Elizabeth Mainolfi

Elevated Levels of Circulating Adhesion Molecules in IDDM Patients and in Subjects at Risk for IDDM

Serum levels of recently discovered circulating forms of adhesion molecules, ICAM-1 and L-selectin, were found to be elevated in IDDM patients and in subjects at risk for developing IDDM compared with 100 normal, nondiabetic blood donors. Both adhesion molecules were determined by sandwich ELISA. Serum concentrations of either clCAM-1 or cL-selectin were >2SD of normal mean in 10 of 14 recent-onset IDDM patients (P < 0.05). Serum levels of clCAM-1 and cL-selectin did not correlate. In first-degree relatives, elevated adhesion molecule levels were observed in the 6 ICA+ individuals and in the ICA− individuals all (n = 14) with a genetic risk of IDDM (sharing HLA-DR3 and/or -DR4− with the diabetic relative) but not in the HLA-DR3− and/or -DR4− relatives (n = 13). We conclude that elevated clCAM-1 and cL-selectin levels occur independently of ICA status and probably reflect ongoing immune processes in recent-onset IDDM patients and first-degree relatives at risk for IDDM.

Circulating intercellular adhesion molecule-1 levels and neutrophil adhesion in stroke

To examine whether changes in leukocyte adhesion properties occur during stroke, we measured circulating serum intercellular adhesion molecule 1 (cICAM-1) levels and neutrophil adhesion in acute stroke, patients at high risk of stroke, and in matched controls. Levels of cICAM-1 were significantly lower in the stroke group (186.2 +/- 15.6 ng ml-1) compared to controls (257.7 +/- 24.8) and risks (257.7 +/- 16.5). Neutrophil adhesion was significantly higher in the stroke group (23.6 +/- 4.3%; n = 14) compared to controls (9.7 +/- 2.3%; n = 12) and risks (12.7 +/- 2.5%; n = 13). These data suggest that changes in leukocyte adhesion dynamics are occurring in acute stroke.

Differential expression and release of CD54 induced by cytokines

Intercellular adhesion molecule‐1 (ICAM‐1, CD54) is upregulated in many cell types stimulated by cytokines. A human hepatoblastoma cell line (C3A, a subclone of HepG2/C3 that is currently being used as a surrogate liver) and human lung adenocarcinoma cells (A549) were stimulated with interleukin‐β (IL‐β), tumor necrosis factor‐α (TNFα), interferon‐γ (EFNγ), or IL‐6 to determine any differences in cell type responsiveness to individual cytokines for ICAM‐1 upregulation. Time courses were performed with each cytokine evaluating ICAM‐1 mRNA, surface expression, and cICAM‐1 in the cell culture media. Between 3 and 6 hours, IL‐β (30 U/mL) stimulated the greatest increase in hepatocyte ICAM‐1 mRNA, followed by IFNγ (100 U/mL), TNFα (30 U/mL), and IL‐6 (100 U/mL) in order of potency. Except for EL‐6, cytokine‐induced hepatocyte surface levels of ICAM‐1 (immunofluorescence flow cytometry, mAb R6.5) were dose dependent, with inhibition at higher concentration. Highest levels followed stimulation with IFNγ (P < .05). Significantly less was found after both EL‐1β and TNFα; none was detected after IL‐6 (P < .05). In contrast, IL‐1β stimulated significantly more cICAM‐1 release from hepatocytes than the other cytokines (P < .001), and IL‐6 stimulated modest cICAM‐1. Between 3 and 6 hours in the A549 cells, IL‐1β stimulated the greatest increase in ICAM‐1 mRNA, followed by TNFα. Both responses were greater than that observed in the hepatocytes. IFNγ‐and IL‐6‐induced ICAM‐1 mRNA synthesis was not different from unstimulated A549 cells. Cytokine‐induced A549 surface levels of ICAM‐1 (immunofluorescence flow cytometry, mAb R6.5) was highest for IL‐1β (peak levels similar to hepatocyte response), modest with TNFα (peak levels less than hepatocytes), detectable with IFNγ (much less than hepatocytes), and nondetectable after IL‐6. No cICAM‐1 release from A549 cells was induced under any condition. In hepatocytes the amount of ICAM‐1 mRNA was best accounted for by considering both cell surface levels of ICAM‐1 and cICAM‐1 levels. In human lung adenocarcinoma cells, the cytokine induction of ICAM‐1 mRNA could potentially be accounted for by observing cell surface levels of ICAM‐1 because no cICAM‐1 was produced. These results suggest that surface ICAM‐1 and cICAM‐1 may be differentially controlled by each cytokine and by each parenchymal cell type. (Hepatology 1995; 22:866–875.)

