The role of inflammatory cytokines in anemia and gastrointestinal mucosal injury induced by foot electric stimulation.

Foot electrical stimulation (FES) has been considered as a classic stressor that can disturb homeostasis. Acute anemia was observed in the model induced by FES. The aim of this study was to explore the role of inflammatory cytokines underlying the acute anemia and gastrointestinal (GI) mucosal injury in the FES. Twenty-four male Kunming mice (20 ± 2 g) were randomly divided into control group and experimental group. The mice were placed in a footshock chamber that can generate 0.5 mA electrical impulse periodically for 0.5 h. After the process, red blood cell count, hemoglobin concentration and hematocrit, the levels of corticotropin releasing hormone (CRH) in serum and hypothalamus, and adrenocorticotropic hormone (ACTH) in serum and pituitary were detected separately. In addition, we investigated the expressions of inflammatory cytokines (IL-1, IL-6, TNF-α, iNOS, and IL-10) in the hypothalamus and duodenum by Polymerase Chain Reaction (PCR). Results showed that this FES model induced anemia, increased CRH and ACTH activity in the serum after the FES. Moreover, the expressions of IL-1ß, IL-6, TNF-α, and iNOS were significantly increased following the process, while IL-10 was not activated. These findings suggest that anemia, the inflammatory cytokines in the hypothalamus and duodenum of the mice in the model induced by FES is closely related to GI mucosal injury/bleeding. Taken together, these results underscore the importance of anemia, GI mucosal injury/bleeding and stress, future studies would be needed to translate these findings into the benefit of affected patients.

A Rationale for Allopregnanolone Treatment of Alcohol Use Disorders: Basic and Clinical Studies.

For many years, research from around the world has suggested that the neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone or 3α,5α-THP) may have therapeutic potential for treatment of various symptoms of alcohol use disorders (AUDs). In this critical review, we systematically address all the evidence that supports such a suggestion, delineate the etiologies of AUDs that are addressed by treatment with allopregnanolone or its precursor pregnenolone, and the rationale for treatment of various components of the disease based on basic science and clinical evidence. This review presents a theoretical framework for understanding how endogenous steroids that regulate the effects of stress, alcohol, and the innate immune system could play a key role in both the prevention and the treatment of AUDs. We further discuss cautions and limitations of allopregnanolone or pregnenolone therapy with suggestions regarding the management of risk and the potential for helping millions who suffer from AUDs.

An anti-CRF antibody suppresses the HPA axis and reverses stress-induced phenotypes.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

Development of an electrochemical impedimetric immunosensor for Corticotropin Releasing Hormone (CRH) using half-antibody fragments as elements of biorecognition.

An electrochemical impedimetric immunosensor was developed for the detection of the neuropeptide Corticotropin Releasing Hormone (CRH) based on the immobilization of half-antibody fragments on gold nanoparticles (AuNp). Then, the optimal conditions for the obtainment of AuNp through electroplating on a bare gold electrode were studied. The results showed that the obtainment of AuNp at a fixed potential of -0.2 V for 330 s, at 80 °C and 2·10-3 mol·L-1 of HAuCl4 generates an adequate nanostructured surface and is a highly reproducible method. Also, the optimal conditions for immobilizing the half-antibody on AuNp were studied. The interaction of the CRH with the recognition layer of the immobilized half-antibody on the nanostructured surface was carried out by incubation at 4 °C for 2 h. A dissolution of [Fe(CN)6]4-/[Fe(CN)6]3- as a redox probe was used to study the electrochemical responses of the nanostructured surface and the immobilization processes of the half-antibody and detection of CRH, using cyclic voltammetry and electrochemical impedance spectroscopy. An immunosensor was obtained for the specific detection of CRH, within a range of 10.0-80.0 µg mL-1, with a limit of detection of 2.7 µg mL-1 and a limit of quantification of 9.2 µg mL-1. Additionally, the association constant between the CRH and the immobilized half-antibody was calculated at 1.96·105 M-1.

Corticotropin-releasing factor induces inflammatory cytokines via the NLRP6-inflammatory cytokine axis in a murine model of irritable bowel syndrome.

OBJECTIVE: This study aimed to determine the effect of corticotropin-releasing factor (CRF) on regulating the NOD-like receptor pyrin domain-containing protein 6 (NLRP6)-inflammatory cytokine axis in a murine model of irritable bowel syndrome (IBS). METHODS: C57BL/6 mice were subjected to water avoidance stress (WAS) for 1 h per day for 10 days, and the abdominal withdrawal reflex (AWR) and colonic inflammation were assessed. We also measured the levels of CRF, NLRP6 inflammasome components, myeloperoxidase, D-lactate, interleukin (IL)-1ß, and IL-18. In vitro experiments with Caco-2 cell line were also performed. In addition, we assessed the effect of Clostridium butyricum (C. butyricum) on IBS mice. RESULTS: IBS mice exhibited visceral hypersensitivity and inflammation, accompanied by increases in CRF, myeloperoxidase, D-lactate, IL-1ß, and IL-18 levels, but a decrease in NLRP6 expression. In vitro data showed that CRF suppressed NLRP6, but induced IL-1ß and IL-18 levels, in Caco-2 cells. C. butyricum restored CRF levels and maintained the NLRP6-inflammatory cytokine axis in IBS mice. CONCLUSIONS: CRF induces the NLRP6-inflammatory cytokine axis in IBS mice. C. butyricum could be beneficial in controlling IBS.

Cannabinoid CB2 Receptor Gene and Environmental Interaction in the Development of Psychiatric Disorders.

CB2 cannabinoid receptor (CB2R) gene is associated with depression. We investigated the gene-environment interaction between CB2R function and diverse stressors. First, anxiety-like behavior during chronic-mild-stress (CMS) was evaluated in C57BL/6JJmsSlc mice following treatment with CB2R agonist JWH015 or inverse-agonist AM630. Second, locomotor activity and anxiety-like behavior were measured following exposure to an immune poly I:C stressor. Gene expressions of HPA axis related molecules, Fkbp5, Nr3c1 and Crf and pro-inflammatory cytokine Il-1b, as well as Bdnf as a key neurotrophin that supports neuron health, function, and synaptic plasticity, were determined in hippocampus of Cnr2 knockout mice, as indicators of stressful environment. CMS-induced anxiety-like behavior was enhanced by AM630 and reduced by JWH015 and fluvoxamine. Poly I:C reduced locomotor activity and increased anxiety-like behavior, and these effects were pronounced in the heterozygote than in the wild type mice. Fkbp5 and Nr3c1 expression were lower in the Cnr2 heterozygotes than in the wild type mice with Poly I:C treatment. These findings indicate that interaction between CB2R gene and stressors increases the risk of depression-like behaviors that may be linked with neuro-immune crosstalk. Further studies in human subjects are necessary to determine the role of CB2R and environmental interaction in the development of depression.

A case of multiple sclerosis and necrotizing autoimmune myopathy with anti-SRP antibodies.

Only few reports exist regarding the coexistence of multiple sclerosis (MS) and autoimmune myopathies. We describe the case of a man with a long history of undiagnosed left lower limb motor impairment who was hospitalized for subacute onset of a myopathic syndrome. In addition, neurological examination revealed sensory impairment and pyramidal signs in the left limbs. Muscle biopsy revealed a necrotizing myopathy and serum anti-signal recognition particle (SRP) antibodies were found. Brain and spinal cord MRI displayed several non-enhancing demyelinating lesions, and CSF-restricted oligoclonal bands were detected. Multimodal evoked potentials showed increased latency of central conduction. Total body PET-CT did not reveal malignancies. A final diagnosis of anti-SRP necrotizing autoimmune myopathy (NAM) and MS was made, and subsequent therapy with azathioprine resulted in a complete stability for both diseases during the follow up. To the best of our knowledge this is the first reported case of concomitant NAM and MS.

Persistent adaptation by chronic alcohol is facilitated by neuroimmune activation linked to stress and CRF.

This review updates the conceptual basis for the association of alcohol abuse with an insidious adaptation that facilitates negative affect during withdrawal from chronic intermittent alcohol (CIA) exposure - a change that later supports sensitization of stress-induced anxiety following alcohol abstinence. The finding that a CRF1-receptor antagonist (CRF1RA) minimized CIA withdrawal-induced negative affect supported an association of alcohol withdrawal with a stress mechanism. The finding that repeated stresses or multiple CRF injections into selected brain sites prior to a single 5-day chronic alcohol (CA) exposure induced anxiety during withdrawal provided critical support for a linkage of CIA withdrawal with stress. The determination that CRF1RA injection into positive CRF-sensitive brain sites prevented CIA withdrawal-induced anxiety provided support that neural path integration maintains the persistent CIA adaptation. Based upon reports that stress increases neuroimmune function, an effort was undertaken to test whether cytokines would support the adaptation induced by stress/CA exposure. Twenty-four hours after withdrawal from CIA, cytokine mRNAs were found to be increased in cortex as well as other sites in brain. Further, repeated cytokine injections into previously identified brain sites substituted for stress and CRF induction of anxiety during CA withdrawal. Discovery that a CRF1RA prevented the brain cytokine mRNA increase induced by CA withdrawal provided critical evidence for CRF involvement in this neuroimmune induction after CA withdrawal. However, the CRF1RA did not block the stress increase in cytokine mRNA increases in controls. The latter data supported the hypothesis that distinct mechanisms linked to stress and CA withdrawal can support common neuroimmune functions within a brain site. As evidence evolves concerning neural involvement in brain neuroimmune function, a better understanding of the progressive adaptation associated with CIA exposure will advance new knowledge that could possibly lead to strategies to combat alcohol abuse.

