Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.     

Small-cell lung carcinoma: inhibition of proliferation by vasoactive intestinal peptide and helodermin and enhancement of inhibition by anti-bombesin antibody

Small-cell lung cancer (SCLC) is a common and highly fatal malignancy for which there is no satisfactory treatment. The amphibian peptide bombesin and its mammalian counterpart, gastrin-releasing peptide, serve as autocrine growth factors for the SCLC cells, but little is known about endogenous substances that inhibit the growth and proliferation of these tumor cells. We report that the neuropeptide vasoactive intestinal peptide (VIP) markedly inhibits the growth and multiplication of SCLC cell lines NCI-H345 and NCI-H69, and that the closely related peptide helodermin inhibits the proliferation of NCI-H345 cells with even higher efficacy. In the latter cells, the inhibition by VIP and isobutyl methyl xanthine paralleled their ability to stimulate cyclic adenosine monophosphate production within the cells. The peptide-induced suppression of SCLC proliferation is enhanced in the presence of an anti-bombesin monoclonal antibody. The antimitogenic activities of VIP and helodermin, and their enhancement by anti-bombesin antibody, offer the potential for a new approach to the pharmacologic control of SCLC.     

Partial inhibition of amplifications by primers of EHEC genes

Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.     

Partial inhibition of oestrogen-induced adenohypophyseal growth by silver nitrate

The administration of silver nitrate to rats in their food (10 mg/rat/day) led to the rapid disappearance of serum polyphenol oxidase activity. After 60 days silver nitrate treatment produced a decrease of adenohypophyseal weight. If given over a period of 40--60 days it also partially inhibited the adenohypophyseal response to oestradiol (a weight decrease and raised thyroxine binding by adenohypophyseal proteins in vitro). The mechanism by which silver nitrate diminishes basal and oestrogen-increased adenohypophyseal weight remains unknown.     

The hemagglutination inhibition antibody responses to an inactivated influenza vaccine among healthy adults: with special reference to the prevaccination antibody and its interaction with age

The immunogenicity of the trivalent split-virus influenza vaccine was investigated among 70 healthy adults (mean age: 48.5, range: 36-68). The vaccine antigens were: A/Yamagata/32/89 (H1N1); A/Beijing/352/89 (H3N2); and B/Bangkok/163/90. Regarding the entire sample, the vaccine induced a tenfold or more rise on the average in the hemagglutination inhibition (HAI) antibody to each antigen. The response rates (greater than or equal to a fourfold rise) were about 90% or more among those with a prevaccination titer < or = 1:64 (equivalent to < or = 1:16 on the Western scale; in Japan, the HAI titers are expressed by the final, and not the initial, dilution of the serum; from hereon our findings will be expressed using the Japanese scale), whereas they were 0-50% at > or = 1:128. Thus, the prevaccination titer was negatively associated with antibody induction. The achievement rates (postvaccination titer > or = 1:128) among those with a prevaccination titer < 1:16 remained at 48-68%. Regarding the analysis of variance, a significant effect on antibody induction was indicated for the prevaccination titer (P < or = 0.002), but not for age (P > or = 0.425). The interaction between the prevaccination titer and age was significant for A/Yamagata (P = 0.030), while it was also suggestive for A/Beijing (P = 0.054): as age increased, those with no preexisting antibody (< 1:16) showed greater titer rises, in contrast to the smaller rises among those with a titer > or = 1:16. Based on the attack survey conducted separately, the vaccine efficacy on influenza-like illnesses with fever < or = 37 degrees C and > or = 37.5 degrees C was calculated to be 16% (95% confidence interval: -66% to 57%) and 37% (-55% to 74%), respectively.     

Improvement in nerve regeneration by monoclonal antibodies to ICAM-1 and LFA-1 in allogeneic mice

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.     

T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells

Transendothelial migration is a crucial step in the complex process of lymphocyte extravasation during lymphocyte homing, immunosurveillance and inflammation. However, little is known about the precise role of cell adhesion molecules (CAM) involved in this particular event. To define the CAM involved in T cell adhesion versus transendothelial migration, we have previously established an in vitro transendothelial migration system using mouse T cells and mouse endothelioma cells. We demonstrate here that, using ICAM-1-deficient endothelioma cells derived from ICAM-1 mutant mice, transendothelial migration of T cells was inhibited to a much greater extent when compared to migration across wild-type cells treated with a blocking anti-ICAM-1 monoclonal antibody. This unexpected result was confirmed by a rescue experiment using retroviral transfer of wild-type ICAM-1 into ICAM-1-deficient endothelial cells. Additional experiments showed that, in the absence of functional ICAM-1, only ICAM-2 was involved in transendothelial migration, but not PECAM-1, VCAM-1, or E-selectin. Taking this novel approach, we show that ICAM-1 and ICAM-2 are essential for transendothelial migration of T cells.     

