Peptide inhibition of acute lung injury in a novel two-hit rat model.

Acute lung injury (ALI) often causes severe trauma that may progress to significant morbidity and mortality. ALI results from a combination of the underlying clinical condition of the patient (e.g., inflammation) with a secondary insult such as viral pneumonia or a blood transfusion. While the secondary insult may be variable, the rapidly progressive disease process leading to pulmonary failure is typically mediated by an overwhelming innate immunological or inflammatory reaction driven by excessive complement and neutrophil-mediated inflammatory responses. We recently developed a 'two-hit' ALI rat model mediated by lipopolysaccharide followed by transfusion of incompatible human erythrocytes resulting in complement activation, neutrophil-mediated ALI and free DNA in the blood indicative of neutrophil extracellular trap formation. The objective of this study was to evaluate the role of peptide inhibitor of complement C1 (RLS-0071), a classical complement pathway inhibitor and neutrophil modulator in this animal model. Adolescent male Wistar rats were infused with lipopolysaccharide followed by transfusion of incompatible erythrocytes in the presence or absence of RLS-0071. Blood was collected at various time points to assess complement C5a levels, free DNA and cytokines in isolated plasma. Four hours following erythrocyte transfusion, lung tissue was recovered and assayed for ALI by histology. Compared to animals not receiving RLS-0071, lungs of animals treated with a single dose of RLS-0071 showed significant reduction in ALI as well as reduced levels of C5a, free DNA and inflammatory cytokines in the blood. These results demonstrate that RLS-0071 can modulate neutrophil-mediated ALI in this novel rat model.

Double anus in an Ixodes scapularis nymph, a medically important tick vector.

BACKGROUND: Ixodes scapularis ticks are medically important arthropod vectors that transmit several pathogens to humans. The observations of morphological abnormalities, including nanism, missing leg, extra leg, and gynandromorphism, have been reported in these ticks. In this study, we report the presence of two anuses in a laboratory-reared I. scapularis nymph. RESULTS: Larval ticks were allowed to feed on mice and to molt to nymphs. Two anuses were observed in one of the freshly molted nymphs. Stereo and scanning electron microscopy confirmed the presence of two anuses in one nymph within a single anal groove. CONCLUSIONS: This report confirms the rare occurrence of double anus in I. scapularis.

Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

OBJECTIVE: Complement activation is instrumental in the pathogenesis of Hypoxic-ischemic encephalopathy (HIE), a significant cause of neonatal mortality and disability worldwide. Therapeutic hypothermia (HT), the only available treatment for HIE, only modestly improves outcomes. Complement modulation as a therapeutic adjunct to HT has been considered, but is challenging due to the wide-ranging role of the complement system in neuroinflammation, homeostasis and neurogenesis in the developing brain. We sought to identify potential therapeutic targets by measuring the impact of treatment with HT on complement effector expression in neurons and glia in neonatal HIE, with particular emphasis on the interactions between microglia and C1q. METHODS: The Vannucci model was used to induce HIE in term-equivalent rat pups. At P10-12, pups were randomly assigned to three different treatment groups: Sham (control), normothermia (NT), and hypothermia (HT) treatment. Local and systemic complement expression and neuronal apoptosis were measured by ELISA, TUNEL and immunofluorescence labeling, and differences compared between groups. RESULTS: Treatment with HT is associated with decreased systemic and microglial expression of C1q, decreased systemic C5a levels, and decreased microglial and neuronal deposition of C3 and C9. The effect of HT on cytokines was variable with decreased expression of pro and anti-inflammatory effectors. HT treatment was associated with decreased C1q binding on cells undergoing apoptosis. CONCLUSION: Our data demonstrate the extreme complexity of the immune response in neonatal HIE. We propose modulation of downstream effectors C3a and C5a as a therapeutic adjunct to HT to enhance neuroprotection in the developing brain.

Incompatible erythrocyte transfusion with lipopolysaccharide induces acute lung injury in a novel rat model.

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype.

Learner Improvement From a Simulation-Enhanced Ultrasonography Curriculum for First-Year Medical Students.

