Behaviour of Vascular Smooth Muscle Cells on Amine Plasma-Coated Materials with Various Chemical Structures and Morphologies.

Amine-coated biodegradable materials based on synthetic polymers have a great potential for tissue remodeling and regeneration because of their excellent processability and bioactivity. In the present study, we have investigated the influence of various chemical compositions of amine plasma polymer (PP) coatings and the influence of the substrate morphology, represented by polystyrene culture dishes and polycaprolactone nanofibers (PCL NFs), on the behavior of vascular smooth muscle cells (VSMCs). Although all amine-PP coatings improved the initial adhesion of VSMCs, 7-day long cultivation revealed a clear preference for the coating containing about 15 at.% of nitrogen (CPA-33). The CPA-33 coating demonstrated the ideal combination of good water stability, a sufficient amine group content, and favorable surface wettability and morphology. The nanostructured morphology of amine-PP-coated PCL NFs successfully slowed the proliferation rate of VSMCs, which is essential in preventing restenosis of vascular replacements in vivo. At the same time, CPA-33-coated PCL NFs supported the continuous proliferation of VSMCs during 7-day long cultivation, with no significant increase in cytokine secretion by RAW 264.7 macrophages. The CPA-33 coating deposited on biodegradable PCL NFs therefore seems to be a promising material for manufacturing small-diameter vascular grafts, which are still lacking on the current market.

Modular synthesis and modification of novel bifunctional dendrons.

Herein, we report the design and synthesis of two novel bifunctional dendrons bearing multiple amine termini at the periphery and an azide at the focal point. Copper-catalyzed alkyne-azide cycloaddition enabled modular dendritic scaffold assembly resulting in a first generation dendron carrying six amines and a second generation dendron carrying eighteen amines. Peripheral amines were labeled with multiple copies of a metal isotope, whereas the azide functionality at the focal point was employed in conjugation to a single anti-human CD4 antibody. We demonstrated that the highly monomeric first generation dendron-antibody conjugate selectively detected CD4+ T cells in the PMBC culture.

Gastrointestinal neuroendocrine peptides/amines in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic recurrent condition whose etiology is unknown, and it includes ulcerative colitis, Crohn's disease, and microscopic colitis. These three diseases differ in clinical manifestations, courses, and prognoses. IBD reduces the patients' quality of life and is an economic burden to both the patients and society. Interactions between the gastrointestinal (GI) neuroendocrine peptides/amines (NEPA) and the immune system are believed to play an important role in the pathophysiology of IBD. Moreover, the interaction between GI NEPA and intestinal microbiota appears to play also a pivotal role in the pathophysiology of IBD. This review summarizes the available data on GI NEPA in IBD, and speculates on their possible role in the pathophysiology and the potential use of this information when developing treatments. GI NEPA serotonin, the neuropeptide Y family, and substance P are proinflammatory, while the chromogranin/secretogranin family, vasoactive intestinal peptide, somatostatin, and ghrelin are anti-inflammatory. Several innate and adaptive immune cells express these NEPA and/or have receptors to them. The GI NEPA are affected in patients with IBD and in animal models of human IBD. The GI NEPA are potentially useful for the diagnosis and follow-up of the activity of IBD, and are candidate targets for treatments of this disease.

Peptide Reactivity of Isothiocyanates--Implications for Skin Allergy.

Skin allergy is a chronic condition that affects about 20% of the population of the western world. This disease is caused by small reactive compounds, haptens, able to penetrate into the epidermis and modify endogenous proteins, thereby triggering an immunogenic reaction. Phenyl isothiocyanate (PITC) and ethyl isothiocyanate (EITC) have been suggested to be responsible for allergic skin reactions to chloroprene rubber, the main constituent of wetsuits, orthopedic braces, and many types of sports gear. In the present work we have studied the reactivity of the isothiocyanates PITC, EITC, and tetramethylrhodamine-6-isothiocyanate (6-TRITC) toward peptides under aqueous conditions at physiological pH to gain information about the types of immunogenic complexes these compounds may form in the skin. We found that all three compounds reacted quickly with cysteine moieties. For PITC and 6-TRITC the cysteine adducts decomposed over time, while stable adducts with lysine were formed. These experimental findings were verified by DFT calculations. Our results may suggest that the latter are responsible for allergic reactions to isothiocyanates. The initial adduct formation with cysteine residues may still be of great importance as it prevents hydrolysis and facilitates the transport of isothiocyanates into epidermis where they can form stable immunogenic complexes with lysine-containing proteins.

