Stimulation of tetanus toxoid-specific immune responses by a traditional Chinese herbal medicine

of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Information Report
Category:

Social Media

Published:

Views: 9 | Pages: 8

Extension: PDF | Download: 0

Share
Description
Stimulation of tetanus toxoid-specific immune responses by a traditional Chinese herbal medicine
Tags
Transcript
  Vaccine 27 (2009) 6634–6641 Contents lists available at ScienceDirect Vaccine  journal homepage: www.elsevier.com/locate/vaccine Stimulation of tetanus toxoid-specific immune responses by a traditional Chineseherbal medicine  John R. Underwood ∗ , Mark Chivers, Thi Thuong Dang, Paul V. Licciardi Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia a r t i c l e i n f o  Article history: Received 15 December 2008Received in revised form 8 March 2009Accepted 19 March 2009Available online 8 April 2009 Keywords: Tetanus toxoidRehmannia six formulaAntibody a b s t r a c t Theimmunomodulatorypropertiesofbotanicalmedicinalsarewell-documented.Inthisstudy,thecapac-ity for the traditional Chinese herbal medicine, Rehmannia Six Formula (R6F), to stimulate anti-tetanustoxoid (TT) immunity following oral administration to mice was examined. A significant rise in serumanti-TT antibody levels were observed in R6F-treated mice immunized with a minimum immunogenicdose of 10  g TT suggesting an oral adjuvant effect. No such enhancement was found for unimmunizedmice treated with R6F. This anti-TT response was preferentially IgG and antigen-specific in relation toantibody reactivity to a panel of unrelated antigens. The R6F used was safe with no adverse effects onmouse weight or survival, providing evidence for the use of R6F as an oral adjuvant.© 2009 Elsevier Ltd. All rights reserved. 1. Introduction The medicinal use of plants has an extensive historical recorddocumented by the ancient civilizations of Mesopotamia, Egypt,ChinaandIndiasome4800yearsago[1–4].Modernmedicineowes much to the plant kingdom, being the source of many commondrugs such as aspirin, morphine, atropine, digoxin and paclitaxel[5,6].Inresource-poorcountrieswhereaccesstomedicaltreatmentis limited, the World Health Organisation (WHO) recommends theuse of botanical medicinal preparations to compensate for this gap[7]. Moreover, it is reported that up to 80% of the World’s popula-tion rely on botanical medicinals as a primary source of healthcare[8,9].TheincreasingpopularityanddemandforcomplementaryoralternativemedicinesinWesternsocietyprovidesastrongimpetusfor the scientific investigation of botanical medicinals [10–12].Botanical medicinals exhibit a diverse range of properties thatcaninfluenceinnateandadaptiveimmunity.Suchexamplesincludethe enhanced macrophage production of TNF   and IL-6 associ-ated with increased NF-  B activity in mice  in vitro  reported for Ganoderma lucidum  and polysaccharides from  Prunella vulgaris stimulating nitric oxide (NO) and TNF   mRNA levels [13–15]. The traditional botanical medicinal preparations, Juzen-taiho-to andSho-saiko-to elicited opposite effects on mouse macrophage NOproduction  in vitro , with NO stimulation by Juzen-taiho-to andinhibitionbySho-saiko-to[16].Popularbotanicalmedicinalprepa- rations such as  Echinacea  and  Ginseng   modulate adaptive immune ∗ Corresponding author. Tel.: +61 3 8344 4292; fax: +61 3 8344 4004. E-mail address:  johnru@unimelb.edu.au (J.R. Underwood). parameters of antibody production, lymphocyte proliferation andcytokinesecretion[17–19].Recentstudiesdemonstrateaugmented ovalbumin(OVA)-specificmousesplenocyteproliferationandspe-cific IgG response following treatment with  Bupleurum chinense  or Glycyrrhiza uralensis  [20,21] increased pertussis antibody responseafterDTPimmunizationofmiceorallyadministered  Asparagusrace-mosa  or  Withania somnifera  and elevated TNF   and IL-2 secretionbyhumanT-lymphocytes invitro byHerbkines,anorientalprepara-tioncontainingeightdifferentherbsincluding Glycyrrhizauralensis ,  Astragalusmembranaceous and Cornusofficinalis [22,23].Incontrast,immunosuppression has also been observed by botanical medici-nals. Immunization of mice with extracts of Pollen Typhae (EEPT)inhibited murine splenocyte proliferation  in vitro  and reducedOVA-specific antibodies [24] whereas the numbers of CD4 + andCD8 + Tlymphocyteswereloweredfollowingtreatmentofcollagen-induced arthritic rats with Triptolide, a compound derived fromthe known immunosuppressive preparation,  Tripterygium wilfordii Hook F [25].These immunomodulatory properties of botanical medicinalsled to the current investigation of the Chinese botanical medic-inal preparation, Rehmannia Six Formula (R6F). This preparationcontains extracts from five herbs including  Rehmannia glutinosa , Cornus officinalis ,  Dioscorea opposita ,  Paeonia suffruticosa ,  Alismaorientalis  and the fungus,  Poria cocos  and is traditionally used forthe maintenance of health and well-being [26,27]. The biological activity of R6F is suggested to result from the synergistic actionofbothimmunostimulatoryandimmunoinhibitoryconstituentsof this complex preparation.This study is the first to our knowledge that examines theimmunomodulatory properties of the complete Rehmannia Six 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2009.03.051   J.R. Underwood et al. / Vaccine 27 (2009) 6634–6641  6635 Formula. Using an  in vivo  model of tetanus toxoid immunity, theantigen-specific immune response of mice was assessed followingoral consumption of R6F. 2. Materials and methods  2.1. Animals Inbred BALB/c mice of 6–8 weeks of age (equal male: femaleratio) were supplied by and conventionally housed in the Depart-ment of Pathology Animal House at the University of Melbourne.All mice were provided with food and water or botanical medic-inal preparations  ad libitum . All experiments involving the use of animals were approved by the University of Melbourne’s AnimalEthics Committee (AEC).  2.2. Vaccine antigen Tetanus toxoid (TT) used in these studies was providedby Commonwealth Serum Laboratories Limited (CSL Ltd., Aus-tralia). Tetanus toxoid used in these experiments represents ahighly pure human vaccine antigen component of the Diphthe-ria:Tetanus:acellularPertussis(DTPa)vaccineadministeredaspartof the routine vaccination program in Australia.  2.3. Preparation of Rehmannia Six Formula (R6F) RehmanniaSixFormula(R6F)iscomprisedofextractsfromfiveherbsandafungus( Rehmanniaglutinosa , Cornusofficinalis , Paeoniasuffruticosa ,  Dioscorea opposita ,  Alisma orientalis  and  Poria cocos ) inthe proportions 32:16:16:12:12:12. The R6F used in this study wasobtainedfromUnisonHerbalPharmaceuticalsPtyLtd(UHPPtyLtd,Melbourne, Australia) and is a commercially available and com-monly prescribed botanical medicinal preparation which meetscurrent standards required for human use.The R6F was prepared in a Class II Safety Cabinet (Email West-inghouse Pty Ltd., Australia) to ensure sterility. The R6F doseadministered to mice was 8.0mg/mL, based on the proportionaldaily human consumption for a 70-kg human and was calculatedonthedailymousefluidconsumptionratespreviouslydetermined.Briefly, Milli-Q  ® ultrapurified water (Millipore Corporation, MA,USA) used for dissolving the R6F preparation was boiled for 10minandtheR6Fdissolvedbystirringforafurther10min.TheR6Fsolu-tionwasaliquotedintosterilewaterbottlesandcappedwithsterilebung-containing sippers. This was replaced with freshly preparedR6F every 2–3 days.  2.4. In vivo experimental model Four groups of 9–10 BALB/c mice per group were used (total N  =38) and were divided according to the treatment regime(Table1).MicewereimmunizedwithtetanustoxoidorPBS(‘sham’ control) intraperitoneally three times at 14-day intervals (Days 0,  Table 1 Experimental strategy to examine Rehmannia Six Formula (R6F) effect on murineanti-tetanus toxoid antibody levels.Treatment  N  a Immunization status Immunogen doseR6F 10 Immunized 10  