Cystatins 9 and C as a Novel Immunotherapy Treatment That Protects against Multidrug-Resistant New Delhi Metallo-Beta-Lactamase-1-Producing Klebsiella pneumoniae.

Multidrug-resistant (MDR) bacterial pneumonia can induce dysregulated pulmonary and systemic inflammation leading to morbidity and mortality. Antibiotics to treat MDR pathogens do not function to modulate the extent and intensity of inflammation and can have serious side effects. Here we evaluate the efficacy of two human cysteine proteinase inhibitors, cystatin 9 (CST9) and cystatin C (CSTC), as a novel immunotherapeutic treatment to combat MDR New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae Our results showed that mice infected intranasally (i.n.) with a 90% lethal dose (LD90) challenge of NDM-1 K. pneumoniae and then treated with the combination of human recombinant CST9 (rCST9) and rCSTC (rCSTs; 50 pg of each i.n. at 1 h postinfection [p.i.] and/or 500 pg of each intraperitoneally [i.p.] at 3 days p.i.) had significantly improved survival compared to that of infected mice alone or infected mice treated with individual rCSTs (P < 0.05). Results showed that both of our optimal rCST treatment regimens modulated pulmonary and systemic proinflammatory cytokine secretion in the serum, lungs, liver, and spleen in infected mice (P < 0.05). Treatment also significantly decreased the bacterial burden (P < 0.05) while preserving lung integrity, with reduced inflammatory cell accumulation compared to that in infected mice. Further, rCST treatment regimens reduced lipid peroxidation and cell apoptosis in the lungs of infected mice. Additionally, in vitro studies showed that rCSTs (50 or 500 pg of each) directly decreased the viability of NDM-1 K. pneumoniae In conclusion, the data showed that rCST9/rCSTC worked synergistically to modulate host inflammation against MDR NDM-1 K. pneumoniae pneumonia, which significantly improved survival. Therefore, rCST9/rCSTC is a promising therapeutic candidate for the treatment of bacterial pneumonia.

Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.

[Protective effect of recombinant Schistosoma japonicum cystatin against acute kidney injury associated with acute liver failure in mice].

OBJECTIVE: To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury. METHODS: Twenty-four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj-Cys control group, lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) model group and LPS/D-GaIN + rSj-Cys treatment group, of 6 mice each group. Mice in the LPS/D-GaIN group and LPS/D-GaIN + rSj-Cys group were intraperitoneally injected with LPS (10 µg/kg) and D-GaIN (700 mg/kg), and mice in the LPS/D-GaIN + rSj-Cys group were additionally administered with rSj-Cys (1.25 mg/kg) by intraperitoneal injection 30 min post-modeling, while mice in the rSj-Cys group were intraperitoneally injected with rSj-Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post-modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin-eosin (HE) staining. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were detected using enzyme-linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis-related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis-related proteins and nuclear factor-kappa B (NF-κB) signaling pathway-associated proteins was determined in mouse kidney specimens using Western blotting assay. RESULTS: HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj-Cys control group, and renal tubular injury was found in LPS/D-GaIN group, while the renal tubular injury was alleviated in LPS/D-GaIN+rSj-Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF-α (F = 102.00, P < 0.001) and IL-6 (F = 202.10, P < 0.001) among the four groups, and lower serum Cr [(85.35 ± 32.05) µmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF-α [(158.27 ± 15.83) pg/mL] and IL-6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF-α (F = 24.16, P < 0.001) and IL-10 (F = 15.07, P < 0.01) expression among the four groups, and lower TNF-α [(106.50 ± 16.57)%] and higher IL-10 expression [(91.83 ± 5.23)%] was detected in the LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL-1ß (F = 19.24 and 22.59, both P values < 0.001) and IL-18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL-1ß and IL-18 expression was quantified in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF-κB signaling pathway-associated proteins p-NF-κB p-P65/NF-κB p65 (F = 71.88, P < 0.001), Toll-like receptor (TLR)-4 (F = 45.49, P < 0.001) and p-IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF-κB signaling pathway-associated proteins was determined in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (all P values < 0.01). CONCLUSIONS: rSj-Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF-κB signaling pathway.

Cystatin cures visceral leishmaniasis by NF-κB-mediated proinflammatory response through co-ordination of TLR/MyD88 signaling with p105-Tpl2-ERK pathway.