The metabolic rationale for a lack of cross-reactivity between sulfonamide antimicrobials and other sulfonamide-containing drugs.

Sulfonamide antimicrobials (sulfamethoxazole) contain an arylamine group, oxidized by CYP2C9 to the hydroxylamine with subsequent auto-oxidation to a highly reactive [-nitroso-] intermediate is a necessary (if not sufficient) cause of drug hypersensitivity. Accordingly, xenobiotics that do not contain an arylamine cannot generate this reactive intermediate and do not cross react with sulfonamide antimicrobials. Despite this well-attested observation, product labeling and direct-to-consumer advertising for non-arylamine therapeutic classes of drugs containing the sulfonamido- functional group persist with a warning of the potential for cross-reactivity. It is hoped that by offering an explicit rationale for the lack of cross-reactivity will provide medical practitioners with a level comfort to proceed with prescribing medications such as thiazide diuretics and celecoxib for patients with a history of hypersensitivity to sulfonamide antimicrobials.

Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid.

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.

Modern laboratory evaluation of peptide and amines: a continuing role for radioimmunoassay?

Modern medicine, and specifically clinical diagnosis, relies, among other diagnostic procedures, on the measurements of the biogenic analytes for elucidation and correlation of specific neuroendocrine markers. Tremendous advances have been made in imaging and radioactive uptake procedures to elucidate tumor presence and characterization. However, such advances only partially provide the fundamental degree of tumor activity and clinical confirmational validity. The author points out in some detail the problems that may arise when the methodological differences presented by each investigational study and investigators are not standardized. This variation causes a concern with the specific objectives of the investigator and the specific aims of the research project at hand, and ultimately for the validity of the published results.

Antigen-containing liposomes engrafted with flagellin-related peptides are effective vaccines that can induce potent antitumor immunity and immunotherapeutic effect.

The bacterial protein flagellin can trigger immune responses to infections by interacting with TLR5 on APCs, and Ag-flagellin fusion proteins can act as effective vaccines. We report that flagellin-related peptides containing a His-tag and sequence related to conserved N-motif (aa 85-111) of FliC flagellin, purportedly involved in the interaction of flagellin with TLR5, can be used to target delivery of liposomal Ag to APCs in vitro and in vivo. When engrafted onto liposomes, two flagellin-related peptides, denoted as 9Flg and 42Flg, promoted strong liposome binding to murine bone marrow-derived dendritic cells and CD11c(+) splenocytes, and cell binding correlated with expression of TLR5. Liposomes engrafted with 9Flg or 42Flg induced functional MyD88-dependent maturation of dendritic cells in vivo. The vaccination of mice with 9Flg liposomes containing OVA induced OVA-specific T cell priming, increased the number of Ag-responsive IFN-gamma-producing CD8(+) T cells, and increased Ag-specific IgG(1) and IgG(2b) in serum. Importantly, the vaccination of C57BL/6 mice with syngeneic B16-OVA-derived plasma membrane vesicles, engrafted with 9Flg or 42Flg, potently inhibited tumor growth/metastasis and induced complete tumor regression in the majority of mice challenged with the syngeneic B16-OVA melanoma, in the lung and s.c. tumor models. Strong antitumor responses were also seen in studies using the s.c. P815 tumor model. Therefore, vaccination with Ag-containing liposomes engrafted with 9Flg or 42Flg is a powerful strategy to exploit the innate and adaptive immune systems for the development of potent vaccines and cancer immunotherapies.

