pumbertot
Active member
yes me too bro. just think when you do get back on and combine them along with igf,mgf,hgh,insulin=**** happens
Peg-anti-gdf-8 [myostatin inhibitor]and CJC-1295
- Thread starter BLACK747
- Start date
yes me too bro. just think when you do get back on and combine them along with igf,mgf,hgh,insulin=**** happens
CryingEmo
Well-known member
I thought you were a real man. No AAS? C'mon man, I wanna see Tren + Test + Anadrol + IGF-1 LR3 + PEG-MGF + Slin + T3 + S4 + gdf-8 + GH
pumbertot
Active member
you just let me get my op out of the way and then you will see pretty much what you posted.
a line out of a song posed to by a very famous BB springs to mind "I wann Freak you!". lol.
Speedbacker
Member
pumbertot
Active member
Bobaslaw
Member
sfearl1
Well-known member
hahahahahahaha
BLACK747
Member
well people its your boy black back again with todays log anyway everything is still the same nothing new to report had and excellent workout today great pump chest and triceps work my way up to 315 for 6 sets of 10s and then drop down to 225 for 2 sets of 40s so it was a real intense workout real good pump today real good i love this s###! lol anyway as i stated nothing out of the ordinary to report just your man black killing the iron and thats real talk peace!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
CryingEmo
Well-known member
BLACK747
Member
like everyone else im looking for that magic potion but with this peg -anti-gdf-8 at 500 mic a week aint it know i could have easy got up here and said this stuff is the best thing since slice bread but its not even though im somewhat dissapointed im still upbeat about anti-gdf-8 if taking at the right dosage i think anti-gdf-8 not gdf-8 should be price like cjc to get the top results know there are stronger products out there that binds to myostatin such as follistatin,myostatin propeptide, the myostatin dna vaccine which will be my next log stay tune i have at max 6 more weeks of this stuff then im done with this experiment so i will continue posting until im bone dry of this stuff !!
pumbertot
Active member
see I have this strong suspicion that the peg antibody is not working as well as the non-peg I just dont think every molecule actually likes the pegylation process so much.
looking forward to the vaccine for sure, if you need another guinea pig to run alongside with you I'm in. as I wont be in the gym from June 16th to middle October or so I could always run it as a non-training guinea pig to see if it can still build muscle in that environment.
looking forward to the vaccine for sure, if you need another guinea pig to run alongside with you I'm in. as I wont be in the gym from June 16th to middle October or so I could always run it as a non-training guinea pig to see if it can still build muscle in that environment.
pumbertot
Active member
What, no gym for months? WTF bro?
flight to Boston 16th June. Reconstruction of left pec tendon(at the musculotendinous junction) 18th June. 6 weeks immobilised in swaithe sling followed by further 10 weeks out of gym
of course im ever hopefull(and quite certain) the post-op therapy of igf,mgf,hgh,slin,exemestane and HRT will speed the recovery but wont hit the gym untill full 4 months as dont want to risk a re-tear. especially so if a tendon graft is needed(from cadaver hamstring) but surgeon will not know untill surgical exploration.
thats why its been 3 years since I last used AAS, I could not 'forgive' the gear for causing the tear(they made me too strong on benchpress for my tendons) but I will finally do so after the op.
hence why im so excited about the future of my peptide use, the AAS will make it oh so good. as im currently 230lbs 5ft11 10% bf, the future will be good. :d
big off topic rant, apologies Black.:good:
disgraziato
Member
If someone has experience with cjc can they post....anyone just take the cjc, not along side anything? So as to clearly state any gains and or sides were attributed solely to the cjc??
BLACK747
Member
alright people another great workout today excellent pump had legs today front squats work up to 315 for a couple sets of 10s and then i perform my leg extensions and leg curls on a side note the front squats felt alot easier then ususual so have to credit the antibody and cjc for increase leg strength nothing else out of the ordinary to report like i said i am getting good pumps from this stuff but not the extraordinary muscle growth i have been expecting this myostatin dna vaccine is extremly potent stay tuned lol
CryingEmo
Well-known member
pumbertot
Active member
hehe cheers bro. :cheers:
BLACK747
Member
ok folks got back in the gym today excellent back and bicep workout great pump could hardly move my arm when i left the gym check my weight today 224 pounds so im holding steady for the moment other than that no unusual muscle growth to report although the fat burning is awesome from this stuff it still a bit dissapointing from a muscle growth aspect anyway i will post again tommorrow peace!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
CryingEmo
Well-known member
pumbertot
Active member
so have you measured your actual bodyfat with calipers at beginning and now? if so why not work out your average lean body mass so we can see how much muscle you have gained. it could be a decent amount if weight is holding steady and bf% is dropping.
please post your starting weight and bf% and what they are now.
BLACK747
Member
to answer your question no i did not get a bodyfat analysis done but when i started getting back on the myostatin band wagon on dec 11 of last year i weighed 215 pounds im know around 10 pounds heavier know from dec 11 until march i was on anti-gdf-8 regular at 40 mics a day and gained alot of weight i actually went up to 235 pounds from eating everything in sight not all was lean but some was but if i had to guess which im not good at i would say i gained about 8 or 10 pounds of lean mass know with that out of the way today more like this week i up the dosage to 1 mg a week to see if i get that real noticeable results and to be honest i been pump all week long and the feeling i got in the gym this week has been incredible to say the least from a pump standpoint lol and today with shoulders my pump was so good it actually hurt a little bit anyway its been a good week to say the least good workout week another chance to share my experiences with the anabolicmind family .
Clarityandfocus
New member
If it was the real mccoy you would be doing much better than you are. It sounds like you are wasting your time.
The genuine polyclonal antibody does not have you saying "this stuff it still a bit dissapointing from a muscle growth aspect." I know this for a fact.
Sorry your results suck.
Clarity
The genuine polyclonal antibody does not have you saying "this stuff it still a bit dissapointing from a muscle growth aspect." I know this for a fact.
Sorry your results suck.
Clarity
sfearl1
Well-known member
If it was the real mccoy you would be doing much better than you are. It sounds like you are wasting your time.
The genuine polyclonal antibody does not have you saying "this stuff it still a bit dissapointing from a muscle growth aspect." I know this for a fact.
Sorry your results suck.
Clarity
clarity maybe you can clarify at what dosage he could be getting results that don't suck, you know, since you know for a fact and all. .....does he need to up the dose?
BLACK747
Member
listen slick when i say its a bit dissapointing doesnt mean its not working just not as fast as i would like this is the pegylated version of the polyclonal anti-body the reason that im not impress i guess i should have said is because of the length of time its taking to work know i have used the reg version of the anti-body and it dont work overnight either so whatever you heard thats just not the case son but believe whatever you must cause frankly i dont give a s###!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
pumbertot
Active member
the regular polyclonal at dosages of 80mcg/day DOES produce very decent muscle gains/fat loss. have witnessed it in more than one person. I would do a course laready if it wasnt for my upcoming surgery. but after that for sure, thats if the DNA vaccine doesnt come my way first.
Bobaslaw
Member
Dosage is definitely the key as with everything. Even taking gear at 1/10 the ideal dosage will lead to the conclusion that "it is useless for muscle growth".
I have posed this question earlier in this thread regarding how the dosages were chosen in this trial? IMO, it is a learning experience, no one "really" knows for sure. It just takes involved testing scenarios like lab studies/clinical trials that use multiple drug dosages ranging from extra small to overdone. Then you can see comparisons.
Who really knows if the dosage Black is using is really ideal or possibly a small fraction of the "sweet spot" dosage.
Just my 2 cents
pumbertot
Active member
Dosage is definitely the key as with everything. Even taking gear at 1/10 the ideal dosage will lead to the conclusion that "it is useless for muscle growth".
I have posed this question earlier in this thread regarding how the dosages were chosen in this trial? IMO, it is a learning experience, no one "really" knows for sure. It just takes involved testing scenarios like lab studies/clinical trials that use multiple drug dosages ranging from extra small to overdone. Then you can see comparisons.
Who really knows if the dosage Black is using is really ideal or possibly a small fraction of the "sweet spot" dosage.
Just my 2 cents
well get on some then matey, whats your excuse?
i have a good one, do you?
BLACK747
Member
well hello muscle freaks its your genetic brother from another mother black wassup anyhow i think i need to clear up a few of my statements this stuff do work but not to the degree i wanted it to at the dose i was taking know i have up the dosage to 1 mg a week know what i can honestly say is it gives you the pump up feeling all day long it also causes the severe hunger pains i was getting on the reg anti-body also
i do feel somewhat stronger know i only been on 1mg a week for going on 2 weeks know i did not want to post until i knew it wasnt a placebo affect another reason is at this dose it would cost a fortune to afford this stuff so i felt no need to imform but like most politician i went back on my word so there you have it the effective dose and you can quote me on this is 1 mg a week possibly split in three dosage like mon-wedn-friday or mondays and fridays i only have 1more week then im gonna be done with the peg version and then im going finish
4 mg of the reg anti-gdf-8 dose in 200 mics a day along with 2 mg a week of peg mgf . the vaccine for myostatin should be available soon in like 3 weeks maybe sooner in which then i will begin a log on it thanks people peace!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
i do feel somewhat stronger know i only been on 1mg a week for going on 2 weeks know i did not want to post until i knew it wasnt a placebo affect another reason is at this dose it would cost a fortune to afford this stuff so i felt no need to imform but like most politician i went back on my word so there you have it the effective dose and you can quote me on this is 1 mg a week possibly split in three dosage like mon-wedn-friday or mondays and fridays i only have 1more week then im gonna be done with the peg version and then im going finish
4 mg of the reg anti-gdf-8 dose in 200 mics a day along with 2 mg a week of peg mgf . the vaccine for myostatin should be available soon in like 3 weeks maybe sooner in which then i will begin a log on it thanks people peace!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
djbombsquad
Board Sponsor
Before and after pictures please
Speedbacker
Member
Here it is:
Main Article
Myostatin DNA vaccine increases skeletal muscle mass and endurance in mice
Liang Tang, MD 1 2, Zhen Yan, MD 1, Yi Wan, MSc 1, Wei Han, MD 1, Yingqi Zhang, MD 1 *
1Biotechnology Center, Fourth Military Medical University, 17 Changle West Road, 710032 Xi'an, China
2Institute of Sports Biology, Shaanxi Normal University, Xi'an, China
email: Yingqi Zhang ([email protected])
*Correspondence to Yingqi Zhang, Biotechnology Center, Fourth Military Medical University, 17 Changle West Road, 710032 Xi'an, China
Funded by:
Changjiang Scholars and Innovative Research Team in University (PCSIRT), China
Faculty of Pathology, Fourth Military Medical University, China
Keywords
autoimmunity • DNA vaccine • myostatin • T-helper epitope
Abstract
Myostatin is a transforming growth factor- family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T-helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting. Muscle Nerve, 2007
--------------------------------------------------------------------------------
Accepted: 28 March 2007
Digital Object Identifier (DOI)
10.1002/mus.20791 About DOI
Article Text
Myostatin, formerly known as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-beta family that plays an essential role in regulating skeletal muscle growth.[1][11][15] Myostatin is a negative regulator of skeletal muscle mass and its sequence has been highly conserved through evolution.[16] Mutations in the myostatin gene lead to dramatic increases in skeletal muscle mass and muscle strength in mice and to less fat accumulation than in wild- type littermates.[6][8][9][22] In humans, mutations in the myostatin gene are correlated with excess muscle mass, whereas expression of myostatin is increased in the setting of muscle loss, including chronic illnesses, infection with human immuodeficiency virus, and during aging.[5][7][20] Systematic administration of exogenous myostatin to adult mice is sufficient to induce severe muscle and fat loss, similar to human cachexia syndromes.[26] Conversely, myostatin antagonists, such as monoclonal antibodies specific to myostatin and follistatin, as well as activin type II receptor antagonists, can significantly increase skeletal muscle mass. Given the highly conserved role of myostatin among animals, improved methods for inhibiting myostatin activity could have important implications not only for human therapeutics, but also other areas such as agriculture.[10]
Although myostatin antagonists are obvious candidates for intervention, obtaining sufficient quantities of purified myostatin antagonist proteins can be costly and time-consuming. By contrast, DNA vaccines are stable, inexpensive, safe, and easy to produce in large quantities and have high levels of purity.[2][4][13][21] Moreover, DNA vaccines stimulate a full spectrum of immune responses, including cytotoxic T lymphocytes generally not induced by protein vaccines, and generate exceptionally long-lasting immune responses.[3][24]
We describe the development of a myostatin-specific DNA vaccine. The pVAC1-cms plasmid is a DNA vaccine vector specifically designed to stimulate a humoral immune response, using the rhesus monkey elongation factor 1-alpha gene promoter to achieve high levels of expression in skeletal muscle cells and antigen-presenting cells. Expression levels are further increased by the addition of the SV40 enhancer, which heightens the ability of the plasmid to be transported into the nucleus. DNA encoding a fusion protein between the T-helper epitope of tetanus toxin (TT) and the mature myostatin peptide was cloned into pVAC1-cms vector (pVAC-TTMs). The immunogenicity of the recombinant DNA vaccine was examined in BALB/c mice. Sera were analyzed from vaccinated mice containing specific antibodies to the recombinant protein. The muscle and function of the mice were evaluated as well.
MATERIALS AND METHODS
A DNA fragment encoding the TT epitope (QYIKANSKFIGITEL),[12] followed by the N terminus of mature myostatin (encoding amino acid residues 267-375, GenBank Accession No. 014793), was synthesized by Shenggong Biotechnology (Shanghai, China). This fragment was subcloned into the EcoRI and BamHI sites of pVAC1-cms (Invitrogen, Carlsbad, California). The recombinant plasmid (pVAC-TTMs) was confirmed by sequencing.
Plasmid pVAC1-cms and pVAC-TTMs were transformed into expression-competent Escherichia coli harboring the DH5 lysogen by heat shock. The transformation mixture (10 l) was added to Luria-Bertani (LB) medium (10 ml) containing 100 g/ml zeocin (Invitrogen) and incubated overnight at 37°C with vigorous shaking. This culture was used to inoculate 200 ml of prewarmed LB medium containing 100 g/ml zeocin for 24 h at 37°C. Cells were harvested by centrifugation at 5000 × g for 20 min and the plasmids were extracted and purified as described elsewhere.[20]
Male BALB/c mice (2-3 weeks old) were purchased from the National Rodent Laboratory Animal Resource (Shanghai, China) and housed in a room controlled for temperature (22 ± 2°C) and humidity (60 ± 5%) and regulated to provide alternating 12-h periods of light and darkness. Mice were allocated to two groups (n = 6 for each group) and injected intramuscularly (IM) with 50 g each of pVAC1-cms or pVAC-TTMs. Mice were vaccinated on days 0, 14, and 28, then boosted (IM) on day 42, and killed on day 67 for serum and tissue analysis. Serum total protein, albumin, globulin, urea nitrogen, glucose, cholesterol, triglycerides, creatine kinase, and antibody titer were analyzed. At the same time, the hindlimb was photographed. The abdominal fat pad, a portion of the quadriceps (rectus femoris), and the gastrocnemius were dissected and weighed.
Detection of Expression of Plasmid-Encoded TT-Ms in Mice by Western Blot.
Tissues kept at -80°C from quadriceps muscle of different groups were homogenized in 10-20 vol of a buffer containing 1% sodium dodecylsulfate, 100 mM Tris-HCl (pH 6.8), 1 mM phenylmethylsulfonylfluoride, and 0.1 mM -mercaptoethanol. The supernatant obtained after centrifugation of homogenized tissue at 15,000 × g for 25 min (4°C) was designated the homogenate, and the same amounts of supernatant quadriceps muscle (20 l) from the mice vaccinated with pVAC-TTMs and pVAC1-cms and purified B7H1 (control protein from our laboratory) were loaded for sodium dodecylsulfate-polyacrylamide gel electrophoresis (15% acrylamide). The proteins were then electrophoretically transferred (80 V, 2 h) onto nitrocellulose (NC) membranes (Bio-Rad Instruments, Hercules, California). The NC membranes were blocked using phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) and 0.05% Tween-20 (PBST) at 4°C overnight. The NC membranes were then incubated with either anti-TT antibody at 1:500 dilution (Abcam, Cambridge, Massachusetts) or anti-GDF-8 antibody at 1:250 dilution (Bethyl, Montgomery, Texas) for 2 h at room temperature. Membranes were washed three times for 10 min in PBST and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Life Technologies, Carlsbad, California) at 1:1000 dilution and goat anti-rabbit IgG (Life Technologies) at 1:1000 dilution in 2% BSA-PBST for 2 h at room temperature. Finally, the membranes were washed as described previously, and developed by adding 10 ml of alkaline phosphatase developer (Life Technologies). The reaction was stopped by rinsing the NC membranes with de-ionized water.
Enzyme-Linked Immunosorbent Assay.
Enzyme-linked immunoassay (ELISA) plates were coated with 100 ng of purified myostatin protein and incubated overnight at 4°C. The plates were washed three times with PBS containing 5% Tween-20. Serially diluted mouse sera (100 l) were added to each well and assayed in duplicate after blocking as described earlier, and then incubated for 1 h at 37°C. After washing, horseradish peroxidase-conjugated goat anti-mouse IgG (diluted 1:1000) was added to each well (100 l per well) and incubated for 1 h at 37°C. The reactions were then visualized with o-phenylenediamine dihydrochloride and H2O2 and stopped by adding 100 l of 2 M H2SO4. The plates were read at OD450 using a Bio-Rad ELISA reader.
Competitive Inhibition Assay.
Ninety-six-well plates were coated with 100 ng of purified myostatin protein and incubated overnight at 4°C. Plates were then washed three times with PBS containing 5% Tween-20 and blocked with BSA for 30 min at 37°C. Anti-GDF-8 polyclonal antibody (100 ng/well) was preincubated with serially diluted sera from mice treated with pVAC-TTMs, pVAC1-cms (negative control), or B7H1 (control antiserum from our laboratory) protein for 30 min at 37°C before addition to myostatin-coated wells. After washing, bound GDF-8 antibody was detected by incubation with horseradish peroxidase-conjugated secondary antibody. The reactions were visualized as described earlier. Inhibition rate was defined as 1 - OD490 (pVAC-TTMs) / OD490 (pVAC1-cms) 100%.
Grip Test.
Grip tests were conducted as described by Peled-Kamar et al,[18] with some modifications. Mice were allowed to grip and hang from a 2-mm horizontal tight-rope, 80 cm above the ground, but the measurement of grip time was different from the method of Peled-Kamar et al. The tails of mice were immobilized when the forelimbs of the mice gripped the rope. The time that elapsed until the forelimbs loosened and the mice fell to the ground was measured. The mice were studied three times each on two different days, and measurements were averaged.
Morphometric Analysis.
The gastrocnemius muscles were dissected rapidly and freed of fat and connective tissue. Muscle tissues were weighed and fixed with 4% polyaldehyde for 24 h. Serial 8-10-m transverse sections, made with a cryostat, were mounted on silanized slides (Dako, Tokyo, Japan). Cross-sectional areas were measured at quadricep midportions following staining with hematoxylin-eosin.
Statistical Analysis.
Statistical analysis of the data was performed with the Student's unpaired t-test. Results are expressed as the mean ± SD. Differences were considered statistically significant at P < 0.05.
Speedbacker
Member
Continued
RESULTS
The average body weights for each group of mice were measured and found to increase during the course of the study. The mice vaccinated with pVAC-TTMs gained more weight than control mice vaccinated with pVAC1-cms (Fig. 1A). Photographs of the bodies and hindlimbs showed that muscle tissues of the mice vaccinated with pVAC-TTMs were obviously larger than those of control mice. Measurements for average mass of the quadriceps and gastrocnemius muscles from mice immunized with pVAC-TTMs were 31.4% and 13.9% greater, respectively, compared with the control group (Fig. 1B). Abdominal fat pad weighs, however, did not differ between the two groups (Fig. 1C).
Figure 1. Changes in body and muscle mass following pVAC-TTMs immunization. (A) Mice immunized with pVAC-TTMs showed a significant increase in body weight compared with controls (31.33 ± 1.93% vs. 28.57 ± 2.35%, n = 6 each, P < 0.05). (B) The average masses of quadricep and gastrocnemius muscles of mice immunized with pVAC-TTMs were 31.4% and 13.9% greater than those of control mice (n = 6, *P < 0.05). (C) Comparison of abdominal fat pad weights. No significant difference was found between the two groups.
[Normal View 20K | Magnified View 61K]
To confirm the expression of recombinant plasmid in muscle cells in vaccinated mice, we performed Western blot with two antibodies: anti-GDF-8 antibody and anti-TT antibody. The expression of myostatin in skeletal muscle cells could be detected by anti-TT antibody (Fig. 2). The results confirm the expression of recombinant protein TT-Ms in muscle cells.
Figure 2. The analysis of expression of recombinant plasmid in muscle cells in vaccinated mice by Western blot. Lane 1, B7H1 protein (unrelated protein); Lane 2, pVAC-TTMs; Lane 3, pVAC1-cms.
[Normal View 22K | Magnified View 37K]
We performed morphometric analyses of the quadricep muscles from the two groups (Fig. 3A) to determine whether the increase in muscle mass in the mice vaccinated with pVAC-TTMs was due to hypertrophy or hyperplasia. A significant increase in whole-muscle cross-sectional area in mice immunized with pVAC-TTMs was observed, as compared with control mice (Fig. 3B). No significant difference was found in the number of muscle fibers per quadricep between the two groups (Fig. 3C), suggesting increased muscle mass was due to hypertrophy rather than hyperplasia.
Figure 3. Morphometric analysis of quadricep muscles. (A) Comparison of the cross-sectional area of muscle. Cross-sectional areas were determined from quadriceps muscles taken at its midportion and stained with hematoxylin-eosin (n = 6 for each group). Bar, 100 m. (B) Mean cross-sectional area of individual fibers from the quadricep muscles. A total of about 1200 fibers from the treated mice were measured. There is a significant increase in the mice immunized with pVAC-TTMs compared with control mice (n = 6 for each group, *P < 0.05). (C) Fiber counts from the quadricep muscle (n = 6 for each group). No significant difference was observed between the two groups.
[Normal View 59K | Magnified View 197K]
We calculated the product of time and body weight for each animal (Fig. 4A). The average grip time/body weight was about 36.5% greater in the mice vaccinated with pVAC-TTMs than in control mice (Fig. 4B). The results show that the grip endurance of the mice vaccinated with pVAC-TTMs was significantly greater than that of control mice.
Figure 4. Grip test in immunized mice. (A) Relationship of grip time to body weight for each animal. (B) The average value of the grip time/body weight. The average value of the time/body weight of the mice immunized with pVAC-TTMs was about 36.5% greater than that of control mice (n = 6, *P < 0.05).
[Normal View 10K | Magnified View 26K]
The immunogenicity of pVAC-TTMs was evaluated in BALB/c mice by ELISA. Sera from pVAC-TTMs-vaccinated mice contained specific antibodies against myostatin protein (Fig. 5). The ability of antibodies induced by pVAC-TTMs to bind myostatin was examined by ELISA in a competitive assay with a polyclonal GDF-8 antibody. Sera from pVAC-TTMs-vaccinated mice were able to reduce binding of the anti-GDF-8 antibody to myostatin (Fig. 6). The inhibition rates were 62.2% at 1:5 dilution and 30.0% at 1:40 dilution, respectively, suggesting that sera from pVAC-TTMs-vaccinated mice are able to interfere with myostatin in vitro.
Figure 5. ELISA analysis of antiserum titers from mice immunized with pVAC-TTMs. Recombinant myostatin was used as the antigen, and was probed with different concentrations of antisera from individual control or experimental mice. The OD450 was measured for each well following reaction with horseradish peroxidase. Average anti-serum titers were calculated from values displayed in the panel.
[Normal View 11K | Magnified View 32K]
Figure 6. Competitive myostatin binding assay using antisera from mice immunized with pVAC-TTMs or pVAC1-cms and commercial anti-GDF-8 antibody. Sera from BALB/c mice immunized with pVAC1-cms were used as negative control. The control anti-serum was B7H1 (unrelated protein).
[Normal View 9K | Magnified View 24K]
Serum levels of total protein, albumin, globulin, urea nitrogen, glucose, cholesterol, triglycerides, and creatine kinase in pVAC-TTMs-vaccinated mice and control mice were not significantly different.
DISCUSSION
Myostatin is highly conserved, both in sequence and in function, across animal species. The amino-acid sequences of mouse and human myostatin are identical.[5][15] Our results indicate that specific antibodies to the recombinant myostatin vaccine were effectively induced and the titers could inhibit myostatin in mice. Importantly, significant increases in body weight and skeletal muscle mass were observed following DNA vaccination. The effect of the myostatin DNA vaccine was comparable with other non-DNA vaccine methods of myostatin interference. For example, a recent study demonstrated that the body weights of mice treated with a neutralizing monoclonal antibody to myostatin increased by 10.0%.[23] Similarly, mice expressing a dominant-negative myostatin variant showed an increase of up to 35.0% in skeletal muscle mass.[25] Our results suggest that a myostatin DNA vaccine could promote an increase in skeletal muscle mass.
Interestingly, there was no significant difference in abdominal fat pad weighs and level of triglycerides and cholesterol in blood in vaccinated mice in our study. These results differ from the reported myostatin knockout phenotype, in which fat accumulation was decreased.[11][17] This discrepancy could be due to differences in loss of myostatin activity during development and during adulthood.[23] Our results suggest that the increase in body weight is caused by the increase in muscle mass but not in fat weight.
Although the muscle mass of mice immunized with the myostatin DNA vaccine was found to be increased significantly relative to the control group, this is not proof of increased muscle function. We therefore quantified functional improvement and found an increase in forelimb grip endurance in the mice immunized with the myostatin DNA vaccine. The increase in muscle endurance was proportional to the increase in muscle mass in the mice immunized with the myostatin DNA vaccine. However, more accurate methods than the grip test are necessary to determine the precise effect of myostatin DNA vaccine on muscle strength.
Our study has provided physiological evidence of functional improvement in muscle growth caused by myostatin inhibition in vivo. Myostatin activates the ubiquitin proteolytic system through a nuclear factor-kappaB (NF-B)-independent, FoxO1-dependent mechanism.[14] The central nervous system (CNS) and peripheral blood flow may play an important role in the activation and performance of myostatin, but it is unclear whether the inhibition of myostatin caused by myostatin DNA vaccine is related to them.
The manner in which muscle mass is increased is important. We found that the number of muscle fibers from mice immunized with the myostatin DNA vaccine did not increase but the muscle fibers were hypertrophic. By contrast, in mice expressing dominant-negative myostatin, muscle fibers showed both hypertrophy and hyperplasia.[15]
The physiological changes observed in the mice immunized with myostatin DNA vaccine were not accompanied by obvious detrimental changes in serum biochemistry, suggesting that vaccine-mediated inhibition of myostatin can increase skeletal muscle size without major side-effects. The functional enhancement of muscle by myostatin DNA vaccine thus provides a novel, pharmacological strategy for the treatment of diseases associated with muscle wasting. However, further safety studies and studies of the long-term effects of induced autoimmunity are required. The present study has provided the foundation for future work on this exciting and promising new strategy.
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Abbreviations
BSA, bovine serum albumin; CNS, central nervous system; ELISA, enzyme-linked immunosorbent assay; GDF-8, differentiation factor-8; LB, Luria-Bertani; NC, nitrocellulose; NF-B, nuclear factor-kappaB; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline/Tween; TT, tetanus toxin
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RESULTS
The average body weights for each group of mice were measured and found to increase during the course of the study. The mice vaccinated with pVAC-TTMs gained more weight than control mice vaccinated with pVAC1-cms (Fig. 1A). Photographs of the bodies and hindlimbs showed that muscle tissues of the mice vaccinated with pVAC-TTMs were obviously larger than those of control mice. Measurements for average mass of the quadriceps and gastrocnemius muscles from mice immunized with pVAC-TTMs were 31.4% and 13.9% greater, respectively, compared with the control group (Fig. 1B). Abdominal fat pad weighs, however, did not differ between the two groups (Fig. 1C).
Figure 1. Changes in body and muscle mass following pVAC-TTMs immunization. (A) Mice immunized with pVAC-TTMs showed a significant increase in body weight compared with controls (31.33 ± 1.93% vs. 28.57 ± 2.35%, n = 6 each, P < 0.05). (B) The average masses of quadricep and gastrocnemius muscles of mice immunized with pVAC-TTMs were 31.4% and 13.9% greater than those of control mice (n = 6, *P < 0.05). (C) Comparison of abdominal fat pad weights. No significant difference was found between the two groups.
[Normal View 20K | Magnified View 61K]
To confirm the expression of recombinant plasmid in muscle cells in vaccinated mice, we performed Western blot with two antibodies: anti-GDF-8 antibody and anti-TT antibody. The expression of myostatin in skeletal muscle cells could be detected by anti-TT antibody (Fig. 2). The results confirm the expression of recombinant protein TT-Ms in muscle cells.
Figure 2. The analysis of expression of recombinant plasmid in muscle cells in vaccinated mice by Western blot. Lane 1, B7H1 protein (unrelated protein); Lane 2, pVAC-TTMs; Lane 3, pVAC1-cms.
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We performed morphometric analyses of the quadricep muscles from the two groups (Fig. 3A) to determine whether the increase in muscle mass in the mice vaccinated with pVAC-TTMs was due to hypertrophy or hyperplasia. A significant increase in whole-muscle cross-sectional area in mice immunized with pVAC-TTMs was observed, as compared with control mice (Fig. 3B). No significant difference was found in the number of muscle fibers per quadricep between the two groups (Fig. 3C), suggesting increased muscle mass was due to hypertrophy rather than hyperplasia.
Figure 3. Morphometric analysis of quadricep muscles. (A) Comparison of the cross-sectional area of muscle. Cross-sectional areas were determined from quadriceps muscles taken at its midportion and stained with hematoxylin-eosin (n = 6 for each group). Bar, 100 m. (B) Mean cross-sectional area of individual fibers from the quadricep muscles. A total of about 1200 fibers from the treated mice were measured. There is a significant increase in the mice immunized with pVAC-TTMs compared with control mice (n = 6 for each group, *P < 0.05). (C) Fiber counts from the quadricep muscle (n = 6 for each group). No significant difference was observed between the two groups.
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We calculated the product of time and body weight for each animal (Fig. 4A). The average grip time/body weight was about 36.5% greater in the mice vaccinated with pVAC-TTMs than in control mice (Fig. 4B). The results show that the grip endurance of the mice vaccinated with pVAC-TTMs was significantly greater than that of control mice.
Figure 4. Grip test in immunized mice. (A) Relationship of grip time to body weight for each animal. (B) The average value of the grip time/body weight. The average value of the time/body weight of the mice immunized with pVAC-TTMs was about 36.5% greater than that of control mice (n = 6, *P < 0.05).
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The immunogenicity of pVAC-TTMs was evaluated in BALB/c mice by ELISA. Sera from pVAC-TTMs-vaccinated mice contained specific antibodies against myostatin protein (Fig. 5). The ability of antibodies induced by pVAC-TTMs to bind myostatin was examined by ELISA in a competitive assay with a polyclonal GDF-8 antibody. Sera from pVAC-TTMs-vaccinated mice were able to reduce binding of the anti-GDF-8 antibody to myostatin (Fig. 6). The inhibition rates were 62.2% at 1:5 dilution and 30.0% at 1:40 dilution, respectively, suggesting that sera from pVAC-TTMs-vaccinated mice are able to interfere with myostatin in vitro.
Figure 5. ELISA analysis of antiserum titers from mice immunized with pVAC-TTMs. Recombinant myostatin was used as the antigen, and was probed with different concentrations of antisera from individual control or experimental mice. The OD450 was measured for each well following reaction with horseradish peroxidase. Average anti-serum titers were calculated from values displayed in the panel.
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Figure 6. Competitive myostatin binding assay using antisera from mice immunized with pVAC-TTMs or pVAC1-cms and commercial anti-GDF-8 antibody. Sera from BALB/c mice immunized with pVAC1-cms were used as negative control. The control anti-serum was B7H1 (unrelated protein).
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Serum levels of total protein, albumin, globulin, urea nitrogen, glucose, cholesterol, triglycerides, and creatine kinase in pVAC-TTMs-vaccinated mice and control mice were not significantly different.
DISCUSSION
Myostatin is highly conserved, both in sequence and in function, across animal species. The amino-acid sequences of mouse and human myostatin are identical.[5][15] Our results indicate that specific antibodies to the recombinant myostatin vaccine were effectively induced and the titers could inhibit myostatin in mice. Importantly, significant increases in body weight and skeletal muscle mass were observed following DNA vaccination. The effect of the myostatin DNA vaccine was comparable with other non-DNA vaccine methods of myostatin interference. For example, a recent study demonstrated that the body weights of mice treated with a neutralizing monoclonal antibody to myostatin increased by 10.0%.[23] Similarly, mice expressing a dominant-negative myostatin variant showed an increase of up to 35.0% in skeletal muscle mass.[25] Our results suggest that a myostatin DNA vaccine could promote an increase in skeletal muscle mass.
Interestingly, there was no significant difference in abdominal fat pad weighs and level of triglycerides and cholesterol in blood in vaccinated mice in our study. These results differ from the reported myostatin knockout phenotype, in which fat accumulation was decreased.[11][17] This discrepancy could be due to differences in loss of myostatin activity during development and during adulthood.[23] Our results suggest that the increase in body weight is caused by the increase in muscle mass but not in fat weight.
Although the muscle mass of mice immunized with the myostatin DNA vaccine was found to be increased significantly relative to the control group, this is not proof of increased muscle function. We therefore quantified functional improvement and found an increase in forelimb grip endurance in the mice immunized with the myostatin DNA vaccine. The increase in muscle endurance was proportional to the increase in muscle mass in the mice immunized with the myostatin DNA vaccine. However, more accurate methods than the grip test are necessary to determine the precise effect of myostatin DNA vaccine on muscle strength.
Our study has provided physiological evidence of functional improvement in muscle growth caused by myostatin inhibition in vivo. Myostatin activates the ubiquitin proteolytic system through a nuclear factor-kappaB (NF-B)-independent, FoxO1-dependent mechanism.[14] The central nervous system (CNS) and peripheral blood flow may play an important role in the activation and performance of myostatin, but it is unclear whether the inhibition of myostatin caused by myostatin DNA vaccine is related to them.
The manner in which muscle mass is increased is important. We found that the number of muscle fibers from mice immunized with the myostatin DNA vaccine did not increase but the muscle fibers were hypertrophic. By contrast, in mice expressing dominant-negative myostatin, muscle fibers showed both hypertrophy and hyperplasia.[15]
The physiological changes observed in the mice immunized with myostatin DNA vaccine were not accompanied by obvious detrimental changes in serum biochemistry, suggesting that vaccine-mediated inhibition of myostatin can increase skeletal muscle size without major side-effects. The functional enhancement of muscle by myostatin DNA vaccine thus provides a novel, pharmacological strategy for the treatment of diseases associated with muscle wasting. However, further safety studies and studies of the long-term effects of induced autoimmunity are required. The present study has provided the foundation for future work on this exciting and promising new strategy.
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Abbreviations
BSA, bovine serum albumin; CNS, central nervous system; ELISA, enzyme-linked immunosorbent assay; GDF-8, differentiation factor-8; LB, Luria-Bertani; NC, nitrocellulose; NF-B, nuclear factor-kappaB; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline/Tween; TT, tetanus toxin
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Clarityandfocus
New member
What Bobaslaw said. I agree with him.
However, yes, if the dose he is using produces questionable results, raising the dose would be the next logical step.
Inhibiting the myostatin gene by even 25% would cause a marked and observable growth of muscle mass because of increased satellite cell activity. Myostatin governs this and is it's primary constraint on hypertrophy/hyperplasia.
Unfortunately even a milligram is unaffordable for most of us and runs between $1500 and $2000 dollars. Black is a fortunate man if it is the real deal.
I knew it would only be a matter of time before the polyclonal antibody would ignite discussion on the boards.
Gene doping would produce the most effective and continuous results.
Clarity
Clarityandfocus
New member
the regular polyclonal at dosages of 80mcg/day DOES produce very decent muscle gains/fat loss. have witnessed it in more than one person. I would do a course laready if it wasnt for my upcoming surgery. but after that for sure, thats if the DNA vaccine doesnt come my way first.
That is cool that you have actually witnessed it yourself.
From what I understand some of the top super heavies have been using anti gdf-8 and IL-15 for quite awhile now. One IFBB pro who was maxed out and had been for some time added a fresh 30 lbs of pure lbm, a 15% increase in just 90 days. He was running the anti gdf-8, IL-15 and the usual regimen (gear, gh, igf, etc)... Author L Rea knows who the pro is but out of respect for the guy's privacy won't say and rightly so.
I don't make enough money to be able to afford polyclonal antibodies and pro- inflammatory cytokines. Props to those who do. :run:
Clarity
Speedbacker
Member
I've seen that posted somewhere before (Author L. Rea talking about IL-15, etc), I think it was in a q and a article.
Speedbacker
Member
Q and A with Author L. Rea
Q 2: One of the huge dude’s at the gym said that IL-15 will make anyone a freak. Is it true?
A 2:
INTERLEUKIN -15 (IL-15)
This is one of the newer drugs appearing on the bodybuilding scene that I would like to comment only briefly on. The human body produces several growth factors that are mediators and intermediates. In short this means they translate or decrease/increase the effect of hormones and other growth factors.
A study published in the Journal of Endocrinology in 1995 showed IL-15 doubled the rate of hypertrophy in skeletal muscle tissue. Interesting? Well the same study showed that stacking IL-15 with IGF-1 (insulin like growth factor-1; the stuff GH is converted into by the liver and other sites) increased muscular hypertrophy (excessive development/growth) by 500%. How is that for mediation?
I have known only a few athletes whom have utilized this stack, and to be honest, I have always believed (and seen that) freaks can be created even from those with below average genetics anyway. Yes, the results were amazing. The down side of IL-15 use is that lack of research. Some have speculated that IL-15 can trigger cancer cell growth. However, available research has not shown a connection between IL-15 and organ growth as of yet. I will not, at this point, explain reported cycles or use. There is not enough research as of yet concerning possible negative side effects. However as more research becomes available, you can bet I will be happy to share the reported results.
Obviously take it with a grain of salt, but that's (above) one man's opinion.
Q 2: One of the huge dude’s at the gym said that IL-15 will make anyone a freak. Is it true?
A 2:
INTERLEUKIN -15 (IL-15)
This is one of the newer drugs appearing on the bodybuilding scene that I would like to comment only briefly on. The human body produces several growth factors that are mediators and intermediates. In short this means they translate or decrease/increase the effect of hormones and other growth factors.
A study published in the Journal of Endocrinology in 1995 showed IL-15 doubled the rate of hypertrophy in skeletal muscle tissue. Interesting? Well the same study showed that stacking IL-15 with IGF-1 (insulin like growth factor-1; the stuff GH is converted into by the liver and other sites) increased muscular hypertrophy (excessive development/growth) by 500%. How is that for mediation?
I have known only a few athletes whom have utilized this stack, and to be honest, I have always believed (and seen that) freaks can be created even from those with below average genetics anyway. Yes, the results were amazing. The down side of IL-15 use is that lack of research. Some have speculated that IL-15 can trigger cancer cell growth. However, available research has not shown a connection between IL-15 and organ growth as of yet. I will not, at this point, explain reported cycles or use. There is not enough research as of yet concerning possible negative side effects. However as more research becomes available, you can bet I will be happy to share the reported results.
Obviously take it with a grain of salt, but that's (above) one man's opinion.
Speedbacker
Member
the regular polyclonal at dosages of 80mcg/day DOES produce very decent muscle gains/fat loss. have witnessed it in more than one person. I would do a course laready if it wasnt for my upcoming surgery. but after that for sure, thats if the DNA vaccine doesnt come my way first.
Just curious P, what kind of gains are you talking about? How long did they use the regular polyclonal for?
BLACK747
Member
my man speed wassup homeboy good find speed and bob hey clarity 650 dollars will get you 2 milligrams so its getting better and its the real deal im trying to get him to lower the price even further i forsee the price of this getting cheaper than igf-1r3 because the dose needed is much higher only time will tell speed pm me man
pumbertot
Active member
15lbs of lean muscle tissue (measured with scales and callipers) over a 2 month period at 80mcg/day of Polyclonal gdf-8 antibody for a couple of guys that are already near maxed out on gear/gh/igf/mgf. yes you can visibly see it. now im not 100% on what happens when they come off it of course but niether of them is planning to do so anytime soon.
and yes its getting cheaper all the time though 'my' supplier is still charging $650/mg so Black really has it cheap.
Bobaslaw
Member
Points of interest on the Myostatin DNA Vaccine Study:
Dosage per mouse was 50 grams once every 2 weeks. At least it took my mind off Gas Prices for a sec.
The results analysis below shows gains vs the control group, however, what surprised me was that it is primarily due to hypertrophy not hyperplasia. Results will most likely be no more permanent than with gear:
Results Analysis:
Dosage per mouse was 50 grams once every 2 weeks. At least it took my mind off Gas Prices for a sec.
The results analysis below shows gains vs the control group, however, what surprised me was that it is primarily due to hypertrophy not hyperplasia. Results will most likely be no more permanent than with gear:
Results Analysis:
pumbertot
Active member
Points of interest on the Myostatin DNA Vaccine Study:
Dosage per mouse was 50 grams once every 2 weeks. At least it took my mind off Gas Prices for a sec.
The results analysis below shows gains vs the control group, however, what surprised me was that it is primarily due to hypertrophy not hyperplasia. Results will most likely be no more permanent than with gear:
Results Analysis:
great post, as usual Prof Boba.
lol who cares if its not permanent, if i can have 4 injections for 2 years of no myostatin i will do it for life and be happy and die big. life is too short to be small.