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Abstract Background Poor medication adherence is an extraordinarily common problem worldwide that contributes to inadequate control of many chronic diseases, including Hypertension (HT). Globally, less than 14% of the estimated 1.4 billion patients with HT achieve optimal control. A myriad of barriers, across patient, healthcare provider, and system levels, contributes to poor medication adherence. Few studies have explored the reasons for poor medication adherence in Tanzania and other African countries. Methods A qualitative study applying grounded theory principles was conducted in the catchment area of two semi-urban clinics in Dar es Salaam, Tanzania, to determine the perceived barriers to HT medication adherence. Ten key informant interviews were conducted with healthcare providers who manage HT patients. Patients diagnosed with HT (SBP ≥ 140 and DBP ≥ 90), were randomly selected from patient registers, and nine focus group discussions were conducted with a total 34 patients. Inductive codes were developed separately for the two groups, prior to analyzing key thematic ideas with smaller sub-categories. Results Affordability of antihypertensive medication and access to care emerged as the most important barriers. Fee subsidies for treatment and medication, along with health insurance, were mentioned as potential solutions to enhance access and adherence. Patient education and quality of physician counseling were mentioned by both providers and patients as major barriers to medication adherence, as most patients were unaware of their HT and often took medications only when symptomatic. Use of local herbal medicines was mentioned as an alternative to medications, as they were inexpensive, available, and culturally acceptable. Patient recommendations for improving adherence included community-based distribution of refills, SMS text reminders, and family support. Reliance on religious leaders over healthcare providers emerged as a potential means to promote adherence in some discussions. Conclusions Effective management of hypertensive patients for medication adherence will require several context-specific measures. These include policy measures addressing financial access, with medication subsidies for the poor and accessible distribution systems for medication refill; physician measures to improve health provider counseling for patient centric care; and patient-level strategies with reminders for medication adherence in low resource settings.

Su Vida, Su Salud/Your Life, Your Health is a community program to increase participation in breast and cervical cancer screening. This program illustrates the diffusion of an innovative outreach strategy from a research environment to two local health departments. The program uses A Su Salud, the communication model in which positive role models are featured in the media, and community volunteers who give positive social reinforcement. Local health departments in Galveston and Corpus Christi, TX, were selected to implement the model because of excessive mortality rates in the region and the departments' adequate level of resources and commitment. Over an 18-month period, 54 stories of role models appeared in the media in Corpus Christi, and 60 appeared in Galveston. There were 490 volunteers active in Corpus Christi and 279 in Galveston. Of 365 inquiries to the Corpus Christi program, 35 percent were from the Mexican American target group of women ages 40-70. Of 1,457 women who contacted the Galveston program, 9 percent were from the African American target group ages 40-70. Local health departments are well-suited to participation in cancer control research applications because they have direct access to high-risk populations and have the authority to institutionalize effective interventions. [ABSTRACT FROM AUTHOR]

Chronic histiocytic intervillositis (CHI) is a recurrent placental lesion where maternal macrophages infiltrate the intervillous space. Its cause is unknown, though due to similarities to rejected allografts one hypothesis is that CHI represents maternal-fetal rejection. Here, virtual crossmatching was applied to healthy pregnancies and those with a history of CHI. Anti-HLA antibodies, measured by Luminex, were present in slightly more controls than CHI (8/17 (47.1%) vs 5/14 (35.7%)), but there was no significant difference in levels of sensitisation or fetal specific antibodies. Quantification of immunohistochemical staining for HLA-Class II was increased in syncytiotrophoblast of placentas with CHI (Grade 0.44 [IQR 0.1-0.7]) compared to healthy controls (0.06 [IQR 0-0.2]) and subsequent pregnancies (0.13 [IQR 0-0.3]) (P = 0.0004). HLA-Class II expression was positively related both to the severity of CHI (r = 0.67) and C4d deposition (r = 0.48). There was no difference in overall C4d and HLA-Class I immunostaining. Though increased anti-HLA antibodies were not evident in CHI, increased expression of HLA-Class II at the maternal-fetal interface suggests that they may be relevant in its pathogenesis. Further investigation of antibodies immediately after diagnosis is warranted in a larger cohort of CHI cases to better understand the role of HLA in its pathophysiology., (© 2024. The Author(s).)

