Propranolol reduces IFN-γ driven PD-L1 immunosuppression and improves anti-tumour immunity in ovarian cancer.

The immune system plays an important role in controlling epithelial ovarian cancer (EOC). EOC is considered to be a "cold tumour," a tumour that has not triggered a strong response by the immune system. However, tumour infiltrating lymphocytes (TILs) and the expression of programmed cell death ligand (PD-L1) are used as prognostic indicators in EOC. Immunotherapy such as PD-(L)1 inhibitors have shown limited benefit in EOC. Since the immune system is affected by behavioural stress and the beta-adrenergic signalling pathway, this study aimed to explore the impact of propranolol (PRO), a beta-blocker, on anti-tumour immunity in both in vitro and in vivo EOC models. Noradrenaline (NA), an adrenergic agonist, did not directly regulate PD-L1 expression but PD-L1 was significantly upregulated by IFN-γ in EOC cell lines. IFN-γ also increased PD-L1 on extracellular vesicles (EVs) released by ID8 cells. PRO significantly decreased IFN-γ levels in primary immune cells activated ex vivo and showed increased viability of the CD8+ cell population in an EV-immune cell co-incubation. In addition, PRO reverted PD-L1 upregulation and significantly decreased IL-10 levels in an immune-cancer cell co-culture. Chronic behavioural stress increased metastasis in mice while PRO monotherapy and the combo of PRO and PD-(L)1 inhibitor significantly decreased stress-induced metastasis. The combined therapy also reduced tumour weight compared to the cancer control group and induced anti-tumour T-cell responses with significant CD8 expression in tumour tissues. In conclusion, PRO showed a modulation of the cancer immune response by decreasing IFN-γ production and, in turn, IFN-γ-mediated PD-L1 overexpression. The combined therapy of PRO and PD-(L)1 inhibitor decreased metastasis and improved anti-tumour immunity offering a promising new therapy.

Smooth muscle relaxation and activation of the large conductance Ca(++)-activated K+ (BK(Ca)) channel by novel oestrogens.

BACKGROUND AND PURPOSE: Oestrogens can interact directly with membrane receptors and channels and can activate vascular BK(Ca) channels. We hypothesized that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and ß1 subunits of the BK(Ca) channel, rather than at an intracellular site. EXPERIMENTAL APPROACH: We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+ß1 subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable. KEY RESULTS: Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol-induced relaxation was iberiotoxin sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloß1 were activated by oestrone, oestrone oxime and Quat DME-oestradiol. CONCLUSION AND IMPLICATIONS: The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on NO and BK(Ca) channel activation. The activation of BK(Ca) currents in HEK 293 cells expressing hSloα+ß1 by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the ß1 subunit. Membrane-impermeant Quat DME-oestradiol lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.

High-density lipoprotein apolipoprotein A-I kinetics: comparison of radioactive and stable isotope studies.

To compare the kinetic determinants of high-density lipoprotein (HDL) apolipoprotein A-I (apoA-I) concentration in lean normolipidaemic subjects using radioisotope and stable isotope studies. We pooled data from 16 radioisotope and 13 stable isotope studies to investigate the kinetics of apoA-I in lean normolipidemic individuals. We also examined the associations of HDL kinetic parameters with age, sex, body mass index (BMI) and concentrations of apoA-I, triglycerides, HDL cholesterol and low-density lipoprotein (LDL) cholesterol. Lean subjects from radioisotope and stable isotope studies were matched for age, gender, BMI and lipid profile. The apoA-I concentration was significantly lower in the radioisotope group than the stable isotope group (P = 0.031). There was no significant difference in HDL apoA-I fractional catabolic rate (FCR) and production rate (PR) between the groups. In the radioisotope group, HDL apoA-I FCR was significantly associated with apoA-I and HDL cholesterol concentrations (r = -0.681, P < 0.001 and r = -0.542, P < 0.001, respectively), whereas in the stable isotope group, only HDL apoA-I PR was significantly associated with apoA-I concentration (r = 0.455, P = 0.004). Our findings suggest that HDL apoA-I FCR is the primary determinant of apoA-I concentrations in lean subjects in studies using radiotracer techniques. By contrast, HDL apoA-I PR is the primary determinant of apoA-I concentration in lean subject in studies employing stable isotope methods. These discrepancies may be reconciled by differences in methodologies and/or study population characteristics.

Changes in the properties of the modulatory cerebral giant cells contribute to aging in the feeding system of Lymnaea.

This study examined whether electrophysiological changes in the endogenous properties and connectivity of the modulatory serotonergic cerebral giant cells (CGCs) contributed to the age-related changes in feeding behavior of the pond snail, Lymnaea. With increasing age there was a decrease in spontaneous CGC firing rates and decreased excitability of the CGCs to both chemosensory stimulation (0.05M sucrose applied to the lips) and direct intracellular current injection. These changes could be accounted for by a decrease in the input resistance of the neuron and an increase in the amplitude and the duration of the after-hyperpolarization. Decreases were also seen in the % of CGC pairs that were electrically coupled causing asynchronous firing. Together these changes would tend to reduce the ability of the CGCs to gate and control the frequency of the feeding behavior. Part of the ability of the CGCs to gate and frequency control the feeding network is to provide a background level of excitation to the feeding motor neurons. Recordings from B1 and B4 motor neurons showed an age-related hyperpolarization of the resting membrane potential consistent with a deficit in CGC function. Increases were seen in the strength of the evoked CGC-->B1 connection, however, this increase failed to compensate for the deficits in CGC excitability. In summary, age-related changes in the properties of the CGCs were consistent with them contributing to the age-related changes in feeding behavior seen in Lymnaea.

Effects of age on feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis.

This study used behavioral and electrophysiological techniques to examine age-related changes in the feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis. Increasing age was associated with a 50% decrease in long-term food consumption. Analysis of short-term sucrose-evoked feeding bouts showed an age-related increase in the number of animals that failed to respond to the stimulus. Of the animals that did respond increasing age was associated with a decrease in the number of sucrose-evoked bites and a increase in the duration of the swallow phase. These changes were observed with both 0.01 and 0.05M sucrose stimuli but were not seen when 0.1M sucrose was used as the stimulus. Electrophysiological analysis of the chemosensory pathway in semi-intact lip-CNS preparations failed to demonstrate a significant change in the neuronal information entering the cerebral ganglia from the lips via the median lip nerve, but did demonstrate an age-related deficit in the neuronal output from the cerebral ganglia. This deficit was also dependent on the sucrose concentration and mirrored the concentration-dependent changes in feeding behavior. In summary, aging appeared to affect central but not peripheral processing of chemosensory information and suggests that this deficit contributes to the age-related changes in feeding behavior.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Discovery of zoniporide: a potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility.

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.

Volume--activated chloride currents in HeLa cells are blocked by tamoxifen but not by a membrane impermeant quaternary analogue.

BACKGROUND/AIMS: Tamoxifen has been shown to inhibit volume activated chloride currents in many cell types. Tamoxifen has also been reported to inhibit a number of cation channels as well as cytosolic proteins such as calmodulin. The mechanism of channel block by tamoxifen is not known but three hypotheses can be proposed: i) a direct effect following binding to the channel protein from the aqueous environment or ii) a direct effect on the channel protein after partitioning into the lipid membrane or iii) an indirect mechanism via binding to intracellular regulatory proteins after diffusion across the lipid membrane. The aim of these experiments was to distinguish between these hypotheses using membrane permeant and impermeant antioestrogens. METHODS: Volume activated chloride currents were recorded from single HeLa cells using whole cell patch clamp technique. The ability of tamoxifen and its membrane impermeant quaternary derivative ethyl bromide tamoxifen (EBT) to inhibit these currents was examined. RESULTS: Extracellular tamoxifen at 3 microM inhibited volume activated chloride currents in HeLa cells whereas EBT had no effect up to 10 microM when applied either to the extracellular bathing solution or the intracellular solution via the patch pipette. CONCLUSION: Eliminating the ability of tamoxifen to cross the plasma membrane abolishes its channel blocking activity against volume activated chloride channels in HeLa cells.

Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the extracellular matrix during corneal wound healing are largely unknown. The following study used an in vitro model of ageing to assess changes in MMP-2 RNA expression and protein secretion. Early passage (EP) EK1.BR keratocyte cultures from 14 to 18 cumulative population doublings (cpds) and late passage (LP) cultures from 40 to 47 cpds were used to isolate protein and mRNA samples. Total protein from EP and LP cultures was measured using the Bradford protein assay. Zymographic analysis of EP and LP samples was carried out to compare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) based chemiluminescent detection system. LP cultures secreted more total protein per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultures. Densitometric analysis of zymograms and calculation of MMP-2 activity indicated a significant increase in MMP-2 activity per cell (P<0.05, n=11). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures suggests that senescent keratocytes increase their degradative capacity. Similar changes in the keratocyte phenotype within the ageing cornea may alter the balanced response necessary for adequate wound healing and may have implications for the therapeutic use of MMP inhibitors in the eye.

Membrane impermeant antioestrogens discriminate between ligand- and voltage-gated cation channels in NG108-15 cells.

Native 5-HT(3) and AChR ligand-gated cation channels can be inhibited (blocked) by the non-steroidal antioestrogen tamoxifen. However, the exact site and mechanism of inhibition by tamoxifen on these channels remain unclear. We have investigated the action of the membrane impermeant quaternary derivative, ethylbromide tamoxifen (EBT), on native ligand-gated 5-HT(3) receptor channels and voltage-gated K(+) channels in NG108-15 cells using whole cell patch clamp. Extracellular EBT inhibited whole cell cationic currents of 5-HT(3) receptors with IC(50) of 0.22+/-0.4 microM (n(H)=1.05+/-0.2). The channel block was characterised by voltage independent and use independent behaviour (similar to that of tamoxifen). EBT was unable to inhibit voltage-gated K(+) currents in NG108-15 cells. This was in contrast to the inhibition by tamoxifen which, at similar concentrations, accelerated the apparent inactivation of these outward K(+) currents. The inhibition of 5-HT(3) receptors by a membrane impermeant derivative of tamoxifen supports the view that the binding site for antioestrogens is extracellular and the inhibition is not mediated through genomic/transcriptional activity.

Human keratocyte migration into collagen gels declines with in vitro ageing.

Although senescence in various cell types has been shown to have detrimental effects on wound repair, the effect of this phenomenon on corneal function with increasing age has yet to be elucidated. This study investigated the effect of in vitro ageing on keratocyte migration into a collagen gel matrix. The keratocyte cell strain EK1. BR was cultured to late passage and a comparison of early passage migration with that of late passage migration was carried out. Early or late passage keratocytes were seeded onto 6 collagen gels (1.75 mg ml(-1)) for each experiment. Gels were incubated at 37 degrees C for 72 h, stained with calcein AM (0.5 mg ml(-1)) and assayed for cell migration using fluorescent microscopy. Changes in the effect of EGF on keratocyte migration with age were assessed by the addition of EGF (20 ng ml(-1)) to 3 of the 6 gels in each experiment. Proliferative lifespan was measured by immunocytochemical detection of Ki67 activity. This study shows for the first time that keratocyte migration, and migration in response to EGF stimulation, significantly declines with increasing age of keratocytes in culture (P<0.001). As keratocyte migration in response to cytokine stimulation is vital for corneal repair, the accumulation of senescent keratocytes with age may impair corneal wound healing.

Novel materials to enhance keratoprosthesis integration.

BACKGROUND: The successful integration of keratoprostheses (KPros) within the cornea depends in part on peripheral host keratocyte adhesion to anchor the implant in place and prevent epithelial downgrowth. The following study incorporated different acrylate co-monomers with poly(hydroxyethyl methacrylate) (p(HEMA)) and measured the suitability of these materials as potential skirt materials in terms of their ability to enhance keratocyte adhesion to p(HEMA). METHODS: p(HEMA) hydrogels incorporating varying amounts of the acrylate co-monomers methacrylic acid (MA), 2-(dimethylamino)ethyl methacrylate (DEM), or phenoxyethyl methacrylate (PEM) were formed by free radical polymerisation. Keratocytes were seeded onto discs of each material and incubated at 37 degrees C for 72 hours. Assays for viable cell adhesion were carried out. A viability/cytotoxicity assay using solutions of calcein-AM (0.5 mM) and ethidium homodimer-1 (EthD-1) (0.5 microM) were used to measure viable and non-viable cell adhesion, respectively. An ATP assay was also used to quantify cell adhesion in terms of the amount of ATP present following lysis of adherent cells. RESULTS: The viability/cytotoxicity assays indicated that the incorporation of 15 mol% of the co-monomer PEM or of 20 mol% DEM increased cell adhesion to p(HEMA) by at least four times. The ATP assays confirmed the results for PEM but absorption of ATP to the DEM containing hydrogel indicated that the assay was not a suitable measure of cell adhesion to this material. CONCLUSIONS: The properties of p(HEMA) may be moderated to enhance keratocyte adhesion by the incorporation of PEM or DEM suggesting that these may be suitable materials for use in the further development of a novel KPro skirt material.

Racial differences in temporal changes in newborn viability and survival by gestational age.

This study examines trends in the rates of very preterm, moderately preterm and gestational age-specific neonatal mortality, and in the gestational age limit of viability in South Carolina (SC) from 1975 to 1994. We also investigate whether trends were similar between African-Americans and Whites. We hypothesised that disproportionate reductions in gestational age-specific mortality, rather than any major changes in the gestational age distributions of either race group, underlie any increasing racial disparity in overall mortality rates. During 1975-94, single livebirths, who were born to mothers resident in SC and were either White or African-American based on recorded maternal race, were selected for the investigation. We define the gestational age limit of viability as the gestational age at which > or = 50% of infants in the population died within 28 days of life. Although preterm percentages have not improved, there was a marked decline in neonatal mortality. Gestational age-specific neonatal mortality decreased for both race groups, although there were greater reductions for White preterm infants. By the end of the study period, the African-American neonatal mortality rate was 2.3 times that of Whites and the gestational age at which 50% of newborns died within 28 days of life was 24.5 weeks for Whites and 23.9 weeks for African-Americans. The ongoing decline in neonatal mortality continues to be mainly due to reductions in gestational age-specific neonatal mortality, probably related to high-risk obstetric and neonatal care. Technological developments in these areas may have differentially benefited Whites, resulting in an increasing racial disparity in neonatal mortality rates. Preterm African-American infants no longer have a marked survival advantage over White infants, even at the gestational age limit of viability.

Developmental outcome of neonatal intensive care: what questions are we asking?

Recent neonatal intensive care outcome studies are asking more focused research questions and incorporating, at least implicitly, pathogenetic models. A few have grappled with the complex issues of health-related quality of life and functional outcomes and the many factors that affect these outcomes. The need to evaluate high-risk obstetrics is increasingly recognized. Studies of risk factors for neurodevelopmental outcomes provide valuable insights into mechanisms of and recovery from central nervous system injury. Ongoing study of the efficacy and effectiveness of interventions must continue amid concern about availability of family and developmental support for increasing numbers of survivors of high-risk obstetric and neonatal intensive care.

A model for the binding of low molecular weight inhibitors to the active site of thrombin.

This paper describes the construction, validation and application of an active site model of the serine protease thrombin. Initial use was made of medium resolution X-ray crystallographic structures of thrombin complexed with low molecular weight, non-specific inhibitors to create a computationally useable active site shell of the enzyme. Molecular mechanics methods were then applied to dock known ligands into the active site region in order to derive a model that would accurately predict binding conformations. Validation of the modelling process was achieved by comparison of the predicted enzyme-bound conformations with their known, crystallographic binding conformations. The resultant model was used extensively for predictive purposes prior to obtaining confirmatory crystal data relating to a ligand possessing a novel and unexpected binding component complexed to thrombin. The data served both to confirm the accuracy of the binding site model and to provide information for the further refinement of the model.

Design and synthesis of thrombin inhibitors: analogues of MD-805 with reduced stereogenicity and improved potency.

Mitsubishi's MD-805, a potent and selective inhibitor of thrombin which contains four stereogenic centers, has been the starting point for an optimization program. A systematic synthetic study resulted in thrombin inhibitors achiral at P2 and P3 but with a 10-fold increase in potency over the original lead. A number of 4-substituted piperidines were synthesized and examined as replacements for 2-carboxy-4-methylpiperidine at P2; 4-fluoroethylpiperidine (FEP) among others provided inhibitors (e.g. 45g) of increased potency. An enantioselective route was developed to 3(R)-methyl-1,2,3,4-tetrahydroquinolinesulfonyl chloride. Inhibitors containing this enantiomerically pure P3 (42d) had similar potency to the racemic material and provided support, with modeling studies, for the preparation of the gem 3,3-disubstituted compounds. A series of inhibitors containing the novel 3, 3-dimethyl-1,2,3,4-tetrahydroquinolinesulfonyl (DMTHQS) P3 (Table 5) were synthesized and showed a similar activity profile as the monomethyl series. The combination of P3-DMTHQS, P2-FEP, and P1-arginine (45g) had a K(i) of 6 nM (MD-805 K(i) = 85 nM). In animal models of both venous and arterial thrombosis, one inhibitor (42e) was shown to produce a dose-dependent inhibition of thrombus formation that in some situations was superior to that of MD-805.

The control of hyperendemic glycopeptide-resistant Enterococcus spp. on a haematology unit by changing antibiotic usage.

The rectal carriage of glycopeptide-resistant Enterococcus spp. (GRE) had been established at approximately 50% in a series of prevalence studies on a busy haematological malignancy unit. The aim of this study was to reduce the chance of patients acquiring GRE. A prospective three-phase sequential study was performed. In Phase 1, the acquisition rate of GRE detectable by rectal swab was measured without any intervention for a period of 4 months. For the following 8 months (Phase 2), the first-line treatment for febrile neutropenic episodes was changed from monotherapy with ceftazidime to piperacillin/tazobactam. In addition, an intense education programme was introduced to improve hygiene to reduce the risk of case-to-case spread. In the final 4 months (Phase 3), ceftazidime was again used as the first-line antimicrobial, while continuing the same level of training in relation to hygiene. The carriage of GRE was measured from rectal swabs done weekly. During the initial 4 months, at any time, 40-50% of patients in the unit were colonized with GRE, and 43 of 75 (57%) new patients initially negative for GRE acquired it within 6 weeks of their admission. In Phase 2, 25 patients out of 129 (19%) acquired GRE, with the acquisition rate falling progressively so that in the last 3 months, only one new patient acquired GRE (logrank comparison of probabilities for cohort 1 vs cohort 2b: P < 0.0001). A return to ceftazidime in Phase 3 was associated with a return of the risk of acquiring detectable GRE colonization, despite continued hygiene teaching and surveillance, with 21 out of 58 patients (36%) acquiring GRE (cohort 1 vs cohort 3: P = 0.08). Glycopeptide usage was not reduced during the period of the study. Clinical cases were seen only in Phases 1 and 3. Although the reduction in the risk of acquiring GRE may have been due in part to hygiene practices as well as to the change in antimicrobial usage, or may have occurred spontaneously for other reasons, the return of the problem with the reintroduction of ceftazidime strongly suggests that this antibiotic was responsible for encouraging the acquisition of detectable GRE.