Clinical features and prognosis in anti-SRP and anti-HMGCR necrotising myopathy.

OBJECTIVE: To elucidate the common and distinct clinical features of immune-mediated necrotising myopathy (IMNM), also known as necrotising autoimmune myopathy associated with autoantibodies against signal recognition particle (SRP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). METHODS: We examined a cohort of 460 patients with idiopathic inflammatory myopathies (IIMs) through a muscle biopsy-oriented registration study in Japan. Study entry was strictly determined by the comprehensive histological assessment to exclude other neuromuscular disorders. Anti-SRP and anti-HMGCR antibodies were detected by RNA immunoprecipitation and ELISA, respectively. RESULTS: Of 460 patients with IIM, we diagnosed 73 (16%) as having inclusion body myositis (IBM). Of 387 patients with IIMs other than IBM, the frequencies of anti-SRP and anti-HMGCR antibodies were 18% and 12%, respectively. One patient had both autoantibodies. Severe limb muscle weakness, neck weakness, dysphagia, respiratory insufficiency and muscle atrophy were more frequently observed in patients with anti-SRP antibodies than in those with anti-HMGCR antibodies. Serum creatine levels were markedly higher in the patients with autoantibodies than in those without. Histology was characterised by necrosis and regeneration of muscle fibres and was consistent with IMNM except in 1 HMGCR-positive IBM patient. Most patients were initially treated with corticosteroids; however, additional immunosuppressive drugs were required, especially in the patients with anti-SRP antibodies. Rates of unsatisfactory neurological outcome were similar in the 2 autoantibody groups. CONCLUSIONS: Anti-SRP antibodies are associated with severe neurological symptoms, more so than are anti-HMGCR antibodies. Although these autoantibodies are independent serological markers associated with IMNM, patients bearing either share common characteristics.

Immune-Mediated Necrotizing Myopathy: Update on Diagnosis and Management.

The idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune disorders that target skeletal muscle. They are characterized by typical laboratory and clinical features including muscle weakness, elevated muscle enzymes, characteristic histopathology of muscle biopsies, as well as electromyography abnormalities. The IIMs are divided into polymyositis, dermatomyositis, inclusion body myositis, nonspecific myositis, and immune-mediated necrotizing myopathy (IMNM). IMNM is distinguished by the absence of primary inflammation on muscle biopsy. IMNM may be associated with myositis-specific autoantibodies (i.e., anti-SRP and anti-HMGCR) and malignancy, in association with viral infections (HIV or hepatitis C), or in relation to other connective tissue diseases (i.e., scleroderma). Typical clinical findings such as severe muscle weakness, highly elevated creatine kinase (CK) levels, as well as resistance to conventional immunosuppressive therapy are associated with this subtype of IIM. This review provides an overview of this disease entity and focuses on its diagnosis and treatment.

Clinical Features and Treatment Outcomes of Necrotizing Autoimmune Myopathy.

IMPORTANCE: Necrotizing autoimmune myopathy (NAM) is characterized pathologically by necrotic muscle fibers with absent or minimal inflammation. It is often accompanied by statin therapy, connective tissue diseases, cancer, and autoantibodies specific for signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). Data are limited concerning differences among etiologic subgroups and treatment outcomes in NAM. OBJECTIVES: To describe the clinical, serologic, and electrophysiologic characteristics of NAM, compare patient subgroups, and determine clinical outcome predictors. DESIGN, SETTING, AND PARTICIPANTS: We conducted a retrospective review of medical records for 63 adult Mayo Clinic patients assigned the clinical and histopathologic diagnosis of NAM from January 1, 2004, through December 31, 2013. Patients were stratified by presumed cause and autoantibody status. MAIN OUTCOMES AND MEASURES: Clinical, electrophysiologic, and pathologic characteristics were collected and compared among patient subgroups. Predictors of response to treatment were identified by univariate logistic regression. RESULTS: Lower extremity weakness predominated (46 [73%]). Distal weakness (26 [41%]), dysphagia (22 [35%]), and dyspnea (23 [37%]) were common. Twenty-two patients (35%) were receiving a statin medication at onset, 6 had cancer, and 3 had a connective tissue disease. The median creatine kinase level was 5326 U/L. In 13 patients (24%), SRP-IgG was detected, and in 17 patients (34%), HMGCR-IgG was detected (one-third of whom had not received statin medication). One patient was dual seropositive. Facial weakness was more common in SRP-IgG-positive patients. Myotonic discharges were more common in statin-associated NAM. Prednisone monotherapy was insufficient to control disease in most patients; 30 (90%) of 32 patients required 2 or more immunotherapeutic agents. Relapse occurred in 16 (55%) of 29 patients during immunosuppressant taper or discontinuation. Predictors of favorable outcome were male sex and use of 2 or more immunotherapeutic agents within 3 months of onset. CONCLUSIONS AND RELEVANCE: Necrotizing autoimmune myopathy was idiopathic in half of this cohort with clinical and histopathologically defined disease. In the remainder, NAM was associated with statin medication, cancer, or connective tissue disease. One in 4 patients was SRP-IgG positive, and 1 in 3 was HMGCR-IgG positive. The disease was usually not controlled by corticosteroid monotherapy. Presentation, course, and outcomes did not differ significantly in seropositive, seronegative, and statin-associated cases. Early aggressive immunosuppressant therapy improved outcomes, and risk of relapse was high during medication dose reduction or withdrawal.

CRH knockout inhibits the murine innate immune responses in association with endoplasmic reticulum stress after thermal injury.

BACKGROUND: Previous studies in our laboratory have demonstrated the hypothalamus destruction and adrenalectomy could blunt the innate immunity while boosting the excessive inflammation after injury. We aimed to investigate the effects of corticotrophin-releasing hormone knockout (CRH KO) on the innate immune responses in macrophages as well as to elucidate the underlying mechanism. METHODS: The chemotaxis and phagocytosis activities of macrophages, bacteria translocation, plasma tumor necrosis factor (TNF)-α secretion, and intestinal injury were observed in the presence of the endoplasmic reticulum stress after thermal injury in CRH KO mice. Meanwhile, the messenger RNA (mRNA) and protein expression of glucose response protein 78 (GRP78), X-box binding protein 1 (XBP1), and activating transcription factor 6 (ATF6) in macrophages was also determined. RESULTS: After thermal injury, the chemotaxis and phagocytosis of peritoneal macrophages were increased, which were both reversed by CRH gene deficiency. The gut-derived bacteria translocation to liver tissues, lung tissues and mesenteric lymph nodes was significantly strengthened in CRH KO mice compared with CRH wild-type littermates. Circulating TNF-α level was increased markedly in response to thermal injury and CRH KO further increased its secretion. Furthermore, the mRNA and protein levels of GRP78, XBP1, and ATF6 in peritoneal macrophages increased, while their expressions in CRH KO mice all decreased significantly. CRH KO mice showed enhancement of inflammatory responses and severe tissue injuries after thermal injury. CONCLUSION: CRH exerted immune defensive actions on immune cells and organs in the early phase of injury, suggesting that the underlying mechanisms are related to endoplasmic reticulum stress.

A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.

This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His6-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹° and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.

Stress and visceral pain: focusing on irritable bowel syndrome.

Recent advances in brain science have shown that the brain function encoding emotion depends on interoceptive signals such as visceral pain. Visceral pain arose early in our evolutionary history. Bottom-up processing from gut-to-brain and top-down autonomic/neuroendocrine mechanisms in brain-to-gut signaling constitute a circuit. Brain imaging techniques have enabled us to depict the visceral pain pathway as well as the related emotional circuit. Irritable bowel syndrome (IBS) is characterized by chronic recurrent abdominal pain or abdominal discomfort associated with bowel dysfunction. It is also thought to be a disorder of the brain-gut link associated with an exaggerated response to stress. Corticotropin-releasing hormone (CRH), a major mediator of the stress response in the brain-gut axis, is an obvious candidate in the pathophysiology of IBS. Indeed, administration of CRH has been shown to aggravate the visceral sensorimotor response in IBS patients, and the administration of peptidergic CRH antagonists seems to alleviate IBS pathophysiology. Serotonin (5-HT) is another likely candidate associated with brain-gut function in IBS, as 5-HT3 antagonists, 5-HT4 agonists, and antidepressants were demonstrated to regulate 5-HT neurotransmission in IBS patients. Autonomic nervous system function, the neuroimmune axis, and the brain-gut-microbiota axis show specific profiles in IBS patients. Further studies on stress and visceral pain neuropathways in IBS patients are warranted.

Assessment of T regulatory cells and expanded profiling of autoantibodies may offer novel biomarkers for the clinical management of systemic sclerosis and undifferentiated connective tissue disease.

In order to identify disease biomarkers for the clinical and therapeutic management of autoimmune diseases such as systemic sclerosis (SSc) and undifferentiated connective tissue disease (UCTD), we have explored the setting of peripheral T regulatory (T reg) cells and assessed an expanded profile of autoantibodies in patients with SSc, including either limited (lcSSc) or diffuse (dcSSc) disease, and in patients presenting with clinical signs and symptoms of UCTD. A large panel of serum antibodies directed towards nuclear, nucleolar, and cytoplasmic antigens, including well-recognized molecules as well as less frequently tested antigens, was assessed in order to determine whether different antibody profiles might be associated with distinct clinical settings. Beside the well-recognized association between lcSSc and anti-centromeric or dcSSC and anti-topoisomerase-I antibodies, we found a significative association between dcSSc and anti-SRP or anti-PL-7/12 antibodies. In addition, two distinct groups emerged on the basis of anti-RNP or anti-PM-Scl 75/100 antibody production among UCTD patients. The levels of T reg cells were significantly lower in patients with SSc as compared to patients with UCTD or to healthy controls; in patients with lcSSc, T reg cells were inversely correlated to disease duration, suggesting that their levels may represent a marker of disease progression.

Immune cells listen to what stress is saying: neuroendocrine receptors orchestrate immune function.

Over the past three decades, the field of psychoneuroimmunology research has blossomed into a major field of study, gaining interests of researchers across all traditionally accepted disciplines of scientific research. This chapter provides an overview of our current understanding in defining neuroimmune interactions with a primary focus of discussing the neuroendocrine receptor activity by immune cells. This chapter highlights the necessity of neuroimmune responses as it relates to a better understanding of the pathophysiological mechanisms of health and disease.

Contribution of stress to asthma worsening through mast cell activation.

OBJECTIVE: To review the available evidence linking stress to asthma and to investigate whether mast cells contribute to the effect of stress through activation by corticotropin-releasing hormone (CRH). DATA SOURCE: The PubMed database was searched for articles (1998-2011) using the keywords anxiety, asthma, exacerbation, inflammation, mast cells, socioeconomic status, stress, violence, and worsening. STUDY SELECTION: Articles were selected based on their relevance to the topic, with emphasis on clinical or epidemiologic data linking stress to asthma and studies that offered possible explanations for how stress may affect asthma. RESULTS: Many articles point to an association between stress (socioeconomic status, interpersonal conflicts, emotional distress, terrorism) and asthma exacerbations but without any distinct pathogenetic mechanism. A few articles have reported reduced circulating cortisol and/or sensitivity to corticosteroids. We propose that mast cells, known to be involved in the pathophysiology of asthma, can be activated by CRH, which is secreted under stress in the lungs, leading to selective release of proinflammatory mediators. This effect may be augmented by neuropeptides or cytokines. CRH also reduces T-regulatory cell production of interleukin 10, which in known to inhibit allergic mast cell activation. CONCLUSION: More studies are required to investigate lung levels of CRH and selective mast cell mediators. Reducing stress and using CRH receptor antagonists and/or mast cell blockers may serve as possible new therapeutic approaches for asthma.

Immune aspects and myometrial actions of progesterone and CRH in labor.

Progesterone and corticotropin-releasing hormone (CRH) have a critical role in pregnancy and labor, as changes related to these hormones are crucial for the transition from myometrial quiescence to contractility. The mechanisms related to their effect differ between humans and other species, thus, despite extensive research, many questions remain to be answered regarding their mediation in human labor. Immune responses to progesterone and CRH are important for labor. Progesterone acts as an immunomodulator which controls many immune actions during pregnancy, and its withdrawal releases the inhibitory action on inflammatory pathways. In humans, a "functional" progesterone withdrawal occurs with onset of labor through changes in progesterone metabolism, progesterone receptors, and other molecules that either facilitate or antagonize progesterone function. Placental CRH acts on the fetal pituitary-adrenal axis to stimulate adrenal production of androgens and cortisol and also acts directly on myometrial cells via its receptors. CRH also affects inflammatory signals and vice versa. Interactions between progesterone and CRH additionally occur during labor. We describe the role of these two hormones in human myometrium and their interactions with the immune system during labor.