Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response.

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

mPGES1-Dependent Prostaglandin E2 (PGE2) Controls Antigen-Specific Th17 and Th1 Responses by Regulating T Autocrine and Paracrine PGE2 Production.

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen-CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1-/- CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.

Polarization independence of laser-induced nucleation in supersaturated aqueous urea solutions.

In a seminal report on laser-induced nucleation in aqueous supersaturated solutions (Phys. Rev. Lett., 1996, 77, 3475) it was noted that needle-shaped crystals of urea were aligned with the direction of the electric field of the linearly polarized laser pulse. The results gave rise to a new mechanism for control of crystal nucleation involving alignment of solute molecules (optical Kerr effect) now commonly known as non-photochemical laser-induced nucleation (NPLIN). Recent theoretical and experimental work has cast doubts on the optical Kerr effect mechanism. In the present letter we present results from digital imaging of urea-crystal growth immediately following laser-induced nucleation. Analysis of the data shows no statistically significant correlation between crystal angle and direction of linear polarization. The results overturn a long-held result that has shaped theoretical and experimental studies of NPLIN.

The human immune system's response to carcinogenic and other infectious agents transmitted by mosquito vectors.

It has been hypothesised that mosquitoes [Diptera: Culicidae] may play more of a role in certain cancers than is currently appreciated. Research links 33 infectious agents to cancer, 27 of which have a presence in mosquitoes, and that, in addition, mosquito saliva downregulates the immune system. The objective of this paper is to review the literature on the immune system and cancer-causing infectious agents, particularly those present in mosquitoes, with a view to establishing whether such infectious agents can, in the long run, defeat the immune system or be defeated by it. Many of the viruses, bacteria and parasites recognised by the International Agency for Research on Cancer (IARC) as carcinogenic and suspected by others as being involved in cancer have evolved numerous complex ways of avoiding, suppressing or altering the immune system's responses. These features, coupled with the multiplicity and variety of serious infectious agents carried by some species of mosquitoes and the adverse effects on the immune system of mosquito saliva, suggest that post-mosquito bite the immune system is likely to be overwhelmed. In such a situation, immunisation strategies offer little chance of cancer prevention, unless a single or limited number of critical infectious agents can be isolated from the 'mosquito' cocktail. If that proves to be impossible cancer prevention will, therefore, if the hypothesis proves to be correct, rest on the twin strategies of environmentally controlling the mosquito population and humans avoiding being bitten. The latter strategy will involve determining the factors that demark those being bitten from those that are not.

Avian and simian malaria: do they have a cancer connection?

It has been claimed that infectious agents transmitted by mosquitoes (Diptera: Culicidae) may have a greater connection to cancer then hitherto supposed and that the immune system struggles to recognize and fight some of these infectious agents. One of the claims made is that there is a connection between human malaria and brain cancers in the USA. However, the USA declared itself free of human malaria in the last century, yet cancer incidences remain high, suggesting any overall cancer connection is slight. Two fundamental questions arise from the possible mosquito-cancer connection. Firstly, if mosquitoes are able to vector some pathogens and parasites linked with cancer pathogenesis, why has the fact not been discovered decades ago? Secondly, if there is a connection (other than in relation to Burkett's lymphoma), what is its extent? The answers may well lie with the various types of malarias known to exist. The discovery in humans of the simian malaria, caused by Plasmodium knowlesi, suggests that other forms of simian or even avian malaria may be capable of survival in humans, albeit at low levels of parasitemia, and humans may be a dead-end host. Other carcinogenic infectious agents transmitted by mosquitoes may also go undetected because either no one is looking for them, or they are looking in wrong anatomical locations and/or with inadequate tools. Research on false negative test results with respect to many infectious agents is sadly lacking, so its extent is unknown. However, electronic and other media provide numerous instances of patients failing to be diagnosed for both human malaria and Lyme's disease, to take just two examples. This review suggests that to shed light on a potential mosquito-cancer connection, more research is required to establish whether other simian and avian forms of malaria play a part. If so, then they potentially provide unique markers for early cancer detection.

Existing But Not Living: Experiences of Long-Term Caregivers of Individuals With Unipolar Depression.

The shift in care from long-term hospitalization of individuals with mental illness to the community places a greater onus of responsibility on informal caregivers. The purpose of the current study was to explore the lived experiences of long-term caregivers of individuals with unipolar depression. A qualitative phenomenological methodology was used and two sets of semi-structured interviews were conducted with nine informal caregivers. Data were transcribed following Giorgi's phenomenological method. The following three themes were identified: Flooded by Emotions, Personal Growth and Satisfaction, and Psychosocial Effects and Challenges. Caregivers described adapting by adjusting their behavior to avoid conflict with care recipients. These adjustments had a detrimental effect on their well-being, where they described that they were "existing but not living." These findings highlight the need for ongoing support, which should be tailored to the unique needs and concerns of individuals who are providing long-term care to individuals with depression. [Journal of Psychosocial Nursing and Mental Health Services, 55(12), 16-21.].

Heat shock transcription factor 1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress.

Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1(+/+) and HSF1(-/-) gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1(-/-) T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B-induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1(-/-) T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1(-/-) mice, but not HSF1(+/+) mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.

Laser-induced nucleation of carbon dioxide bubbles.

A detailed experimental study of laser-induced nucleation (LIN) of carbon dioxide (CO2) gas bubbles is presented. Water and aqueous sucrose solutions supersaturated with CO2 were exposed to single nanosecond pulses (5 ns, 532 nm, 2.4-14.5 MW cm(-2)) and femtosecond pulses (110 fs, 800 nm, 0.028-11 GW cm(-2)) of laser light. No bubbles were observed with the femtosecond pulses, even at high peak power densities (11 GW cm(-2)). For the nanosecond pulses, the number of bubbles produced per pulse showed a quadratic dependence on laser power, with a distinct power threshold below which no bubbles were observed. The number of bubbles observed increases linearly with sucrose concentration. It was found that filtering of solutions reduces the number of bubbles significantly. Although the femtosecond pulses have higher peak power densities than the nanosecond pulses, they have lower energy densities per pulse. A simple model for LIN of CO2 is presented, based on heating of nanoparticles to produce vapor bubbles that must expand to reach a critical bubble radius to continue growth. The results suggest that non-photochemical laser-induced nucleation of crystals could also be caused by heating of nanoparticles.

A periodicity of approximately 1 hour in X-ray emission from the active galaxy RE J1034+396.

Active galactic nuclei and quasars are thought to be scaled-up versions of Galactic black hole binaries, powered by accretion onto supermassive black holes with masses of 10(6)-10(9) M[symbol: see text], as opposed to the approximately 10 M [symbol: see text] in binaries (here M [symbol: see text] is the solar mass). One example of the similarities between these two types of systems is the characteristic rapid X-ray variability seen from the accretion flow. The power spectrum of this variability in black hole binaries consists of a broad noise with multiple quasi-periodic oscillations superimposed on it. Although the broad noise component has been observed in many active galactic nuclei, there have hitherto been no significant detections of quasi-periodic oscillations. Here we report the discovery of an approximately 1-hour X-ray periodicity in a bright active galaxy, RE J1034+396. The signal is highly statistically significant (at the 5.6 sigma level) and very coherent, with quality factor Q > 16. The X-ray modulation arises from the direct vicinity of the black hole.

Immunologic profiles of red blood cells using in vitro models of transfusion.

BACKGROUND: Transfusion of packed red blood cells (RBCs) produces a myriad of immunologic derangements, from suppressive to stimulatory. Proliferation of human T cells is suppressed in vitro after exposure to processed red blood cells (PRBCs). We hypothesized that this effect would be mitigated by using fresh RBCs. We also hypothesized that this suppressive effect was a generalized effect on lymphocyte proliferation and would be observed in both CD4+ and CD8+ T-cell subpopulations as well as B cells. MATERIALS AND METHODS: We isolated human T cells from donor peripheral blood mononuclear cells and exposed them to either blood bank PRBCs or fresh RBCs from volunteer donors and stimulated them with anti-CD3/anti-CD28. Human B cells were stimulated with lipopolysaccharide and exposed to PRBCs or fresh RBCs. We measured proliferation of B cells by thymidine incorporation assays. We also treated RBCs with citrate-phosphate-dextrose (CPD) at different time points before culture them with stimulated T cells to determine the role of this common RBC storage solution in lymphocyte proliferation. RESULTS: In vitro proliferation of CD4+ and CD8+ T cells was suppressed by blood bank RBCs. This suppression is eliminated when fresh RBCs were used. The B cells showed inhibition of proliferation when exposed to similar conditions, which appeared to be consistent over serial dilutions. Fresh RBCs exposed to CPD did not appear suppressive in the first 6 h after exposure. CONCLUSIONS: T-cell and B-cell proliferation inhibition by blood banked RBCs suggests a generalized effect of RBCs on cellular proliferation. The lack of suppression by fresh RBCs further suggests that something involved in blood banking alters RBC properties such that they attain a suppressive phenotype. One such blood banking component, CPD, does not appear to affect this suppressive phenotype within the first 6 h.

Exploring the effects of high pressure on hydrogen bonding in pharmaceutical cocrystals: A systematic study of pyridine dicarboxylic acid systems using synchrotron and neutron diffraction.

Pharmaceutical cocrystals use common robust hydrogen bonding synthons to create novel materials with different physicochemical properties. In this systematic study of a series of cocrystals, we explore the effect of high pressure on one of these commonly used motifs, the acid-pyridine motif, to assess the commonality of behaviour under extreme conditions. We have surveyed five pyridine dicarboxylic acid systems using both synchrotron and neutron diffraction methods to elucidate the changes in structure. We observe that the hydrogen bonding in these systems compress at a similar rate despite the changes to the molecular make-up of the solids and that on compression the changes in structure are indicative that the layers move along the major slip planes in the structure. We have observed two phase transitions to new forms of the pyrazine:malonic acid system, one for each stoichiometric ratio. This study demonstrates that the combination of two complementary diffraction approaches is key to understanding polymorphic behaviour at high pressure.

Exploring the thermal behaviour of the solvated structures of nifedipine.

Understanding the solvation and desolvation of pharmaceutical materials is an important part of materials discovery and development. In situ structural data are vital to understand the changes to crystal form that may occur in the system. In this study, the isolation and characterization of seven solvates of the L-type calcium channel antagonist, nifedipine, is described using variable-temperature powder X-ray diffraction so that the structural evolution as a function of temperature can be followed. The solvates reported herein can be split into those that are structurally similar to the previously reported dimethyl sulfoxide (DMSO) and dioxane solvates and those that have a novel packing arrangement. Of particular note is the solvate with tetrahydrofuran (THF) which has a hydrogen-bonding motif between the nifedipine molecules very similar to that of metastable ß-nifedipine. In addition to variable-temperature X-ray diffraction, the stability of the solid forms was assessed using differential scanning calorimetry and thermogravimetric analysis and indicates that in all cases desolvation results in the thermodynamically stable α-polymorph of nifedipine even with the THF solvate. From the diffraction data the pathway of desolvation during heating of the DMF solvate showed conversion to another likely 1:1 polymorph before desolvation to α-nifedipine. The desolvation of this material indicated a two-stage process; first the initial loss of 90% of the solvent before the last 10% is lost on melting. The methanol solvate shows interesting negative thermal expansion on heating, which is rarely reported in organic materials, but this behaviour can be linked back to the winerack-type hydrogen-bonding pattern of the nifedipine molecules.

Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Pushing Technique Boundaries to Probe Conformational Polymorphism.

We present an extensive exploration of the solid-form landscape of chlorpropamide (CPA) using a combined experimental-computational approach at the frontiers of both fields. We have obtained new conformational polymorphs of CPA, placing them into context with known forms using flexible-molecule crystal structure prediction. We highlight the formation of a new polymorph (ζ-CPA) via spray-drying experiments despite its notable metastability (14 kJ/mol) relative to the thermodynamic α-form, and we identify and resolve the ball-milled η-form isolated in 2019. Additionally, we employ impurity- and gel-assisted crystallization to control polymorphism and the formation of novel multicomponent forms. We, thus, demonstrate the power of this collaborative screening approach to observe, rationalize, and control the formation of new metastable forms.

Pressure-induced postsynthetic cluster anion substitution in a MIL-53 topology scandium metal-organic framework.

Postsynthetic modification of metal-organic frameworks (MOFs) has proven to be a hugely powerful tool to tune physical properties and introduce functionality, by exploiting reactive sites on both the MOF linkers and their inorganic secondary building units (SBUs), and so has facilitated a wide range of applications. Studies into the reactivity of MOF SBUs have focussed solely on removal of neutral coordinating solvents, or direct exchange of linkers such as carboxylates, despite the prevalence of ancillary charge-balancing oxide and hydroxide ligands found in many SBUs. Herein, we show that the µ2-OH ligands in the MIL-53 topology Sc MOF, GUF-1, are labile, and can be substituted for µ2-OCH3 units through reaction with pore-bound methanol molecules in a very rare example of pressure-induced postsynthetic modification. Using comprehensive solid-state NMR spectroscopic analysis, we show an order of magnitude increase in this cluster anion substitution process after exposing bulk samples suspended in methanol to a pressure of 0.8 GPa in a large volume press. Additionally, single crystals compressed in diamond anvil cells with methanol as the pressure-transmitting medium have enabled full structural characterisation of the process across a range of pressures, leading to a quantitative single-crystal to single-crystal conversion at 4.98 GPa. This unexpected SBU reactivity - in this case chemisorption of methanol - has implications across a range of MOF chemistry, from activation of small molecules for heterogeneous catalysis to chemical stability, and we expect cluster anion substitution to be developed into a highly convenient novel method for modifying the internal pore surface and chemistry of a range of porous materials.

Non-photochemical laser-induced nucleation of supercooled glacial acetic acid.

Non-photochemical laser-induced nucleation (NPLIN) of glacial acetic acid (GAA) is demonstrated. The fraction of samples nucleated depends linearly on peak laser power density at low powers (<100 MW cm(-2)) with a threshold of (9.0 ± 4.2) MW cm(-2); at higher laser powers the fraction reaches a plateau of 0.75 ± 0.24 (2σ uncertainties). A simple model based on polarizability of pre-nucleating clusters gives a value of the solid-liquid interfacial tension γ(SL) = 15.5 mJ m(-2). It is hoped that the results will stimulate new developments in experimental and theoretical studies of cluster structure and nucleation in liquids.

Heat shock factor 1 protects mice from rapid death during Listeria monocytogenes infection by regulating expression of tumor necrosis factor alpha during fever.

Heat shock factor 1 (HSF1) is a stress-induced transcription factor that promotes expression of genes that protect mammalian cells from the lethal effects of severely elevated temperatures (>42°C). However, we recently showed that HSF1 is activated at a lower temperature (39.5°C) in T cells, suggesting that HSF1 may be important for preserving T cell function during pathogen-induced fever responses. To test this, we examined the role of HSF1 in clearance of Listeria monocytogenes, an intracellular bacterial pathogen that elicits a strong CD8(+) T cell response in mice. Using temperature transponder microchips, we showed that the core body temperature increased approximately 2°C in L. monocytogenes-infected mice and that the fever response was maintained for at least 24 h. HSF1-deficient mice cleared a low-dose infection with slightly slower kinetics than did HSF1(+/+) littermate controls but were significantly more susceptible to challenges with higher doses of bacteria. Surprisingly, HSF1-deficient mice did not show a defect in CD8(+) T cell responses following sublethal infection. However, when HSF1-deficient mice were challenged with high doses of L. monocytogenes, increased levels of serum tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) compared to those of littermate control mice were observed, and rapid death of the animals occurred within 48 to 60 h of infection. Neutralization of TNF-α enhanced the survival of HSF1-deficient mice. These results suggest that HSF1 is needed to prevent the overproduction of proinflammatory cytokines and subsequent death due to septic shock that can result following high-dose challenge with bacterial pathogens.

Narrow emission linewidths of positioned InAs quantum dots grown on pre-patterned GaAs(100) substrates.

We report photoluminescence measurements on a single layer of site-controlled InAs quantum dots (QDs) grown by molecular beam epitaxy (MBE) on pre-patterned GaAs(100) substrates with a 15 nm re-growth buffer separating the dots from the re-growth interface. A process for cleaning the re-growth interface allows us to measure single dot emission linewidths of 80 µeV under non-resonant optical excitation, similar to that observed for self-assembled QDs. The dots reveal excitonic transitions confirmed by power dependence and fine structure splitting measurements. The emission wavelengths are stable, which indicates the absence of a fluctuating charge background in the sample and confirms the cleanliness of the re-growth interface.

Chiral hide-and-seek: retention of enantiomorphism in laser-induced nucleation of molten sodium chlorate.

We report the observation of non-photochemical laser-induced nucleation (NPLIN) of sodium chlorate from its melt using nanosecond pulses of light at 1064 nm. The fraction of samples that nucleate is shown to depend linearly on the peak power density of the laser pulses. Remarkably, we observe that most samples are nucleated by the laser back into the enantiomorph (dextrorotatory or levorotatory) of the solid prior to melting. We do not observe a significant dependence on polarization of the light, and we put forward symmetry arguments that rule out an optical Kerr effect mechanism. Our observations of retention of chirality can be explained by decomposition of small amounts of the sodium chlorate to form sodium chloride, which provide cavities for retention of clusters of sodium chlorate even 18 °C above the melting point. These clusters remain sub-critical on cooling, but can be activated by NPLIN via an isotropic polarizability mechanism. We have developed a heterogeneous model of NPLIN in cavities, which reproduces the experimental data using simple physical data available for sodium chlorate.

Pressure-induced superelastic behaviour of isonicotinamide.

Dynamic organic crystals have come to the fore as potential lightweight alternatives to inorganic actuators providing high weight-to-force ratios. We have observed pressure-induced superelastic behaviour in Form I of isonicotinamide. The reversible single-crystal to single-crystal transformation exhibited by the system is an important component for functioning actuators. Crucially, our observations have enabled us to propose a mechanism for the molecular movement supported by Pixel energy calculations, that may pave the way for the future design and development of functioning dynamic crystals.