Mahnaz Badamchian

Thymosin β4 and a synthetic peptide containing its actin‐binding domain promote dermal wound repair in db/db diabetic mice and in aged mice

Impaired wound healing is a problem for immobilized patients, diabetics, and the elderly. Thymosin β4 has previously been found to promote dermal and corneal repair in normal rats. Here we report that thymosin β4 was also active in accelerating wound repair in full‐thickness dermal wounds in both db/db diabetic and aged mice. We found that thymosin β4 in either phosphate‐buffered saline or a hydrogel formulation is active in promoting dermal wound repair in normal rats. In diabetic mice, where healing is delayed, we found that wound contracture and collagen deposition were significantly increased in the mice treated with thymosin β4 in either phosphate buffered saline solution or a hydrogel formulation. No difference was observed in keratinocyte migration, with all of the diabetic animals showing almost complete coverage of the wound at 8 days. Wound healing in 26‐month‐old (aged) animals was significantly delayed. Thymosin β4 accelerated wound healing in these aged mice, with increases in keratinocyte migration, wound contracture, and collagen deposition. The hydrogel formulation generally showed similar wound healing activity with thymosin β4 in PBS. The actin‐binding domain of thymosin β4 duplicated in a seven‐amino acid synthetic peptide, LKKTETQ, was able to promote repair in the aged animals comparable to that observed with the parent molecule. These studies show that thymosin β4 is active for wound repair in models of impaired healing and may have efficacy in chronic wounds in humans. (WOUND REP REG 2003;11:19–24)

Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum ☆

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3-6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship.

Thymosin β4 reduces lethality and down-regulates inflammatory mediators in endotoxin-induced septic shock

Thymosin beta(4) (Tbeta(4)), a highly conserved peptide with immunomodulatory properties, is the major actin-sequestering peptide in mammalian cells. Recent studies have established that Tbeta(4) can accelerate wound healing in full thickness skin wounds and following burn injuries to the cornea. In the eye studies, the accelerated healing due to Tbeta(4) was accompanied by a significant reduction in polymorphonuclear leukocyte (PMN) infiltration and a several-fold decrease in interleukin-1beta (p< or =0.015) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF1alpha, p< or =0.05). Given the recognized role of proinflammatory cytokines in septic shock and of extracellular F- and G-actin in the pathophysiology of multiple organ dysfunction, we have investigated the role of Tbeta(4) in sepsis. We report that an LD(50) dose of LPS (24 mg/kg) in rats resulted in a significant reduction of Tbeta(4) levels in the blood. Furthermore, administration of 100 microg of Tbeta(4) immediately following and at 2 and 4 h after an LD(50) dose of LPS (60 mg/kg) in mice significantly reduced mortality rates (p< or =0.024) and lowered blood levels of a number of inflammatory cytokines, eicosanoids, and other molecules that are highly elevated following endotoxin administration. In studies in human subjects given low doses of endotoxin (4 ng/kg LPS) and in patients with septic shock, we have also observed significant decreases in blood levels of Tbeta(4). The rapid disappearance of Tbeta(4) in the blood following LPS administration or during septic shock suggests that Tbeta(4) may be involved in early events leading to activation of the inflammatory cascade and ultimately the clinical sequelae of sepsis. The results of this study indicate that Tbeta(4) may have utility in the clinic in the treatment of septic shock and in syndromes associated with actin toxicities.

Thymosins: chemistry and biological properties in health and disease

This paper will review the historical background that has generated our present interest in the actions of the thymosins in biological therapy. It will also discuss the multiple actions of the thymosins in the immune, endocrine and central nervous systems. The isolation from the thymus gland of the thymosins, a family of biologically active molecules with hormone-like properties, was first described in 1966 by AL Goldstein and A White. Since that time, significant progress has been made in understanding the role of the thymosins in immunity and the nature of the growth factors, cytokines and chemokines they modulate. The thymosins include a family of biochemically and functionally distinct polypeptides with clinically important physiological properties. In the early 1970s, preclinical studies establishing the immunorestorative effects of a partially purified thymosin preparation termed thymosin fraction 5 (TF5) provided the scientific foundation for the first clinical trials with TF5 in 1974. TF5 was effective in turning on the immune systems of a number of children with DiGeorge syndrome and other thymic dysplasias. These trials led to further interest in the active components in TF5 and to the chemical characterisation of the biologically active thymosins. Several of these molecules are showing significant promise in the clinic in the areas of cancer, infectious diseases and wound healing.

Thymosin α1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways

It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.

Biodistribution of synthetic thymosin β4 in the serum, urine, and major organs of mice

Thymosin beta 4 (T beta 4) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. T beta 4 functions mainly as an actin-sequestering molecule in nonmuscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of T beta 4 in human and other mammals have not been reported so far. In the present study, we have measured T beta 4 concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 micrograms synthetic T beta 4. Using a modified enzymatic immunoassay, our data show a significant increase of T beta 4 in serum starting 2 min after injection and lasting for 40 min (average: 2.34 +/- 0.54 micrograms/ml). High concentrations were found in urine (59.3 +/- 7.54 micrograms/ml) at three different points after injection (20 min, 40 min, and 2 h). Of the 400 micrograms T beta 4 administered to mice 83% was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of T beta 4 increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 micrograms/g), heart (80 micrograms/g), liver (15 micrograms/g vs 9 micrograms/g), kidneys (65 micrograms/g vs 28 micrograms/g), and peritoneal fat (47 micrograms/g vs 13 micrograms/g). Wet weight concentrations increased in the thymus (196 micrograms/g vs 147 micrograms/g) and muscle (45 micrograms/g vs 0 micrograms/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 micrograms/g vs 161 micrograms/g). Ovaries had a biphasic increase at 40 min (72 micrograms/g vs 62 micrograms/g) and 24 h (92 micrograms/g vs 62 micrograms/g) after T beta 4 administration. In lungs, the highest wet weight increase after injection (149 micrograms/g at timepoint 6 h) was not higher than its basal wet weight concentration (153 micrograms/g). These pharmacokinetic studies of T beta 4 in mice have established that high levels of T beta 4 are found in blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of T beta 4 during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.

Identification and Quantification of Thymosin β4 in Human Saliva and Tears

Abstract:  Thymosin β4 (Tβ4) is a ubiquitous, naturally occurring, 43‐amino acid peptide that takes part in several biological activities including angiogenesis, inhibition of inflammation, wound healing, chemotaxis, and endothelial cell migration. Recent studies also indicate that Tβ4 accelerates corneal wound healing and downregulates several proinflamatory chemokines and cytokines. In this study, we sought to determine whether Tβ4 is naturally occurring in human tears and other human bodily fluids, such as saliva. Tear and saliva samples were analyzed by EIA to identify and quantify the amount of Tβ4 present. Around 10–20 samples were collected from each of three different age groups: 15–20, 25–35, and >50 years old with n= 30 and n= 60 for tears and saliva, respectively. Exclusion criteria included the use of any topical ophthalmic or topical oral medication and/or history of ocular or oral surgery within the past 6 months. Tears were collected from both eyes using Schirmers strips. Saliva samples were collected in sterile tubes and were then centrifuged to remove solid particles. Tβ4 was found in tear and saliva samples in all age groups. The concentrations ranged from 0.5–7 μg/mL in tears and 0.2–3.6 μg/mL in saliva. In both fluids, Tβ4 concentration varied with age and appeared to peak at ages 25–35 years. Studies are in progress to determine if Tβ4 levels in saliva and tears demonstrate a circadian rhythm during a 24‐h period, as well as to confirm that they vary with age and to explore if they vary with diseased states. This is the first study to report the presence of Tβ4 in human tears and saliva. This finding raises the possibility that Tβ4 acts as an endogenous agent contributing to the rapid healing of corneal and oral wounds. Considering that Tβ4 facilitates reepithelialization and modulates anti‐inflammatory mediators, Tβ4 could potentially be used therapeutically in the treatment of (a) ocular surface disease and injury of eye and (b) various oral disorders, such as periodontal disease.

Thymosin α1 is a time and dose-dependent antagonist of dexamethasone-induced apoptosis of murine thymocytes in vitro

It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptosis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin alpha1 (Talpha1) antagonizes dexamethasone-induced apoptosis of CD4+CD8+ thymocytes. In the present study, we further characterize the dose and time dependence of Talpha1s antagonism of dexamethasone-induced thymocyte apoptosis. Talpha1 is effective at concentrations ranging from 2 to 100 microg/10(6) thymocytes. Talpha1 pre-treatment is necessary to achieve its anti-apoptotic activity. Talpha1 provides temporary protection to thymocytes by slowing dexamethasones apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Talpha1s activity is not sensitive to cycloheximide treatment, suggesting Talpha1s activity is independent of protein synthesis. Finally, Talpha1 is unable to antagonize apoptosis induced by the reactive oxygen species, H2O2, suggesting Talpha1s antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Talpha1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.

Characterization of Choline O-Acetyltransferase (ChAT) in the BALB/C Mouse Spleen

The synthesizing enzyme, Choline-O-acetyl transferase (ChAT) (EC 2.3.1.6) and the degradation enzyme, acetylcholinesterase (EC 3.1.1.7) for the neurotransmitter acetylcholine, have been anatomically and biochemically characterized in the thymus of the BALB/C mouse. In the present study we continue to analyze the possibility of cholinergic immunomodulation of immune tissues by determining if ChAT is present in the BALB/C mouse spleen. Our enzymatic evaluation of ChAT activity in splenic extracts revealed .05 nmoles/min/mg protein as compared to .1 nmoles/min/mg of protein activity in controls prepared from whole brain extracts. No detectable levels of ChAT activity were observed in the serum. Immunoblotting and immunoprecipitating using the anti ChAT monoclonal antibody, MB16, demonstrated two bands in the brain and one band in the spleen. Membrane bound ChAT in the brain was composed of two subunits with apparent molecular weights of 28 and 50 kDa. The spleen demonstrated only one form of ChAT with an apparent molecular weight of 28 kDa. Immunoprecipitation of the enzyme from both the brain and spleen resulted in a recovery of 59% and 60% of the activity respectively.

Isolation of a vitamin E analog from a green barley leaf extract that stimulates release of prolactin and growth hormone from rat anterior pituitary cells in vitro

Abstract We have previously reported that green barley leaf extract, a dried extract of young green barley leaves that is widely used in Japan and other countries as a nutritional supplement, contains a molecule(s) that enhances the release of growth hormone and/or prolactin from rat anterior pituitary cells in vitro. Using reverse-phase high performance liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS) we have now isolated and chemically characterized the molecule possessing this neuroendocrine activity. The molecule is α-tocopherol succinate, an analog of α-tocopherol or vitamin E. This is the first report documenting a role for this vitamin E analog on pituitary endocrine release.