Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution tracks compounds or molecules of interest in vivo to understand a compound's anticipated efficacy and safety. Nanoparticles deliver nucleic acid and drug payloads and enhance tumor permeability due to multiple properties such as high surface area to volume ratio, surface functionalization, and modifications. Studying the in vivo biodistribution of nanoparticles documents the effectiveness and safety of nanoparticles and facilitates a more application-driven approach for nanoparticle development that allows for more successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. In vitro, cells take up branched amphiphilic peptide-coated magnetic nanobeads (BAPc-MNBs) like their counterparts, i.e., branched amphiphilic peptide capsules (BAPCs) with a hollow water-filled core. Both BAPc-MNBs and BAPCs have widespread applications as a nanodelivery system. We evaluated the BAPc-MNBs tissue distribution in wild-type mice injected intravenously (i.v.), intraperitoneally (i.p.), or orally gavaged to understand the biological interactions and to further the development of branched amphiphilic peptide-based nanoparticles. The magnetic nanoparticles allowed collection of the BAPc-MNBs from multiple organs by magnetic bead sorting, followed by a high-throughput screening for iron content. When injected i.v., nanoparticles were distributed widely to various organs before elimination from the system via the intestines in feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered NPs via i.v. and i.p. but not via oral gavage. Taken together, these data demonstrate that the magnetic sorting not only allowed quantification of the BAPc-MNBs but also identified the distribution of BAPc-MNBs after distinct administration methods.

Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution is the tracking of compounds or molecules of interest in the subject which is integral to understanding their anticipated efficacy and safety. Nanoparticles are highly desirable delivery systems which have the ability to deliver higher nucleic acid and drug payloads and they have enhanced tumor permeability due to their unique properties such as high surface area to volume ratio. Studying the biodistribution of nanoparticles is crucial to understand their effectiveness and safety in vivo, facilitate a more application driven approach for nanoparticle development which will lead to their successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. Branched Amphiphilic Peptide coated Magnetic Nanobeads BAPc-MNBs like their counterpart i.e., Branched Amphiphilic Peptide capsules (BAPCs) with a hollow water-filled core, are readily taken up by cells in vitro and have widespread application as a nanodelivery systems. We evaluated the BAPc-MNBs tissue distribution in wildtype mice injected intravenously (i.v.), intraperitoneally (i.p.) or orally gavaged to understand the biological interactions of the peptide nanoparticles and to further the development of branched amphiphilic peptides-based nanoparticles. BAPc-MNBs were distributed widely to various organs when injected i.v. and were eliminated from the system via the intestines in feces. The spleen was found to accumulate the highest amount of BAPc-MNBs in mice administered the NPs i.v. and i.p. while they were not absorbed into the system via oral gavage. This study not only presents a relatively simple quantification method to determine in vivo biodistribution of magnetic iron nanoparticles that can be widely applied but also demonstrates the potential of Branched Amphiphilic Peptides in the form of BAPCs or BAPc-MNBs as a delivery system.

Tick Intrastadial Feeding and Its Role on IgE Production in the Murine Model of Alpha-gal Syndrome: The Tick "Transmission" Hypothesis.

Recent studies have provided strong evidence indicating that lone star tick bites are a cause of AGS (alpha-gal syndrome, also known as red meat allergy RMA) in humans. AGS is characterized by an increase in IgE antibody production against galactose-alpha-1,3-galactose (aGal), which is a common glycan found in mammalian tissue, except in Old World monkeys and humans. The main causative factor of AGS, the lone star tick (Amblyomma americanum), is broadly distributed throughout the east and midwest of the United States and is a vector of a wide range of human and animal pathogens. Our earlier glycomics study of the salivary glands of partially fed male and female ticks revealed relatively high levels of aGal epitopes. In this study, we found that partially fed males of A. americanum on bovine blood, which engage in multiple intrastadial feedings, carry a large amount of aGal in the salivary glands. In our current work, we aimed to test whether ticks mediate the transmission of the aGal sensitizer acquired from nonhuman blood to humans in the intrastadial host switch (referred to as the "transmission" hypothesis). To test this hypothesis, we used an alpha-galactosyltransferase knockout mutant mouse (aGT-KO) model system infested with ticks that were unfed or partially fed on bovine blood. Based on the levels of total IgE and specific IgG and IgE antibodies against aGal after tick feedings, aGT-KO mice significantly responded to tick feeding and injection of aGal (Galα1-3Galß1-4GlcNAc) conjugated to human serum albumin or mouse serum albumin (aGal-HSA or aGal-MSA) by increasing total IgE and aGal-specific IgE levels compared to those in C57BL/6 control mice. All of the treatments of aGT-KO mice involving the feeding of partially fed and unfed ticks functioned as sensitizers that increased the levels of specific IgE against aGal, with large individual variations. The data in this study do not support the "transmission" component of AGS, although they confirmed that aGT-KO mice can be used as a model for RMA studies.