OBJECTIVES: We describe a simulation-enhanced ultrasonography (US) curriculum for first-year medical students as part of a comprehensive curricular integration of US skills. Our goal was to assess student knowledge and performance of US and determine their satisfaction with the integrated curriculum. METHODS: A committee of basic science, clinical, and interinstitutional faculty developed 7 educational US modules integrated into existing anatomy and physiology courses. First-year students in years 2012 through 2014 were administered a demographic survey and a knowledge-based pretest at the outset of the US program and assessed with a posttest, satisfaction survey, and their image acquisition abilities in an objective structured clinical examination with standardized patients on completion of the program. RESULTS: Data from 390 students showed a significant increase in knowledge from the pretest to the posttest [t(389) = 58.027; P < .0001]. Students with higher spatial abilities or some previous US experience performed better on the posttest. The objective structured clinical examination results showed that about 83% of the students were able to capture acceptable or marginally acceptable images. Ninety-five percent of students indicated that the US educational experience enhanced their medical education. CONCLUSIONS: Initial results show that we were able to successfully develop, implement, and evaluate performance of first-year medical students on their fundamental knowledge and performance of basic US using a model that emphasized hands-on simulation-enhanced training. Furthermore, most students found the experience to be a beneficial component of their education and indicated a desire for more US training in the medical curricula.

Therapeutic hypothermia modulates complement factor C3a and C5a levels in a rat model of hypoxic ischemic encephalopathy.

BACKGROUND: Therapeutic hypothermia (HT) is the only intervention that improves outcomes in neonatal hypoxic-ischemic encephalopathy (HIE). However, the multifactorial mechanisms by which HT impacts HIE are incompletely understood. The complement system plays a major role in the pathogenesis of ischemia-reperfusion injuries such as HIE. We have previously demonstrated that HT modulates complement activity in vitro. METHODS: Term equivalent rat pups were subjected to unilateral carotid ligation followed by hypoxia (8% O2) for 45 min to simulate HIE. A subset of animals was subjected to HT (31-32°C for 6 h). Plasma and brain levels of C3a and C5a were measured. Receptors for C3a (C3aR) and C5a (C5aR) along with C1q, C3, and C9 were characterized in neurons, astrocytes, and microglia. RESULTS: We found that HT increased systemic expression of C3a and decreased expression of C5a after HIE. In the brain, C3aR and C5aR are predominantly expressed on microglia after HIE. HT increased local expression of C3aR and decreased expression on C5aR after HIE. Furthermore, HT decreased local expression of C1q, C3-products, and C9 in the brain. CONCLUSION: HT is associated with significant alteration of complement effectors and their cognate receptors. Complement modulation may improve outcomes in neonatal HIE.

Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats.

BACKGROUND: Acute hemolytic transfusion reactions have a broad clinical presentation from mild and transitory signs and symptoms to shock, disseminated intravascular coagulation, renal failure, and death. We have recently developed a rat model of acute intravascular hemolysis showing that the classical complement pathway mediates antibody-dependent hemolysis. The objective of this study was to evaluate the role of the classical pathway inhibitor peptide inhibitor of complement C1 (PIC1) in this animal model. STUDY DESIGN AND METHODS: Male Wistar rats received a 15% transfusion of human red blood cells (RBCs) and blood was isolated from the animals up to 120 minutes. Animals received PIC1 either 2 minutes before or 0.5 minutes after transfusion. Sham-, vehicle-, and cobra venom factor (CVF)-treated animals were used as control groups with a subset of rats also receiving an equivalent dose of intravenous immunoglobulin (IVIG) before transfusion. Blood was analyzed for transfused RBC survival by flow cytometry and free hemoglobin (Hb) in isolated plasma by spectrophotometry. RESULTS: Vehicle-treated rats showed decreased human RBC survival and increased free Hb as expected. Rats receiving PIC1 before transfusion showed increased human RBC survival and decreased Hb similar to CVF-treated rats. Notably, rats receiving PIC1 after initiation of transfusion showed similar decreases in hemolysis as animals receiving PIC1 before transfusion. Compared to IVIG and saline controls, PIC1-treated animals demonstrated decreased hemolysis and protection from acute kidney injury. CONCLUSIONS: These results demonstrate that PIC1 has efficacy in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios.

Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis.

BACKGROUND: Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. STUDY DESIGN AND METHODS: Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. RESULTS: In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. CONCLUSIONS: Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis.

A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility.

Previous experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals' serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (polar assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24h post-injection. CPP's ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease.

Accelerated atherosclerosis in Apoe-/- mice heterozygous for the insulin receptor and the insulin receptor substrate-1.

OBJECTIVE: Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. METHODS AND RESULTS: To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr(+/-)Irs1(+/-)Apoe(-/-)) mice. Insr(+/-)Irs1(+/-)Apoe(-/-) mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr(+/-)Irs1(+/-)Apoe(-/-) and Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr(+/-)Irs1(+/-)Apoe(-/-) compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr(+/-)Irs1(+/-) to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. CONCLUSIONS: These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr(+/-)Irs1(+/-)Apoe(-/-) mice by promoting vascular dysfunction and inflammation.

Efficacy of a phosphorodiamidate morpholino oligomer antisense compound in the inhibition of corneal transplant rejection in a rat cornea transplant model.

PURPOSE: The cornea is one of the most commonly transplanted tissues. The morpholino-oligomer antisense compound AVI-5126 suppresses expression of proto-oncogene c-myc, a key factor in transplant rejection. AVI-5126 was evaluated in a rat cornea transplant model. METHODS: Donor corneas obtained from August x Copenhagen Irish rats were stored in Optisol™ containing 1.0 or 0.5 mg/mL AVI-5126 or Optisol alone for 24 h before transplant. Recipient Lewis rats were treated topically 3x/d with TobraDex™ and with 1.0 or 0.5 mg/mL of AVI-5126 or saline with daily monitoring for rejection using a modified McDonald-Shadduck Slit Lamp Scale. Using the high-performance liquid chromatography technique, the stability of AVI-5126 (0.5 mg/mL) in Optisol was evaluated for 30 days. AVI-5126 corneal transport was measured using Ussing chamber mounted rabbit corneas. The potential ocular toxicity of AVI-5126 (0.5 mg/mL) was evaluated after 8 days of 3x/d topical application in rats and in-vitro by incubation of human corneas for 8 days. RESULTS: Cornea storage in Optisol containing 1.0 mg/mL AVI-5126 plus post-transplant topical tid AVI-5126 (1.0 mg/mL) application significantly increased graft survival (7.0±1.6 days) versus 5.0±0.8 days for Optisol alone storage plus post-transplant topical tid saline application (P<0.001). After 30 days of storage, no significant degradation of AVI-5126 in Optisol was noted by high-performance liquid chromatography analysis. After 24 h, 5 µg/mL (1% of total dose) crossed the corneas mounted in Ussing chambers. Neither extended topical application of AVI-5126 to rats nor incubation of human corneas in AVI-5126 decreased endothelial cell density. CONCLUSIONS: Graft rejection was significantly delayed after pretransplantation storage of graft corneas in Optisol containing AVI-5126 followed by topical application of AVI-5126 post-transplantation. AVI-5126 was well tolerated, stable, and effectively penetrated the cornea.

Lacritin, a novel human tear glycoprotein, promotes sustained basal tearing and is well tolerated.

PURPOSE: Lacritin is a novel human tear glycoprotein that promotes basal tear peroxidase secretion by rat lacrimal acinar cells in vitro. This study investigates whether lacritin is prosecretory when added topically to the ocular surface of normal living rabbits, and if so, what is its efficacy and tolerability versus cyclosporine and artificial tears. METHODS: Purified recombinant human lacritin (1, 10, 50, or 100 µg/mL), inactive lacritin truncation mutant C-25 (10 µg/mL), cyclosporine (0.05%), or artificial tears were topically administered to eyes of normal New Zealand White rabbits either as a single dose or three times daily for 14 days with monitoring of basal tear production. Basal tearing under proparacaine anesthesia was repeatedly assessed throughout and 1 week after chronic treatment ceased. Eyes were examined weekly by slit-lamp biomicroscopy. RESULTS: Lacritin acutely increased basal tearing to 30% over vehicle at 240 minutes. Three times daily treatment with 10-100 µg/mL lacritin was well tolerated. Basal tearing became progressively elevated 4, 7, and 14 days later and was 50% over baseline (50 µg/mL lacritin) 1 week after treatment had ceased. Cyclosporine elevated tearing to a similar level on days 4 and 7 but had little or no effect on day 14 and had returned to baseline 1 week after ending treatment. C-25 and artificial tears had no effect. CONCLUSIONS: Lacritin acutely stimulates basal tear flow that is sustained for at least 240 minutes. Two weeks of lacritin treatment three times daily was well tolerated and progressively elevated the basal tear flow. One week after treatment ended, basal tearing was still 50% over baseline. In contrast, cyclosporine triggered mild to moderate corneal irritation and a temporary elevation in tearing.

3,4-Methylenedioxymethamphetamine alters left ventricular function and activates nuclear factor-Kappa B (NF-κB) in a time and dose dependent manner.

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-κB) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, duration of relaxation, duration of cardiac cycle, and mean left ventricular pressure. Pretreatment with L-NAME followed by treatment with MDMA caused significant dysfunction in additional parameters that were not abnormal upon exposure to either compound in isolation: duration of contraction, inotropy, and pulse pressure. Exposure to 1.0 mM MDMA for 6 h or 2.0 µM MDMA for 12 h caused increased nuclear localization of NF-κB in cultured H9c2 cells. The current results suggest that MDMA is acutely detrimental to heart function and that an intact cardiovascular NOS system is important to help mitigate early sequelae in some functional parameters. The delayed timing of NF-κB activation suggests that this factor may be relevant to MDMA induced cardiomyopathy of later onset.

Comparison of topical and intravenous administration of WIN 55-212-2 in normotensive rabbits.

OBJECTIVE: This study compares the effect of topical versus intravenous (IV) administration of synthetic WIN 55-212-2 (WIN) or timolol on intraocular pressure (IOP). METHODS: WIN or timolol were administered either topically or by IV in normotensive New Zealand white rabbits. IOP was measured at baseline and 30, 60, and 120 min after administration (n = 4 per group). Blood pressure (BP) and heart rate (HR) were measured concomitantly with IOP. RESULTS: IV administration of 0.1 mg/kg WIN reduced IOP by 30% after 30 min, which continued to decline for up to 120 min. Timolol injection (25 mu g/kg) also reduced IOP by 25% after 30 min but was not sustained. In comparison, both topical WIN (1.0%) and timolol (0.5%) reduced IOP by 20% from baseline after 30 min. IV injection of either WIN or timolol significantly reduced HR to 155.4 +/- 11.4 bpm and 165.9 +/- 11.1 bpm, respectively, from a baseline of 256.3 +/- 9.9 bpm. Topical administration was well tolerated and did not affect behavior, BP, or HR. CONCLUSION: Topical administration of either WIN or timolol did not decrease IOP as much as IV administration, but the lack of systemic or local toxicity could make it the safer alternative.

Topical WIN55212-2 alleviates intraocular hypertension in rats through a CB1 receptor mediated mechanism of action.

INTRODUCTION: Systemically administered cannabinoids can reduce intraocular pressure (IOP), but produce undesirable cardiovascular and central nervous system effects. In a chronic model of ocular hypertension, we examined the efficacy of acute topical administration of WIN55212-2 (WIN) in a novel commercially available vehicle and in combination with timolol. METHODS: IOP was chronically elevated by the surgical ligature of vortex veins in Sprague Dawley rats. IOP was measured by using Goldmann applanation tonometry. IOP, blood pressure (BP), and heart rate (HR) were measured at baseline and 30, 60, 90, and 120 min after the topical administration of WIN 1.0%, 0.25%, 0.06%, or 0.015%, the commercially available vehicle, timolol 0.5%, or a combination of WIN and timolol. SR141716 (CB1 antagonist) or SR144528 (CB2 antagonist) was administered topically 30 min before WIN to determine receptor specificity. To determine ocular and systemic penetration, 3H WIN 55212-2 was administered topically and tissues were collected at 60 and 120 min. Ocular irritation was evaluated by slit-lamp examination (SLE) at baseline and 120 min. RESULTS: WIN significantly decreased IOP in the hypertensive eye, with no BP or HR effects. SR141716 pretreatment significantly inhibited the IOP effects of WIN 1.0% in a dose-dependent manner, while SR 144528 was not as effective. No significant additive effects were observed by combining WIN (0.5% or 1.0%) with timolol 0.5%. WIN was retained in ocular tissue with a t1/2 of 80-100 min. SLE at 120 min revealed no solvent or drug-related toxic effects. CONCLUSIONS: In a chronic ocular hypertensive rat model, topically applied WIN is an effective, nontoxic ocular hypotensive agent with no hemodynamic side-effects. This effect was predominantly CB1 receptor mediated, but some CB2 contribution could not be ruled out.

Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system.

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.

Primate granulosa cell response via prostaglandin E2 receptors increases late in the periovulatory interval.

Successful ovulation requires elevated follicular prostaglandin E2 (PGE2) levels. To determine which PGE2 receptors are available to mediate periovulatory events in follicles, granulosa cells and whole ovaries were collected from monkeys before (0 h) and after administration of an ovulatory dose of hCG to span the 40-h periovulatory interval. All PGE2 receptor mRNAs were present in monkey granulosa cells. As assessed by immunofluorescence, PTGER1 (EP1) protein was low/nondetectable in granulosa cells 0, 12, and 24 h after hCG but was abundant 36 h after hCG administration. PTGER2 (EP2) and PTGER3 (EP3) proteins were detected by immunofluorescence in granulosa cells throughout the periovulatory interval, and Western blotting showed an increase in PTGER2 and PTGER3 levels between 0 h and 36 h after hCG. In contrast, PTGER4 (EP4) protein was not detected in monkey granulosa cells. Granulosa cell response to PGE2 receptor agonists was examined 24 h and 36 h after hCG administration, when elevated PGE2 levels present in periovulatory follicles initiate ovulatory events. PGE2 acts via PTGER1 to increase intracellular calcium. PGE2 increased intracellular calcium in granulosa cells obtained 36 h, but not 24 h, after hCG; this effect of PGE2 was blocked by a PTGER1 antagonist. A PTGER2-specific agonist and a PTGER3-specific agonist each elevated cAMP in granulosa cells obtained 36 h, but not 24 h, after hCG. Therefore, the granulosa cells of primate periovulatory follicles express multiple receptors for PGE2. Granulosa cells respond to agonist stimulation of each of these receptors 36 h, but not 24 h, after hCG, supporting the hypothesis that granulosa cells are most sensitive to PGE2 as follicular PGE2 levels peak, leading to maximal PGE2-mediated periovulatory effects just before ovulation.

Argon laser photodynamic therapy of human corneal neovascularization after intravenous administration of dihematoporphyrin ether.

PURPOSE: Long-term evaluation of dihematoporphyrin ether (DHE) safety and efficacy as photodynamic therapy (PDT) for patients with corneal neovascularization (KNV). DESIGN: Prospective multi-center interventional case series. METHODS: Seven patients were enrolled after Institutional Review Board approval and a detailed informed consent were obtained. Eligible patients presented with clinically stable KNV without active vessel progression or inflammation. All patients with severe hypertension, history of renal or hepatic disease, or sensitivity to porphyrins, and those with active keratitis or uncontrolled ocular surface disease were excluded. DHE was administered as an intravenous bolus (2 mg/kg). Seventy-two hours later, PDT was carried out using argon green laser (514 nm). The main outcome measure, extent of vascular thrombosis, was estimated during postoperative follow-up examinations performed at day 1, 1 week, 6 months, and up to 12 years postoperative. RESULTS: All patients obtained an immediate reduction in measurable corneal vascularization. With at least 6 months of follow-up, six of seven patients maintained a significant reduction (52.5% +/- 19.6%, P < .01) in KNV. The mean length of followup was 5.4 years (Range = 6 months to 12 years) during which time there were no other ocular changes attributable to laser treatment. Three patients suffered significant systemic short-term phototoxicity reactions. CONCLUSIONS: Intravenous DHE followed by photodynamic treatment in humans is effective for the reduction of inactive, established KNV. However, the significant short-term adverse effects related to systemic administration of this drug are of particular concern and warrant further investigation.

Differential O-glycosylation of a conserved domain expressed in murine and human ZP3.

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.