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. Author manuscript; available in PMC: 2009 Mar 30.
Published in final edited form as: Circulation. 2008 Sep 30;118(14 Suppl):S38–S45. doi: 10.1161/CIRCULATIONAHA.107.756890

Mechanisms of Sex Differences in TNFR2 Mediated Cardioprotection

Meijing Wang 1, Paul R Crisostomo 1, Troy A Markel 1, Yue Wang 1, Daniel R Meldrum 1,2,3,✉
PMCID: PMC2655136  NIHMSID: NIHMS81011  PMID: 18824767

Abstract

Background

TNFR1/TNFR2 signaling may mediate different cellular and molecular responses (injury vs. protection) and the balance may be affected by sex hormones. Previous studies have shown that females have improved myocardial functional recovery, TNFR1 signaling resistance, and increased SOCS3 expression following acute I/R when compared to males. However, it is unknown whether the TNFR2 pathway protects the myocardium from I/R injury, and if so, whether sex differences exist in TNFR2-mediated cardioprotection. Therefore, we hypothesized that: 1) TNFR2 mediates myocardial protection from I/R through STAT3, SOCS3 and VEGF in both genders; and 2) TNFR2 elicits greater protective signaling in females compared to males.

Methods and Results

Isolated male and female mouse hearts from TNFR2 knockout (TNFR2 KO), TNFR1/2KO and wild type (WT: C57BL/6J or B6129SF2/J) (n=5-6/group) were subjected to 20 minutes ischemia followed by 60 minutes reperfusion. TNFR2 deficiency decreased post-ischemic myocardial recovery in both genders, but had a greater effect on females. The deleterious effects of TNFR2 ablation were associated with a decrease in mRNA and protein levels of SOCS3, STAT3, and VEGF, as well as an increase in myocardial IL-1beta production in female hearts. However, a significant increase in JNK activation and IL-1beta protein levels were noted in male TNFR2KO hearts following I/R. Additionally, TNFR1/2 KO decreased myocardial function in female hearts, but not males. This observation was associated with a decrease in mRNA levels of SOCS3, STAT3 and VEGF, and an increase in myocardial p38 MAPK activation in females.

Conclusions

Sex differences in the mechanisms of TNFR2 mediated cardioprotection occur by increasing STAT3, SOCS3, VEGF in females and by decreasing JNK in males.

Keywords: gender, tumor necrosis factor receptor, ischemia

INTRODUCTION

Proinflammatory signaling has been implicated in the pathophysiology of a number of conditions related to cardiothoracic surgery1-4. Interestingly, sex differences have been observed in myocardial proinflammatory signaling following ischemia and reperfusion injury (I/R). Cardiothoracic surgeons recognize that men and women respond differently to cardiothoracic surgical procedures. Indeed, clinical differences in sex related outcomes appear to be multifactorial and complex. Clinically, women have a lower overall incidence of heart failure, slower heart failure progression, better age-matched cardiac contractility, and better preservation of myocardial mass as they age compared to men 5. Indeed, our previous animal studies have demonstrated that females have better myocardial functional recovery, decreased proinflammatory cytokine production and reduced apoptotic signaling following I/R 6, 7.

Ischemia and reperfusion injury results in substantial amounts of myocardial proinflammatory cytokine production, such as tumor necrosis factor alpha (TNF). Numerous studies have demonstrated that TNF contributes to alterations in calcium homeostasis, stimulation of cardiomyocyte apoptosis, as well as induction of other cytokines associated with myocardial dysfunction following acute ischemia 8. Interestingly, while animal studies performed by several different investigators have shown that decreasing the bioavailability of TNF has beneficial effects following I/R 9, 10, others have suggested that TNF may actually be protective 11. Indeed, clinical studies have indicated that simply decreasing the bioavailability of TNF in heart failure patients appears to lack benefit 12, 13. These observations have led to the important appreciation that TNF may mediate different cellular and molecular responses (injury vs. protection) depending on which of its receptors are activated.

TNF initiates its biological actions by binding to a 55-KDa receptor (TNFR1) and/or a 75-KDa receptor (TNFR2), both of which are present on cardiac myocytes 14. The majority of TNF-induced myocardial dysfunction and myocyte apoptosis is initiated by the activation of TNFR18. Ablation of TNFR1 has been reported to improve myocardial function and survival in mice after myocardial infarction15, while ablation of TNFR2 has been associated with increased heart failure rates and a reduction in survival following infarction 16. A previously published study from our laboratory has also shown that improved myocardial functional recovery in females was associated with TNFR1 signaling resistance following I/R 6. However, it remains unknown whether TNFR2 plays a role in the sex differences observed in the myocardial response to I/R. Therefore, we hypothesized that TNFR2 signaling plays a cardioprotective role in the myocardial response to I/R via STAT3, SOCS3 and VEGF in both genders, but with a relative greater benefit in females.

MATERIALS AND METHODS

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Animals

C57BL/6J mice with and without targeted deletion of TNFR2 (TNFR2KO and WT) and B6129SF2/J mice with and without targeted deletion of TNFR1/2 (TNFR1/2KO and WT') of both genders (Jackson Laboratories, Bar Harbor, ME)] were fed a standard diet and acclimated in a quiet quarantine room for two months before the experiments. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 85-23, revised 1985).

Isolated mouse hearts were divided into: 1. Male WT (M WT), 2. M TNFR2KO, 3. M WT', 4. M TNFR1/2KO, 5. Female WT (F WT), 6. F TNFR2KO, 7. F WT' and 8. F TNFR1/2KO, and subjected to: 15 minutes equilibration period, 20 minutes of global ischemia (37°C), and 60 minutes of reperfusion (n=5-6 animals/group).

Isolated heart preparation (Langendorff)

Experiments were performed with the use of a Langendorff apparatus as described previously for use in mouse heart 6. Briefly, mice were anesthetized (sodium pentobarbital, 60 mg/kg i.p.) and heparinized (500 U i.p.), and hearts were rapidly excised via median sternotomy and placed in 4°C Krebs-Henseleit solution. The aorta was cannulated and the heart was perfused (90 mm Hg) with oxygenated (95% O2 / 5% CO2) Krebs-Henseleit solution (37° C). Data was continuously recorded using a PowerLab 8 preamplifier/digitizer (AD Instruments Inc., Milford, MA) and an Apple G4 PowerPC computer (Apple Computer Inc., Cupertino, CA).

Real-Time RT-PCR

Total RNA was extracted from each heart's left ventricle using RNA STAT-60 (TEL-TEST, Friendswood, TX). 0.5 μg of total RNA was subjected to cDNA synthesis using cloned AMV first-strand cDNA synthesis kit (Invitrogen life technologies, Carlsbad, CA). cDNA from each sample was analyzed for 18S (assay ID# Hs99999901_s1), TNF (assay ID# Mm00443258_m1), IL-6 (assay ID# Mm00446190_m1), VEGFa (assay ID# Mm00437304_m1), SOCS3 (assay ID# Mm00545913_s1) and STAT3 (assay ID# Mm00456961_m1) by using TaqMan gene expression assay (Real-time PCR) (Applied Biosystems, Foster City, CA). Densitometry was performed to assess relative quantity and represented as a ratio to male WT control (without I/R).

Enzyme Linked Immunosorbent Assay

Myocardial TNF, IL-6, IL-1β and VEGF in the cardiac tissue were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA set (BD Opt EIA ELISA set, BD Biosciences Pharmingen, San Diego, CA and Duo set ELISA Development System, R&D Systems Inc., Minneapolis, MN). ELISA was performed according to the manufacturer's instructions. All samples and standards were measured in duplicate.

Western blotting

Heart tissue was homogenized in cold buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-Glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin, 1 mM PMSF, and centrifuged at 12000 rpm for 5 minutes. The protein extracts (20 μg/lane) were subjected to electrophoresis on a 4-12% tris-Hepes gel from Pierce and transferred to a nitrocellulose membrane. The membrane was incubated in 5% dry milk for 1 hour and then incubated with the following primary antibodies: STAT3, phosphor-STAT3 (Tyr705), p38 MAPK, phosphor-p38 MAPK (Thr180/Tyr182), JNK, phosphor-JNK (Thr183/Tyr185), ERK1/2 and phosphor-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA), caspase-3, -8 and SOCS-3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and GAPDH antibody (Biodesign International, Saco, Maine) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody and detection using Supersignal West Pico Stable Peroxide solution (Pierce, Rockford, IL). Films were scanned using an Epson Perfection 3200 Scanner (Epson America, Long Beach, CA) and band density was analyzed using ImageJ software (NIH).

Presentation of data and statistical analysis

All reported values are mean ± SEM. Data were compared using two-way analysis of variance (ANOVA) with post-hoc Bonferroni test or Student's t-test. A two-tailed probability value of less than 0.05 was considered statistically significant.

RESULTS

Effects of TNFR2 and TNFR1/2 on myocardial function following ischemia-reperfusion

No differences in baseline absolute +/- dP/dt were observed during equilibration between all groups. TNFR2 deficiency further impaired myocardial function after I/R, which was exhibited by depressed +dP/dt and -dP/dt in both male and female mouse hearts. Additionally, TNFR2KO neutralized sex differences in myocardial functional recovery (figure 1). Interestingly, ablation of both TNFR1 and TNFR2 genes decreased recovery of post-ischemic myocardial function in females, but not in males (figure 2), which resulted in a lower recovery of + dP/dt and - dP/dt in female double knockouts compared to males following I/R.

Figure 1.

Figure 1

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Deficiency of the TNFR2 gene decreased post-ischemic myocardial function in both genders. Myocardial function was recorded following I/R in male wild type (n=6), male TNFR2 knockout (n=5), female wild type (n=6), and female TNFR2 knockout (n=5) mouse hearts. Results are represented as % of equilibration (eq). A: Male +dP/dt; B: Male -dP/dt; C: Female +dP/dt; D: Female -dP/dt; E, F: +dP/dt, -dP/dt at the end of reperfusion. Results are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. wild type, #p<0.05 vs. M WT

Figure 2.

Figure 2

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TNFR1/2KO reduced myocardial functional recovery in females following I/R. Changing of myocardial function in male wild type (n=5), male TNFR1/2 knockout (n=6), female wild type (n=5), and female TNFR1/2 knockout (n=5) mouse hearts subjected to I/R. Results are represented as % of equilibration (eq). A: Male +dP/dt; B: Male -dP/dt; C: Female +dP/dt; D: Female -dP/dt; E, F: +dP/d, -dP/dt at the end of reperfusion. Results are mean ± SEM, *p<0.05, **p<0.01 vs. wild type

Effects of TNF receptors on myocardial TNF, IL-6 and IL-1β, and VEGF production

Ischemia-induced production of myocardial TNF, IL-6 and IL-1β has been observed in our previous studies, as well as the present one (data not shown). Ablation of TNFR2 or TNFR1/2 did not significantly affect mRNA and protein levels of myocardial TNF and IL-6 in male and female hearts following I/R (data not shown). However, deficiency of TNFR2 increased myocardial IL-1β protein levels by two- fold in males, and by three-fold in females (figure 3A) while TNFR1/2KO hearts did not show any significant difference in IL-1β levels (figure 3B).

Figure 3.

Figure 3

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Effects of TNF receptor ablation on myocardial IL-1b following I/R. A: Deficiency of TNFR2 (TNFR2KO) increased IL-1b protein levels in both genders (ELISA); B: TNFR1/2KO had no effects on myocardial IL-1β production in male and female hearts subjected to I/R. Results are mean ± SEM, n= 4 mouse hearts/group, *p<0.05 vs. MWT

Ablation of TNFR2 or TNFR1/2 significantly decreased VEGFa mRNA by 43% or 56% in female hearts, but not in males (figure 4A, B). Additionally, markedly reduced VEGF protein levels were noted in female TNFR2KO compared to female WT and male TNFR2KO (figure 4C). Further, a trend of lower protein levels of VEGF was observed in female TNFR1/2KO hearts after I/R (figure 4D). However, this observation was not seen in males (figure 4C, D).

Figure 4.

Figure 4

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Effects of TNF receptors on myocardial growth factor VEGF production after I/R. Relative quantitation of VEGFa mRNA compared to male wild type control (RT Real-time PCR) is shown in A (WT and TNFR2KO hearts) and B (WT' and TNFR1/2KO). Myocardial VEGF production is analyzed by ELISA in C (WT and TNFR2KO) and D (WT' and TNFR1/2KO). Results are mean ± SEM, n=5-6 hearts/group *p<0.05, **p<0.01 vs. F WT or F WT', #p<0.05 vs. MWT', @p<0.005 vs. MTNFR2KO

Effects of TNF receptors on myocardial SOCS3 and STAT3 following ischemia and reperfusion

Our results indicated that TNFR2 and TNFR1/2 ablation significantly decreased mRNA levels of SOCS3 and STAT3 in females, but not in males (figure 5A, B, E and F). Female WT (C57BL/6J) had higher protein levels of SOCS3 and p-STAT3 compared to male WT, which was neutralized by deficiency of TNFR2 in female hearts, whereas ablation of TNFR2 did not significantly reduce SOCS3 levels and STAT3 activation in males (figure 5C, G). In contrast, TNFR1/2KO increased SOCS3 protein levels and had no effect on myocardial STAT3 activation in both genders (figure 5D, H).

Figure 5.

Figure 5

Figure 5

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Relative mRNA and protein levels of myocardial SOCS3 and STAT3 after I/R in TNFR2KO, TNFR1/2KO and wild type of both genders. A, B: Relative quantitation of SOCS3 mRNA compared to male wild type control (RT Real-time PCR) in TNFR2KO and TNFR1/2KO hearts, respectively; C, D: Representative immunoblots (2 lanes/group) and densitometry data of SOCS3 (% of GAPDH) in TNFR2KO and TNFR1/2KO myocardium after I/R; E, F: Relative quantitation of STAT3 mRNA compared to male wild type control (RT Real-time PCR) in TNFR2KO and TNFR1/2KO; G, H: Representative immunoblots (2 lanes/group) and densitometry data of STAT3 (% of total-STAT3) in TNFR2KO and TNFR1/2KO. Mean ± SEM, n= 5-6/group, * p<0.05 vs. F WT or F WT', **p<0.01 vs. WT', #p<0.05 vs. MWT or MWT', @p<0.05 vs. MTNFR2KO

Effects of TNF receptors on mitogen activated protein kinases (MAPKs) following ischemia and reperfusion

Ablation of the TNFR2 gene did not change activation of p38 MAPK in the myocardium subjected to I/R in either gender (figure 6A). However, double knockout of TNFR1 and TNFR2 increased myocardial p38 MAPK activation in female hearts, but not in males (figure 6B). Interestingly, significantly augmented myocardial JNK activation was noted in male TNFR2KO hearts, but not in male TNFR1/2KO (figure 6C, D). In addition, neither TNFR2 nor TNFR1/2 deficiency affected myocardial JNK activation in females following I/R. Furthermore, ablation of TNFR2 or TNFR1/2 had no effects on myocardial ERK activation in both genders (figure 6E, F).

Figure 6.

Figure 6

Figure 6

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The expression of activated myocardial p38 MAPK, JNK, ERK1/2, and total p38 MAPK, JNK, ERK1//2 after I/R in TNFR2KO, TNFR1/2KO and wild type of both genders. Representative immunoblots (2 lanes/group) and densitometry data of phosphorylated p38 MAPK (% of total p38 MAPK) indicate p38 MAPK activation in TNFR2KO (A) and TNFR1/2KO hearts (B) after I/R. Phosphorylated JNK (activation) and nonphosphorylated (total) JNK are shown in C (TNFR2KO) and D (TNFR1/2KO). Representative immunoblots (2 lanes/group) and densitometry bar graph of phosphorylated ERK (% of total ERK) demonstrate active myocardial ERK in TNFR2KO (E) and TNFR1/2KO (F). Mean ± SEM, n=4-6/group, * p<0.05 vs. M WT, **p<0.01 vs. F WT', #p<0.05 vs. MWT' and @p<0.05 vs. MTNFR2KO

Effects of TNF receptors on pro-apoptotic signaling following ischemia and reperfusion

To study the role of TNF receptors in the myocardial response to I/R, it was necessary to determine the degree of apoptosis given that the TNF receptor is usually recognized as a death receptor. However, our experimental period was too brief to detect significant apoptosis in the myocardium. Therefore, we examined myocardial expression of caspase-8 and caspase-3 by western blot. Deficiency of TNFR2 and TNFR1/2KO had no significant effects on caspase-8 and caspase-3 expression in males after I/R (figure 7A, B, C, D). Conversely, TNFR2 and TNFR1/2 ablation appeared to increase myocardial caspase-3 levels in females (figure 7C, D).

Figure 7.

Figure 7

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The expression of myocardial caspase-8 and caspase-3 after I/R in TNFR2KO, TNFR1/2KO and wild type of both genders. Representative immunoblots (2 lanes/group) and relative level of caspase-8 p20 vs. GAPDH indicate active caspase-8 expression in TNFR2KO (A) and TNFR1/2KO (B). Representative immunoblots (2 lanes/group) and densitometry bar graph of caspase-3 p20 (% of GAPDH) demonstrate active caspase-3 in C (TNFR2KO) and D (TNFR1/2KO). Mean ± SEM, n=5-6/group

DISCUSSION

Sex differences in cardiothoracic surgical outcomes and heart failure progression is widely accepted as a clinical given 5. To investigate the potential signaling mechanisms involved, we conducted the present studies and the results indicated the following: 1) ablation of the TNFR2 gene decreased myocardial functional recovery in both males and females; 2) double knockout of TNFR1 and TNFR2 reduced post-ischemic functional recovery only in female hearts; 3) sex differences in the mechanisms of TNFR2 mediated cardioprotection following ischemia occur by increasing STAT3, SOCS3, VEGF in females and by decreasing JNK in males.

Cardiac myocytes and macrophages produce substantial amounts of TNF in response to I/R. The biological function of TNF is regulated through two structurally distinct receptors: TNFR1 and TNFR2, both expressed in cardiac myocytes. The majority of the deleterious effect of TNF on the myocardium is clearly mediated by TNFR1, including short term negative inotropic effects, and long term TNF-induced apoptosis 8. Conversely, TNFR2 signaling appears to conduct protective effects 15, 16. The results of this study, as well as that of previous literature, would suggest that the balance of TNFR1 and TNFR2 signaling in female hearts may be shifted to favor salutary effects of TNF conducted by TNFR2 during I/R. Our data suggest that enhanced TNFR2 signaling may exist in female hearts subjected to acute I/R. However, it is unclear as to which downstream signals TNFR2 targets in order to improve myocardial function.

Inflammatory cytokines undoubtedly play a critical role in mediating post-ischemic myocardial dysfunction 17. In this regard, TNF is also important in initiating the reperfusion-dependent cytokine release seen in myocardial I/R 18. Thus, it can be postulated that TNFR2 may mediate myocardial protection through downregulation of other proinflammatory cytokines or the upregulation of beneficial growth factors. Indeed, there is evidence that ablation of TNFR2 exaggerated myocardial dysfunction accompanied by its upregulation of IL-6 and IL-1β in noninfarcted myocardium compared to WT in a murine myocardial infarction model 16. In addition, impaired post-ischemic recovery has been associated with a decrease in both mRNA and protein levels of VEGF in TNFR2KO muscle tissue 19. Here, we found that deficiency of the TNFR2 gene increased myocardial IL-1β protein levels in both male (by ∼2 fold) and female (by ∼3 fold) hearts after I/R. However, there were no significant differences in expression of TNF and IL-6 between TNFR2KO and WT mice (data not shown). Interestingly, ablation of TNFR2 reduced myocardial VEGF expression in females, but not in males. Therefore, it is possible that the combination of upregulated IL-1β and decreased VEGF production in females caused a greater degree of myocardial dysfunction in female hearts compared to WT. However, TNFR1/2KO did not show similar effects on myocardial IL-1β and VEGF production (except VEGFa mRNA in female hearts) following I/R.

Emerging evidence suggests that suppressor of cytokine signaling protein 3 (SOCS3) may be induced by various stimuli 20 and may participate in the important process of controlling proinflammatory signals. Our previous study has demonstrated that a deficiency of TNFR1 increased myocardial SOCS3 protein levels in males, but not in females following I/R 6. Herein, we further found that ablation of TNFR2 significantly reduced SOCS3 protein in females while double knockout of TNFR1 and TNFR2 augmented myocardial SOCS3 levels in both genders. These data suggest that TNFR1 signaling may downregulate SOCS3 expression in both genders, whereas TNFR2 signaling may represent a compensatory mechanism to upregulate SOCS3 expression in females.

On the other hand, the signal transducer and activator of transcription3 (STAT3) pathway exerts cardioprotective signaling in the ischemic heart 21, most likely via the induction of growth factors, the suppression of apoptosis, and the upregulation of SOCS3. In this study, females exhibited higher levels of myocardial SOCS3 and STAT3 activation compared to male hearts (C57BL/6J) in response to I/R. In addition, ablation of TNFR2 significantly decreased SOCS3 and STAT3 (mRNA and protein) in female hearts after I/R. These results suggest that improved post-ischemic myocardial function in females is likely associated with TNFR2-upregulated STAT3 activation and SOCS3 expression in females. Furthermore, overexpression or activation of STAT3 in cardiomyocytes has been shown to increase myocardial VEGF production 22. Therefore, it is possible that TNFR2 ablation decreased the STAT3 pathway and thereby, reduced myocardial VEGF production (both mRNA and protein) in females. These data also result in an appreciation that TNF receptors might regulate VEGF expression differently between male and female hearts following I/R.

Recent evidence also suggests that TNF mediated activation of p38 MAPK and JNK occurs via both TNFR1 and TNFR2 in bronchial smooth muscle 23. Additionally, our previous work has indicated that deficiency of TNFR1 decreased myocardial p38 activation in males 6. Herein, we further demonstrated that ablation of TNFR2 did not change p38 activation following I/R. Taken together, these data would suggest that I/R-induced p38 MAPK activation is more likely mediated via TNFR1 in both genders. However, noting that females have an inherent resistance to TNFR1 signaling following I/R, it is not surprising that TNFR1/2 ablation increased myocardial p38 MAPK activation in females, but not males. This result is consistent with impaired myocardial function in female TNFR1/2KO hearts following I/R. On the other hand, TNFR2KO significantly increased myocardial JNK activation, while TNFR1/2 ablation appeared to reduce activation of JNK in male hearts. These data suggest that TNFR1 may be responsible for activation of JNK or TNFR2 may downregulate myocardial JNK activation in males following I/R. Moreover, we found that neither TNFR2 ablation nor TNFR1/2 ablation affected myocardial ERK activation in either gender following I/R.

TNF binding to TNFR1 results in recruitment and activation of procaspase-8. Caspase-8 then activates downstream caspase-3 and induces the classic extrinsic death pathway. In this study, our data suggest that TNFR2 ablation and TNFR1/2KO had no significant effects on caspase-8 and caspase-3 expression in males after I/R. Those results are inconsistent with previous studies in that TNFR1 is required for caspase-8 mediated extrinsic proapoptotic signaling. This discrepancy likely resulted from a limitation of our experimental model- a short experimental time period.

ACKNOWLEDGEMENTS

None.

FUNDING SOURCES This work was supported in part by NIH K99/R00 HL0876077 (MW), NIH R01GM070628 (DRM), NIH R01HL085595 (DRM), and American Heart Association Post-doctoral Fellowship 0725663Z (PC).

Footnotes

This is an un-copyedited author manuscript that was accepted for publication in Circulation, copyright The American Heart Association. This may not be duplicated or reproduced, other than for personal use or within the “Fair Use of Copyrighted Materials” (section 107, title 17, U.S. Code) without prior permission of the copyright owner, The American Heart Association. The final copyedited article, which is the version of record, can be found at http://circ.ahajournals.org/cgi/reprint/118/14_suppl_1/S38. The American Heart Association disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties.

DISCLOSURES None.

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