Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro.

BACKGROUND: Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. METHODS: Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1. CONCLUSIONS: Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.

Cysteine protease inhibitor of Schistosoma japonicum - A parasite-derived negative immunoregulatory factor.

Studies have shown that cysteine protease inhibitors from some parasites have immunosuppressive effects on the host. We previously have cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant version (protein named rSj-C). Its possible inhibitory effect on the host immune response has not been described.This study shows that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). After DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on the surface of DCs decreased significantly by flow cytometry. These results indirectly proved that rSj-C can suppress exogenous-antigen presentation by DCs. The flow cytometric assay revealed that in comparison with control groups, the proportion of CD4+CD25+Foxp3+ T cells among CD4+CD25+ T cells of Schistosom-infected mice increased significantly 8 weeks after the infected mice were injected with rSj-C (p Ë‚ 0.05). Additionally, the expression levels of cytokines IL-4 and TGF-ß produced by T cells increased significantly as compared with these levels in the normal group (p Ë‚ 0.05). These results clearly show that the cysteine protease inhibitor from S. japonicum is a new parasite-derived immunosuppressive factor.

Insight into the biochemical, kinetic and spectroscopic characterization of garlic (Allium sativum) phytocystatin: Implication for cardiovascular disease.

Phytocystatins are cysteine proteinase inhibitors present in plants. They play crucial role in maintaining protease-anti protease balance and are involved in various endogenous processes. Thus, they are suitable and convenient targets for genetic engineering which makes their isolation and characterisation from different sources the need of the hour. In the present study a phytocystatin has been isolated from garlic (Allium sativum) by a simple two-step process using ammonium sulphate fractionation and gel filtration chromatography on Sephacryl S-100HR with a fold purification of 152.6 and yield 48.9%. A single band on native gel electrophoresis confirms the homogeneity of the purified inhibitor. The molecular weight of the purified inhibitor was found to be 12.5kDa as determined by SDS-PAGE and gel filtration chromatography. The garlic phytocystatin was found to be stable under broad range of pH (6-8) and temperature (30°C-60°C). Kinetic studies suggests that garlic phytocystatins are reversible and non-competitive inhibitors having highest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy revealed significant conformational change upon garlic phytocystatin-papain complex formation. Secondary structure analysis was performed using CD and FTIR. Garlic phytocystatin possesses 33.9% alpha-helical content as assessed by CD spectroscopy.

Physico-chemical and in-silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host.

OBJECTIVES: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. MATERIALS AND METHODS: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. RESULTS: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). CONCLUSIONS: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response.

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.

Vaccination with a genetically modified Brugia malayi cysteine protease inhibitor-2 reduces adult parasite numbers and affects the fertility of female worms following a subcutaneous challenge of Mongolian gerbils (Meriones unguiculatus) with B. malayi infective larvae.

Vaccination of Mongolian gerbils with Brugia malayi cysteine protease inhibitor-2 in which the amino acid Asn66 was mutated to Lys66 (Bm-CPI-2M) resulted in reduced parasite numbers of 48.6% and 48.0% at 42 and 90 days p.i. with B. malayi L3s. Fertility of female worms was also affected at 90 days p.i. In vitro killing of L3s observed in the presence of gerbil peritoneal exudate cells and anti-Bm-CPI-2M sera suggests antibody-dependent cell-mediated cytotoxicity as a putative protective mechanism. These observations suggest that Bm-CPI-2M is a promising prophylactic and anti-fecundity vaccine candidate.

Modulation of dendritic cell function and immune response by cysteine protease inhibitor from murine nematode parasite Heligmosomoides polygyrus.

Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively and the cysteine protease inhibitor (CPI or cystatin) is identified as one of the major immunomodulators. In the present study, we cloned and produced recombinant CPI protein from the murine nematode parasite Heligmosomoides polygyrus (rHp-CPI) and investigated its immunomodulatory effects on dendritic cell (DC) function and immune responses in mice. Bone-marrow-derived CD11c(+) DC (BMDC) that were exposed to rHp-CPI during the differentiation stage showed reduced MHC-II molecule expression compared with BMDC that were generated in normal culture conditions. The BMDC generated in the presence of rHp-CPI also exhibited reduced expression of CD40, CD86 and MHC-II molecules and reduced interleukin-6 and tumour necrosis factor-α cytokine production when stimulated with Toll-like receptor ligand CpG. Activation of BMDC generated in normal conditions induced by lipopolysaccharide and CpG was also suppressed by rHp-CPI, as shown by reduced co-stimulatory molecule expression and cytokine production. Furthermore, BMDC treated with rHp-CPI before ovalbumin (OVA) antigen pulsing induced a weaker proliferation response and less interferon-γ production of OVA-specific CD4(+) T cells compared with BMDC without rHp-CPI pre-treatment. Adoptive transfer of rHp-CPI-treated and OVA-loaded BMDC to mice induced significantly lower levels of antigen-specific antibody response than the BMDC loaded with antigen alone. These results demonstrated that the CPI from nematode parasites is able to modulate differentiation and activation stages of BMDC. It also interferes with antigen and MHC-II molecule processing and Toll-like receptor signalling pathway, resulting in functionally deficient DC that induce a suboptimum immune response.

Quantitative salivary proteomic differences in oral chronic graft-versus-host disease.

PURPOSE: Chronic graft-versus-host disease (cGVHD) is a severe immunological complication that occurs after allogeneic hematopoietic stem cell transplantation (HSCT). Although oral cGVHD occurs in >25% of cGVHD patients and leads to decreased quality of life, its etiology is poorly understood. The present retrospective cross-sectional analysis of oral cGVHD patients sought to (1) test the feasibility of liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein biomarkers of oral cGVHD and (2) to gain a clearer understanding of salivary proteins impacted by oral cGVHD. METHODS: Using unstimulated whole saliva, we compared pooled saliva from five patients with a diagnosis of moderate or severe oral cGVHD, with a gender-and age- matched pool of five cGVHD patients with no oral mucosal findings. LC-MS/MS was used to identify salivary proteins, followed by Ingenuity Pathway Analysis (IPA). Selected mass spectrometric findings, including lactotransferrin, lactoperoxidase, and albumin, were confirmed by targeted label-free quantification. RESULTS: LC-MS/MS led to confident identification of 180 proteins. Of these proteins, 102 changed in abundance at least 2 fold, including 12 proteins identified only in the No oral cGVHD group. Downregulation of ~0.4 fold was confirmed for both lactotransferrin and lactoperoxidase in Oral cGVHD saliva using targeted label-free quantification. IPA analysis implicated pathways involved in cellular metabolism and immunoregulation. CONCLUSIONS: Reduction of salivary lactoperoxidase, lactotransferrin, and several cysteine proteinase inhibitor family proteins suggests impaired oral antimicrobial host immunity in cGVHD patients. This shotgun proteomic analysis of oral cGVHD saliva using targeted label-free quantification of select proteins supports the use of mass spectrometry for future validation in a large patient population as noninvasive tests for screening, early detection, and monitoring of cGVHD.

A helminth immunomodulator exploits host signaling events to regulate cytokine production in macrophages.

Parasitic worms alter their host's immune system to diminish the inflammatory responses directed against them, using very efficient immunomodulating molecules. We have previously shown that the helminth immunomodulator cystatin (AvCystatin) profoundly reduces the progression of inflammatory diseases via modulation of macrophages. Here we elucidate the signaling events in macrophages triggered by AvCystatin. Labeled AvCystatin was predominantly taken up by macrophages and subsequently induced the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38. IL-10 expression induced by AvCystatin in macrophages was tyrosine kinase sensitive and dependent on activation of both MAP kinases, in clear contrast to expression of IL-12/23p40. In addition, phosphorylation of the transcription factors CREB and STAT3 was induced by AvCystatin and regulated by phospho-ERK. Chemical inhibition of phosphoinositide 3-kinase (PI3K) reduced AvCystatin-induced cytokine release; however, AKT, the downstream target of PI3K, was not activated following AvCystatin exposure. To characterize signaling elements involved in alteration of the macrophage phenotype we applied mathematical modeling. Experimental testing of the in silico generated hypotheses identified dual specificity phosphatase (DUSP) 1 and 2, as regulators in AvCystatin triggered macrophages in vitro and in vivo. In particular, DUSP1 was subsequently found to be responsible for regulation of ERK- and p38-phosphorylation and controlled the IL-10 expression in macrophages by AvCystatin. Thus, we show that AvCystatin exploits activation and deactivation pathways of MAP kinases to induce regulatory macrophages. This study provides insights into molecular mechanisms of macrophage manipulation by parasites and highlights the utility of mathematical modeling for the elucidation of regulatory circuits of immune cells.

[Construction of eukaryotic recombinant plasmid of periodic Brugia malayi CPI gene and its immunity].

OBJECTIVE: To observe the immune responses elicited in BALB/c mice by DNA vaccine encoding cysteine protease inhibitor (CPI) of periodic Brugia malayi cloned in vector pcDNA3.1. METHODS: Specific primers were designed on the basis of known sequences of CPI gene from periodic B. malayi. The desired gene fragment was amplified by PCR from cDNA, inserted into cloning vector, pGEM-T, and sub-cloned into pcDNA3.1 to construct pcDNA3.1-BmCPI Forty-eight mice were randomly divided into 4 groups, i.e. normal control group, pcDNA3.1(+) group, pcDNA3.1-BmCPI group, and pcDNA3.1-BmCPI/CpG group injected with PBS 100 microl, pcDNA3.1 100 microg, pcDNA3.1-BmCPI 100 microg and pcDNA3.1-BmCPI 100 microg+CpG 30 microg, respectively on left hind leg of each mouse. All mice received three immunizations with 2-week interval. At the 4th week after the last immunization the muscle around injection spot was collected, in which the level of BmCPI mRNA was detected by RT-PCR. The stimulation index (SI) of spleen lymphocytes was measured by MTI method and the levels o f secreted IL-4 and IFN-gamma in serum were detected by ELISA. RESULTS: The recombinant plasmid pcDNA3.1-BmCPI was constructed and the length of the gene fragment was 621 bp. The results showed that BmCPI gene in the muscle of the immunized mice was detected by PCR. At the 4th and 6th weeks after immunization, the SI of the two immunized groups was significantly higher than normal control group and pcDNA3.1(+) group (53.789 +/- 1.937, 59.735 +/- 4.139, and 61.975 +/- 1.029) (P < 0.05). No significant difference existed between pcDNA3.1BmCPI group and pcDNA3.1-BmCPI/CpG group (P > 0.05). Serum IFN-gamma in pcDNA3.1-BmCPI group and pcDNA3.1-BmCPI/ CpG group increased from the 2nd to the 6th week after the last immunization with the value of 69.544 +/- 3.145 and 106.069 +/- 7.518, 120.019 +/- 5.968 and 136.229 +/- 7.198, 149.109 +/- 2.700 and 178.429 +/- 1.126, respectively. The levels of IFN-gamma in serum from the immunized mice were significantly higher than those of normal control group and pcDNA3.1(+) group (28.264 +/- 1.129, 35.179 +/- 1.029, and 40.110 +/- 1.176, respectively) (P < 0.05). There was a significant difference between the two immunized groups at the 2nd and the 6th weeks after the last immunization (P < 0.05). The level of IL-4 in serum from the immunized mice was significantly higher than those of normal control group and pcDNA3.1(+) group at the 4th and the 6th weeks after the last immunization (P < 0.05). No significant difference was noted in IL-4 level between pcDNA3.1-BmCPI group and pcDNA3.1-BmCPI/CpG group (P > 0.05). CONCLUSION: The recombinant eukaryotic plasmid pcDNA3.1-BmCPI was transcribed in vivo and elicited immune responses in mice.

The Onchocerca volvulus cysteine proteinase inhibitor, Ov-CPI-2, is a target of protective antibody response that increases with age.

BACKGROUND: Despite considerable efforts, a suitable vaccine against Onchocerca volvulus infection has remained elusive. Herein, we report on the use of molecular tools to identify and characterize O. volvulus antigens that are possibly associated with the development of concomitant immunity in onchocerciasis. METHODOLOGY/PRINCIPAL FINDINGS: Third-stage larvae (L3) and molting L3 (mL3) O. volvulus stage-specific cDNA libraries were screened with a pool of sera from chronically infected patients who had likely developed such immunity. The 87 immunoreactive clones isolated were grouped into 20 distinct proteins of which 12 had already been cloned and/or characterized before and 4 had been proven to be protective in a small O. volvulus animal model. One of these, onchocystatin (Ov-CPI-2), a previously characterized O. volvulus cysteine proteinase inhibitor was, overall, the most abundant clone recognized by the immune sera in both the L3 and mL3 cDNA libraries. To further characterize its association with protective immunity, we measured the IgG subclass and IgE class specific responses to the antigen in putatively immune (PI) and infected (INF) individuals living in a hyperendemic area in Cameroon. It appeared that both groups had similar IgG3 and IgE responses to the antigen, but the INF had significantly higher IgG1 and IgG4 responses than the PI individuals (p<0.05). In the INF group, the IgG3 levels increased significantly with the age of the infected individuals (r = 0.241; p<0.01). The IgG1 responses in the INF were high regardless of age. Notably, culturing L3 in vitro in the presence of anti-Ov-CPI-2 monospecific human antibodies and naïve neutrophils resulted in almost complete inhibition of molting of L3 to L4 and to cytotoxicity to the larvae. CONCLUSIONS/SIGNIFICANCE: These results add to the knowledge of protective immunity in onchocerciasis and support the possible involvement of anti-Ov-CPI-2 IgG1 and/or IgG3 cytophilic antibodies in the development of protective immunity in the PI and the INF. The results further support the consideration of Ov-CPI-2 as a leading target for an anti-L3 vaccine.

Autoantibody against survivin in patients with systemic sclerosis.

OBJECTIVE: In patients with systemic sclerosis (SSc), to determine concentrations of antibodies against survivin and their clinical association with SSc, and to evaluate serum survivin concentrations. METHODS: Anti-survivin antibody was examined by ELISA and immunoblotting using human recombinant survivin. Serum survivin levels were assessed by ELISA. RESULTS: IgG but not IgM anti-survivin antibody levels in patients with SSc were significantly higher than those in healthy controls and patients with systemic lupus erythematosus (SLE). When cutoff values were set as mean + 2 SD of control, IgG anti-survivin antibodies were positive in 41% (25/61) of patients with SSc, while they were detected in only 1 healthy individual (3%, 1/29) and 1 patient with SLE (5%, 1/20). Regarding the clinical correlation, patients with SSc who were positive for IgG anti-survivin antibody exhibited significantly longer disease duration than those who were negative. Immunoblotting analysis confirmed the presence of anti-survivin antibody in sera from patients with SSc. Serum survivin levels in patients with SSc were also significantly higher than in controls and patients with SLE. CONCLUSION: Our results suggest that autoantibody against survivin is generated in patients with SSc, especially those with long disease duration.

Cysteine proteinase inhibitor Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.

Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to 11 IUIS allergens, with Act d 1, Act d 2 and Act d 5 characterized in extenso. Evaluation of the allergenic properties of Act d 4, a cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa) was performed in this study. Identity of the purified glycoprotein was determined by Edman degradation and by mass fingerprint whereby more than 90% of the primary structure of the mature kiwifruit cystatin was confirmed. Using MALDI TOF analysis, molecular masses of 10902.5 and 11055.2 Da were detected for Act d 4, respectively. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as the upregulation of CD63 and CD203c molecules in the basophile activation assay. The IgE reactivity was detected in dot blot analysis while Western blot analysis was negative using sera from six kiwifruit patients, suggesting the presence of conformational IgE epitopes on the Act d 4 molecule. As activator of effector cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis.

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.

A nonsynonymous substitution of cystatin A, a cysteine protease inhibitor of house dust mite protease, leads to decreased mRNA stability and shows a significant association with atopic dermatitis.

BACKGROUND: Cystatin A (CSTA) is a strong candidate for atopic dermatitis (AD) because it maps to AD susceptibility locus on chromosome 3q21 and it does inhibit Der p 1 and Der f 1, major house dust mite cysteine proteases and environmental triggers for AD and asthma. OBJECTIVE: To examine any association between polymorphisms in CSTA and AD and study the effect on the CSTA mRNA expression level. METHODS: We identified three polymorphisms and characterized the linkage disequilibrium mapping of the CSTA gene. All three CSTA polymorphisms were genotyped in 100 AD patients and 203 matched controls. Subsequently, we performed transfection-based RNA stability assays. RESULTS: We found a significant association between the CSTA +344C variant and AD [odds ratio (OR) = 1.91; P = 0.024]. When further 61 control samples were genotyped. The association with CSTA +344C allele was enhanced OR = 2.13; P = 0.006. To test whether the CSTA +344 affected the CSTA transcriptional activity, the decay rates of RNAs transcribed from the CSTA +344C and CSTA +344T variants were investigated. COS-7 cells were transfected with a pcDNA3.1-CSTA+344C or a pcDNA3.1-CSTA+344T construct and cultured in the presence or absence of actinomycin D. Real-time RT-PCR revealed that CSTA +344C mRNA is more than two times less stable than the CSTA +344T mRNA (P < 0.001). CONCLUSION: These results suggest that the CSTA +344C allele associated with unstable mRNA could result in failing to protect the skin barrier in AD patients from both exogenous and endogenous proteases.

Microinjection of an antibody against the cysteine-protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle.

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development.

Follicular dendritic cell immunohistochemical markers in angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma is characterized by a paracortical proliferation of medium to large neoplastic T cells, often with clear cytoplasm, in a background of arborizing high endothelial venules, many surrounded by follicular dendritic cells (FDCs). IHC staining may be applied to highlight these extrafollicular FDCs, traditionally using CD21, or CD23. Several alternative FDC markers have been described, including CNA.42, cystatin A/acid cysteine proteinase inhibitor (ACPI, involved in antigen presentation), and fascin (an actin binding protein). The authors stained a collection of 45 angioimmunoblastic T-cell lymphomas with CD21, CD23, CNA.42, cystatin A, and fascin for direct comparison of FDC staining characteristics in this setting. CD21 highlighted the expected dendritic network of cell processes, within residual follicles and outside of follicles, often adjacent to proliferating vessels. CD23 exhibited similar staining quality but was less sensitive than CD21. CNA.42 showed only diffuse weak labeling of FDCs. Cystatin A stained the cytoplasm of follicular dendritic cells within and outside of follicles; however, staining was often not sharply localized to dendritic cell processes, and scoring was further complicated by reactivity with other cell types in over half of the cases. Likewise, fascin stained a variety of cell types, including strong staining of interdigitating dendritic-like cells, moderate staining of endothelial cells, and only weak staining of follicular dendritic cells within and outside of follicles. Thus, CD21 remains the most reliable marker of follicular dendritic cells in angioimmunoblastic T-cell lymphoma.