Context.—: Chronic histiocytic intervillositis (CHI) is a rare condition characterized by maternal immune cell infiltration into the human placenta. CHI is strongly associated with fetal growth restriction, miscarriage, and stillbirth, and knowledge of its etiology, and consequently effective treatment, is limited. Currently, diagnosis is largely subjective and varies between centers, making comparison between studies challenging., Objective.—: To objectively quantify and interrelate inflammatory cells and fibrin in placentas with CHI compared with controls and determine how pathology may be altered in subsequent pregnancies following diagnosis. Macrophage phenotype was also investigated in untreated cases of CHI., Design.—: Computerized analysis was applied to immunohistochemically stained untreated (index) cases of CHI, subsequent pregnancies, and controls. Index placentas were additionally stained by immunofluorescence for M1 (CD80 and CD86) and M2 macrophage markers (CD163 and CD206)., Results.—: Quantification revealed a median 32-fold increase in macrophage infiltration in index cases versus controls, with CHI recurring in 2 of 11 (18.2%) subsequent pregnancies. A total of 4 of 14 placentas (28.6%) with a diagnosis of CHI did not exhibit infiltration above controls. Macrophages in index pregnancies strongly expressed CD163. There was no significant difference in fibrin deposition between index cases and controls, although subsequent pregnancies displayed a 2-fold decrease compared with index pregnancies. CD3+ T cells were significantly elevated in index pregnancies; however, they returned to normal levels in subsequent pregnancies., Conclusions.—: In CHI, intervillous macrophages expressed CD163, possibly representing an attempt to resolve inflammation. Computerized analysis of inflammation in CHI may be useful in determining how treatment affects recurrence, and alongside pathologist expertise in grading lesion severity., (© 2024 College of American Pathologists.)

Objectives: Despite the use of post-mortem investigations, approximately 20% of stillbirths remain unexplained. Cardiac ion channelopathies have been identified as a cause of death in Sudden Infant Death Syndrome (SIDS) and could be associated with unexplained stillbirths. This study aimed to understand if the expression or localisation of cardiac ion channels associated with channelopathies were altered in cases of unexplained stillbirths., Methods: A case control study was conducted using formalin-fixed cardiac tissue from 20 cases of unexplained stillbirth and a control group of 20 cases of stillbirths from intrapartum hypoxia. 4 µm tissue sections were stained using haematoxylin and eosin, Masson's trichrome (MT) and Elastic van Gieson (EVG). Immunohistochemistry (IHC) was performed using antibodies against CACNA1G, KCNJ2, KCNQ1, KCNH2 and KCNE1. The cardiac conduction system in samples stained with MT and EVG could not be identified. Therefore, the levels of immunoperoxidase staining were quantified using QuPath software., Results: The nuclear-cytoplasmic ratio of sections stained with haematoxylin and eosin was higher for the hypoxia group (hypoxia median 0.13 vs. 0.04 unexplained, p < 0.001). CACNA1G (unexplained median 0.26 vs. hypoxia 0.30, p=0.009) and KCNJ2 (unexplained median 0.35 vs. hypoxia 0.41, p=0.001) had lower staining intensity in the unexplained stillbirth group. There were no statistically significant differences in the staining intensity of KCNQ1, KCNH2 and KCNE1., Conclusions: Two ion channels associated with channelopathies demonstrated lower levels of expression in cases of unexplained stillbirth. Further genetic studies using human tissue should be performed to understand the association between channelopathies and otherwise unexplained stillbirths., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Chronic histiocytic intervillositis (CHI) is a rare, but highly recurrent inflammatory placental lesion wherein maternal macrophages infiltrate the intervillous space. Pregnancies with CHI are at high risk of fetal growth restriction, miscarriage or stillbirth. Presently, the diagnosis can only be made after histopathological examination of the placenta. Given its proposed immunological etiology, current treatments include aspirin, heparin, and immunomodulatory agents. However, the rationale for these medications is largely based upon small case series and reports as there is a lack of larger studies investigating treatment efficacy. Therefore, this study sought to determine whether inclusion of immunomodulatory medications was effective at reducing the severity of lesions and improving pregnancy outcomes in subsequent pregnancies. Thirty-three women with a history of CHI in at least one pregnancy (index case) were identified retrospectively through medical records. Twenty-eight participants presented with a first subsequent pregnancy and a further 11 with a second subsequent pregnancy at a specialist clinic for pregnancy after loss. Data on maternal demographics, medical history, medication, pregnancy outcome, and placental pathology was collected and compared between pregnancies. Twenty-seven (69%) subsequent pregnancies were treated with at least one or both of prednisolone and hydroxychloroquine. Inclusion of at least one immunomodulatory agent in treatment regimen resulted in an almost 25% increase in overall livebirth rate (61.5 vs. 86.2%). In women treated with immunomodulatory medication a greater proportion of placentas had reduced severity of lesions compared to those treated without (86.7 vs. 33.3%, respectively). A reduction in CHI severity was associated with a 62.3% improvement in livebirth rate compared to those where severity remained unchanged in relation to the index case. These data provide preliminary evidence that the use of immunomodulatory medication in the management of CHI improves histopathological lesions and the chance of livebirth in subsequent pregnancies. Due to CHI's rarity and ethical and feasibility issues, randomized controlled trials in affected women are challenging to conduct. As a result, collaboration between centers is required in future to increase study sample sizes and elucidate the mechanisms of hydroxychloroquine and prednisolone in reducing pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Brady, Williams, Batra, Church, Tower, Crocker and Heazell.)