Proteolytic cleavage of ICAM-1 by human neutrophil elastase

Human leukocyte elastase (HLE) participates in tissue destruction in a number of inflammatory disorders, including rheumatoid arthritis and cystic fibrosis. Since HLE has been shown to bind to Mac-1, and ICAM-1 plays a key role during the recruitment and the activation of leukocytes at inflamed sites, we investigated the capacity of HLE to cleave ICAM-1. Flow-cytometric analyses showed a dose-dependent cleavage of ICAM-1 by HLE on different human cell lines. The cleavage was completely inhibited by alpha1-antitrypsin, a natural HLE protease inhibitor. The ability of HLE to degrade ICAM-1 was further confirmed by electrophoretic analysis using a soluble form of ICAM-1 (D1-D5). Enzymatic removal of N-linked glycosylation did not significantly modulate ICAM-1 cleavage by HLE, while removal of sialic acid residues partially reduced the sensitivity of ICAM-1 to HLE. We further showed that sputum of cystic fibrosis patients contains high levels of HLE activity capable of cleavage of cell surface ICAM-1. The cleavage induced by incubation of cells with the sputum sample was totally inhibited by alpha1-antitrypsin and the specific peptidic HLE inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone. Moreover, the cleavage of ICAM-1 was concomitant to that of CD4 at the surface of the same cell, at the same amplitude, and at all HLE concentrations. The capacity of HLE to modulate the expression of ICAM-1 on the surface of leukocytes by proteolytic cleavage brings support to the hypothesis that overproduction of HLE can cause severe immunologic lung disorders by affecting intercellular adhesion.     

Identification of Merkel cells by an antibody to villin

Merkel cells represent a population of epithelial cells in the skin and oral mucosa. Although Merkel cells are reliably distinguishable from other epithelial cells at the ultrastructural level, these cells are usually not discernible by standard light microscopy and need special techniques for their identification. Villin is an actin-crosslinking protein that is associated with the actin filament cores of brush border microvilli. In this study we show that an antibody against villin is an excellent marker of Merkel cells and their microvilli even at the light microscopic level. The surrounding keratinocytes and subepithelial connective tissue cells do not show any significant affinity for the antibody against villin. Confocal laser micrographs reconstructed from serial images 0.5 microm thick of Merkel cells that were immunostained with villin clearly reveal the three-dimensional morphology of Merkel cells and their microvilli. The presence of villin in Merkel cell microvilli lends support to the idea that these cells might have a mechanoreceptor function.     

Trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism

When the infected mammalian host of Trypanosoma brucei brucei is injected with a solution of the iron chelator salicyl hydroxamic acid and glycerol, the aerobic and anaerobic glucose catabolism of the parasite is blocked and the parasite is rapidly destroyed.     

Subchronic neurotoxicity screening studies with six organophosphate insecticides: an assessment of behavior and morphology relative to cholinesterase inhibition

Sulprofos, disulfoton, azinphos-methyl, methamidophos, trichlorfon, and tebupirimphos were screened for neurotoxic potential, in accordance with U.S. EPA (FIFRA) requirements. Each organophosphate was administered through the diet for 13 weeks to separate groups of Fischer 344 rats at four dose levels, including a vehicle control. For each study, 12 rats/sex/dietary level were tested using a functional observational battery (FOB), automated measures of activity (figure-8 maze), and detailed clinical observations, with half of the animals perfused at term for microscopic examination of neural and muscle tissues. Separate groups of satellite animals (6/sex/dietary level) were used to measure the effect of each treatment on plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity. The results show that measures of ChE activity were consistently the most sensitive indices of exposure and assisted in the interpretation of findings. All treatment-related neurobehavioral findings were ascribed to cholinergic toxicity, occurring only at dietary levels that produced more than 20% inhibition of plasma, RBC, and brain ChE activity. Neurobehavioral tests provided no evidence of additional cumulative toxicity after 8 weeks of treatment. The FOB and motor activity findings did not alter the conclusions and generally did not reduce the neurobehavioral no-observed-effect level (NOEL) for any of the six compounds, relative to the results from detailed clinical observations as conducted in these studies. The one exception occurred with tebupirimphos, where the NOEL for motor activity was one dose level lower than the NOEL for the FOB and clinical observations. These results support the value of detailed clinical observations to screen for the neurotoxic potential of organophosphates and a general standard of more than 20% inhibition of brain ChE activity for cholinergic neurotoxicity.     

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.     

Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4, 5-bisphosphate

Ezrin is a cytoplasmic linker molecule between plasma membrane components and the actin-containing cytoskeleton. We studied whether ezrin is associated with intercellular adhesion molecule (ICAM)-1, -2, and -3. In transfected cells, ICAM-1 and ICAM-2 colocalized with ezrin in microvillar projections, whereas an ICAM-1 construct attached to cell membrane via a glycophosphatidylinositol anchor was uniformly distributed on the cell surface. An interaction of ICAM-2 and ezrin was seen by affinity precipitation, microtiter binding assay, coimmunoprecipitation, and surface plasmon resonance methods. The calculated KD value was 3.3 x 10(-7) M. Phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) induced an interaction of ezrin and ICAM-1 and enhanced the interaction of ezrin and ICAM-2, but ICAM-3 did not bind ezrin even in the presence of PtdIns(4,5)P2. PtdIns(4, 5)P2 was shown to bind to cytoplasmic tails of ICAM-1 and ICAM-2, which are the first adhesion proteins demonstrated to interact with PtdIns(4,5)P2. The results indicate an interaction of ezrin with ICAM-1 and ICAM-2 and suggest a regulatory role of phosphoinositide signaling pathways in regulation of ICAM-ezrin interaction.     

Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization

Incorporation of the intercellular adhesion molecule ICAM-1 into human immunodeficiency virus type 1 (HIV-1) particles increased virus infectivity on peripheral blood mononuclear cells (PBMCs) by two- to sevenfold. The degree of ICAM-1-mediated enhancement was greater for viruses bearing envelope glycoproteins derived from primary HIV-1 isolates than for those bearing envelope glycoproteins from laboratory-adapted strains. Treatment of target PBMCs with an antibody against LFA-1, a principal ICAM-1 receptor, was able to nullify the ICAM-1-mediated enhancement. The incorporation of ICAM-1 rendered HIV-1 virions less susceptible to neutralization by a monoclonal antibody directed against the viral envelope glycoproteins. Surprisingly, an antibody against ICAM-1 completely neutralized infection by ICAM-1-containing viruses, reducing the efficiency of virus entry by almost 100-fold. Thus, HIV-1 neutralization by an ICAM-1-directed antibody involves more than an inhibition of the contribution of ICAM-1 to virus entry.     

The domain structure of ICAM-1 and the kinetics of binding to rhinovirus

Fragments of intercellular adhesion molecule 1 (ICAM- 1) containing only the two most N terminal of its five immunoglobulin SF domains bind to rhinovirus 3 with the same affinity and kinetics as a fragment with the entire extracellular domain. The fully active two-domain fragments contain 5 or 14 more residues than a previously described fragment that is only partially active. Comparison of X-ray crystal structures show differences at the bottom of domain 2. Four different glycoforms of ICAM- 1 bind with identical kinetics.     

Tyrosine kinase inhibition: an approach to drug development

Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.     

Expression of CD11b and ICAM-1 in an in vivo model of transplant arteriosclerosis

Cachexia consists of a constellation of metabolic changes that occur in cancer patients, including the reduction of muscle and fat tissue, asthenia, anorexia, hypoglycemia and hypercalcemia. These syndromes complicate therapeutic intervention and decrease the quality of life of the patient. This review discusses the involvement of cytokines in cancer cachexia and describes the contribution of IL-6 and other cytokines to the wasting of C-26-bearing mice. The neutralization of IL-6 by antibody, or IL-6 receptor antagonism by suramin, significantly reduce the severity of key parameters of cachexia. The participation of several other factors (PGE2, IL-1, IL-10 and TNF-alpha) in the cellular communication between the C-26 tumor cell and tumor-infiltrating macrophages is also described.