Cystatin could completely cure experimental visceral leishmaniasis by switching the differentiation of Th2 cells to Th1 type, as well as upregulating NO, and activation of NF-κB played a major role in these processes. Analysis of upstream signaling events revealed that TLR 2/4-mediated MyD88-dependent participation of IL-1R-activated kinase (IRAK)1, TNF receptor-associated factor (TRAF)6 and TGFß-activated kinase (TAK)1 is essential to induce cystatin-mediated IκB kinase (IKK)/NF-κB activation in macrophages. Cystatin plus IFN-γ activated the IKK complex to induce phosphorylation-mediated degradation of p105, the physiological partner and inhibitor of the MEK kinase, tumor progression locus 2 (Tpl-2). Consequently, Tpl-2 was liberated from p105, thereby stimulating activation of the MEK/ERK MAPK cascade. Cystatin plus IFN-γ-induced IKK-ß post-transcriptionally modified p65/RelA subunit of NF-κB by dual phosphorylation in infected phagocytic cells. IKK induced the phosphorylation of p65 directly on Ser-536 residue whereas phosphorylation on Ser 276 residue was by sequential activation of Tpl-2/MEK/ERK/MSK1. Collectively, the present study indicates that cystatin plus IFN-γ-induced MyD88 signaling may bifurcate at the level of IKK, leading to a divergent pathway regulating NF-κB activation by IκBα phosphorylation and by p65 transactivation through Tpl-2/MEK/ERK/MSK1.

New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action.

OBJECTIVE: A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). METHODOLOGY: In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. RESULTS: Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). CONCLUSIONS: Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.

Trichinella spiralis cystatin alleviates polymicrobial sepsis through activating regulatory macrophages.

BACKGROUND: Sepsis is a life-threateningorgandysfunction caused by the cytokine storm induced by the severe bacterial infection. Excessive inflammatory responses are responsible for the lethal organ damage during the early stage of sepsis. Helminth infection and helminth-derived proteins have been identified to have the ability to immunomodulate the host immune system by reducing inflammation against inflammatory diseases. Trichinella spiralis cystatin (Ts-Cys) is a cysteine protease inhibitor with strong immunomodulatory functions on host immune system. Our previous studies have shown that excretory-secretory proteins of T. spiralis reduced sepsis-induced inflammation and Ts-Cys was able to inhibit macrophages to produce inflammatory cytokines. Whether Ts-Cys has a therapeutic effect on polymicrobial sepsis and related immunological mechanism are not yet known. METHODS: Sepsis was induced in BALB/c mice using cecal ligation and puncture (CLP), followed by intraperitoneal injection of 15 µg recombinant Ts-Cys (rTs-Cys). The therapeutic effect of rTs-Cys on sepsis was evaluated by observing the 72-hour survival rates of CLP-induced septic mice and the acute injury of lung and kidney through measuring the wet/dry weight ratio of lung, the levels of blood urea nitrogen (BUN) and creatinine (Cr) in sera and the tissue section pathology. The potential underlying mechanism was investigated using mouse bone marrow-derived macrophages (BMDMs) by observing the effect of rTs-Cys on LPS-stimulated macrophage polarization. The expression of genes associated with macrophage polarization in BMDMs and tissues of septic mice was measured by Western Blotting and qPCR. RESULTS: In this study, we demonstrated the treatment with rTs-Cys alleviated CLP-induced sepsis in mice with significantly reduced pathological injury in vital organs of lung and kidney and reduced mortality of septic mice. The further study identified that treatment with rTs-Cys promoted macrophage polarization from classically activated macrophage (M1) to alternatively activated macrophage (M2) phenotype via inhibiting TLR2/MyD88 signal pathway and increasing expression of mannose receptor (MR), inhibited pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) and increased regulatory anti-inflammatory cytokines (IL-10 and TGF-ß) in sera and tissues (lung and kidney) of mice with polymicrobial sepsis. CONCLUSIONS: Our results demonstrated that rTs-Cys had a therapeutic effect on sepsis through activating regulatory macrophages possibly via suppressing TLR2/MyD88 signal pathway. We also identified that rTs-Cys-induced M2 macrophage differentiation was associated with increased expression of MR on the surface of macrophages. Our results underscored the importance of MR in regulating macrophages during the treatment with rTs-Cys, providing another immunological mechanism in which helminths and their derived proteins modulate the host immune system. The findings in this study suggest that rTs-Cys is a potential therapeutic agent for the prevention and treatment of sepsis and other inflammatory diseases.

Recombinant sugarcane cystatin CaneCPI-5 down regulates inflammation and promotes angiogenesis and collagen deposition in a mouse subcutaneous sponge model.

Cystatins are natural inhibitors of cysteine peptidases that are found practically in all living organisms. CaneCPI-5 is a sugarcane cystatin with inhibitory activity against human cathepsins B, K and L, which are cysteine proteases highly expressed in a variety of pathological conditions, usually marked by persistent inflammation and processing of the extracellular matrix. This work evaluated the effects of daily administration of the recombinant cystatin CaneCPI-5 [0.01, 0.1 or 1.0 µg in 10 µL of Phosphate-Buffered Saline (PBS)] on the inflammatory, angiogenic and fibrogenic components during chronic inflammatory response induced by subcutaneous sponge implants. The anti-inflammatory effect of treatment with CaneCPI-5 was confirmed by reduction of the levels of the pro-inflammatory mediators TNF-α, CXCL1 and CCL2/JE/MCP-1, as well as the activity of the myeloperoxidase and n-acetyl-ß-D-glucosaminidase. Treatment with CaneCPI-5 promoted angiogenesis in the implants, increasing the production of cytokines VEGF and FGF and the formation of new blood vessels. Finally, the administration of the recombinant cystatin favored the production of the pro-fibrogenic cytokine TGF-ß1 and collagen deposition next to the implants. Together, these results show the potential therapeutic application of CaneCPI-5 as an anti-inflammatory agent, capable of favoring angiogenesis and fibrogenesis processes, necessary for tissue repair.

Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: involvement of ERK1/2, NF-kappaB and JAK/STAT pathways.

Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-gamma in inducing ERK1/2 phosphorylation and NF-kappaB DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-kappaB or ERK1/2 pathway blocked the cystatin plus IFN-gamma-inducible NF-kappaB activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-kappaB-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-gamma-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-kappaB, including nuclear translocation of NF-kappaB subunits, IkappaB phosphorylation and IkappaB kinase, are all remarkably enhanced in Leishmania-infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general.

Therapeutic efficacy of Schistosoma japonicum cystatin on sepsis-induced cardiomyopathy in a mouse model.

BACKGROUND: Myocardial dysfunction is one of the most common complications of multiple organ failure in septic shock and significantly increases mortality in patients with sepsis. Although many studies having confirmed that helminth-derived proteins have strong immunomodulatory functions and could treat inflammatory diseases, there is no report on the therapeutic effect of Schistosoma japonicum-produced cystatin (Sj-Cys) on sepsis-induced cardiac dysfunction. METHODS: A model of sepsis-induced myocardial injury was established by cecal ligation and puncture (CLP) in mice. Upon CLP operation, each mouse was intraperitoneally treated with 10 µg of recombinant Sj-Cys (rSj-Cys). Twelve hours after CLP, the systolic and diastolic functions of the left ventricular were examined by echocardiography. The levels of myoglobin (Mb), cardiac troponin I (cTnI), N-terminal pro-Brain Natriuretic peptide (NT-proBNP) in sera, and the activity of myeloperoxidase (MPO) in cardiac tissues were examined as biomarkers for heart injury. The heart tissue was collected for checking pathological changes, macrophages and pro-inflammatory cytokine levels. To address the signaling pathway involved in the anti-inflammatory effects of rSj-Cys, myeloid differentiation factor 88 (MyD88) was determined in heart tissue of mice with sepsis and LPS-stimulated H9C2 cardiomyocytes. In addition, the therapeutic effects of rSj-Cys on LPS-induced cardiomyocyte apoptosis were also detected. The levels of M1 biomarker iNOS and M2 biomarker Arg-1 were detected in heart tissue. The pro-inflammatory cytokines TNF-α and IL-6, and regulatory cytokines IL-10 and TGF-ß were measured in sera and their mRNA levels in heart tissue of rSj-Cys-treated mice. RESULTS: After rSj-Cys treatment, the sepsis-induced heart malfunction was largely improved. The inflammation and injury of heart tissue were significantly alleviated, characterized as significantly decreased infiltration of inflammatory cells in cardiac tissues and fiber swelling, reduced levels of Mb, cTnI and NT-proBNP in sera, and MPO activity in heart tissue. The therapeutic efficacy of rSj-Cys is associated with downregulated pro-inflammatory cytokines (TNF-α and IL-6) and upregulated regulatory inflammatory cytokines (IL-10 and TGF-ß), possibly through inhibiting the LPS-MyD88 signal pathway. CONCLUSIONS: RSj-Cys significantly reduced sepsis-induced cardiomyopathy and could be considered as a potential therapeutic agent for the prevention and treatment of sepsis associated cardiac dysfunction.

[The statin dosage for achieving goal of cholesterol-lowering based on risk stratification in patients with ischemic cerebrovascular diseases].

OBJECTIVE: To explore statin dosages for targeting goal of LDL-C lowering on the basis of stroke risk stratification and the dosage-effective relation of statin and LDL-C lowering in Chinese patients with ischemic stroke and transient ischemic attack (TIA). METHODS: This is a prospective and open clinical trial patients with ischemic stroke/TIA within 6 months were enrolled and the dosages of atorvastatin were calculated based on risk stratification according to "Chinese Consensus for Prevention of Ischemic Stroke/TIA with Statin" (Chinese Consensus). A dose of 10 mg of atorvastatin daily to target LDL-C goal was taken as the standard dosage targeting goal (SDTG). Patients taking this dosage of atorvastatin constituted a SDTG group. Those who needed a daily dose of 20 mg or more of atorvastatin were randomized into an intensive dosage targeting goal (IDTG) group (atorvastatin 20 - 80 mg/d) and a standard dosage non-targeting goal (SDNTG) group (atorvastatin 10 mg/d without targeting goal). All patients took atorvastatin for 12 weeks. The primary outcome was the rate of targeting goal for LDL-C lowering at 2, 4 and 12 weeks, respectively and the secondary outcome was the occurence of recurrent stroke and other vascular events within 12 weeks. The main safety endpoint was serial adverse events including symptomatic intracranial hemorrhage. RESULTS: Altogether 102 cases were enrolled and 99 cases were followed up for 12 weeks. According to the Chinese Consensus, the rate of high risk, very high risk-I and very high risk-II was 44%, 28% and 28%, respectively. Targeting rate for LDL-C lowering was 77% - 85% at each time point in the SDTG and IDTG groups, being significantly higher than those in the SDNTG group (12% - 16%, P < 0.01). No significant difference was found concerning the occurrence of recurrent stroke, other vascular events and safety endpoints among the three groups. The amplitude of LDL-C lowering was 32% - 35%, 46% - 49%, 51% - 52% and 60% - 65% with corresponding to daily dosage of 10 mg, 20 mg, 40 mg and 80 mg atorvastatin. CONCLUSIONS: At least more than half of the patients after ischemic stroke/TIA need intensive statin therapy to target the LDL-C lowering goal. The dosage-effective relation of atorvastatin and LDL-C lowering in Chinese is similar to the reported data in other races.

Cystatins: biochemical and structural properties, and medical relevance.

The cystatin superfamily comprises a large group of the cystatin domain containing proteins, present in a wide variety of organisms, including humans. Cystatin inhibitory activity is vital for the delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases such as the papain (C1) family, including cysteine cathepsins. Some of the cystatins also inhibit the legumain (C13) family of enzymes. Failures in biological mechanisms controlling protease activities result in many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis, arthritis, and cancer. Cystatins have been classified into three types: the stefins, the cystatins and the kininogens, although other cystatin-related proteins, such as CRES proteins, are emerging. The stefins are mainly intracellular proteins, whereas the cystatins and the kininogens are extracellular. The cystatins are tight binding and reversible inhibitors. The basic mechanism of interaction between cystatins and their target proteases has been established, based mainly on the crystal structures of various cathepsins, stefins and cystatins and their enzyme-inhibitor complexes. Cystatins, as rather non-selective inhibitors, discriminate only slightly between endo- and exopeptidases. They are also prone to form amyloids. The levels of some stefins and cystatins in tissue and body fluids can serve as relatively reliable markers for a variety of diseases. In this review we summarize present knowledge about cystatins and their role in some diseases.

[Effect of cystatin from Schistosoma japonicum on DSS - induced ulcerative colitis in mice].

OBJECTIVE: To investigate the effect of cysteine protease inhibitor derived from Schistosoma japonicum (SjCystatin) on dextran sodium sulfate (DSS)-induced acute ulcerative colitis in mice. METHODS: Eighteen C57BL/6 mice were randomly divided into three groups: a control group treated with PBS (Group A), a DSS-induced-colitis group treated with PBS (Group B), and a DSS-induced-colitis group treated with SjCystatin (Group C). Colitis was induced in mice by giving 3% DSS orally for 7 days. During this period, the mice were daily injected with 10 µg of SjCystatin or PBS only as a control intraperitoneally. The mice were monitored daily for their clinical manifestations and given scores based on disease activity index (DAI). The severity of colonic inflammation was monitored by the macroscopic score and pathological change. The cytokine profile including TNF-α, IL-4, IL-6 and IL-10 in the supernatants of colon homogenate was detected by ELISA. RESULTS: Compared with Group A (0.50 ± 0.28), the DAI score increased significantly in Group B (9.30 ± 1.30) (F = 86.86, P < 0.01), with remarkable pathological damages seen in colon tissues. and the levels of TNF-α and IL-6 were (321.33±67.01) and (403.58 ±180.51) pg/mL. The DAI score significantly reduced in Group C (6.67±1.57) as compared to Group B (F = 86.86, P < 0.01), with improvements in the macroscopic and microscopic pathology in mouse colon specimens. As compared to Group B, the levels of TNF-α [(188.14 ± 40.14) pg/mL] and IL-6 ([ 209.71 ± 48.47) pg/mL] significantly decreased (F = 17.46 and 9.89, both P < 0.01). CONCLUSIONS: SjCystatin has a significantly inhibitory effect for alleviating DSS-induced acute ulcerative colitis in C57BL/6 mice.

Protective effect of recombinant Schistosoma japonicum cystatin against acute kidney injury associated with acute liver failure in mice / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the protective effect of recombinant Schistosoma japonicum cystatin (rSj-Cys) against acute kidney injury induced by acute liver failure and unravel the underlying mechanism, so as to provide insights into the clinical therapy of acute kidney injury.@*METHODS@#Twenty-four male C57BL/6J mice at ages of 6 to 8 weeks were randomly divided into the normal control group, rSj-Cys control group, lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) model group and LPS/D-GaIN + rSj-Cys treatment group, of 6 mice each group. Mice in the LPS/D-GaIN group and LPS/D-GaIN + rSj-Cys group were intraperitoneally injected with LPS (10 μg/kg) and D-GaIN (700 mg/kg), and mice in the LPS/D-GaIN + rSj-Cys group were additionally administered with rSj-Cys (1.25 mg/kg) by intraperitoneal injection 30 min post-modeling, while mice in the rSj-Cys group were intraperitoneally injected with rSj-Cys (1.25 mg/kg), and mice in the normal control group were injected with the normal volume of PBS. All mice were sacrificed 6 h post-modeling, and mouse serum and kidney samples were collected. Serum creatinine (Cr) and urea nitrogen (BUN) levels were measured, and the pathological changes of mouse kidney specimens were examined using hematoxylin-eosin (HE) staining. Serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were detected using enzyme-linked immunosorbent assay (ELISA), and the expression of inflammatory factors and pyroptosis-related proteins was quantified in mouse kidney specimens using immunohistochemistry. In addition, the expression of pyroptosis-related proteins and nuclear factor-kappa B (NF-κB) signaling pathway-associated proteins was determined in mouse kidney specimens using Western blotting assay.@*RESULTS@#HE staining showed no remarkable abnormality in the mouse kidney structure in the normal control group and the rSj-Cys control group, and renal tubular injury was found in LPS/D-GaIN group, while the renal tubular injury was alleviated in LPS/D-GaIN+rSj-Cys treatment group. There were significant differences in serum levels of Cr (F = 46.33, P < 0.001), BUN (F = 128.60, P < 0.001), TNF-α (F = 102.00, P < 0.001) and IL-6 (F = 202.10, P < 0.001) among the four groups, and lower serum Cr [(85.35 ± 32.05) μmol/L], BUN [(11.90 ± 2.76) mmol/L], TNF-α [(158.27 ± 15.83) pg/mL] and IL-6 levels [(56.72 ± 4.37) pg/mL] were detected in the in LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (all P values < 0.01). Immunohistochemical staining detected significant differences in TNF-α (F = 24.16, P < 0.001) and IL-10 (F = 15.07, P < 0.01) expression among the four groups, and lower TNF-α [(106.50 ± 16.57)%] and higher IL-10 expression [(91.83 ± 5.23)%] was detected in the LPS/D-GaIN + rSj-Cys group than in the LPS/D-GaIN group (both P values < 0.01). Western blotting and immunohistochemistry detected significant differences in the protein expression of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (F = 24.57 and 30.72, both P values < 0.001), IL-1β (F = 19.24 and 22.59, both P values < 0.001) and IL-18 (F = 16.60 and 19.30, both P values < 0.001) in kidney samples among the four groups, and lower NLRP3, IL-1β and IL-18 expression was quantified in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (P values < 0.05). In addition, there were significant differences in the protein expression of NF-κB signaling pathway-associated proteins p-NF-κB p-P65/NF-κB p65 (F = 71.88, P < 0.001), Toll-like receptor (TLR)-4 (F = 45.49, P < 0.001) and p-IκB/IκB (F = 60.87, P < 0.001) in mouse kidney samples among the four groups, and lower expression of three NF-κB signaling pathway-associated proteins was determined in the LPS/D-GaIN + rSj-Cys treatment group than in the LPS/D-GaIN group (all P values < 0.01).@*CONCLUSIONS@#rSj-Cys may present a protective effect against acute kidney injury caused by acute liver failure through inhibiting inflammation and pyroptosis and downregulating the NF-κB signaling pathway.

Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction.

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

Therapeutic effect of Schistosoma japonicum cystatin on bacterial sepsis in mice.

BACKGROUND: Sepsis is a life-threatening complication of an infection and remains one of the leading causes of mortality in surgical patients. Bacteremia induces excessive inflammatory responses that result in multiple organ damage. Chronic helminth infection and helminth-derived materials have been found to immunomodulate host immune system to reduce inflammation against some allergic or inflammatory diseases. Schistosoma japonicum cystatin (Sj-Cys) is a cysteine protease inhibitor that induces regulatory T-cells and a potential immunomodulatory. The effect of Sj-Cys on reducing sepsis inflammation and mortality was investigated. METHODS: Sepsis was induced in BALB/c mice using cecal ligation and puncture (CLP), followed by intraperitoneal injection of different doses (10, 25 or 50 µg) of recombinant Sj-Cys (rSj-Cys). The therapeutic effect of rSj-Cys on sepsis was evaluated by observing the survival rates of mice for 96 h after CLP and the pathological injury of liver, kidney and lung by measuring the levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in sera and the tissue sections pathology, and the expression of MyD88 in liver, kidney and lung tissues. The immunological mechanism was investigated by examining pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß) and IL-10 and TGF-ß1 in mice sera and in culture of macrophages stimulated by lipopolysaccharides (LPS). RESULTS: rSj-Cys treatment provided significant therapeutic effects on CLP-induced sepsis in mice demonstrated with increased survival rates, alleviated overall disease severity and tissue injury of liver, kidney and lung. The rSj-Cys conferred therapeutic efficacy was associated with upregualted IL-10 and TGF-ß1 cytokines and reduced pro-inflammatory cytokines TNF-α, IL-6, IL-1ß. MyD88 expression in liver, kidney and lung tissues of rSj-Cys-treated mice was reduced. In vitro assay with macrophages also showed that rSj-Cys inhibited the release of pro-inflammatory cytokines and mediator nitric oxide (NO) after being stimulated by lipopolysaccharide (LPS). CONCLUSIONS: The results suggest the anti-inflammatory potential of rSj-Cys as a promising therapeutic agent on sepsis. The immunological mechanism underlying its therapeutic effect may involve the downregulation of pro-inflammatory cytokines and upregulation of IL-10 and TGF-ß1 cytokines possibly via downregulation of the TLR adaptor-transducer MyD88 pathway. The findings suggest rSj-Cys is a potential therapeutic agent for sepsis and other inflammatory diseases.

Towards novel anti-cancer strategies based on cystatin function.

Cystatins have recently emerged as important players in a multitude of physiological and patho-physiological settings that range from cell survival and proliferation, to differentiation, cell signaling and immunomodulation. This group of cysteine protease inhibitors forms a large super-family of proteins composed of one, two, three, and, in some species, more than three cystatin domains. Over the last 20 years or so, members of the cystatin super-family have been primarily explored with respect to their capacity to inhibit intracellular cysteine proteases. Yet, this classical mode of action does not fully explain their remarkably diverse biological functions. Due to the space limitations, the author will discuss here the most recent findings that suggest that some of the single-domain, cytoplasmic and cell-secreted cystatins may play important roles in the promotion or suppression of tumor growth, invasion and metastasis. Based on the present understanding of cystatin function, novel avenues for anti-cancer strategies are proposed.

Protein therapy for Unverricht-Lundborg disease using cystatin B transduction by TAT-PTD. Is it that simple?

In this work we analysed the characteristics of the cell-permeable peptide TAT-PTD fused to cystatin B (CSTB) to evaluate its potential for protein therapy of Unverricht-Lundborg (UL) epilepsy. TAT-PTD-CSTB does not penetrate the cells despite initial evidence of time and concentration-dependent transduction. Therefore, it cannot be used as a form of replacement of the intracytoplasmic protein missing in UL. Importantly, we discuss precautions to avoid false-positive results when working with TAT-PTD for protein therapy of neurological diseases.

Therapeutic potential of recombinant cystatin from Schistosoma japonicum in TNBS-induced experimental colitis of mice.

BACKGROUND: Helminth infections and their components have been shown to have a protective effect on autoimmune diseases. The isolated purified protein from Schisotosoma japonicum and its potential therapeutic effect on trinitrobenzene sulfonic acid (TNBS)-induced colitis could provide an alternative way to treat inflammatory bowel disease (IBDs). METHODS: Colitis was induced in Balb/c mice by rectal administration of 2.5% TNBS, followed by intraperitoneal injection of rSjcystatin 50 µg at 6 h and 24 h afterwards. The inflammation was monitored by recording weight change, stool character and bleeding, colon length, macroscopic score (MAO), microscopic score (MIO), myeloperoxidase activity (MPO) and disease activity index (DAI). The potential underlying mechanism was investigated by examining cytokine profiles including Th1 (IFNγ), Th2 (IL-4), Th17 (IL-17A) and Treg subsets from lymphocytes of spleen, mesenteric lymph nodes (MLN) and intestinal lamina propria mononuclear cells (LPMCs) by flow cytometry. The mRNA relative expressions of the cytokines in splenocytes and MLN were analysed by quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR). Simultaneously, the concentrations of the cytokines in the colon homogenate supernatants were tested by enzyme-linked immunosorbent assay (ELISA) and key transcription factors were detected by Western blotting. RESULTS: Administration of rSjcystatin significantly reduced inflammatory parameters and ameliorated the severity of the TNBS-induced colitis through decreasing IFNγ in three organs and lifting the level of IL-4, IL-13, IL-10, and TGF-ß in the colon tissues, with uptrending Tregs in the MLN and LPMC. CONCLUSION: The findings provide evidence that rSjcystatin has a therapeutic potential for diminishing colitis inflammation in Balb/c mice. The immunological mechanism may involve the down-regulation of Th1 response and up-regulation of Th2 and Tregs in the MLN and colon.

Brugia malayi cystatin therapeutically ameliorates dextran sulfate sodium-induced colitis in mice.

OBJECTIVE: Helminth immunomodulation in the host has been shown to have therapeutic implications in inflammatory bowel diseases. In this study we aimed to evaluate the therapeutic effect of Brugia malayi recombinant cystatin (rBmCys) in a dose-dependent manner on dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: The anti-inflammatory activity of rBmCys on mice peritoneal exudate cells was initially analyzed in vitro. BALB/c mice were fed with 5% DSS for 7 days to induce colitis. The colitis mice were treated intraperitoneally with rBmCys (10, 25 or 50 µg for the three different groups of mice) on days 1, 3 and 5 of the DSS administration. Disease severity was assessed by the disease activity index (DAI) and macroscopic and histopathological scores of colon and myeloperoxidase activity in colonic mucosa. Cytokine profiles were measured in sera and cultured splenocytes of treated mice followed by stimulation with rBmCys. RESULTS: rBmCys showed anti-inflammatory activity in vitro. Treatment of DSS-induced colitis with rBmCys in mice ameliorated the overall disease severity as reflected by a significant reduction in weight loss, the DAI, mucosal edema, colon damage and myeloperoxidase activity of the colonic mucosa. While the mRNA expressions of IFN-γ, TNF-α, interleukin (IL)-5, IL-6 and IL-17 were downregulated, IL-10 expression was upregulated in the splenocytes of colitis mice treated with rBmCys. The amelioration of DSS-induced colitis occurred in a dose-dependent manner. CONCLUSION: The results of this study indicate an anti-inflammatory potential of rBmCys and provide evidence for using this protein as a promising therapeutic agent in ulcerative colitis.

A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation.

Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.