Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of "second generation" antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.</p

Submicroscopic PM does not induce inflammatory immune responses, but does dysregulate hemostasis.

<p>(A) Monocyte levels in IVB as detected by flow cytometry. (B) TNF levels in IVB. (C) D-dimer levels in IVB. (D) PAI-1 levels in IVB. (E) TAT complex levels in IVB. TNF and coagulation/fibrinolysis markers were measured by ELISA. Statistical results in panels C, D and E represent analysis of submicroscopic (PM^sub) and microscopic (PM+) groups combined versus PM− samples. Bars represent the median.</p

Coagulation factor gene expression is elevated in IP mice and malaria-exposed murine trophoblasts.

<p>(A) RNA was isolated from conceptuses removed from ED 10 UP (n = 5) and IP (n = 6) mice. Primers specific for the genes indicated were utilized to measure cDNA expression levels in IP relative to UP mice. Data are normalized against murine <i>18S</i> RNA. Data are expressed as the ratio of fold increase in IP mice to that of UP mice ± SEM. (B) SM9-1 trophoblasts were stimulated with <i>P. chabaudi</i> AS-iRBCs and RNA isolated over the time course indicated. QRT-PCR was conducted as in panel A. Data are expressed as the ratio of fold increase relative to time matched SM9-1 trophoblasts stimulated with uninfected RBC ± SEM and are representative of four separate experiments.</p

Clinical and sociological attributes of human participants.

<p>Data are presented as mean ± standard deviation or percentage with sample size in parentheses.</p>a<p>PM = placental malaria; PM^sub indicates microscopy negative, PCR positive participants.</p>b<p>defined as <36 weeks gestation.</p>c<p>LBW = low birth weight.</p>d<p>SP = reported use of sulfadoxine-pyrimethamine.</p>e<p>geometric placental parasitemia from intervillous blood thin smear.</p>f<p>Hz = hemozoin; WBC = white blood cell; indicates percent of WBCs bearing hemozoin on intervillous blood thick smear.</p>g<p>indicates chronic or past infection as evidenced by the presence of any Hz in fibrin observed by histology.</p>h<p>indicates chronic or past infection as evidenced by the presence of any Hz in intervillous WBCs observed by histology.</p><p>Statistics by one-way ANOVA (<i>P</i> values shown in table) with Tukey's post-hoc test for continuous variables:</p>†<p><i>P</i><0.05,</p>‡,§<p><i>P</i><0.001.</p><p>Fisher's exact test was used for pairwise comparison of proportions (<i>P</i> values shown in table with *^,# symbols indicating significant comparisons).</p

LMWH treatment improves midgestational embryo survival in IP mice.

<p>(A) Viable embryos per mouse among UP (n = 18), IP (n = 15), IP LMWH-treated (n = 11), and IP enoxaparin-treated mice (n = 5) on ED 12. Bars represent the mean. (B) Mean (± SEM) viable embryos as a proportion of total embryos within each group as described in panel A. *<i>P</i><0.0001, **<i>P</i> = 0.0008, ***<i>P</i> = 0.0270. (C) Gross pathological view of UP uterus. (D) Gross pathological view of IP uterus, showing active embryonic expulsion (arrow), diminished vascularization (black blunt arrow), and intrauterine hemorrhage (white blunt arrow). (E) Gross pathological view of LMWH-treated IP uterus with one resorption (arrow). (F, I) Hematoxylin and eosin (H&E)-stained thin section of a UP conceptus. (G, J) H&E-stained thin section of an IP conceptus; arrow indicates fibrin deposition. (H, K) H&E-stained thin section of an IP LMWH-treated conceptus. Enlargements (panels I, J and K) delineate the three principle regions of the murine placenta, decidua (d), junctional zone (j), labyrinth (l), and also identify the embryo (e). Gross macroscopic pictures were taken with a Kodak Easyshare DX7630 digital camera at 6 MP. Micrographs were captured on an Olympus BX41TF light microscope using an Olympus D70 digital camera. Panels F, G, and H depict magnification with a 2× objective and panels I, J, and K with a 4× objective. Images were resized, cropped as appropriate, and in some cases brightened using GNU Image Manipulation Program v2.6.</p