Black Friday Sale at HorsePreRace.com

Tomorrow we will be having our annual Black Friday Sale. If you are not already signed up for our Newsletter you can do so by going to our homepage. Once you are signed up you will receive emails with coupons & discounts.

TB-500 CAMEL RACING

This is the most amazing supplement for Camel Racing ever created! If you want to win you have to try Thymosin Beta 4. Use the six injection series and see increased performance and endurance in your racing camel. Blood counts sore, muscles grow, ligaments and tendons heal, no more stomach ulcers! Truly amazing. Try it out by go here http://www.horseprerace.com/tb500-thymosin-beta-4-peptide-2ml-vial-p-157.html

TB 500 – In Stock

Don’t wait long as this product is sure to sell out quickly once again. This is the greatest performance product on the market today bar none.
-increase endurance
-stop bleeding
-eliminate ulcers
-reduce tie up
-repair soft tissue
-open airways
-increase red blood count

This is truly an amazing product that our customers have had amazing results with! Don’t miss out. You can read more and purchase right here!

TB-500 Benefits in Horses, Greyhounds, & Camels!

TB-500 has gained popularity faster than any other supplement in recent history. The list of effects goes on and on. We want to take this time to share the results of what our clients are telling us to better help you gain the maximum effect out of your product. First, most of our clients give there first shot the day after the animal has done hard work or raced. The shot is best given in the neck with a subcutaneous injection. Shots should be spaced 7 days apart. The most critical thing we have seen is hydration. You must provide ample water and if possible give up to 4 liters of fluids prior to event via IV This insures the animal is sufficiently hydrated. Some clients are still using there normal routine with vitamins and supplements throughout the week. We caution that TB500 will increase the blood count on it’s own so you need to watch the blood closely as too not get it too high. One last thing, some clients are reporting that after three consecutive injections skipping a week might be necessary due to excitability in the animal. Once we have more data on this amazing one of a kind product we will be sure to pass that along. Thank again

TB500

Thymosin Beta-4 and Chronic Wound Healing
First human trial tests thymic hormone in pressure sores

About three million people in the U.S., primarily elderly and bedridden, suffer from chronic pressure sores (“bedsores”) that do not heal. These kinds of infections can result in loss of limbs or, in some cases, even death. Because they require such long-term care, the cost of treating just one pressure wound can range from $14,000 to $50,000.

When a chronic wound occurs, it is because the normal process of healing has been disrupted. Many factors may be responsible for such interruption, including infection, systemic causes such as diabetes, and the use of certain medications such as corticosteroids.

Standard treatments for pressure sores and diabetic ulcers include agents to debride them (remove dead tissue), topical agents such as antimicrobials and enzymes, and various types of dressings. Other options include treatment by Vacuum-Assisted Closure or surgery. But even with these advanced strategies, the recurrence rate for chronic wounds remains high. What is needed is a way to help the body heal once infections develop.

NewYork-Presbyterian Hospital/Columbia University Medical Center is addressing this challenge in a study on thymosin beta-4, a naturally occurring protein that can reduce inflammation and help wounds to heal. “Thymosin beta-4 is a major activator of actin, which improves the process of wound healing,” says Mark A. Hardy, MD. Dr. Hardy is Auchincloss Professor of Surgery at Columbia University College of Physicians and Surgeons, and Director of Islet Transplantation at NewYork-Presbyterian Hospital/ Columbia University Medical Center.

In collaboration with Alan Goldstein, PhD, Dr. Hardy was the first to test new synthesized thymic hormones in 1968. Since then the hormones have been studied by several groups in adult patients with cancer, children with mucocutaneous candidiasis, and now in the healing of chronic wounds. “As a surgeon, I have had a long-term interest in finding ways of helping to speed up wound healing,” says Dr. Hardy.

Although thymosin has not proven as successful in treating cancer as researchers had hoped, studies have found that it does improve the healing of chronic wounds. Initial tests on pressure sores in animal models found that thymosin is active in several wound healing processes, according to June K. Wu, MD, Assistant Professor of Clinical Surgery at Columbia University College of Physicians and Surgeons, and Co-Principal Investigator with Dr. Hardy. The next phase of the current study will be among the first to test thymosin beta-4 in people with pressure sores.

The thymosin beta-4 trial, sponsored by RegeneRx Biopharmaceuticals, Inc., is a randomized, double-blind, placebo-controlled study. About 20 patients will be enrolled at NewYork-Presbyterian/Columbia, and three-quarters of these will receive topical thymosin for their chronic wounds. The safety of three doses will be evaluated in this phase of study. NewYork-Presbyterian/Columbia is one among four participating institutions.

“Finding an agent that can promote healing of chronic wounds, reduce the time it takes for them to heal, or reduce the rate of recurrence of wounds, would have the potential to improve the lives of many disabled patients,” says Dr. Hardy.

Based on the results of this phase of the trial, the investigators plan to expand the study to examine the effect of thymosin beta-4 on the rate of postoperative wound healing, and to study the mechanism of its activity. This could lead to the clinical application of this novel hormonal approach to the recovery of surgical patients whose wound healing is impaired by various diseases.

Researchers Report that Thymosin beta 4 Improves Neurological Function after Stroke

ROCKVILLE, Md.–(BUSINESS WIRE)–Mar 5, 2010 – REGENERX BIOPHARMACEUTICALS, INC. (NYSE Alternext US:RGN) announced that a research team from the Henry Ford Hospital in Detroit, MI reported that Thymosin beta 4 (Tβ4), administered to rats one day after embolic stroke, improved neurological functional outcome compared to control animals. Improvement in neurological function was measured at various time intervals over a seven week period and was statistically significant.

An increase in remyelination of axons (regeneration of the nerve sheath) was observed in rats receiving Tβ4 compared to control animals, likely due to an increased mobilization of oligodendrocyte progenitors (stem cells surrounding axons) that differentiate into mature myelin-producing oligodendrocytes. In cell culture, Tβ4 treated neuronal progenitor cells isolated from normal and stroke rats demonstrated increased mRNA levels of epidermal growth factor receptor. This receptor has previously been shown to be a regulator of oligoprogenitor cell expansion and tissue regeneration in response to brain injury and further supports the role of Tβ4 in stem cell-mediated tissue repair.

“These data are compelling and are consistent with previously reported data in EAE mice (experimental models for multiple sclerosis) showing that Tβ4 stimulates oligoprogenitor cells after injury. In this recent experiment, after an ischemic stroke, neurological function in the rat models was significantly improved, apparently by remyelination of neuronal axons induced by Tβ4. The fact that Tβ4 helps repair and regenerate tissue after a brain injury is not only remarkable, but strongly correlates with data previously published showing Tβ4′s ability to regenerate cardiac tissue after an ischemic event,” stated Dr. Hynda Kleinman, chief of the Cell Biology Section at the National Institute of Dental and Craniofacial Research, NIH, and a consultant to RegeneRx.

“We are very pleased with these results, which provide a foundation to further explore Tβ4 as a treatment for neurological injury,” commented Daniel C. Morris, MD, senior staff physician, Department of Emergency Medicine, Henry Ford Health Systems.

The research was presented by Dr. Morris, representing the Departments of Neurology and Emergency Medicine, Henry Ford Health System, Detroit, MI, at the International Stroke Conference, San Antonio, TX, February 23-26, 2010. The research was performed under a Material Transfer Agreement between RegeneRx Biopharmaceuticals, Inc. and the Henry Ford Health System.

About RegeneRx Biopharmaceuticals, Inc.

RegeneRx is focused on the discovery and development of novel peptides to accelerate tissue and organ repair. Currently, RegeneRx is developing three product candidates, RGN-137, RGN-259 and RGN-352 for dermal, ophthalmic, and cardiovascular tissue repair, respectively. These product candidates are based on Tβ4, a synthetic copy of a 43-amino acid, naturally occurring peptide, in part, under an exclusive world-wide license from the National Institutes of Health. RegeneRx holds over 60 world-wide patents and patent applications related to novel peptides. It is currently in Phase 2 clinical development for dermal and ophthalmic wound healing and has now completed a Phase 1 clinical trial supporting delivery of RGN-352 for acute cardiovascular and other indications requiring systemic administration. RegeneRx is also developing novel peptides for the cosmeceutical industry based on its experience with Tβ4 and its biological activities in the skin. It is currently in Phase 2 clinical development for dermal and ophthalmic wound healing and has now completed a Phase 1 clinical trial supporting delivery of RGN-352 for acute cardiovascular and other indications requiring systemic administration. RegeneRx is also developing novel peptides for the cosmeceutical industry based on its experience with Tβ4 and its biological activities in the skin.

RegeneRx Technology Background

Tβ4 is a synthetic version of a naturally occurring peptide present in virtually all human cells. It is a first-in-class, multi-faceted molecule that promotes endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, and collagen deposition, while down-regulating inflammation. RegeneRx has identified several molecular variations of Tβ4 that may affect the aging of skin, among other properties, and could be important candidates as active ingredients in pharmaceutical and consumer products. Researchers at the National Institutes of Health, and at other academic institutions throughout the world, have published numerous scientific articles indicating Tβ4′s in vitro and in vivo efficacy in accelerating wound healing and tissue protection under a variety of conditions. Abstracts of scientific papers related to Tβ4′s mechanisms of action may be viewed at RegeneRx’s web page: www.regenerx.com

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells

KM Malinda, AL Goldstein and HK Kleinman
National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.

Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.

Researchers Report Thymosin Beta 4 Significantly Reduces Damage From Traumatic Brain Injury And Improves Brain Function In Experimental Animals
RegeneRx Biopharmaceuticals, Inc. (NYSE Amex:RGN) announced today that in a preclinical research paper published in the May 2010 issue of the Journal of Neurosurgery, (online ahead of publication), scientists found that the systemic administration of thymosin beta 4, or Tβ4, significantly reduced brain tissue damage and improved brain function in rats with traumatic brain injury, or TBI. In the study, 10 rats were injected with Tβ4 one day following the inducement of TBI and four times thereafter over a 12-day period, while 9 rats were injected with a placebo or saline solution. In the group of rats treated with Tβ4, researchers observed reduced cell loss in the hippocampus, a part of the brain that plays an important role in long-term memory, as compared to the placebo group. The rats treated with Tβ4 also experienced growth of new blood vessels and neurons in the injured cerebral cortex, growth of brain cells known as oligodendrocytes in the CA3 field of the hippocampus, and recovery of sensory and motor functions as well as spatial learning. The researchers noted that the data for the first time demonstrate that delayed administration of Tβ4 significantly improves histological and functional outcomes in rats with TBI, indicating that Tβ4 has considerable therapeutic potential for patients with TBI.

“We believe these results are very encouraging. The fact that Tβ4 was administered beginning one day following injury and was still able to improve outcomes is significant,” added Dr. Allan L. Goldstein, professor of biochemistry and molecular biology at the George Washington University Medical School, and chairman of the board of directors and chief scientific advisor for RegeneRx. “We have now seen compelling data using Tβ4 in three different animal models – an EAE mouse model for multiple sclerosis, a rat model for embolic stroke, and this new study in traumatic brain injury – that have each demonstrated Tβ4′s ability to promote angiogenesis, regenerate neuronal tissue, and improve functional outcome. These data are also consistent with previously published studies showing regeneration of heart tissue after ischemic injuries to the myocardium.”

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes
A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.

PNAS
Proceedings of the National Academy of Sciences of the United States of America

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells
Abstract

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

Thymosin β4 (Tβ4) is a polypeptide involved in cellular proliferation, differentiation, and migration, over-expressed in several tumor entities. We evaluated its expression and function in 298 newly diagnosed multiple myeloma patients and the murine 5TMM model. Mean Tβ4 expression was significantly lower in myeloma cells compared to normal plasma cells (P<0.001). The same observation can be made in the 5TMM-mouse model by qRT-PCR and ELISA. Here, Tβ4 overexpression by lentiviral transduction of 5T33MMvt-cells led to significantly decreased proliferative and migratory capacities and increased sensitivity to apoptosis-induction. Mice injected with Tβ4 over-expressing myeloma cells showed a longer survival compared to mice injected with controls (88,9 vs. 65,9 days, P<0.05). In 209 MM patients treated with high-dose therapy and autologous stem cell transplantation, expression of Tβ4 below the median was associated with a significantly shorter event free survival (37.6 vs. 26.2 months, P<0.05). In conclusion, our results indicate a possible tumor suppressive function of Tβ4.

β-thymosins are a family of small peptides that were originally proposed to be thymic hormones.1 They were identified as actin monomer binding proteins, controlling the availability of actin for polymerization. They may, therefore, have a crucial role in regulating cellular functions involving actin polymerization/depolymerization cycles. Currently, 15 β-thymosins have been identified and characterized as highly conservative 5-kDa peptides containing 40 to 44 amino acid residues. In most mammalian tissues, thymosin-β 4 (Tβ4), the most abundant thymosin peptide, Tβ10 and Tβ15, have been studied as important members of the β-thymosin family.2 Several studies reported that these genes are over-expressed in solid tumors, which could be correlated to the angiogenic and metastatic potential of the studied tumors.3

Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells (PC) in the bone marrow (BM). MM cell biology can be dissected into the interactions of MM cells with their surrounding stroma (matrix proteins, cytokines and BM cells) and in the acquisition of essential changes in cell behavior, such as self-sufficiency in growth signals, evasion of apoptosis and acquisition of invasive and spreading capacities.4 Earlier reports indicated that Tβ4 was down-regulated in RNA from primary human MM cells and cell lines.5

This observation is in contrast to the results obtained in most solid tumors where an upregulation is seen in malignant cells compared to their normal counterparts. Cha et al. showed that overexpression of Tβ4 resulted in an increased metastatic capacity of lung cancer cells and increased angiogenic response.6

Since migration, invasion and associated angiogenesis are key features in MM biology, we were interested in studying Tβ4 expression in a large panel of MM patients and its functionality in the murine 5TMM model.

Gene expression analysis on human myeloma cells
Tβ4 expression was analyzed in purified PCs from BM samples obtained from 14 healthy donors, 11 patients with monoclonal gammopathy of unknown significance (MGUS) and 298 previously untreated multiple MM patients at the University Hospitals of Heidelberg or Montpellier.7 Of these, 209 MM patients were treated by high-dose therapy and autologous stem cell transplantation (ASCT). Biotinylated complementary RNA (cRNA) was amplified according to the Affymetrix labeling protocol (Affymetrix, Santa Clara, CA, USA). cRNA from a first group of patients (7 normal donors, 7 MGUS and 65 MM patients) was hybridized to the human U133 A and B. This group will be referred to as the HM1-group. A second independent validation group of patients (7 normal donors, 16 MGUS and 233 MM patients) was named the HM2 group. For these patients, the U133 2.0 GeneChip was used. These micro-array data had been previously used for several analyses, but thymosin β4 expression had never been analyzed before.8,9 HM2 data were corrected for batch effect due to the usage of different labeling kits according to Johnson et al.10 Expression data were gcrma-normalized and analyzed by the bioconductor packages for R. For patients’ characteristic see Online Supplementary Table S1.

The 5T2MM and 5T33MM murine models of myeloma
The 5TMM models originated in elderly C57Bl/KaLwRij mice.11 The 5T33MMvivo (5T33MMvv) cells grow in vitro stroma-dependently with a limited survival while the 5T33MMvitro (5T33MMvt) cell line is a clonally identical variant that originated from an in vitro culture of 5T33MMvv cells, growing BM stroma-independently in RPMI-1640 supplemented with 10% bovine serum 1% natriumpyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (all from Biowhittaker, Verviers, Belgium).12

Quantification of intracellular protein levels of Tβ4 and F-Actin G-Actin
Enzyme-Linked Immunosorbent Assays (ELISA) for measuring Tβ4 concentrations were performed according to the manufacturer’s instructions (Immundiagnostik, Bensheim, Germany). Cells (107) were lyzed in a phosphate buffer containing 0.14 M NaCl, 2.6 M KCl, 8 mM Na2HPO4, 1.4 M KH2PO4 and 1% Triton X100 and sonicated with an ultrasound finger. Protein levels and ratios between F-Actin and G-Actin were determined using the G-actin/F-actin in vivo assay kit (Cytoskeleton Inc, Denver, USA).

Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7700 Sequence Detection System. For the detection of both human and mouse Tβ4 mRNA and the endogenous reference gene GUS, Assays on Demand (Applied Biosystems) were used. To verify the results obtained with the microarrays studies, Tβ4 expression was measured in 3 cell lines and in 3 patient samples and their correlations statistically verified using a Spearman correlation test.

Generation of 5T33MMvt cells over-expressing Tβ4
A lentiviral transferplasmid encoding mouse Tβ4 (m Tβ4) was constructed. The mTβ4 gene was obtained from HJ Cha (NIDCR, NIH, Bethesda, USA)6 and inserted into the transferplasmid pHR’tripCMV-IRES-tNGFR-SIN.13 mTβ4-encoding lentiviral vector particles were produced in 293T cells, collected, ultracentrifugated and their viral titer determined.14 After transduction, 5T33MMvt cells were surface stained using an in-house biotinylated anti-tNGFR antibody and purified by FACS sorting into a 6-well plate (Becton Dickinson, FACSVantage). Next, they were analyzed for Tβ4 expression by RT-PCR. The 5T33MMvt cells over-expressing Tβ4 will be referred to as 5T33MMvtTβ4+.

In vitro and in vivo effects of Tβ4 overexpression
In vitro proliferation was assessed by measuring DNA synthesis using a 3H-thymidine incorporation assay, as described earlier.15 Apoptosis sensitivity of the MM cells was analyzed by staining with FITC labeled-annexin V and propidium iodide according to the manufacturers’ instructions (BD Biosciences, Erembodegem, Belgium). In vitro migration studies were performed using Transwell chambers and 10% fetal calf serum as chemoattractant and were quantified through flow cytometry. To determine the effect of Tβ4 overexpression on survival, groups of 10 C57BLKaLwRij mice were intravenously injected with either 5×105 5T33MMvtTβ4+ or wild-type 5T33MMvt cells. Animals were sacrificed when they showed signs of morbidity, namely hind limb paralysis. Kaplan-Meier analysis was used to determine a difference in the survival.

Different studies indicated a pivotal role of Tβ4 in the metastatic process of solid tumors.16,17 An adenoviral-based overexpression of Tβ4 was applied in a colon cancer and melanoma model showing increased growth, motility and invasive capacities in vitro and a larger tumor load in vivo.18,19 Since proliferation, migration and invasion are part of the hallmarks of the biology of MM, we were interested in investigating an involvement of Tβ4 in this disease. We first investigated the Tβ4 expression pattern in 298 primary MM-cell samples and 14 normal plasma cell samples from healthy donors. Tβ4 expression is significantly lower in MM cells of the HM1 group (P<0.05) and HM2 group (P<0.001) compared to normal plasma cells. This holds true for a significantly lower Tβ4 expression in its pre-malignant stage (MGUS), its early (Durie Salmon stage I) or late stage (Durie Salmon II and III) in both HM1 and HM2 groups (P<0.001) (Figure 1A). No relevant correlation could be found between Tβ4 expression and percentage of plasma cell infiltration in the bone marrow smear. Gene expression assessed by DNA-microarray correlates well with qRT-PCR performed on MM patient samples (coefficient of correlation r=0.993, P<0.001). These data are in agreement with results from Gondo et al. showing a decrease in Tβ4 expression in a small number of MM samples by Northern blot analysis.5

To assess the functional involvement of differential Tβ4 expression we used the 5T33MMvt and 5T33MMvtTβ4+ cell lines. In a 3H thymidine assay, 5T33MMvtTβ4+ cells showed a significant decrease in DNA synthesis compared to control cells (P<0.05). 5T33MMvtTβ4+ cells showed a significantly increased sensitivity to vinca-alkaloids (vinblastin) and bortezomib (Figure 2B; P<0.001 for both bortezomib and vinblastin).

Likewise, bortezomib induced apoptosis was higher in 5T33MMvtTβ4+ compared with 5T33MMvt cells (P<0.05; Figure 2C). In addition to affect survival pathways, Tβ4 overexpression reduced migratory capacities of 5T3MM cells; the percentages of cells that migrated in basal conditions and in 10%FCI was significantly lower in 5T33MMvtTβ4+ compared to control cells (P<0.05; Figure 2D). The relative increase after stimulation (compared to basal conditions) was, however, similar in both populations. We further examined the effects of Tβ4 expression on tumor development and survival of diseased mice by injecting mice intravenously with 5T33MMvtTβ4+ or control cells. In this study, the mean survival of mice injected with control cells was significantly shorter 65.9 days (SD 6.6 days), compared to 88.9 days (SD 9.3 days) for mice injected with 5T33MMvtTβ4+ cells (P<0.05; Figure 2F). These in vivo results confirm data obtained using the in vitro proliferation and apoptosis assays.

In solid tumors, Tβ4 expression is frequently upregulated in malignant and metastatic cells. In these cancers, higher Tβ4 expression resulted in increased metastatic and invasive capacities of tumor cells, while proliferation remained unaffected.6 In hematologic disorders, malignant plasma cell disorders, such as plasma cell leukemia and MM were the rare disorders that showed a decreased Tβ4 expression.5,20 In contrast to solid tumors, publications on the function of Tβ4 in hematologic conditions are scanty but indicate some inhibitory activity. Tβ4 was initially isolated and purified from a thymic protein preparation, called thymosin fraction-5. Addition of this protein fraction to different leukemic cell lines resulted in a decrease in growth responses.21 Similar inhibitory effects were recently described for Tβ4 on hematopoietic stem cells,22 bone marrow derived mast cells23 and human promyelocytic leukemia cells,24 in agreement with the results presented here. Whereas a mechanistic explanation of this discrepancy is beyond the scope of this paper, further investigations are clearly merited.

Since Tβ4 has been shown to bind G-actin in a 1:1 manner and thus affects the polymerization of G-Actin into F-Actin, we analyzed in a semi-quantitative way, intracellular G-actin and F-Actin. This quantification showed a lowered G-Actin-F-Actin ratio after Tβ4 overexpression (Figure 2E). F-Actin is of particular importance in cytoskeleton changes involved in cellular migration and in microtubuli organization controlling the mitotic spindle.25,26 In line with these results, vinca-alkaloids (e.g. vinblastine used here) with micro-tubulin (polymerization) inhibitory activity, had more affect on the proliferation capacities of 5T33MMvtTβ4+ cells than on control cells (Figure 2B). Since immunohistochemical studies also showed a nuclear staining of Tβ4 in 5TMM cells (results not shown), involvement of other pathways might also be implicated. Supervized gene analysis comparing Tβ4high with Tβ4low found different groups of genes differently expressed, including genes involved in cytoskeleton organization, nuclear homeostasis, lymphocyte differentiation and protein metabolism, which might indicate that the role of Tβ4 is more complicated than initially supposed.

In conclusion, our results propose a tumor suppressive function of Tβ4 expression in MM with impact on survival. Tβ4 was down-regulated in MM cells of patients compared to the normal BM plasma cells and studies with the murine 5T33MM model show a decreased in vitro and in vivo tumor growth for cells over-expressing the Tβ4 gene.

Blood Builders

Instead of posting information already on the net we will try to give you the information we send to our customers. Many of you ask, “What are the best Blood Builders?” First off, you can only make the blood so good. There is a limit to everything. Getting a good baseline is very important so you do not cause “sledging” This occurs when the blood count and packed cell volume goes too high.
After you have determined your baseline and still feel that the blood needs to be increased you can begin with the basics. The group of basics includes Cacco Copper, Folic Acid, B12, & B Complex. These are all good products to help a “normal” horse increase there red blood count.
The next group we will call the moderately aggressive group. This group is used for horses that are under the stress of racing and are having a difficult time producing red blood cells. This group includes Hemo 15, Hemoplex, Hippiron, & Newcells.
The final group is the aggressive blood builders. These are products that increase the blood dramatically but can also be detrimental if the blood is already in a good state. This group includes Blood Building Peptide, Liquid Aranesp, & Redtide.
You can click on any one of these products to read more about them and purchase them.

Baycox

How are you using Baycox? We receive many questions on this subject so we thought it be best to give you the most popular information we have. Many trainers like to use Baycox as a pre race by giving it 48hrs. prior to the event. Most believe this works well because the Toltrazuril paralyzes the EPM and also helps the horse to breath better.
If the horse has a severe case of EPM and displays signs “the wobbles” you can give one bottle seven days prior to the event then again two days prior to the event. This will work 98% of the time in our opinion. If after two treatments within 7 days you don’t see noticeable results you should consult your veterinarian. If you have any other ways you use Toltrazuril or Baycox please feel free to share below. You can purchase Baycox/ Toltrazuril by clicking on this link.

ITPP

Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate

1. Andreia Bioloa,
2. Ruth Greferathb,c,
3. Deborah A. Siwika,
4. Fuzhong Qina,
5. Eugene Valskya,
6. Konstantina C. Fylaktakidouc,1,
7. Srinivasu Pothukanuric,
8. Carolina D. Duartec,
9. Richard P. Schwarzb,
10. Jean-Marie Lehnc,2,
11. Claude Nicolaub,c,d,2 and
12. Wilson S. Coluccia,2

+ Author Affiliations

1.
aCardiovascular Section, Department of Medicine, Boston University Medical Center, and Myocardial Biology Unit, Boston University School of Medicine, Boston, MA 02115;
2.
bNormOxys, Inc., 200 Boston Avenue, Medford, MA 02155;
3.
cInstitut de Science et d’Ingénierie Supramoléculaires, Université Louis Pasteur, 8 Allée Gaspard Monge, 67000 Strasbourg, France; and
4.
dFriedman School of Nutrition Science and Policy, Tufts University, Boston, MA 02115

1.

Contributed by Jean-Marie Lehn, December 31, 2008 (received for review December 1, 2008)

Next Section
Abstract

A major determinant of maximal exercise capacity is the delivery of oxygen to exercising muscles. myo-Inositol trispyrophosphate (ITPP) is a recently identified membrane-permeant molecule that causes allosteric regulation of Hb oxygen binding affinity. In normal mice, i.p. administration of ITPP (0.5–3 g/kg) caused a dose-related increase in the oxygen tension at which Hb is 50% saturated (p50), with a maximal increase of 31%. In parallel experiments, ITPP caused a dose-related increase in maximal exercise capacity, with a maximal increase of 57 ± 13% (P = 0.002). In transgenic mice with severe heart failure caused by cardiac-specific overexpression of Gαq, i.p. ITPP increased exercise capacity, with a maximal increase of 63 ± 7% (P = 0.005). Oral administration of ITPP in drinking water increased Hb p50 and maximal exercise capacity (+34 ± 10%; P < 0.002) in normal and failing mice. Consistent with increased tissue oxygen availability, ITPP decreased hypoxia inducible factor-1α mRNA expression in myocardium. It had no effect on myocardial contractility in isolated mouse cardiac myocytes and did not affect arterial blood pressure in vivo in mice. Thus, ITPP decreases the oxygen binding affinity of Hb, increases tissue oxygen delivery, and increases maximal exercise capacity in normal mice and mice with severe heart failure. ITPP is thus an attractive candidate for the therapy of patients with reduced exercise capacity caused by heart failure.
Keywords:

* hypoxia
* oxygen delivery

A major determinant of exercise capacity is the amount of oxygen available to the exercising muscles. Heart failure is characterized by a reduction in exercise capacity caused primarily to the inability of the cardiovascular system to increase cardiac output and hence, blood flow, to exercising muscles (1–5). Accordingly, most previous efforts to improve exercise capacity in patients with heart failure have relied on interventions that increase the pumping function of the heart and/or the distribution of blood flow to exercising muscles. Increasing the concentration of Hb also enhances the delivery of oxygen to exercising muscles, and in patients with concurrent heart failure and anemia the administration of erythropoietin to increase the red cell mass may increase maximal exercise capacity (6, 7).

The increased delivery of oxygen to exercising muscles might also be achieved at a constant Hb concentration by decreasing the oxygen binding affinity of Hb so as to release more oxygen in hypoxic tissues. The organic phosphate 2,3-bisphosphoglycerate (2,3-BPG) is the natural allosteric effector that decreases the oxygen binding affinity of human Hb, and increased levels of 2,3-BPG appear to play a compensatory role in a variety of circumstances including high altitude and chronic pulmonary disease (8, 9). It has been noted that red blood cell 2,3-BPG levels are increased in patients with low output heart failure, leading to the suggestion that interventions to further decrease Hb oxygen binding affinity might be of clinical value in such patients (10).

myo-Inositol hexakisphosphate (IP6) is a powerful allosteric effector of Hb that increases the regulated oxygen-releasing capacity of RBC. In piglets, transfusion of RBC loaded ex vivo with IP6 led to physiologic effects consistent with increased oxygen delivery (11, 12). Because at neutral pH, IP6 is a partially dissociated polyanion bearing 8 negative charges (13), it is unable to cross the RBC membrane and ex vivo physical methods are required to load erythrocytes (11, 14). However, we recently showed that myo-inositol trispyrophosphate (ITPP) hexasodium salt is capable of crossing the RBC plasma membrane and acting as an allosteric effector of Hb, shifting the oxyhemoglobin dissociation curve to higher oxygen pressures (pO2) (15, 16), and leading to a marked inhibition of blood vessel formation processes in vitro (16). Accordingly, we tested the hypothesis that systemic administration of ITPP would increase exercise capacity in normal mice and in mice with severe exercise limitation caused by reduced cardiac output as a result of myocardial failure.
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Results and Discussion
ITPP Decreases the Oxygen Binding Affinity of Hb in Vivo.

To determine whether ITPP increases the tissue availability of oxygen in vivo, ITPP was administered to normal mice by i.p. injection and the oxygen binding affinity of Hb was measured 18–24 h later. In blood from control mice, the pO2, at which Hb is 50% saturated with oxygen (p50) averaged 37.3 ± 1.5 torr. ITPP caused a dose-related right shift in the oxygen dissociation curve, resulting in a mean 22% increase in p50 at a dose of 1 g/kg, and a mean 37% increase with the 2 g/kg dose (Fig. 1A). There was little or no effect of ITPP on maximal oxygen saturation at pO2 of >100 torr (Fig. 1B). Thus, although ITPP had no significant effect on total oxygen carrying capacity, the right shift in the oxygen dissociation curve at O2 tensions in the physiologic range suggests that a larger fraction of O2 would be released at the lower oxygen tension in the tissue. After a single ITPP administration, the p50 increase was sustained for 48 h, had decreased by ≈50% in 5 days, and was no longer present after 12 days. The prolonged duration of the effect, which is consistent with the half-life of RBC in mice, suggests that the effect of ITTP on Hb is relatively long-lived.
Fig. 1.
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Fig. 1.

ITPP increases Hb oxygen dissociation in vivo. ITPP was administrated by i.p. injection to normal mice, and Hb p50 (the pO2 at which Hb is 50% saturated with oxygen) was measured 24 h later by using a HEMOX analyzer. (A) Mean p50 values measured in blood samples from mice receiving placebo (n = 5), 1 g/kg ITPP (n = 5), or 2 g/kg ITPP (n = 4), demonstrating a dose-related increase in p50. (B) Representative Hb oxygen dissociation curves for the 3 treatment groups. ITPP administration caused a parallel right-shift in the dissociation curve, thus increasing the O2-releasing capacity at physiologic pO2 levels while having little or no effect on total O2 carrying capacity.
ITPP Suppresses Hypoxia-Inducible Factor (HIF) 1α in Myocardium in Vivo.

To further test the ability of ITPP to increase oxygen delivery to tissues in vivo, we measured the expression of HIF 1α mRNA in myocardium from normal mice. In normal mice, ITPP administration (2 g/kg, i.p.) decreased the level of myocardial HIF mRNA, measured 3 days after administration, from 5.5 ± 1.4 to 1.7 ± 0.6 arbitrary units (P = 0.06; n = 3 per group). These data are consistent with the thesis that the ITPP-induced decrease in Hb oxygen affinity, as reflected by the increase in p50, results in increased availability of oxygen at the tissue level.
ITPP Increases Exercise Capacity in Normal Mice.

Because ITPP should increase the availability of oxygen at the tissue level, we hypothesized that ITPP would increase exercise capacity in vivo in mice. We therefore measured the maximal exercise capacity of normal mice by using a progressive workload motorized treadmill with air puff motivation to ensure maximal effort. Maximal exercise capacity was measured in a blinded manner at baseline and again 24 h after the i.p. injection of ITPP or placebo. The exercise capacity was similar in all groups at baseline (Fig. 2). Whereas placebo had no effect on maximal exercise capacity, ITPP caused a dose-related increase of up to ≈50%, with a plateau in effect between the 2 highest doses of 2 and 3 g/kg.
Fig. 2.
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Fig. 2.

ITPP increases exercise capacity in normal mice. Maximal exercise capacity was determined as the maximal distance run (meters) until exhaustion on a motorized treadmill by using air-puff stimulation. Bars represent distance run at baseline (B) and 16–24 h after i.p. administration of ITPP (T) in doses ranging from 0.5 to 3 g/kg or placebo. *, P < 0.05 vs. baseline; n = 22 in baseline group, 4–7 in each treatment group.
ITPP Improves Exercise Function in Mice with Severe Heart Failure.

To test the hypothesis that ITPP would increase exercise capacity in animals with heart failure, we used transgenic mice with dilated cardiomyopathy caused by cardiac-specific overexpression of Gαq, as described (17). These mice have severe left ventricular (LV) dilation (LV end-diastolic dimension = 4.3 ± 0.4 mm vs. 3.0 ± 0.1 mm in normal mice; P < 0.001; n = 10) and a markedly reduced LV fractional shortening (29 ± 4% vs. 62 ± 2% in normal mice; P < 0.001; n = 10). Baseline maximal exercise capacity is consequently severely depressed to ≈60% of that in normal mice (Fig. 3A). ITPP administration caused striking dose-related increases in maximal exercise capacity of 34% and 71% for the 1 and 2 g/kg doses, respectively, whereas placebo had no effect (Fig. 3A). The magnitude of the ITPP-induced increase in exercise capacity was related to the increase in Hb p50 (Fig. 3B).
Fig. 3.
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Fig. 3.

ITPP restores exercise capacity in mice with severe heart failure. Transgenic mice with cardiac-specific overexpression of Gαq have an ≈40% reduction in maximal exercise capacity compared with normal mice. (A) Distance run at baseline (B) and 16–24 h after administration of ITPP (T) or placebo. *, P < 0.05 vs. baseline distance; n = 5–6 per group. (B) Relationship between the ITPP-induced changes in p50 and maximal exercise capacity, as displayed in A.

It has recently been reported that, in mice, bone marrow transplantation with a Hb variant having a low oxygen affinity or i.v. administration of another type of allosteric Hb modifier increased the voluntary running time (19). Whereas measurement of voluntary running time precludes conclusions about possible effects on maximal exercise capacity, we used here an exercise protocol designed to measure maximal exercise capacity, which is directly related to the maximal oxygen delivery.

Our findings suggest that increasing the availability of oxygen with ITPP may be a means of improving maximal exercise in patients with heart failure who have impaired exercise capacity caused by reduced cardiac output. However, it should be noted that the causes of reduced exercise in patients with heart failure are incompletely understood and may involve multiple factors other than oxygen delivery, including deconditioning, atrophy, and/or metabolic dysfunction of the skeletal (18). It is also possible that the Gαq mouse model of dilated heart failure may not fully reflect these and other causes of exercise impairment in humans.
Oral ITPP Increases p50 and Exercise Capacity.

Previous efforts to modify RBC Hb affinity pharmacologically have been limited by the failure of effector molecules to cross RBC membranes (11, 14), short duration of effects (19), and poor solubility in water (20). Because ITPP is both membrane permeant and readily soluble in water (15, 16), we considered the possibility that ITPP would be absorbed after oral ingestion.

To determine whether oral ingestion of ITPP causes a shift in Hb oxygen affinity, normal mice were allowed to drink ad libitum water in which ITPP or IP6 was dissolved at a concentration of 20 mg/mL. Hb p50 increased by ≈16% in mice that drank ITPP, but was unchanged in mice that drank water (+3%) or water with IP6 (+3%), which is membrane impermeant. To determine whether oral ingestion would also improve exercise function, maximal exercise capacity was determined in normal mice (n = 4) and mice with heart failure caused by Gq overexpression (n = 2) before and after drinking water containing ITPP (20 mg/mL, ad libitum) for 4–8 days. Oral ingestion of ITPP increased maximal exercise capacity by an average of 34 ± 10% (P < 0.002; n = 6).
Lack of Direct Vascular and Cardiac Effects of ITPP.

Pharmacologic agents that exert vasodilator and/or positive inotropic effects may increase cardiac output, blood flow to skeletal muscle, and exercise capacity in heart failure (21). To determine whether ITPP exerts vasodilator effects, blood pressure was measured in 12 normal mice at baseline and 24 h after i.p. injection of ITPP (2 g/kg) or placebo. Neither ITPP nor placebo affected systolic blood pressure (before ITPP = 134 ± 6 mm Hg; after ITPP = 135 ± 4 mm Hg; P not significant), diastolic blood pressure (before ITPP = 76 ± 12 mm Hg; after ITPP = 73 ± 4 mm Hg; P not significant), or heart rate (before ITPP = 706 ± 12; after ITPP = 720 ± 17 beats per min; P not significant).

To determine whether ITPP exerts a direct positive inotropic effect, contractile properties were measured in freshly-isolated adult rat ventricular myocytes, as described (22). ITPP (500 μM; 10 min) had no effect on baseline myocyte sarcomere shortening (−9 ± 29%; P not significant) or on the rate of sarcomere shortening (−17 ± 27%; P not significant), whereas the positive control norepinephrine caused 4- to 6-fold increases in sarcomere shortening (+405 ± 155%, P < 0.01) and the rate of shortening +670 ± 170%; P < 0.01).
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Conclusion

The present results show that i.p. and oral administration of ITPP cause a decrease in Hb-oxygen affinity that is associated with a parallel, dose-related increase in maximal exercise capacity in both normal mice and mice with reduced exercise capacity caused by severe myocardial failure. These observations demonstrate that allosteric modulation of Hb oxygen binding affinity can exert clinically meaningful effects on maximal exercise capacity. Accordingly, ITPP is an attractive therapeutic candidate to alleviate symptoms in patients with reduced exercise capacity caused by low cardiac output heart failure. ITPP may also enhance physical performance of otherwise healthy individuals, in particular, under extreme conditions such as high attitude or intense physical exercise.
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Methods
Synthesis of ITPP.

This membrane-permeant allosteric effector of Hb was synthesized as reported (15), by using an improved procedure. Briefly, IP6 dodecasodium salt (Sigma) was converted via its perprotonated form, obtained by passage over Dowex 50 resin (Sigma–Aldrich) in its H+ form, into its triethylammonium salt and then, upon heating with dicyclohexylcarbodiimide (Sigma–Aldrich) in acetonitrile/water 2/1 solution, triply cyclized to give the triethylammonium salt of ITPP. Thereafter, an aqueous solution of the latter was passed over Dowex Marathon C Na+ resin (Sigma–Aldrich) until cation exchange of triethylammonium against sodium was complete. Evaporation of the aqueous solution gave the sodium salt of ITPP in high purity (≈90% yield).
p50 Measurements.

The p50 value (pO2 at which 50% of Hb is saturated with O2), a measure of the affinity of Hb for oxygen, was determined by using a HEMOX Analyzer (TCS Scientific), by constructing Hb dissociation curves based on dual wavelength spectrophotometry as described (16).
RT-PCR for HIF-1α mRNA.

Total RNA was extracted from mouse hearts by using a Total RNA Purification System (Invitrogen). Quantitative RT-PCR was performed by using cybergreen and the I-cycler iQ RT-PCR (Bio-Rad). The primers for HIF-1α were: 5′-TCAAGTCAGCAACGTGGAAG-3′ and 5′-TATCGAGGCTGTGTCGACTG-3′.
Exercise Testing.

Maximum exercise capacity was measured by using a rodent treadmill equipped with an air puff motivator (Columbus Instruments) as described (23). Animals were familiarized with running on the treadmill. For tests, the treadmill was set at a constant incline of 15°, and the initial speed of 15 m/min was increased by 3 m/min every 2 min. Total exercise time was recorded as the elapsed time to exhaustion and then converted to distance. Exhaustion was determined by an observer blinded to treatment group and was defined as the point at which the animals could not keep pace with the treadmill and had no response to the air puff stimulus. All exercise evaluations were performed twice and the results were averaged.
Gαq Transgenic Mice.

Gαq transgenic mice were kindly provided by Gerald W. Dorn II (University of Cincinnati, Cincinnati) (17) and subsequently bred at Boston University Medical Center. This mouse overexpresses Gαq exclusively in the myocardium under the myosin heavy chain promoter. The present study used the Gαq line that overexpresses 40 (Gαq40) copies of the transgene on a FVB background, which leads to a 5-fold increase in the transgene protein levels. Age-matched controls were bred by using heterozygote Gαq male mice with WT females obtained from Charles River. Animals were studied at 12–14 weeks of age. The Institutional Animal Care and Use Committee at Boston University School of Medicine approved all study procedures and use of animals.
Statistical Analysis.

Data are shown as mean ± SEM. Group comparisons were performed by using Student’s t test (2 groups) or 1-way ANOVA (multiple groups). Baseline versus treatment comparisons were performed by paired t test. All tests were 2-tailed, and P < 0.05 was considered significant.

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