Exercise intensity and duration affect blood soluble HSP72.

Soluble heat shock protein 72 (sHSP72) is suggested to play a role as a signalling molecule in the immune response to exercise. We were interested in whether duration and intensity of endurance running affect the level of inducible sHSP72 in the plasma/serum of endurance athletes. In the first part of the study, the influence of a continuous treadmill run of 60 min (CR) with an intensity of 75 % VO2max, a long treadmill run of 120 min (LR) with an intensity of 60 % VO2max, an extensive interval training program (IT; 10 x 1000 m, ca. 35 min, VO2max 88 %), and a competitive marathon run (MA) within 260 +/- 39 min (VO2max ca. 65 %) on the release of sHSP72 into the peripheral blood was tested. Blood samples were drawn before and directly after exercise, as well as 0.5, 1, 3, 24 h after exercise to determine sHSP72 levels. Secondly, we compared the effects of two exercise bouts with identical duration (23.7 +/- 7 min) but different intensities (Exhaustive exercise (ET) at 80 % VO2max vs. moderate exercise (MT) at 60 % VO2max) on sHSP72 concentration. The sHSP72 levels in plasma/serum were analyzed using an enzyme immunoassay specific for inducible HSP72 (Stressgen,Victoria, Canada). Early, significant increases of sHSP72 were detected immediately after all types of exercise with highest levels after MA. ET induced significantly higher levels of sHSP72 compared with MT. Long-lasting, competitive endurance exercise induced a more pronounced response of sHSP compared with more intensive but shorter exercise. Exercise intensity was also an important influencing factor. A duration- and intensity-dependent role for sHSP72 in the exercise-induced changes of the immune response may be assumed.

Antioxidant intervention does not affect the response of plasma erythropoietin to short-term normobaric hypoxia in humans.

Recent research has demonstrated that reactive oxygen species (ROS) participate in intracellular signaling processes initiated during hypoxia. We investigated the role of ROS in the response of plasma erythropoietin (Epo) to short-term normobaric hypoxia in humans. Twelve male subjects were exposed twice to 4 h of normobaric hypoxia (H; inspired oxygen fraction 12.5%) with a period of 6 wk between both experiments (H1 and H2). With the use of a randomized placebo-controlled crossover design, the subjects received orally a combination of the antioxidants all-rac-alpha-tocopherol (800 mg/day for 3 wk) and alpha-lipoic acid (600 mg/day for 2 wk) or placebo before H1 and H2, respectively. Three weeks before H1, the subjects underwent one control experiment in normoxia (N; inspired oxygen fraction 20.9%) without any treatment. Serum alpha-tocopherol was significantly higher after treatment with antioxidants compared with placebo. Capillary Po(2) declined during H without significant differences between antioxidants and placebo. Plasma peroxide levels were lower under antioxidant treatment but not affected by hypoxia. The response of Epo to H did not show significant differences between antioxidant [maximum increase (means, 95% confidence interval): +121%, +66 to +176%] and placebo conditions (+108%, +68 to +149%). Similarly, hypoxia-induced increase of Epo corrected for diurnal variations, as revealed during N, did not differ between antioxidants and placebo. Individual variability of Epo in response to H was not related to the individual degree of hypoxemia during H. Our results do not support the assumption that ROS play a major modulating role in the response of Epo to short-term normobaric hypoxia in humans.

Microarray technology--the future analyses tool in exercise physiology?

Microarray analysis offers a set of analytical platforms that provide rapid, affordable and substantial information at the DNA, RNA or protein level. It enables the analysis of thousands of genes simultaneously in a parallel manner across samples derived from various biological sources and treatment regimens. This development became possible as a result of the combination of three technological advances achieved at the beginning of the 1990's, namely parallelism, miniaturization and automation. In regular physical checkups the microarray technology could be used to supplement the current spectrum of tests and therefore enhance the quality of the data obtained. Arrays for analyses of RNA expression will allow gene expression profiling also in exercise physiology. DNA chips may also be used for genetic screening and diagnostics to analyze polymorphisms and mutations which may underlie genetic diseases or interindividual variations between subjects. Using microarrays in exercise physiology will provide new insights into the complex molecular mechanisms of the exercise-induced stress response, adaptation to training and modulation of immune function. Gene expression profiling and genetic screening will probably help to characterize and predict the individually variable response to and efficiency of training.

Influence of different types of exercise on the expression of haem oxygenase-1 in leukocytes.

Haem-oxygenase-1 (HO-1) is an antioxidant stress protein that is mainly induced by reactive oxygen species, inflammatory cytokines and hyperthermia. We assessed the influence of different types of exercise on HO-1 expression in leukocytes of the peripheral blood in three groups of male participants: a short exhaustive run above the lactate steady state (n = 15), eccentric exercise (n = 12) and an intensive endurance run (half-marathon, n = 12). Blood samples were taken at rest and up to 24 h after exercise. Blood lactate concentration after exercise was 9.0 +/- 2.1, 3.8 +/- 1.6 and 5.1 +/- 2.2 mmol x l(-1) (mean +/- s) for the exhaustive run, eccentric exercise and half-marathon groups, respectively (P < 0.05). Creatine kinase concentration was highest 24 h after exercise: 133 +/- 91, 231 +/- 139 and 289 +/- 221 U x l(-1) for the exhaustive run, eccentric exercise and half-marathon groups, respectively (P < 0.05). The maximal increase in leukocyte counts after exercise was 11.5 +/- 19.2, 6.2 +/- 1.4 and 14.7 +/- 2.1 x 10(9) x l(-1). There was no change in HO-1 as a result of the short exhaustive run or the eccentric exercise, whereas the half-marathon had a significant stimulatory effect on HO-1-expression in lymphocytes, monocytes and granulocytes (P < 0.001) using flow cytometry analyses. In conclusion, eccentric exercise alone or short-term heavy exercise are not sufficient to stimulate the antioxidative stress protein HO-1 in peripheral leukocytes

Impact of elevated ambient temperatures on the acute immune response to intensive endurance exercise.

To date, there has been little research examining how elevated ambient temperatures exert an additional effect on the acute immune response to endurance exercise. Seven endurance-trained, non-heat-acclimated men [mean (95% confidence interval): 29.7 (25.9-33.5) years, .VO(2max) 66.3 (61.3-71.3) ml.kg(-1).min(-1)] performed two 60-min treadmill runs (75% .VO(2max)) in two different environments (EX1: 18 degrees C/50% room temperature/relative humidity and EX2: 28 degrees C/50% room temperature/relative humidity) with a 7-day interval between the runs. Blood samples were drawn at rest and 0, 0.5, 3, 24, and 48 h after exercise. Compared to EX1, exercise-induced increases in core temperature, sweating rate, heart rate, plasma norepinephrine, cortisol, human growth hormone, and neutrophil and monocyte counts were significantly (5% level) more pronounced after EX2. In contrast, responses of plasma epinephrine, myeloperoxidase, interleukin (IL)-6 as well as lymphocyte counts were similar in EX1 and EX2. Plasma concentrations of IL-8 and C-reactive protein were affected by neither exercise nor by additional heat exposure. Our results suggest that the additional impact of elevated ambient temperatures on stress responses to endurance exercise in trained subjects seems to affect primarily the cardiocirculatory and hormonal systems, and resulting changes in neutrophil and monocyte cell-trafficking. In contrast, heat stress does not seem to exert large additional effects on the acute immune response to endurance exercise as performed in the present study.

Effects of RRR-alpha-tocopherol on leukocyte expression of HSP72 in response to exhaustive treadmill exercise.

Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.

Basal expression of leukocyte iNOS-mRNA is attenuated in moderately endurance-trained subjects.

Nitric oxide (NO) is generated in immunocompetent cells by inducible NO-synthase (iNOS), and plays an important role in host defense. However, when produced in large amounts, NO can also exert damaging effects, a scenario that is observed during several inflammatory processes. In the study presented here, we investigated the impact of moderate endurance training (running volume: mean 53.1 km x week(-1), 95% confidence interval 41.2-65.1 km week(-1)) on the leukocyte expression of iNOS mRNA. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to examine iNOS mRNA expression in total leukocyte samples from 12 male trained subjects (TR) and a control group of 12 untrained men (UT) at rest. Relative iNOS mRNA levels (iNOS/beta-actin) were higher in UT (0.88, 0.73-1.03) when compared with TR (0.34, 0.09-0.58, P<0.001). iNOS mRNA was not detectable in 5 of the 12 TR subjects. These initial results show that the basal expression of iNOS mRNA is downregulated by moderate endurance training. Further research should clarify whether regular training also affects the responsiveness of leukocyte iNOS gene expression to stimulatory signals. It would be of interest to establish whether moderate training can exert a suppressive and therefore therapeutic effect on the elevated levels of expression of iNOS observed in, for example, several inflammatory disorders.

Free radicals, exercise, apoptosis, and heat shock proteins.

Free radicals are an integral part of metabolism and are formed continuously in the body. Many sources of stress heat, irradiation, hyperoxia, inflammation and any increases in metabolism including exercise, injury, and even repair processes lead to increased production of free radicals and associated reactive oxygen or nitrogen species (ROS/RNS). Evidence is accumulating that free radicals have important functions in the signal network of cells, including induction of growth and apoptosis and as killing tools of immunocompetent cells. Endogenous and nutritional antioxidant systems have to be adjusted to ensure adequate removal of radicals during stress to prevent damage to membranes, proteins, or nucleic acids. Excessive stress will induce DNA damage in the form of oxidized nucleosides, strand breaks, or DNA-protein crosslinks. Possible consequences of DNA damage are repair, apoptosis/necrosis, or defective repair leading to DNA sequence alterations and possibly to the development of cancer or, in case of mitochondrial DNA, to metabolic dysfunction. Excessive exercise will also induce DNA damage in peripheral leukocytes. The good message is that moderate stress in form of regular exercise/training may have protective effects against exercise-induced DNA damage. Up-regulation of endogenous antioxidant defense systems and complex regulation of repair systems such as heat shock proteins (HSP 70, HSP 27, HO 1) are seen in response to training and exercise. Up-regulation of antioxidants and modulation of the repair response may be mechanisms by which exercise can beneficially influence our health. Massive intervention into the redox state by pharmaceutical doses of exogenous antioxidants should be regarded with caution due to the ambiguous role of free radicals in regulation of growth, apoptosis, and cytotoxicity by immunocompetent cells.

Changes of HSP72-expression in leukocytes are associated with adaptation to exercise under conditions of high environmental temperature.

Overexpression of the heat shock protein HSP72 provides thermotolerance. We asked if two consecutive endurance runs 1 week apart (CR1, CR2) and additional environmental heat stress affect HSP72-expression in leukocytes of nonheat-acclimated endurance athletes. Twelve subjects were allocated randomly into two groups. Group HH completed both runs at 28 degrees C ambient temperature, and group NH performed CR1 at 18 degrees C and CR2 at 28 degrees C. HSP72-expression was determined by flow cytometry and RT-PCR before and 0, 24, and 48 h after exercise. Additionally, post-exercise cells were exposed to in vitro heat shock (HS; 2 h, 42 degrees C). The prolonged, high HSP72 protein level after CR1 in HH compared with NH may reflect thermotolerance induced by endurance exercise at high ambient temperature. Adaptation of cardiocirculatory/thermoregulatory capacity after CR2 in HH went along with a more rapid down-regulation of HSP72 compared with CR1. HSP72 mRNA demonstrated temperature-related changes after exercise. The reduced HS response in vitro after CR2 may represent exercise-related adaptation mechanisms. HSP72 concentrations in leukocytes may indicate previous exercise- and temperature-related stress conditions and adaptation in immunocompetent cells.

Transcriptional and translational regulation of heat shock proteins in leukocytes of endurance runners.

Heat shock proteins (HSP) represent cell-protective and antioxidant systems that may be induced by reactive oxygen species, cytokines, and hyperthermia. In the present study, we evaluated the influence of heavy endurance exercise and training on HSP27 and HSP70 in peripheral leukocytes of 12 athletes (before and at 0, 3, and 24 h after a half-marathon) and 12 untrained controls on protein and mRNA levels by flow cytometry and RT/PCR, respectively. HSP transcripts increased significantly immediately after acute exertion accompanied by elevated levels of corresponding proteins. HSP protein expression remained high until 24 h postexercise. Significant increases of plasma interleukin-8, myeloperoxidase, and creatine kinase occurred after exercise. Basal HSP expression was usually lower in trained compared with untrained subjects. Applying in vitro heat shock to resting blood samples of all subjects significantly stimulated HSP mRNA, showing higher increases in trained individuals. The exercise-induced alterations indicate that immunocompetent cells became activated. In addition to heat stress, other exercise-associated stress agents (oxidants, cytokines) may have also participated in stimulation of HSP expression in leukocytes. The expression pattern of HSP due to training status may be attributed to adaptive mechanisms.

Expression of the inducible nitric oxide synthase (iNOS) in human leukocytes: responses to running exercise.

INTRODUCTION: We examined the influence of two different bouts of vigorous running exercise on the expression of the inducible nitric oxide synthase (iNOS) in leukocytes (LE). METHODS: In study 1, 10 trained runners competed in a half marathon (HM) lasting 90.5 +/- 11.0 min. In study 2, 8 untrained subjects performed a graded treadmill test followed by a continuous run (CR) until exhaustion (11.3 +/- 1.3 min). iNOS mRNA levels were assessed by RT/PCR at rest, 0, 3, and 24 h after HM and CR. In study 2, iNOS was additionally analyzed at the protein level in lympho- (L), mono- (M), and granulocytes (G) by flow cytometry at rest and up to 48 h after CR. RESULTS: Analysis revealed a rise of the iNOS transcript directly after the HM in 8 of 10 subjects. In study 2, the expression of iNOS protein at rest differed between L (mean +/- SE: 30.9 +/- 4.5% iNOS positive cells), M (91.3 +/- 4.0%), and G (64.9 +/- 10.3%): 3 h after CR, expression of iNOS increased in L (67.3 +/- 7.4%) and G (90.3 +/- 2.9%) and was still elevated 48 h post-exercise. However, our measurements failed to detect significant changes of leukocyte iNOS mRNA in response to CR. After the HM, our findings were paralleled by elevated plasma levels of interleukin-8, myeloperoxidase (MPO), and partly of TNF-alpha, whereas CR only induced a low rise of MPO. CONCLUSION: Our investigations revealed an increased expression of iNOS at the transcriptional and translational level in response to vigorous exercise. This reflects an inflammatory response and may contribute to an exercise-induced rise of endogenous nitric oxide production. It remains unclear if these effects serve an in-vivo immunoregulatory or cell-damaging role.

HSP expression in human leukocytes is modulated by endurance exercise.

PURPOSE: Temperature increase, oxidative stress, and inflammatory reactions after endurance exercise were expected to stimulate the synthesis of heat shock proteins (HSP) in peripheral blood leukocytes. Furthermore, it was of interest whether regular endurance training influences HSP expression. METHODS: The expression of HSP27, HSP60, HSP70, constitutive HSC70, and HSP90 in the cytoplasma and surface of lymphocytes, monocytes, and granulocytes of 12 trained athletes was analyzed by flow cytometry before and after (0, 3, and 24 h) a half marathon. Twelve untrained persons at rest were included as control. RESULTS: After the race, there was a significantly greater percentage of leukocytes expressing cytoplasmic HSP27, HSP60, and HSP70 (P < 0.01), whereas HSC70 and HSP90 remained unchanged. The fluorescence intensity increased significantly in monocytes for HSP27 (0 and 3 h) and HSP70 (0, 3, and 24 h) and in granulocytes, only 24 h postexercise for HSP70. The percent values of trained athletes at rest were significantly lower compared with untrained persons (P < 0,01). CONCLUSIONS: Strenuous exercise increased HSP expression in blood immediately after the run, indicating a protective function of HSP in leukocytes of athletes to maintain function after heavy exercise. The downregulation of HSP-positive cells in trained athletes at rest seems to be a result of adaptation mechanisms to regular endurance training.

Physical exercise-induced expression of inducible nitric oxide synthase and heme oxygenase-1 in human leukocytes: effects of RRR-alpha-tocopherol supplementation.

This study evaluated the effects of RRR-alpha-tocopherol (500 IU/day, 8 days) on in vivo cytokine response and cytoplasmic expression of inducible nitric oxide synthase (iNOS) and the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes after exhaustive exercise. Thirteen men were investigated in a double-blind, placebo-controlled, cross-over study with a wash-out period of 28 days. The exercise procedure consisted of an incremental treadmill test followed by a continuous run until exhaustion at 110% of the individual anaerobic threshold (total duration 28.5 +/- 0.8 min). HO-1 and iNOS protein were assessed in mono- (M), lympho-, and granulocytes (G) using flow cytometry. Plasma interleukin-6 (IL-6) and IL-8 were measured by ELISA. IL-6 rose significantly whereas IL-8 did not exhibit significant changes after exercise. Changes of IL-6 were not affected by RRR-alpha-tocopherol. Exercise induced an increase of iNOS protein primarily in M and G. A small, but significant, increase of HO-1 protein was measured in M and G. RRR-alpha-Tocopherol did not show any significant effects on cytoplasmic expression of iNOS and HO-1 at rest and after exercise. In conclusion, exhaustive exercise induces expression of iNOS and HO-1 in human leukocytes by a mechanism that is not sensitive to RRR-alpha-tocopherol supplementation.

Free radicals and oxidative stress in exercise--immunological aspects.

Reactive oxygen (ROS) and nitrogen species (RNS) are continuously generated in the biological system and play an important role in a variety of physiological and pathological processes. There is evidence that physical exercise augments the generation of ROS/RNS. The present review discusses and compares insights into the generation and function of ROS/RNS such as superoxide, hydrogen peroxide, hypochloric acid, and nitric oxide released by leukocytes in response to exercise. Emphasis is placed on: (a) mechanisms and regulation of ROS/RNS generation in immunocompetent cells with respect to acute exercise and regular training; (b) damaging effects of ROS/RNS in terms of oxidative stress which may be causally involved in features such as exercise-induced damage to muscle tissue and leukocyte DNA; (c) (immuno-) modulating effects of ROS/RNS which include activation of transcription factors; (d) responses of antioxidant stress proteins to acute exercise and regular training; and (e) effects of antioxidants on exercise-induced changes in immune function. Available data suggests that ROS/RNS are involved in the inflammatory response to heavy exercise and therefore exert damaging effects. Several immune functions are influenced by actions of ROS/RNS, and it is hypothesized that adaption to regular training is also modulated in part by free radicals. Furthermore, regular training seems to reduce the capacity of leukocytes for oxidant release and leads to an adaptation of antioxidative mechanisms, which may contribute to a limitation of exercise-induced oxidative stress.

Role of heat shock proteins in the exercise response.

Heat shock proteins (HSP) are a characteristic set of highly conserved proteins that are synthesized shortly after the organism is exposed to external stress, including physical activity. HSP help to maintain cellular homeostasis and protein conformation, reactivate denatured or malformed proteins, and provide "housekeeping," translocase, and chaperone functions. Some of the conditions known to elicit the cellular stress reaction are similar to those experienced by cells in response to physical exercise. Hyperthermia, ischemia, oxidative, cytokine and muscular stress, glucose deprivation, and alterations in calcium and pH are potent inducers of HSP expression in different types of cells and tissues. This review provides an overview of the cellular heat shock response to exercise. The presently known exercise related HSP are introduced. Their possible roles in response to acute exercise, thermotolerance, adaptation to training, aging, and immunological reactions are discussed.

Expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in human leukocytes.

Inducible heme oxygenase (HO-1) is an antioxidant stress protein, that is mainly induced by reactive oxygen species (ROS), cytokines and hyperthermia. By using flow cytometry the present investigation demonstrated a rise in the cytoplasmic expression of HO-1 in lympho- (L), mono- (M) and granulocytes (G) of 9 endurance-trained male subjects after a half marathon run. The expression was more pronounced in M (median: 98.3% HO-1 positive cells/4.31 mfc) and G (94.8%/1.93 mfc) than in L (80.1%/1.51 mfc) when measured 3 h post-exercise. Additionally the exercise protocol caused a rise in the plasma levels of myeloperoxidase, TNF alpha and interleukin-8 (IL-8), indicating an inflammatory response. We could detect a correlation between IL-8 and HO-1, directly after exercise, that was apparent in G (r = 0.67, p < .05) and L (r = 0.80, p < .05), but did not reach significance in M (r = 0.65, p = 0.06). An additional detection of HO-1 at rest in 12 untrained subjects showed a higher baseline expression of HO-1 compared to the athletes. The regulatory pathways leading to an increased expression of HO-1 after endurance exercise are not completely clear, but a causal involvement of a cytokine-mediated generation of ROS must be discussed. We supposed that the down-regulation of the baseline expression of HO-1 in athletes reflects an adaptional mechanism to regular exercise training.

Induction of cytokines and expression of surface receptors in Mono Mac 6 cells after infection with different Legionella species.

The ability of Legionella species to multiply within human mononuclear phagocytes is usually regarded as being associated with their pathogenicity. Activation of host cells results in inhibition of intracellular Legionella multiplication. The most effective substance to induce macrophage activation, both in vivo and in vitro, is interferon-gamma. In addition, some evidence exists that macrophage-derived cytokines may contribute to the host defense against L. pneumophila, but the production of pro- and antiinflammatory cytokines by monocytes after infection with different Legionella species has not been reported with regard to their ability to multiply within the host cells. We therefore examined the production of TNF-alpha, IL-1, IL-6, IL-8, IL-10 and TGF-beta by Mono Mac 6 cells after infection with Legionella species of different human prevalence that differ in their ability to replicate within this macrophage-like cell line. After infection, Mono Mac 6 cells showed a cytokine response with time kinetics characteristic for the cytokine. Maximum cytokine levels produced differed with Legionella species, but were not related to intracellular multiplication rates. Moreover, LPS-tolerant Mono Mac 6 cells, which failed to produce cytokines, showed intracellular increase or decrease of bacterial numbers identical to that of untreated Mono Mac 6 cells. By FACS analysis, an up-regulation of CD14 (LPS receptor) and CD54 (ICAM-1) could be demonstrated. We conclude that, in the Mono Mac 6 cell line, induction of macrophage-derived cytokines after infection with members of the genus Legionella mimics an inflammatory reaction without association with intracellular multiplication rate.

Effects of a new bone-inducing biomaterial on mesenchymal cells in vitro.

A purified bone-inducing protein complex (BIC), isolated from bovine bone and causing de novo bone formation in vivo, induces defined effects on rat mesenchymal cells in vitro. Spindle-like mesenchymal cells growing in monolayers change to polygonal cells, forming a multilayered growth pattern. The mesenchymal cells acquire alkaline phosphatase activity. Upon culture with BIC, the typical collagen Type III deposition of these mesenchymal cells is remarkably reduced whereas the collagen Type I expression remains unaffected. All these in vitro effects are consistent with the strong bone-forming capacity of BIC in vivo. A combination of two cytokines, transforming growth factor beta 1 (TGF beta 1) and epidermal growth factor (EGF), shows a similar activity to BIC. Neutralizing anti-TGF beta antibodies interfere with all in vitro effects of BIC. The neutralization of BIC and the inductive capacity of the combination of TGF beta 1 plus EGF point to the substantial role of TGF beta or TGF beta-like molecules in BIC; whether the active polypeptides are identical to TGF beta or somewhat structurally homologous to TGF beta remains to be elucidated.