Increase in Autoantibodies-Abzymes with Peroxidase and Oxidoreductase Activities in Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

The cytosolic tryparedoxin peroxidase from Trypanosoma cruzi induces a pro-inflammatory Th1 immune response in a peroxidatic cysteine-dependent manner.

Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin (Prx) with an important role in detoxifying host cell oxidative molecules during parasite infection. c-TXNPx is a virulence factor, as its overexpression enhances parasite infectivity and resistance to exogenous oxidation. As Prxs from other organisms possess immunomodulatory properties, we studied the effects of c-TXNPx in the immune response and analysed whether the presence of the peroxidatic cysteine is necessary to mediate these properties. To this end, we used a recombinant c-TXNPx and a mutant version (c-TXNPxC52S) lacking the peroxidatic cysteine. We first analysed the oligomerization profile, oxidation state and peroxidase activity of both proteins by gel filtration, Western blot and enzymatic assay, respectively. To investigate their immunological properties, we analysed the phenotype and functional activity of macrophage and dendritic cells and the T-cell response by flow cytometry after injection into mice. Our results show that c-TXNPx, but not c-TXNPxC52S, induces the recruitment of IL-12/23p40-producing innate antigen-presenting cells and promotes a strong specific Th1 immune response. Finally, we studied the cellular and humoral immune response developed in the context of parasite natural infection and found that only wild-type c-TXNPx induces proliferation and high levels of IFN-γ secretion in PBMC from chronic patients without demonstrable cardiac manifestations. In conclusion, we demonstrate that c-TXNPx possesses pro-inflammatory properties that depend on the presence of peroxidatic cysteine that is essential for peroxidase activity and quaternary structure of the protein and could contribute to rational design of immune-based strategies against Chagas disease.

Sestrins teach old T cells new tricks.

Sestrin proteins dampen TCR signaling and induce antigen-independent natural killer-like cytotoxicity in highly differentiated CD8 T cells.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes.

BACKGROUND: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. METHODS: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. RESULTS: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. CONCLUSIONS: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Evaluation of the immune response against Trypanosoma cruzi cytosolic tryparedoxin peroxidase in human natural infection.

Trypanosoma cruzi, the aetiological agent of Chagas disease, has a highly efficient detoxification system to deal with the oxidative burst imposed by its host. One of the antioxidant enzymes involved is the cytosolic tryparedoxin peroxidase (c-TXNPx), which catalyses the reduction to hydrogen peroxide, small-chain organic hydroperoxides and peroxynitrite. This enzyme is present in all parasite stages, and its overexpression renders parasites more resistant to the oxidative defences of macrophages, favouring parasite survival. This work addressed the study of the specific humoral and cellular immune response triggered by c-TXNPx in human natural infection. Thus, sera and peripheral blood mononuclear cells (PBMC) were collected from chronically infected asymptomatic and cardiac patients, and non-infected individuals. Results showed that levels of IgG antibodies against c-TXNPx were low in sera from individuals across all groups. B-cell epitope prediction limited immunogenicity to a few, small regions on the c-TXNPx sequence. At a cellular level, PBMC from asymptomatic and cardiac patients proliferated and secreted interferon-γ after c-TXNPx stimulation, compared with mock control. However, only proliferation was higher in asymptomatic patients compared with cardiac and non-infected individuals. Furthermore, asymptomatic patients showed an enhanced frequency of CD19+ CD69+ cells upon exposure to c-TXNPx. Overall, our results show that c-TXNPx fails to induce a strong immune response in natural infection, being measurable only in those patients without any clinical symptoms. The low impact of c-TXNPx in the human immune response could be strategic for parasite survival, as it keeps this crucial antioxidant enzyme activity safe from the mechanisms of adaptive immune response.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host's cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.

Trans-sialidase overcomes many antigens to be used as a vaccine candidate against Trypanosoma cruzi.

