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.

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.

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.

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.

[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.

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.

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.

Immunochemical studies on monoclonal antibodies to stearyl-isomaltotetraose from C58/J and a C57BL/10 nude mouse.

Six hybridomas, five from C58/J and one from C57BL/10 nu/nu, immunized with stearyl-isomaltotetraose (S-IM4) were established. One produced IgG3, one IgM and four IgA. The specificities and sizes of the antibody combining sites were determined by quantitative precipitin and ELISA quantitative inhibition assays. All cross-react with alpha(1----6)dextran B512. Their combining sites were complementary to five to seven glucose residues. Association constants of hybridoma antibodies for dextran B512 ranged from 10(3) to 10(5) ml/g, and for isomaltoheptaose (IM7) from 10(3) to 10(4)M-1. Two hybridoma antibodies, one IgA and another IgM derived from different fusions have identical inhibition curves and combining sites, and very close association constants. It will be important to establish whether they have very similar or identical nucleotide sequences in their variable regions. These studies provide further insight into the specificity and repertoire of antidextran combining sites.

Delayed onset of experimental allergic neuritis in rats treated with reserpine.

In delayed-type hypersensitivity most of the invading cells are not specifically sensitized against the initiating antigen but are augmenting cells called in by inflammatory mediators. It has been suggested that vasoactive amines, such as the monoamine serotonin, released by the action of sensitized T-cells on mast cells, may participate in the perivascular emigration of these cells that do not normally leave the blood. To test this hypothesis in experimental allergic neuritis (EAN), Lewis rats sensitized on day zero were treated with a single dose of the monoamine-depleting drug reserpine (2.5 mg/kg) immediately before the onset of early clinical signs on day nine (reserpine day 9, Rd9); during the onset of early clinical signs on day ten (Rd10); or immediately after the onset of early clinical signs on day 11 (Rd11). The results showed that the onset of early clinical signs was delayed in the Rd9 treated rats until approximately day thirteen whereas no effect on the course of the disease was observed in the Rd11 rats, and variable results were obtained in the Rd10 animals. Infiltration of mononuclear cells and leakage of 125I-albumin into the peripheral nerve was reduced in the Rd9 rats killed during the suppressed period. The delay in the onset of early clinical signs in the Rd9 rats correlated well with the time-course of serotonin depletion as reflected by levels in the peripheral blood of reserpine-treated normal animals. Although a role for histamine could not be demonstrated, the results suggest that other vasoactive amines are involved in the reaction. These results, therefore, would support the hypothesis that vasoactive amines play a role in the perivascular transit of inflammatory cells in EAN.

Photochemical linking of primary aromatic amines to carrier proteins to elicit antibody response against the amine haptens.

Two chemical methods, diazocoupling and reaction with isocyanates, are commonly used to conjugate primary aromatic amines with carrier proteins in order to elicit antibody responses against the aromatic amine haptenic group. Limitations of these conjugation techniques include the requirement for specific functional groups on the carrier protein which generally limits the degree of haptenic substitution obtainable, the many possible side reactions yielding hapten-hapten and carrier-carrier conjugates which waste valuable materials and lower desired hapten-carrier conjugate yields, and, in some cases, conjugation conditions which may denature the carrier protein (e.g., alkaline coupling conditions). We report here a photolabeling approach for conjugating primary aromatic amines to carrier proteins which avoids some of the problems of other conjugation methods and which was used to elicit antibodies against the primary aromatic amine hapten. The method described here is of general application for coupling primary aromatic amines to the carrier proteins and circumvents many of the problems inherent in the isocyanate or diazocoupling methods. 3-Azido-N-ethylcarbazole (ANEC), the azido analog of 3-amino-N-ethylcarbazole, was conjugated to bovine serum albumin (BSA), human transferrin (TR), thyroglobulin (TH), poly-(lysine X tyrosine), and poly-(lysine X phenylalanine) using standard photolabeling procedures. After photolysis, the conjugated proteins or polypeptides were separated from the unbound products of ANEC photolysis on a Sephadex G-10 column. The conjugated proteins were extracted with isobutanol which demonstrated that approximately 20% of the ANEC was covalently coupled to the protein carriers and that the larger portion of the aromatic haptens was non-covalently and hydrophobically bound to the carriers. The ANEC-protein conjugates used for immunization demonstrated a total covalently and non-covalently bound ANEC epitope density of 90 per BSA, 107 per TR and 800 per TH molecule. Rabbits were immunized with the three conjugated proteins and the production of antibody specific for the 3-amino-N-ethylcarbazole hapten was demonstrated by enzyme-linked immunosorbent assay and by inhibition studies using hapten-carrier conjugates of free hapten. The results demonstrate that antibodies against aromatic amine haptens may be raised by immunizing animals with hapten-carrier protein conjugates produced by photolabeling. Since the coupling conditions are very mild and the functional group requirements are so general (requiring only the presence of C-H, N-H, C = O, C = S, or S-H bonds) most carrier proteins should be suitable for use in this method.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Evaluation of the influence of epoxide resins and their hardeners on the female body. I. Skin tests.

Skin tests of hypersensitivity were performed in 175 women and 16 men having direct contact during work with epoxide resinsand their hardeners. The tests were applied for 24 hours, and results were recorded after 24, 48 and 72 hours. The percentages of positive skin tests and numbers of skin lesions were found to increase with time of employment in contact with epoxide resins and their hardeners.

Immunopathology of renal disease.

The reviewed information implicates immune mechanisms in a variety of renal glomerular and tubulointerstitial diseases. Antibodies reactive with intrinsic structural or planted endogenous or exogenous antigens, and with circulating endogenous or exogenous antigens can initiate inflammatory capillary injury by localization in glomerular capillary tufts or along tubular basement membranes. This results in activation of mediator systems, including complement, neutrophils and other leukocytes, amines, peptides, and proteases, which result in vascular and tissue alterations. In some instances, nonimmune activation of complement (for instance, by the properdin system) and other mediators of vascular injury may be involved. A role of cellularly mediated immunologic injury in glomerular disease is not clear and remains the subject of considerable current research. More clearly, there is involvement of lymphocytes in tubulointerstitial and interstitial diseases as well as allograft rejection reactions. A rich armamentarium of in-vitro immunologic tests for specific antibodies, immune-complexes, serum complement levels, and renal tissue analysis provides opportunity for enhanced precision of diagnosis and monitoring progress of disease or treatment. Unfortunately, at the present time, there are not many effective therapies specific for most renal glomerular diseases; perhaps in the future better identification of offending environmental or host antigens will result in more effective prevention and treatment. Application of knowledge concerning renal glomerular diseases to the study of hypersensitivity induced tubulointerstitial injury has resulted in increasing understanding of the pathogenesis of interstitial inflammatory disease of the kidney.

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.

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.

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.