TPE, including thrombotic thrombocytopenic purpura, heparin-induced thrombocytopenia, induction of immunotolerance in heart and lung transplant patients, and treatment of antibody-mediated rejection in heart and lung transplant patients. The review is based on published literature and the authors’ institutional experience with perioperative TPE in cardiac surgical patients

Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis.

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvβ6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvβ6 and FMDV empty capsids in FMD diagnostic assays

In Vitro Comparison of Recombinant and Plasma-Derived von Willebrand Factor Concentrate for Treatment of Acquired von Willebrand Syndrome in Adult Extracorporeal Membrane Oxygenation Patients

BACKGROUND: Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation. METHODS: Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples. RESULTS: ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P \u3c .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P \u3c .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, \u3e.99, and \u3e.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P \u3c .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P \u3e .99 for both comparisons). CONCLUSIONS: In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients

Platelet factor-4 concentration in adult veno-arterial ECMO patients

Background: Heparin induced thrombocytopenia (HIT) is reported at a variable rate in extracorporeal membrane oxygenation (ECMO) patients. A critical factor impacting platelet factor-4 (PF4)-heparin antibody formation is plasma PF4 concentration. We hypothesized that PF4 concentration would be increased during veno-arterial (VA) ECMO. Methods: Plasma PF4 concentration was measured during the first 5 ECMO days in 20 VA ECMO patients and 10 control plasma samples. PF4-heparin ratios were estimated using an assumed heparin concentration of 0.4 IU/mL. This correlates with an activated partial thromboplastin time of 60 to 80 seconds, which is the anticoagulation target in our center. Results: Twenty VA ECMO patients were enrolled, 10 of which had pulmonary embolism. Median PF4 concentration was 0.03 µg/mL [0.01, 0.13] in control plasma. Median PF4 concentration was 0.21 µg/mL [0.12, 0.34] on ECMO day 1 or 2, 0.16 µg/mL [0.09, 0.25] on ECMO day 3, and 0.12 µg/mL [0.09, 0.22] on ECMO day 5. Estimated median PF4-heparin ratios were 0.04, 0.03, and 0.02 respectively. Two patients (10%) developed HIT that was confirmed by serotonin release assay. PF4 concentration did not differ significantly in these patients compared to non-HIT patients (p = 0.37). No patient had an estimated PF4-heparin ratio between 0.7 and 1.4, which is the reported optimal range for PF4-heparin antibody formation. Conclusion: Our data suggest that PF4 concentration is mildly elevated during VA ECMO compared to control plasma. Estimated PF4-heparin ratios were not optimal for HIT antibody formation. These data support epidemiologic studies where HIT incidence is low during VA ECMO

Platelet Transfusion and In-Hospital Mortality in Veno-Arterial Extracorporeal Membrane Oxygenation Patients

Thrombocytopenia is common during extracorporeal membrane oxygenation (ECMO), and platelets are sometimes transfused to meet arbitrary goals. We performed a retrospective cohort study of veno-arterial (VA) ECMO patients from a single academic medical center and explored the relationship between platelet transfusion and in-hospital mortality using multivariable logistic regression. One hundred eighty-eight VA ECMO patients were included in the study. Ninety-one patients (48.4%) were transfused platelets during ECMO. Patients who received platelet transfusion had more coronary artery disease, lower platelet counts at cannulation, higher predicted mortality, lower nadir platelet counts, more ECMO days, and more red blood cell (RBC) and plasma transfusion. Mortality was 19.6% for patients who received no platelets, 40.8% for patients who received 1-3 platelets, and 78.6% for patients who received 4 or more platelets ( P \u3c 0.001). After controlling for confounding variables including baseline severity of illness, central cannulation, postcardiotomy status, RBC and plasma transfusion, major bleeding, and total ECMO days, transfusion of 4 or more platelets remained associated with in-hospital mortality; OR = 4.68 (95% CI = 1.18-27.28), P = 0.03. Our findings highlight the need for randomized controlled trials that compare different platelet transfusion triggers, so that providers can better understand when platelet transfusion is indicated in VA ECMO patients

Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus

Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus