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Depot-medroxyprogesterone acetate (DMPA) and Norethisterone Enanthate (NET-EN) are progestin-only injectable contraceptives widely used by women in sub-Sharan Africa, where incidence of HIV-1 and HSV-2 infection remains high. Studies indicate that DMPA usage can increase the risk of HSV-2 infection, but limited data indicate no increased risk with use of NET-EN. We therefore investigated the effects of NET-EN and DMPA on susceptibility to vaginal HSV-2 infection in ovariectomized (OVX) mice and effects on immune responses, particularly in the vaginal tract (VT). OVX mice, when treated with NET-EN and infected intravaginally, had delayed genital pathology, decreased viral shedding, and extended survival compared to DMPA- or untreated OVX mice. CD4+ T cells isolated from VT showed no significant change in frequency with either contraceptive. However, DMPA significantly decreased the total number of VT CD4+ and CD8+ T cells and the number of IFN-γ producing CD4 and CD8 T cells and increased the percentage of CD4 and CD8 T cells producing TNF-α compared to untreated mice. In contrast, NET-EN significantly enhanced percentages of CD8+ T cells compared to DMPA treated mice, and frequencies of IFN-γ+ CD4 and CD8 T cells in the VT compared to untreated mice. Comparative analysis of splenic lymphocytes indicated that DMPA treatment resulted in reduction of CD4+ T cell frequency, but enhanced TNF-α+ CD4 T cells compared to untreated mice. NET-EN enhanced the frequency of CD8 T cells, as well as IFN-γ+ and TNF-α+ CD4, and IFN-γ+ CD8 T cells in the spleen compared to untreated mice. Importantly, we found DMPA treatment that significantly reduced mucin production, whereas NET-EN enhanced expression of cell-associated mucin in VT. High levels of mucin in NET-EN mice were associated with lower levels of HSV-2 virus detected in the vaginal tract. This study provides the first evidence that NET-EN treatment can delay HSV-2 infection compared to DMPA.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1β (p

The ability to expand and activate natural Killer (NK) cells ex vivo has dramatically changed the landscape in the development of novel adoptive cell therapies for treating cancer over the last decade. NK cells have become a key player for cancer immunotherapy due to their innate ability to kill malignant cells while not harming healthy cells, allowing their potential use as an “off-the-shelf” product. Furthermore, recent advancements in NK cell genetic engineering methods have enabled the efficient generation of chimeric antigen receptor (CAR)-expressing NK cells that can exert both CAR-dependent and antigen-independent killing. Clinically, CAR-NK cells have shown promising efficacy and safety for treating CD19-expressing hematologic malignancies. While the number of pre-clinical studies using CAR-NK cells continues to expand, it is evident that solid tumors pose a unique challenge to NK cell-based adoptive cell therapies. Major barriers for efficacy include low NK cell trafficking and infiltration into solid tumor sites, low persistence, and immunosuppression by the harsh solid tumor microenvironment (TME). In this review we discuss the barriers posed by the solid tumor that prevent immune cell trafficking and NK cell effector functions. We then discuss promising strategies to enhance NK cell infiltration into solid tumor sites and activation within the TME. This includes NK cell-intrinsic and -extrinsic mechanisms such as NK cell engineering to resist TME-mediated inhibition and use of tumor-targeted agents such as oncolytic viruses expressing chemoattracting and activating payloads. We then discuss opportunities and challenges for using combination therapies to extend NK cell therapies for the treatment of solid tumors.

Abstract Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4+ and CD8+ T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications.

Stripped-back representative VCSEL devices with a simple fabrication process that very closely approaches the performance of standard BCB-planarised devices have been produced. These VCSEL Quick Fabrication (VQF) devices achieve threshold currents only 0.3 mA higher than that of a standard device produced from the same material. The predictability of standard performance from VQF performance is also robustly assessed in terms of temperature effects to account for the observed disparities. These VQF devices are then processed across a 6-inch (152 mm) wafer and the resulting device-level characteristics are mapped. From this, it is apparent that there is an approximately radial decrease in oxide aperture diameter from centre to edge, found to be driven by the strain-induced wafer bow. After corrections, a residual spatial variation across the wafer remains, which, in conjunction with temperature dependent measurements, is shown to be a result of epi-material variation. By observation at 50 °C, that is, at a temperature closely resembling that of intended application, the residual centre-to-edge variation in threshold current density is found to be only 0.2 kA/cm2, compared to 1.3 kA/cm2 when observing the room temperature variation of devices of nominally equivalent active volumes.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1β (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1β and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes. [ABSTRACT FROM AUTHOR]

In people living with HIV, Mycobacterium tuberculosis (Mtb) is the major cause of death. Due to the increased morbidity/mortality in co-infection, further research is urgently required. A limiting factor to research in HIV and HIV/Mtb co-infection is the lack of accessible in vivo models. Next-generation humanized mice expressing HLA transgenes report improved human immune reconstitution and functionality, which may better recapitulate human disease. This study compares well-established huNRG mice and next-generation HLA I/II-transgenic (huDRAG-A2) mice for immune reconstitution, disease course, and pathology in HIV and TB. HuDRAG-A2 mice have improved engraftment of key immune cell types involved in HIV and TB disease. Upon intravaginal HIV-1 infection, both models developed significant HIV target cell depletion in the blood and tissues. Upon intranasal Mtb infection, both models sustained high bacterial load within the lungs and tissue dissemination. Some huDRAG-A2 granulomas appeared more classically organized, characterized by focal central necrosis, multinucleated giant cells, and foamy macrophages surrounded by a halo of CD4+ T cells. HIV/Mtb co-infection in huNRG mice trended towards worsened TB pathology and showed potential for modeling co-infection. Both huNRG and huDRAG-A2 mice are viable options for investigating HIV and TB, but the huDRAG-A2 model may offer advantages.

