Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development.

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

A microfluidic colorimetric immunoassay for sensitive detection of altenariol monomethyl ether by UV spectroscopy and smart phone imaging.

A novel microfluidic colorimetric immunoassay was developed using gold nanoparticles (GNPs) for indicating different concentrations of altenariol monomethyl ether (AME), and UV spectroscopy and smart phone imaging for monitoring color change of the GNPs. Norland Optical Adhesive 81 (NOA 81) was used for simple and rapid fabrication of the microfluidic chip. AME-BSA modified magnetic nanoparticles (MNPs-BSA-AME) were used as capture probe and the self-magnetism for rapid separation and purification. AME monoclonal antibodies modified gold nanoparticles (GNP-mAbs) which dried on conjugate pad were used as detection probe and the self-catalyst for signal amplification. Under the optimal conditions, the proposed microfluidic colorimetric immunoassay was able to detect AME as low as 12.5 pg/mL for UV spectroscopy (574 nm), and 200 pg/mL for smart phone imaging. The total analysis time is less than 15 min. The immunoassay also has a lower cross-reactivity to AME analogues. It was also evaluated by analyzing fruit samples spiked with AME. The recoveries ranged from 91.19% to 94.15% for UV spectroscopy, and from 90.63% to 93.9% for smart phone imaging. This method can be used for rapid, sensitive, low-cost and portable point-of care testing (POCT) of other mycotoxins or haptens in food samples.

High affinity antibody based on a rationally designed hapten and development of a chemiluminescence enzyme immunoassay for quantification of Alternariol in fruit Juice, maize and flour.

Alternariol (AOH), a secondary metabolites of the genus Alternaria, is a genotoxic mycotoxin. Because the previous "Mannich antigen" for AOH readily underwent unstable induction, a new AOH hapten was designed for inducing a high-quality antibody against AOH using a conjugate of the AOH carboxymethyl derivative with a carrier protein as the immunogen. A competitive indirect chemiluminescence enzyme immunoassay (ciCLEIA) for AOH was optimized and showed good sensitivity (limit of detection 0.068 ng/mL), a linear range (0.11-1.23 ng/mL), and negligible cross-reactivity with analogues. Average rates of recovery from spiked samples (juice and cereal) were between 72.7% and 115.8%, with coefficients of variation <14.3%. Results from ciCLEIA correlated well with those of high-performance liquid chromatography coupled with tandem mass spectrometry, meaning the proposed method might be an effective screening tool for detection of AOH in food samples.

Screening and Identification for Immunological Active Components from Andrographis Herba Using Macrophage Biospecific Extraction Coupled with UPLC/Q-TOF-MS.

The method of cell biospecific extraction coupled with UPLC/Q-TOF-MS has been developed as a tool for the screening and identification of potential immunological active components from Andrographis Herba (AH). In our study, a macrophage cell line (RAW264.7) was used to extract cell-combining compounds from the ethanol extract of AH. The cell binding system was then analyzed and identified by UPLC/Q-TOF-MS analysis. Finally, nine compounds, which could combine with macrophages, in an ethanol extract of AH were detected by comparing basic peak intensity (BPI) profiles of macrophages before and after treatment with AH. Then they were identified as Andrographidine E (1), Andrographidine D (2), Neoandrographolide (3), Dehydroandrographolide (4), 5, 7, 2', 3'-tetramethoxyflavone (5), ß-sitosterol (7), 5-hydroxy-7, 2', 3'-trimethoxyflavone (8) and 5-hydroxy-7, 8, 2', 3'-tetramethoxyflavone (9), which could classified into five flavonoids, three diterpene lactones, and one sterol. Their structures were recognized by their characteristic fragment ions and fragmentations pattern of diterpene lactones and flavonoids. Additionally, the activity of compounds 3, 4, and 7 was tested in vitro. Results showed that these three compounds could decrease the release of NO (p < 0.01) in macrophages remarkably. Moreover, 3, 4, and 7 showed satisfactory dose-effect relationships and their IC50 values were 9.03, 18.18, and 13.76 µg/mL, respectively. This study is the first reported work on the screening of immunological active components from AH. The potential immunological activity of flavonoids from AH has not been reported previously.

Development of an Immunochromatographic Strip Test for the Rapid Detection of Alternariol Monomethyl Ether in Fruit.

A rapid, portable, and semi-quantitative immunochromatographic strip was developed for rapid and visual detection of alternariol monomethyl ether (AME). For this purpose, the anti-AME monoclonal antibody (mAb) was prepared and identified. AME coupled to bovine serum albumin (BSA) via methyl 4-bromobutanoate was prepared as immunogen. The recoveries of AME in spiked cherry and orange fruits determined by competitive ELISA were 86.1% and 80.7%, respectively. A colloidal gold nanoparticle (CGN) and CGNs-mAb conjugate were synthesized, and on this basis, a competitive colloidal gold immunochromatographic strip was developed and applied to the detection of AME toxin in fruit samples. The intensity of red density of the test line (T line) is inversely proportional to AME concentration in the range 0.1-10 ng/mL. The visual limit of detection (LOD) of AME was found to be about 10 ng/mL. The semi-quantitative detection can be completed in 10 min. Moreover, the immunochromatographic strip has lower cross-reactivity with AME analogues, and it has a good stability performance (following 3 months of storage). Hence, the colloidal gold immunochromatographic strip could be used as a semi-quantitative tool for the on-site, rapid, and visual detection of AME in fruit.

Performance of a commercially available plant allergen series in the assessment of suspected occupational contact dermatitis to plants in north Indian patients.

BACKGROUND: Parthenium hysterophorus is the leading cause of phytogenic allergic contact dermatitis in India. The Indian Standard Series currently supplied by Systopic Laboratories Ltd and manufactured by Chemotechnique Diagnostics ® contains parthenolide as the only allergen representing plant allergens. AIM: The study was conducted to assess the performance of the Chemotechnique plant series (PL-1000), consisting of 14 allergens, in patients with clinically suspected occupational contact dermatitis to plant allergens. METHODS: Ninety patients were patch tested with the Chemotechnique plant series from 2011 to 2013. Demographic details, clinical diagnosis and patch test results were recorded in the contact dermatitis clinic proforma. RESULTS: Of 90 patients, 24 (26.7%) showed positive reactions to one or more allergens in the plant series. Positive patch tests were elicited most commonly by sesquiterpene lactone mix in 19 (78.6%) patients, followed by parthenolide in 14 (57.1%), Achillea millefolium in 10 (42.9%) and others in decreasing order. CONCLUSION: The plant allergen series prepared by Chemotechnique Diagnostics is possibly not optimal for diagnosing suspected allergic contact dermatitis to plants in north Indians. Sesquiterpene lactone mix should replace parthenolide as the plant allergen in the Indian Standard Series until relevant native plant extracts are commercially available for patch testing.

Sunflower seeds as eliciting agents of Compositae dermatitis.

BACKGROUND: Sunflowers may cause dermatitis because of allergenic sesquiterpene lactones (SLs). Contact sensitization to sunflower seeds has also been reported, but the allergens are unknown. OBJECTIVES: To analyse sunflower seeds for the presence of SLs and to assess the prevalence of sunflower sensitization in Compositae-allergic individuals. PATIENTS/MATERIALS/METHODS: Sunflower-sensitive patients were identified by aimed patch testing. A dichloromethane extract of whole sunflower seeds was analysed by liquid chromatography-mass spectrometry and high-performance liquid chromatography. RESULTS: The prevalence of sensitivity to sunflower in Compositae-allergic individuals was 56%. A solvent wash of whole sunflower seeds yielded an extract containing SLs, the principal component tentatively being identified as argophyllin A or B, other SLs being present in minute amounts. CONCLUSIONS: The concentration of SLs on the sunflower seeds is considered high enough to elicit dermatitis in sensitive persons, and it seems appropriate to warn Compositae-allergic subjects against handling sunflower seeds.

Parthenium dermatitis in India: past, present and future.

Parthenium dermatitis is an immuno-inflammatory disease caused by Parthenium hysterophorus and is the commonest cause of plant dermatitis in India. It is caused by airborne dry and friable plant particles including trichomes, and the most important allergens responsible for allergic contact dermatitis are sesquiterpene lactones. The combined type IV and type I hypersensitivity to parthenium has been recently postulated. In sensitized individuals, it can cause a spectrum of clinical patterns, such as classical airborne pattern, chronic actinic dermatitis-like presentation, mixed pattern dermatitis, exfoliative dermatitis, widespread dermatitis, and other rare patterns. There is definite trend towards change from airborne pattern to chronic actinic pattern in natural history of parthenium dermatitis. Contact sensitivity to parthenium is everlasting, and hence the disease runs a chronic course with exacerbation during summers. Patch testing with acetone or aqueous plant extract is the simplest way of confirming parthenium contact allergy. Management includes avoiding contact with allergen, managing dermatitis with topical corticosteroids/tacrolimus, and other immunosupressives like azathioprine. In future, we expect parthenium dermatitis to become less prevalent due to rapid urbanization and possible development of new biological methods to eradicate the parthenium. Genetic factors associated with susceptibility to parthenium dermatitis need to be studied.

Relating skin sensitizing potency to chemical reactivity: reactive Michael acceptors inhibit NF-κB signaling and are less sensitizing than S(N)Ar- and S(N)2- reactive chemicals.

The skin sensitization potency of chemicals is partly related to their reactivity to proteins. This can be quantified as the rate constant of the reaction with a model peptide, and a kinetic profiling approach to determine rate constants was previously proposed. A linear relationship between the skin sensitization potency in the local lymph node assay (LLNA) and the rate constant for Michael acceptors was reported, characterized by a relatively flat regression line. Thus, a 10-fold increase of reactivity correlates to an increase of the sensitization potential of only 1.7-fold. Here, we first validate this model by repeating previous data and testing additional Michael acceptors and prove that the model is both reproducible and robust to the addition of new data. Chemicals of different mechanistic applicability domains, namely, S(N)Ar- and S(N)2-reactive sensitizers, were then tested with the same kinetic profiling approach. A linear relationship between sensitization potency in the LLNA and rate constants was also found, yet with a much steeper slope, i.e., for S(N)Ar- and S(N)2-reactive sensitizers, increasing reactivity correlates to a much stronger increase in sensitization potency. On the basis of the well-known inhibitory activity of some Michael acceptors on IKK kinase, it was hypothesized that the difference in the slopes is due to the specific anti-inflammatory potential of Michael acceptor chemicals. Therefore, all chemicals were tested for anti-inflammatory activity in a reporter gene assay for the inhibition of NF-κB activation. Increasingly reactive Michael acceptors have increasing anti-inflammatory potential in this assay, whereas no such biological activity was detected for the S(N)Ar and S(N)2 reactive sensitizers. Thus, the increasing reactivity of Michael acceptors confers both anti-inflammatory and skin sensitizing/pro-inflammatory potential, which may partially neutralize each other. This may be the reason for the relatively weak relationship between the potency in the LLNA and the rate constant of this particular group of chemicals.

Immunotoxic sesquiterpene lactone from Carpesium rosulatum Miq.