AIM: The development of vaccines against Trypanosoma cruzi remains in an exploratory stage. Despite several antigen candidates have been evaluated, a comparison among the performance of the immunogens cannot be carried out because the available reports differ in formulations and infection model. In this work, we compared the protective capacity of seven T. cruzi antigens in the same model of five new antigens and two well-established candidates. Materials & methods: We evaluated highly immunogenic proteins that contain tandem repeats (FRA [flagelar repetitive protein], Tc3, Tc6); enzymes involved in metabolic pathways critical for parasite survival (cytosolic tryparedoxin peroxidase and tryparedoxin peroxidase); and enzymes involved in parasite invasion (trans-sialidase [TS] and cruzipain). All these antigens were formulated with Freund's adjuvant and protection against the parasite infection was assessed in BALB/c mice. RESULTS: Tc3, cytosolic tryparedoxin peroxidase, cruzipain and TS showed the best outcome after infection in survival level and parasitemia. According to these data, these groups were also assessed using the ISCOMATRIX™ adjuvant which is being used in clinical trials. CONCLUSION: Taken together, our results showed that the TS overcomes the performance of other antigens when the same model is employed, confirming that TS is a promising antigen that could be used as a vaccine against T. cruzi.

Serum Helicobacter pylori KatA and AhpC antibodies as novel biomarkers for gastric cancer.

AIM: To investigate catalase (KatA) and alkyl hydroperoxide reductase (AhpC) antibodies of Helicobacter pylori as biomarkers for gastric cancer (GC). METHODS: This study included 232 cases and 264 controls. Recombinant KatA and AhpC proteins were constructed and the levels of antibodies were tested by indirect enzyme-linked immunosorbent assay (ELISA). Logistic regression was applied to analyze the relationships between KatA, AhpC and GC. The χ(2) trend test was used to evaluate the dose-response relationships between serum KatA and AhpC antibody levels and GC. Receiver operating characteristic (ROC) curve was used to evaluate the screening accuracy of KatA and AhpC as biomarkers. Combined analysis was used to observe screening accuracy of predictors for GC. RESULTS: In all subjects, the association between KatA and AhpC and GC risk was significant (P < 0.001) with odds ratio (OR) = 12.84 (95%CI: 7.79-21.15) and OR = 2.4 (95%CI: 1.55-3.73), respectively. KatA and AhpC antibody levels were strongly related to GC risk with a dose-dependent effect (P for trend < 0.001). The area under the ROC (AUC) for KatA was 0.806, providing a sensitivity of 66.81% and specificity of 86.36%; and the AUC for AhpC was 0.615, with a sensitivity of 75.65% and specificity of 45.49%. The AUC was 0.906 for KatA and flagella protein A (FlaA) combined analysis. CONCLUSION: Serum KatA and AhpC antibodies are associated with GC risk and KatA may serve as a biomarker for GC. KatA/FlaA combined analysis improved screening accuracy.

An unusual cause of cerebellovestibular symptoms.

Hashimoto encephalopathy (HE) is a controversial autoimmune disorder, probably underdiagnosed, that causes a wide variety of neurological manifestations. Symptoms differ among patients and may be very severe in some cases. However, it can be treated, with a very good prognosis. In our case, a teenaged girl with a family history of migraine, vitiligo and benign paroxysmal positional vertigo presented with severe ataxia, vomiting and hypotension. She had a history of similar, but milder, symptoms and was misdiagnosed several times. She had subclinical hypothyroidism, and high levels of antithyroid antibodies. There were abnormal MRI and visual evoked potential findings. After excluding other more common causes, we diagnosed her as having 'Hashimoto Encephalopathy', and started treatment with corticosteroids, on which she showed dramatic improvement. After about 2 years of presentation, the patient is able to continue her life independently.

Differential innate immune response of European seabass (Dicentrarchus labrax) against Streptococcus iniae.