Although antiretroviral therapy has transformed human immunodeficiency virus-type 1 (HIV-1) from a deadly infection into a chronic disease, it does not clear the viral reservoir, leaving HIV-1 as an uncurable infection. Currently, 1.2 million new HIV-1 infections occur globally each year, with little decrease over many years. Therefore, additional research is required to advance the current state of HIV management, find potential therapeutic strategies, and further understand the mechanisms of HIV pathogenesis and prevention strategies. Non-human primates (NHP) have been used extensively in HIV research and have provided critical advances within the field, but there are several issues that limit their use. Humanized mouse (Hu-mouse) models, or immunodeficient mice engrafted with human immune cells and/or tissues, provide a cost-effective and practical approach to create models for HIV research. Hu-mice closely parallel multiple aspects of human HIV infection and disease progression. Here, we highlight how innovations in Hu-mouse models have advanced HIV-1 research in the past decade. We discuss the effect of different background strains of mice, of modifications on the reconstitution of the immune cells, and the pros and cons of different human cells and/or tissue engraftment methods, on the ability to examine HIV-1 infection and immune response. Finally, we consider the newest advances in the Hu-mouse models and their potential to advance research in emerging areas of mucosal infections, understand the role of microbiota and the complex issues in HIV-TB co-infection. These innovations in Hu-mouse models hold the potential to significantly enhance mechanistic research to develop novel strategies for HIV prevention and therapeutics.

BackgroundBoth active and passive cigarette smoking have previously been associated with orofacial cleft aetiology. We aimed to analyse the impact of declining active smoking prevalence and the implementation of smoke-free legislation on the incidence of children born with a cleft lip and/or palate within the United Kingdom.Methods and findingsWe conducted regression analysis using national administrative data in the United Kingdom between 2000-2018. The main outcome measure was orofacial cleft incidence, reported annually for England, Wales and Northern Ireland and separately for Scotland. First, we conducted an ecological study with longitudinal time-series analysis using smoking prevalence data for females over 16 years of age. Second, we used a natural experiment design with interrupted time-series analysis to assess the impact of smoke-free legislation. Over the study period, the annual incidence of orofacial cleft per 10,000 live births ranged from 14.2-16.2 in England, Wales and Northern Ireland and 13.4-18.8 in Scotland. The proportion of active smokers amongst females in the United Kingdom declined by 37% during the study period. Adjusted regression analysis did not show a correlation between the proportion of active smokers and orofacial cleft incidence in either dataset, although we were unable to exclude a modest effect of the magnitude seen in individual-level observational studies. The data in England, Wales and Northern Ireland suggested an 8% reduction in orofacial cleft incidence (RR 0.92, 95%CI 0.85 to 0.99; P = 0.024) following the implementation of smoke-free legislation. In Scotland, there was weak evidence for an increase in orofacial cleft incidence following smoke-free legislation (RR 1.16, 95%CI 0.94 to 1.44; P = 0.173).ConclusionsThese two ecological studies offer a novel insight into the influence of smoking in orofacial cleft aetiology, adding to the evidence base from individual-level studies. Our results suggest that smoke-free legislation may have reduced orofacial cleft incidence in England, Wales and Northern Ireland.

Chemotherapeutic intervention remains the primary strategy in treating and controlling tuberculosis (TB). However, a complex interplay between therapeutic and patient-related factors leads to poor treatment adherence. This in turn continues to give rise to unacceptably high rates of disease relapse and the growing emergence of drug-resistant forms of TB. As such, there is considerable interest in strategies that simultaneously improve treatment outcome and shorten chemotherapy duration. Therapeutic vaccines represent one such approach which aims to accomplish this through boosting and/or priming novel anti-TB immune responses to accelerate disease resolution, shorten treatment duration, and enhance treatment success rates. Numerous therapeutic vaccine candidates are currently undergoing pre-clinical and clinical assessment, showing varying degrees of efficacy. By dissecting the underlying mechanisms/correlates of their successes and/or shortcomings, strategies can be identified to improve existing and future vaccine candidates. This mini-review will discuss the current understanding of therapeutic TB vaccine candidates, and discuss major strategies that can be implemented in advancing their development.

A systematic analysis of the performance of VCSELs, fabricated with a decreasing number of structural elements, is used to assess the complexity of fabrication (and therefore time) required to obtain sufficient information on epitaxial wafer suitability. Initially, sub-mA threshold current VCSEL devices are produced on AlGaAs-based material, designed for 940 nm emission, using processing methods widely employed in industry. From there, stripped-back Quick Fabrication (QF) devices, based on a bridge-mesa design, are fabricated and this negates the need for benzocyclcobutane (BCB) planarisation. Devices are produced with three variations on the QF design, to characterise the impact on laser performance from removing time-consuming process steps, including wet thermal oxidation and mechanical lapping used to reduce substrate thickness. An increase in threshold current of 1.5 mA for oxidised QF devices, relative to the standard VCSELs, and a further increase of 1.9 mA for unoxidised QF devices are observed, which is a result of leakage current. The tuning of the emission wavelength with current increases by ~0.1 nm/mA for a VCSEL with a 16 μm diameter mesa when the substrate is unlapped, which is ascribed to the increased thermal resistance. Generally, relative to the standard VCSELs, the QF methods employed do not significantly impact the threshold lasing wavelength and the differences in mean wavelengths of the device types that are observed are attributed to variation in cavity resonance with spatial position across the wafer, as determined by photovoltage spectroscopy measurements.

Abstract Background Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. Methods We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. Results After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. Conclusion NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.