The whole plants of Carpesium rosulatum were chloroform extracted and the isolated sesquiterpene lactones and immunotoxicity effects were studied. The structures and stereochemistry of these compounds were established on the basis of analysis of spectra including mp, [α](D)(25), IR, UV, EI-MS, MS, (1)H-NMR, (13)C-NMR and some chemical transformations as follows: 1 (4ß,10α-dihydroxy-guaia-8α,12-olide), 2 (4ß,10α-dihydroxy-1(2),11 (13)-guaiadien -8α,12-olide), 3 (3ß,8ß-dihydroxy-1α,5α-guaian-10(14)-ene-6α,12-olide). 4 (2ß,5-epoxy-5,10-dihydroxy-6α,9ß-diangeloyloxy-germacran-8α,12-olide) The chloroform extracted had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 13.11 ppm and an LC(90) value of 20.33 ppm. The results could be useful in search for newer, safer, and more effective natural immunotoxicity agents against A. aegypti.

Detection of bacterial signaling molecules in liquid or gaseous environments.

The detection of bacterial signaling molecules in liquid or gaseous environments has been occurring in nature for billions of years. More recently, man-made materials and systems has also allowed for the detection of small molecules in liquid or gaseous environments. This chapter will outline some examples of these man-made detection systems by detailing several acoustic-wave sensor systems applicable to quorum sensing. More importantly though, a comparison will be made between existing bacterial quorum sensing signaling systems, such as the Vibrio harveyi two-component system and that of man-made detection systems, such as acoustic-wave sensor systems and digital communication receivers similar to those used in simple cell phone technology. It will be demonstrated that the system block diagrams for either bacterial quorum sensing systems or man-made detection systems are all very similar, and that the established modeling techniques for digital communications and acoustic-wave sensors can also be transformed to quorum sensing systems.

Contact and photocontact sensitization in chronic actinic dermatitis: a changing picture.

BACKGROUND: Patients with chronic actinic dermatitis (CAD) frequently have positive patch or photopatch tests. In our previous study (period 1987-1992), the most prominent contact allergen was the sesquiterpene lactone mix (36% of patients with CAD). OBJECTIVE: To assess whether contact allergy profiles in CAD patients between 2000 and 2005 have changed in respect to our previous data (1987-1992). PATIENTS AND METHODS: Fifty CAD patient records from 2000 to 2005 for patch and photopatch testing were retrospectively analysed and data were compared with that from 86 patients seen between 1987 and 1992. RESULTS: Thirty-two (64%) and 64 (74%) patients had positive patch or photopatch tests in 2000-2005 and 1987-1992, respectively. The allergen profile has altered. A decline in sesquiterpene lactone mix positive reactions was noted: 29 (36%) patients were positive in 1987-1992 and 10 (20%) patients in 2000-2005, but this was not significant (P = 0.08). Reactions to non-fragrance consumer allergens (i.e. p-phenylenediamine and preservatives) had risen from 7 reactions (1987-1992) to 21 reactions in 13 individuals (2000-2005) (P < 0.001). Of these allergens, p-phenylenediamine was the most common (12%; P = 0.004). CONCLUSIONS: A significant rise in positive patch tests to non-fragrance consumer allergens, particularly p-phenylenediamine, was seen in CAD patients in 2000-2005. We speculate this alteration of allergen profile may be partly due to changes in exposure patterns.

Anti-inflammatory and immune-regulatory mechanisms prevent contact hypersensitivity to Arnica montana L.

Sesquiterpene lactones (SL), secondary plant metabolites from flowerheads of Arnica, exert anti-inflammatory effects mainly by preventing nuclear factor (NF)-kappaB activation because of alkylation of the p65 subunit. Despite its known immunosuppressive action, Arnica has been classified as a plant with strong potency to induce allergic contact dermatitis. Here we examined the dual role of SL as anti-inflammatory compounds and contact allergens in vitro and in vivo. We tested the anti-inflammatory and allergenic potential of SL in the mouse contact hypersensitivity model. We also used dendritic cells to study the activation of NF-kappaB and the secretion of interleukin (IL)-12 in the presence of different doses of SL in vitro. Arnica tinctures and SL potently suppressed NF-kappaB activation and IL-12 production in dendritic cells at high concentrations, but had immunostimulatory effects at low concentrations. Contact hypersensitivity could not be induced in the mouse model, even when Arnica tinctures or SL were applied undiluted to inflamed skin. In contrast, Arnica tinctures suppressed contact hypersensitivity to the strong contact sensitizer trinitrochlorobenzene and activation of dendritic cells. However, contact hypersensitivity to Arnica tincture could be induced in acutely CD4-depleted MHC II knockout mice. These results suggest that induction of contact hypersensitivity by Arnica is prevented by its anti-inflammatory effect and immunosuppression as a result of immune regulation in immunocompetent mice.