Complement Initiation Varies by Sex in Intestinal Ischemia Reperfusion Injury.

Intestinal ischemia reperfusion (IR)-induced tissue injury represents an acute inflammatory response with significant morbidity and mortality. The mechanism of IR-induced injury is not fully elucidated, but recent studies suggest a critical role for complement activation and for differences between sexes. To test the hypothesis that complement initiation differs by sex in intestinal IR, we performed intestinal IR on male and female WT C57B6L/, C1q-/-, MBL-/-, or properdin (P)-/- mice. Intestinal injury, C3b and C5a production and ex vivo secretions were analyzed. Initial studies demonstrated a difference in complement mRNA and protein in male and female WT mice. In response to IR, male C1q-, MBL- and P-deficient mice sustained less injury than male WT mice. In contrast, only female MBL-/- mice sustained significantly less injury than female wildtype mice. Importantly, wildtype, C1q-/- and P-/- female mice sustained significant less injury than the corresponding male mice. In addition, both C1q and MBL expression and deposition increased in WT male mice, while only elevated MBL expression and deposition occurred in WT female mice. These data suggested that males use both C1q and MBL pathways, while females tend to depend on lectin pathway during intestinal IR. Females produced significantly less serum C5a in MBL-/- and P-/- mice. Our findings suggested that complement activation plays a critical role in intestinal IR in a sex-dependent manner.

Eicosanoid production varies by sex in mesenteric ischemia reperfusion injury.

Intestinal ischemia/reperfusion (I/R)-induced injury is an inflammatory response with significant morbidity and mortality. The early inflammatory response includes neutrophil infiltration. However, the majority of rodent studies utilize male mice despite a sexual dimorphism in intestinal I/R-related diseases. We hypothesized that sex may alter inflammation by changing neutrophil infiltration and eicosanoid production. To test this hypothesis, male and female C57Bl/6 mice were subjected to sham treatment or 30 min intestinal ischemia followed by a time course of reperfusion. We demonstrate that compared to male mice, females sustain significantly less intestinal I/R-induced tissue damage and produced significant LTB4 concentrations. Male mice release PGE2. Finally, treatment with a COX-2 specific inhibitor, NS-398, attenuated I/R-induced injury, total peroxidase level, and PGE2 production in males, but not in similarly treated female mice. Thus, I/R-induced eicosanoid production and neutrophil infiltration varies between sexes suggesting that distinct therapeutic intervention may be needed in clinical ischemic diseases.

Beta2 glycoprotein I-derived therapeutic peptides induce sFlt-1 secretion to reduce melanoma vascularity and growth.

Melanoma, a form of skin cancer, is one of the most common cancers in young men and women. Tumors require angiogenesis to provide oxygen and nutrients for growth. Pro-angiogenic molecules such as VEGF and anti-angiogenic molecules such as sFlt-1 control angiogenesis. In addition, the serum protein, Beta2 Glycoprotein I (ß2-GPI) induces or inhibits angiogenesis depending on conformation and concentration. ß2-GPI binds to proteins and negatively charged phospholipids on hypoxic endothelial cells present in the tumor microenvironment. We hypothesized that peptides derived from the binding domain of ß2-GPI would regulate angiogenesis and melanoma growth. In vitro analyses determined the peptides reduced endothelial cell migration and sFlt-1 secretion. In a syngeneic, immunocompetent mouse melanoma model, ß2-GPI-derived peptides also reduced melanoma growth in a dose-dependent response with increased sFlt-1 and attenuated vascular markers compared to negative controls. Importantly, administration of peptide with sFlt-1 antibody resulted in tumor growth. These data demonstrate the therapeutic potential of novel ß2-GPI-derived peptides to attenuate tumor growth and endothelial migration is sFlt-1 dependent.

Essential Role of Complement in Pregnancy: From Implantation to Parturition and Beyond.

The complement cascade was identified over 100 years ago, yet investigation of its role in pregnancy remains an area of intense research. Complement inhibitors at the maternal-fetal interface prevent inappropriate complement activation to protect the fetus. However, this versatile proteolytic cascade also favorably influences numerous stages of pregnancy, including implantation, fetal development, and labor. Inappropriate complement activation in pregnancy can have adverse lifelong sequelae for both mother and child. This review summarizes the current understanding of complement activation during all stages of pregnancy. In addition, consequences of complement dysregulation during adverse pregnancy outcomes from miscarriage, preeclampsia, and pre-term birth are examined. Finally, future research directions into complement activation during pregnancy are considered.

A Study of the Cellular Uptake of Magnetic Branched Amphiphilic Peptide Capsules.

Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e., avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules in vitro and in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water-filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery, and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their applications both in vitro and in vivo.

Drivers and regulators of humoral innate immune responses to infection and cancer.

The complement cascade consists of cell bound and serum proteins acting together to protect the host from pathogens, remove cancerous cells and effectively links innate and adaptive immune responses. Despite its usefulness in microbial neutralization and clearance of cancerous cells, excessive complement activation causes an immune imbalance and tissue damage in the host. Hence, a series of complement regulatory proteins present at a higher concentration in blood plasma and on cell surfaces tightly regulate the cascade. The complement cascade can be initiated by B-1 B cell production of natural antibodies. Natural antibodies arise spontaneously without any known exogenous antigenic or microbial stimulus and protect against invading pathogens, clear apoptotic cells, provide tissue homeostasis, and modulate adaptive immune functions. Natural IgM antibodies recognize microbial and cancer antigens and serve as an activator of complement mediated lysis. This review will discuss advances in complement activation and regulation in bacterial and viral infections, and cancer. We will also explore the crosstalk of natural antibodies with bacterial populations and cancer.

Interactions of viruses and the humoral innate immune response.

The innate immune response is crucial for defense against virus infections where the complement system, coagulation cascade and natural antibodies play key roles. These immune components are interconnected in an intricate network and are tightly regulated to maintain homeostasis and avoid uncontrolled immune responses. Many viruses in turn have evolved to modulate these interactions through various strategies to evade innate immune activation. This review summarizes the current understanding on viral strategies to inhibit the activation of complement and coagulation cascades, evade natural antibody-mediated clearance and utilize complement regulatory mechanisms to their advantage.

Role of B1 and B2 lymphocytes in placental ischemia-induced hypertension.

Preeclampsia is a prevalent pregnancy complication characterized by new-onset maternal hypertension and inflammation, with placental ischemia as the initiating event. Studies of others have provided evidence for the importance of lymphocytes in placental ischemia-induced hypertension; however, the contributions of B1 versus B2 lymphocytes are unknown. We hypothesized that peritoneal B1 lymphocytes are important for placental ischemia-induced hypertension. As an initial test of this hypothesis, the effect of anti-CD20 depletion on both B-cell populations was determined in a reduced utero-placental perfusion pressure (RUPP) model of preeclampsia. Anti-murine CD20 monoclonal antibody (5 mg/kg, Clone 5D2) or corresponding mu IgG2a isotype control was administered intraperitoneally to timed pregnant Sprague-Dawley rats on gestation day (GD)10 and 13. RUPP or sham control surgeries were performed on GD14, and mean arterial pressure (MAP) was measured on GD19 from a carotid catheter. As anticipated, RUPP surgery increased MAP and heart rate and decreased mean fetal and placental weight. However, anti-CD20 treatment did not affect these responses. On GD19, B-cell populations were enumerated in the blood, peritoneal cavity, spleen, and placenta with flow cytometry. B1 and B2 cells were not significantly increased following RUPP. Anti-CD20 depleted B1 and B2 cells in peritoneum and circulation but depleted only B2 lymphocytes in spleen and placenta, with no effect on circulating or peritoneal IgM. Overall, these data do not exclude a role for antibodies produced by B cells before depletion but indicate the presence of B lymphocytes in the last trimester of pregnancy is not critical for placental ischemia-induced hypertension.NEW & NOTEWORTHY The adaptive and innate immune systems are implicated in hypertension, including the pregnancy-specific hypertensive condition preeclampsia. However, the mechanism of immune system dysfunction leading to pregnancy-induced hypertension is unresolved. In contrast to previous reports, this study reveals that the presence of classic B2 lymphocytes and peritoneal and circulating B1 lymphocytes is not required for development of hypertension following third trimester placental ischemia in a rat model of pregnancy-induced hypertension.

Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia.

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Transforming Growth Factor Beta Signaling in Dendritic Cells Is Required for Immunotolerance to Sperm in the Epididymis.

The epididymis exhibits a less restrictive physical blood-tissue barrier than the testis and, while numerous immunosuppressive factors have been identified in the latter, no mechanisms for epididymal immunotolerance have been identified to date. Therefore, data are currently insufficient to explain how the immune system tolerates the extremely large load of novel antigens expressed on sperm, which become present in the male body after puberty, i.e., long after central tolerance was established. This study tested the hypothesis that transforming growth factor beta (TGFß) signaling in dendritic cells (DCs) is required for immunotolerance to sperm located in the epididymis, and that male mice lacking TGFß signaling in DCs would develop severe epididymal inflammation. To test this, we employed adult Tgfbr2ΔDC males, which exhibit a significant reduction of Tgfbr2 expression and TGFß signaling in DCs, as reported previously. Results show that Tgfbr2ΔDC males exhibit sperm-specific immune response and severe epididymal leukocytosis. This phenotype is consistent with epididymal loss of immunotolerance to sperm and suggests that TGFß signaling in DCs is a factor required for a non-inflammatory steady state in the epididymis, and therefore for male tract homeostasis and function.