The metabolism of l-tryptophan to N -formyl-l-kynurenine by indoleamine-2,3-dioxygenase 1 (IDO1) is thought to play a critical role in tumour-mediated immune suppression. Whilst there has been significant progress in elucidating the overall enzymatic mechanism of IDO1 and related enzymes, key aspects of the catalytic cycle remain poorly understood. Here we report the design, synthesis and biological evaluation of a series of tryptophan analogues which have the potential to intercept putative intermediates in the metabolism of 1 by IDO1. Functionally-relevant binding to IDO1 was demonstrated through enzymatic inhibition, however no IDO1-mediated metabolism of these compounds was observed. Subsequent T m -shift analysis shows the most active compound, 17, exhibits a distinct profile from known competitive IDO1 inhibitors, with docking studies supporting the hypothesis that 17 may bind at the recently-discovered S i site. These findings provide a start-point for development of further mechanistic probes and more potent tryptophan-based IDO1 inhibitors., Competing Interests: NJC was a PhD student sponsored by Celentyx Ltd. TT, OQ, CAB are employees of Celentyx Ltd with share options in the company. JG and NMB are Directors of Celentyx Ltd with shareholdings in the company. There are no other conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Chronic histiocytic intervillositis (CHI) is a pregnancy disorder characterized by infiltration of maternal macrophages into the intervillous space of the human placenta, often with accompanying perivillous fibrin deposition. CHI is associated strongly with foetal growth restriction and increased risk of miscarriage and stillbirth. Although rare, affecting 6 in every 10 000 pregnancies beyond 12 weeks' gestation, the rate of recurrence is high at 25%-100%. To date, diagnosis of CHI can only be made post-delivery upon examination of the placenta due to a lack of diagnostic biomarkers, and criteria vary across publications. No treatment options have shown proven efficacy, and CHI remains a serious obstetric conundrum. Although its underlying aetiology is unclear, due to the presence of maternal macrophages and the reported increased incidence in women with autoimmune disease, CHI is hypothesized to be an inappropriate immune response to the semi-allogeneic foetus. Given this lack of understanding, treatment approaches remain experimental with limited rationale. However, there is recent evidence that immunosuppression and antithrombotic therapies may be effective in preventing recurrence of associated adverse pregnancy outcomes. With similarities noted between the pathological features of CHI and acute rejection of solid organ transplants, further investigation of this hypothesis may provide a basis for tackling CHI and other immune-related placental conditions. This review will explore parallels between CHI and allograft rejection and identify areas requiring further confirmation and exploitation of this comparison., (© 2020 The Authors. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, was first identified after a cluster of cases in Wuhan, China in December 2019. Whether vertical transmission or placental pathology might occur following maternal infection during pregnancy remains unknown. This review aimed to summarise all studies that examined the placenta or neonates following infection with SARS-CoV-2, or closely related highly pathogenic coronavirus (SARS-CoV-1, or the Middle East respiratory syndrome coronavirus (MERS-CoV)). Structured literature searches found 50 studies that met the inclusion criteria. Twenty studies reported placental histopathology findings in third trimester placentas following maternal SARS-CoV-2 infection. Using the Amsterdam Consensus criteria to categorise the histopathology results, evidence of both fetal vascular malperfusion (35.3% of cases; 95% Confidence Interval (CI) 27.7-43.0%) and maternal vascular malperfusion (46% of cases; 95% CI 38.0-54.0%) were reported, along with evidence of inflammation in the placentas (villitis 8.7% cases, intervillositis 5.3% of cases, chorioamnionitis 6% of cases). The placental pathologies observed in SARS-CoV-2 were consistent with findings following maternal SARS-CoV-1 infection. Of those tested, a minority of neonates (2%) and placental samples tested positive for SARS-CoV-2 infection (21%). Limited conclusions can be drawn about the effect of maternal SARS-CoV-2 infection on placental pathology as most lack control groups and the majority of reports followed third trimester infection. Collaboration to maximise the number of samples examined will increase the reliability and generalisability of findings. A better understanding of the association between maternal SARS-CoV-2 infection and placental pathology will inform maternity care during the coronavirus pandemic., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The 5-hydroxytryptamine (5-HT) (serotonin) 5-HT 3 receptor represents a clinical target for antagonists to deliver symptomatic relief to patients with diarrhea-predominant irritable bowel syndrome (IBS-d) or carcinoid syndrome. Unfortunately, this pharmacological strategy can present side effects (e.g., severe constipation). The present study investigates the potential of a novel 5-HT 3 receptor partial agonist, CSTI-300, to treat patients with IBS-d and other conditions associated with discomfort from colonic distension, with a predicted reduced side-effect profile. The in vitro and in vivo preclinical pharmacology of the drug CSTI-300 was investigated to explore the potential to treat patients with IBS-d. CSTI-300 displayed selective high affinity for the human and rat 5-HT 3 receptor (K i approximately 2.0 nM) and acted as a partial agonist (approximately 30%-50% intrinsic efficacy) in vitro. In an in vivo model of IBS-d, the rat colon distension model, CSTI-300 displayed dose-dependent efficacy. In addition, oral administration of CSTI-300 to dogs that achieved plasma levels of the drug exceeding the K i value for the 5-HT 3 receptor failed to either evoke emesis or alter the state of feces. Pharmacokinetics for CSTI-300 in rat and dog identified high levels of oral availability with t 1/2 range of 1.6-4.4 hours. The preclinical pharmacology of the lead candidate drug, CSTI-300, supports the potential of this novel drug to offer symptomatic relief to patients with irritable bowel syndrome and carcinoid syndrome with a rationale for a reduced "on-target" side-effect profile relative to 5-HT 3 receptor antagonists, such as alosetron. SIGNIFICANCE STATEMENT: There is a lack of effective current treatment for diarrhea-predominant irritable bowel syndrome and carcinoid syndrome, and in both conditions, overactivity of the 5-hydroxytryptamine (5-HT) 5-HT 3 receptor is thought to be implicated in the pathophysiology. Because 5-HT 3 receptor blockade with antagonists results in significant side effects, we present evidence that treatment with a suitable 5-HT 3 receptor partial agonist will alleviate some symptoms associated with these conditions yet, without fully inhibiting the receptor, predict a less pronounced side-effect profile associated with this therapeutic strategy., Competing Interests: Conflicts of interest. D.X. and J.H. are executives of Chengdu SciMount Pharmatech Co. Ltd. S.L. is an executive of ConSynance Therapeutics, Inc. P.R.G. was an employee of Albany Molecular Research, Inc. and an executive of ConSynance Therapeutics, Inc. and is currently an advisor to Chengdu SciMount Pharmatech Co. Ltd. D.D.M., N.A.M., and B.J.S. are current/former employees of Albany Molecular Research, Inc. N.M.B. was a former consultant of Albany Molecular Research, Inc and an advisor to ConSynance Therapeutics, Inc. and Chengdu SciMount Pharmatech Co. Ltd., (Copyright © 2020 The Author(s).)

Inosine Acedoben Dimepranol (IAD), licensed for the treatment of cell-mediated immune deficiencies associated with viral infections, has been reported to impact a variety of immune parameters both in vitro and in vivo. Here we report the results from a clinical trial where multiple lymphocyte subsets - CD19+ B cells, CD3+ T cells, CD4+ T-helper cells, FoxP3 hi /CD25 hi /CD127 lo regulatory T cells (Tregs), CD3-/CD56+ NK cells, and CD3+/CD56+ NKT cells - were, together with serum immunoglobulins and IgG subclasses, followed during 14days of IAD administration to ten healthy volunteers; these selected from 27 individuals pre-screened in vitro for their capacity to respond to IAD as gauged by increases in the percentage of Treg and/or NKT cells arising in PHA-stimulated cultures. While a transient spike and dip in Treg and T-helper fractions, respectively, was noted, the outstanding consequence of IAD administration (1g po, qds) was an early and durable rise in NK cells. For half the cohort, NK cells increased as a percentage of total peripheral blood lymphocytes within 1.5h of receiving drug. By Day 5, all but one of the volunteers displayed higher NK cell percentages, such elevation - effectively a doubling or greater - being maintained at termination of study. The IAD-induced populations were as replete in Granzyme A and Perforin as basal NK cells. The novel finding of IAD boosting phenotypically competent NK numbers in healthy individuals supports the drug's indicated benefit in conditions associated with viral infection and reinforces the potential for uplift where immune performance may be compromised., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The p53 tumor suppressor is a stress sensor, driving cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19(Arf) tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19(Arf)-independent. Accordingly, p19(Arf)-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19(Arf) is required for replicative senescence, a condition associated with an activated DNA damage response, as p19(Arf)-/- MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19(Arf) is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19(Arf) has a specific role. We therefore examined the role of p19(Arf) in cellular responses to chronic, low-dose DNA-damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 G y γ-irradiation daily or 150 μM hydroxyurea, a replication stress inducer. In contrast to their response to acute DNA damage, p19(Arf)-/- MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19(Arf) in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19(Arf)-/- MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19(Arf) in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA-damaging agent treatment is insufficient to promote senescence in p19(Arf)-/- MEFs, suggesting that the role of p19(Arf) in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19(Arf) for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19(Arf) serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19(Arf) and DNA damage-signaling pathways in tumor suppression.

Phenotypic small molecule screens in zebrafish have gained popularity as an unbiased approach to probe biological processes. In this chapter we outline basic methods for performing chemical screens with larval zebrafish including breeding large numbers of embryos, plating larval fish into multi-well dishes, and adding small molecules to these wells. We also highlight important considerations when designing and interpreting the results of a phenotypic screen and possible follow-up approaches, including popular methods used to identify the mechanism of action of a chemical compound.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage., (© 2014. Published by The Company of Biologists Ltd.)

Serotonin type 3 (5-HT3) receptor partial agonists have been targeted as potential new drugs for the symptomatic relief of irritable bowel syndrome (IBS). Multiple diazepinone-based compounds have been discovered, which exhibit nanomolar binding affinity for the h5-HT3A receptor and display a range of intrinsic activities (IA=7-87% of 5-HT Emax) in HEK cells heterologously expressing the h5-HT3A receptor. Favorable physicochemical properties and in vitro ADME profile coupled with oral activity in the murine von Bezold-Jarisch reflex model demonstrates the series has promise for producing low to moderate IA partial agonists suitable for an IBS indication., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background and Purpose: The 5-HT₃ receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT₃ receptor., Experimental Approach: 5-HT₃ receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT₃A receptor and also the mouse native 5-HT₃ receptor that increases neuronal contraction of bladder smooth muscle., Key Results: Cl-indole (1-100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT₃A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT₃ receptor. Radioligand-binding studies identified that Cl-indole induced a small (≈ twofold) increase in the apparent affinity of 5-HT for the h5-HT₃A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT₃ receptors. In contrast to its effect on the 5-HT₃ receptor, Cl-indole did not alter human nicotinic α7 receptor responses., Conclusions and Implications: The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT₃ receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT₃ receptor and may assist the discovery of novel therapeutic drugs targeting this receptor., (© 2013 The British Pharmacological Society.)

The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology, we used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53-bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53-bound genes is transcriptionally regulated, yielding a list of 432 p53-bound and regulated genes. Interestingly, we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53, the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53-induced autophagy does not affect cell cycle arrest in response to DNA damage, it is important for both robust p53-dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together, our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53-dependent apoptosis and cancer suppression.

Over half of all human cancers, of a wide variety of types, sustain mutations in the p53 tumor suppressor gene. Although p53 limits tumorigenesis through the induction of apoptosis or cell cycle arrest, its molecular mechanism of action in tumor suppression has been elusive. The best-characterized p53 activity in vitro is as a transcriptional activator, but the identification of numerous additional p53 biochemical activities in vitro has made it unclear which mechanism accounts for tumor suppression. Here, we assess the importance of transcriptional activation for p53 tumor suppression function in vivo in several tissues, using a knock-in mouse strain expressing a p53 mutant compromised for transcriptional activation, p53(25,26). p53(25,26) is severely impaired for the transactivation of numerous classical p53 target genes, including p21, Noxa, and Puma, but it retains the ability to activate a small subset of p53 target genes, including Bax. Surprisingly, p53(25,26) can nonetheless suppress tumor growth in cancers derived from the epithelial, mesenchymal, central nervous system, and lymphoid lineages. Therefore, full transactivation of most p53 target genes is dispensable for p53 tumor suppressor function in a range of tissue types. In contrast, a transcriptional activation mutant that is completely defective for transactivation, p53(25,26,53,54), fails to suppress tumor development. These findings demonstrate that transcriptional activation is indeed broadly critical for p53 tumor suppressor function, although this requirement reflects the limited transcriptional activity observed with p53(25,26) rather than robust transactivation of a full complement of p53 target genes.

The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p53(25,26), is severely compromised for transactivation of most p53 target genes, and, moreover, p53(25,26) cannot induce G(1)-arrest or apoptosis in response to acute DNA damage. Surprisingly, p53(25,26) retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p53(25,26,53,54) mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression-a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Serotonin type 3 (5-HT(3)) receptor partial agonists are being targeted as potential new drugs for the treatment of irritable bowel syndrome (IBS). Two new chemical series bearing indazole and indole cores have exhibited nanomolar binding affinity for the h5-HT(3)A receptor. A range of partial agonist activities in HEK cells heterologously expressing the h5-HT(3)A receptor were measured for the indazole series. Excellent 5-HT(3) receptor selectivity, favorable in vitro metabolic stability and CYP inhibition properties, and good oral in vivo potency in the murine von Bezold-Jarisch reflex model is exemplified thereby indicating the series to have potential utility as improved IBS agents., (Copyright © 2010. Published by Elsevier Ltd.)

The pentameric 5-HT(3) receptor complex is a ligand-gated ion channel that mediates fast synaptic transmission in the brain. Expression of two subunits (5-HT(3A) and 5-HT(3B) subunits) gives rise to at least two receptor isoforms (homomeric 5-HT(3A) and heteromeric 5-HT(3A/3B) receptors), which differ in their biophysical characteristics, although expression of these proteins has not been investigated in human brain. The expression of h5-HT(3A) and 5-HT(3B) subunits in the human hippocampus was investigated using selective polyclonal antibodies (SDS-PAGE/Western blotting, immunohistochemistry), with expression of each subunit verified by PCR detection of subunit transcripts. 5-HT(3A) and 5-HT(3B) subunit immunoreactivity was identified within the human hippocampus. The cellular pattern of expression for each subunit was similar, with predominant immunoreactivity associated with pyramidal neurones in CA fields 2 and 3, and also the relatively large neurones within the hilus (CA4 field). Transcripts for each subunit were also identified in human hippocampal tissue. These findings indicate that human hippocampal neurones are capable of forming at least two, functionally different, isoforms of the 5-HT(3) receptor. Furthermore the expression pattern of 5-HT(3A) and 5-HT(3B) subunits in human hippocampus appears to differ with the rodent counterpart, which may underlie the differences in some of the behavioural effects of 5-HT(3) receptor antagonists between these species.

Background & Aims: Serotonin (5-hydroxytryptamine [5-HT]) is a key signaling molecule in the gut. Recently, the neural 5-HT3 receptor received a lot of attention as a possible target in functional bowel diseases. Yet, the 5-HT3 receptor-mediated changes in properties of human enteric neurons is unknown., Methods: We used a fast imaging technique in combination with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine to monitor directly the membrane potential changes in neurons of human submucous plexus from surgical specimens of 21 patients. An Ussing chamber technique was used to study 5-HT3 receptor involvement in chloride secretion., Results: Local microejection of 5-HT directly onto ganglion cells resulted in a transient excitation of enteric neurons characterized by increased spike discharge. This response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, and blocked by the 5-HT3 receptor antagonist, tropisetron. The proportions of 5-HT-responsive nerve cells per ganglion ranged from 25.5% +/- 18.4% in the duodenum to 54.2% +/- 46.9% in the colon. Interestingly, 2-methyl-5-HT did not evoke chloride secretion in the human intestine but it did in the guinea-pig intestine. Specific 5-HT3A and 5-HT3B receptor subunit immunoreactivity as well as 5-HT3A and 5-HT3B receptor-specific messenger RNA were detected in the tissue samples. Based on co-labeling with the pan-neuronal marker HuC/D we conclude that submucous nerve cells potentially express heteromeric 5-HT3A/B receptors., Conclusions: We show that 5-HT excited human enteric neurons via 5-HT3 receptors, which may comprise both 5-HT3A and 5-HT3B receptor subunits.

This case report describes a cat that was presented with infected oral tissues and signs of systemic inflammatory response. Incomplete dental extractions had been performed 10-days earlier During a second dental procedure, 22 tooth root remnants were removed. Diabetic ketoacidosis and possible thromboembolism complicated the cat's recovery period After 13-days of hospitalization, the patient was stable enough to be sent home. Postoperative examinations at 1, 3, 6, 12, 16, 24, and 52-weeks indicated continued improvement with moderate glycemic control and chronic but stable renal failure. The patient died from further complications of diabetic ketoacidosis 20-months following root remnant extractions.

Polycyclic aromatic hydrocarbons (PAHs) have long been recognised as potential carcinogens in animals in which biotransformation into reactive metabolites can lead to DNA damage. In animals PAHs metabolism mainly occurs in hepatic microsomes and is associated with the cytochrome p-450 mediated mixed functional oxidase (MFO) system. PAH metabolism in plants has been shown to occur via a similar enzyme system, but has received relatively little attention. This study is looking at how the plant species Plantago lanceolata metabolizes benzo(a)pyrene (B(a)P), which is one of the PAHs whose metabolism has been studied extensively in animals. The aim of the work is to establish firstly that the B(a)P is taken up and secondly that it is biotransformed by the plant to products possibly similar to those found in animals. This work is achieved by using C-14-B(a)P along with whole body autoradiography, scintillation analysis and chromatography techniques to locate the B(a)P and its metabolites.

Objective: To identify risk factors associated with development of pyothorax in cats, assess survival rates for cats that are treated, determine prognostic indicators, and determine recurrence rates., Design: Retrospective study., Animals: 80 cats with pyothorax and 212 control cats., Procedure: History; month of evaluation; physical examination findings; results of hematologic, serum biochemical, and retrovirus testing; radiographic findings; outcome; recurrence rate; and necropsy findings were recorded. For control cats, age, sex, breed, indoor versus outdoor status, vaccination history, and single- versus multi-cat household status were recorded., Results: Cats from multi-cat households were 3.8 times as likely (95% confidence interval, 1.9 to 8.2) to develop pyothorax, compared with cats from single-cat households. Indoor or outdoor status was not a risk factor. Cats with pyothorax were significantly younger (mean, 3.83 +/- 3.43 years) than controls (mean, 5.62 +/- 5.27 years). Nonsurvivors had significantly lower heart rates than survivors. Hypersalivation was significantly more common in nonsurvivors (11/39; 26.8%) than survivors (1/39; 3%). Overall, 48.8% (39/80) of cats survived. When cats that were euthanatized without treatment were excluded from analyses, the survival rate was 66.1% (39/59). Pyothorax recurred in 1 of 17 cats for which follow-up information was obtained., Conclusions and Clinical Relevance: Cats with pyothorax that received treatment had a fair to good prognosis, with low recurrence rates in survivors. Hypersalivation and low heart rate were associated with worse clinical outcome. Cats with pyothorax were likely to come from multi-cat households.

The present study generated a polyclonal antibody (AP86/3) that recognises a peptide sequence of the h5-HT(3B) receptor subunit. Western blot analysis of homogenates prepared from cell lines expressing either homomeric (h5-HT(3A)) or heteromeric (h5-HT(3A/3B)) receptors, as well as immunocytochemical studies with the same cell lines, indicated that AP86/3 recognised, selectively, the 5-HT(3B) subunit. Immunohistochemical labelling was also apparent in cells in the rat hippocampus that displayed the distribution and morphology of interneurones.

Companion animals with sepsis and multiple organ dysfunction can be the most challenging of all patients to treat. Current research in humans and laboratory models offers some exciting insights into the pathophysiology behind some of our most frustrating clinical challenges. This article applies several current concepts to a clinical case of pancreatitis and secondary sepsis to illustrate some of the cardiovascular, immune, and coagulation abnormalities commonly seen.

The present study determined the detailed pharmacological profile of heterologously expressed human (h) homomeric 5-HT3A receptors in direct comparison to heteromeric h5-HT3A/3B receptors. The very minor differences in their respective pharmacological profiles indicates that the 5-HT3B receptor subunit alters, predominantly, the biophysical rather than the pharmacological properties of the 5-HT3 receptor.

Objective: To document the clinical, clinicopathologic, and pathologic findings in cats with severe sepsis, identify abnormalities unique to this species, and identify criteria that could be used antemortem to diagnose the systemic inflammatory response syndrome in cats., Design: Retrospective study., Animals: 29 cats confirmed to have severe sepsis at necropsy., Procedure: Pertinent history, physical examination findings, and results of hematologic and biochemical testing were extracted from medical records., Results: Clinical diagnoses included pyothorax, septic peritonitis, bacteremia secondary to gastrointestinal tract disease, pneumonia, endocarditis, pyelonephritis, osteomyelitis, pyometra, and bite wounds. Physical examination findings included lethargy, pale mucous membranes, poor pulse quality, tachypnea, hypo- or hyperthermia, signs of diffuse pain on abdominal palpation, bradycardia, and icterus. Clinicopathologic abnormalities included anemia, thrombocytopenia, band neutrophilia, hypoalbuminemia, low serum alkaline phosphatase activity, and hyperbilirubinemia. Necropsy findings included multi-organ necrosis or inflammation with intralesional bacteria., Conclusions and Clinical Relevance: Results suggest that severe sepsis in cats is characterized by lethargy, pale mucous membranes, signs of diffuse abdominal pain, tachypnea, bradycardia, weak pulses, anemia, hypoalbuminemia, hypothermia, and icterus. Recognition of this combination of clinical findings should facilitate the diagnosis of severe sepsis in cats.

Hypovolemia, hypothermia, and hypotension are common postoperative findings that predispose the critically ill patient to secondary complications. This patient population is especially vulnerable to sepsis, hypoxia, and immune dysfunction. Careful monitoring is essential for early recognition of potentially life-threatening physiologic derangements. Early and aggressive intervention may help minimize systemic insult before it progresses to acute respiratory distress syndrome, acute renal failure, disseminated intravascular coagulation, or multiple organ failure.

A 3-year-old mixed-breed dog was evaluated for lethargy, weakness, anorexia, and vomiting. The dog was dehydrated, hyponatremic, hypochloremic, and hypoglycemic. Results of an ACTH stimulation test indicated hypoadrenocorticism. Treatment to restore cardiovascular stability and serum electrolyte balance caused serum sodium concentration to increase by 32 mEq/L within 48 hours, and the dog developed severe neurologic signs that persisted for approximately 3 weeks. Magnetic resonance imaging revealed cerebrocortical lesions on day 6 and more severe lesions, including diffuse atrophy of the cerebral hemispheres, at 23 weeks after initial evaluation; however, the dog recovered complete neurologic function. Serum sodium concentration should be monitored during treatment for hypoadrenal crisis to avoid rapid increases that can cause CNS damage.

