Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

INTRODUCTION: The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface. METHODS: We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding. RESULTS: Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells. CONCLUSION: We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential

Identification of the Microsporidian Encephalitozoon cuniculi as a New Target of the IFNγ-Inducible IRG Resistance System

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.Grants from the Deutsche Forschungsgemeinschaft: SFB635, 670, 680, SPP1399

cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Effects of alirocumab on types of myocardial infarction: insights from the ODYSSEY OUTCOMES trial

Aims  The third Universal Definition of Myocardial Infarction (MI) Task Force classified MIs into five types: Type 1, spontaneous; Type 2, related to oxygen supply/demand imbalance; Type 3, fatal without ascertainment of cardiac biomarkers; Type 4, related to percutaneous coronary intervention; and Type 5, related to coronary artery bypass surgery. Low-density lipoprotein cholesterol (LDL-C) reduction with statins and proprotein convertase subtilisin–kexin Type 9 (PCSK9) inhibitors reduces risk of MI, but less is known about effects on types of MI. ODYSSEY OUTCOMES compared the PCSK9 inhibitor alirocumab with placebo in 18 924 patients with recent acute coronary syndrome (ACS) and elevated LDL-C (≥1.8 mmol/L) despite intensive statin therapy. In a pre-specified analysis, we assessed the effects of alirocumab on types of MI. Methods and results  Median follow-up was 2.8 years. Myocardial infarction types were prospectively adjudicated and classified. Of 1860 total MIs, 1223 (65.8%) were adjudicated as Type 1, 386 (20.8%) as Type 2, and 244 (13.1%) as Type 4. Few events were Type 3 (n = 2) or Type 5 (n = 5). Alirocumab reduced first MIs [hazard ratio (HR) 0.85, 95% confidence interval (CI) 0.77–0.95; P = 0.003], with reductions in both Type 1 (HR 0.87, 95% CI 0.77–0.99; P = 0.032) and Type 2 (0.77, 0.61–0.97; P = 0.025), but not Type 4 MI. Conclusion  After ACS, alirocumab added to intensive statin therapy favourably impacted on Type 1 and 2 MIs. The data indicate for the first time that a lipid-lowering therapy can attenuate the risk of Type 2 MI. Low-density lipoprotein cholesterol reduction below levels achievable with statins is an effective preventive strategy for both MI types.For complete list of authors see http://dx.doi.org/10.1093/eurheartj/ehz299</p

Effect of alirocumab on mortality after acute coronary syndromes. An analysis of the ODYSSEY OUTCOMES randomized clinical trial

Background: Previous trials of PCSK9 (proprotein convertase subtilisin-kexin type 9) inhibitors demonstrated reductions in major adverse cardiovascular events, but not death. We assessed the effects of alirocumab on death after index acute coronary syndrome. Methods: ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) was a double-blind, randomized comparison of alirocumab or placebo in 18 924 patients who had an ACS 1 to 12 months previously and elevated atherogenic lipoproteins despite intensive statin therapy. Alirocumab dose was blindly titrated to target achieved low-density lipoprotein cholesterol (LDL-C) between 25 and 50 mg/dL. We examined the effects of treatment on all-cause death and its components, cardiovascular and noncardiovascular death, with log-rank testing. Joint semiparametric models tested associations between nonfatal cardiovascular events and cardiovascular or noncardiovascular death. Results: Median follow-up was 2.8 years. Death occurred in 334 (3.5%) and 392 (4.1%) patients, respectively, in the alirocumab and placebo groups (hazard ratio [HR], 0.85; 95% CI, 0.73 to 0.98; P=0.03, nominal P value). This resulted from nonsignificantly fewer cardiovascular (240 [2.5%] vs 271 [2.9%]; HR, 0.88; 95% CI, 0.74 to 1.05; P=0.15) and noncardiovascular (94 [1.0%] vs 121 [1.3%]; HR, 0.77; 95% CI, 0.59 to 1.01; P=0.06) deaths with alirocumab. In a prespecified analysis of 8242 patients eligible for ≥3 years follow-up, alirocumab reduced death (HR, 0.78; 95% CI, 0.65 to 0.94; P=0.01). Patients with nonfatal cardiovascular events were at increased risk for cardiovascular and noncardiovascular deaths (P<0.0001 for the associations). Alirocumab reduced total nonfatal cardiovascular events (P<0.001) and thereby may have attenuated the number of cardiovascular and noncardiovascular deaths. A post hoc analysis found that, compared to patients with lower LDL-C, patients with baseline LDL-C ≥100 mg/dL (2.59 mmol/L) had a greater absolute risk of death and a larger mortality benefit from alirocumab (HR, 0.71; 95% CI, 0.56 to 0.90; Pinteraction=0.007). In the alirocumab group, all-cause death declined wit h achieved LDL-C at 4 months of treatment, to a level of approximately 30 mg/dL (adjusted P=0.017 for linear trend). Conclusions: Alirocumab added to intensive statin therapy has the potential to reduce death after acute coronary syndrome, particularly if treatment is maintained for ≥3 years, if baseline LDL-C is ≥100 mg/dL, or if achieved LDL-C is low. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01663402

Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients

A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti-DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)-prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 µg/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti-dsDNA autoantibodies, anti-tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA-specific antibodies. Treatment with hCDR1 down-regulated the latter significantly. No significant effect of the treatment on the levels of anti-tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients