Functionally selective inhibition of the oxytocin receptor by retosiban in human myometrial smooth muscle

Context: Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared to first generation oxytocin antagonists when used for the prevention of preterm birth. Objective: To determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban in comparison to the currently used peptide-based compound atosiban. Design: Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Results: Retosiban antagonism of oxytocin action in human myometrium was potent, rapid and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single site competitive binding kinetics for epelsiban, retosiban and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban, but not retosiban inhibited forskolin and calcitonin stimulated cAMP production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated ERK1/2 activity in a time a concentration dependent manner. Oxytocin and atosiban stimulated cyclo oxygenase 2 (COX2) activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gαq G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gαi G-proteins. Conclusions: Retosiban and epelsiban demonstrate distinct pharmacology when compared to atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production

The spectrum of resistance in SR/CR mice: the critical role of chemoattraction in the cancer/leukocyte interaction

<p>Abstract</p> <p>Background</p> <p>Spontaneous regression/complete resistance (SR/CR) mice are a unique colony of mice that possess an inheritable, natural cancer resistance mediated primarily by innate cellular immunity. This resistance is effective against sarcoma 180 (S180) at exceptionally high doses and these mice remain healthy.</p> <p>Methods</p> <p>In this study, we challenged SR/CR mice with additional lethal transplantable mouse cancer cell lines to determine their resistance spectrum. The ability of these transplantable cancer cell lines to induce leukocyte infiltration was quantified and the percentage of different populations of responding immune cells was determined using flow cytometry.</p> <p>Results</p> <p>In comparison to wild type (WT) mice, SR/CR mice showed significantly higher resistance to all cancer cell lines tested. However, SR/CR mice were more sensitive to MethA sarcoma (MethA), B16 melanoma (B16), LL/2 lung carcinoma (LL/2) and J774 lymphoma (J774) than to sarcoma 180 (S180) and EL-4 lymphoma (EL-4). Further mechanistic studies revealed that this lower resistance to MethA and LL/2 was due to the inability of these cancer cells to attract SR/CR leukocytes, leading to tumor cell escape from resistance mechanism. This escape mechanism was overcome by co-injection with S180, which could attract SR/CR leukocytes allowing the mice to resist higher doses of MethA and LL/2. S180-induced cell-free ascites fluid (CFAF) co-injection recapitulated the results obtained with live S180 cells, suggesting that this chemoattraction by cancer cells is mediated by diffusible molecules. We also tested for the first time whether SR/CR mice were able to resist additional cancer cell lines prior to S180 exposure. We found that SR/CR mice had an innate resistance against EL-4 and J774.</p> <p>Conclusions</p> <p>Our results suggest that the cancer resistance in SR/CR mice is based on at least two separate processes: leukocyte migration/infiltration to the site of cancer cells and recognition of common surface properties on cancer cells. The infiltration of SR/CR leukocytes was based on both the innate ability of leukocytes to respond to chemotactic signals produced by cancer cells and on whether cancer cells produced these chemotactic signals. We found that some cancer cells could escape from SR/CR resistance because they did not induce infiltration of SR/CR leukocytes. However, if infiltration of leukocytes was induced by co-injection with chemotactic factors, these same cancer cells could be effectively recognized and killed by SR/CR leukocytes.</p

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf^-/-), superoxide (Cybb^-/), or inducible nitric oxide (Nos2^-/).</p> <p>Methods</p> <p>SR/CR mice were bred into individual Prf^-/-, Cybb^-/-, or Nos2^-/-genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined.</p> <p>Results</p> <p>When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf^-/-, Cybb^-/-, or Nos2^-/-did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2^-/-background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls.</p> <p>Conclusion</p> <p>Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.</p

Towards understanding the myometrial physiome: approaches for the construction of a virtual physiological uterus

Premature labour (PTL) is the single most significant factor contributing to neonatal morbidity in Europe with enormous attendant healthcare and social costs. Consequently, it remains a major challenge to alleviate the cause and impact of this condition. Our ability to improve the diagnosis and treatment of women most at risk of PTL is, however, actually hampered by an incomplete understanding of the ways in which the functions of the uterine myocyte are integrated to effect an appropriate biological response at the multicellular whole organ system. The level of organization required to co-ordinate labouring uterine contractile effort in time and space can be considered immense. There is a multitude of what might be considered mini-systems involved, each with their own regulatory feedback cycles, yet they each, in turn, will influence the behaviour of a related system. These include, but are not exclusive to, gestational-dependent regulation of transcription, translation, post-translational modifications, intracellular signaling dynamics, cell morphology, intercellular communication and tissue level morphology. We propose that in order to comprehend how these mini-systems integrate to facilitate uterine contraction during labour (preterm or term) we must, in concert with biological experimentation, construct detailed mathematical descriptions of our findings. This serves three purposes: firstly, providing a quantitative description of series of complex observations; secondly, proferring a database platform that informs further testable experimentation; thirdly, advancing towards the establishment of a virtual physiological uterus and in silico clinical diagnosis and treatment of PTL

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.</p> <p>Results</p> <p>In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing.</p> <p>Conclusion</p> <p>Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases.</p

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility

Are caregiving responsibilities associated with non-attendance at breast screening?

<p>Abstract</p> <p>Background</p> <p>Previous research showed that deprived individuals are less likely to attend breast screening and those providing intense amounts of informal care tend to be more deprived than non-caregivers. The aim of this study was to examine the relationship between informal caregiving and uptake of breast screening and to determine if socio-economic gradients in screening attendance were explained by caregiving responsibilities.</p> <p>Methods</p> <p>A database of breast screening histories was linked to the Northern Ireland Longitudinal Study, which links information from census, vital events and health registration datasets. The cohort included women aged 47 - 64 at the time of the census eligible for breast screening in a three-year follow-up period. Cohort attributes were recorded at the Census. Multivariate logistic regression was used to examine the relationship between informal caregiving and uptake of screening using STATA version 10.</p> <p>Results</p> <p>37,211 women were invited for breast screening of whom 27,909 (75%) attended; 23.9% of the cohort were caregivers. Caregivers providing <20 hours of care/week were more affluent, while those providing >50 hours/week were more deprived than non-caregivers. Deprived women were significantly less likely to attend breast screening; however, this was not explained by caregiving responsibilities as caregivers were as likely as non-caregivers to attend (Odds Ratio 0.97; 95% confidence intervals 0.88, 1.06).</p> <p>Conclusions</p> <p>While those providing the most significant amounts of care tended to be more deprived, caregiving responsibilities themselves did not explain the known socio-economic gradients in breast screening attendance. More work is required to identify why more deprived women are less likely to attend breast screening.</p

Bioactive Hydrogel Substrates: Probing Leukocyte Receptor–Ligand Interactions in Parallel Plate Flow Chamber Studies

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe(X)) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine–glycine–aspartic acid–serine (RGDS) and leucine–aspartic acid–valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering

A Computational Model of the Ionic Currents, Ca2+ Dynamics and Action Potentials Underlying Contraction of Isolated Uterine Smooth Muscle

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: currents (L- and T-type), current, an hyperpolarization-activated current, three voltage-gated currents, two -activated current, -activated current, non-specific cation current, - exchanger, - pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area∶volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/inactivation gating variables and temporal changes in intracellular computed from known fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltage-clamp. Phasic contraction is modeled in relation to the time constant of changing . This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels