Characterization of residue-specific glutathionylation of CSF proteins in multiple sclerosis - A MS-based approach.

Reduction of a disulfide linkage between cysteine residues in proteins, a standard step in the preanalytical preparation of samples in conventional proteomics approach, presents a challenge to characterize S-glutathionylation of proteins. S-glutathionylation of proteins has been reported in medical conditions associated with high oxidative stress. In the present study, we attempted to characterize glutathionylation of CSF proteins in patients with multiple sclerosis which is associated with high oxidative stress. Using the nano-LC/ESI-MS platform, we adopted a modified proteomics approach and a targeted database search to investigate glutathionylation at the residue level of CSF proteins. Compared to patients with Intracranial hypertension, the following CSF proteins: Extracellular Superoxide dismutase (ECSOD) at Cys195, α1-antitrypsin (A1AT) at Cys232, Phospholipid transfer protein (PLTP) at Cys318, Alpha-2-HS-glycoprotein at Cys340, Ectonucleotide pyrophosphate (ENPP-2) at Cys773, Gelsolin at Cys304, Interleukin-18 (IL-18) at Cys38 and Ig heavy chain V III region POM at Cys22 were found to be glutathionylated in patients with multiple sclerosis during a relapse. ECSOD, A1AT, and PLTP were observed to be glutathionylated at the functionally important cysteine residues. In conclusion, in the present study using a modified proteomics approach we have identified and characterized glutathionylation of CSF proteins in patients with multiple sclerosis.

Assessment of Cysteine Reactivity of Human Hemoglobin at Its Residue Level: A Mass Spectrometry-Based Approach.

In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α2ß2) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site-specific reactivities of cysteine residues of Hb were investigated using alkylation kinetics with iodoacetamide (IAM). The observed reactivities corroborated well with the differential surface accessibilities of cysteine residues in the crystal structures of human Hb. The proposed method might be used to investigate cysteine reactivities of all the genetic and post-translational variants of Hb discovered to date. In addition, this method can be extended to explore cysteine reactivities of proteins, irrespective of the presence of chromophores and availability of crystal structures.

Clinico-immunological changes post-immunotherapy with Periplaneta americana.

BACKGROUND: Cockroach proteins induce allergies including asthma in predisposed individuals. Well-designed controlled studies are required to show the effect of cockroach immunotherapy (IT). This study is aimed to assess changes in clinical and immunological parameters post-IT with Periplaneta americana extract. MATERIALS AND METHODS: A double-blind, placebo-controlled trial of cockroach IT was performed for 1year in 50 patients of asthma, rhinitis or both. The efficacy of IT was assessed by change in skin reactivity and clinical parameters such as symptom/drug score, airway reactivity and immunological parameters namely IgE, IgG1 and IgG4, IL-4 and IFN-γ by enzyme-linked immunosorbent assay and western blotting using patients' sera at baseline and after 1year of treatment. RESULTS: Immunotherapy with cockroach extract demonstrated significant improvement in clinical parameters of active group patients compared with baseline values and placebo group. Specific IgE levels showed a modest reduction, while IgG4 levels increased significantly in active IT group after 1year. IgE immunoblotting demonstrated reduction in intensity and number of specific bands, whereas IgG4 binding showed more number and distinct bands following IT. Active group patients showed correlation between increase in IgG4/IgG1 ratio and reduction in symptom score post-IT. CONCLUSIONS: Immunotherapy with cockroach extract improved clinical and immunological status of asthma and rhinitis patients. Clinical improvement in patients after IT is associated with immunological changes.

Proteolytically inactive per a 10 allergen of Periplaneta americana modulates Th2 response and enhances IL-10 in mouse model.

BACKGROUND: Purified allergens with reduced IgE reactivity are required to improve the safety and efficacy of allergen-specific immunotherapy (IT). OBJECTIVE: The present study investigates the efficacy of purified cockroach allergen immunotherapy with proteolytically active and inactive Per a 10 in allergic mouse model. METHODS: Balb/c mice were sensitized intraperitoneally with cockroach extract (CE) and purified allergen Per a 10 in separate groups. Mice were treated subcutaneously with phosphate-buffered saline (PBS), CE, active and inactive Per a 10 and challenged intranasally. Antigen specific IgE, IgG1 and IgG2a in serum and cytokines IL-4, IL-13, IFN-gamma, IL-10, TGF-beta in bronchoalveolar lavage (BAL) fluid and spleen culture supernatant (CS) were estimated by enzyme-linked immunosorbent assay. Lung histology was analyzed by hematoxylin and eosin staining. RESULTS: IT with Per a 10 demonstrated significant reduction in IgE levels in serum, IL-4 levels in BAL fluid, CS, and eosinophilic infiltration in lungs than PBS-treated mice. This was associated with significantly increased IL-10 secretion in BAL fluid and CS. IT with Per a 10 effectively suppressed T-helper type 2 (Th2) response in mice sensitized with Per a 10 than CE group. Further, IT with inactive Per a 10 showed maximum reduction in systemic and airway inflammation and induced maximum IL-10 release in BAL fluid and CS than other antigens. CONCLUSIONS: IT with Per a 10 effectively suppressed Th2 response and lung inflammation in Per a 10- or CE-sensitized mice. The beneficial effects of IT with inactive Per a 10 are more pronounced than active Per a 10.

