Antigenic Peptide-Thioredoxin Fusion Chimeras for In Vitro Stimulus of CD4+ TCR+ Jurkat T Cells.

Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.

The thioredoxin-1 inhibitor Txnip restrains effector T-cell and germinal center B-cell expansion.

Thioredoxin-1 (Trx1) is a vital component for cellular redox homeostasis. In T cells, Trx1 donates electrons for the de novo synthesis of deoxyribonucleotides to allow rapid cell proliferation. The Trx-interacting protein (Txnip) binds to the reduced Trx1 and inhibits its activity. However, the role of Txnip in adaptive immunity in vivo is unknown. Here, we show that absence of Txnip increased proliferation of effector T cells and GC B-cell responses in response to lymphocytic choriomeningitis virus and Qß virus-like particles, respectively, but did not affect development and homeostasis of T and B cells. While downregulation of Txnip and concomitant upregulation of Trx1 is critical for rapid T-cell expansion upon viral infection, re-expression of Txnip and consequently inhibition of Trx1 is important to restrain late T-cell expansion. Importantly, we demonstrated that T-cell receptor (TCR) engagement but not CD28 costimulation is critically required for Txnip downregulation. Thus, this study further uncovers positive and negative control of lymphocyte proliferation by the Trx1 system.

Thioredoxin-interacting protein links oxidative stress to inflammasome activation.

The NLRP3 inflammasome has a major role in regulating innate immunity. Deregulated inflammasome activity is associated with several inflammatory diseases, yet little is known about the signaling pathways that lead to its activation. Here we show that NLRP3 interacted with thioredoxin (TRX)-interacting protein (TXNIP), a protein linked to insulin resistance. Inflammasome activators such as uric acid crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner and allowed it to bind NLRP3. TXNIP deficiency impaired activation of the NLRP3 inflammasome and subsequent secretion of interleukin 1beta (IL-1beta). Akin to Txnip(-/-) mice, Nlrp3(-/-) mice showed improved glucose tolerance and insulin sensitivity. The participation of TXNIP in the NLRP3 inflammasome activation may provide a mechanistic link to the observed involvement of IL-1beta in the pathogenesis of type 2 diabetes.

Trifolirhizin regulates the balance of Th17/Treg cells and inflammation in the ulcerative colitis mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Ulcerative colitis (UC) is a chronic and recurrent autoimmune disease, characterized by recurrence and remission of mucosal inflammation. Although the understanding of the pathogenesis of UC has been improved, effective therapeutic drugs are required for treating patients with UC. In current work, the mouse model of colitis was established. Trifolirhizin was demonstrated to improve symptom in dextran sulfate sodium (DSS)-induced colitis mice. The body weight of mice was elevated, whereas the disease activity index (DAI) was reduced. Moreover, trifolirhizin was involved in inhibition of inflammation and regulation of the balance of T helper 17 (Th 17) cells and regulatory T (Treg) cells in DSS-induced colitis mice. Further, the activation NLRP3 inflammasome was suppressed by trifolirhizin in DSS-induced colitis mice. Trifolirhizin was also identified to regulate AMP-activated protein kinase (AMPK)-thioredoxin-interacting protein (TXNIP) pathway. The trifolirhizin-mediated anti-inflammatory effect was inhibited by suppressing AMPK in DSS-induced UC mice. In summary, the research suggested that administration of trifolirhizin significantly improved the symptoms and the pathological damage in DSS-induced UC mice. Trifolirhizin regulated the balance of Th17/Treg cells and inflammation in the UC mice through inhibiting the TXNIP-mediated activation of NLRP3 inflammasome.

Molecular characterization and immune regulatory, antioxidant, and antiapoptotic activities of thioredoxin domain-containing protein 17 (TXNDC17) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin domain-containing protein 17 (TXNDC17) is an important, highly conserved oxidoreductase protein, ubiquitously expressed in all living organisms. It is a small (~14 kDa) protein mostly co-expressed with thioredoxin 1 (TRx1). In the present study, we obtained the TXNDC17 gene sequence from a previously constructed yellowtail clownfish (Amphiprion clarkii) (AcTXNDC17) database and studied its phylogeny as well as the protein's molecular characteristics, antioxidant, and antiapoptotic effects. The full length of the AcTXNDC17 cDNA sequence was 862 bp with a 372 bp region encoding a 123 amino acid (aa) protein. The predicted molecular mass and isoelectric point of AcTXNDC17 were 14.2 kDa and 5.75, respectively. AcTXNDC17 contained a TRX-related protein 14 domain and a highly conserved N-terminal Cys43-Pro44-Asp45-Cys46 motif. qPCR analysis revealed that AcTXNDC17 transcripts were ubiquitously and differently expressed in all the examined tissues. AcTXNDC17 expression in the spleen tissue was significantly upregulated in a time-dependent manner upon stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), and Vibrio harveyi. Besides, LPS-induced intrinsic apoptotic pathway (TNF-α, caspase-8, Bid, cytochrome C, caspase-9, and caspase-3) gene expression was significantly lower in AcTXNDC17-overexpressing RAW264.7 cells, as were NF-κB activation and nitric oxide (NO) production. Furthermore, the viability of H2O2-stimulated macrophages was significantly improved under AcTXNDC17 overexpression. Collectively, our findings indicate that AcTXNDC17 is involved in the innate immune response of the yellowtail clownfish.

Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin.

Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a kcat/KM value of 8.1 µM-1⋅min-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.

Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation.

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.

Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs.IMPORTANCE Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so-called neutralization epitopes within its N terminus. We designed polytopes comprising the L2 epitope amino acids 20 to 38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile archaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising a heptamerized L2 8-mer thioredoxin single-peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.

Characterization and immune function of the thioredoxin-interacting protein (TXNIP) from Litopenaeus vannamei.

The thioredoxin (Trx) system plays essential roles in maintenance and regulation of the redox state of cysteine residues in cellular proteins. The Trx-interacting protein (TXNIP) is a TRX inhibitory protein that works as a negative regulator in the TRX system. The function of TXNIP in invertebrates, in particular in immunity, remains unclear to date. In the current study, a novel TXNIP from Pacific white shrimp Litopenaeus vannamei was identified and characterized and its roles in immune responses was investigated. TXNIP could interact with Trx and inhibit its redox regulatory activity, suggesting that TXNIP was involved in regulation of the cellular redox state in shrimp. The expression of TXNIP was high in the stomach, gill, scape, eyestalk, epithelium, pyloric and muscle and low in the hepatopancreas, intestine, nerve, hemocytes and heart. Stimulations with pathogens white spot syndrome virus (WSSV) and Vibrio parahaemolyticus and immune stimulants poly (I:C) and LPS could significantly increase the expression of TXNIP in vivo. Silencing of TXNIP using RNAi strategy significantly facilitated the infection of V. parahaemolyticus but inhibited the infection of WSSV in shrimp. These indicated that TXNIP could be positively involved in antibacterial responses but negatively involved in antiviral responses in shrimp. Moreover, knockdown of TXNIP in vivo exerted opposite effects on expression of antimicrobial peptides anti-lipopolysaccharide factors and penaeidins and enhanced the phagocytic activity of hemocytes against bacteria. These suggested that TXNIP could play a complex role in regulation of humoral and cellular immune responses in shrimp.

Cloning and functional characterization of thioredoxin gene from kuruma shrimp Marsupenaeus japonicus.

As an important disulfide reductase of the intracellular antioxidant system, Thioredoxin (Trx) plays an important role in maintaining oxidative stress balance and protecting cells from oxidative damage. In recent years, there is increasing evidence that Trx is a key molecule in the pathogenesis of various diseases and a potential therapeutic target for major diseases including lung, colon, cervical, gastric and pancreatic cancer. However, few knowledge is known about the function of Trx in virus infection. In this study, we reported the cloning and functional investigation of a Trx homologue gene, named MjTrx, in shrimp Marsupenaeus japonicus suffered white spot syndrome virus (WSSV) infection. MjTrx is a 105-amino acid polypeptide with a conservative Cys-Gly-Pro-Cys motif in the catalytic center. Phylogenetic trees analysis showed that MjTrx has a higher relationship with Trx from other invertebrate and clustered with Trx1 from arthropod. MjTrx transcripts is abundant in the gill and intestine tissues and can be detected in the hemocytes, heart, stomach, and hepatopancreas tissues. The transcription levels of MjTrx in hemocytes, gills and intestine tissues of shrimp were significantly up-regulated after white spot syndrome virus infection. MjTrx was recombinant expressed in vitro and exhibited obvious disulfide reductase activity. In addition, overexpression MjTrx in shrimp resulted in the increase of hydrogen peroxide (H2O2) concentration in vivo. All these results strongly suggested that MjTrx functioned in redox homeostasis regulating and played an important role in shrimp antiviral immunity.

Identification of thioredoxin domain-containing protein 17 from big-belly seahorse Hippocampus abdominalis: Molecular insights, immune responses, and functional characterization.

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses.

Hypoxia potentiates LPS-induced inflammatory response and increases cell death by promoting NLRP3 inflammasome activation in pancreatic ß cells.

Hypoxia and islet inflammation are involved in ß-cell failure in type 2 diabetes (T2D). Elevated plasma LPS levels have been verified in patients with T2D, and hypoxia occurs in islets of diabetic mice. Activation of inflammasomes in ischemic or hypoxic conditions was identified in various tissues. Here, we investigated whether hypoxia activates the inflammasome in ß cells and the possible mechanisms involved. In mouse insulinoma cell line 6 (MIN6), hypoxia (1% O2) primes the NLRP3 inflammasome along with NF-κB signaling activation. Our results demonstrate that hypoxia can activate the NLRP3 inflammasome in LPS-primed MIN6 to result in initiating the ß cell inflammatory response and cell death in vitro. Reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) are up-regulated in response to hypoxia. Finally, the role of the ROS-TXNIP axis in mediating the activation of the NLRP3 inflammasome and cell death was characterized by pretreating with the ROS scavenger N-acetylcysteine (NAC) and performing TXNIP knockdown experiments in MIN6. Our data indicate for the first time that the inflammasome is involved in the inflammatory response and cell death in hypoxia-induced ß cells through the ROS-TXNIP-NLRP3 axis in vitro. This provides new insight into the relationship between hypoxia and inflammation in T2D.

Differential responses of a thioredoxin-like protein gene to Vibrio parahaemolyticus challenge in the noble scallop Chlamys nobilis with different total carotenoids content.

Being lack of specific immune system, both enzymes and non-enzymatic antioxidants play crucial roles in immune of invertebrates. In the present study, in order to investigate immune roles of enzyme (thioredoxin, TRX) and antioxidants (carotenoids), Golden scallops with golden shell and golden muscle rich in carotenoids content and Brown scallops with brown shell and white muscle less carotenoids content of the noble scallop Chlamys nobilis were challenged by Vibrio parahaemolyticus for 48 h. Firstly, a cDNA of TRX protein gene from the scallop (named as CnTRX) was cloned and characterized. The cDNA contains 1280 bp, consisting of a 5' -UTR of 99 bp, a long 3' -UTR of 860 bp and a 321 bp open reading frame (ORF) encoding 106 amino acids. Phylogenetic analysis showed that CnTRX had a closer evolution relationship with TRX from Chlamys farreri. CnTRX was ubiquitously expressed in all examined tissues including intestine, adductor, mantle, gonad, gill, kidney, hepatopancreas and hemolymph, and the highest expression level was detected in the hemolymph. Next, CnTRX transcripts were significantly up-regulated in V. parahaemolyticus group in comparison with PBS control group. Moreover, CnTRX transcripts were significantly higher in Golden scallops than that of Brown ones at 6 h, 12 h and 24 h with bacteria challenge (P < 0.05). The present result indicates that both CnTRX and carotenoids are important factors involved in the immune defense against bacteria challenge in the noble scallop.

Cloning and functional characterization of thioredoxin genes from large yellow croaker Larimichthys crocea.

Thioredoxin(Trx)with a redox-active disulfide/dithiol in the active site, is an ubiquitous disulfide reductase majorly responsible for maintaining the balance of reactive oxygen species. In this study, the complete thioredoxin-like protein 1 (designated as LcTrx) was cloned from large yellow croaker Larimichthys crocea through rapid amplification of cDNA ends. The full-length cDNA of LcTrx was 1295 bp in length containing a 131 bp 5' untranslated region (UTR) ,a 3'UTR of 294bp with a poly (A) tail, and an 870 bp open reading frame (ORF) encoding a polypeptide of 289 amino acids. Protein sequence analysis revealed that LcTrx contains the evolutionarily conserved redox motif CRPC (Cys-Arg-Pro-Cys-). Multiple alignments revealed that LcTrx is highly identical to Trx from other organisms, especially in the CRPC motifs. The recombinant LcTrx showed obvious insulin reduction activity in vitro. The LcTrx transcripts were constitutively expressed in all examined tissues with the highest levels found in the muscles and the lowest in the head kidney. Results of Vibrio parahaemolyticus infection experiment showed that the expression levels of LcTrx were tissue and time dependent. In the liver and kidney, LcTrx was down-regulated both at 12 h and 48 h post-infection. In contrast, LcTrx showed induced expression in the spleen and head kidney at same post-infection time points. The different responses to pathogen stimulation indicated the diversified physiological function of LcTrx in the four examined tissues.

Molecular characterization of thioredoxin-like protein 1 (TXNL1) from big-belly seahorse Hippocampus abdominalis in response to immune stimulation.

Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (ShTXNL1). Evolutionary identification showed that ShTXNL1 protein belongs to the thioredoxin superfamily comprising 289 amino acids. It possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of ShTXNL1 which is a component of 26S proteasome and binds to the matrix or cell. Pairwise alignment results showed 99.0% identity and 99.7% similarity with the sequence of Hippocampus species. Conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif may be found in the first few amino acids in the second beta sheet starting from the N-terminus. This motif can be discovered in ShTXNL1 as 14CRPC17 and comprised two N-linked glycosylation sites at 72NISA75 and 139NESD142. According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest ShTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of ShTXNL1 was observed in gill and blood tissues after bacterial stimuli. Thus, the ShTXNL1 gene may be identified as an immunologically important gene in seahorse.

Identification of the thioredoxin-related protein of 14 kDa (TRP14) from Litopenaeus vannamei and its role in immunity.

The thioredoxin system plays essential roles in maintenance and regulation of the redox state of cysteine residues in cellular proteins. The thioredoxin-related protein of 14 kDa (TRP14) is an important member of the TRX superfamily which acts on various substrate proteins, some of which are not overlapped with those of thioredoxin. The knowledge on the function of TRP14 in invertebrates is limited to date. In this study, a TRP14 gene was identified from Pacific white shrimp Litopenaeus vannamei (LvTRP14) and its role in immune responses was investigated. We demonstrated that the expression level of LvTRP14 was high in hepatopancreas and intestine, low in eyestalk, and medium in other tissues of healthy shrimp. The transcription of LvTRP14 in vivo was significantly down-regulated in Relish-silencing shrimp but up-regulated in STAT-silencing shrimp, indicating a complex regulation of LvTRP14 expression. Although the LvTRP14 expression showed little change after immune stimulation with different type of pathogens, knockdown of LvTRP14 expression using RNAi strategy could significantly facilitate the infection of white spot syndrome virus (WSSV) and Vibrio parahaemolyticus in shrimp. Dual luciferase reporter assays demonstrated that LvTRP14 enhanced the transcription factor activity of Relish but attenuated that of Dorsal. Furthermore, silencing of LvTRP14 in vivo had opposite effects on expression of different type of antimicrobial peptides. These suggested that LvTRP14 could play a complex role in shrimp immunity.

Upregulation of thioredoxin-1 in activated human NK cells confers increased tolerance to oxidative stress.