[Synthesis and immunotropic activity of 2-alkylaminomethylene-19beta,28-epoxyolean-3-ones].

2-Alkylaminomethylene-19beta,28-epoxyolean-3-ones were obtained by interaction of 2-hydroxymethylene-19beta,28-epoxyolean-3-one with aliphatic amines. Some of the resulting substances exhibit immunotropic activity.

Complement activation on surfaces carrying amino groups.

The complement system is strongly activated by surfaces carrying nucleophilic groups, such as hydroxyl (OH) groups, and triggered by deposition of complement protein fragment, C3b. Surfaces carrying amino groups, the other representative nucleophilic group, are expected to be potential activators of the complement system through the alternative pathway. Few studies thus far have examined the potential of artificial materials carrying amino groups in activating the complement system. In this study, we employed a self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (NH2-SAM) and a polyethyleneimine (PEI)-coated surface as model surfaces to study interactions between amino groups and serum complement pathway. SAMs of 11-mercaptoundecanol (OH-SAM) and 1-dodecanethiol (CH3-SAM) were used as control surfaces, respectively. Although much protein was adsorbed from serum solutions on the two types of amino surfaces, amounts of C3b deposition were much less than those observed on OH-SAM. Amounts of C3a released on the amino surfaces were same levels as that of CH3-SAM, but significantly smaller than that on OH-SAM. These facts suggest that the nucleophilic amino groups on NH2-SAM and PEI-coated surfaces do not directly activate the alternative pathway, but the protein adsorbed layers formed on amino surfaces activate it, but to an extent much smaller than that on OH-SAM. In addition, we found no deposition of C1q molecules on the amino surfaces, suggesting that these surfaces fail to activate the classical pathway. However, more careful studies are needed to conclude it, because it is known that C1q is only transiently detected at typical classical activation interfaces.

Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine.

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

Antigen recognition by human gammadelta T lymphocytes.

Mature T lymphocytes carrying the conventional alphabeta T cell receptor (TCR) recognize peptide antigens in the context of MHC class I (CD8+) and MHC class II (CD4+) antigens, respectively. In striking contrast, human gammadelta T lymphocytes recognize unconventional antigens via their heterodimeric TCR in a non-MHC-restricted fashion. In this brief review, we discuss recent progress in the identification of ligands that are specifically recognized by human gammadelta T cells. It appears that gammadelta T cells have evolved to supplement the cellular immune response towards antigens that are not seen by alphabeta T lymphocytes.

Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity.

Approximately 4% of peripheral blood T cells in humans express a T cell receptor with markedly restricted germline gene segment usage (V gamma 2 V delta 2). Remarkably, these T cells expand 2- to 10-fold (8%-60% of all circulating T cells) during many microbial infections. We show here that these T cells recognize a family of naturally occurring primary alkylamines in a TCR-dependent manner. These antigenic alkylamines are secreted to millimolar concentrations in bacterial supernatants and are found in certain edible plants. Given the large numbers of memory V gamma 2 V delta 2 T cells in adult humans, recognition of alkylamine antigens offers the immune system a response of the magnitude of major superantigens for alpha beta T cells and may bridge the gap between innate and adaptive immunity.

Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups.

The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethyl-maleic anhydride prior to processing. Immobilon AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected epsilon-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzene sulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 microliter) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

Ontogeny, distribution and amine/peptide colocalization of chromogranin A- and B-immunoreactive cells in the chicken gizzard and antrum.

