Inhibition of cell proliferation by CD44: Akt is inactivated and EGR-1 is down-regulated.

OBJECTIVE: CD44 is a transmembrane glycoprotein and can facilitate signal transduction by serving as a platform for molecular recruitment and assembly. A number of studies have suggested that CD44 can either positively or negatively regulate cell proliferation. The purpose of this study was to investigate how CD44 can inhibit cell proliferation. MATERIALS AND METHODS: We engineered E6.1 Jurkat cells to express CD44. Importantly, these cells lack endogenous CD44 expression. Molecular pathways involved with cell proliferation were studied using RT(2)-PCR array, siRNA, Western blotting and by employing pharmacological inhibitors of ERK1/2, p38 and the PI3K/Akt pathways. RESULTS: We found that CD44 expression significantly inhibited cell proliferation and down-regulated EGR-1 expression and EGR-1 targets cyclin D1 and cyclin D2. Transfection of control E6.1 Jurkat cells with EGR-1 siRNA also inhibited cell proliferation, confirming its role. Disruption of the PI3K/Akt pathway with pharmacological inhibitors reduced both EGR-1 expression and cell proliferation, recapitulating the properties of CD44 expressing cells. Akt was hypophosphorylated in cells expressing CD44 showing its potential role in negatively regulating Akt activation. Strikingly, constitutively active Akt rescued the proliferation defect showing requirement for active Akt, in our system. CONCLUSION: Our results suggest a novel pathway by which CD44 inactivates Akt, down-regulates EGR-1 expression and inhibits cell proliferation.

New technologies to prevent and treat contact hypersensitivity responses.

Allergic contact dermatitis is a common inflammatory skin disease caused by T cells that recognize environmental and industrial allergens (i.e., haptens). Langerhans' cells (LC), which are skin-specific and "immature" members of the dendritic cell (DC) family of antigen-presenting cells, play crucial roles in the induction of contact hypersensitivity (CH) responses. Upon exposure to haptens, LC migrate from the epidermis to draining lymph nodes, mature into T cell-stimulatory DC, and activate hapten-reactive T cells. Therefore, CH responses should be preventable at the sensitization phase by interfering with one of these changes that occur in LC. Our objective is to develop new technologies for the prevention and treatment of allergic contact dermatitis. In this article, we will introduce three technologies that we have recently developed. First, using a phage display strategy, we have identified a 12-mer peptide (termed "peptide 1") that binds and blocks the function of hyaluronan (HA), which is known to serve as an adhesive substrate for LC migration. Local injection of peptide 1 in mice before topical application of DNFB blocked almost completely the emigration of LC from the epidermis to the draining lymph node, where antigen presentation takes place. Peptide 1 represents a new strategy that is designed to inhibit the initial event of CH. Second, we have established an in vitro experimental system to study the terminal maturation of LC during antigen-specific interaction with T cells. This experimental system, which employs a long-term LC line and T cell clones, should provide a unique tool for the identification of new immunosuppressive agents that block LC terminal maturation selectively. Finally, under the hypothesis that LC, which are engineered to overexpress a death ligand, would deliver apoptotic signals instead of activation signals to T cells, we created a "killer" LC clone by introducing CD95L cDNA into our long-term LC line XS106. In vivo administration of DNFB-pulsed killer LC into mice, either before or after sensitization, resulted in marked suppression of CH responses to DNFB. The killer LC technology represents an entirely new immunosuppressive therapy that is designed to eliminate only the pathogenic T cells.

Development of a peptide inhibitor of hyaluronan-mediated leukocyte trafficking.

Hyaluronan (HA), a high molecular weight glycosaminoglycan, is expressed abundantly in the extracellular matrix and on cell surfaces. Although HA is known to bind many adhesion molecules, little information has been available with respect to its direct physiological role. In this study, we developed a novel 12-mer (GAHWQFNALTVR) peptide inhibitor of HA, termed "Pep-1," by using phage display technology. Pep-1 showed specific binding to soluble, immobilized, and cell-associated forms of HA, and it inhibited leukocyte adhesion to HA substrates almost completely. Systemic, local, or topical administration of Pep-1 inhibited the expression of contact hypersensitivity responses in mice by blocking skin-directed homing of inflammatory leukocytes. Pep-1 also inhibited the sensitization phase by blocking hapten-triggered migration of Langerhans cells from the epidermis. These observations document that HA plays an essential role in "two-way" trafficking of leukocytes to and from an inflamed tissue, and thus provide technical and conceptual bases for testing the potential efficacy of HA inhibitors (e.g., Pep-1) for inflammatory disorders.

Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site.

Recent data have indicated that the MHC class I-related receptor, FcRn, regulates the half-lives of serum IgG in addition to its known role in transferring IgG from mother to young. In the current study, the activity of rat IgG (rIgG) isotypes in FcRn-mediated functions has been analyzed. The serum half-life and maternofetal transfer in mice decreased in the order rIgG2a > rIgG1 > rIgG2c > rIgG2b. This decrease in activity correlates well with reduced binding affinity for soluble mouse FcRn, and site-directed mutagenesis of a recombinant Fc-hinge fragment has been used to investigate the molecular basis for the differences in activities of the rIgG. Analysis of the serum half-lives of the mutated Fc-hinge fragments demonstrated that, in addition to Ile253, His310, His435 and His436 that were identified in earlier studies, amino acids at positions 257, 307 and 309 play a role in building the FcRn interaction site of IgG. The study also excludes the involvement of amino acids in a fourth loop located at the CH2-CH3 domain interface that encompasses residues 386-387 in FcRn binding. Sequence differences at positions 257, 307 and 309 between rIgG most likely account for the reduced affinity of rIgG2b and IgG2c relative to rIgG1 and rIgG2a for binding to FcRn.

Effects of secondary forces on a high affinity monoclonal IgM anti-fluorescein antibody possessing cryoglobulin and other cross-reactive properties.

The effects of secondary forces on monoclonal IgM anti-fluorescein antibody 18-2-3 reactivity were investigated and the results correlated with similar studies characterizing anti-fluorescein mAbs 4-4-20 and 9-40. mAb 18-2-3 was considered an important model for further elucidation of secondary forces since it possessed ligand binding properties similar to mAb 4-4-20, such as a similar affinity, but due to a very different primary structure it was idiotypically and metatypically distinct. mAb 18-2-3 also possessed cryoglobulin (anti-Ig) and extensive cross-reactive properties (e.g. anti-phenyloxazolone) suggestive of an atypical anti-fluorescein active site. The reactivity of mAb 18-2-3 with model fluorescein-peptides was modulated by secondary forces in a manner that differed from both mAbs 4-4-20 and 9-40. Thus, the effects of secondary forces seemed to vary with each monoclonal antibody even though each of the immunoglobulins studied were specific for the same homologous ligand. Results indicated that secondary forces impacted immune complex stability, variable domain conformation and protein dynamics. Models were postulated to account for secondary effects on the mAb 18-2-3 active site relative to mAbs 4-4-20 and 9-40. Levels of hydration, active site architecture and local amino acid dynamics were among the models cited.

Secondary force-mediated perturbations of antifluorescein monoclonal antibodies 4-4-20 and 9-40 as determined by circular dichroism.

Secondary forces have been defined as those interactions between antibody and antigen that occur external to the antibody active site. Previous investigations indicated that non-active-site secondary interactions can modulate immune complex stability and may influence antibody variable domain conformation and/or dynamics. To assess secondary force-induced perturbations of monoclonal antibodies 4-4-20 and 9-40 a series of monofluoresceinated peptides was reacted and the various interactions analyzed by circular dichroism (CD). The mAbs 4-4-20 and 9-40 vary by nearly 1000-fold in their respective affinities for the fluorescein ligand, yet both immunoglobulins are highly related at the primary structural (idiotype) level. Near-UV CD spectra were evaluated as well as the induced optical activity (visible CD) of the antibody-bound fluorescein moiety when covalently attached to various peptide carriers. Comparative spectral studies revealed significant differences in the near-UV CD spectra of mAbs 9-40 and 4-4-20 relative to the various peptide antigens and to one another. CD spectra were interpreted as reflecting differential secondary force-induced perturbations of the antibody variable domains as well as intrinsic differences between the two mAbs.

Effects of secondary forces on the ligand binding and conformational state of antifluorescein monoclonal antibody 9-40.

Biochemical interactions that occur external to the antibody active site have been termed secondary forces. Secondary forces are supplemental to interactions within the antibody active site (i.e., primary interactions) and can affect ligand binding efficiency as well as variable domain conformation. The antifluorescein antibody system has been determined to be a superior method for delineating primary from secondary interactive components due to the active site-filling properties of the fluorescyl ligand. To date, all studies of secondary forces within the context of the antifluorescein system have been with the high-affinity monoclonal antibody 4-4-20 (mAb 4-4-20) (Mummert & Voss, 1995, 1996, 1997). In order to determine the generality of experimental observations and proposed models, we investigated the effects of secondary forces on the antifluorescein mAb 9-40. In addition to assessing the results of former studies, mAb 9-40 possesses properties unique from those of mAb 4-4-20, namely, a decreased affinity for fluorescein and increased conformational dynamics relative to mAb 4-4-20 (Carrero & Voss, 1996). Results of fluorescein and intrinsic mAb 9-40 tryptophan quenching as well as differential scanning calorimetric (DSC) studies indicated that secondary forces modulated the conformational (metatypic) state in accordance with previous investigations with mAb 4-4-20. Unlike mAb 4-4-20, mAb 9-40 did not exhibit altered ligand binding efficiency due to the inclusion of secondary interactive components. Models were developed that proposed that the increased malleability of mAb 9-40 variable domains could account for functional differences in properties between mAb 9-40 and mAb 4-4-20.

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS.

SR 49059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3, 4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut cartridges. The extracted samples were analyzed on a C18 HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (2H6-SR 49059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2-20 ng ml-1, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20-5000 ng ml-1, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).

Effects of secondary forces on the ligand binding properties and variable domain conformations of a monoclonal anti-fluorescyl antibody.

Biochemical interactions occurring external to the antibody active site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4-4-20 active site as a series of thermally averaged substates is proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.

Transition-state theory and secondary forces in antigen--antibody complexes.

Secondary forces, defined as those interactions between the antigen (epitope including the surrounding environment) and areas immediately adjacent to the antibody active site, were investigated using monofluorescein-derivatized synthetic peptides of varying electrostatic properties. Secondary forces were quantitated by measuring the unimolecular rate constants at two different temperatures using the high-affinity anti-fluorescein monoclonal antibody 4-4-20 complexed with fluorescein-derivatized synthetic peptides. Unimolecular rate constants were correlated with transition-state theory to explain secondary effects. An acidic peptide produced a large temperature-dependent effect upon binding including a significant enthalpic factor (+33.28 kcal/mol) relative to the binding of fluorescein ligand (+23.96 kcal/mol). Binding of a basic peptide produced both a relatively smaller temperature effect and enthalpy factor than fluorescein ligand. The antibody-ligand binding results were interpreted invoking the concepts of thermally averaged metatypic (liganded) states of the antibody as well as potential biochemical interactions between the antigen and accessible surface regions of the antibody's complementarity determining regions.

Detection of protease activity using a fluorescence-enhancement globular substrate.

Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase resulted in fluorescence enhancement of 4300%. Both alpha-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.

Effects of secondary forces on the primary antibody-ligand interaction.

The binding efficiency of high affinity monoclonal antifluorescyl antibody 4.4-20 with the homologous ligand situated in different protein environments has been investigated to quantitate the effect of non-active site secondary factors. To synthesize monofluoresceinated proteins, fluorescein 5-isothiocyanate was reacted with a 100-fold molar excess of ribonuclease, lysozyme, lactalbumin and bovine serum albumin. Absorption and emission spectra, as well as fluorescence life-time measurements which yielded discrete components and proteolytic studies suggested that fluorescein was conjugated to a specific lysine residue consistent with a non-random distribution of lysines within each protein population. The derivatized residue was probably a surface moiety based on accessibility analyses with iodide as a dynamic quencher. Dissociation rate analyses indicated that the relative release time of 4.4-20 with each monofluoresceinated protein was Fl-RNAse > or = Fl-lyso > or = FDS > Fl-lact > or = Fl-BSA which correlated with changes in free energy of binding. Relative fluorescence quenching measurements of the fluorescein moiety indicated that 4.4-20 showed decreasing quenching in the order FDS > Fl-RNAse > Fl-lyso > or = Fl-lact > Fl-BSA. Because spectral data indicated that fluorescein was conjugated to a specific residue or a non-random distribution of residues in each protein population, the results represented the effect of a single distinct environment or a weighted average of different microenvironments. Results have been interpreted within the theoretical framework of a dynamic antibody model involving conformer selection and the relative effects of primary and secondary interactions.

Hydrolysis of the prodrug, 2',3',5'-triacetyl-6-azauridine.

PURPOSE: The purposes were to study the kinetics of hydrolysis of 2',3',5'-triacetyl-6-azauridine (1) in aqueous solution (mu = 0.5) and to identify the main intermediates and products of the reaction. METHODS: A stability indicating isocratic LC assay was used to study the rate of degradation of 1. A gradient LC assay was used to study the time courses of the degradants. The products of hydrolysis were isolated by preparative liquid chromatography and identified by 1H-NMR and CI-MS. The pKa value was obtained by potentiometric titration. RESULTS: At 36.8 degrees C, the pH-rate profile of 1 in water was adequately described by a four-term rate equation. The intermediates were identified as the primary and secondary di-acetates, and the primary and secondary mono-acetates. The final product was 6-azauridine. CONCLUSIONS: A simplified kinetic scheme could be used to describe the concentration-time profiles of 1, the intermediates and the final product.

Collections of adult Ixodes dammini in Indiana, 1987-1990, and the isolation of Borrelia burgdorferi.

The collection records for Ixodes dammini Spielman, Clifford, Piesman & Corwin in Indiana are summarized for the period 1987-1990. In 1990, 13 of 729 deer examined were found to harbor adult I. dammini ticks. Eleven of these ticks were collected from 10 deer at a site in Newton County in northwestern Indiana. Borrelia burgdorferi spirochetes were isolated from a single female I. dammini tick collected from this site.