A Component-Resolved Therapeutic Vaccine for Cockroach Allergy Made of Per a 9 and Transforming Growth Factor-ß Homologue, an Immunosuppressive Protein of Brugia malayi.

Allergen-specific-immunotherapy (ASIT) can cause long-term resolution of allergic diseases, reduces drug use and chances of new allergen sensitization. Nevertheless, therapeutic vaccine and data on ASIT efficacy for cockroach (CR) allergy are relatively scarce. In this study, efficacy and mechanism of a novel intranasal vaccine consisting of liposome (L)-entrapped mixture of American CR (Periplaneta americana) major allergen (Per a 9) and immunosuppressive protein of Brugia malayi nematode named transforming growth factor-beta homologue (TGH) in treatment of CR allergy were investigated along with two other vaccines (L-Per a 9 alone and L-TGH alone). All three vaccines could reduce pathogenic type 2 response and lung immunopathology in the vaccines-treated CR-allergic mice, but by different mechanisms. L-Per a 9 caused a deviation of the pathogenic type 2 to type 1 response (IFN-γ-upregulation), whereas the L-(TGH + Per a 9) and L-TGH generated regulatory immune responses including up-expression of immunosuppressive cytokine genes and increment of serum adenosine and lung indoleamine-2,3-dioxygenase-1 which are signatures of regulatory T cells (Tregs) and tolerogenic dendritic cells, respectively. The L-(TGH + Per a 9) should be further evaluated towards clinical application, as this vaccine has a propensity to induce broadly effective therapeutic effects for inhalant allergies.

Development of a highly sensitive IgG-ELISA based on recombinant arginine kinase of Toxocara canis for serodiagnosis of visceral larva migrans in the murine model.

Toxocariasis is a worldwide zoonotic disease caused by the ascarid nematode Toxocara canis. The most common method available for serodiagnosis of toxocariasis is an enzyme-linked immunosorbent assay (ELISA) test using Toxocara excretory-secretory antigen (TES). The present study describes the development of IgG-ELISA based on antiserum prepared against the recombinant arginine kinase of Toxocara canis. Antiserum was prepared against the purified recombinant arginine kinase (AK) using 6-week-old female Japanese white rabbits. Serum samples were collected from experimentally infected BALB/c and C57BL/6 mice at different time periods. The IgG-ELISA was performed using serum samples from mice (infected/uninfected) and TES antigen with antiserum prepared against the recombinant-AK. The optical density (OD450) was measured at 450 nm using a micro-plate ELISA reader. There were significant differences (P<0.01) in the absorbance between infected and control serum samples. Further, we obtained 100% sensitivity for the serum samples from T. canis-infected mice. Therefore, it is suggested that the recombinant-AK based IgG-ELISA could be applied for immunodiagnosis of human toxocariasis. However, it is necessary to evaluate the specificity of this recombinant antigen with similar geohelminth infections.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Molecular cloning and response to laminarin stimulation of arginine kinase in haemolymph in Chinese shrimp, Fenneropenaeus chinensis.

Arginine kinase (AK) was previously reported as a phosphagen-ATP phosphotransferase found in invertebrates. In this study, an 1184 bp cDNA was cloned and sequenced. It contained an open reading frame of 1068 bp that coded for 356 deduced amino acids of AK in Fenneropenaeus chinensis. The calculated molecular mass of AK is 40129.73 Da and pI is 5.92. The predicted protein showed a high level of identity to known AK in invertebrates and creatine kinase from vertebrates, which belong to a conserved family of ATP:guanidino phospho-transferases. In addition, AK protein in plasma of F. chinensis was identified using two-dimensional electrophoresis (2DE) and electrospray ionization mass spectrometry (ESI-MS) according to the calculated molecular mass and pI. AK was significantly decreased in the plasma of F. chinensis at 45 min and recovered at 3 h after laminarin injection as confirmed by 2DE and ESI-MS. The results showed that AK was one of the most significantly changed proteins on two-dimensional gel in the plasma proteins of F. chinensis at 45 min and 3 h after simulation.

Creatine kinase measurement in the 1977 CAP Enzymology Survey: anomalies explained.

In the recently published results of the 1977 College of American Pathologists Enzymology Survey, Some anomalous results were obtained in measuring the creatine kinase (EC 2.7.3.2) activity of the samples. The cause of these anomalies was discovered to be an arginine kinase (EC 2.7.3.3) contaminant in one of the enzyme pools used to make the samples. The contaminant shows cross reactivity with creatine phosphate and is activated by dithioerythritol but not the other commonly used thiol activators.