Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Regulation of invariant NKT cell development and function by a 0.14 Mbp locus on chromosome 1: a possible role for Fcgr3.

Invariant NKT (iNKT) cells are tissue-resident innate-like T cells critical to the host immune response. We previously identified a 6.6 Mbp region on chromosome 1 as a major regulator of iNKT cell number and function in C57BL/6 and 129X1/SvJ mice. Here, we fine-mapped this locus by assessing the iNKT cell response to alpha-galactosylceramide (αGalCer) in a series of B6.129 congenic lines. This analysis revealed the presence of at least two genetic elements that regulate iNKT cell cytokine production in response to αGalCer. While one of these genetic elements mapped to the B6.129c6 interval containing Slam genes, the dominant regulator in this region mapped to the 0.14 Mbp B6.129c3 interval. In addition, we found that numbers of thymic iNKT cells and DP thymocytes were significantly lower in B6.129c3 mice, indicating that this interval also regulates iNKT cell development. Candidate gene analysis revealed a fivefold increase in Fcgr3 expression in B6.129c3 iNKT cells, and we observed increased expression of FcγR3 protein on B6.129c3 iNKT cells, NK cells, and neutrophils. These data identify the B6.129c3 interval as a novel locus regulating the response of iNKT cells to glycosphingolipid, revealing a link between this phenotype and a polymorphism that regulates Fcgr3 expression.

Evaluation of medetomidine-ketamine and medetomidine-ketamine-butorphanol for the field anesthesia of free-ranging dromedary camels (Camelus dromedarius) in Australia.

Abstract We report the clinical course and physiologic and anesthetic data for a case series of 76 free-ranging dromedary camels (Camelus dromedarius) chemically restrained, by remote injection from a helicopter, in the rangelands of Western Australia and South Australia, 2008-11, to attach satellite-tracking collars. Fifty-five camels were successfully anesthetized using medetomidine-ketamine (MK, n=27) and medetomidine-ketamine-butorphanol (MKB, n=28); the induction of anesthesia in 21 animals was considered unsuccessful. To produce reliable anesthesia for MK, medetomidine was administered at 0.22 mg/kg (± SD=0.05) and ketamine at 2.54 mg/kg (± 0.56), and for MKB, medetomidine was administered at 0.12 mg/kg (± 0.05), ketamine at 2.3 mg/kg (± 0.39), and butorphanol at 0.05 mg/kg (± 0.02). Median time-to-recumbency for MKB (8.5 min) was 2.5 min shorter than for MK (11 min) (P=0.13). For MK, the reversal atipamezole was administered at 0.24 mg/kg (± 0.10), and for MKB, atipamezole was administered at 0.23 mg/kg (± 0.13) and naltrexone at 0.17 mg/kg (± 0.16). Median time-to-recovery was 1 min shorter for MK (5 min) than MKB (6 min; P=0.02). Physiologic parameters during recumbency were not clinically different between the two regimes. Both regimes were suitable to safely anesthetize free-ranging camels; however, further investigation is required to find the safest, most consistent, and logistically practical combination.

Optically transparent piezoelectric transducer for ultrasonic particle manipulation.

We report an optically transparent ultrasonic device, consisting of indium-tin-oxide-coated lithium niobate (LNO), for use in particle manipulation. This device shows good transparency in the visible and near-infrared wavelengths and, acoustically, compares favorably with conventional prototype devices with silver electrodes.

Polymorphisms in the CD1d promoter that regulate CD1d gene expression are associated with impaired NKT cell development.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow-derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions -331 and -164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

Should oral misoprostol be used to prevent postpartum haemorrhage in home-birth settings in low-resource countries? A systematic review of the evidence.

BACKGROUND: Using misoprostol to prevent postpartum haemorrhage (PPH) in home-birth settings remains controversial. OBJECTIVES: To review the safety and effectiveness of oral misoprostol in preventing PPH in home-birth settings. SEARCH STRATEGY: The Cochrane Library, PubMed, and POPLINE were searched for articles published until 31 March 2012. SELECTION CRITERIA: Studies, conducted in low-resource countries, comparing oral misoprostol with a placebo or no treatment in a home-birth setting. Studies of misoprostol administered by other routes were excluded. DATA COLLECTION AND ANALYSIS: Data were extracted by two reviewers and independently checked for accuracy by a third. The quality of evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria. Data were sythesised and meta-analysis was performed where appropriate. MAIN RESULTS: Ten papers describing two randomised and four non randomised trials. Administration of misoprostol was associated with a significant reduction in the incidence of PPH (RR 0.58, 95% CI 0.38-0.87), additional uterotonics (RR 0.34, 95% CI 0.16-0.73), and referral for PPH (RR 0.49, 95% CI 0.37-0.66). None of the studies was large enough to detect a difference in maternal mortality, and none reported neonatal mortality. Shivering and pyrexia were the most common side effects. AUTHOR'S CONCLUSIONS: The finding that the distribution of oral misoprostol through frontline health workers is effective in reducing the incidence of PPH could be a significant step forwards in reducing maternal deaths in low-resource countries. However, given the limited number of high-quality studies in this review, further randomised controlled trials are required to confirm the association, particularly in different implementation settings. Adverse effects have not been systematically captured, and there has been limited consideration of the potential for inappropriate or inadvertent use of misoprostol. Further evidence is needed to inform the development of implementation and safety guidelines on the routine availability of misoprostol.

Slam haplotypes modulate the response to lipopolysaccharide in vivo through control of NKT cell number and function.

CD1d-restricted NKT cells make up an innate-like T cell subset that plays a role in amplifying the response of innate immune leukocytes to TLR ligands. The Slam locus contains genes that have been implicated in innate and adaptive immune responses. In this study, we demonstrate that divergent Slam locus haplotypes modulate the response of macrophages to the TLR4 ligand LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2(+) 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison with macrophage TNF production in Slam haplotype-1(+) C57BL/6J mice. Although no cell-intrinsic differences in macrophage responses to LPS were observed between strains, 129 mice were found to be deficient in liver NKT cell number, in NKT cell cytokine production in response to the CD1d ligand alpha-galactosylceramide, and in NKT cell IFN-gamma production after LPS challenge in vivo. Using B6.129c1 congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled the response to LPS in vivo, as well as the diminished NKT cell number and function, and that these phenotypes were associated with differential expression of signaling lymphocytic activation molecule family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivo through their effect on NKT cells.

Quality of care for maternal and newborn health: the neglected agenda.

The quality of care received by mothers and babies in developing countries is often reported as poor. Yet efforts to address this contributory factor to maternal and newborn mortality have received less attention compared with barriers of access to care. The current heightened concern to achieve Millennium Development Goals 4 & 5 has illuminated the neglected quality agenda. Whilst there is no universally-accepted definition of "quality care", it is widely acknowledged to embrace multiple levels--from patient to health system, and multiple dimensions, including safety as well as efficiency. Quality care should thus lie at the core of all strategies for accelerating progress towards MDG4 &5. Interventions to measure and improve quality need themselves to be evidence-based. Two promising approaches are maternal and perinatal death reviews and criterion-based audit. These and other quality improvement tools have a crucial role to play in the implementation of effective maternal and newborn care.

Professional assistance during birth and maternal mortality in two Indonesian districts.

OBJECTIVE: To examine determinants of maternal mortality and assess the effect of programmes aimed at increasing the number of births attended by health professionals in two districts in West Java, Indonesia. METHODS: We used informant networks to characterize all maternal deaths, and a capture-recapture method to estimate the total number of maternal deaths. Through a survey of recent births we counted all midwives practising in the two study districts. We used case-control analysis to examine determinants of maternal mortality, and cohort analysis to estimate overall maternal mortality ratios. FINDINGS: The overall maternal mortality ratio was 435 per 100,000 live births (95% confidence interval, CI: 376-498). Only 33% of women gave birth with assistance from a health professional, and among them, mortality was extremely high for those in the lowest wealth quartile range (2303 per 100,000) and remained very high for those in the lower middle and upper middle quartile ranges (1218 and 778 per 100,000, respectively). This is perhaps because the women, especially poor ones, may have sought help only once a serious complication had arisen. CONCLUSION: Achieving equitable coverage of all births by health professionals is still a distant goal in Indonesia, but even among women who receive professional care, maternal mortality ratios remain surprisingly high. This may reflect the limitations of home-based care. Phased introduction of fee exemption and transport incentives to enable all women to access skilled delivery care in health centres and emergency care in hospitals may be a feasible, sustainable way to reduce Indonesia's maternal mortality ratio.

Criterion-based clinical audit in obstetrics: bridging the quality gap?

The Millennium Development Goal 5 - reducing maternal mortality by 75% - is unlikely to be met globally and for the majority of low-income countries. At this time of heightened concern to scale-up services for mothers and babies, it is crucial that not only shortfalls in the quantity of care - in terms of location and financial access - are addressed, but also the quality. Reductions in maternal and perinatal mortality in the immediate term depend in large part on the timely delivery of effective practices in the management of life-threatening complications. Such practices require a functioning health system - including skilled and motivated providers engaged with the women and communities whom they serve. Assuring the quality of this system, the services and the care that women receive requires many inputs, including effective and efficient monitoring mechanisms. The purpose of this article is to summarise the practical steps involved in applying one such mechanism, criterion-based clinical audit (CBCA), and to highlight recent lessons from its application in developing countries. Like all audit tools, the ultimate worth of CBCA relates to the action it stimulates in the health system and among providers.

Measuring maternal mortality: an overview of opportunities and options for developing countries.

BACKGROUND: There is currently an unprecedented expressed need and demand for estimates of maternal mortality in developing countries. This has been stimulated in part by the creation of a Millennium Development Goal that will be judged partly on the basis of reductions in maternal mortality by 2015. METHODS: Since the launch of the Safe Motherhood Initiative in 1987, new opportunities for data capture have arisen and new methods have been developed, tested and used. This paper provides a pragmatic overview of these methods and the optimal measurement strategies for different developing country contexts. RESULTS: There are significant recent advances in the measurement of maternal mortality, yet also room for further improvement, particularly in assessing the magnitude and direction of biases and their implications for different data uses. Some of the innovations in measurement provide efficient mechanisms for gathering the requisite primary data at a reasonably low cost. No method, however, has zero costs. Investment is needed in measurement strategies for maternal mortality suited to the needs and resources of a country, and which also strengthen the technical capacity to generate and use credible estimates. CONCLUSION: Ownership of information is necessary for it to be acted upon: what you count is what you do. Difficulties with measurement must not be allowed to discourage efforts to reduce maternal mortality. Countries must be encouraged and enabled to count maternal deaths and act.

Universal reporting of maternal mortality: an achievable goal?

This paper aims to highlight the importance of aspiring to achieve universal reporting of maternal deaths as a part of taking responsibility for these avoidable tragedies. The paper first discusses the reasons for reporting maternal deaths, distinguishing between individual case notification and aggregate statistics. This is followed by a summary of the status of reporting at national and international levels, as well as major barriers and facilitators to this process. A new framework is then proposed - the REPORT framework, designed to highlight six factors essential to universal reporting. Malaysia is used to illustrate the relevance of these factors. Finally, the paper makes a Call to Action by FIGO to promote REPORT and to encourage health professionals to play their part in improving the quality of reporting on all maternal deaths - not just those directly in their care.

Describing safe motherhood programs for priority setting: the case of Burkina Faso.

OBJECTIVE: This study was implemented to describe safe motherhood programs in Burkina Faso for planning and programming purposes. METHODS: Twenty safe motherhood programs were described from November 2003 through May 2004 using a structured questionnaire, interviews with safe motherhood program managers and document reviews. RESULTS: Only 2 of the 20 programs were designed to improve the availability of comprehensive emergency obstetric care, and only 2 comprehensively addressed all components of skilled attendance at delivery. Other gaps identified included poor availability of baseline data, few monitoring measures, and lack of planning for evaluation needs. National geographical coverage was also uneven. CONCLUSION: A systematic overview of safe motherhood programs in a country can help to set priorities and aid in decision making for the allocation of resources towards contextually relevant strategies to curtail maternal mortality and severe morbidity. Planning for program design and evaluation may also be aided by such a process.

A randomised trial comparing local versus general anaesthesia for microwave endometrial ablation.

OBJECTIVE: To compare the acceptability of microwave endometrial ablation using a local anaesthesia/sedation regime or general anaesthesia. To compare recovery following treatment with each type of anaesthetic. DESIGN: Prospective randomised controlled trial with follow up of women who declined randomisation. SETTING: The gynaecology department of a large teaching hospital in the UK. POPULATION: All women referred for microwave endometrial ablation at the Aberdeen Royal Infirmary between July 1999 and September 2000 without a medical reason to favour one or other type of anaesthetic. METHODS: 191 women were equally randomised to undergo microwave endometrial ablation under general or local anaesthesia. Details were also collected for women not randomised because of an anaesthetic preference. All procedures were undertaken in an operating theatre. MAIN OUTCOME MEASURES: Data collected by questionnaire including the woman's view of treatment acceptability, operative details and post-operative recovery. RESULTS: Sixty-nine percent of eligible women would consider treatment under local anaesthesia. Ninety-one percent of microwave endometrial ablation procedures that started under local anaesthesia were completed without conversion to general anaesthesia. Anaesthetic type and allocation by randomisation or preference made no significant difference to the proportion of women describing treatment as totally or generally acceptable at two weeks. Neither parity nor cavity size predicted acceptability. Women allocated general anaesthesia were more likely to describe the procedure as totally acceptable and to choose the same anaesthetic again. There was no significant difference between anaesthetic groups regarding post-operative pain, nausea or recovery time. CONCLUSIONS: Microwave endometrial ablation under local anaesthesia was acceptable to the majority of women referred for treatment. There was no recovery advantage from local anaesthesia and almost 1 in 10 women who starting treatment under local anaesthesia needed a general anaesthetic because of discomfort. The incidence of post-operative pain and nausea means that treatment with this local anaesthetic/sedation regime remains a day case rather than an outpatient procedure.