g TT b Water 9 Immunized 10  g TTR6F 10 Unimmunized PBS c Water 9 Unimmunized PBS a Number of BALB/c mice in each experimental group. b BALB/c mice immunized three times with 10  g tetanus toxoid(TT)/mouse/immunization. c BALB/c mice ‘sham’-immunized (unimmunized) with PBS. 14 and 28) in the absence of conventional adjuvants. Retro-orbitalsinus bleeds (eye bleeds) occurred 10 days post-immunization(Days 10, 24 and 38) and were performed in a Class I Safety Fume-hoodonPenthrane(Methoxyflurane)-anaesthetisedmice(MedicalDevelopments Australia Pty Ltd., Springvale). Sera were harvestedfollowing overnight incubation of collected blood at 4 ◦ C by cen-trifugation at 11,000 ×  g   for 10min.RehmanniaSixFormulawasorallyadministeredtoBALB/cmicevia the drinking vessels for 8–9h per day at a proportionallyequivalent human dose. Consumption of R6F and water (controlmice) was monitored daily throughout the experimental period.Mice were provided with standard drinking water at all othertimes.  2.5. Tetanus toxoid total and isotype-specific antibody levels Tetanustoxoid-specificantibodiesintheseraofmiceweredeter-mined by ELISA. Briefly, tetanus toxoid was adsorbed onto 96-wellELISA microtitre plates (Greiner Bio-one GmBH Microlon, Frick-enhausen, Germany) at a standard concentration of 250ng/welland incubated overnight at room temperature (RT). Plates wereblocked with 1% (w/v) bovine serum albumin (BSA) for 2h at37 ◦ C.FollowingwashingofELISAplateswithPBScontaining0.05%(v/v) Tween 20 (PBS-T), sera were serially diluted (initial dilu-tion=1:50) and total anti-tetanus toxoid antibodies present weredetected with a horseradish peroxidase conjugated rabbit anti-mouse immunoglobulin reagent (DAKO Corporation, Denmark).The reaction was developed with incubation of an enzyme sub-strate solution containing H 2 O 2 / o- phenylenediamine and totalantibody levels quantified by optical density measurement at490nm (OD Units) using an ELISA reader (Biorad Corporation, Cal-ifornia, USA).Measurement of tetanus toxoid-specific IgG, IgM and IgA lev-els in serum were measured as above except with the useof horseradish peroxidase conjugated immunoglobulin isotype-specific reagents. These consisted of goat anti-mouse IgG(Kirkeegard and Perry Laboratories, MD, USA), IgM (JacksonImmunolabs, Pennsylvannia, USA) and IgA (Southern Biotechnol-ogy Associates Inc., AL, USA) reagents.Measurement of serum anti-TT antibody titres was calculatedat a pre-defined ELISA threshold optical density value for anti-TTantibody reactivity using SigmaPlot 8.0 linear regression analysis(SPSS Inc., Chicago, USA). This ELISA threshold limit was based onthe mean+3 standard deviations (S.D.) of all pre-immune mousesera and responses above this level were considered positive witha 99.7% confidence level.  2.6. Specificity of the anti-tetanus toxoid immune response Thereactivitytoapanelofunrelatedantigenswasusedtoassessthespecificityofthemurineanti-tetanustoxoidantibodyresponse.Antigens in this panel included diphtheria toxoid, DNP-OVA andOVA (Sigma–Aldrich Company, St. Louis, USA).The ELISA method used was similar to that for the total andisotype-specific measurements with some minor modifications.Antigens were adsorbed separately onto 96-well ELISA plates(Greiner Bio-one GmBH Microlon, Frickenhausen, Germany) andincubated at RT overnight. Plates were blocked with 1% (w/v) BSAand incubated at 37 ◦ C for 2h. Sera was assessed in duplicate at a1:250 dilution in PBS-T containing 0.5% (w/v) BSA. A serum dilu-tion of 1:250 was selected as only low volumes were available andsincehightitreanti-TTantibodieswerestilldetectableatthispoint(Fig. 2A). Bound antibodies were detected with a HRP-conjugated rabbit anti-mouse immunoglobulin (DAKO Corporation, Denmark)and developed by addition of an enzyme substrate solution of   6636  J.R. Underwood et al. / Vaccine 27 (2009) 6634–6641  Table 2 Summary of ELISA threshold limits used in each experiment.Experiment TT threshold limits (OD units)TT-specific total antibody titre 0.23TT Isotype-specific antibody titre 0.13 (IgG)0.54 (IgM)0.09 (IgA)Threshold limits calculated as mean+3 S.D. of ELISA optical density values of allpre-immune mice ( N  =56) for each experiment (Day 0). H 2 O 2 / o- phenylenediamine for 1h at RT. The reaction was stoppedwith8MH 2 SO 4  andplateswerereadat490nmtomeasureopticaldensitiesofthereactions(BioradCorporation,CA,USA).Aresponsewasconsideredpositiveifabovethepre-immunethresholdlimitforeachantigencalculatedasmean+3S.D.forallpre-immuneBALB/cmouseresponsesfordiphtheriatoxoid,tetanustoxoid,DNP-OVAorOVA.  2.7. Adverse effects Forassessmentofpotentialadversebotanicalmedicinaleffects,the weight of each BALB/c mouse was monitored daily. Any weightchange in botanical medicinal-treated mice was compared towater-treated mice to determine whether any deviation from nor-mal growth rates occurred during the experiment. Survival rateswerealsoundertakenasanindicatorofbotanicalmedicinaltoxicityrelative to water-treated mice.  2.8. Statistical analyses Analysisoftotalandisotype-specificimmunoglobulinlevelswasundertaken by the Mann-Whitney Rank Sum test. Results from theantigenspecificitystudieswereanalysedusingthestudent’s t  -test.In all cases, the level of significance was defined by  p <0.05. 3. Results  3.1. Rehmannia Six Formula significantly enhances anti-TT immunity Athresholdlimitof0.23ODUnitswasdeterminedformeasure-ment of anti-TT antibody titres in this study based on pre-immunemouse serum reactivity ( N  =56). A summary of ELISA thresholdlimits for this study is presented in Table 2. Pre-immune analy- sis revealed no detectable anti-TT antibody response above thisthreshold level suggesting no prior contact with TT. Treatmentof mice with R6F over the 38-day treatment regimen stimulateda significant increase in anti-TT total antibody levels comparedto water-treated mice (Fig. 1). Immunization of mice with one or two doses of 10  g TT/mouse did not elevate the anti-TTantibody response at Day 10 or 24 compared to water-treatedmice. At Day 24, the mean anti-TT antibody titre for R6F- andwater-treated mice was 6156 (S.E.=1959) and 6378 (S.E.=3375),respectively(Fig.1).AtDay38followingthreeimmunizationswith 10  g TT/mouse, a significant increase in the anti-TT total anti-body response was detected for the first time, with R6F-treatedmice producing a mean anti-TT titre of 511,797 (S.E.=362,419)compared with 33,873 (S.E.=18,297) for water-treated mice repre-senting a statistically significant 15.1-fold enhancement of murineanti-TT antibody levels (  p <0.01; Fig. 1). A high degree of variabil- ity was associated with the anti-TT antibody titre response at Day38 for R6F-treated mice as not all mice in this group respondedequally to R6F treatment (Fig. 2). No effect was observed in unim- munized mice following R6F treatment, with very low levels of  Fig. 1.  Mean ± S.E. serum anti-tetanus toxoid (TT) total (IgG+IgM+IgA) antibodytitresatDay0(pre-immune)andfollowingone(Day10),two(Day24)andthree(Day38) immunizations with 10  g TT/mouse/immunization. Antibody titres calculatedusingdefinedthresholdlimitsbasedonpre-immunereactivity(referTable2).Exper- imental groups are Rehmannia Six Formula (R6F)-treated, TT-immunized (closedcircle;  N  =10), water-treated, TT-immunized (closed triangle;  N  =9), R6F-treated,unimmunized (sham PBS; open circle;  N  =10) and water-treated, unimmunized(shamPBS;opentriangle; N  =9).*Significantdifferenceinrelationtowater-treated,TT-immunized group (  p <0.05). anti-TT total antibody detected at all stages of this experiment(Fig. 1).  3.2. Rehmannia Six Formula preferentially stimulates serum IgGresponses Analysis of the immunoglobulin isotype response was based onpre-determined threshold limits for IgG (0.13 OD Units), IgM (0.54OD Units) and IgA (0.09 OD Units) in pre-immune mouse serum( N  =56). A lack of anti-TT IgG, IgM and IgA antibody was detectedinthepre-immuneserumofallmiceinthisexperiment,consistentwith the total antibody analysis (Fig. 3A–C). Again, R6F treatment of mice produced significant increases in the anti-TT IgG responsebut not IgM or IgA in the serum of mice after three immunizations(Day 38). No significant enhancement of anti-TT antibody levels of any isotype were observed on Days 10 or 24 following primary orsecondaryimmunizationofmicewith10  gTT/mouse(Fig.3).The mean anti-TT IgG titre on Day 24 for R6F- and water-treated micewere 43,970 (S.E.=18,072) and 30,921 (S.E.=15,438), respectively(Fig. 3A). Low levels of serum anti-TT IgM and no anti-TT IgA titres were detected (Fig. 3B and C). Treatment with R6F significantly elevated the anti-TT IgG antibody response of mice immunizedthreetimeswith10  gTT/mouse,consistentwiththetotalantibodyresponse detected. The R6F-treated mice produced a significant4-fold higher mean anti-TT IgG titre of 373,630 (S.E.=212,043)comparedto95,680(S.E.=60,654)forwater-treatedmice(  p <0.05;Fig. 3A). Only low anti-TT IgM (mean titre <1000) and no anti-TTIgA titres were detected by Day 38 (Fig. 3B and C). A high level of  specificity for each immunoglobulin isotype was apparent in theseexperiments with negligible cross-reactivity detected (Fig. 3D). For unimmunized mice treated with R6F, low levels of anti-TTIgG, IgM and IgA titres were observed throughout this study andsupports an antigen-driven mechanism of action for this botanicalmedicinal preparation.   J.R. Underwood et al. / Vaccine 27 (2009) 6634–6641  6637 Fig. 2.  Mean ± S.D. serum anti-tetanus toxoid (TT) total (IgG+IgM+IgA) antibody titration analysis at Day 38 following three immunizations with 10  gTT/mouse/immunization. Panel A–D represents R6F-treated, TT-immunized ( N  =10), water-treated, TT-immunized ( N  =9), R6F-treated, unimmunized (sham PBS;  N  =10)and water-treated, unimmunized (sham PBS;  N  =9) mice, respectively. Dashed line in Panel 2A indicates the presence of high-titre anti-TT antibodies at the 1:250 serumdilution used in the specificity analysis. Fig. 3.  Mean ± S.E. serum anti-tetanus toxoid (TT) isotype-specific titres at Day 0 (pre-immune) and following one (Day 10), two (Day 24) and three (Day 38) immunizationswith 10  g TT/mouse/immunization. Isotype-specific titres calculated using defined threshold limits based on pre-immune serum IgG, IgM or IgA reactivity (refer Table 2).Panels A, B and C represent the IgG-, IgM- and IgA-specific response. Panel D shows the specificity of the anti-immunoglobulin IgG (black bars), IgM (light gray bars) and IgA(darkgraybars)antibodiestoeachIgisotypeattheconcentrationsusedintheseexperiments.ExperimentalgroupsareRehmanniaSixFormula(R6F)-treated,TT-immunized(closed circle;  N  =10), water-treated, TT-immunized (closed triangle;  N  =9), R6F-treated, unimmunized (sham PBS; open circle;  N  =10) and water-treated, unimmunized(sham PBS; open triangle; N=9). *Significant difference only for IgG response in relation to water-treated, TT-immunized group (  p <0.05).  3.3. Rehmannia Six Formula induces an anti-tetanus toxoidresponse that is antigen-specific  Thespecificityoftheanti-tetanustoxoidantibodyresponsewasassessed following oral administration of R6F to mice. A panel of structurally unrelated antigens including diphtheria toxoid (DT),DNP-OVA and OVA were used to probe the specificity of thisresponse. Threshold optical density values of 0.09 OD Units (DT),0.19 OD Units (DNP-OVA) and 0.05 OD Units (OVA) were derivedfrom pre-immune serum reactivity ( N  =56) at a 1:250 dilution(Fig. 2A) and for comparison, a threshold of 0.06 OD Units for TT was established.Responses of mice to DT, DNP-OVA and OVA in the pre-immuneserum were all below the threshold limits. Immunization with10  g/mouse TT did not induce positive reactivity to DT, DNP-OVAor OVA by Day 38 irrespective of mice treated with R6F or waterdespite significant increases in the anti-TT response by Day 38 fol-lowing three immunizations of mice treated with R6F comparedto water (  p <0.05; Fig. 4). In contrast, unimmunized mice did not produce any detectable reactivity to TT or to DT, DNP-OVA andOVA.  3.4. Rehmannia Six Formula treatment was safe During the 38-day experimental period, no difference wasobserved in the mean weights of mice treated with R6F or water(Table 1). Treatment of TT-immunized mice with R6F did not cause any significant change in mouse health status, with a meanweight of 26.0 ± 0.6g compared to 21.1 ± 1.2g for water-treatedmice while for unimmunized mice, mean weights of 24.6 ± 0.8gand 19.0 ± 2.5g were recorded for R6F- and water-treated mice,respectively (Table 3). Two mice ( N  =56) did not survive the38-day experimental period, equally distributed between the TT-immunized and unimmunized groups, giving an overall survivalrate of 96.4%. There was no significant R6F treatment effect onmouse survival (Fig. 5). Together, these results suggest the safe  6638  J.R. Underwood et al. / Vaccine 27 (2009) 6634–6641 Fig. 4.  Specificity of the anti-tetanus toxoid response. Mean ± S.D. ELISA optical density (OD) values for serum antibody responses to tetanus toxoid (TT), diphtheria toxoid(DT),DNP-OVAandOVAatDay0(pre-immune)andfollowingone(Day10),two(Day24)andthree(Day38)immunizationswith10  gTT/mouse/immunization.Toppanelsrepresent R6F-treated groups and the bottom panels represent the water-treated groups. Experimental groups in the top panel are Rehmannia Six Formula (R6F)-treated,TT-immunized (closed circle;  N  =10) and R6F-treated, unimmunized (sham PBS; open circle;  N  =10) while the bottom panel are water-treated, TT-immunized (closed circle; N  =9) and water-treated, unimmunized (sham PBS; open circle;  N  =9). *Significant difference only for R6F-stimulated TT-specific response in relation to the TT-immunized,water-treated group at Day 38 (  p <0.05);  # Significant difference between TT-immunized groups compared to counterpart unimmunized (sham PBS) groups for R6F- andwater-treated mice on Days 24 and 38 (  p <0.05). treatmentofmicewithR6Fbotanicalmedicinalpreparationinthisstudy. 4. Discussion Botanical medicinals have been used for thousands of yearsfor treatment of simple infections and wounds to more severecases of illness including cancer and immunodeficiency [8]. The continued widespread use of botanical medicinals in develop-ing countries and the increasing use in Western society haveemphasized the need to understand the scientific basis for thesebiological activities [28,29]. Several studies have demonstrated the effects of individual herbal extracts on the immune system[17,30–39]. Using a traditional Chinese botanical medicinal prepa-ration,R6F,asignificant15-foldenhancementofthemurineanti-TTtotal antibody response was observed over water-treated miceusing an ELISA with anti-mouse IgG, IgM and IgA-reactive poly-clonal antibodies. Using isotype-specific reagents, this responsewas found to be IgG-restricted, with negligible TT-specific serum  Table 3 Effect of Rehmannia Six Formula (R6F) on mouse weights.Experimental group  N  a Weight (g)post-treatment  p -ValueTT-immunized, R6F-treated 10 26.0  ±  0.6 ns b TT-immunized, water-treated 9 21.1  ±  1.2 nsPBS (sham)-immunized, R6F-treated 10 24.6  ±  0.8 nsPBS (sham)-immunized, water-treated 9 19.0  ±  2.5 ns a Number of BALB/c mice in each experimental group. b Notsignificant(comparedtorelevanttreatmentorimmunizationcontrolgroup). IgM and IgA levels detected. Detection of a predominately IgGresponse in the serum of mice could be explained by the par-enteral administration of the TT antigen. Indeed, protective serumIgGantibodiesareinducedfollowinghumanvaccinationprograms Fig. 5.  Kaplan–Meier survival curves of mice during the experimental period. Per-centage of mice in the TT-immunized and unimmunized (sham PBS) groups thatsurvived over the 38-day period following treatment with Rehmannia Six Formula(R6F) or water.
Recommended
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x