Adoptive transfer of immune cells, such as T lymphocytes and NK cells, has potential to control cancer growth. However, this can be counteracted by immune escape mechanisms within the tumor microenvironment, including those mediated by reactive oxygen species (ROS). Here, we determined the levels of anti-oxidant molecules in NK cells and their capacity to overcome ROS-induced immune suppression. We investigated the effect of H2O2 on resting NK cells, IL-2-activated NK cells and NK cells expanded by coculture with the K562 leukemia cell line genetically modified to express membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Expression of anti-oxidant and anti-apoptotic genes was evaluated by expression array, and protein levels of anti-oxidant molecules by Western blot. Activated NK cells, IL-2-activated NK cells and NK cells expanded by K562-mb15-41BBL were significantly more resistant to H2O2-induced cell death than resting NK. Thioredoxin-1 (TXN1) and peroxiredoxin-1 (PRDX1) were also up-regulated in activated NK cells. Moreover, H2O2-induced cell death after IL-2 activation was significantly induced in the presence of an anti-TXN1-neutralising antibody. Collectively, these data document that activated NK cells can resist to H2O2-induced cell death by up-regulation of TXN1.

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application.

BACKGROUND: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). RESULTS: The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. CONCLUSIONS: E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.

Protection and immunological study on two tetraspanin-derived vaccine candidates against schistosomiasis japonicum.

Tetraspanins (TSPs) are proteins found on the surface of helminth parasites of the genus Schistosoma and are regarded as potentially protective antigens. The large extracellular loop of Schistosoma mansoni tetraspanin-2, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. It is well recognized that CD4(+) T-cell-dependent immunity might play an important role against schistosomes; however, the contribution of CD8(+) T cells against multicellular pathogen is still uncertain. The exogenous protein-pulsed dendritic cells (DCs) can easily activate CD4(+) T cells response, while CD8(+) T cells response was relatively difficult to be induced. In this study, we evaluated the immunogenicity of TSP2HD antigen (hydrophilic domain of the S. japonicum tetraspanin-2) and TAT (the protein transduction domain of HIV-1)-coupled TSP2HD protein. As TAT-fused protein could promote major histocompatibility complex class I-dependent antigen presentation in vitro, TAT-TSP2HD-pulsed DCs induced stronger proliferation of schistosome-specific CD8(+) T cells compared with DCs incubated with TSP2HD alone. Vaccination with TAT-TSP2HD-pulsed DCs in vivo could improve disease outcome in S. japonicum-infected mice and was slightly superior to vaccination with DCs treated with TSP2HD. In summary, these data showed that TAT fusion proteins could help activate CD8(+) cells and Th1 cells and provide part protection against schistosome.

Nature and Implications of Oxidative and Nitrosative Stresses in Autoimmune Hepatitis.

Oxidative and nitrosative stresses can damage cellular membranes, disrupt mitochondrial function, alter gene expression, promote the apoptosis and necrosis of hepatocytes, and increase fibrosis in diverse acute and chronic liver diseases, including autoimmune hepatitis. The objectives of this review are to describe the mechanisms of oxidative and nitrosative stresses in inflammatory liver disease, indicate the pathogenic implications of these stresses in autoimmune hepatitis, and suggest investigational opportunities to develop interventions that counter them. The principal antioxidant defenses, including glutathione production, the activities of antioxidant enzymes, and the release of the nuclear factor erythroid 2-related factor 2, may be inadequate or suppressed by transforming growth factor beta. The generation of reactive oxygen species can intensify nitrosative stress, and this stress may not be adequately modulated by the thioredoxin-thioredoxin reductase system and induce post-translational modifications of proteins that further disrupt hepatocyte function. The unfolded protein response and autophagy may be unable to restore redox stability, meet metabolic demands, and maintain hepatocyte survival. Emerging interventions with highly selective site- and organelle-specific actions may improve outcomes, and they include inhibitors of nicotinamide adenine dinucleotide phosphate oxidase, nitric oxide synthase, and transforming growth factor beta. Pharmacological manipulation of nuclear transcription factors may favor expression of antioxidant genes, and stimulation of chaperone proteins within the endoplasmic reticulum and modulation of autophagy may prevent hepatic fibrosis and enhance cell survival. These interventions constitute investigational opportunities to improve the management of autoimmune hepatitis.