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.

Identification of a Potent Sodium Hydrogen Exchanger Isoform 1 (NHE1) Inhibitor with a Suitable Profile for Chronic Dosing and Demonstrated Cardioprotective Effects in a Preclinical Model of Myocardial Infarction in the Rat

Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.

Circulating intercellular adhesion molecule-1 in amniotic fluid, maternal serum α-fetoprotein levels, and intrauterine growth retardation

OBJECTIVE Our purpose was to determine if circulating intercellular adhesion molecule-1, a marker of chronic inflammation, is present in amniotic fluid in midtrimester, is increased in patients with elevated maternal serum alpha-fetoprotein level, and is associated with intrauterine growth retardation. STUDY DESIGN Amniotic fluid circulating intercellular adhesion molecule-1 levels were assayed by enzyme-linked immunoassay in 273 samples obtained by midtrimester amniocentesis in gestations involving a single, structurally normal fetus. The control group consisted of 108 patients with normal maternal serum alpha-fetoprotein levels and 165 patients with elevated levels. Intrauterine growth retardation was diagnosed if birth weight was < 10th percentile for the clinically estimated gestational age. RESULTS Circulating intercellular adhesion molecule-1 was detectable in amniotic fluid in 105 of 273 samples (38%). In the control group it was detectable in amniotic fluid in seven of 108 (6%). In patients with elevated maternal serum alpha-fetoprotein 97 of 164 (59%) had detectable levels (p < 0.001). Of the 273 cases 38 (14%) had intrauterine growth retardation. Of these 23 (59%) had detectable circulating intercellular adhesion molecule-1 levels (p < 0.001). Of the seven cases of intrauterine growth retardation with normal maternal serum alpha-fetoprotein levels, one (14%) had detectable circulating intercellular adhesion molecule-1. Of the 31 cases of intrauterine growth retardation with elevated maternal serum alpha-fetoprotein 22 (71%) had detectable circulating intercellular adhesion molecule-1. When circulating intercellular adhesion molecule-1 was detectable in amniotic fluid, increasing levels was significantly related to decreasing gestational age at delivery (p < 0.005). CONCLUSIONS Midtrimester amniotic fluid from normal pregnancies does not generally contain detectable circulating intercellular adhesion molecule-1. Detectable amniotic fluid levels are significantly related to a birth weight < 10th percentile at delivery and to elevated midtrimester maternal serum alpha-fetoprotein levels. Increasing circulating intercellular adhesion molecule-1 levels are related to shortened length of gestation. This test may contribute to risk assessment for intrauterine growth retardation and prematurity. Circulating intercellular adhesion molecule-1 is a known marker of inflammatory processes; its further study may also improve understanding of the pathophysiologic mechanisms of certain cases of intrauterine growth retardation and prematurity.

Deconstruction of sulfonamide inhibitors of the urotensin receptor (UT) and design and synthesis of benzylamine and benzylsulfone antagonists

Potent small molecule antagonists of the urotensin receptor are described. These inhibitors were derived via systematically deconstructing a literature inhibitor to understand the basic pharmacophore and key molecular features required to inhibit the protein receptor. The series of benzylamine and benzylsulfone antagonists herein reported display a combination of nanomolar molecular and cellular potency as well as acceptable in vitro permeability and metabolic stability.

Detection and Characterization of Circulating ICAM-1

Over the past several years, the expression of intercellular adhesion molecule-1 (ICAM-1) on cells from normal and diseased tissue has been extensively studied. Almost as soon as ICAM-1 was identified, it was found to be a cell-surface adhesion molecule that was inducible in vitro with inflammatory cytokines such as IL-1, TNFα, and IFNγ on a multitude of cell types. Furthermore, ICAM-1’s expression on cells from normal and diseased tissue has been and continues to be a focus of much research from many laboratories. To date, it has been reported that there is a low constitutive expression of ICAM-1 on venule endothelial cells; however, its expression is markedly increased at inflammatory sites (1). Furthermore, there is an increased ICAM-1 expression on multiple cell types including keratinocytes in inflammatory skin lesions (2,3), transplanted liver bile duct and perivenular hepatocytes during rejection (4), and in cases of primary biliary cirrhosis (5), endothelium in the brain surrounding MS plaques and EAE lesions in man and rodent, respectively (6–12), transplanted kidney glomeruli and tubules during rejection (13), as well as lung epithelial cells following antigen provocation (14). Increased ICAM-1 is also found on melanoma cells following metastasis (15,16) and recently ICAM-1 expression has been shown to be expressed on placental macrophages and decidua during pregnancy.