Mechanical and Biological Properties of Magnesium- and Silicon-Substituted Hydroxyapatite Scaffolds

Magnesium (Mg)- and silicon (Si)-substituted hydroxyapatite (HA) scaffolds were synthesized using the sponge replica method. The influence of Mg²⁺ and SiO₄⁴⁻ ion substitution on the microstructural, mechanical and biological properties of HA scaffolds was evaluated. All synthesized scaffolds exhibited porosity >92%, with interconnected pores and pore sizes ranging between 200 and 800 μm. X-ray diffraction analysis showed that β-TCP was formed in the case of Mg substitution. X-ray fluorescence mapping showed a homogeneous distribution of Mg and Si ions in the respective scaffolds. Compared to the pure HA scaffold, a reduced grain size was observed in the Mg- and Si-substituted scaffolds, which greatly influenced the mechanical properties of the scaffolds. Mechanical tests revealed better performance in HA-Mg (0.44 ± 0.05 MPa), HA-Si (0.64 ± 0.02 MPa) and HA-MgSi (0.53 ± 0.01 MPa) samples compared to pure HA (0.2 ± 0.01 MPa). During biodegradability tests in Tris-HCl, slight weight loss and a substantial reduction in mechanical performances of the scaffolds were observed. Cell proliferation determined by the MTT assay using hBMSC showed that all scaffolds were biocompatible, and the HA-MgSi scaffold seemed the most effective for cell adhesion and proliferation. Furthermore, ALP activity and osteogenic marker expression analysis revealed the ability of HA-Si and HA-MgSi scaffolds to promote osteoblast differentiation.     

Pharmacological inhibition of MDA-9/Syntenin blocks breast cancer metastasis through suppression of IL-1β

Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1β pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.

Breast cancer remains the second leading cause of death among women in the United States (1). Prognosis for early-stage disease is favorable, whereas late-stage disease with tumor cell spread beyond the primary site (i.e., metastasis) frequently heralds poorer outcomes (1). Therapy of metastatic disease usually involves systemic chemotherapy combined with radiation, providing mostly palliative options to reduce metastatic outgrowth (2). Multiple unique and distinct biological steps and an interplay between transformed and nontransformed cells highlight complexities of the metastatic process, which habitually thwarts clinical intervention. In principle, targeting these processes independently or collectively could culminate in effective antimetastatic therapies.Melanoma differentiation-associated gene-9 (mda-9), also known as Syntenin-1 or syndecan binding protein (SDCBP), was cloned in our laboratory using subtraction hybridization from terminal differentiating metastasis-derived human melanoma cells treated with interferon (IFN)-β and the protein kinase C activator, mezerein (3, 4) (designated as mda-9/Syntenin). Preferential elevated expression of mda-9/Syntenin is evident in histologically distinct tumors and contributes to several steps in the metastatic process (5). These include tumor cell invasion and migration (6, 7), induction of angiogenesis through secretion of proangiogenic factors (8–10), enhancement of epithelial–mesenchymal transition (EMT) (11, 12), regulation of the expression of integrins affecting cell-adhesion processes (13), exosome biogenesis and exosome-mediated signaling in cell–cell communication (14), and recently immune-modulation suppressing host-immune surveillance (15). Cancer cell-independent functions of MDA-9/Syntenin also contribute to metastatic progression by regulating immunosuppressive cell infiltration in the metastatic niche (16). Based on its relevance to the invasive and metastatic phenotype of cancers, MDA-9/Syntenin represents a prospective target for rational design of antimetastatic drugs.Differential expression of MDA-9/Syntenin in cancer versus adjacent normal tissue is often a predictor of poor clinical outcomes (8). A relationship exists between MDA-9/Syntenin (SDCBP) and breast cancer in rat mammary tumors (genomic localization) (17) and in metastasis and clinical situations in human triple negative and other human breast cancers (11, 15, 18). MDA-9/Syntenin plays a pivotal role in EMT induction that includes initiation of Smad-dependent EMT through interaction with TGF-βR1, disrupting receptor internalization (11). Physical interaction between MDA-9/Syntenin and TGF-β activates small GTPases, Rho A, and CDC 42 (12). In addition, MDA-9/Syntenin enhances primary tumor growth and lung metastasis through immune evasion by up-regulating PD-L1 (program death ligand 1) through STAT3 activation, causing T cell apoptosis (15). In breast cancer, MDA-9/Syntenin affects tumor cell proliferation in estrogen receptor-negative breast cancer, causing cells to bypass the G1/S checkpoint promoting S-phase entry (19). MDA-9/Syntenin is also considered a potential antigen in breast cancer (20). These observations endorse MDA-9/Syntenin as a prospective target for the therapy of breast cancer metastasis.Disturbing MDA-9/Syntenin protein:protein interactions is viewed as a viable strategy to disrupt key downstream signaling pathways regulating cancer cell invasion and metastasis (reviewed in ref. 5). Fragment-based drug discovery guided by NMR identified a first in-class interaction inhibitor of the PDZ1 domain of MDA-9/Syntenin, PDZ1i (21), displaying efficacy against glioblastoma multiforme, neuroblastoma, and prostate cancer (5, 13, 22, 23). PDZ1i suppresses cancer cell-autonomous and nonautonomous functions of MDA-9/Syntenin, culminating in strong antiinvasive and antimetastatic properties in vitro and in vivo, without inducing overt cytostatic or toxic effects in normal or most cancer cells. Informed by the crystal structure of MDA-9/Syntenin, peptide-based inhibitory molecules have been developed and validated in cell-based assays (24). Additionally, a genetic approach using adenovirus-mediated delivery of shmda-9 has shown efficacy in xenografted human melanoma (8) and prostate cancer (25) in nude mice. These investigations confirm MDA-9/Syntenin as a viable target for suppressing both primary and metastatic tumor growth and support further evaluation of PDZ1i on breast cancer metastasis.Evidence from both experimental models and clinical studies show a relationship between abundance of tumor-infiltrating immune cells and metastasis (26). To create a permissive environment in a secondary site, disseminated tumor cells employ multiple strategies, including reducing host immune surveillance (27). In several mouse tumor models, myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells with immunosuppressive properties, are expanded in the blood, lymph nodes, and spleen (28). They help shape the microenvironment and metastatic niches by regulating both innate and adaptive immunity (29). In breast cancer mouse models, MDSCs accumulate in the lungs prior to metastatic spread (30) and promote immune suppression by producing reactive oxygen species and arginase (Arg-1) (31). Not surprisingly, various chemotherapeutic agents, such as gemcitabine (32), 5-flurouracil (33), and docetaxel (34) decrease MDSC accumulation in the tumors’ stroma, thereby enhancing antitumor immune responses (29). Similar soluble factors are operational in primary tumors and metastases, including granulocyte-macrophage colony-stimulating factor, interleukins (e.g., IL-6, IL-1β), and vascular endothelial growth factor (VEGF), causing MDSC infiltration. Tumor cells in the metastatic niche that produce various cytokines or growth factors also regulate this process.We have now explored a potential role of MDA-9/Syntenin in breast cancer progression with specific emphasis on a relevant interleukin, IL-1β, representing an important inflammatory cytokine mediating cancer pathogenesis and tumor progression (35). Inflammation regulates fundamental pathways that are causative of the cancer phenotype, including proliferation, survival, and migration (36). IL-1β regulates tumor initiation/progression, angiogenesis, Th17 cell differentiation, and expansion of MDSCs (35). Additionally, IL-1β controls macrophage recruitment and invasion, and metastasis of cancer cells (35). Based on these seminal roles in orchestrating the neoplastic process, IL-1β represents a potential therapeutic target and its regulation deserves further analysis. We now confirm that MDA-9/Syntenin, which can be obstructed by the small-molecule inhibitor PDZ1i, regulates IL-1β, thereby directly controlling breast cancer pathogenesis.     

Two cases of hepatic zygomycosis in allogeneic stem cell transplant recipients and review of literature

Gastrointestinal zygomycosis is a rare condition with a high mortality rate. We present 2 fatal cases of hepatic zygomycosis following allogeneic hematopoietic stem cell transplantation and review the literature.     

Nonclassical secretory dynamics of LH revealed by hypothalamo-hypophyseal portal sampling of sheep

Continuous withdrawal of hypophyseal portal blood from unrestrained sheep has permitted detailed assessments of the pulsatile secretion of gonadotrophin-releasing hormone (GnRH). To determine if this blood can also be used to characterize the secretory dynamics of pituitary hormones, patterns of luteinizing hormone (LH) in the hypophyseal portal blood of ovariectomized ewes was compared with previous patterns of GnRH and peripheral LH. Hypophyseal portal blood and jugular vein blood were collected every 5 min from six ovariectomized ewes over 6–12 h. Hypophyseal portal blood contained GnRH-associated, sharply defined LH pulses that were much larger than in the periphery. Pulses of secreted LH (hypophyseal portal LH less peripheral LH) showed much faster rates of rise and fall than peripheral and followed pulses of GnRH by an average of 1.26 min. In contrast to pulses in jugular blood, secreted LH pulses often reached a relatively unchanging interpulse nadir-plateau and thereby approached closely algorithm-estimated, extrapolated baselines. The interpulse baseline concentrations of secreted LH (99.6 ng/mL) in hypophyseal portal blood were 31-fold higher than those for jugular LH (3.23 ng/mL). These elevated concentrations also exceeded mean jugular peak concentrations (11.1 ng/mL) and, thus, primarily must represent newly secreted LH. The non-Gaussian profiles of this secreted LH were substantially more complex than the inputs predicted from jugular LH measurements by deconvolution. Furthermore, regardless of the analytical approach, estimations of the mass of secreted LH in each pulse did not correlate well with inputs predicted by deconvolution or Kushler-Brown pulsefit analysis of corresponding pulses in jugular blood (r² ranging 0.40–0.48). Among alternative explanations is the possibility of heterogeneity in concentrations of GnRH in the portal vessels and variable distribution within the hypophysis. In summary, assay of hypophyseal portal blood obtained directly from the pituitary provides a method for direct assessment of secretory responses to hypothalamic peptides, and thereby serves as an unmatched method for studying the dynamics of LH secretion in vivo. With this approach, LH is revealed to be secreted as complex, non-Gaussian pulses that are far more sharply defined than those in the periphery, include non-GnRH-dependent, secretory components that cannot be predicted by deconvolution and are followed by periods of relatively constant, basal secretion.     

Clinical significance of RBC alloantibodies and autoantibodies in sickle cell patients who received transfusions

BACKGROUND: The clinical significance of alloimmunization to RBC antigens in sickle cell patients was analyzed by a retrospective review of the records of pediatric and adult sickle cell patients who received transfusions and who were followed over a 10-year period. STUDY DESIGN AND METHODS: Charts of pediatric and adult sickle cell patients followed at Schneider Children's Hospital (SCH) and Long Island Jewish Medical Center between 1989 and 1999 were retrieved. Patients followed at SCH were classified as pediatric, regardless of age. Data on transfusion history, alloimmunization, and transfusion reactions from 1990 were retrieved from computerized blood bank records. Transfusion history, development of alloantibodies and autoantibodies, and transfusion reactions were correlated with clinical evidence of hemolysis or other adverse reactions from the charts. All patients received ABO- and Rh-compatible blood transfusions for which a partial or extended antigen match was not performed. RESULTS: Among pediatric patients, 29 percent developed clinically significant alloantibodies, and 8 percent developed autoantibodies. Seven patients developed delayed hemolytic and/or serologic transfusion reactions, two with hyperhemolysis, two with clinical evidence of hemolysis, and three with serologic evidence only. The two patients with hyperhemolysis had received extended antigen-matched RBC transfusions to provide blood compatible with their existing antibodies. Among adult patients, 47.0 percent developed significant alloantibodies, and 9.7 percent developed autoantibodies. Five incidences of delayed hemolytic and/or serologic transfusion reactions occurred, one with hyperhemolysis and four with serologic evidence only. CONCLUSION: The alloimmunization rate is 29 percent in pediatric and 47 percent in adult sickle cell patients when partial or extended RBC antigen match is not performed. However, the delayed serologic and/or hemolytic transfusion reactions did not result in severe clinical outcome in most instances. The most important adverse event was hyperhemolysis, which may be triggered by a transfusion, but was not prevented by matching for RBC antigens. In most instances, the cause of hyperhemolysis was multifactorial.