We evaluated the use of a community-based intervention model to increase Pap smear and mammogram screening in minority women. The community programs were based on the A Su Salud (To Your Health) model, which includes the presentation of positive role models in the media and positive social reinforcement by community volunteers. Mexican-American women 40-70 years of age in Corpus Christi, Texas, and black women 40-70 years of age in Galveston, Texas, were targeted. Role models from the target community told their personal stories about cancer screening through local media (television, radio, and newspapers). Volunteers reinforced media messages among the target audience by distributing newsletters. Preintervention and postintervention cross-sectional surveys among the target group measured relative increases in knowledge, attitude, and practice. Pap smear use (past two years) was not substantially improved. Mammogram use (past two years) increased 57% among Mexican-American women (prevalence rate ratio [PRR] = 1.57; 90% confidence interval [CI] = 1.19, 2.08) and 30% among black women (PRR = 1.30; 90% CI = 1.04, 1.63). We demonstrated the feasibility of implementing a peer role model intervention at the local health department level. The improvement in mammogram use among high-risk women is encouraging, but further controlled research is needed to test the effectiveness and cost-efficiency of this approach.

Lymphocytotoxic (LCT) and platelet reactive (PR) antibody (Ab) responses were serially determined in 49 patients with acute leukaemia. LCTAb were found in 20 patients and occurred in 13 patients with acute myeloid leukaemia and 7 patients with acute lymphoblastic leukaemia. Four differing patterns of LCTAb responses could be defined. Thirteen of 22 subjects showed marked reduction or loss of LCTAb. Indirect platelet immunofluorescence, measured by flow cytometry, provided the most convenient means of detecting PRAb which were found in 11 subjects and generally showed moderate or weak reactivity.

To assess the protective efficacy of the Haemophilus b polysaccharide vaccine, a case-control study was conducted in Connecticut, Dallas County, and greater Pittsburgh. Seventy-six children 24 to 72 months of age who had H influenzae type b isolated from normally sterile sites from August 1985 through July 1987 were enrolled. For each case two controls, matched by age and place of residence, were randomly selected from the records of all live births in the area. Antecedent receipt of the Haemophilus b polysaccharide vaccine was ascertained from the records of all physicians and clinics at which the subjects received medical care. Overall, 12% of the cases and 39% of the controls had received the vaccine. The estimate of the protective efficacy of the vaccine was 88% overall (95% confidence interval, 74% to 96%) and 91% (95% confidence interval, 71% to 99%), 92% (95% confidence interval, 76% to 99%), and 81% (95% confidence interval, 45% to 96%) in Connecticut, Dallas County, and greater Pittsburgh, respectively. The estimates were not substantially affected by adjusting with logistic regression for differences between the cases and controls in race and the attendance of group day care. We conclude that the Haemophilus b polysaccharide vaccine is highly effective in these areas among children who receive the vaccine when they are 24 months of age or older.

To determine whether clinicians correctly identify newborn infants who are at high risk of child maltreatment, we examined the outcomes of high-risk and non-high-risk children. Infants who were born at Yale-New Haven Hospital from 1979 to 1981 and who were referred by clinicians during the postpartum period to the hospital's child abuse registry because they were considered at high risk of child abuse or neglect became the high-risk group. For each high-risk infant, a comparison infant was selected and matched according to date of birth, gender, race, and method of payment for the hospitalization. For both groups, the occurrence of maltreatment was ascertained by reviewing the medical records until the child's fourth birthday. Each injury for which medical care was sought was classified into one of seven categories (from definite child abuse to accident) by a pediatrician who was unaware of the child's risk status. Information also was recorded about nonorganic failure to thrive and changes in the child's caretaker. Maltreatment (defined as abuse or neglect) occurred more frequently in the high-risk group than the comparison group (adjusted matched odds ratio = 4.3; 95% confidence interval = 1.41, 6.93; p less than 0.001), as did poor weight gain from a nonorganic cause (matched odds ratio = 7.0; 95% confidence interval = 1.59, 30.79; p less than 0.01) and changes in the child's caretaker (matched odds ratio = 9.0; 95% confidence interval = 3.80; 20.55; p less than 0.001). We conclude that as early as the postpartum period, clinicians can identify some families who are at high risk of maltreatment and other major adverse outcomes resulting from poor parenting.

Thirty-eight of 98 strains of Streptococcus uberis were shown to be carrying lysogenic phage. Although propagating strains were rare, host modification by field strains sensitive to phage was used to increase the lytic spectra. When 120 nationally-collected strains were challenged with 25 phages, selected on the basis of differing lytic spectra and propagating strains, 30% were susceptible to at least one phage, increasing to 42% when 480 strains from a single farm were considered. A typing system based on susceptibility to lytic phage was considered feasible.

21 members of a staff process group at a mental health treatment and research center were administered the Job Descriptive Index and the Work Environmental Scale once a month for a 3-mo. period. A two-day analysis of variance (job satisfaction x time) was computed for each of the 10 subscales. The three administrations did not significantly vary on any of the dimensions, nor were significant interaction effects found. Subjects high in satisfaction significantly differed from those who scored low on the dimensions of involvement and peer cohesion, staff support, autonomy, and innovation. However, no significant differences were found on the dimensions of task orientation, work pressure, clarity, control, or physical comfort. Results were discussed in terms of the importance of the variables, relationship and responsibility, to job satisfaction in a mental health setting.