Cutting Edge: The Use of Topical Aminoglycosides as an Effective Pull in "Prime and Pull" Vaccine Strategy.

The presence of tissue-resident memory T cells at barrier tissues is critical for long-lasting protective immune responses. Previous work has shown that tissue-resident memory T cells can be established by "pulling" virus-specific effector T cells from circulation to the genital mucosa via topical vaginal application of chemokines in mice. Once established, these cells protect hosts against genital herpes infection. We recently showed that vaginal application of aminoglycoside antibiotics induces robust activation of the IFN signaling pathway, including upregulation of chemokine expression within the tissue in mice. In this study, we show that a single topical application of neomycin, an inexpensive and vaginally nontoxic antibiotic, is sufficient to pull CD8 T cells to the vaginal mucosa and provide protection against genital herpes infection in mice.

Optical Fiber-Mediated Immunosensor with a Tunable Detection Range for Multiplexed Analysis of Veterinary Drug Residues.

We describe herein a newly developed chemiluminescent optical fiber immunosensor (OFIS) with a tunable detection range for multiplexed analysis of veterinary drug residues with vastly different concentrations in milk samples. The optical fiber probe is used as a carrier of biorecognition element as well as a transducer, enabling a low-cost compact design, which makes this system suitable for cost-effective on-site detection of the target analytes. Importantly, the synergy between modulation of the length of the optical fiber sensing region and the number of fibers allows performing multiplexed immunoassays in an easily controllable manner over a tunable detection range from pg/mL to µg/mL analyte concentrations. By combining the optical fiber sensor with a nanocomplex signal amplification system, a highly sensitive chemiluminescent OFIS system is demonstrated for the multiplexed assaying of veterinary drug residues in milk samples with linear ranges of 10-(2 × 104) pg/mL for chloramphenicol, 0.5-500 ng/mL for sulfadiazine, and 0.1-300 µg/mL for neomycin. This controllable strategy, based on modulation of the fiber probe, provides a versatile platform for multiplexed quantitative detection of both low-abundance and high-abundance targets, which shows great potential for on-site testing in food safety.

A SERS-based multiple immuno-nanoprobe for ultrasensitive detection of neomycin and quinolone antibiotics via a lateral flow assay.

The authors describe an ultrasensitive method for simultaneous detection of neomycin (NEO) and quinolones antibiotics (QNS). It is based on the use of (a) two immuno-nanoprobes (a probe for NEO and a probe for QNS), (b) surface-enhanced Raman scattering (SERS) detection, and (c), a portable lateral flow assay (LFA). The two probes consist of gold nanoparticles (AuNPs) conjugated to the Raman active molecule 4-aminothiophenol (PATP), and to monoclonal antibody against NEO (NEO mAb) or against NOR (NOR mAb). Quantitative detection of NEO and QNS was realized via SERS of the PATP-coated AuNPs captured in the test line of a LFA. Under optimized condition, the visual limits of LFA are 10 ng·mL-1 for NEO and 200 ng·mL-1 for NOR, and with LODs down to 0.37 pg·mL-1 and 0.55 pg·mL-1 by using SERS. The NEO test line is not interfered by the NEO analogues gentamycin, streptomycin and tobramycin, but the NOR test line suffers from different degrees of cross-reactivity (CR) to 12 common other QNS, the CRs ranging from 1.5% to 136%. The recoveries of NEO and NOR from spiked milk samples ranged between 86% and 121%, with relative standard deviations (RSD) from 3% to 6%. The method is highly sensitive, accurate and effective. It may be applied to simultaneous detection of NEO and 8 QNS, including NOR, enoxacin, ciprofloxacin, ofloxacin, fleroxacin, marbofloxacin, enrofloxacin, and pefloxacin. Graphical abstract Schematic of a lateral flow assay (LFA) based on an indirect competitive model. By using two test lines, the LFA can detect the neomycin and quinolones antibiotics simultaneously. Based on the surface-enhanced Raman scattering (SERS), the LFA shows high sensitivity to antibiotics with low limit of detection.

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Contact dermatitis to topical medicaments: a retrospective chart review from the Ottawa Hospital Patch Test Clinic.

BACKGROUND: Topical medicaments are a common cause of allergic contact dermatitis. This study will evaluate the prevalence of contact allergy to a wide array of topical medicaments at the Ottawa Patch Test Clinic. OBJECTIVES: The objectives of this study are to report the results of positive patch testing to topical medicaments at the Ottawa Patch Test Clinic and identify common sensitizers in topical medicaments. METHODS: Patients were tested with the standard North American Contact Dermatitis screening series of 70 allergens plus supplementary allergens when indicated. A retrospective chart review of patients positive to topical medicaments between January 1, 2000, and September 30, 2010, was undertaken. RESULTS: The average age of patients was 49.5 years. Thirty-four percent were atopic. Common sensitizers included topical antibiotics (58%), steroids (30%), anesthetics (6%), and antifungals (6%). Patch testing showed that 61% of patients tested positive to antibiotics, 21% to topical steroids, 17% tested positive to topical anesthetics, and 1% tested positive to topical antifungals. The most common reactions were to bacitracin (44%) and neomycin (29%). The most common steroid screener was tixocortol-17-pivalate (group A) (19%), and the most common local anesthetic was lidocaine (12%). CONCLUSIONS: Topical medicaments of all kinds are common causes of allergic contact dermatitis. Those that are more readily available, in over-the-counter preparations, are the most frequent culprits.

[Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk].

As a result of immunization of rabbits with neomycin B (N M) conjugated to periodate-oxidized transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5-10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/1 06), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the M RL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.

Amplification strategy based on gold nanoparticle-decorated carbon nanotubes for neomycin immunosensors.

A novel amperometric immunosensor with an enhanced sensitivity for the detection of neomycin (Neo) was prepared by covalently immobilizing a monoclonal Neo antibody onto a new conducting polymer, poly-[2,5-di-(2-thienyl)-1H-pyrrole-1-(p-benzoic acid)] (pDPB), as a sensor probe. The probe was used to detect Neo in a sandwich-type approach, where the secondary antibody was attached to gold nanoparticle-decorated multi-wall carbon nanotubes labeled with hydrazine (Hyd-MWCNT(AuNP)-Ab(2)). Hydrazine on the conjugate served as a catalyst for the reduction of hydrogen peroxide, and the catalytic current was monitored at -0.45 V vs. Ag/AgCl. The performance of the immunosensor with and without AuNPs on the probe and the conjugate was compared. The parameters affecting the immunosensor response in terms of antibody dilution ratio, incubation time, pH, applied potential, and temperature were optimized. A linear dynamic range for Neo analysis was obtained between 10 ng/mL and 250 ng/mL with a detection limit of 6.76 ± 0.17 ng/mL. The proposed immunosensor was successfully applied to detect Neo content in real meat samples.

Frequency of sensitization to common allergens: comparison between Europe and the USA.

BACKGROUND: Previously, contact allergy to formaldehyde and quaternium-15 was found to be more prevalent in the United States than in Europe. No such data have been assessed for other contact allergens. OBJECTIVE: Determine any differences in frequencies of sensitization to contact allergens in the United States and Europe. METHODS: Literature study. Comparison of reported frequencies of sensitization to contact allergens routinely tested both in the United States and Europe in recent, large, multicentre studies. Because of the heterogeneity of studies and background parameters, statistical evaluation was not attempted. RESULTS: Major differences were found only for neomycin (USA 10.0-11.8%, mean 11.4%; Europe 1.2-5.4%, mean 2.6%). Most allergens had somewhat higher prevalence in the United States, with rates versus Europe ranging from 1.3 to 1.9. CONCLUSIONS: Contact allergy to neomycin is much more prevalent in the United States. Stricter selection of patients for patch testing in United States tertiary referral centres may result in 50% more positive reactions compared to European studies.

FCS-based sensing for the detection of ochratoxin and neomycin in food.

In this work, we present an advanced fluorescence assay for the detection of traces of ocratoxin A and neomycin in food. The described assay is based on measurement of the fluctuations of the fluorescein-labeled analytes by a focused laser beam in the absence and in the presence of the specific antibodies anti-analytes. A competitive assay based on the utilization of unlabeled analytes was developed. The obtained results indicated that the combination of high-avidity IgG antibodies together with an innovative fluorescence immunoassay strategy resulted in the detection limit of 0.0078 ng and 0.0156 ng for ochratoxin A and neomycin, respectively.

Reduced frequency of atopic dermatitis in quinoline-allergic patients: the 'hapten-atopy hypothesis'.

BACKGROUND: While allergy to food proteins is almost exclusively found in association with atopy, it has been our experience that contact allergy to some contact allergens/haptens with both cutaneous and gastrointestinal exposures is reduced in atopic dermatitis (AD) patients as a group. OBJECTIVE: To assess the contact allergy rates of two classes of antimicrobial haptens, one with both cutaneous and gastrointestinal exposures (quinolines) and one with only significant cutaneous exposure (aminoglycosides), with respect to the presence or absence of AD. METHODS: Contact allergy rates to neomycin (aminoglycoside) and quinoline mix/clioquinol in patients attending the St John's Institute of Dermatology for diagnostic patch testing were retrospectively analysed; current AD and history of AD were noted. RESULTS: In comparison to neomycin-allergic subjects, there was a highly significant negative association between quinoline contact allergy and current presence of AD (P = 0.0028); negative association between quinoline contact allergy and a history of AD did not reach significance (P = 0.07). CONCLUSIONS: In comparison to an antimicrobial with no significant gastrointestinal exposure (neomycin), contact allergy to quinolines is negatively associated with the presence of AD. This is in contrast to food protein allergy, which is strongly associated with atopy. Possible explanations could include (i) confounding factors or (ii) AD patients are efficient at orally tolerizing haptens and inefficient at orally tolerizing proteins, secondary to their atopic status or (iii) oral tolerance of haptens antagonizes tolerance of food proteins and also leads to an immunological shift towards atopy (hapten-atopy hypothesis).

Fixed exanthema from systemic tobramycin.

Eye drops contain several ophthalmic medications which can produce allergic reactions. We report the case of a patient with contact dermatitis from neomycin and a probable fixed exanthema after parenteral administration of tobramycin who tolerated topical tobramycin and other aminoglycosides.

Development of ELISA and immunochromatographic assay for the detection of neomycin.

BACKGROUND: Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields. METHODS: Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk. RESULTS: No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50-200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk. CONCLUSIONS: Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

Reacciones adversas producidas por vacunas infantiles / Adverse reactions to childhood vaccines

Las vacunas han cambiado la vida de millones de personas en el mundo. Enfermedades como la viruela han sido erradicadas, la poliomielitis está en camino de serlo y otras han disminuido drásticamente. La inmunización activa consiste en estimular al organismo para producir anticuerpos, mediante la administración de una vacuna, lo que origina una respuesta similar a la infección natural pero sin riesgo para el vacunado. Este artículo examina las reacciones adversas producidas tras la vacunación y en especial las reacciones de hipersensibilidad. La incidencia de algunas de ellas se aproxima a 1:100.000 sujetos vacunados. Estas reacciones por vacunas se pueden clasificar atendiendo a la causa que las induce en: reacciones debidas a la propia vacuna, idiosincrásicas, asociadas a errores de manipulación, almacenaje o administración y reacciones coincidentes con la vacunación. Como las vacunas consisten en una mezcla del microorganismo activo, antibióticos, conservantes, proteínas del medio de cultivo y aditivos, es fácil adivinar que tras la vacunación se pueden producir reacciones de hipersensibilidad. Las vacunas incluidas en el programa de vacunación son extraordinariamente seguras y eficaces. Por tanto, los niños que presenten alguna reacción adversa al recibir las vacunas, que normalmente son combinadas, deben someterse a un estudio antes de interrumpir su calendario de vacunación, ya que los efectos adversos que producen se presentan con una frecuencia muy baja. Las nuevas técnicas de biología y genética molecular abren un campo nuevo para la elaboración de vacunas. Las áreas más prometedoras son: la tecnología del ADN recombinante, la expresión controlada de genes y la síntesis de péptidos. Estas técnicas crearán vacunas nuevas y más seguras, lo que reducirá las reacciones adversas

Use of vaccines has altered life for millions of world´s citizens. Smallpox has been eradicated. Poliomyelitis is on the way to potential eradication and other childhood diseases have been dramatically reduced. Vaccination is an attempt to replace the natural primary contact with a safer artificial contact so that natural contact first takes place in the face of heightened immunity. This report examines adverse reactions to vaccines after immunization, specially hypersensitivity reactions. The incidence of some reactions may approximate 1:100.000 individual vaccinated. These reactions can be classified according to probable cause as vaccination-induced reactions (due to an effect of the vaccine itself or an idiosyncrasia), reactions associated with faulty production, storage or administration and coincidental reactions Because vaccines consist of mixture of the active agent, antibiotics, preservatives, culture medium proteins and other additives, it is not surprising that hypersensitivity reactions occur after vaccination. Vaccines currently used for routine preventive care in children are extraordinarily safe and effective. So children should not be excluded from the normal vaccine schedule when they suffer from a reaction after receipt of such a combination. So that a logical analysis has been performed due to adverse reactions occur at a very low frequency. It seems likely that advances in vaccines will occur through the application of molecular biology and, in particular, molecular genetics. The three most promising areas are recombinant DNA technology, controlled gene expression and synthetic peptide chemistry. These techniques will create new and safer vaccines and will reduce the possibility of adverse reactions to vaccines

Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin, and neomycin in milk.

A broad-specific ELISA using one antibody preparation for the detection of gentamicin, kanamycin, and neomycin in milk is reported for the first time. For the immunization of rabbits, neamin was used as the generic hapten on the basis of the facts that it is a two-ring fragment of neomycin and, in shape and charge, it resembles parts of kanamycin and gentamicin. Neamin was linked to the preactivated carrier protein keyhole limpet hemocyanin by EDC coupling. The specificity and sensitivity of the polyclonal antibodies for the aminoglycoside antibiotics were tested in a competitive assay using homologous and heterologous conjugates coupled by various conjugation procedures as the ELISA solid phase. In contrast to the homologous assay recognizing only neomycin, the heterologous assay could be used for the detection of the whole subclass of deoxystreptamin antibiotics in buffer and raw milk. Gentamicin, kanamycin, and neomcyin were detected in artificially contaminated undiluted raw milk (without sample pretreatment) with 50% inhibition levels at 9, 21, and 113 ng mL(-)(1), respectively. Neomycin levels were also measured in milk samples obtained from a cow suffering from mastitis and treated with an antibiotic cocktail including neomycin. Levels below the EU maximum residue levels for deoxystreptamin antibiotics could readily be measured in this generic ELISA.

Comparison of the sensitivities of the Buehler test and the guinea pig maximization test for predictive testing of contact allergy.

International test guidelines, such as the Organisation for Economic Cooperation and Development (OECD) guideline #406, recommend 2 guinea pig methods for testing of the contact allergenic potential of chemicals: the Guinea Pig Maximization Test (GPMT) and the Buehler test. Previous comparisons between the methods suggested that the Buehler test was less sensitive than the GPMT although modified Buehler test protocols were used. Parallel GPMT and Buehler tests were conducted according to OECD guideline #406 using a multiple-dose design and test results were analysed using a standard logistic dose-response model. To compare the sensitivity of the 2 test procedures the test conditions were kept identical and the following chemicals with a range of sensitization potentials were tested: chloraniline, chlorhexidine, eugenol, formaldehyde, mercaptobenzothiazole and neomycin sulphate. Formaldehyde and neomycin sulphate were strong sensitizers in both tests. Mercaptobenzothiazole, eugenol and chloraniline were all strong sensitizers in the GPMT, eugenol and mercaptobenzothiazole were negative in the Buehler test and equivocal results were obtained with chloraniline. Chlorhexidine was negative in the GPMT and equivocal responses were obtained with the Buehler test. Higher induction concentrations were needed to show allergenicity in the Buehler test and for some allergens the Buehler test protocol was not sensitive enough to demonstrate allergenic potential.

Lymph node cell proliferation assay in guinea pigs for the assessment of sensitizing potentials of chemical compounds.

The efficacy of a lymph node cell proliferation assay in the guinea pig as a first stage screening method of predicting sensitizing potentials of chemicals was studied by using several haptens. Animals were sensitized by a single 24-hr occlusive patch (24 cp), intradermal injection (id) and a combination of id and 24 cp, at a concentration used for guinea pig conventional contact hypersensitivity assay methods. Control animals were treated with vehicle(s) only. Suspensions of the lymph node cells (LNC) were individually prepared and cultured with [3H]methyl thymidine ([3H]TdR). [3H]TdR incorporation was measured and a stimulation index (SI) was calculated as a ratio of the mean [3H]TdR incorporation in sensitized animals and the mean [3H]TdR incorporation in control animals. LNC sensitized by 24 cp with 2,4-dinitrochlorobenzene proliferated maximally and significantly at day 5, whereas this occurred at day 7 after id sensitization. Significant LNC proliferation and high SI values were obtained successively by a combination of 24 cp and id. Moreover, strongly sensitizing chemicals increased significant LNC proliferation (SI > 2.0); weakly to moderately sensitizing chemicals also induced significant LNC proliferation (SI = 1.3-1.7). On the other hand, a primary irritant, sodium dodecyl sulfate, failed to encourage LNC proliferation (SI approximately 1.0).

[Immunogenic properties of neomycin conjugates with macromolecular carriers]. / Issledovanie immunogennykh svoistv kon"iugatov antibiotika neomitsina s makromolekuliarnym nositeliami.

To obtain diagnostic antibodies to neomycin, immunogenic properties of the neomycin conjugates with macromolecular carriers were studied. Bovine serum albumin and copolymers of N-vinylpyrrolidone with crotonic acid and N-hydroxyphthalimide ether of crotonic acid were used as carriers. It was shown that immunization of mice by the conjugates in combination with Freund's adjuvant resulted in production of neomycin antibodies, the titer being 1/80 to 1/130. When the antibiotic conjugates with the copolymers of N-vinylpyrrolidone were used and not the neomycin conjugates with the carrier of the protein nature, the neomycin antibodies were produced in the absence of Freund's full adjuvant. With the use of the isolated antibodies to neomycin a method for indirect solid phase enzyme immunoassay of neomycin was developed at the minimum detectable level of 25 ng/ml.

The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells.

The gene encoding neomycin resistance (neo) mediates a cis-acting negative effect on expression from promoters in transient and stable transfectants of mammalian cell lines. Inserting the neo gene either into retroviral vectors or into plasmids containing reporter genes results in a five- to tenfold decrease of expression from proximal promoters like the simian virus 40 early or the retroviral myeloproliferative sarcoma virus promoter. The silencing effect is not dependent on the insertion site or the orientation of the fragment. The neo gene is frequently used in eukaryotic vectors as a dominant selectable gene. Other selectable genes were tested for potential cis-activity. We found that the gene conferring resistance to puromycin from Streptomyces alboniger does not influence adjacent promoters.

Contact dermatitis sparing the eruption of herpes zoster and its periphery.

A 63-year-old male developed allergic contact dermatitis with antibiotic ointment applied to the skin eruptions of herpes zoster. From the result of patch test, fradiomycin sulfate contained in the ointment was identified as the contact sensitizing antigen. Strangely, this contact dermatitis was confined to the area surrounding the sores, sparing the lesions and their periphery. We postulated that a decrease in Langerhans cell activity in the herpes zoster lesions and their peripheral area was primarily responsible for this phenomenon, since an important role of Langerhans cells in host defence against herpes virus infection has recently been demonstrated.