Streptococcus iniae is a Gram-positive bacteria that causes invasive infections with severe septicemia and meningitis, producing high economic losses in marine and continental aquaculture. Head kidney leukocytes of European sea bass (Dicentrarchus labrax) were used to measure the differential innate immune response upon infection with S. iniae. The complete inhibition in the production of intracellular superoxide radicals and total peroxidase content was observed in infected cells. This study also elucidates changes in the relative expression of some immune-related genes. Interleukin 1ß, tumor necrosis factor-α and interleukin-6 reached a peak of expression at 4-8 h post-infection, subsequently decreasing significantly up to 48 h post-infection. However, interleukin-10 and Mx protein increased over time, reaching the pick of expression at 48 h post-infection, whereas caspase-3 showed down regulation until 48 h post-infection. The in vivo study of immune related genes show the same kinetics of mRNAs expression as in vitro experience. The proinflammatory cytokines mRNA transcription levels peaked at an earlier time in vivo than in vitro system. Our findings indicate that there is a direct relationship between the dissemination of bacteria and the resulting infection-associated inhibition of respiratory burst, apoptosis, and the pro- and anti-inflammatory gene expression profiles.

Biochemical mechanisms and therapeutic potential of pseudohalide thiocyanate in human health.

Thiocyanate (SCN(-)) is a ubiquitous molecule in mammalian biology, reaching up to mM concentrations in extracellular fluids. Two- electron oxidation of SCN(-) by H2O2 produces hypothiocyanous acid (HOSCN), a potent anti-microbial species. This reaction is catalyzed by chordate peroxidases (e.g., myeloperoxidase and lactoperoxidase), occurring in human secretory mucosa, including the oral cavity, airway, and alimentary tract, and regulates resident and transient flora as part of innate immunity. Increasing SCN(-) levels limits the concentrations of a family of 2-electron oxidants (H2O2, hypohalous acids, and haloamines) in favor of HOSCN formation, altering the oxidative impact on host tissue by substitution of repairable thiol and selenol oxidations instead of biomolecule degradation. This fine-tuning of inflammatory oxidation paradoxically associates with maintained host defense and decreased host injury during infections, due in part to phylogenetic differences in the thioredoxin reductase system between mammals and their pathogens. These differences could be exploited by pharmacologic use of SCN(-). Recent preclinical studies have identified anti-microbial and anti-inflammatory effects of SCN(-) in pulmonary and cardiovascular animal models, with implications for treatment of infectious lung disease and atherogenesis. Further research is merited to expand on these findings and identify other diseases where SCN(-) may be of use. High oral bioavailability and an increased knowledge of the biochemical effects of SCN(-) on a subset of pro-inflammatory reactions suggest clinical utility.

Immunological consequences of stress-related proteins--cytosolic tryparedoxin peroxidase and chaperonin TCP20--identified in splenic amastigotes of Leishmania donovani as Th1 stimulatory, in experimental visceral leishmaniasis.

In earlier studies, proteomic characterization of splenic amastigote fractions from clinical isolates of Leishmania donovani, exhibiting significant cellular responses in cured Leishmania subjects, led to the identification of cytosolic tryparedoxin peroxidase (LdcTryP) and chaperonin-TCP20 (LdTCP20) as Th1-stimulatory proteins. Both the proteins, particularly LdTCP20 for the first time, were successfully cloned, overexpressed, purified and were found to be localized in the cytosol of purified splenic amastigotes. When evaluated against lymphocytes of cured Leishmania-infected hamsters, the purified recombinant proteins (rLdcTryP and rLdTCP20) induced their proliferations as well as nitric oxide production. Similarly, these proteins also generated Th1-type cytokines (IFN-γ/IL-12) from stimulated PBMCs of cured/endemic Leishmania patients. Further, vaccination with rLdcTryP elicited noticeable delayed-type hypersensitivity response and offered considerably good prophylactic efficacy (~78% inhibition) against L. donovani challenge in hamsters, which was well supported by the increased mRNA expression of Th1 and Th2 cytokines. However, animals vaccinated with rLdTCP20 exhibited comparatively lesser prophylactic efficacy (~55%) with inferior immunological response. The results indicate the potentiality of rLdcTryP protein, between the two, as a suitable anti-leishmanial vaccine. Since, rLdTCP20 is also an important target, for optimization, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up.

Blockade of CD40-TRAF2,3 or CD40-TRAF6 is sufficient to inhibit pro-inflammatory responses in non-haematopoietic cells.

Inhibition of the CD40-CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti-CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40-driven inflammatory disorders. Blocking tumour necrosis factor receptor-associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II(+) cells diminishes inflammation. However, CD40-TRAF6 blockade may cause immunosuppression. We examined the role of CD40-TRAF2,3 and CD40-TRAF6 signalling in the development of pro-inflammatory responses in human non-haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild-type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non-haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154-induced up-regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic protein 1 (MCP-1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM-1 up-regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP-1 and tissue factor up-regulation in these cells. Treatment of endothelial and smooth muscle cells with cell-permeable peptides that block CD40-TRAF2,3 or CD40-TRAF6 signalling impaired pro-inflammatory responses. In contrast, while the CD40-TRAF2,3 blocking peptide did not reduce CD154-induced dendritic cell maturation, the CD40-TRAF6 blocking peptide impaired this response. Hence, preventing CD40-TRAF2,3 or CD40-TRAF6 interaction inhibits pro-inflammatory responses in human non-haematopoietic cells. In contrast to inhibition of CD40-TRAF6 signalling, inhibition of CD40-TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40-TRAF2,3 signalling may control CD40-CD154-dependent inflammatory disorders.

[Effect of chitooligosaccharides with different degrees of acetylation on the activity of wheat pathogen-inducible anionic peroxidase].

The effect of chitooligosaccharides (CHOSs) with a molecular weight of 5-10 kDa and a degree of acetylation (DA) of 65 and 13% at a concentration of 1.0 mg/L on the expression of the TC151917 gene, which encodes wheat anionic peroxidase, and the activity of "anionic" isoperoxidases in common wheat plants infected with Septoria nodorum Berk.--the causative agent of septoriosis. Treatment with CHOSs with a 65% DA and infection promoted the transcription of the anionic peroxidase gene and increased the enzymatic activity of the anionic peroxidase with an isoelectric point of 3.5 in soluble and ion-bound to cell walls protein fractions. Chitooligosaccharides with a 13% DA change these parameters to a lesser extent. These data suggest the importance of the degree of acetylation of CHOSs in the development of immune responses of wheat with the involvement of peroxidases.

Leishmania amazonensis amastigotes highly express a tryparedoxin peroxidase isoform that increases parasite resistance to macrophage antimicrobial defenses and fosters parasite virulence.

Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO-), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO- cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania's ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.

Antioxidative and immunogenic properties of catalase-peroxidase protein in Penicillium marneffei.

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.

Ciona intestinalis peroxinectin is a novel component of the peroxidase-cyclooxygenase gene superfamily upregulated by LPS.

Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reaction. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase-cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the outgroup of mammalian MPO, EPO and TPO clades. The CiPxt molecular structure model resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid gland.

Influence of melatonin on the immune system of fish: a review.

Endocrine-immune system interactions have been widely demonstrated in mammals, whereas in fish, these relationships remain unclear. Of the organs that constitute the endocrine system, the pineal gland and its secretory product melatonin act in the synchronization of daily and seasonal rhythms in most vertebrates, including fish. Seasonal differences in immunocompetence and disease prevalence have been well documented in humans. Seasonality also strongly influences the life history of fish by controlling the timing of physiological events, such as reproduction, food intake, locomotor activity, and growth performance. Apart from its synchronizing capabilities, the role of melatonin in physiological processes in fish is not thoroughly understood. The purpose of this review is to summarize current studies on the effects of melatonin on the fish immune system. These studies suggest that melatonin represents an important component of fish endocrine-immune system interactions. The elucidation of the defense mechanisms of fish will facilitate the development of health management tools to support the growing finfish aquaculture industry as well as address questions concerning the origins and evolution of the immune system in vertebrates.

Crystal structure of two anti-porphyrin antibodies with peroxidase activity.

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααß-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection.