Selecting disease-modifying medications in 5q spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an inherited lower motor neuron disease. SMA occurs secondary to alterations in the survival motor neuron 1 gene (SMN1), which is the main driver of SMN protein production. The severity of the disease is determined by the number of copies of the SMN2 gene, which is a homolog to SMN1 but not as efficient in protein production. Three medications have recently been approved for the treatment of SMA. Nusinersen is an intrathecal antisense oligonucleotide that alters SMN2 pre-mRNA, onasemnogene abeparvovec-xioi is an intravenous SMN1 gene replacement therapy, and risdiplam is an oral small molecule splicing modifier of SMN2. No head-to-head studies have been conducted comparing these medications, so selection of one of these medications for an individual with SMA can be challenging. In this article we outline the efficacy, safety, and other pertinent factors to consider when selecting a therapy for an individual with SMA. The age of the individual and comorbidities, such as liver or kidney disease, help guide treatment choices. All three of these medications are efficacious, and early initiation is critical for obtaining the best outcomes.

Quantification of Ponceau 4R in Foods by Indirect Competitive Enzyme-Linked Immunosorbent Assay (icELISA).

As one of the rarely allowable azo dyes, ponceau 4R can be added in some foods in some countries. However, it is necessary to develop a credible and rapid analytical method for its monitoring, because of its potentially harmful risk. The hapten of ponceau 4R was first designed and synthesized by introducing a primary amine group into the structure of ponceau 4R. Based on the well-prepared hapten, the immunogen of ponceau 4R was prepared using glutaraldehyde to link ponceau 4R to the carrier protein. The triggered polyclonal antibody was obtained and tested by ELISA to optimize the proper dilution. An icELISA was developed for ponceau 4R, and the IC50 of the method is 36.82 ng/mL. The limit of detection is 0.80 ng/mL, and the linear range is 1-10000 ng/mL. Five selected structural analogues have no cross-reactivity with the anti-ponceau 4R polyclonal antibody (<0.3). In three food samples (grape juice, carbonated beverage, and RIO cocktail), the assay exhibits good stability and reproducibility with a recovery range of 93.87-103.77%, and the intra- and interassay coefficients of variation were <11.73%. The results indicate that the proposed icELISA is sensitive, accurate, specific, and simple, which provides an alternative for the detection of ponceau 4R in foods.

A label-free impedimetric immunosensor for direct determination of the textile dye Disperse Orange 1.

A strategy for a label-free impedimetric immunosensor is described for detection of the textile dye Disperse Orange 1 (DO1). The compounds 1,12-diaminododecane (DADD) and then 1,7-diaminoheptane (DAH) were firstly successively grafted onto a glassy carbon electrode (GCE) surface by electro-oxidation of one amino group, while the other terminal amino group was modified with the antibody anti-DO1. The construction process of the immunosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and capacitance measurements. The electron transfer resistance (Rct) exhibited an effective response to the affinity between the immobilized antibody and the antigen in solution. The linear range for the target compound was from 5.0 nmol L(-1) to 0.5 µmol L(-1) (R=0.9980), and the limit of detection (LOD) was 7.56 nmol L(-1). The proposed impedimetric immunosensor has the advantages of simplicity, cost-effectiveness, and sensitivity.

Immune reactivity to food coloring.

Artificial food dyes are made from petroleum and have been approved by the US Food and Drug Administration (FDA) for the enhancement of the color of processed foods. They are widely used in the food and pharmaceutical industries to increase the appeal and acceptability of their products. Synthetic food colorants can achieve hues not possible for natural colorants and are cheaper, more easily available, and last longer. However, since the use of artificial food coloring has become widespread, many allergic and other immune reactive disorders have increasingly been reported. During the past 50 y, the amount of synthetic dye used in foods has increased by 500%. Simultaneously, an alarming rise has occurred in behavioral problems in children, such as aggression, attention deficit disorder (ADD), and attention-deficit/hyperactivity disorder (ADHD). The ingestion of food delivers the greatest foreign antigenic load that challenges the immune system. Artificial colors can also be absorbed via the skin through cosmetic and pharmaceutical products. The molecules of synthetic colorants are small, and the immune system finds it difficult to defend the body against them. They can also bond to food or body proteins and, thus, are able to act in stealth mode to circumvent and disrupt the immune system. The consumption of synthetic food colors, and their ability to bind with body proteins, can have significant immunological consequences. This consumption can activate the inflammatory cascade, can result in the induction of intestinal permeability to large antigenic molecules, and could lead to cross-reactivities, autoimmunities, and even neurobehavioral disorders. The Centers for Disease Control (CDC) recently found a 41% increase in diagnoses of ADHD in boys of high-school age during the past decade. More shocking is the legal amount of artificial colorants allowed by the FDA in the foods, drugs, and cosmetics that we consume and use every day. The consuming public is largely unaware of the perilous truth behind the deceptive allure of artificial color.

Bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides.

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.

Positive relationship-intensity of response to p-phenylenediamine on patch testing and cross-reactions with related allergens.

BACKGROUND: Hair dye exposure is the most common cause of sensitization to p-phenylenediamine (PPD). Cross-reactions with structurally related allergens occur. OBJECTIVES: It is suggested that a stronger patch test reaction (3+ rather than 1+) to PPD (usually tested as 1% petrolatum) is associated with an increased propensity for cross-reactions. In this article we will demonstrate this association. METHODS: Of 230 patients with allergic reactions to PPD on patch testing identified during 2007-2012 from clinical records, notes for 221 were available for review. Data were collected regarding age, sex, and grade of reaction [International Contact Dermatitis Research Group (ICDRG) criteria] to PPD. Cross-reactions with the following allergens, found in our baseline series, were recorded: Disperse Yellow 3, N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD), and caine mix. Having excluded 23 doubtful reactions, the reactions from 198 patients were further considered. RESULTS: Of the patients, 75.3% (n = 149) were female, and the mean age was 48.6 years (12-82 years). Of the patients allergic to PPD, 16.6% (n = 33) showed cross-reactions with one or more related allergens. Cross-reactions were seen in 16% with a grade of 1+, 14.5% with a grade of 2+, 28.6% with a grade of 3+ when PPD was tested 1% pet., and 50.0% when PPD was tested at 0.1-0.001%, arbitrarily considered to be 4+ (p = 0.02; Cramér's V = 0.23). CONCLUSION: An increasing likelihood of reactions to Disperse Yellow 3, IPPD or caine mix was seen with increasing strength of patch test reaction to PPD. The clinical relevance of these cross-reactions is unclear.

Trends in patch-test results and allergen changes in the standard series: a Mayo Clinic 5-year retrospective review (January 1, 2006, to December 31, 2010).

BACKGROUND: Patch testing is essential for identification of culprits causing allergic contact dermatitis. OBJECTIVE: We sought to identify trends and allergen changes in our standard series during 2006 to 2010, compared with our previous report (2001-2005). METHODS: We conducted a retrospective review of patch-test results. RESULTS: A total of 3115 patients were tested with a mean of 73.0 allergens. Since our prior report, 8 allergens were added to the standard series; 14 were deleted. Significantly higher rates of allergic positive reaction were documented for carba mix, 3%, and Disperse Orange 3, 1%. Rates were lower for 10 allergens: neomycin sulfate, 20%; gold sodium thiosulfate, 0.5%; hexahydro-1,3,5-tris(2-hydroxyethyl)triazine, 1%; disperse blue 124, 1%; disperse blue 106, 1%; diazolidinyl urea, 1%; hexylresorcinol, 0.25%; diazolidinyl urea, 1% aqueous; 2-bromo-2-nitropropane-1,3-diol, 0.25%; and lidocaine, 5%. Many final patch-test readings for many allergens were categorized as mild reactions (erythema only). Overall allergenicity and irritancy rates declined significantly since our prior report. Results were generally comparable with those in a North American Contact Dermatitis Group report from 2005 to 2006. LIMITATIONS: This was a retrospective study; there is a lack of long-term follow-up. CONCLUSIONS: Since our previous report, our standard series composition has changed, and overall rates of allergenicity and irritancy have decreased. Notably, many final patch-test readings showed mild reactions.

A sensitive and selective enzyme-linked immunosorbent assay for the analysis of Para red in foods.

Para red is a synthetic dye and a potential genotoxic carcinogen. A hapten mimicking Para red structure was synthesized by introducing a carboxyl to the naphthol part of Para red and coupled to carrier protein to form an immunogen for the production of specific antibodies. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Para red in food samples. The limit of detection and inhibition half-maximum concentrations of Para red in phosphate buffered saline with 10% methanol were 0.06 and 2.2 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested compounds including Sudan red I, II, III, IV, and G, sunset yellow, 2-naphthol, and 4-nitroaniline were ≤0.2%. This assay was used to determine Para red in tomato sauce, chilli sauce, chilli powder and sausage samples after ultrasonic extraction, cleanup and concentration steps. The average recoveries, repeatability (intraday extractions and analysis), and intra-laboratory reproducibility (interday extractions and analysis) were in the range 90-108%, 4-12% and 8-17%, respectively. This assay was compared to a high-performance liquid chromatographic method for 28 samples, displaying a good correlation (R(2) = 0.95). Para red residues in 53 real world samples determined by ELISA were below the limit of detection.

Production of the polyclonal antibody against Sudan 3 and Immunoassay of Sudan dyes in food samples.

In this study, 4-aminophenylacetic acid was covalently coupled to aniline to synthesize an intermediate hapten and the intermediate hapten was coupled to ß-naphthol to synthesize a tentative hapten of Sudan 3. The hapten was coupled to bovine serum albumin as the immunogen to produce the polyclonal antibody. The obtained antibody was highly specific to Sudan 3, Sudan 1, and Para red, but showed relative low binding ability to Sudan 2, Sudan 4, and Sudan red G. After evaluation of different coating antigens, a heterologous indirect competitive immunoassay was developed to multidetermine the six red dyes in food samples. The cross reactivities to the six analytes were in a range of 21-105%, and the limits of detection were in a range of 0.1-0.8 ng/mL depending on the compound. Intra- and interassay recoveries from the standard fortified blank samples were in a range of 74.5-96.3% with coefficients of variation lower than 15.1%.

Production of the monoclonal antibody against Sudan 2 for immunoassay of Sudan dyes in egg.

Many methods have been reported to determine the residues of Sudan dyes in food samples. Among the reported methods, enzyme-linked immunosorbent assay (ELISA) was a frequently used practical screen tool. In this study, a novel hapten of Sudan 2 was synthesized by coupling 4-amino-3-methylbenzoic acid to ß-naphthol, and the monoclonal antibody against Sudan 2 was produced. The obtained antibody can recognize Sudan 1, 2, 3, and 4, Sudan red G, and Para red simultaneously. After evaluation of different coating antigens, a heterologous indirect competitive ELISA was then developed to determine the six Sudan dyes in egg. The cross-reactivities for the six analytes were in a range of 63% to 100%, and the limits of detection were in a range of 0.2 to 0.5 ng/g, depending on the compound. Intra- and interassay recoveries from the standard fortified blank eggs were in a range of 71.7% to 97.6% with coefficients of variation lower than 17.1%.

In vitro studies on immunotoxic potential of Orange II in splenocytes.

Orange II, an azo dye, is not permitted in food preparations, but high levels of the dye have been detected in different food commodities. Though there are reports on the toxicity of Orange II but knowledge based on the immunomodulatory properties of Orange II is scanty. The present investigation was undertaken to study the in vitro immunotoxic potential of Orange II in splenocytes. Splenocytes were isolated, cultured and subjected to immunophenotypic analysis, mixed lymphocyte reaction (MLR) assay or stimulated with lipopolysaccharide (LPS) or concanavalin A (Con A) for 72 h. The supernatant was collected for cytokine assays. Orange II showed cytotoxic effects at 100-1000µg/ml concentrations and 50µg/ml was determined as the highest non-cytotoxic dose. Orange II at the non-cytotoxic dose (50µg/ml) significantly altered the relative distribution of T and B-cells, MLR response and the mitogen induced proliferative response of T-cells and B-cells. Consistent with the hypo-responsiveness of the T and B-lymphocytes, Orange II induced a concomitant decline in the secretion of cytokines IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17. On the contrary, there was an increase in the production of IL-10, an anti-inflammatory regulatory cytokine, which may be one of the causative factor for immunosuppressive property of Orange II. These results suggest that non-cytotoxic dose of Orange II may have immunomodulatory effects.

Preparation of anti-Sudan red monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of Sudan red in chilli jam and chilli oil.

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC(50) value of 1.7 µg L(-1) in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (<1%), Sudan IV (<1%) and para red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 µg L(-1) and 19.6 µg L(-1), respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%-110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

Para-phenylenediamine-specific lymphocyte activation test: a sensitive in vitro assay to detect para-phenylenediamine sensitization in patients with severe allergic reactions.

Patients sensitized to para-phenylenediamine (PPD) by semi-permanent tattoos increasingly develop threatening allergic reactions in response to black hair dye. The gold standard to diagnose allergic contact dermatitis is to perform epicutaneous patch tests, however, iatrogenic sensitizations and severe patch test reactions to PPD have been described, the latter especially in patients with severe allergic reactions. We examined nine patients with severe allergic reactions in response to permanent hair dyes. Patch tests using the standard concentration of 1% or 0.5% PPD resulted in severe and sometimes even bullous reactions in all patients responsive to PPD. Titration revealed that at 1% of the standard concentration (0.01% PPD), patch test sensitivity decreased and only 50% of patients responded. Consequently, we established an in vitro assay to diagnose PPD allergy. Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured with titrated concentrations of PPD with or without IL-2 supplementation, and cell proliferation was determined by [3H]-thymidine incorporation. Lymphocyte activation test (LAT) detected PBMC cell proliferation specific to PPD, with at least 3.5-fold increase in [3H]-thymidine uptake in all PPD allergic patients. Most importantly, PPD-LAT without IL-2 supplementation remained negative in three out of eight PPD allergic patients. Thus, PPD-LAT with IL-2 supplementation demonstrated a sensitivity of 100%, remained unresponsive in controls not sensitized to PPD, and in one patient sensitive to other p-amino compounds. These data demonstrate that LAT with PPD can be used to detect PPD sensitization as a possible alternative to patch testing at least in patients with severe allergic reactions to PPD.

Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red.

To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01ngmL(-1) in phosphate-buffered saline (PBS) buffer and 0.5ngg(-1) in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.

Gold nanoparticles used as a carrier enhance production of anti-hapten IgG in rabbit: a study with azobenzene-dye as a hapten presented on the entire surface of gold nanoparticles.

The azobenzene moiety, well-known not only for its reversible cis-to-trans photoisomerization but also as a hapten, does not induce antibodies on its own, but it reacts with antibodies raised against conjugates with protein carriers. Hence we selected azobenzene dye as an indicator to assess the possibility of having gold nano-particles act as an immunological carrier instead of protein carriers. In rabbits, we confirmed an in vivo response against azobenzene dye presented on the entire surface of gold nanoparticles (azo-nanoparticles), where the gold nanoparticles appeared to play a role as a carrier for the hapten. A high yield of immunoglobulin G (IgG) against the azobenzene derivative took place in rabbits injected with azo-nanoparticles, whereas no increase in IgG was recognized in other rabbits treated solely with chemically equivalent azobenzene dye instead of azo-nanoparticles. Electron microscopy and surface plasmon resonance spectroscopy indicated that the IgG obtained specifically recognized the difference between the isomer conformations of the azobenzene moiety.

Multiple chemical sensitivities following intolerance to azo dye in sweets in a 5-year-old girl.

BACKGROUND: Cases of multiple chemical sensitivities (MCS) have been reported predominantly in adult patients, but pediatric cases have rarely been reported. METHODS: We present a 5-year-old girl who suffered from recurrent reactions accompanied by urticaria, angioedema, headaches, dyspnea, loss of consciousness, and abdominal pain that were not eradicated, but were instead exacerbated, by various treatments with antihistamines and intravenous corticosteroids. Her diet diary revealed that symptoms occurred after ingestion of colorful sweets such as candies and jellybeans. Open challenge tests with food additives and nonsteroidal anti-inflammatory drugs (NSAIDs) were performed after elimination of these items. Skin prick tests using additives and NSAIDs, which were dissolved in saline, and prick- prick tests using candies and jellybeans, were carried out. RESULTS: Open challenge tests with Tartrazine, aspirin and acetaminophen were positive, whereas skin prick tests using additives and NSAIDs and prick-prick tests using candies and jellybeans were all negative. Consequently, intolerance to azo dyes and NSAIDs such as aspirin was diagnosed. However, she appeared to react to multiple chemical odors such as those of cigarette smoke, disinfectant, detergent, cleaning compounds, perfume, and hairdressing, all while avoiding additives and NSAIDs. On the basis of her history and the neuro-ophthalmological abnormalities, a diagnosis of severe MCS was made and she was prescribed multiple vitamins and glutathione. CONCLUSIONS: The present results suggest that in pediatric MCS, food and drug additives containing azo dyes might play important roles as elicitors.

Experimental study on skin sensitization potencies and cross-reactivities of hair-dye-related chemicals in guinea pigs.

In screening patch testing of hairdressers with occupational contact dermatitis, multiple positive reactions to hair dye-related chemicals, such as p-phenylenediamine (PPD), p-toluenediamine x 2HCl (PTD) and p-aminophenol (PAP), a fabric dye p-aminoazobenzene (PAB), and a tar dye Sudan III, were frequently encountered. To investigate individual skin sensitization potency and the cross-reactivities among above chemicals, a guinea pig maximization test with the above 5 chemicals was performed. In each group, 6 animals were induced with one of the chemicals at 0.1% concentration by intradermal injection and at 1.0% by topical application. The animals were challenged with all 5 chemicals in concentrations of dilution by 10 from 0.1% to 0.001%. Under the conditions of 0.1% challenges, similar sensitization potencies were observed in PPD (6/6), PTD (6/6), PAP (5/6) and PAB (6/6) groups, but no positive reactions were elicited in the Sudan III group. The cross-reactivities to PPD were confirmed in the animals challenged with PTD (6/6), PAP (6/6), PAB (6/6) and Sudan III (3/6). In the PTD-induced group, positive responses to cross-challenges were elicited by PPD (5/6), PAP (3/6), PAB (5/6) and Sudan III (1/6). The cross-reactivities to PAP were observed only with PPD (2/5) and PAB (5/5). PAB-induced animals responded only to PPD (1/6). The results indicate that all these chemicals except Sudan III are strong sensitizers. Their cross-reactivities are different in sensitized conditions, respectively. The cross-reactivities to PPD were higher than those to PTD, PAP and PAB.

A novel phosphocholine antigen protects both normal and X-linked immune deficient mice against Streptococcus pneumoniae. Comparison of the 6-O-phosphocholine hydroxyhexanoate-conjugate with other phosphocholine-containing vaccines.

A novel form of phosphocholine (PC), p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC) coupled to keyhole limpet hemocyanin (KLH) has been compared with unencapsulated, avirulent Streptococcus pneumoniae (R36a) and with the traditional thymus-dependent form of PC, diazophenylphosphocholine (DPPC)-conjugated KLH for its vaccine potential against virulent S. pneumoniae. Immunization with any of these three PC-containing Ags protects normal mice against a lethal challenge with 10(4) S. pneumoniae, whereas only EPC-KLH provides total protection to Xid mice. DPPC-KLH and unencapsulated S. pneumoniae confer less than 40% protection in Xid mice. Passive transfer of a PC-specific hybridoma Ab made from EPC-KLH-immunized Xid mice also provided protection against lethal challenge with S. pneumoniae. Protective anti-PC Ab were capable of binding to the surface of virulent bacteria, whereas anti-PC Ab incapable of binding to the bacterial surface failed to protect. Furthermore, serum Ab from EPC-KLH immunized and protected mice bound to S. pneumoniae, whereas secondary Abs from DPPC-KLH- or R36a-immunized mice failed to bind to the bacteria. EPC-KLH is potentially a vaccine candidate for pneumococcal prophylaxis in settings of immune compromise.

A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae.

Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.