The Complement System and Preeclampsia.

PURPOSE OF REVIEW: Preeclampsia affects 3-4% of pregnancies with few treatment options to reduce maternal and fetal harm. Recent evidence that targeting the complement system may be an effective therapeutic strategy in prevention or treatment of preeclampsia will be reviewed. RECENT FINDINGS: Studies in humans confirm the safety and efficacy of C5 blockade in complement-mediated disorders of pregnancy, including preeclampsia. Animal models mimic the placental abnormalities and/or the maternal symptoms which characterize preeclampsia. These models in mouse and rat have defined a role for complement and its regulators in placental dysfunction, hypertension, proteinuria, endothelial dysfunction, fetal growth restriction, and angiogenic imbalance, thus informing future human studies. Targeting excessive complement activation, particularly the terminal complement complex (C5b-9) and C5a may be an effective strategy to prolong pregnancy in women with preeclampsia. Continued research is needed to identify the initiator(s) of activation, the pathways involved, and the key component(s) in the pathophysiology to allow development of safe and effective therapeutics to target complement without compromising its role in homeostasis and host defense.

Role of IgM and angiotensin II Type I receptor autoantibodies in local complement activation in placental ischemia-induced hypertension in the rat.

Preeclampsia is characterized by development of hypertension during pregnancy and reduced placental perfusion. Previous studies in a rat model of placental ischemia-induced hypertension demonstrated that inhibiting complement activation attenuated increased maternal blood pressure with C3a and C5a identified as the important products of complement activation. Given that in other forms of ischemia both natural IgM and antigen antibody complexes initiate complement activation, we hypothesized that placental ischemia exposes neoepitopes recognized by IgM to cause local complement activation and hypertension. Alternatively, we postulated that autoantibody to angiotensin II Type 1 receptor (AT1-AA) interacts with AT1 receptors to cause complement activation. Since complement activation occurs in kidney and placenta in preeclampsia, we used immunohistochemistry to determine IgM deposition and local complement activation in each organ (C3 deposition), and quantitative real-time polymerase chain reaction (qRT-PCR) to quantitate mRNA for endogenous regulators of complement activation CD55, CD59 and Complement receptor 1-related gene/protein y (Crry). On gestation day (GD)14.5, timed pregnant Sprague Dawley rats underwent Sham surgery or placement of clips on inferior abdominal aorta and ovarian arteries to create placental ischemia using the reduced utero-placental perfusion pressure (RUPP) model. As previously reported, RUPP surgery increased mean arterial pressure and circulating C3a on GD19.5. In placenta, IgM and C3 deposition increased, whereas mRNA for complement regulators Crry and CD59 decreased along with Crry protein in RUPP compared to Sham treated animals. In kidney, IgM deposition increased in animals subjected to RUPP vs Sham surgery without a significant change in C3 deposition and coincident with an increase in mRNA for CD55 and CD59. The AT1 receptor antagonist losartan prevents placental ischemia-induced hypertension as well as AT1-AA interaction with AT1 receptors. However, losartan did not attenuate complement activation as measured by circulating C3a or placental C3 deposition. Importantly, our studies indicate that following placental ischemia, complement activation is not due to AT1-AA but is associated with IgM deposition. These studies suggest a role for natural antibodies interacting with placental ischemia-induced neoepitopes to activate complement and contribute to hypertension.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

TLR2 Regulates Complement-Mediated Inflammation Induced by Blood Loss During Hemorrhage.

Hemorrhagic shock resulting from blood loss directs the majority of the blood to the vital organs, dramatically reducing blood flow to the intestines and resulting in damage and inflammation. The excessive intestinal inflammatory response includes pro-inflammatory cytokines and complement activation, although the mechanism is not clear. Toll-like receptors play a vital role in the innate immune response and toll-like receptor 2 (TLR2) is required for intestinal ischemia/reperfusion-induced injury. We hypothesized that TLR2 plays an integral role in the intestinal inflammatory response after hemorrhage and subjected C57Bl/6 wild-type and Tlr2(-/-) mice to atraumatic loss of ∼30% total blood volume. Two hours after blood removal, the intestinal injury and inflammation were assessed. We demonstrate that compared with wild-type control mice, Tlr2(-/-) mice sustain less intestinal damage and inflammation. Importantly, TLR2 regulated eicosanoid and complement activation and IL-12 and TNFα secretions, indicating interactions between TLR2 and complement in response to significant blood loss.