Detection of heat-stable antibacterial activity in cotyledonary and discoid placentas.

The placenta serves in immunological defense of the fetus, providing proteins essential for innate immunity. Maternal and fetal portions of two mammalian placenta types, discoid, and cotyledonary, were separated and analyzed for antibacterial activity using a culture-independent method. Antibacterial activity was detected in both maternal and fetal portions of all placenta types tested. Protease resistance and increased activity after boiling suggests that the factor is activated upon release from a larger molecule. Identification of this factor and the mechanism of activation will lead to a better understanding of the innate immune function provided by the placenta.

Intergenerational Talking Circles Exploring Psychosocial Stressors for Preterm Birth and Strategies for Resilience among American Indian Women.

INTRODUCTION: This study used phenomenological qualitative research design to understand the primary psychosocial stressors American Indian (AI) women face during pregnancy-factors that may be overlooked as contributing to the community's heightened preterm birth rate-and to identify mechanisms of resilience. METHOD: Using Lakota talking circles (TCs) as the data collection method, researchers collected young mothers' and tribal elders' narratives about pregnancy and parenting. Themes were derived and verified from the transcripts. RESULTS: During the TCs, the women identified transgenerational/historical trauma and instability in housing, childcare, and relationships as major contributors to pregnancy and parenting stress. DISCUSSION: Having overcome obstacles while bearing and raising children, AI women exhibit resilience. Participants indicated that the TCs provided support and a cultural connection, thus serving as both a data collection method and an intervention. Future work will involve using TCs to support young mothers, transmit elder wisdom, and increase resilience among AI women.

Creating and Teaching Science Lessons in K-12 Schools Increases Undergraduate Students' Science Identity.

Success and persistence in the life sciences is influenced by a student's self-efficacy, sense of belonging, and science identity. It has already been demonstrated that outreach experiences and service learning by graduate students in K-12 schools aid in the graduate students' confidence and intrinsic satisfaction. Others have shown the importance of engaging scientists in outreach activities, both for the benefit of the K-12 student and as a way to engage scientists with the community. We predicted it would also be beneficial for undergraduates to engage in service-learning activities during their coursework because working with K-12 students would solidify their scientific identity and sense of belonging while deepening their understanding of the course content. Consequently, we implemented service projects in our upper-level molecular biology and human physiology courses at a primarily undergraduate institution that focuses on five core values: Christian, Liberal Arts, Excellence, Community, and Service. Outcomes such as the undergraduate students' value of service, confidence in their knowledge of course content, ability to create effective lesson plans, and science identity were measured using anonymous surveys. Overall, students reported that they highly valued and enjoyed this unique experience. This type of activity could be used to increase future scientists' awareness of synergistic activities such as academic service and of the joy found in such activities. Future plans include measuring the effects on the participating high school and elementary school students and visiting schools with a high proportion of students from underserved populations.

Sushi Domain Containing 2 (SUSD2) inhibits platelet activation and binding to high-grade serous ovarian carcinoma cells.

Platelets play a central role in primary hemostasis affecting tumor survival and metastases. Tumors induce platelets to aggregate and bind to the cancer cells, resulting in protection from immune surveillance and often leading to thrombocytosis. In ovarian cancer (OvCa), one-third of patients present with thrombocytosis, a diagnosis that correlates with shorter survival. SUSD2 (SUShi Domain containing 2), a type I transmembrane protein, shown to inhibit metastatic processes in high-grade serous ovarian carcinoma (HGSOC), is expressed on endothelial cells and thus may influence platelet reactivity. As such, we hypothesized that SUSD2 levels in ovarian cancer-derived cell lines influence platelet activation. We incubated OvCa non-targeting (NT) and SUSD2 knockdown (KD) cell lines with labeled platelets and quantified platelet binding, as well as GPIIb/IIIa integrin activation. The role of GPIIb/IIIa in tumor cell/platelet interaction was also examined by measuring cell-cell adhesion in the presence of eptifibatide. We found that platelets exposed to OvCa cells with low SUSD2 expression display increased tumor cell-platelet binding along with an increase in GPIIb/IIIa receptor activation. As such, platelet activation and binding to HGSOC cells was inversely correlated with the presence of SUSD2. This represents one of the first tumor proteins known to provide differential platelet interaction based on protein status.

The mucin MUC16 (CA125) binds to NK cells and monocytes from peripheral blood of women with healthy pregnancy and preeclampsia.

PROBLEM: MUC16 (CA125) released from ovarian tumors binds to NK cells and monocytes via the inhibitory receptor Siglec-9. Here, we investigate whether MUC16 also binds to circulating immune cells during pregnancy and in women with preeclampsia. METHOD OF STUDY: MUC16 binding was monitored by flow cytometry and immunoprecipitation, and RT-PCR was used to monitor indigenous expression in immune cells. Serum CA125 levels were measured by a clinical assay. RESULTS: MUC16 was equally distributed on Siglec-9(pos) CD16(pos)/CD56(dim) and CD16(neg)/CD56(br) NK cells in the healthy pregnant and preeclampsia groups. While serum CA125 levels and number of NK and monocytes were similar, increased binding of MUC16 was observed on these immune cells in the preeclampsia cohort as compared to the healthy pregnant samples. CONCLUSION: MUC16 binding to NK cells and monocytes likely contributes to tolerance of the fetal allograft from maternal responses and may also serve as a novel biomarker for preeclampsia.

The immunomodulating roles of glycoproteins in epithelial ovarian cancer.

The complexity of the immune system demands an intricate defense mechanism by tumors. Ovarian and other tumors employ specific glycoproteins and the associated glycan sequences to modulate immune responses. Glycoproteins enable tumor cells that express or secrete these molecules to evade immune cell attack and induce the immune system to promote tumor growth. This review focuses first on the immune environment in ovarian cancer, and the mechanisms of activation and inhibition that immune cells undergo in order to either attack or ignore a target cell. Next we illustrate the immunomodulatory roles of ovarian cancer-associated glycans and glycoproteins in 1. preventing immune synapse formation, 2. serving as ligands of immune cell receptors, 3. scavenging cytokines and chemokines, and 4. participating in the formation of autoantibodies against the tumor. The importance of these immunomodulating strategies from the view points of understanding the tumor immunology of ovarian tumors, potential origin of such mechanisms, and specific strategies to circumvent the glycoconjugate-mediated suppression of immune responses is discussed in this review.

Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface.

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

Differential internalization of hu14.18-IL2 immunocytokine by NK and tumor cell: impact on conjugation, cytotoxicity, and targeting.

The hu14.18-IL2 (EMD 273063) IC, consisting of a GD(2)-specific mAb genetically engineered to two molecules of IL-2, is in clinical trials for treatment of GD(2)-expressing tumors. Anti-tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25(+)CD16(-) NK cell lines (NKL and RL12) and GD(2)(+) M21 melanoma after IC binding to the cells via IL-2R and GD(2), respectively. For NK cells, ∼ 50% of IC was internalized by 3 h and ∼ 90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody-dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼ 70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21-bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.

MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells.

BACKGROUND: Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition. RESULTS: Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16(low) targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors. CONCLUSION: MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.

Effect of acetyl-l-carnitine on ovarian cancer cells' proliferation, nerve growth factor receptor (Trk-A and p75) expression, and the cytotoxic potential of paclitaxel and carboplatin.

OBJECTIVES: The incidence of chemotherapy induced peripheral neuropathy (CIPN) is 15-25% with platinum and taxanes. CIPN can be permanent and often requires dose reduction or change in chemotherapy. Acetyl-l-carnitine (ALCAR), an ester of l-carnitine, is used to treat CIPN in humans and in animal models. The goals of this study are: 1) examine the effects of ALCAR on ovarian cancer cells, 2) determine if ALCAR affects the cytotoxicity of standard chemotherapy on ovarian cancer cells. METHODS: OVCAR-3 and SKOV-3 ovarian cancer lines were incubated in ALCAR containing media. Viability, proliferation, and expression of the nerve growth factor receptors (NGFR) Trk-A and p-75 were determined by flow cytometry. Cytotoxicity assays examining ALCAR's effect on paclitaxel and carboplatin were done by flow cytometry and infrared plate-reader. RESULTS: Flow cytometry showed no change in percent live (p = 0.87) or proliferation (p = 0.95) of OVCAR-3 cells when comparing controls with up to 100 microM ALCAR. However, there was a slight but significant decrease in the proliferation of SKOV-3 cells incubated at higher ALCAR concentrations (p = < 0.01). Flow cytometry showed no difference in the viability of OVCAR-3 cells when comparing ALCAR: +/- paclitaxel (p = 1), +/- carboplatin (p = 0.8), or both (p = 0.4). Proliferation assays indicated that paclitaxel's cytotoxicity on OVCAR-3 and SKOV-3 cells was unchanged at higher ALCAR concentrations (p = < 0.01-0.4). ALCAR did not affect the expression of NGFR on OVCAR-3 or SKOV-3 cells. CONCLUSION: ALCAR does not affect the cytotoxicity of paclitaxel or carboplatin. There was no increase in proliferation, or NGFR of OVCAR-3 or SKOV-3 cells exposed to ALCAR.

Peritoneal natural killer cells from epithelial ovarian cancer patients show an altered phenotype and bind to the tumour marker MUC16 (CA125).

The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down-regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16(-) CD56(br) whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16(-) CD56(br) population. This shift in phenotype is not caused by increased apoptosis of the CD16(+) CD56(dim) cells or selective proliferation of the CD16(-) CD56(br) NK cells. Thus, the terminal differentiation of the CD16(-) CD56(br) NK cells to CD16(+) CD56(dim) subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down-regulated in the PFNK. MUC16 binds selectively to 30-40% of CD16(+) CD56(dim) NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour-derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto-maternal tolerance and immune evasion by ovarian cancers.

Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors.

BACKGROUND: The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. RESULTS: In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5-10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+). Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer. CONCLUSION: The strong binding kinetics of the mesothelin-MUC16 interaction and the cell adhesion between ovarian tumor cells and A431-Meso+ even in the presence of peritoneal fluid strongly support the importance of these two glycoproteins in the peritoneal metastasis of ovarian tumors. The demonstration that N-linked glycans are essential for mediating mesothlein-MUC16 binding may lead to novel therapeutic targets to control the spread of ovarian carcinoma.