Can obstetric complications explain the high levels of obstetric interventions and maternity service use among older women? A retrospective analysis of routinely collected data.

OBJECTIVE: To determine whether the higher levels of obstetric intervention and maternity service use among older women can be explained by obstetric complications. DESIGN: A retrospective analysis of routinely collected data from the Aberdeen Maternity and Neonatal Databank. PARTICIPANTS: All residents of Aberdeen city district delivering singleton infants at the Maternity Hospital 1988-1997 (28,484 deliveries). MAIN OUTCOME MEASURES: Odds ratios for each intervention in older maternal age groups compared with women aged 20-29. Interventions considered include obstetric interventions (induction of labour, augmentation, epidural use, assisted delivery, caesarean section) and raised maternity service use (more than two prenatal scans, amniocentesis, antenatal admission to hospital, admission at delivery of more than five days, infant resuscitation, and admission to the neonatal unit). METHODS: Logistic regression was used to investigate the association between maternal age and the incidence of interventions. The odds ratios for each intervention were then adjusted for relevant obstetric complications and maternal socio-demographic characteristics. RESULTS: Levels of amniocentesis, caesarean section, assisted delivery, induction, and augmentation (in primiparae) are all higher among older women. Maternity service use also increases significantly with age: older women are more likely to have an antenatal admission, more than two scans, a hospital stay at delivery of more than five days, and have their baby admitted to a neonatal unit. Controlling for relevant obstetric complications reveals several examples of effect modification, but does not eliminate the age effect for most interventions in most groups of women. CONCLUSIONS: Higher levels of intervention among older women are not explained by the obstetric complications we considered.

Holding up a mirror: changing obstetric practice through criterion-based clinical audit in developing countries.

The objective of the study described is to assess the feasibility and effectiveness of using a criterion-based clinical audit to measure and improve the quality of obstetric care at the district hospital level in developing countries. The focus is on the management of five life-threatening obstetric complications--hemorrhage, eclampsia, genital tract infection, obstructed labor and uterine rupture was audited using a "before and after" design. The five steps of the audit cycle were followed: establish criteria of good quality care; measure current practice (Review I); feedback findings and set targets; take action to change practice; and re-evaluate practice (Review II). Systematic literature review, panel discussions and pilot work led to the development of 31 audit criteria. Review I included 555 life-threatening complications occurring over 66 hospital-months; Review II included 342 complications over 42 hospital-months. Many common areas for improvement were identified across the four hospitals. Agreed mechanisms for achieving these improvements included clinical protocols, reviews of staffing, and training workshops. Some aspects of clinical monitoring, drug use and record keeping improved significantly between Reviews I and II. Criterion-based clinical audit in four typical district hospitals in Ghana and Jamaica is a feasible and acceptable method for quality assurance and appears to have improved the management of life-threatening obstetric complications.

An investigation of women's involvement in the decision to deliver by caesarean section.

OBJECTIVE: To assess the degree and nature of women's involvement in the decision to deliver by caesarean section, and women's satisfaction with this involvement. DESIGN: Observational study. SETTING: The maternity unit in a large teaching hospital. SAMPLE: One hundred and sixty-six women undergoing caesarean section. METHODS: Interviews with the women on the third or fourth day postpartum, questionnaires sent to the women at 6 weeks and at 12 weeks postpartum, and extraction of information from the women's medical records. MAIN OUTCOME MEASURES: Women's knowledge, satisfaction, and involvement in making the decision concerning their caesarean section. RESULTS: The majority of the women were satisfied with the information they received during pregnancy on caesarean section and with their involvement in making the decision, but the proportions were significantly higher for elective than emergency sections. For 7% of the women, maternal preference for caesarean section was a direct factor in making the decision. Just over half of the 166 women reported that they were not debriefed on the reasons for their caesarean section before their discharge from hospital. Almost a third of the women undergoing emergency caesarean section expressed negative feelings towards their delivery, compared with 13% of those undergoing elective caesarean sections. CONCLUSION: Women are not a homogeneous group in terms of their requirements for information, nor their desire to be involved in the decision on mode of delivery. Health professionals need to be responsive to this variability and to agree on standards for communicating with women during pregnancy about the possibility of operative delivery and for debriefing women after caesarean section.