Isolation, characterization and biological evaluation of datura lactones as potential immunomodulators.

Phytochemical investigation of Datura quercifolia (Solanaceae) plant yielded a new datura lactone, 1beta,5alpha,12alpha-trihydroxy-6alpha,7alpha,24alpha,25alpha-diepoxy-20S,22R with-2-enolide (1), along with two known compounds, 2 and 3. The structure of 1 was established on the basis of spectral analysis, as well as by its chemical transformation into known datura lactones. These compounds have been evaluated for immunomodulatory activity by observing their effect on antibody production, T-cell and B-cell activation, and cytokine production from splenocytes. Compound 2 was found to be the most promising immunostimulator in the present study.

Haemophilus influenzae luxS mutants form a biofilm and have increased virulence.

To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5alpha expressing a cloned luxSHi contained 350-fold more AI-2 activity per cell than E. coli DH5alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxSHi mutants. Two clinical H. influenzae and their luxSHi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxSHi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxSHi mutants tended to be more invasive. The luxSHi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.

Sesquiterpene lactone mix patch testing supplemented with dandelion extract in patients with allergic contact dermatitis, atopic dermatitis and non-allergic chronic inflammatory skin diseases.

We investigated the value of patch testing with dandelion (Compositae) extract in addition to sesquiterpene lactone (SL) mix in selected patients. After we detected a case of contact erythema multiforme after patch testing with dandelion and common chickweed (Caryophyllaceae), additional testing with common chickweed extract was performed. A total of 235 adults with a mean age of 52.3 years were tested. There were 66 men and 169 women: 53 consecutive patients with allergic contact dermatitis (ACD); 43 with atopic dermatitis (AD); 90 non-atopics suffering from non-allergic chronic inflammatory skin diseases; 49 healthy volunteers. All were tested with SL mix 0.1% petrolatum (pet.) and diethyl ether extracts from Taraxacum officinale (dandelion) 0.1 and 3.0% pet. and from Stellaria media (common chickweed) 0.1 and 3% pet. A total of 14 individuals (5.9%) showed allergic reaction (AR) to at least 1 of the plant allergens, 4 (28.6%) to common chickweed extract, and 11 (78.6%) to Compositae allergens. These 11 persons made the overall prevalence of 4.7%: 8 (3.4%) were SL-positive and 3 (1.3%) reacted to dandelion extract. 5 persons (45.5%) had AD, 2 had ACD, 2 had psoriasis and 2 were healthy controls. The Compositae allergy was relevant in 8 cases (72.7%). The highest frequency of SL mix sensitivity (9.3%) was among those with AD. Half the SL mix-sensitive individuals had AD. ARs to dandelion extract were obtained only among patients with eczema. A total of 9 irritant reactions (IRs) in 9 individuals (3.8%) were recorded, 8 to SL mix and 1 to common chickweed extract 3.0% pet. No IR was recorded to dandelion extract (P = 0.007). Among those with relevant Compositae allergy, 50.0% had AR to fragrance mix and balsam of Peru (Myroxylon pereirae resin) and colophonium. SLs were detected in dandelion but not in common chickweed. Our study confirmed the importance of 1 positive reaction for emerging, not fully established, Compositae allergy. In conclusion, the overall prevalence of 4.7% in our study represents a basal SL mix detection rate of 3.4% reinforced and safely supplemented by testing with the dandelion extract.