The ontogeny and the distribution of chromogranin A (CgA)- and chromogranin B (CgB)-immunoreactive endocrine cells was studied in the chicken gizzard and gizzard-duodenal junction (also called pylorus or antrum) during embryonic and postnatal life. The same tissue sections were then double-immunostained to identify the CgA-and CgB-immunoreactive cells, with a panel of polyclonal antibodies raised against main gut amine/peptides. In the gizzard, positive cells were observed only in its two diverticula (proximal and distal caeca), where the first CgA- and CgB-immunoreactive cells were found on day 12 of incubation. They always remained moderate in number and co-stored mainly serotonin, gastrin/CCK and neurotensin. A few also co-stored somatostatin, but only during the embryonic period. Others co-stored PYY, but only after hatching. Co-localization with motilin was rare and never occurred with bombesin. In the chicken antrum, the first CgA- and CgB-immunoreactive cells were observed on day 12 of incubation and soon reached very high numbers. Antral positive cells showed almost the same co-localization pattern as the gizzard diverticula. Despite their high chromogranin content, the antral cells had weak argyrophilia, whereas in the gizzard diverticula the two staining patterns corresponded.

Therapeutic potentiation of the immune system by costimulatory Schiff-base-forming drugs.

Immune responses are orchestrated by CD4 T lymphocytes, which receive a cognitive signal when clonally distributed receptors are occupied by major histocompatibility complex (MHC) class II-bound peptides on antigen-presenting cells (APCs). The APCs provide costimulatory signals, through macromolecules such as CD80, that regulate outcomes in terms of T-cell activation or anergy. We have studied essential complementary chemical events in the form of Schiff base formation between carbonyls and amines that are constitutively expressed on presenting cell and T-cell surfaces and provide a new target for manipulation of immune responses. Here we show that small Schiff base-forming molecules can substitute for the physiological donor of carbonyl groups and provide a costimulatory signal to CD4 Th-cells through a mechanism that activates clofilium-sensitive K+ and Na+ transport. One such molecule, tucaresol, enhances CD4 Th-cell responses, selectively favouring a Th1-type profile of cytokine production. In vivo tucaresol potently enhances CD4 Th-cell priming and CD8 cytotoxic T-cell priming to viral antigens, and has substantial therapeutic activity in murine models of disease.

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies.

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.

Shark cytotoxic macrophages interact with target membrane amino groups.

The types of target structures recognized by cytotoxic macrophages have been described for various microorganisms, but have not been defined for tumor cells. Tumoricidal macrophages are selective in their destructive mechanisms, sparing normal cells while directing their lytic machinery toward neoplastic targets. The cytotoxic activity of macrophages from a primitive vertebrate, the nurse shark, closely resembles the activity of mammalian tumoricidal macrophages. Host defense mechanisms of these animals appear to rely on antigen nonspecific cellular effector systems, and it has been postulated that macrophage-mediated cytotoxicity plays a dominant role in protection during periods of decreased environmental temperatures when lymphocyte responses of poikilothermic vertebrates are compromised. Similar to mammalian tumoricidal macrophages shark macrophages display selective recognition of target cells. Previous studies showed that TNP modification of targets was protective, preventing recognition by the shark spontaneously cytotoxic macrophage. Additionally, it was shown that cytotoxic activity was inhibited in a dose dependent fashion by the addition of excess unlabeled targets. In the present study, similar inhibition experiments with hapten-modified targets have been used to determine the nature of the target structures recognized by the shark cytotoxic macrophage. Cold targets modified with haptens which react covalently with free amino groups on cell membranes, trinitrobenzene sulfonic acid (TNBS) and flourescein isothiocyanate (FITC), are not recognized by the cytotoxic macrophage. The relative amount of membrane bound TNP was correlated with inhibition of cytotoxicity. Conversely, target cells modified with sulfhydryl reacting reagents, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine and dithionicotinic acid, are recognized similarly to untreated targets. Moreover, TNP-containing lipids, permitted to diffuse into target membranes without covalent binding, do not alter target recognition, indicating that TNP itself has no effect on macrophage:target interaction. From these data, it is concluded that the shark cytotoxic macrophage interacts with membrane bound amino, but not sulfhydryl groups. The ability to distinguish between membrane structures may have appeared early in evolution as a means of preserving self cells while retaining protective nonspecific cytotoxic mechanisms.