Clinico-immunologic study on immunotherapy with mixed and single insect allergens.

BACKGROUND: Immunotherapy (IT) is practiced mainly with mixed and single allergen vaccines. But studies are rare with mixed allergen preparations. OBJECTIVE: The objective of this study is to study mix and single insect allergen IT in patients of allergic rhinitis and asthma. METHODS: We performed a double-blind placebo-controlled trial of mix and single allergen IT for 1 year in 99 patients of asthma or rhinitis or both. There were two groups, (1) active allergen IT (n = 61) with three subgroups single insect extract (cockroach, housefly, or mosquito) and mix allergen IT (two or three insect extracts) and (2) placebo (n = 38). Clinical (skin reactivity, airway reactivity, and symptom score) and immunological (IgE/IgG4 and IgG1/IgG4 ratio) parameters were assessed at baseline and after 1 year of IT. RESULTS: Eighty-five patients completed 1 year of IT. The active allergen IT group patients showed a significant improvement compared to baseline values (p < 0.05) and placebo group patients (p < 0.05) with regard to symptom scores, FEV1 values, and immunological parameters (IgG4). No significant difference was found between mixed and single IT group patients for changes in clinical and immunological parameters. Positive correlation was observed between increase in IgG4 and clinical improvement. The changes in above parameters in placebo group were nonsignificant after 1 year of treatment. CONCLUSION: IT with two to three mix extract from the same allergen group is effective for insect hypersensitivity.

Current immunological approaches for management of allergic rhinitis and bronchial asthma.

A large population world over is affected with allergic diseases and asthma. Pharmacotherapy for allergic diseases and asthma is effective in controlling symptoms but on discontinuation of medication, symptoms reoccur. In contrast, immunotherapy modifies and corrects the underlying pathological immune responses in an antigen-specific manner. Immunotherapy shows an increase in IgG (blocking antibody) that competes with IgE for allergen, inhibiting the release of inflammatory mediators. Recent studies suggest that immunotherapy acts by modifying CD4+ T-cell responses either by immune deviation, T-cell anergy and/or both. Current immunological approaches for management of allergies and asthma involve immunization with native allergen, modified allergen, peptides/cDNA of allergen, anti-IgE, adjuvants coupled allergen, including immunostimulatory DNA sequences, cytokines, and bacterial products. These approaches modulate the immune response and are intended to give long-term benefit.

Immunotherapy with mosquito (Culex quinquefasciatus) extract: a double-blind, placebo-controlled study.

BACKGROUND: Mosquito allergy is well established, but mosquito immunotherapy requires validation using clinical and immunologic variables. OBJECTIVE: To evaluate the tolerability and efficacy of specific immunotherapy with Culex quinquefasciatus (mosquito) extract. METHODS: We performed a randomized, double-blind, placebo-controlled trial of immunotherapy for 1 year in 40 patients with asthma, rhinitis, or both. Patients were evaluated by means of intradermal testing, symptom and drug scores, and histamine provocation testing before and after 1 year of immunotherapy. Mosquito specific IgE and IgG subclass antibody responses were evaluated at the basal level and after 1 year. RESULTS: Patients receiving allergen immunotherapy for 1 year showed a significant improvement compared with baseline and patients receiving placebo regarding skin reactions, symptom scores (rhinitis and asthma), and forced expiratory volume in 1 second. Provocation concentration of histamine that caused a decrease in forced expiratory volume in 1 second of 20% by inhalation was elevated in the group receiving immunotherapy. In the active group serologic analysis showed a slight reduction in IgE levels (P = .02) but a significant elevation in IgG4 levels (P = .001), with a significant decrease in the IgE/IgG4 ratio (P = .001). All these changes in the placebo group were nonsignificant. CONCLUSIONS: Allergen immunotherapy with mosquito extract was well tolerated, with improvement in symptoms and airway reactivity. Good clinical outcome was associated with increased IgG4 antibody levels.

Stability of protease-rich periplaneta Americana allergen extract during storage: formulating preservatives to enhance shelf life.

Allergenic proteins in extracts degrade rapidly and lose potency on storage. Hence, formulation of optimum conditions is required to enhance shelf life of extracts for proper allergy diagnosis and immunotherapy. In the present study, allergenic potency of P. americana proteins was evaluated after storage with epsilon-aminocaproic acid (EACA), sucrose, glycerol, pepstatin A, and aprotinin, individually for 1, 3, 6, and 12 months at 4, 25, and 37 degrees C. P. americana extract stored with EACA and sucrose individually retained potency comparable to proteins in standard extract (freeze-dried extract, stored at-70 degrees C) upto 6 months at 4 degrees C. The extracts without preservatives or with glycerol, pepstatin A, aprotinin, or stored at 37/25 degrees C were severely degraded and lost potency by 3 months. A formulation containing a combination of EACA and sucrose enhanced the shelf life of P. americana proteins upto 12 months at 4 degrees C. Hence, EACA and sucrose together show better potential for stabilization of protease-rich extracts.